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Library of Congress Cataloging-in-Publication Data
Biomaterials fabrication and processing handbook / [edited by] Paul K. Chu and Xuanyong Liu.
p. ; cm.
“A CRC title.”
Includes bibliographical references and index.
ISBN 978-0-8493-7973-4 (alk. paper)
1. Biomedical materials. 2. Biomedical engineering. I. Chu, Paul K. II. Liu, Xuanyong. III. Title.
[DNLM: 1. Biocompatible Materials. 2. Biosensing Techniques. 3. Nanotechnology--methods. 4.
Tissue Engineering--methods. QT 37 B61413 2008]
R857.M3B5696 2008
610.284--dc22 2007042613
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![4 Biomaterials Fabrication and Processing Handbook
1.4.2.4 Microsphere Sintering................................................................................... 31
1.4.2.5 Foam Coating................................................................................................ 31
1.5 Surface Functionalization.......................................................................................................32
1.5.1 Protein Adsorption ......................................................................................................32
1.5.2 Silane-Modified Surfaces (Silanization Technique)....................................................32
1.5.3 Topography (Roughness) Modification .......................................................................33
1.5.4 Polymer Coatings ........................................................................................................33
1.6 Conclusions .............................................................................................................................33
References........................................................................................................................................34
1.1 INTRODUCTION
Being a modern discipline, tissue engineering encounters various challenges, such as the develop-
ment of suitable scaffolds that temporarily provide mechanical support to cells at an early stage of
implantation until the cells are able to produce their own extracellular matrix (ECM) [1]. Numerous
biomaterials and techniques to produce three-dimensional (3-D) tissue-engineering scaffolds have
been considered; biomaterials include polymers, ceramics, and their composites, as discussed in the
literature [1–3]. In this chapter, we present an up-to-date summary of the fabrication technologies
for tissue-engineering scaffolds, including the choice of suitable materials and related fabrication
techniques, with a focus on the development of synthetic scaffolds based on bioceramics, glasses,
and their composites combined with biopolymers for bone regeneration. Being one of the most
promising technologies, the replication method for the production of highly porous, biodegrad-
able, and mechanically competent Bioglass®
-derived glass-ceramic scaffolds is highlighted. The
enhancement of scaffold properties and functions by surface modification is also discussed, and
examples of novel approaches are given.
1.2 DESIGN OF 3-D SCAFFOLDS
In an organ, cells and their ECM are organized into 3-D tissues. Therefore, in tissue engineering
a highly porous 3-D matrix (i.e., scaffold) is necessary to accommodate cells and to guide their
growth and tissue regeneration in 3-D structures. This is particularly relevant in the field of bone
tissue engineering and regeneration, bone being a highly hierarchical 3-D composite structure.
Moreover, the structure of bone tissue varies with its location in the body. So the selection of
configurations as well as appropriate biomaterials depends on the anatomic site for regeneration,
the mechanical loads present at the site, and the desired rate of incorporation. Ideally, the scaffold
should be porous enough to support cell penetration, tissue ingrowth, rapid vascular invasion, and
nutrient delivery. Moreover, the matrix should be designed to guide the formation of new bones in
anatomically relevant shapes, and its degradation kinetics should be such that the biodegradable
scaffold retains its physical (e.g., mechanical) properties for at least 6 months (for in vitro and in vivo
tissue regeneration) [1,3]. Important scaffold design parameters are summarized in Table 1.1.
The design of highly porous scaffolds involves a critical issue related to their mechanical prop-
erties and structural integrity, which are time dependent. For example, it has been reported that
the compressive strength of hydroxyapatite scaffolds increases from ∼10 to ∼30 MPa because of
tissue ingrowth in vivo [5]. This finding leads to a conclusion that it might not be necessary to have
a starting scaffold with a mechanical strength equal to that of a bone, because cultured cells on the
scaffold in vitro will create a biocomposite and increase the strength of the scaffold significantly.
Another factor that affects scaffold design is the need for vascularization and angiogenesis
in the constructs [6]. In vitro engineering approaches face the problem of critical thickness while
regenerating tissue in the absence of true vascularization: mass transportation into tissue is dif-
ficult beyond a thin peripheral layer of a tissue construct even if artificial means are used to supply
nutrients and oxygen [7]. Diffusion barriers that are present in vitro are most likely to become more
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 5
deleterious in vivo due to lack of vascularization. Once the engineered tissue construct is placed in
the body, vascularization becomes a key issue for further remodeling in the in vivo environment.
Thus, angiogenesis is an essential step in the colonization of macroporous biomaterials during
osteointegration. Capillaries bring osteoprogenitor cells and the nutriments that are required for
their growth. They transport especially numerous angiogenic growth factors [8].
The main critical factors affecting bone formation are the pore size and pore interconnection
of the scaffold. Pore size is related to the in vivo bone tissue ingrowth, allowing migration and
proliferation of osteoblasts and mesenchymal cells, and matrix deposition in the empty spaces [9].
Pore interconnection provides the channel for cell distribution and migration allowing efficient
in vivo blood vessel formation. An incomplete pore interconnection could limit blood vessels
invasion. Small pore size could obstruct cell adhesion and bone ingrowth. Bone vascularization,
besides providing nutrients essential for tissue survival, plays also a crucial role in coordinating
the activity of bone cells and their migration for new bone formation [10].
Several studies have investigated the minimum pore size required to regenerate mineralized
bone. The minimum requirement for pore size is considered to be around 100 µm due to cell size,
migration requirements, and transport. However, pore sizes >300 µm are recommended due to
enhanced growth rate of a new bone and the formation of capillaries [3,4,11]. Pore size in the range
of 300–500 µm would promote vascularization and mass transportation of nutrients and waste
products, while the scaffold would maintain good mechanical integrity during in vitro culture and
in vivo transplantation [12].
It is equally important to notice that tissue-engineering scaffolds should have enhanced biologi-
cal functions. Therefore, the incorporation of growth factors, such as bone growth factors (BGF)
and vascularization growth factors (VGF), or specific peptide sequences into the scaffolds or on
their surface is being considered as part of the integral design of scaffolds. Moreover, to improve
cell attachment and growth, the surface of scaffolds’ struts needs to be pretreated (a process called
surface functionalization) [13–15]. The design of the surface properties of scaffolds is an important
step to achieve their successful in vitro and in vivo applications. A few approaches to surface modi-
fication of scaffolds are discussed below.
TABLE 1.1
Scaffold Design Parameters for Bone Tissue Engineering [4]
Parameters Requirements
Porosity Maximum possible without compromising mechanical
properties
Pore size 200–400 µm
Pore structure Interconnected
Mechanical properties of the cancellous bone
Tension and compression Strength: 5–10 MPa
Modulus: 50–100 MPa
Mechanical properties of the cortical bone
Tension Strength: 80–150 MPa
Modulus: 17–20 GPa
Compression Strength: 130–220 MPa
Modulus: 17–20 GPa
Fracture toughness: 6–8 MPa√
__
m
Degradation properties
Degradation time Must be tailored to match the application in patients
Degradation mechanism Bulk dissolution in medium
Biocompatibility No chronic inflammation
Sterilizability Sterilizable without altering material properties
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![6 Biomaterials Fabrication and Processing Handbook
1.3 SCAFFOLD MATERIALS FOR BONE TISSUE ENGINEERING
The first step in achieving a successful scaffold is to choose a suitable biomaterial. Natural bone
matrix is a composite of biological ceramic (a natural apatite) and biological polymer. Carbonated
hydroxyapatite Ca10(PO4)6(OH)2 accounts for nearly two-thirds of the weight of a bone. The inor-
ganic component provides compressive strength to the bone. Roughly one-third of the weight of
a bone is from collagen fibers. Collagen fibers are tough and flexible, and thus tolerate stretching,
twisting, and bending. It is not surprising that polymers, ceramics, or their composites have been
chosen for bone repair [16]. They can be either synthetic or naturally occurring ones. Table 1.2 lists
synthetic and natural scaffold biomaterials that have been most widely investigated for bone regen-
eration, some of which are well-established and clinically applicable. In this section, the biocompat-
ibility, biodegradability, and mechanical properties of these scaffold materials, which are the most
essential factors to be considered in the fabrication of bone regeneration scaffold, are reviewed
concisely. Particular attention is paid to a key issue that remains with almost all existing scaffold
biomaterials, that is, mechanically strong materials (in crystalline structure) tend to be bioinert, and
biodegradable materials (in amorphous structure) are, in general, mechanically weak. An excep-
tion, 45S5 Bioglass-derived glass-ceramic, is considered in more detail because the issue associated
with the two apparently irreconcilable properties (mechanical strength and biodegradability) have
been successfully addressed in this material [17].
1.3.1 BIOCERAMICS: CALCIUM PHOSPHATES
1.3.1.1 Biocompatibility
Since almost two-thirds of the weight of a bone is hydroxyapatite Ca10(PO4)6(OH)2, it seems logi-
cal to use this ceramic as a major component of scaffold materials for bone tissue engineering.
Actually, hydroxyapatite and related calcium phosphates (e.g., β-tricalcium phosphate [β-TCP])
have been intensively investigated [16,18,21]. As expected, calcium phosphates have an excellent
biocompatibility due to their close chemical and crystal resemblance to bone mineral [19,20].
Although they have not shown osteoinductive ability, they certainly possess osteoconductive prop-
erties as well as a remarkable ability to bind directly to bone [32–35]. A high number of in vivo
and in vitro assessments have concluded that calcium phosphates, no matter which forms (bulk,
coating, powder, or porous) and which phases (crystalline or amorphous) they are in, always sup-
port the attachment, differentiation, and proliferation of cells (such as osteoblasts and mesenchy-
mal cells), with hydroxyapatite being the best among these scaffold materials [36]. Although the
excellent biological performance of hydroxyapatite and related calcium phosphates has been well-
documented, the slow biodegradation of their crystalline phases and the weak mechanical strength
of their amorphous states limit their application in engineering of new bone tissue, especially at
load-bearing sites.
1.3.1.2 Degradability
Typically, crystalline calcium phosphates have a long degradation time in vivo, often of the order of
years [37]. The dissolution rate of synthetic hydroxyapatite depends on the type and concentration
of the buffered or unbuffered solutions, pH of the solution, degree of the saturation of the solution,
solid and solution ratio, length of suspension in the solution, as well as composition and crystallinity
of the hydroxyapatite. In the case of crystalline hydroxyapatite, the degree of micro and macropo-
rosities, defect in the structure, and amount and type of other phases present also have significant
influence [39]. Crystalline hydroxyapatite exhibits the slowest degradation rate, compared with
other calcium phosphates. The dissolution rate decreases in the following order [38]:
Amorphous hydroxyapatite > all other calcium phosphates (e.g., TCP) >
> crystalline
hydroxyapatite.
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 7
1.3.1.3 Mechanical Properties
The properties of synthetic calcium phosphates vary significantly with their crystallinity, grain
size, porosity, and composition (e.g., calcium deficiency). In general, the mechanical properties of
synthetic calcium phosphates decrease significantly with increasing content of amorphous phase,
microporosity, and grain size. High crystallinity, low porosity, and small grain size tend to give
TABLE 1.2
List of Promising Scaffold Biomaterials for Bone Regeneration
Biomaterials Abbreviation Application
Ceramics [16,18]
Calcium phosphates [19–21]
Hydroxyapatite
Tricalcium phosphate
Biphasic calcium phosphate: HA and TCP
CaP
HA
TCP
BCP
Dental
Drug delivery
Scaffolds
Bioactive glasses [22–25]
Bioglass
Phosphate glasses
Dental
Drug delivery
Scaffolds
Bioactive Glass-Ceramics [26,27]
Apatite-Wollastonite
Ceravital
A/W Dental
Drug delivery
Scaffolds
Polymers [28–31]
Synthetic degradable polymers
Bulk biodegradable polymers
Aliphatic polyester
Poly(lactic acid)
Poly(d-lactic acid)
Poly(l-lactic acid)
Poly(d,l-lactic acid)
Poly(glycolic acid)
Poly(lactic-co-glycolic acid)
Poly(ε-caprolactone)
Poly(hydroxyalkanoate)
Poly(3- or 4-hydroxybutyrate)
Poly(3-hydroxyoctanoate)
Poly(3-hydroxyvalerate)
Polydioxanone
Poly(propylene fumarate)
PLA
PDLA
PLLA
PDLLA
PGA
PLGA
PCL
PHA
PHB
PHO
PHV
PPF
Sutures
Dental
Orthopedic
Drug delivery
Scaffolds
Surface bioerodible polymers
Poly(ortho esters)
Poly(anhydrides)
Poly(phosphazene)
POE
PPHOS
Drug delivery
Natural degradable polymers
Polysaccharides
Hyaluronan
Alginate
Chitosan
Proteins
Collagen
Fibrin
HyA
Composites [12]
Composed of the above-mentioned ceramics and polymers
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![8 Biomaterials Fabrication and Processing Handbook
higher stiffness, higher compressive and tensile strength, and greater fracture toughness [39,40].
It has been reported that the flexural strength and fracture toughness of dense hydroxyapatite are
much lower in a dry condition than in a wet condition [41]. The mechanical properties of hydroxy-
apatite and related calcium phosphates, as well as those of bone, are given in Table 1.3.
In brief, hydroxyapatite and related calcium phosphates exhibit excellent biocompatibility and
osteoconductivity. However, these materials are poorly degradable in case of crystalline structures,
and their amorphous counterparts are mechanically too fragile to be used for fabrication of highly
porous tissue-engineering scaffolds.
1.3.2 BIOCERAMICS: BIOACTIVE SILICATE GLASSES
1.3.2.1 Biocompatibility
As early as in 1969, Hench and colleagues discovered that certain silicate glass compositions had
excellent biocompatibility as well as the ability of bone bonding [23–25]. Through interfacial and
cell-mediated reactions, bioactive glass develops a calcium-deficient, carbonated calcium phosphate
surface layer that allows it to chemically bond to the host bone. This bone-bonding behavior is
referred to as “bioactivity” and has been associated with the formation of a carbonated hydroxy-
apatite layer on the glass surface when implanted or when in contact with biological fluids [47–50].
Bioactivity is not an exclusive property of bioactive silicate glasses. Hydroxyapatite and related
calcium phosphates also show an excellent bone-bonding ability, as discussed above. The capability
of a material to form a biological interface with the surrounding tissue is critical in avoiding scaf-
fold loosening in vivo.
Bioactive glasses have also been found to support enzyme activity [51–54], vascularization
[55,56], as well as foster osteoblast adhesion, growth, and differentiation. Bioactive glasses were
also shown to induce the differentiation of mesenchymal cells into osteoblasts [57–59] and to pro-
vide osteoconductivity [60].
A significant finding for the development of bone engineering is that the dissolution products
from bioactive glasses exert a genetic control over osteoblast cycle and rapid expression of genes
that regulate osteogenesis and the production of growth factors [61,62]. Silicon has been found to
play a key role in the bone mineralization and gene activation, which has led to the substitution
of silicon for calcium into synthetic hydroxyapatite. Investigations in vivo have shown that bone
ingrowth into silicon-substituted hydroxyapatite granules was remarkably greater than that into
pure hydroxyapatite [62,63].
The above-mentioned advantages make 45S5 Bioglass a very successful material in clinical
applications, for example, for the treatment of periodontal disease (PerioGlas) and as a bone-filler
material (NovaBone) [63,64]. Bioglass implants have also been used to replace damaged middle
ear bones, restoring auditory capabilities of patients [64]. Recently bioactive glasses have gained
attention as promising scaffold materials for bone tissue engineering [64–69]. Similar to calcium
phosphates, the application of this material, particularly in tissue engineering, has encountered a
TABLE 1.3
Comparison of Mechanical Properties of Calcium Phosphates and Human Bone
Ceramics
Compressive
Strength
(MPa)
Tensile Strength
(MPa)
Elastic Modulus
(GPa)
Fracture
Toughness
(MPa√
__
m) References
Calcium phosphates 20–900 30–200 30–103 <1.0 39,42
Hydroxyapatite >400 ∼40 ∼100 ∼1.0 39,42
Cortical bone 130–180 50–151 12–18 6–8 28,43–46
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 9
hurdle caused by the conflict between the properties of biodegradability and mechanical reliability,
which is discussed in Sections 1.3.2.3 and 1.3.3.4.
1.3.2.2 Biodegradability
The basic constituents of most bioactive glasses are SiO2, Na2O, CaO, and P2O5. The well-known
45S5 Bioglass contains 45% SiO2, 24.5% Na2O, 24.4% CaO, and 6% P2O5, in weight percent. The
bioreactivity of the material is composition-dependent. Hench and coworkers [22] have systemati-
cally studied a series of glasses in the four-component systems with a constant 6 wt.% P2O5 content.
This work is summarized in the ternary SiO2–Na2O–CaO diagram shown in Figure 1.1. In region A,
the glasses are bioactive and bond to bone. In region B, glasses are nearly inert when implanted.
Compositions in region C are resorbed within 10–30 days in tissue. In region D, the compositions
are not technically practical.
The key advantage of bioactive glasses that makes them promising scaffold materials is the
possibility of controlling a range of chemical properties and thereby the rate of bioresorption. The
structure and chemistry of glasses, in particular sol–gel derived glasses [47,48], can be tailored at
a molecular level by varying either composition, or thermal or environmental processing history. It
is possible to design glasses with degradation properties specific to a particular application of bone
tissue engineering.
1.3.2.3 Mechanical Properties
A primary disadvantage of bioactive glasses is their low fracture toughness (Table 1.4) because of
their amorphous structure. Hence, many researchers sintered bioactive glasses at their crystalliza-
tion temperatures in order to improve the mechanical performance of these materials. However,
it was reported that crystallization of bioactive glasses could decrease the level of bioactivity [70]
FIGURE 1.1 Compositional dependence (in wt.%) of bone bonding and soft tissue bonding of bioactive
glasses and glass-ceramics. Bioactivity index IB is defined as IB = 100/t0.5, where t0.5 is the time taken for 50%
of the interface to bond to bone. All compositions have a constant 6 wt.% of P2O5. In region A, the glasses are
bioactive and bond to bone. In region B, glasses are nearly inert when implanted. Compositions in region C are
resorbed within 10–30 days in tissue. In region D, the compositions are not technically practical. In the region
where IB > 8 (called region E), soft tissue bonding occurs. Apatite-wollastonite glass-ceramic (A-WGC) has
higher P2O5 content [22].
CaO Na2O
SiO2
A-WGC
(high P2O5)
D
B
C
1
8
5
2
0
A
I
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![10 Biomaterials Fabrication and Processing Handbook
and even turn a bioactive glass into an inert material [71]. This is one of the disadvantages that
limit the application of bioactive glasses as scaffold materials, as full crystallization occurs prior to
significant densification upon heat treatment (i.e., sintering) [72]. Extensive sintering is necessary
to densify the struts of a scaffold, which would otherwise be made up of loosely packed particles
and thus the structure would be too fragile to handle. Most recently, Boccaccini’s group at Imperial
College London [17] reported on a phase transformation from a mechanically competent crystalline
phase to a biodegradable amorphous calcium phosphate in 45S5 Bioglass-derived scaffolds. This
phase transition, which takes place in a biological environment at body temperature, couples the
two required properties (mechanical strength and biodegradability) in a single scaffold. A detailed
characterization of this material is given in Section 1.3.3.4.
In summary, like hydroxyapatite and related calcium phosphates, bioactive glasses exhibit good
biocompatibility and osteoconductivity. At the same time, all these materials, except 45S5 Bioglass-
derived glass-ceramics, encounter a similar disadvantage, that is, a mechanically strong scaffold
has to be achieved through crystallization, which unfortunately hampers the biodegradability of
these materials.
1.3.3 BIOCERAMICS: GLASS-CERAMICS
Glasses can be strengthened by the formation of crystalline particles in the glass matrix upon heat
treatment in the relevant glass-crystal region of its phase diagram. The resultant glass-ceramics usually
exhibit better mechanical properties than both the parent glass and sintered crystalline ceramics (e.g.,
sintered hydroxyapatite) (Table 1.4). There are many biomedical glass-ceramics available for the repair
of damaged bones. Among them, apatite-wollastonite (A-W), Ceravital, and Bioverit glass-ceramics
have been intensively investigated [16,18]. Recently, a 45S5 Bioglass-derived glass-ceramic showed a
great potential as a tissue-engineering scaffold material, as mentioned above (Section 1.3.2.3).
1.3.3.1 A-W Glass-Ceramics
In A-W glass-ceramic, the glass matrix is reinforced by β-wollastonite (CaSiO3) crystals and a
small amount of apatite phase, which precipitate successively at 870°C and 900°C, respectively
[75]. Some mechanical properties of this glass-ceramic have been listed in Table 1.4. The high
bending strength (215 MPa) of A-W glass-ceramic is due to the precipitation of wollastonite as well
as apatite. These two precipitates also give the glass-ceramic a higher fracture toughness than that
of both the glass and ceramic phases. It is believed that wollastonite effectively prevents straight
propagation of cracks, causing them to deflect or branch out [26,75–77].
A-W glass-ceramic is capable of binding tightly to a living bone in a few weeks after implanta-
tion, and the implants do not deteriorate in vivo [78]. The excellent bone-bonding ability of A-W
TABLE 1.4
Mechanical Properties of Hydroxyapatite, 45S5 Bioglass, Glass-Ceramics, and Human
Cortical Bone
Ceramics
Compression
Strength (MPa)
Tensile Strength
(MPa)
Elastic Modulus
(GPa)
Fracture
Toughness
(MPa√
__
m) References
45S5 Bioglass 500 42 35 0.5–1 42,73
A-W 1080 215 (bend) 118 2.0 26
Parent glass of A-W NA 72 (bend) NA 0.8 26
Bioverit I 500 140–180 (bend) 70–90 1.2–2.1 74
Cortical bone 130–180 50–151 12–18 6–8 28,43–46
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 11
glass-ceramic is attributed to the glass matrix and apatite precipitates, whereas the in vivo stability
as a whole is due to the inertness of β-wollastonite. Although the long-term integrity in vivo is desir-
able in the application of nonresorbable prosthesis, the material does not match the goal of tissue
engineering, which demands biodegradable scaffolds.
1.3.3.2 Ceravital Glass-Ceramics [79]
“Ceravital” was coined to mean a number of different compositions of glasses and glass-ceramics
and not only one product. Their basic network components include SiO2, Ca(PO2)2, CaO, Na2O,
MgO, and K2O, with ceramic additions being Al2O3, Ta2O5, TiO2, B2O3, Al(PO3)3, SrO, La2O3, or
Gd2O3. This material system was developed as solid fillers in the load-bearing conditions for the
replacement of bone and teeth. It turned out, however, that their mechanical properties do not serve
the purpose, and there has been virtually no research on the application of this material in tissue-
engineering scaffolds.
1.3.3.3 Bioverit Glass-Ceramics [74]
Bioverit products are mica-apatite glass-ceramics. Mica crystals (aluminum silicate minerals) give
the materials good machinability, and apatite crystals ensure the bioactivity of the implants. The
mechanical properties of Bioverit materials (Table 1.4) allow them to be used as fillers in dental
application. As regards bioreactivity, Bioverit implants show a hydrolytic stability in vivo. As for
Ceravital glass-ceramics, no significant research has been carried out regarding the use of this
glass-ceramic in tissue engineering.
1.3.3.4 45S5 Bioglass-Derived Glass-Ceramics
In 2005, Chen et al. [80] fabricated a 3-D, highly porous, mechanically competent, bioactive and
biodegradable scaffold for the first time by the replication technique using 45S5 Bioglass pow-
der. Under an optimum sintering condition (1000°C/h), nearly full densification of the foam struts
occurred and fine crystals of Na2Ca2Si3O9 are formed, which conferred the scaffolds the highest
possible compressive and flexural strength for this foam structure. Important findings in this work
are that the mechanically strong crystalline phase Na2Ca2Si3O9 can transform into an amorphous
calcium phosphate phase after immersion in simulated body fluid (SBF) for 28 days and that the
transformation kinetics can be tailored by controlling the crystallinity of the sintered 45S5 Bio-
glass. As such, it was demonstrated that the goal of an ideal scaffold that provides good mechanical
support temporarily while maintaining bioactivity and that can biodegrade at later stages at a tailor-
able rate can be achieved with these Bioglass-based scaffolds [17].
1.3.4 NATURALLY OCCURRING BIOPOLYMERS
Much research effort has been focused on naturally occurring polymers such as demineralized bone
ECM [81], purified collagen [82,83], and chitosan [84] for tissue engineering applications. Theoreti-
cally, naturally occurring polymers should not cause response of foreign materials when implanted.
They provide a natural substrate for cellular attachment, proliferation, and differentiation in their
native state. For these reasons, naturally occurring polymers could be a favorite substrate for tis-
sue engineering [28]. Table 1.5 provides a list of some of the naturally occurring polymers, their
sources, and applications. Among them, collagen and chitosan are most widely investigated for
bone engineering and are briefly discussed here.
1.3.4.1 Collagen and ECM-Based Materials
The most commonly used naturally occurring polymer is the structural protein collagen. Bioma-
terials derived from ECM include collagen and other naturally occurring structural and functional
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proteins. Natural polymers must be modified and sterilized before clinical use. All methods of
stabilization and sterilization can moderately or severely alter the rate of in vivo degradation and
change the mechanical and physical properties of the native polymers. Each method has certain
advantages and disadvantages, and thus should be selectively utilized for scaffolds of specifically
sited bone tissue engineering [86].
1.3.4.2 Chitosan
The use of chitosan for bone tissue engineering has been widely investigated [84,87]. This is in
part due to the apparent osteoconductive properties of chitosan. Mesenchymal stem cells cultured
in the presence of chitosan have demonstrated an increased differentiation to osteoblasts compared
with cells cultured in the absence of chitosan [88]. It is also speculated that chitosan may enhance
osteoconduction in vivo by entrapping growth factors at the wound site [89].
1.3.5 SYNTHETIC POLYMERS
Although naturally occurring polymers possess the above-mentioned advantages, their poor
mechanical properties and variable physical properties with different sources of protein matrices
have hampered their progress in broad applications in tissue engineering. Concerns have also
been expressed regarding immunogenic problems associated with the introduction of foreign
collagen [37].
Following the developmental efforts regarding the use of naturally occurring polymers as scaf-
folds, much attention has been paid to synthetic polymers. Synthetic polymers have high potential in
tissue engineering not only because of their excellent processing characteristics, which can ensure
their off-the-shelf availability, but also because of their advantage of being biocompatible and bio-
degradable [37,90]. Synthetic polymers have predictable and reproducible mechanical and physical
properties (e.g., tensile strength, elastic modulus, and degradation rate) and can be manufactured
with great precision. Although they are unfamiliar to cells and many have some shortcomings,
such as eliciting persistent inflammatory reactions, being eroded, not being compliant or able to
TABLE 1.5
List of Naturally Occurring Polymers, Their Sources, and Applications [85]
Polymers Source Application
Collagen Tendons and ligament Multiapplications, including bone
tissue engineering
Collagen-Glycosaminoglycan (GAG)
(alginate) copolymers
Artificial skin grafts for skin
replacement
Albumin In blood Transporting protein used as coating
to form a thromboresistant surface
Hyaluronic acid In the ECM of all higher animals An important starting material for
preparation of new biocompatible
and biodegradable polymers that
have applications in drug delivery,
tissue engineering, and
viscosupplementation
Fibrinogen–Fibrin Purified from plasma in blood Multiapplications, including bone
tissue engineering
Chitosan Shells of shrimps and crabs Multiapplications, including bone
tissue engineering
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 13
integrate with the host tissues, they may be replaced in vivo in a timely fashion by native constructs
built by the cells seeded into them. It has been widely accepted that an ideal tissue-engineered bone
substitute should be a synthetic scaffold, which is biocompatible and provides for cell attachment,
proliferation and maturation, has mechanical properties to match those of the tissues at the site of
implantation, and degrades at rates to match tissue replacement. Table 1.6 lists selected properties
of synthetic, biocompatible, and biodegradable polymers that have been intensively investigated as
scaffold materials for tissue engineering, type I collagen fibers being included for comparison.
1.3.5.1 Bulk Degradable Polymers
1.3.5.1.1 Saturated Poly-α-Hydroxyesters (PLA, PGA, and PCL)
The biodegradable synthetic polymers most often utilized for 3-D scaffolds in tissue engineer-
ing are the poly(α-hydroxyacids), including poly(lactic acid) (PLA) and poly(glycolic acid) (PGA),
as well as poly(lactic-co-glycolide) (PLGA) copolymers [91]. PLA exists in three forms: l-PLA
(PLLA), d-PLA (PDLA), and racemic mixture of d,l-PLA (PDLLA).
These polymers are popular for various reasons, among which biocompatibility and biode-
gradability stand out. These materials have chemical properties that allow hydrolytic degradation
through de-esterification. After the process of degradation is over, the monomeric components of
each polymer are removed through natural pathways: PGA can be converted to other metabolites
or eliminated by other mechanisms, and PLA can be cleared through tricarboxylic acid cycle. The
body already contains highly regulated mechanisms for completely removing monomeric compo-
nents of lactic and glycolic acids. Due to these properties, PLA and PGA have been used in products
such as degradable sutures and have been approved by the U.S. Food and Drug Administration
(FDA) [28]. Other significant properties of these polymers are their very good processability, and
their ability to exhibit a wide range of degradation rates, physical, mechanical, and other proper-
ties, which can be achieved by PLA and PGA of various molecular weights and their copolymers.
However, these polymers undergo a bulk erosion process in contact with body fluids such that they
can cause scaffolds to fail prematurely. In addition, abrupt release of these acidic degradation prod-
ucts can cause a strong inflammatory response [92,93].
In general, PGA degrades faster than PLA, as listed in Table 1.6. Their degradation rates
decrease in the following order.
PGA>PDLLA>PLLA
Degradation rates decrease
Table 1.6 also lists the mechanical properties of type I collagen, which is the major organic
component of ECM in bone. The strength and ductility (e.g., ultimate elongation) of PLA and PGA
are comparable to those of type I collagen fibers.
PDLLA has been extensively investigated as a biomedical coating material because of its excel-
lent features with respect to implant surface [28,104]. In addition to its high mechanical stabil-
ity [105], PDLLA also shows excellent biocompatibility in vivo and good osteoinductive potential
[106]. PDLLA of low molecular weight can be combined with drugs like growth factors [106],
antibiotics [107], or thrombin inhibitors [108] to establish a locally acting drug-delivery system. It
is due to these desirable features that much more attention has recently been paid to PDLLA for
applying it as a scaffold material for tissue engineering.
Highly porous 3-D scaffolds made of Bioglass-filled PDLLA and PLGA were fabricated by
Boccaccini et al. [59]. Since then an increasing number of publications have emerged on this subject,
as reviewed recently [12]. Porous PDLLA foams and Bioglass-filled PDLLA composite foams have
both been fabricated, using thermally induced–phase separation (TIPS) technique [109,110]. Bioglass-
filled PDLLA composite foams exhibit high bioactivity, assessed by the formation of hydroxyapatite
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 15
on the strut surfaces upon immersion in SBF [111]. It has also been shown that the foams support the
migration, adhesion, spreading, and viability of MG-63 cells (osteosarcoma cell line) [112].
Poly(ε-caprolactone) (PCL) is also an important member of the aliphatic polyester family. It
has been used to effectively entrap antibiotic drugs and thus a construct made with PCL can be
considered as a drug-delivery system, being used to enhance bone ingrowth and regeneration in
the treatment of bone defects [113]. The degradation of PCL and its copolymers involves similar
mechanisms to PLA, proceeding in two stages: random hydrolytic ester cleavage and weight loss
through the diffusion of oligometric species from the bulk. It has been found that the degradation of
PCL system with a high molecular weight (
__
Mn of 50,000) is remarkably slow, requiring 3 years for
complete removal from the host body [114].
1.3.5.1.2 Polyhydroxyalkanoates (PHB, PHBV, P4HB, PHBHHx, PHO)
Recently, polyhydroxyalkanoates (PHAs), another type of polyesters, have been suggested for tis-
sue engineering because of their controllable biodegradation and high biocompatibility [115]. They
are aliphatic polyesters as well, but produced by microorganisms under unbalanced growth condi-
tions [116,117]. They are generally biodegradable (via hydrolysis) and thermoprocessable, making
them attractive as biomaterials for application in medical devices and tissue engineering. Over
the past years, PHA, particularly poly-3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate
and 3-hydroxyvalerate (PHBV); poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate
and 3-hydroxyhexanoate (PHBHHx); and poly 3-hydroxyoctanoate (PHO) were demonstrated to be
suitable for tissue engineering and are reviewed in detail in Refs. 115,116.
Depending on the property requirement of different applications, PHA polymers can be either
blended, surface modified, or composed with other polymers, enzymes, or inorganic materials to
further adjust their mechanical properties or biocompatibility. The blending among the several PHA
themselves can dramatically change their material properties and biocompatibility [115,116].
PHB is of particular interest for bone tissue application as it was demonstrated to produce a
consistent favorable bone tissue adaptation response with no evidence of an undesirable chronic
inflammatory response after an implantation period of up to 12 months [116]. The bone is formed
close to the material and subsequently becomes highly organized, with up to 80% of the implant
surface lying in direct apposition to the new bone. The materials showed no evidence of extensive
structural breakdown in vivo during the implantation period of the study [118].
However, a drawback of some PHA polymers is their limited availability and the time-consuming
extraction procedure from bacterial cultures that is required for obtaining sufficient processing
amounts as described in the literature [115,119]. Therefore, the extraction process might be a chal-
lenge to a cost-effective industrial upscale production for large amounts of some PHA polymers.
1.3.5.1.3 Polypropylene Fumarate
Poly(propylene fumarate) (PPF) is an unsaturated linear polyester. Similar to PLA and PGA, the
degradation products of PPF through hydrolysis (i.e., propylene glycol and fumaric acid) are bio-
compatible and readily removed from the body. The double bond along the backbone of the polymer
permits cross-linking in situ, which causes a moldable composite to harden within 10–15 min.
Mechanical properties and degradation time of the composite may be controlled by varying the
PPF molecular weight. Therefore, preservation of the double bonds and control of molecular weight
during PPF synthesis are critical issues [120]. PPF has been suggested for use as scaffold for guided
tissue regeneration, often as part of an injectable bone replacement composite [121], and has been
used as a substrate for osteoblast culture [122].
1.3.5.2 Surface Bioeroding Polymers
There is a family of hydrophobic polymers that undergo a heterogeneous hydrolysis process, which
is predominantly confined to the polymer–water interface. This property is referred to as surface
eroding as opposed to bulk degrading behavior. These surface bioeroding polymers have been
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intensively investigated as drug-delivery vehicles. The surface-eroding characteristic offers three
key advantages over bulk degradation when used as scaffold materials: (1) retention of mechani-
cal integrity over the degradative lifetime of the device, owing to the maintenance of mass to vol-
ume ratio; (2) minimal toxic effects (i.e., local acidity), owing to lower solubility and concentration
of degradation products; and (3) significantly enhanced bone ingrowth into the porous scaffolds,
owing to the increment in pore size as the erosion proceeds [123].
1.3.5.2.1 Poly(anhydrides)
Poly(1,3-bis-p-carboxyphenoxypropane anhydride) [124] and poly(erucic acid dimer anhydride)
[125] are biodegradable polymers for controlled drug delivery in a form of implant or injectable
microspheres. Studies in rabbits have shown that the osteocompatibility of poly(anhydrides) that
undergo photocuring are comparable to PLA and that the implants of poly(anhydrides) show
enhanced integration with the surrounding bones in comparison to PLA controls [126].
1.3.5.2.2 Poly(ortho-esters)
Poly(ortho-esters) (POE) scaffolds were coated with cross-linked acidic gelatine to improve surface
properties for cell attachment. Preliminary in vitro and in vivo results revealed that POE did not
show any inflammation and had little or no effect on bone formation while PLA provoked a chronic
inflammatory response and inhibited bone formation [127,128].
1.3.5.2.3 Polyphosphazenes
These polymers seem to be potential bioerodible materials capable of controlled degradation and
sustained drug delivery for therapeutic use [101,129] and bone regeneration [130]. Their tailored
side groups enable a wide variety of hydrolytic properties to be designed into selected polymers
for application in biological environments without the release of harmful degradation products at
physiological concentration.
1.3.6 BIOCOMPOSITES
From a biological perspective, it is a natural strategy to combine polymers and ceramics to fabricate
scaffolds for bone tissue engineering because native bone is the combination of a naturally occurring
polymer and a biological apatite. From the point of view of materials science, a single material type
does not always provide the necessary mechanical and chemical properties desired for a particular
application. In these instances, composite materials designed to combine the advantages of both
components may be most appropriate. Polymers and ceramics that degrade in vivo should be cho-
sen for designing biocomposites for tissue-engineering scaffolds. While massive release of acidic
degradation from polymers can cause inflammatory reactions [4,92,131], the basic degradation of
calcium phosphate or bioactive glasses would buffer the acidic by-products of polymers and may
thereby help to avoid the formation of an unfavorable environment for cells due to a decreased pH
level. Mechanically, bioceramics are much stronger than polymers and play a critical role in provid-
ing mechanical stability to constructs prior to the synthesis of a new bone matrix by cells. However,
ceramics and glasses are very fragile because of their intrinsic brittleness and flaw sensitivity. To
capitalize on their advantages and minimize their shortcomings, ceramic and glass materials have
been combined with various biopolymers to form composite biomaterials for osseous regenera-
tion. Table 1.7 lists selected ceramic/glass–polymer composites, which were designed as biomedical
devices or scaffold materials for bone tissue engineering, and their mechanical properties.
In general, all these synthetic composites have good biocompatibility. Kikuchi et al. [132], for
instance, combined TCP with PLA to form a polymer–ceramic composite, which was found to pos-
sess the osteoconductivity of β-TCP and the degradability of PLA [132].
The research team led by Laurencin [147] synthesized porous scaffolds containing PLGA and
hydroxyapatite, which were reported to combine the degradability of PLGA with the bioactiv-
ity of hydroxyapatite, fostering cell proliferation and differentiation as well as mineral formation
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[147,157,158]. Similarly, composites of bioactive glass and PLA were observed to form calcium
phosphate layers on their surfaces and support rapid and abundant growth of human osteoblasts and
osteoblast-like cells when cultured in vitro [109–112,148–154].
A comparison between the dense composites and cortical bone indicates that the most promis-
ing synthetic composite seems to be hydroxyapatite fiber–reinforced PLA composite [134], which,
however, exhibit mechanical property values close to the lower values of the cortical bone. Other
promising composite scaffolds reported in literature are those from Bioglass and PLLA or PDLLA
[149–152]. They have a well-defined porous structure, for example obtained by thermally induced
phase separation [151], at the same time their mechanical properties are close to (but lower than)
those of cancellous bone.
1.3.7 SUMMARY
To design an ideal scaffold, which is bioresorbable, biocompatible, provides for cell attachment,
proliferation, and maturation, and which disappears whenever a new bone forms allowing the new
bone to undergo remodeling, it is necessary to weight up the pros and cons of the potential precursor
materials, as summarized in Table 1.8.
Among the bioactive ceramics and glasses listed in Table 1.8, bioactive (silicate) glasses have
remarkable advantages. The ability to enhance vascularization, the role of silicon in upregulating
TABLE 1.8
Advantages and Disadvantages of Synthetic Scaffold Biomaterials in Bone
Tissue Engineering
Biomaterials Positive Negative
Calcium phosphates
(e.g., HA, TCP, and BPCP)
1. Excellent biocompatibility
2. Supporting cell activity
3. Good osteoconductivity
1. Too fragile in amorphous
structure
2. Nearly bioinert in crystalline
phase
Bioactive glasses and glass-ceramics 1. Excellent biocompatibility
2. Supporting cell activity
3. Good osteconductivity
4. Vascularization
5. Upregulation of gene expression
6. Tailorable degradation rate
1. Mechanically brittle and weak
in the glass state
2. Degrade slowly in crystalline
structures, except for 45S5
Bioglass-derived glass-ceramics
Bulk biodegradable polymers
(e.g., PLA, PGA, PLGA, PPF)
1. Good biocompatibility
2. Biodegradable with a wide range of
degradation rates
3. Bioresorbable
4. Good processability
5. Good ductility
1. Inflammation caused by acid
degradation products
2. Accelerated degradation rates
cause collapse of scaffolds
Surface bioerodible polymers
(e.g., POE, poly(anhydrides),
poly(phosphazene))
1. Good biocompatibility
2. Retention of mechanical integrity
over the degradative life of the device
3. Significantly enhanced bone ingrowth
into the porous scaffolds, owing to
the increment in pore size
1. They cannot be completely
replaced by new bone tissue
Composites 1. Excellent biocompatibility
2. Supporting cell activity
3. Good osteconductivity
4. Tailorable degradation rate
5. Improved mechanical properties
1. Fabrication techniques can
be complex
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 19
gene expression, and the tailorable degradation rate make bioactive glasses promising scaffold
materials over others, and so they could be the material of choice as the inorganic component of
composite scaffolds. Although bioactive glasses are brittle with low fracture toughness (Table 1.4),
they can be used in combination with polymers to form composite materials. The ability to couple
mechanical strength with tailorable biodegradability makes 45S5 Bioglass-derived glass-ceramics
advantageous over calcium phosphates (including hydroxyapatite), as well as other bioactive glasses
and related glass-ceramics.
Between the two types of polymers, the bulk degradable type is more promising than the
surface-erosive group, considering that being replaced by new bone tissue is one of the important
criteria of an ideal scaffold material (Table 1.1). Finally, it is obvious that composites can be consid-
ered ideal scaffolding materials for bone tissue engineering if fabrication processes suitable for the
production of 3-D structures of the required size and shape and amenable to commercialization are
further developed and optimised.
1.4 FABRICATION OF TISSUE-ENGINEERING SCAFFOLDS
1.4.1 FABRICATION OF INORGANIC SCAFFOLDS
Porous ceramics can be produced by a variety of different processes [2,159], which may be classi-
fied into two main categories: (1) manual-based processing techniques and (2) computer-controlled
fabrication processes, such as solid free-form (SFF) technology, which is also commonly known as
rapid prototyping (RP) [160]. Most manual-based processing techniques can further be divided into
two groups: conventional powder-forming processes and sol–gel techniques [161].
1.4.1.1 Powder-Forming Processes
A flowchart that is common to all powder-forming processes is shown in Figure 1.2, and the differ-
ent steps involved in these processes are discussed in this section.
1.4.1.1.1 Preparation of Slurries
Slurry is a suspension of ceramic particles in a suitable liquid (e.g., water or ethanol) used to prepare
green bodies. The inherent mechanism of pore formation in a powder compact is illustrated in
Figure 1.3. Attractive forces that consist of hydrogen bonds, van der Waals forces, Coulomb’s forces,
and physical friction between particles cause agglomeration of particles. Addition of fillers to the
FIGURE 1.2 Flowchart of the powder-sintering method to produce porous ceramic scaffolds.
Heat treatment of the green body
to sinter the ceramic structure
Prepare slurry from the powder
Form a green body from the slurry
Start with a ceramic powder
Porous ceramic
Add Additives
(e.g., porogen, binder)
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slurry, such as sucrose, gelatine, and PMMA microbeads, and a wetting agent (i.e., a surfactant)
can increase porosity. These chemicals, which are called porogens, are evaporated or burned out
during sintering, and as a result pores are formed [2,159]. One successful formulation has been the
use of hydroxyapatite powder slurries (dispersed with vegetable oil) added with gelatine solution
[162], which has led to porous scaffolds with interconnected pore structure with pore diameters of
∼100 µm. A similar process has been used to prepare melt-derived Bioglass scaffolds using cam-
phor (C10H16O) as the porogen [163].
Binders are also added to slurries. The most important function of a binder is to improve the
strength of the green body in order to provide structural integrity for handling (green strength)
before the product is sintered [164]. Polysaccharides [165], polyvinyl alcohol (PVA) [166], and poly-
vinyl butyl (PVB) [167] are the frequently added binders in bioceramic slurries.
1.4.1.1.2 Formation of Green Bodies
In ceramic production, a green body is always porous, and its structure largely determines that of
the sintered product. Table 1.9 lists different methods of obtaining green bodies for 3-D porous
ceramics. These methods can be classified into two categories: dry and wet processes [159].
They lead to different porous structures and pore volume fractions. Certain techniques, such as
tape casting, extrusion, slurry dipping, and spraying, are not included here; because they aim at
achieving a predetermined geometric shape of ceramic parts (such as rods, tubes, sheets, and
coating on films), instead of a given porous structure. Except injection molding, all conventional
processes listed in Table 1.9 have been applied to synthesize ceramic scaffolds for tissue engi-
neering as discussed below.
FIGURE 1.3 Schematic illustration of pores among agglomerates and particles [159].
Primary particle
Agglomerate of
primary particles
Pore among agglomerates
Pore among primary particles
TABLE 1.9
Methods for Obtaining Ceramic Bodies for 3-D Porous Ceramics [159]
Dry processes
Loose packing
Compaction
Uniaxial pressing
Cold isostatic pressing (CIPing)
Wet processes
Slip casting
Injection molding
Phase separation/freeze-drying
Polymer replication
Gelcasting
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 21
Dry methods. The simplest way to prepare ceramic green bodies is the dry powder method
where powders are directly compressed by pressing (uniaxially or isostatically) into molds, thereby
forming green bodies. Pore diameters decrease and mechanical properties increase as the packing
density of the particles in the green bodies increases. A densification step by sintering at high tem-
perature is required (see Section 1.4.1.1.3). Mechanical properties can be increased further by hot-
isostatic pressing (HIP) [168] or by uniaxial hot pressing. These pressure-assisted methods decrease
the pore diameter as well. The addition of porogens, such as sucrose and camphor, enhances the
formation of pores [159].
Slip casting. Slip is a creamy (relatively thick) slurry. In this method, the slurry is cast into a
porous mold. The liquid of the slurry is absorbed into the porous mold, and as a result the particles
in the slurry are filtered, which adhere to the mold surface. After this process, a porous green body
is obtained through further drying [161,169].
Phase separation/freeze-drying. In this method, a ceramic slurry is poured into a container,
which is immersed in a freezing bath. Thus, ice is stimulated to grow and ceramic particles are piled
up between the columns of the growing ice. After the slurry is completely frozen, the container is
dried in a drying vessel, usually under vacuum [170]. The pores are created by the ice crystals that
sublimate at a reduced pressure. Freeze-drying removal of ice crystals creates 3-D interconnected
pore channels with complex structures. The porous structure can be customized by the variation of
the slurry concentration, freezing temperature, and pressure.
Replication technique. This method, which is also called the polymer-sponge method, was
patented for the manufacturing of ceramic foams [171]. In the polymer-replication process, the
green bodies of ceramic foams are prepared by coating a polymer (e.g., polyurethane) foam
with a ceramic slurry. The polymer foam, which already has the desired macrostructure, simply
serves as a sacrificial template for the ceramic coating. The polymer template is immersed in
the slurry, which subsequently infiltrates the structure, and so the ceramic particles adhere to
the surface of the polymer substrate. Excess slurry is squeezed out leaving a ceramic coating on
the foam struts. After it is dried, the polymer is slowly burned out in order to minimize dam-
age to the porous ceramic coating. After the removal of the polymer, the ceramic is sintered to
the desired density. The process replicates the macroporous structure of the polymer foam and
results in a rather distinctive microstructure within the struts. A flowchart of the process is given
in Figure 1.4 [172]. This method has been applied for the preparation of foam-like scaffolds for
tissue engineering, including porous calcium phosphates [173], Bioglass [80], and other inert
bioceramics [172,174].
FIGURE 1.4 Flowchart of the replication process to produce a ceramic foam.
Dry, burn out the polymer substrate,
and sinter the green body
Coat a polymer foam with the slurry
Prepare slurry from the powder
Ceramic powder
Ceramic foam
Add
Binder
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Apart from the slurry-immersion coating, electrospray coating techniques have also been
applied together with the polymer-sponge process to produce ceramic foams, for example, Al2O3
[175] and ZrO2 foams [176]. Unlike the foams produced by the slurry-immersion method, the struts
of the ceramic foams produced by the process of electrospray coating contain fewer holes and
cracks. This microstructure can lead to improved mechanical properties of the foams [176].
Another possibility investigated to improve the mechanical properties of foams made by the rep-
lication method is to apply a thin polymer coating on the porous structure. For example, to improve
the mechanical stability of highly porous Bioglass-derived scaffolds produced by the replication
technique [80], a polymer coating, such as poly(d,l-lactic acid) (PDLLA), was applied [177]. The
coating thickness was approximately 3 µm on an average. Although the thin coating layer did not
increase the mechanical strength of the foams considerably, it significantly improved the mechani-
cal stability of the structure. The fracture energy of the coated foams was ∼20 times higher than
that of uncoated foams. More importantly, upon immersion in SBF, nanofibers of hydroxyapatite
deposited within the PDLLA coating layer, eventually a nanocomposite layer, formed biomimeti-
cally on the strut surfaces. This method has remarkably improved the mechanical performance of
the scaffolds in a biological environment [177].
Gelcasting. This method adopts one of the direct-foaming techniques mentioned in Table 1.10
to achieve highly porous green bodies. The foamed suspension is set through a direct-consolidation
technique, listed in Table 1.10, that is, polymerization of organic monomers (i.e., gelation), in which
the particles of the slurry are consolidated through polymerization reaction. A green body is formed
after the gel is cast in a mold [178–180]. Figure 1.5 gives the flowchart of the gelcasting process.
Two factors are critical in the gelcasting process: (1) the gelation speed must be fast enough to
prevent foam collapse, and (2) the gel rheology is important because the process involves casting.
Systems of high fluidity are required in order to enable easy filling of small details in molds to allow
production of high-complexity shapes. Gelcasting techniques have been applied to produce hydroxy-
apatite foams [181–183]. Gelcasting has also been combined with the replication process (described
above in this section) to produce hydroxyapatite scaffolds with interconnected pores [184].
1.4.1.1.3 Sintering
The final step in the production of a ceramic foam is the densification of the green bodies by con-
ducting a high temperature sintering process. Foams are normally dried at room temperature for at
least 24 h prior to sintering. In this step, controlled heating is important to prevent collapse of the
ceramic network. The heating rate, sintering temperature, and holding time depend on the ceramic
starting materials. For example, values are in the range of 0.5–2°C/min, 1200–1350°C, and 2–5 h,
TABLE 1.10
Techniques of Direct Foaming and Direct Consolidation
Techniques References
Direct foaming
1. Injection of gases through the fluid medium
2. Mechanically agitating particulate suspension
3. Blowing agents
4. Evaporation of compounds
5. Evaporation of gas by in situ chemical reaction
20
Direct consolidation
1. Gelcasting
2. Direct coagulation consolidation (DCC)
3. Hydrolysis-assisted solidification (HAS)
4. Freezing (quick set)
178
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 23
respectively, in the case of porous hydroxyapatite [173,181,183,185]. It is worthwhile noticing that
there is a narrow time–temperature window for densification of foams made from bioactive glasses,
which are prone to crystallize while sintering by viscous flow. Hence the production of bioactive
glass foams by powder-based methods presents difficulties [80].
1.4.1.2 Sol–Gel Techniques
1.4.1.2.1 Sol–Gel Process and Synthesis of Aerogel Ceramics
The sol–gel process is a well-developed, robust, and versatile “wet” technique for the synthesis of
ceramics and glasses. By applying the sol–gel process, it is possible to fabricate inorganic mate-
rials in various forms: ultrafine or spherical shaped powders, thin film coatings, ceramic fibers,
microporous inorganic membranes, monolithic ceramics and glasses, and extremely porous aerogel
materials [186].
The processing path of aerogel ceramics starts with an alkoxide precursor. Alkoxide precursors,
such as tetraethyl orthosilicate (TEOS) and triethoxyl orthophosphate (TEP), undergo hydrolysis
and condensation reactions to form a sol. In case of silicate precursors, polymerization of –Si–OH
groups continues after hydrolysis is complete, beginning the formation of the silicate (–Si–O–Si–)
network. The network connectivity increases until it spans throughout the solvent medium. Eventually
a wet gel forms. The wet gel is then subjected to controlled thermal processes of aging to strengthen
the gel, drying to remove the liquid by-product of the polycondensation reaction, and thermal sta-
bilization (or sintering) to remove organic species from the surface of the material; and as a result,
a porous aerogel forms [2,187].
1.4.1.2.2 Production of Highly Porous Glasses
Highly porous glasses (or glass foams) have been developed by a slightly modified sol–gel pro-
cess [188]. The sol–gel process is based on the polymerization reactions of metal alkoxide precur-
sors (usually TEOS and TEP). These precursors are dissolved in a solvent, and a gel is formed by
hydrolysis and condensation reactions. The gel is then subjected to controlled thermal processes of
aging to strengthen the gel, drying to remove the liquid by-product of the polycondensation reac-
tion, and thermal stabilization/sintering to remove organic species from the surface of the material
(500–800°C). Sol–gel derived glass scaffolds are obtained by directly foaming the sol with the use
Prepare suspension from the powder
Ceramic powder
Add Dispersant, surfactant,
monomer, cross-linker
Foam the suspension using one of the
foaming techniques in Table 2.16
While the foamed suspension is poly
merized to form a gel, cast the gel
Ceramic foam
Dry and sinter the green body
Initiator, catalyst
Add
FIGURE 1.5 Flowchart of the gelcasting method to produce a ceramic foam.
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![24 Biomaterials Fabrication and Processing Handbook
of a surfactant and catalysts [188–190]. Therefore, after sol hydrolysis, the surfactant (e.g., Teepol,
a detergent containing a low-concentration mixture of anionic and nonionic surfactants), water
(improves foamability of surfactant), and the catalyst for polycondensation (e.g., HF) are added by
vigorous agitation. A flowchart of the process is given in Figure 1.6. Porosity of the foam scaffolds
is influenced by the foaming temperature, water content, and catalyst content. Sol–gel derived bio-
active glass foams [191,192] and gelcast hydroxyapatite scaffolds [181,183] have shown favorable
results in both in vitro and in vivo tests for bone regeneration.
1.4.1.3 Solid Free-Form Techniques
SFF techniques, also known as RP, are computer-controlled fabrication processes. They can rapidly
produce highly complex 3-D objects using data generated by computer-aided design (CAD) systems.
In a typical case, an image of a bone defect in a patient can be taken, which is used to develop a
3-D CAD model. The computer can then reduce the model to slices or layers. The 3-D objects are
constructed layer-by-layer using RP techniques such as fused deposition modeling (FDM), selective
laser sintering (SLS), 3-D printing (3-DP), or stereolithography [160]. Calcium phosphate scaffolds
have been produced using the FDM process [193,194], SLS, 3-DP processes [160], stereolithogra-
phy [195,196], and RP combined with replication technique [197]. The typical process chain for all
SFF techniques is presented in Figure 1.7.
To date, only a small number of SFF techniques, such as 3-DP, FDM, and SLS, have been
adopted for tissue-engineering scaffolds. The following paragraphs give brief descriptions of the
principles on which these three techniques are based. Comprehensive technical details can be found
in previous detailed reviews [160,198–201].
1.4.1.3.1 Three-Dimensional Printing
Three-dimensional printing employs ink-jet printing technology for processing materials from pow-
ders. Therefore, this technique is a combination of SFF and powder sintering. During fabrication, a
printer head is used to print a liquid binder onto thin layers of powder following the object’s profile
being generated by the system computer. The subsequent stacking and printing layer recreates the
full structure of the desired object.
Alkoxides: TEOS and TEP
Prepare a sol from the alkoxides and
Ca(NO3)2 in deionized water solvent
Add Catalysis (HNO3) to
speed up hydrolysis
Foam the sol by vigorous agitation
When the gelation of the foamed sol is
nearly completed, cast the gel in molds
Glass foam
Age, dry, and sinter the gel
Surfactant for foaming,
catalyst (HF) for gelation
Add
FIGURE 1.6 Flowchart of the production of bioactive glass foams using sol–gel technology.
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 25
1.4.1.3.2 Fused Deposition Modeling
FDM employs the concept of melt extrusion to deposit a parallel series of material rods that forms a
material layer. In FDM, filament material stock (generally thermoplastic) is fed and melted inside a
heated liquefier head before being extruded through a nozzle with a small orifice.
Indirect fabrication methods involving FDM have been applied for producing porous bioc-
eramic implants. In this method, FDM was employed to fabricate wax molds containing the nega-
tive profiles of the desired scaffold microstructure. Ceramic scaffolds were then cast from the mold
through a lost mold technique [193,194].
1.4.1.3.3 Selective Laser Sintering
SLS employs a CO2 laser beam to selectively sinter polymer, ceramic, or polymer-ceramic com-
posite powders to form material layers. The laser beam is directed onto the powder bed by a high
precision laser scanning system. The fusion of material layers that are stacked on top of one another
replicates the object’s height [202,203].
1.4.1.4 Comparison of Fabrication Techniques for Ceramic or Glass Scaffolds
Table 1.11 lists the porosity, pore size, and mechanical properties of several porous ceramics pro-
duced by different techniques. Figure 1.8 shows typical pore structures produced by different tech-
niques. Comparing the pore structures of ceramic scaffolds shown in Figure 1.8 with the structure
of cancellous bone, it is evident that the pore morphology produced by the replication technique is
the most similar one, containing completely interconnecting pores and solid material forming only
the struts. The ceramic foams synthesized by gelcasting and sol–gel techniques come next in terms
of structural similarity to cancellous bone, however, it is expected that these foams exhibit lower
pore interconnectivity than foams made by the replication method.
The advantages of replication method over other ceramic foaming techniques are summarized
in Table 1.12. In brief, the replication technique meets all criteria posed on the fabrication process of
tissue-engineering scaffolds: suitable for commercialization, reproducible, cost-effective, safe, and
capable of producing irregular or complex shapes. Contemporary authors consider the replication
technique as the optimal technique for production of novel bioactive glass-ceramic scaffolds for
bone tissue engineering [204].
FIGURE 1.7 Flowchart of the typical rapid prototyping (RP) process [160].
Medical imaging
• CT, MRI, etc.
3-D solid model creation in CAD
• Pro/engineer (PTC)
SFF system computer
• Generation of slice data, etc.
SFF fabrication
• SLS, FDM, etc.
Post-processing
• Finishing and cleaning
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![28 Biomaterials Fabrication and Processing Handbook
e VFP = 0.35
a b c d
f g h
500 µm
100 µm
FIGURE 1.8 Typical structures of porous ceramics produced by different techniques: (a) porous hydroxya-
patite produced by the powder method combined with PVA as a porogen additive [205], (b) porous alumina by
freeze-drying method [170], (c) porous hydroxyapatite by gelcasting method [181], (d) bioactive glass foams by
sol–gel technique [188], (e) porous β-TCP by solid free-form technique [211], (f) β-TCP+hydroxyapatite foam
produced by the replication technique [207], (g) porous bioglass-based glass-ceramic foam by the replication
technique [80], and (h) porous structure of cancellous bone [215].
TABLE 1.12
Advantages of the Replication Technique over Other Methods
1. Cancellous bone-like macroporous structure
The porous structure produced by the replication technique is very similar to cancellous bone: highly porous network
with open and highly interconnected porosity, compared with the rest of the techniques (see Figures 1.8 (f, g, h)).
2. High commercialization potential
This technique is the simplest and most cost-effective method, and thus most suitable for commercialization, for
example, compared with SFF. SFF-RP, which are expensive processes, it may be a method for producing specific and
complex scaffold architectures.
3. Safety
It does not involve any toxic chemicals, compared with sol–gel and gelcasting techniques, which use HF to accelerate
polymerization.
4. Irregular or complex shape production ability
It can produce scaffolds of irregular or complex shapes, compared with standard dry powder processing or sol–gel-based
methods.
1.4.2 FABRICATION OF COMPOSITE SCAFFOLDS
The fabrication of polymer–ceramic composite scaffolds is based on conventional processes used
for neat polymeric scaffolds. Numerous techniques have been developed to process porous poly-
mer scaffolds for use in tissue engineering. Table 1.13 lists currently applied 3-D polymer scaf-
fold fabrication technologies, based on Ref. 1. Excellent reviews can be found in the literature
[1,216–219].
While intensive efforts have been made to develop the processing technologies of polymer scaf-
folds, relatively less attention has been paid to the fabrication of porous composite scaffolds. Among
the technologies in Table 1.13, solvent casting with or without particle leaching [146,150,153,154]
and TIPS combined with freeze-drying [109–112,144,145] seem to be the most applied method to
the fabrication of polymer–ceramic composite scaffolds. In addition to these methods, there are
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 29
TABLE
1.13
3-D
Fabrication
Technologies
of
Polymer
Scaffolds
[1]
Fabrication
Technology
Required
Properties
of
Materials
Reproducibility
Available
Pore
Size
(µm)
Porosity
(%)
Architecture
Solvent
casting/particle
leaching
Soluble
User,
material
and
technique
sensitive
30–300
20–50
Spherical
pores
Membrane
lamination
Soluble
User,
material
and
technique
sensitive
30–300
<85
Irregular
pores
Textile
technology
Fibers
Machine
controlled
20–100
<95
Melt
molding
Thermoplastic
Machine
controlled
50–500
<80
Extrusion/particle
leaching
Thermoplastic
Machine
controlled
<100
<84
Spherical
pores
Emulsion
freeze-drying
Soluble
User,
material
and
technique
sensitive
<200
<97
High
volume
of
interconnected
micropores
Thermally
induced
phase
separation
Soluble
User,
material
and
technique
sensitive
<200
<97
High
volume
of
interconnected
micropores
Gas
foaming
Amorphous
Material
and
technique
sensitive
<100
10–30
High
volume
of
noninterconnected
micropores
Gas
foaming/particle
leaching
Amorphous
Material
and
technique
sensitive
Micropores
<50
Macropores
<400
<97
Low
volume
of
noninterconnected
micropores
combined
with
high
volume
of
interconnected
macropores
Three-dimensional
printing
Soluble
Machine
and
computer
controlled
45–150
<60
100%
interconnected
macrospores
Fused
deposition
modeling
Thermoplastic
Machine
and
computer
controlled
>150
<80
100%
interconnected
macrospores
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![30 Biomaterials Fabrication and Processing Handbook
two other methods: microsphere sintering and foam coating, which have been considered exten-
sively for the combination of ceramic and polymeric materials, as listed in Table 1.14 and shown
in Figure 1.9. This section will briefly introduce the processing techniques for polymer-ceramic
composite scaffolds.
1.4.2.1 Solvent Casting
Solvent casting of biocomposite scaffolds involves the dissolution of the polymer in an organic solvent,
mixing it with ceramic granules, and casting the solution into a predefined 3-D mold. The solvent is
subsequently allowed to evaporate. The main advantage of this processing technique is the ease of
fabrication without the need of specialized equipment. The primary disadvantages of solvent cast-
ing are (1) the limitation in the shapes (typically flat sheets and tubes are the only shapes that can
be formed), (2) the possible retention of toxic solvent within the polymer, and (3) the denaturation
of the proteins and other molecules incorporated into the polymer by the use of solvents. The use
of organic solvents to cast the polymer may decrease the activity of bioinductive molecules (e.g.,
protein). The detailed processing steps can be found in Ref. 65.
1.4.2.2 Solvent Casting or Particle Leaching and Microsphere Packing
Polymer-ceramic constructs can be fabricated by the solvent aggregation method. The polymer
microspheres are first formed from traditional water oil/water emulsions. Solvent-aggregated
polymer-ceramic scaffolds can then be constructed by mixing solvent, salt or sugar particles,
ceramic granules, and prehardened microspheres [220]. A 3-D structure of controlled porosity
TABLE 1.14
Fabrication Methods of Three-Dimensonal Porous Composite Scaffolds
Fabrication Technique
Biocomposites
Percentage
of Ceramic
(%)
Porosity
(%)
Pore Size
(μm) References
Ceramic Polymer
Solvent casting/particle
leaching
HA PLGA 60–75 (wt.) 81–91 800–1800 146
Bioglass PLLA 20–50 (wt.) 77–80 ∼100 (macro)
∼10 (micro)
150
Phosphate
glass
PLA–PDLLA 40 (wt.) 93–97 153
A/W PDLLA 20–40 (wt.) 85.5–95.2 98–154 154
Thermally induced phase
separation/freeze-drying
β-TCP Chitosan–
gelatin
10–70 (wt.) 322–355 144
HA PLLA 50 (wt.) 85–95 100×300 145
Bioglass PDLLA 5–29 (wt.) 94 ∼100 (macro)
10–50
(micro)
109–112,
152, 221
Microsphere/sintering Amorphous
CaP
PLGA 28–75 (wt.) 75 >100 142, 143
Bioglass PLGA 75 (wt.) 43 89 149
Polymer foam/ceramic
coating
HA
Bioglass
PLGA
PDLLA
40–85 (vol.) 135, 136
109, 110,
222, 223
Ceramic foam/polymer
coating
HA foam PDLLA 173,
221–223
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 31
is formed based on this method combined with particle leaching and microsphere packing. This
method shares similar advantages and disadvantages with the solvent casting technique. Details
of the method are presented in Ref. 65.
1.4.2.3 Thermally Induced Phase Separation or Freeze-Drying
Three-dimensional porous structures can also be achieved through phase separation and evapora-
tion. An approach to induce phase separation is to lower the temperature of the suspension of poly-
mer and ceramic materials. The solvent is solidified first, forcing the polymer and ceramic mixture
into the interstitial spaces. The frozen mixture is then lyophilized using a freeze dryer in which the
ice solvent evaporates [109,144,145].
1.4.2.4 Microsphere Sintering
In this process, microspheres of a ceramic and polymer composite are synthesized first, using emul-
sion or solvent evaporation technique. Sintering the composite microspheres yields a 3-D porous
scaffold [142,143].
1.4.2.5 Foam Coating
An alternative approach to address the combination of polymeric and ceramic materials is to coat
bioactive ceramics onto polymeric foams [220–222]. The inverse method, known as polymer-coated
ceramic scaffolds, has also been investigated [173,177,223], as discussed in Section 1.4.1.1.2.
FIGURE 1.9 Typical structures of porous biocomposites by various techniques: (a) solvent casting or parti-
cle leaching [146,150], (b) phase separation or freeze-drying [145], (c) microsphere sintering [143], (d) polymer
coating of Bioglass foam [177].
c
a b
d
200 µm
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![32 Biomaterials Fabrication and Processing Handbook
1.5 SURFACE FUNCTIONALIZATION
The biocompatibility of biomaterials is related to cell behavior in contact with the biomaterial
surface and particularly to cell adhesion to the surface. Surface characteristics of materials, such as
topography (roughness), chemistry, or surface energy, play a key role in the cell adhesion behavior
on biomaterials. The first stage in cell or material interactions involves cell attachment, adhesion,
and spreading. The quality of this first phase will influence the cell’s capacity to proliferate and to
differentiate (second stage) in contact with the scaffold [224–228].
The process of bone tissue regeneration involves expression of genes and synthesis of proteins
known to be important in the mineralization process. To ensure the in vivo biocompatibility of an
implant, it is often necessary to modify the material surface, either physically by surface roughen-
ing, for example, or chemically, such as by attachment of chemically active species [228,229].
To improve the cell–substrate interaction, different strategies including surface modification
have been developed. The functionalized surfaces can control not only the initial protein adsorp-
tion and production, but also the differentiation potential of different cells (such as human stem
cells) [229]. The main strategies developed for surface modification are presented in Sections 1.5.1
through 1.5.4.
1.5.1 PROTEIN ADSORPTION
This first phase of cell–implant interaction depends on protein adhesion. The proteins currently
used for chemical surface modification of biomaterials are growth factors (or related proteins) and
adhesion proteins (or related peptides). Members of the transforming growth factor-β family are
widely studied: TGF-β1, bone morphogenetic proteins BMP-2, BMP-7, or osteogenic protein OP-1
[230–233]. Among adhesion proteins, RGD-peptides (contain a sequence of the amino acids: argi-
nine R, glycine G, and aspartic acid D) have a high efficacy in promoting osteoblast adhesion
[226,234,235].
All these proteins can be adsorbed in vitro from the serum containing media or in vivo from
biological fluids. The pH, ionic composition of biological solution, temperature, and the functional
group of proteins are the main factors determining protein adsorption on a specific substrate [226].
Another important factor that influences the protein adsorption is the surface energy. Positively
and negatively charged substrates adsorb different proteins [226]. In general, proteins that have a
number of positively charged residues are expected to show a high affinity for the anionic surfaces,
due to electrostatic attractions. By an appropriate pretreatment of the ceramic substrate (e.g., in
phosphate solution), the ionic charges from the surface can be modified, providing an increasing
affinity of the proteins or other biomolecules [236].
1.5.2 SILANE-MODIFIED SURFACES (SILANIZATION TECHNIQUE)
The ability of organosilanes to bond to surfaces arises from the fact that the ethoxy groups (–Si–
(OCH2CH3)3) of aminosilanes, such as aminopropyltriethoxysilane (APTES), form silanols (–Si–
(OH)3) in aqueous solution. These silanol groups can then bond covalently to a suitable substrate,
usually inorganic solids displaying appropriate surface chemical groups (such as –OH), thus leaving
terminal (nonbonded) amino groups free to serve as attachment sites for biological modifiers like
peptides and proteins. Attachment of biological modifiers to the terminal amino groups can be
accomplished by either of the two processes: physisorption or chemisorption [226,227].
The functional groups such as methyl (–CH3), hydroxyl (–OH), carboxyl (–COOH), and
amino (–NH2) groups are present in many biological molecules and have specific physical and
chemical properties that influence the cellular process. By silanization, the substrate surface can
be modified in order to immobilize specific biomolecules. The inclusion of all these functional
groups, using silane modification techniques, especially on glass surfaces, provides a method that
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![Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 33
TABLE 1.15
Functional Group Abbreviations, Chemical Formulae, and Silane Precursors
for Silane-Modified Surfaces [235]
Functional Group Formula Precursor
NH2
COOH
OH
C6H5
CH3
–(CH2)3NH3
+
–COO−
–CH2CH2OH
–C6H5
–CH3
3-Aminopropyltrimethoxysilane
Trichlorovinylsilanea
Trichlorovinylsilanea
Phenyltrichlorosilane
Dimethyldichlorosilane
a
The COOH and OH surfaces were produced by a postsilanization modification of the vinyl surfaces.
produces well-defined and organized substrates with different surface chemistries and energies
[225–228,235,236]. Table 1.15 presents the typical silane precursors for silane-modified surfaces.
1.5.3 TOPOGRAPHY (ROUGHNESS) MODIFICATION
The cell adhesion on ceramic substrates can be enhanced by modifying the surface roughness (e.g.,
by sandblasting, heat treatment, acid etching, etc.). Increased surface wettability or hydrophilicity
has been associated with enhanced protein adsorption and, consequently, cell adhesion on bioma-
terials [237]. There has been, however, only limited work on developing techniques to modify the
surface topography of 3-D ceramic or composite scaffolds [238–240], these techniques being devel-
oped especially for metallic implant for orthopedics.
Another strategy consists of immersing the ceramic substrate in an SBF to mimic the first stage
of bone tissue integration on the in vivo implants. The integration of bone tissue on the bioactive
ceramic or composite surfaces takes place by biomineralization of a thin layer of calcium phos-
phate at the interface between the implant and the bone tissue. Therefore, by soaking the bioactive
biomaterial in SBF, a uniform thick-film composed of nanocrystallites of biologically active cal-
cium phosphate is produced [241]. During this biomimetic process, the topography of the ceramic
implant is modified by the precipitation of nanocrystals of calcium phosphate on its surface. The
new nanoscaled texture of the bioactive ceramic implant can improve the cellular adhesion.
1.5.4 POLYMER COATINGS
Another strategy to improve the cell–scaffold interaction is coating the substrate with an organic
phase, usually a biodegradable polymer [177]. Moreover, the strong interfacial adhesion between
the ceramic biomaterial and the organic polymer is a key parameter in generating composites with
good mechanical properties. A wide variety of polymers have been investigated for this application,
including PLA, PGA, PLGA, PDLLA, PHA, and PCL [173,177,223,242–244]. It has been antici-
pated that the combination of ceramic scaffolds and appropriate biodegradable polymer coatings
can enhance the interfacial adhesion of proteins.
1.6 CONCLUSIONS
Significant developments have been achieved in the design and fabrication of a variety of bioceramic
and composite scaffolds, which have demonstrated outstanding properties for applications in bone
tissue engineering. However, there are several challenges ahead for material scientists and tissue engi-
neers, associated with, in particular, the improvement of biological functions and mechanical integrity
of synthetic scaffolds. Vascularization is the single most important issue to be addressed prior to
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Biomaterials Fabrication And Processing Handbook Paul K Chu
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- 6. Biomaterials Fabrication and Processing HANDBOOK CRC_7973_FM.indd i CRC_7973_FM.indd i 2/26/2008 2:29:23 PM 2/26/2008 2:29:23 PM
- 7. CRC_7973_FM.indd ii CRC_7973_FM.indd ii 2/26/2008 2:29:24 PM 2/26/2008 2:29:24 PM
- 8. CRC Press is an imprint of the Taylor & Francis Group, an informa business Boca Raton London New York Biomaterials Fabrication and Processing HANDBOOK Edited by Paul K. Chu Xuanyong Liu CRC_7973_FM.indd iii CRC_7973_FM.indd iii 2/26/2008 2:29:24 PM 2/26/2008 2:29:24 PM
- 9. CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2008 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-13: 978-0-8493-7973-4 (Hardcover) This book contains information obtained from authentic and highly regarded sources Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the valid- ity of all materials or the consequences of their use. The Authors and Publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or uti- lized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopy- ing, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For orga- nizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Biomaterials fabrication and processing handbook / [edited by] Paul K. Chu and Xuanyong Liu. p. ; cm. “A CRC title.” Includes bibliographical references and index. ISBN 978-0-8493-7973-4 (alk. paper) 1. Biomedical materials. 2. Biomedical engineering. I. Chu, Paul K. II. Liu, Xuanyong. III. Title. [DNLM: 1. Biocompatible Materials. 2. Biosensing Techniques. 3. Nanotechnology--methods. 4. Tissue Engineering--methods. QT 37 B61413 2008] R857.M3B5696 2008 610.284--dc22 2007042613 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com CRC_7973_FM.indd iv CRC_7973_FM.indd iv 2/26/2008 2:29:24 PM 2/26/2008 2:29:24 PM
- 10. v Contents Preface..............................................................................................................................................ix Editors..............................................................................................................................................xi Contributors................................................................................................................................. xiii PART I Tissue Engineering Scaffold Materials Chapter 1 Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering..............3 Qi-Zhi Chen, Oana Bretcanu, and Aldo R. Boccaccini Chapter 2 Design, Fabrication, and Characterization of Scaffolds via Solid Free-Form Fabrication Techniques...............................................................................................45 Dietmar W. Hutmacher and Maria Ann Woodruff Chapter 3 Control and Monitoring of Scaffold Architecture for Tissue Engineering................69 Ying Yang, Cassilda Cunha-Reis, Pierre Olivier Bagnaninchi, and Halil Murat Aydin Chapter 4 Rapid Prototyping Methods for Tissue Engineering Applications............................95 Giovanni Vozzi and Arti Ahluwalia Chapter 5 Design and Fabrication Principles of Electrospinning of Scaffolds ........................ 115 Dietmar W. Hutmacher and Andrew K. Ekaputra PART II Drug Delivery Systems Chapter 6 Nanoparticles in Cancer Drug Delivery Systems .................................................... 143 So Yeon Kim and Young Moo Lee Chapter 7 Polymeric Nano/Microparticles for Oral Delivery of Proteins and Peptides .......... 171 S. Sajeesh and Chandra P. Sharma Chapter 8 Nanostructured Porous Biomaterials for Controlled Drug Release Systems...........193 Yang Yang Li, Jifan Li, and Bunichiro Nakajima Chapter 9 Inorganic Nanostructures for Drug Delivery........................................................... 217 Ying-Jie Zhu CRC_7973_FM.indd v CRC_7973_FM.indd v 2/26/2008 2:29:25 PM 2/26/2008 2:29:25 PM
- 11. vi Contents PART III Nano Biomaterials and Biosensors Chapter 10 Self-Assembly of Nanostructures as Biomaterials..................................................237 Hua Ai, Yujiang Fan, and Zhongwei Gu Chapter 11 Electrohydrodynamic Processing of Micro- and Nanometer Biological Materials ................................................................................................................. 275 Yiquan Wu and Robert Lewis Clark Chapter 12 Fabrication and Function of Biohybrid Nanomaterials Prepared via Supramolecular Approaches ............................................................................. 335 Katsuhiko Ariga Chapter 13 Polypyrrole Nano- and Microsensors and Actuators for Biomedical Applications ............................................................................................................367 Yevgeny Berdichevsky and Yu-Hwa Lo Chapter 14 Processing of Biosensing Materials and Biosensors...............................................401 Yingchun Zhu, Yu Yang, and Yanyan Liu PART IV Other Biomaterials Chapter 15 Synthetic and Natural Degradable Polymeric Biomaterials ................................... 457 Sanjukta Deb Chapter 16 Electroactive Polymers as Smart Materials with Intrinsic Actuation Properties: New Functionalities for Biomaterials...................................................483 Federico Carpi and Danilo De Rossi Chapter 17 Blood-Contacting Surfaces.....................................................................................505 Menno L.W. Knetsch Chapter 18 Improving Blood Compatibility of Biomaterials Using a Novel Antithrombin–Heparin Covalent Complex............................................................. 535 Leslie Roy Berry and Anthony Kam Chuen Chan Chapter 19 Surface Modification of Biomaterials Using Plasma Immersion Ion Implantation and Deposition................................................................................... 573 Xuanyong Liu, Ricky K.Y. Fu, and Paul K. Chu CRC_7973_FM.indd vi CRC_7973_FM.indd vi 2/26/2008 2:29:25 PM 2/26/2008 2:29:25 PM
- 12. Contents vii Chapter 20 Biomaterials for Gastrointestinal Medicine, Repair, and Reconstruction .............633 Richard M. Day Chapter 21 Biomaterials for Cartilage Reconstruction and Repair...........................................659 Wojciech Swieszkowski, Miroslawa El Fray, and Krzysztof J. Kurzydlowski Index..............................................................................................................................................679 CRC_7973_FM.indd vii CRC_7973_FM.indd vii 2/26/2008 2:29:25 PM 2/26/2008 2:29:25 PM
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- 14. ix Preface Biomaterials are used in the biomedical industry to replace or repair injured and nonfunctional tissues. The worldwide biomaterials market was worth over $300 billion in 2005. This market is projected to grow at a rate of 20% per year, and a growing number of scientists and engineers are engaged in fabrication and research of biomaterials. Recognizing the ever increasing importance of biomaterials, a number of books on biomaterials were published in the past 20 years. The Biomaterials Fabrication and Processing Handbook is different from these published books in that it brings together the various aspects of fabrication and processing of the latest biomateri- als, including tissue engineering scaffold materials, drug delivery systems, and nanobiomaterials and biosensors. Some common implant materials including hard tissue materials, blood-contacting materials, and soft tissue materials are also described in this book. Tissue engineering involves the development of new materials or devices capable of interacting specifically with biological tissues. The key to tissue engineering is the preparation of scaffolds using materials with the appropriate composition and structure. In the drug industry, advances in drug delivery systems are very important. Controlled release can be obtained by selecting the appropriate materials to produce the drug delivery system. Attempts have been made to incorporate drug reservoirs into implantable devices for sustained and preferably controlled release. Nanotech- nology also plays an important role in the biomedical and biotechnology industries and has been used in the preparation of drugs for protein delivery, tissue engineering, bones, cardiovascular biomaterials, hard tissue replacements, biosensors, and biological microelectromechanical systems (Bio-MEMS). This book covers the latest information pertaining to tissue engineering scaffold materials, drug delivery systems, and nanobiomaterials and biosensors. The book has 21 chapters describing different types of biomaterials, and is divided into four sections, namely tissue engineering scaffold materials, drug delivery systems, nanobiomaterials and biosensors, and other biomaterials. The section on tissue engineering describes inorganic and composite bioactive scaffolds for bone tissue engineering, design, fabrication, and characterization of scaffolds via solid free-form fabrication techniques, control and monitoring of scaffold architec- ture for tissue engineering, rapid prototyping methods for tissue engineering applications, as well as design and fabrication principles of electrospinning of scaffolds. The section on drug delivery systems discusses nanoparticles in cancer drug delivery systems, polymeric nano/microparticles for oral delivery of proteins and peptides, nanostructured porous biomaterials for controlled drug release systems, and inorganic nanostructures for drug delivery. The section on nanobiomaterials and bio- sensors includes self-assembly of nanostructures as biomaterials, electrohydrodynamic processing of micro- and nanometer biological materials, fabrication and functions of biohybrid nanomaterials prepared via supramolecular approaches, polypyrrole nano- and microsensors and actuators for biomedical applications, as well as processing of biosensing materials and biosensors. The last section, which deals with other biomaterials, includes synthetic and natural degradable polymeric biomaterials, electroactive polymers as smart materials with intrinsic actuation properties such as new functionalities for biomaterials, blood-contacting surfaces, improvement of blood compatibility of biomaterials using a novel antithrombin–heparin covalent complex, surface modification of bio- materials using plasma immersion ion implantation and deposition, biomaterials for gastrointestinal medicine, repair, and reconstruction, and biomaterials for cartilage reconstruction and repair. These chapters have been written by renowned experts in their respective fields, and this book is valuable to the biomaterials and biomedical engineering community. It is intended for a broad and diverse readership including bioengineers, materials scientists, physicians, surgeons, research students, practitioners, and researchers in materials science, bioengineering, and medicine. CRC_7973_FM.indd ix CRC_7973_FM.indd ix 2/26/2008 2:29:25 PM 2/26/2008 2:29:25 PM
- 15. x Preface Readers will be able to familiarize themselves with the latest techniques in biomaterials and processing. In addition, each chapter is accompanied by an extensive list of references for readers interested in pursuing further research. The outstanding cooperation from contributing authors who devoted their valuable time and effort to write excellent chapters for this handbook is highly appreciated. We are also indebted to all our colleagues who have made this book a reality. Paul K. Chu Xuanyong Liu CRC_7973_FM.indd x CRC_7973_FM.indd x 2/26/2008 2:29:25 PM 2/26/2008 2:29:25 PM
- 16. xi Editors Paul K. Chu is a professor (chair) of materials engineering at the City University of Hong Kong. He received a BS in mathematics from The Ohio State University in 1977 and an MS and a PhD in chemis- try from Cornell University in 1979 and 1982, respectively. Professor Chu’s research activities are quite diverse, encompassing plasma sur- face engineering and various types of materials and nanotechnology. He has published over 550 journal papers and has been granted eight U.S. and three Chinese patents. He is a fellow of the IEEE, AVS, and HKIE, senior editor of IEEE Transactions on Plasma Science, asso- ciate editor of International Journal of Plasma Science and Engi- neering, and a member of the editorial board of Materials Science & Engineering: Reports, Surface and Interface Engineering, and Biomolecular Engineering. He is a member of the Plasma-Based Ion Implantation and Deposition International Committee, Ion Implantation Technology International Committee, and IEEE Plasma Science and Application Executive Committee. Xuanyong Liu is an associate professor of materials engineering at the Shanghai Institute of Ceramics, Chinese Academy of Sciences (SICCAS), and a professor at Hunan University. He received a BS and an MS in materials science and engineering from Hunan University in 1996 and 1999, respectively, and a PhD in materials science and engineering from SICCAS in 2002. His doctoral dis- sertation was awarded the National Excellent Doctoral Disserta- tion of People’s Republic of China in 2004. Professor Liu’s primary research focus is on surface modification of biomaterials. He has founded the Surface Engineering of Biomaterials Group in SICCAS and has published over 70 journal papers, including 14 papers on biomaterials. CRC_7973_FM.indd xi CRC_7973_FM.indd xi 2/26/2008 2:29:25 PM 2/26/2008 2:29:25 PM
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- 18. xiii Arti Ahluwalia Interdepartmental Research Center “E. Piaggio” and Department of Chemical Engineering University of Pisa Pisa, Italy Hua Ai National Engineering Research Center for Biomaterials Sichuan University Chengdu, China Katsuhiko Ariga WPI Center for Materials Nanoarchitectonics National Institute for Materials Science Tsukuba, Japan Halil Murat Aydin Institute for Science and Technology in Medicine Keele University Staffordshire, U.K. Pierre Olivier Bagnaninchi Institute for Science and Technology in Medicine Keele University Staffordshire, U.K. Yevgeny Berdichevsky Electrical and Computer Engineering Department University of California San Diego, California, U.S.A. Leslie Roy Berry Henderson Research Centre Hamilton, Ontario, Canada Aldo R. Boccaccini Department of Materials Imperial College London, U.K. Oana Bretcanu Department of Materials Imperial College London, U.K. Federico Carpi Interdepartmental Research Centre “E. Piaggio” University of Pisa Pisa, Italy Anthony Kam Chuen Chan Henderson Research Centre Hamilton, Ontario, Canada Qi-Zhi Chen Department of Materials Imperial College London, U.K. Paul K. Chu Department of Physics and Materials Science City University of Hong Kong Hong Kong, China Robert Lewis Clark Center for Biologically Inspired Materials and Material Systems Pratt School of Engineering Duke University Durham, North Carolina, U.S.A. Cassilda Cunha-Reis Institute for Science and Technology in Medicine Keele University Staffordshire, U.K. Richard M. Day Department of Medicine University College London, U.K. Contributors CRC_7973_FM.indd xiii CRC_7973_FM.indd xiii 2/26/2008 2:29:26 PM 2/26/2008 2:29:26 PM
- 19. xiv Contributors Danilo De Rossi Interdepartmental Research Centre “E. Piaggio” University of Pisa Pisa, Italy Sanjukta Deb Department of Biomaterials Dental Institute, King’s College London, U.K. Andrew K. Ekaputra Graduate Program in Bioengineering National University of Singapore Singapore Miroslawa El Fray Division of Biomaterials and Microbiological Technologies Szczecin University of Technology Polymer Institute Szczecin, Poland Yujiang Fan National Engineering Research Center for Biomaterials Sichuan University Chengdu, China Ricky K.Y. Fu Department of Physics and Materials Science City University of Hong Kong Hong Kong, China Zhongwei Gu National Engineering Research Center for Biomaterials Sichuan University Chengdu, China Dietmar W. Hutmacher Division of Regenerative Medicine Institute of Health and Biomedical Innovation Queensland University of Technology Brisbane, Australia So Yeon Kim Division of Engineering Education College of Engineering Chungnam National University Daejeon, South Korea Menno L.W. Knetsch Centre for Biomaterials Research University of Maastricht Maastricht, The Netherlands Krzysztof J. Kurzydlowski Division of Materials Design Faculty of Materials Science and Engineering Warsaw University of Technology Warsaw, Poland Young Moo Lee School of Chemical Engineering Hanyang University Seoul, South Korea Jifan Li Hitachi Chemical Research Center Irvine, California, U.S.A. Yang Yang Li Hitachi Chemical Research Center Irvine, California, U.S.A. and Department of Physics and Materials Science City University of Hong Kong Hong Kong, China Xuanyong Liu Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai, China and Department of Physics and Materials Science City University of Hong Kong Hong Kong, China Yanyan Liu Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai, China and Laboratory of Special Functional Materials Henan University Kaifeng, China Yu-Hwa Lo Electrical and Computer Engineering Department University of California San Diego, California, U.S.A. CRC_7973_FM.indd xiv CRC_7973_FM.indd xiv 2/26/2008 2:29:26 PM 2/26/2008 2:29:26 PM
- 20. Contributors xv Bunichiro Nakajima Hitachi Chemical Research Center Irvine, California, U.S.A. S. Sajeesh Division of Biosurface Technology Sree Chitra Tirunal Institute for Medical Sciences and Technology Thiruvananthapuram, India Chandra P. Sharma Division of Biosurface Technology Sree Chitra Tirunal Institute for Medical Sciences and Technology Thiruvananthapuram, India Wojciech Swieszkowski Division of Materials Design Faculty of Materials Science and Engineering Warsaw University of Technology Warsaw, Poland Giovanni Vozzi Interdepartmental Research Center “E. Piaggio” and Department of Chemical Engineering University of Pisa Pisa, Italy Maria Ann Woodruff Division of Regenerative Medicine Institute of Health and Biomedical Innovation Queensland University of Technology Brisbane, Australia Yiquan Wu Center for Biologically Inspired Materials and Material Systems Pratt School of Engineering Duke University Durham, North Carolina, U.S.A. Ying Yang Institute for Science and Technology in Medicine School of Medicine Keele University Staffordshire, U.K. Yu Yang Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai, China Yingchun Zhu Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai, China Ying-Jie Zhu State Key Laboratory of High Performance Ceramics and Superfine Microstructures Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai, China CRC_7973_FM.indd xv CRC_7973_FM.indd xv 2/26/2008 2:29:26 PM 2/26/2008 2:29:26 PM
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- 22. Part I Tissue Engineering Scaffold Materials CRC_7973_S001.indd 1 CRC_7973_S001.indd 1 12/10/2007 9:12:24 AM 12/10/2007 9:12:24 AM
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- 24. 3 1 Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering Qi-Zhi Chen, Oana Bretcanu, and Aldo R. Boccaccini CONTENTS 1.1 Introduction...............................................................................................................................4 1.2 Design of 3-D Scaffolds............................................................................................................4 1.3 Scaffold Materials for Bone Tissue Engineering......................................................................6 1.3.1 Bioceramics: Calcium Phosphates ................................................................................6 1.3.1.1 Biocompatibility..............................................................................................6 1.3.1.2 Degradability ..................................................................................................6 1.3.1.3 Mechanical Properties ....................................................................................7 1.3.2 Bioceramics: Bioactive Silicate Glasses........................................................................8 1.3.2.1 Biocompatibility..............................................................................................8 1.3.2.2 Biodegradability..............................................................................................9 1.3.2.3 Mechanical Properties ....................................................................................9 1.3.3 Bioceramics: Glass-Ceramics .....................................................................................10 1.3.3.1 A-W Glass-Ceramics ....................................................................................10 1.3.3.2 Ceravital Glass-Ceramics ............................................................................. 11 1.3.3.3 Bioverit Glass-Ceramics ............................................................................... 11 1.3.3.4 45S5 Bioglass-Derived Glass-Ceramics ....................................................... 11 1.3.4 Naturally Occurring Biopolymers............................................................................... 11 1.3.4.1 Collagen and ECM-Based Materials ............................................................ 11 1.3.4.2 Chitosan ........................................................................................................12 1.3.5 Synthetic Polymers......................................................................................................12 1.3.5.1 Bulk Degradable Polymers ...........................................................................13 1.3.5.2 Surface Bioeroding Polymers .......................................................................15 1.3.6 Biocomposites.............................................................................................................. 16 1.3.7 Summary ..................................................................................................................... 18 1.4 Fabrication of Tissue-Engineering Scaffolds..........................................................................19 1.4.1 Fabrication of Inorganic Scaffolds ..............................................................................19 1.4.1.1 Powder-Forming Processes...........................................................................19 1.4.1.2 Sol–Gel Techniques ......................................................................................23 1.4.1.3 Solid Free-Form Techniques.........................................................................24 1.4.1.4 Comparison of Fabrication Techniques for Ceramic or Glass Scaffolds......25 1.4.2 Fabrication of Composite Scaffolds ............................................................................28 1.4.2.1 Solvent Casting..............................................................................................30 1.4.2.2 Solvent Casting or Particle Leaching and Microsphere Packing..................30 1.4.2.3 Thermally Induced Phase Separation or Freeze-Drying .............................. 31 CRC_7973_Ch001.indd 3 CRC_7973_Ch001.indd 3 1/25/2008 7:14:30 PM 1/25/2008 7:14:30 PM
- 25. 4 Biomaterials Fabrication and Processing Handbook 1.4.2.4 Microsphere Sintering................................................................................... 31 1.4.2.5 Foam Coating................................................................................................ 31 1.5 Surface Functionalization.......................................................................................................32 1.5.1 Protein Adsorption ......................................................................................................32 1.5.2 Silane-Modified Surfaces (Silanization Technique)....................................................32 1.5.3 Topography (Roughness) Modification .......................................................................33 1.5.4 Polymer Coatings ........................................................................................................33 1.6 Conclusions .............................................................................................................................33 References........................................................................................................................................34 1.1 INTRODUCTION Being a modern discipline, tissue engineering encounters various challenges, such as the develop- ment of suitable scaffolds that temporarily provide mechanical support to cells at an early stage of implantation until the cells are able to produce their own extracellular matrix (ECM) [1]. Numerous biomaterials and techniques to produce three-dimensional (3-D) tissue-engineering scaffolds have been considered; biomaterials include polymers, ceramics, and their composites, as discussed in the literature [1–3]. In this chapter, we present an up-to-date summary of the fabrication technologies for tissue-engineering scaffolds, including the choice of suitable materials and related fabrication techniques, with a focus on the development of synthetic scaffolds based on bioceramics, glasses, and their composites combined with biopolymers for bone regeneration. Being one of the most promising technologies, the replication method for the production of highly porous, biodegrad- able, and mechanically competent Bioglass® -derived glass-ceramic scaffolds is highlighted. The enhancement of scaffold properties and functions by surface modification is also discussed, and examples of novel approaches are given. 1.2 DESIGN OF 3-D SCAFFOLDS In an organ, cells and their ECM are organized into 3-D tissues. Therefore, in tissue engineering a highly porous 3-D matrix (i.e., scaffold) is necessary to accommodate cells and to guide their growth and tissue regeneration in 3-D structures. This is particularly relevant in the field of bone tissue engineering and regeneration, bone being a highly hierarchical 3-D composite structure. Moreover, the structure of bone tissue varies with its location in the body. So the selection of configurations as well as appropriate biomaterials depends on the anatomic site for regeneration, the mechanical loads present at the site, and the desired rate of incorporation. Ideally, the scaffold should be porous enough to support cell penetration, tissue ingrowth, rapid vascular invasion, and nutrient delivery. Moreover, the matrix should be designed to guide the formation of new bones in anatomically relevant shapes, and its degradation kinetics should be such that the biodegradable scaffold retains its physical (e.g., mechanical) properties for at least 6 months (for in vitro and in vivo tissue regeneration) [1,3]. Important scaffold design parameters are summarized in Table 1.1. The design of highly porous scaffolds involves a critical issue related to their mechanical prop- erties and structural integrity, which are time dependent. For example, it has been reported that the compressive strength of hydroxyapatite scaffolds increases from ∼10 to ∼30 MPa because of tissue ingrowth in vivo [5]. This finding leads to a conclusion that it might not be necessary to have a starting scaffold with a mechanical strength equal to that of a bone, because cultured cells on the scaffold in vitro will create a biocomposite and increase the strength of the scaffold significantly. Another factor that affects scaffold design is the need for vascularization and angiogenesis in the constructs [6]. In vitro engineering approaches face the problem of critical thickness while regenerating tissue in the absence of true vascularization: mass transportation into tissue is dif- ficult beyond a thin peripheral layer of a tissue construct even if artificial means are used to supply nutrients and oxygen [7]. Diffusion barriers that are present in vitro are most likely to become more CRC_7973_Ch001.indd 4 CRC_7973_Ch001.indd 4 1/25/2008 7:14:31 PM 1/25/2008 7:14:31 PM
- 26. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 5 deleterious in vivo due to lack of vascularization. Once the engineered tissue construct is placed in the body, vascularization becomes a key issue for further remodeling in the in vivo environment. Thus, angiogenesis is an essential step in the colonization of macroporous biomaterials during osteointegration. Capillaries bring osteoprogenitor cells and the nutriments that are required for their growth. They transport especially numerous angiogenic growth factors [8]. The main critical factors affecting bone formation are the pore size and pore interconnection of the scaffold. Pore size is related to the in vivo bone tissue ingrowth, allowing migration and proliferation of osteoblasts and mesenchymal cells, and matrix deposition in the empty spaces [9]. Pore interconnection provides the channel for cell distribution and migration allowing efficient in vivo blood vessel formation. An incomplete pore interconnection could limit blood vessels invasion. Small pore size could obstruct cell adhesion and bone ingrowth. Bone vascularization, besides providing nutrients essential for tissue survival, plays also a crucial role in coordinating the activity of bone cells and their migration for new bone formation [10]. Several studies have investigated the minimum pore size required to regenerate mineralized bone. The minimum requirement for pore size is considered to be around 100 µm due to cell size, migration requirements, and transport. However, pore sizes >300 µm are recommended due to enhanced growth rate of a new bone and the formation of capillaries [3,4,11]. Pore size in the range of 300–500 µm would promote vascularization and mass transportation of nutrients and waste products, while the scaffold would maintain good mechanical integrity during in vitro culture and in vivo transplantation [12]. It is equally important to notice that tissue-engineering scaffolds should have enhanced biologi- cal functions. Therefore, the incorporation of growth factors, such as bone growth factors (BGF) and vascularization growth factors (VGF), or specific peptide sequences into the scaffolds or on their surface is being considered as part of the integral design of scaffolds. Moreover, to improve cell attachment and growth, the surface of scaffolds’ struts needs to be pretreated (a process called surface functionalization) [13–15]. The design of the surface properties of scaffolds is an important step to achieve their successful in vitro and in vivo applications. A few approaches to surface modi- fication of scaffolds are discussed below. TABLE 1.1 Scaffold Design Parameters for Bone Tissue Engineering [4] Parameters Requirements Porosity Maximum possible without compromising mechanical properties Pore size 200–400 µm Pore structure Interconnected Mechanical properties of the cancellous bone Tension and compression Strength: 5–10 MPa Modulus: 50–100 MPa Mechanical properties of the cortical bone Tension Strength: 80–150 MPa Modulus: 17–20 GPa Compression Strength: 130–220 MPa Modulus: 17–20 GPa Fracture toughness: 6–8 MPa√ __ m Degradation properties Degradation time Must be tailored to match the application in patients Degradation mechanism Bulk dissolution in medium Biocompatibility No chronic inflammation Sterilizability Sterilizable without altering material properties CRC_7973_Ch001.indd 5 CRC_7973_Ch001.indd 5 1/25/2008 7:14:31 PM 1/25/2008 7:14:31 PM
- 27. 6 Biomaterials Fabrication and Processing Handbook 1.3 SCAFFOLD MATERIALS FOR BONE TISSUE ENGINEERING The first step in achieving a successful scaffold is to choose a suitable biomaterial. Natural bone matrix is a composite of biological ceramic (a natural apatite) and biological polymer. Carbonated hydroxyapatite Ca10(PO4)6(OH)2 accounts for nearly two-thirds of the weight of a bone. The inor- ganic component provides compressive strength to the bone. Roughly one-third of the weight of a bone is from collagen fibers. Collagen fibers are tough and flexible, and thus tolerate stretching, twisting, and bending. It is not surprising that polymers, ceramics, or their composites have been chosen for bone repair [16]. They can be either synthetic or naturally occurring ones. Table 1.2 lists synthetic and natural scaffold biomaterials that have been most widely investigated for bone regen- eration, some of which are well-established and clinically applicable. In this section, the biocompat- ibility, biodegradability, and mechanical properties of these scaffold materials, which are the most essential factors to be considered in the fabrication of bone regeneration scaffold, are reviewed concisely. Particular attention is paid to a key issue that remains with almost all existing scaffold biomaterials, that is, mechanically strong materials (in crystalline structure) tend to be bioinert, and biodegradable materials (in amorphous structure) are, in general, mechanically weak. An excep- tion, 45S5 Bioglass-derived glass-ceramic, is considered in more detail because the issue associated with the two apparently irreconcilable properties (mechanical strength and biodegradability) have been successfully addressed in this material [17]. 1.3.1 BIOCERAMICS: CALCIUM PHOSPHATES 1.3.1.1 Biocompatibility Since almost two-thirds of the weight of a bone is hydroxyapatite Ca10(PO4)6(OH)2, it seems logi- cal to use this ceramic as a major component of scaffold materials for bone tissue engineering. Actually, hydroxyapatite and related calcium phosphates (e.g., β-tricalcium phosphate [β-TCP]) have been intensively investigated [16,18,21]. As expected, calcium phosphates have an excellent biocompatibility due to their close chemical and crystal resemblance to bone mineral [19,20]. Although they have not shown osteoinductive ability, they certainly possess osteoconductive prop- erties as well as a remarkable ability to bind directly to bone [32–35]. A high number of in vivo and in vitro assessments have concluded that calcium phosphates, no matter which forms (bulk, coating, powder, or porous) and which phases (crystalline or amorphous) they are in, always sup- port the attachment, differentiation, and proliferation of cells (such as osteoblasts and mesenchy- mal cells), with hydroxyapatite being the best among these scaffold materials [36]. Although the excellent biological performance of hydroxyapatite and related calcium phosphates has been well- documented, the slow biodegradation of their crystalline phases and the weak mechanical strength of their amorphous states limit their application in engineering of new bone tissue, especially at load-bearing sites. 1.3.1.2 Degradability Typically, crystalline calcium phosphates have a long degradation time in vivo, often of the order of years [37]. The dissolution rate of synthetic hydroxyapatite depends on the type and concentration of the buffered or unbuffered solutions, pH of the solution, degree of the saturation of the solution, solid and solution ratio, length of suspension in the solution, as well as composition and crystallinity of the hydroxyapatite. In the case of crystalline hydroxyapatite, the degree of micro and macropo- rosities, defect in the structure, and amount and type of other phases present also have significant influence [39]. Crystalline hydroxyapatite exhibits the slowest degradation rate, compared with other calcium phosphates. The dissolution rate decreases in the following order [38]: Amorphous hydroxyapatite > all other calcium phosphates (e.g., TCP) > > crystalline hydroxyapatite. CRC_7973_Ch001.indd 6 CRC_7973_Ch001.indd 6 1/25/2008 7:14:32 PM 1/25/2008 7:14:32 PM
- 28. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 7 1.3.1.3 Mechanical Properties The properties of synthetic calcium phosphates vary significantly with their crystallinity, grain size, porosity, and composition (e.g., calcium deficiency). In general, the mechanical properties of synthetic calcium phosphates decrease significantly with increasing content of amorphous phase, microporosity, and grain size. High crystallinity, low porosity, and small grain size tend to give TABLE 1.2 List of Promising Scaffold Biomaterials for Bone Regeneration Biomaterials Abbreviation Application Ceramics [16,18] Calcium phosphates [19–21] Hydroxyapatite Tricalcium phosphate Biphasic calcium phosphate: HA and TCP CaP HA TCP BCP Dental Drug delivery Scaffolds Bioactive glasses [22–25] Bioglass Phosphate glasses Dental Drug delivery Scaffolds Bioactive Glass-Ceramics [26,27] Apatite-Wollastonite Ceravital A/W Dental Drug delivery Scaffolds Polymers [28–31] Synthetic degradable polymers Bulk biodegradable polymers Aliphatic polyester Poly(lactic acid) Poly(d-lactic acid) Poly(l-lactic acid) Poly(d,l-lactic acid) Poly(glycolic acid) Poly(lactic-co-glycolic acid) Poly(ε-caprolactone) Poly(hydroxyalkanoate) Poly(3- or 4-hydroxybutyrate) Poly(3-hydroxyoctanoate) Poly(3-hydroxyvalerate) Polydioxanone Poly(propylene fumarate) PLA PDLA PLLA PDLLA PGA PLGA PCL PHA PHB PHO PHV PPF Sutures Dental Orthopedic Drug delivery Scaffolds Surface bioerodible polymers Poly(ortho esters) Poly(anhydrides) Poly(phosphazene) POE PPHOS Drug delivery Natural degradable polymers Polysaccharides Hyaluronan Alginate Chitosan Proteins Collagen Fibrin HyA Composites [12] Composed of the above-mentioned ceramics and polymers CRC_7973_Ch001.indd 7 CRC_7973_Ch001.indd 7 1/25/2008 7:14:32 PM 1/25/2008 7:14:32 PM
- 29. 8 Biomaterials Fabrication and Processing Handbook higher stiffness, higher compressive and tensile strength, and greater fracture toughness [39,40]. It has been reported that the flexural strength and fracture toughness of dense hydroxyapatite are much lower in a dry condition than in a wet condition [41]. The mechanical properties of hydroxy- apatite and related calcium phosphates, as well as those of bone, are given in Table 1.3. In brief, hydroxyapatite and related calcium phosphates exhibit excellent biocompatibility and osteoconductivity. However, these materials are poorly degradable in case of crystalline structures, and their amorphous counterparts are mechanically too fragile to be used for fabrication of highly porous tissue-engineering scaffolds. 1.3.2 BIOCERAMICS: BIOACTIVE SILICATE GLASSES 1.3.2.1 Biocompatibility As early as in 1969, Hench and colleagues discovered that certain silicate glass compositions had excellent biocompatibility as well as the ability of bone bonding [23–25]. Through interfacial and cell-mediated reactions, bioactive glass develops a calcium-deficient, carbonated calcium phosphate surface layer that allows it to chemically bond to the host bone. This bone-bonding behavior is referred to as “bioactivity” and has been associated with the formation of a carbonated hydroxy- apatite layer on the glass surface when implanted or when in contact with biological fluids [47–50]. Bioactivity is not an exclusive property of bioactive silicate glasses. Hydroxyapatite and related calcium phosphates also show an excellent bone-bonding ability, as discussed above. The capability of a material to form a biological interface with the surrounding tissue is critical in avoiding scaf- fold loosening in vivo. Bioactive glasses have also been found to support enzyme activity [51–54], vascularization [55,56], as well as foster osteoblast adhesion, growth, and differentiation. Bioactive glasses were also shown to induce the differentiation of mesenchymal cells into osteoblasts [57–59] and to pro- vide osteoconductivity [60]. A significant finding for the development of bone engineering is that the dissolution products from bioactive glasses exert a genetic control over osteoblast cycle and rapid expression of genes that regulate osteogenesis and the production of growth factors [61,62]. Silicon has been found to play a key role in the bone mineralization and gene activation, which has led to the substitution of silicon for calcium into synthetic hydroxyapatite. Investigations in vivo have shown that bone ingrowth into silicon-substituted hydroxyapatite granules was remarkably greater than that into pure hydroxyapatite [62,63]. The above-mentioned advantages make 45S5 Bioglass a very successful material in clinical applications, for example, for the treatment of periodontal disease (PerioGlas) and as a bone-filler material (NovaBone) [63,64]. Bioglass implants have also been used to replace damaged middle ear bones, restoring auditory capabilities of patients [64]. Recently bioactive glasses have gained attention as promising scaffold materials for bone tissue engineering [64–69]. Similar to calcium phosphates, the application of this material, particularly in tissue engineering, has encountered a TABLE 1.3 Comparison of Mechanical Properties of Calcium Phosphates and Human Bone Ceramics Compressive Strength (MPa) Tensile Strength (MPa) Elastic Modulus (GPa) Fracture Toughness (MPa√ __ m) References Calcium phosphates 20–900 30–200 30–103 <1.0 39,42 Hydroxyapatite >400 ∼40 ∼100 ∼1.0 39,42 Cortical bone 130–180 50–151 12–18 6–8 28,43–46 CRC_7973_Ch001.indd 8 CRC_7973_Ch001.indd 8 1/25/2008 7:14:32 PM 1/25/2008 7:14:32 PM
- 30. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 9 hurdle caused by the conflict between the properties of biodegradability and mechanical reliability, which is discussed in Sections 1.3.2.3 and 1.3.3.4. 1.3.2.2 Biodegradability The basic constituents of most bioactive glasses are SiO2, Na2O, CaO, and P2O5. The well-known 45S5 Bioglass contains 45% SiO2, 24.5% Na2O, 24.4% CaO, and 6% P2O5, in weight percent. The bioreactivity of the material is composition-dependent. Hench and coworkers [22] have systemati- cally studied a series of glasses in the four-component systems with a constant 6 wt.% P2O5 content. This work is summarized in the ternary SiO2–Na2O–CaO diagram shown in Figure 1.1. In region A, the glasses are bioactive and bond to bone. In region B, glasses are nearly inert when implanted. Compositions in region C are resorbed within 10–30 days in tissue. In region D, the compositions are not technically practical. The key advantage of bioactive glasses that makes them promising scaffold materials is the possibility of controlling a range of chemical properties and thereby the rate of bioresorption. The structure and chemistry of glasses, in particular sol–gel derived glasses [47,48], can be tailored at a molecular level by varying either composition, or thermal or environmental processing history. It is possible to design glasses with degradation properties specific to a particular application of bone tissue engineering. 1.3.2.3 Mechanical Properties A primary disadvantage of bioactive glasses is their low fracture toughness (Table 1.4) because of their amorphous structure. Hence, many researchers sintered bioactive glasses at their crystalliza- tion temperatures in order to improve the mechanical performance of these materials. However, it was reported that crystallization of bioactive glasses could decrease the level of bioactivity [70] FIGURE 1.1 Compositional dependence (in wt.%) of bone bonding and soft tissue bonding of bioactive glasses and glass-ceramics. Bioactivity index IB is defined as IB = 100/t0.5, where t0.5 is the time taken for 50% of the interface to bond to bone. All compositions have a constant 6 wt.% of P2O5. In region A, the glasses are bioactive and bond to bone. In region B, glasses are nearly inert when implanted. Compositions in region C are resorbed within 10–30 days in tissue. In region D, the compositions are not technically practical. In the region where IB > 8 (called region E), soft tissue bonding occurs. Apatite-wollastonite glass-ceramic (A-WGC) has higher P2O5 content [22]. CaO Na2O SiO2 A-WGC (high P2O5) D B C 1 8 5 2 0 A I CRC_7973_Ch001.indd 9 CRC_7973_Ch001.indd 9 1/25/2008 7:14:32 PM 1/25/2008 7:14:32 PM
- 31. 10 Biomaterials Fabrication and Processing Handbook and even turn a bioactive glass into an inert material [71]. This is one of the disadvantages that limit the application of bioactive glasses as scaffold materials, as full crystallization occurs prior to significant densification upon heat treatment (i.e., sintering) [72]. Extensive sintering is necessary to densify the struts of a scaffold, which would otherwise be made up of loosely packed particles and thus the structure would be too fragile to handle. Most recently, Boccaccini’s group at Imperial College London [17] reported on a phase transformation from a mechanically competent crystalline phase to a biodegradable amorphous calcium phosphate in 45S5 Bioglass-derived scaffolds. This phase transition, which takes place in a biological environment at body temperature, couples the two required properties (mechanical strength and biodegradability) in a single scaffold. A detailed characterization of this material is given in Section 1.3.3.4. In summary, like hydroxyapatite and related calcium phosphates, bioactive glasses exhibit good biocompatibility and osteoconductivity. At the same time, all these materials, except 45S5 Bioglass- derived glass-ceramics, encounter a similar disadvantage, that is, a mechanically strong scaffold has to be achieved through crystallization, which unfortunately hampers the biodegradability of these materials. 1.3.3 BIOCERAMICS: GLASS-CERAMICS Glasses can be strengthened by the formation of crystalline particles in the glass matrix upon heat treatment in the relevant glass-crystal region of its phase diagram. The resultant glass-ceramics usually exhibit better mechanical properties than both the parent glass and sintered crystalline ceramics (e.g., sintered hydroxyapatite) (Table 1.4). There are many biomedical glass-ceramics available for the repair of damaged bones. Among them, apatite-wollastonite (A-W), Ceravital, and Bioverit glass-ceramics have been intensively investigated [16,18]. Recently, a 45S5 Bioglass-derived glass-ceramic showed a great potential as a tissue-engineering scaffold material, as mentioned above (Section 1.3.2.3). 1.3.3.1 A-W Glass-Ceramics In A-W glass-ceramic, the glass matrix is reinforced by β-wollastonite (CaSiO3) crystals and a small amount of apatite phase, which precipitate successively at 870°C and 900°C, respectively [75]. Some mechanical properties of this glass-ceramic have been listed in Table 1.4. The high bending strength (215 MPa) of A-W glass-ceramic is due to the precipitation of wollastonite as well as apatite. These two precipitates also give the glass-ceramic a higher fracture toughness than that of both the glass and ceramic phases. It is believed that wollastonite effectively prevents straight propagation of cracks, causing them to deflect or branch out [26,75–77]. A-W glass-ceramic is capable of binding tightly to a living bone in a few weeks after implanta- tion, and the implants do not deteriorate in vivo [78]. The excellent bone-bonding ability of A-W TABLE 1.4 Mechanical Properties of Hydroxyapatite, 45S5 Bioglass, Glass-Ceramics, and Human Cortical Bone Ceramics Compression Strength (MPa) Tensile Strength (MPa) Elastic Modulus (GPa) Fracture Toughness (MPa√ __ m) References 45S5 Bioglass 500 42 35 0.5–1 42,73 A-W 1080 215 (bend) 118 2.0 26 Parent glass of A-W NA 72 (bend) NA 0.8 26 Bioverit I 500 140–180 (bend) 70–90 1.2–2.1 74 Cortical bone 130–180 50–151 12–18 6–8 28,43–46 CRC_7973_Ch001.indd 10 CRC_7973_Ch001.indd 10 1/25/2008 7:14:33 PM 1/25/2008 7:14:33 PM
- 32. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 11 glass-ceramic is attributed to the glass matrix and apatite precipitates, whereas the in vivo stability as a whole is due to the inertness of β-wollastonite. Although the long-term integrity in vivo is desir- able in the application of nonresorbable prosthesis, the material does not match the goal of tissue engineering, which demands biodegradable scaffolds. 1.3.3.2 Ceravital Glass-Ceramics [79] “Ceravital” was coined to mean a number of different compositions of glasses and glass-ceramics and not only one product. Their basic network components include SiO2, Ca(PO2)2, CaO, Na2O, MgO, and K2O, with ceramic additions being Al2O3, Ta2O5, TiO2, B2O3, Al(PO3)3, SrO, La2O3, or Gd2O3. This material system was developed as solid fillers in the load-bearing conditions for the replacement of bone and teeth. It turned out, however, that their mechanical properties do not serve the purpose, and there has been virtually no research on the application of this material in tissue- engineering scaffolds. 1.3.3.3 Bioverit Glass-Ceramics [74] Bioverit products are mica-apatite glass-ceramics. Mica crystals (aluminum silicate minerals) give the materials good machinability, and apatite crystals ensure the bioactivity of the implants. The mechanical properties of Bioverit materials (Table 1.4) allow them to be used as fillers in dental application. As regards bioreactivity, Bioverit implants show a hydrolytic stability in vivo. As for Ceravital glass-ceramics, no significant research has been carried out regarding the use of this glass-ceramic in tissue engineering. 1.3.3.4 45S5 Bioglass-Derived Glass-Ceramics In 2005, Chen et al. [80] fabricated a 3-D, highly porous, mechanically competent, bioactive and biodegradable scaffold for the first time by the replication technique using 45S5 Bioglass pow- der. Under an optimum sintering condition (1000°C/h), nearly full densification of the foam struts occurred and fine crystals of Na2Ca2Si3O9 are formed, which conferred the scaffolds the highest possible compressive and flexural strength for this foam structure. Important findings in this work are that the mechanically strong crystalline phase Na2Ca2Si3O9 can transform into an amorphous calcium phosphate phase after immersion in simulated body fluid (SBF) for 28 days and that the transformation kinetics can be tailored by controlling the crystallinity of the sintered 45S5 Bio- glass. As such, it was demonstrated that the goal of an ideal scaffold that provides good mechanical support temporarily while maintaining bioactivity and that can biodegrade at later stages at a tailor- able rate can be achieved with these Bioglass-based scaffolds [17]. 1.3.4 NATURALLY OCCURRING BIOPOLYMERS Much research effort has been focused on naturally occurring polymers such as demineralized bone ECM [81], purified collagen [82,83], and chitosan [84] for tissue engineering applications. Theoreti- cally, naturally occurring polymers should not cause response of foreign materials when implanted. They provide a natural substrate for cellular attachment, proliferation, and differentiation in their native state. For these reasons, naturally occurring polymers could be a favorite substrate for tis- sue engineering [28]. Table 1.5 provides a list of some of the naturally occurring polymers, their sources, and applications. Among them, collagen and chitosan are most widely investigated for bone engineering and are briefly discussed here. 1.3.4.1 Collagen and ECM-Based Materials The most commonly used naturally occurring polymer is the structural protein collagen. Bioma- terials derived from ECM include collagen and other naturally occurring structural and functional CRC_7973_Ch001.indd 11 CRC_7973_Ch001.indd 11 1/25/2008 7:14:33 PM 1/25/2008 7:14:33 PM
- 33. 12 Biomaterials Fabrication and Processing Handbook proteins. Natural polymers must be modified and sterilized before clinical use. All methods of stabilization and sterilization can moderately or severely alter the rate of in vivo degradation and change the mechanical and physical properties of the native polymers. Each method has certain advantages and disadvantages, and thus should be selectively utilized for scaffolds of specifically sited bone tissue engineering [86]. 1.3.4.2 Chitosan The use of chitosan for bone tissue engineering has been widely investigated [84,87]. This is in part due to the apparent osteoconductive properties of chitosan. Mesenchymal stem cells cultured in the presence of chitosan have demonstrated an increased differentiation to osteoblasts compared with cells cultured in the absence of chitosan [88]. It is also speculated that chitosan may enhance osteoconduction in vivo by entrapping growth factors at the wound site [89]. 1.3.5 SYNTHETIC POLYMERS Although naturally occurring polymers possess the above-mentioned advantages, their poor mechanical properties and variable physical properties with different sources of protein matrices have hampered their progress in broad applications in tissue engineering. Concerns have also been expressed regarding immunogenic problems associated with the introduction of foreign collagen [37]. Following the developmental efforts regarding the use of naturally occurring polymers as scaf- folds, much attention has been paid to synthetic polymers. Synthetic polymers have high potential in tissue engineering not only because of their excellent processing characteristics, which can ensure their off-the-shelf availability, but also because of their advantage of being biocompatible and bio- degradable [37,90]. Synthetic polymers have predictable and reproducible mechanical and physical properties (e.g., tensile strength, elastic modulus, and degradation rate) and can be manufactured with great precision. Although they are unfamiliar to cells and many have some shortcomings, such as eliciting persistent inflammatory reactions, being eroded, not being compliant or able to TABLE 1.5 List of Naturally Occurring Polymers, Their Sources, and Applications [85] Polymers Source Application Collagen Tendons and ligament Multiapplications, including bone tissue engineering Collagen-Glycosaminoglycan (GAG) (alginate) copolymers Artificial skin grafts for skin replacement Albumin In blood Transporting protein used as coating to form a thromboresistant surface Hyaluronic acid In the ECM of all higher animals An important starting material for preparation of new biocompatible and biodegradable polymers that have applications in drug delivery, tissue engineering, and viscosupplementation Fibrinogen–Fibrin Purified from plasma in blood Multiapplications, including bone tissue engineering Chitosan Shells of shrimps and crabs Multiapplications, including bone tissue engineering CRC_7973_Ch001.indd 12 CRC_7973_Ch001.indd 12 1/25/2008 7:14:33 PM 1/25/2008 7:14:33 PM
- 34. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 13 integrate with the host tissues, they may be replaced in vivo in a timely fashion by native constructs built by the cells seeded into them. It has been widely accepted that an ideal tissue-engineered bone substitute should be a synthetic scaffold, which is biocompatible and provides for cell attachment, proliferation and maturation, has mechanical properties to match those of the tissues at the site of implantation, and degrades at rates to match tissue replacement. Table 1.6 lists selected properties of synthetic, biocompatible, and biodegradable polymers that have been intensively investigated as scaffold materials for tissue engineering, type I collagen fibers being included for comparison. 1.3.5.1 Bulk Degradable Polymers 1.3.5.1.1 Saturated Poly-α-Hydroxyesters (PLA, PGA, and PCL) The biodegradable synthetic polymers most often utilized for 3-D scaffolds in tissue engineer- ing are the poly(α-hydroxyacids), including poly(lactic acid) (PLA) and poly(glycolic acid) (PGA), as well as poly(lactic-co-glycolide) (PLGA) copolymers [91]. PLA exists in three forms: l-PLA (PLLA), d-PLA (PDLA), and racemic mixture of d,l-PLA (PDLLA). These polymers are popular for various reasons, among which biocompatibility and biode- gradability stand out. These materials have chemical properties that allow hydrolytic degradation through de-esterification. After the process of degradation is over, the monomeric components of each polymer are removed through natural pathways: PGA can be converted to other metabolites or eliminated by other mechanisms, and PLA can be cleared through tricarboxylic acid cycle. The body already contains highly regulated mechanisms for completely removing monomeric compo- nents of lactic and glycolic acids. Due to these properties, PLA and PGA have been used in products such as degradable sutures and have been approved by the U.S. Food and Drug Administration (FDA) [28]. Other significant properties of these polymers are their very good processability, and their ability to exhibit a wide range of degradation rates, physical, mechanical, and other proper- ties, which can be achieved by PLA and PGA of various molecular weights and their copolymers. However, these polymers undergo a bulk erosion process in contact with body fluids such that they can cause scaffolds to fail prematurely. In addition, abrupt release of these acidic degradation prod- ucts can cause a strong inflammatory response [92,93]. In general, PGA degrades faster than PLA, as listed in Table 1.6. Their degradation rates decrease in the following order. PGA>PDLLA>PLLA Degradation rates decrease Table 1.6 also lists the mechanical properties of type I collagen, which is the major organic component of ECM in bone. The strength and ductility (e.g., ultimate elongation) of PLA and PGA are comparable to those of type I collagen fibers. PDLLA has been extensively investigated as a biomedical coating material because of its excel- lent features with respect to implant surface [28,104]. In addition to its high mechanical stabil- ity [105], PDLLA also shows excellent biocompatibility in vivo and good osteoinductive potential [106]. PDLLA of low molecular weight can be combined with drugs like growth factors [106], antibiotics [107], or thrombin inhibitors [108] to establish a locally acting drug-delivery system. It is due to these desirable features that much more attention has recently been paid to PDLLA for applying it as a scaffold material for tissue engineering. Highly porous 3-D scaffolds made of Bioglass-filled PDLLA and PLGA were fabricated by Boccaccini et al. [59]. Since then an increasing number of publications have emerged on this subject, as reviewed recently [12]. Porous PDLLA foams and Bioglass-filled PDLLA composite foams have both been fabricated, using thermally induced–phase separation (TIPS) technique [109,110]. Bioglass- filled PDLLA composite foams exhibit high bioactivity, assessed by the formation of hydroxyapatite CRC_7973_Ch001.indd 13 CRC_7973_Ch001.indd 13 1/25/2008 7:14:33 PM 1/25/2008 7:14:33 PM
- 35. 14 Biomaterials Fabrication and Processing Handbook TABLE 1.6 Physical Properties of Synthetic, Biocompatible, and Biodegradable Polymers Investigated as Scaffold Materials Polymers Melting Point, T m (°C) Glass Transition Point, T g (°C) Degradation Time (months) Tensile or Compressive* Strength (MPa) Modulus (GPa) Ultimate Elongation (%) References Bulk degradable polymers PDLLA Amorphous 55–60 12–16 Pellet: 35–150* Film or disk: 29–35 Film or disk: 1.9–2.4 Pellet: 0.5–8.0 Film or disk: 5.0–6.0 90,94,95 PLLA 173–178 60–65 >24 Pellet: 40–120 Film or disk: 28–50 Fiber: 870–2300 Film or disk: 1.2–3.0 Fiber: 10–16 Pellet: 2.0–10.0 Film or disk: 2.0–6.0 Fiber: 12–26 90,94 PGA 225–230 35–40 6–12 Fiber: 340–920 Fiber: 7–14 Fiber: 15–25 90,96,97 PLGA Amorphous 45–55 Adjustable 41.4–55.2 1.4–2.8 3–10 28 PPF Bulk 2–30* 28,30 PCL 58 −72 Bulk 700 98 Surface erosive polymers Poly(anhydrides) 150–200 Surface 25–27 30–40* 0.14–1.4 28,30,99 Poly(ortho-esters) 30–100 Surface 4–16* 2.5–4.4 28,100 Polyphosphazene −66–50 242 Surface 101,102 Type I collagen Bulk Uncross-linked fi ber: 0.91–7.2 Cross-linked fi ber: 46.8–68.8 Uncross-linked fi ber: 1.8–46×10 –3 Cross-linked fi ber: 0.383–0.766 Uncross-linked fi ber: 24.1–68.0 Cross-linked fi ber: 11.6–15.6 103 CRC_7973_Ch001.indd 14 CRC_7973_Ch001.indd 14 1/25/2008 7:14:34 PM 1/25/2008 7:14:34 PM
- 36. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 15 on the strut surfaces upon immersion in SBF [111]. It has also been shown that the foams support the migration, adhesion, spreading, and viability of MG-63 cells (osteosarcoma cell line) [112]. Poly(ε-caprolactone) (PCL) is also an important member of the aliphatic polyester family. It has been used to effectively entrap antibiotic drugs and thus a construct made with PCL can be considered as a drug-delivery system, being used to enhance bone ingrowth and regeneration in the treatment of bone defects [113]. The degradation of PCL and its copolymers involves similar mechanisms to PLA, proceeding in two stages: random hydrolytic ester cleavage and weight loss through the diffusion of oligometric species from the bulk. It has been found that the degradation of PCL system with a high molecular weight ( __ Mn of 50,000) is remarkably slow, requiring 3 years for complete removal from the host body [114]. 1.3.5.1.2 Polyhydroxyalkanoates (PHB, PHBV, P4HB, PHBHHx, PHO) Recently, polyhydroxyalkanoates (PHAs), another type of polyesters, have been suggested for tis- sue engineering because of their controllable biodegradation and high biocompatibility [115]. They are aliphatic polyesters as well, but produced by microorganisms under unbalanced growth condi- tions [116,117]. They are generally biodegradable (via hydrolysis) and thermoprocessable, making them attractive as biomaterials for application in medical devices and tissue engineering. Over the past years, PHA, particularly poly-3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV); poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx); and poly 3-hydroxyoctanoate (PHO) were demonstrated to be suitable for tissue engineering and are reviewed in detail in Refs. 115,116. Depending on the property requirement of different applications, PHA polymers can be either blended, surface modified, or composed with other polymers, enzymes, or inorganic materials to further adjust their mechanical properties or biocompatibility. The blending among the several PHA themselves can dramatically change their material properties and biocompatibility [115,116]. PHB is of particular interest for bone tissue application as it was demonstrated to produce a consistent favorable bone tissue adaptation response with no evidence of an undesirable chronic inflammatory response after an implantation period of up to 12 months [116]. The bone is formed close to the material and subsequently becomes highly organized, with up to 80% of the implant surface lying in direct apposition to the new bone. The materials showed no evidence of extensive structural breakdown in vivo during the implantation period of the study [118]. However, a drawback of some PHA polymers is their limited availability and the time-consuming extraction procedure from bacterial cultures that is required for obtaining sufficient processing amounts as described in the literature [115,119]. Therefore, the extraction process might be a chal- lenge to a cost-effective industrial upscale production for large amounts of some PHA polymers. 1.3.5.1.3 Polypropylene Fumarate Poly(propylene fumarate) (PPF) is an unsaturated linear polyester. Similar to PLA and PGA, the degradation products of PPF through hydrolysis (i.e., propylene glycol and fumaric acid) are bio- compatible and readily removed from the body. The double bond along the backbone of the polymer permits cross-linking in situ, which causes a moldable composite to harden within 10–15 min. Mechanical properties and degradation time of the composite may be controlled by varying the PPF molecular weight. Therefore, preservation of the double bonds and control of molecular weight during PPF synthesis are critical issues [120]. PPF has been suggested for use as scaffold for guided tissue regeneration, often as part of an injectable bone replacement composite [121], and has been used as a substrate for osteoblast culture [122]. 1.3.5.2 Surface Bioeroding Polymers There is a family of hydrophobic polymers that undergo a heterogeneous hydrolysis process, which is predominantly confined to the polymer–water interface. This property is referred to as surface eroding as opposed to bulk degrading behavior. These surface bioeroding polymers have been CRC_7973_Ch001.indd 15 CRC_7973_Ch001.indd 15 1/25/2008 7:14:34 PM 1/25/2008 7:14:34 PM
- 37. 16 Biomaterials Fabrication and Processing Handbook intensively investigated as drug-delivery vehicles. The surface-eroding characteristic offers three key advantages over bulk degradation when used as scaffold materials: (1) retention of mechani- cal integrity over the degradative lifetime of the device, owing to the maintenance of mass to vol- ume ratio; (2) minimal toxic effects (i.e., local acidity), owing to lower solubility and concentration of degradation products; and (3) significantly enhanced bone ingrowth into the porous scaffolds, owing to the increment in pore size as the erosion proceeds [123]. 1.3.5.2.1 Poly(anhydrides) Poly(1,3-bis-p-carboxyphenoxypropane anhydride) [124] and poly(erucic acid dimer anhydride) [125] are biodegradable polymers for controlled drug delivery in a form of implant or injectable microspheres. Studies in rabbits have shown that the osteocompatibility of poly(anhydrides) that undergo photocuring are comparable to PLA and that the implants of poly(anhydrides) show enhanced integration with the surrounding bones in comparison to PLA controls [126]. 1.3.5.2.2 Poly(ortho-esters) Poly(ortho-esters) (POE) scaffolds were coated with cross-linked acidic gelatine to improve surface properties for cell attachment. Preliminary in vitro and in vivo results revealed that POE did not show any inflammation and had little or no effect on bone formation while PLA provoked a chronic inflammatory response and inhibited bone formation [127,128]. 1.3.5.2.3 Polyphosphazenes These polymers seem to be potential bioerodible materials capable of controlled degradation and sustained drug delivery for therapeutic use [101,129] and bone regeneration [130]. Their tailored side groups enable a wide variety of hydrolytic properties to be designed into selected polymers for application in biological environments without the release of harmful degradation products at physiological concentration. 1.3.6 BIOCOMPOSITES From a biological perspective, it is a natural strategy to combine polymers and ceramics to fabricate scaffolds for bone tissue engineering because native bone is the combination of a naturally occurring polymer and a biological apatite. From the point of view of materials science, a single material type does not always provide the necessary mechanical and chemical properties desired for a particular application. In these instances, composite materials designed to combine the advantages of both components may be most appropriate. Polymers and ceramics that degrade in vivo should be cho- sen for designing biocomposites for tissue-engineering scaffolds. While massive release of acidic degradation from polymers can cause inflammatory reactions [4,92,131], the basic degradation of calcium phosphate or bioactive glasses would buffer the acidic by-products of polymers and may thereby help to avoid the formation of an unfavorable environment for cells due to a decreased pH level. Mechanically, bioceramics are much stronger than polymers and play a critical role in provid- ing mechanical stability to constructs prior to the synthesis of a new bone matrix by cells. However, ceramics and glasses are very fragile because of their intrinsic brittleness and flaw sensitivity. To capitalize on their advantages and minimize their shortcomings, ceramic and glass materials have been combined with various biopolymers to form composite biomaterials for osseous regenera- tion. Table 1.7 lists selected ceramic/glass–polymer composites, which were designed as biomedical devices or scaffold materials for bone tissue engineering, and their mechanical properties. In general, all these synthetic composites have good biocompatibility. Kikuchi et al. [132], for instance, combined TCP with PLA to form a polymer–ceramic composite, which was found to pos- sess the osteoconductivity of β-TCP and the degradability of PLA [132]. The research team led by Laurencin [147] synthesized porous scaffolds containing PLGA and hydroxyapatite, which were reported to combine the degradability of PLGA with the bioactiv- ity of hydroxyapatite, fostering cell proliferation and differentiation as well as mineral formation CRC_7973_Ch001.indd 16 CRC_7973_Ch001.indd 16 1/25/2008 7:14:34 PM 1/25/2008 7:14:34 PM
- 38. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 17 TABLE 1.7 Biocomposites Designed for Bone Tissue Engineering Biocomposites Percentage of Ceramic (%) Porosity (%) Pore Size (µm) Compressive (C), Tensile(T), Flexural Strength (F) (MPa) Modulus (MPa) Ultimate Strain (%) Toughness (kJ/m 2) References Ceramic Polymer Dense composites HA fi ber PDLLA 2–10.5 (vol.) Not applicable 45 (F) 1.75–2.47×10 3 133 PLLA 10–70 (wt.) 50–60 (F) 6.4–12.8×10 3 0.7–2.3 134 HA PLGA 40–85 (vol.) 22 (F) 1.1×10 3 5.29 135,136 Chitosan Chitosan+PLGA 40–85 (vol.) 40–85 (vol.) 12 (F) 43 (F) 2.15×10 3 2.6×10 3 0.092 9.77 136 136 PPhos 85–95 (wt.) 137 Collagen 50–72 (wt.) 138 β-TCP PLLA-co-PEH PPF 75 (wt.) 25 (wt.) 51 (F) 7.5–7.7 (C) 5.18×10 3 191–134 132 139 A/W PE 10–50 (vol.) 18–28 (F) 0.9–5.7×10 3 140 Ca 3 (CO 3 ) 2 PLLA 30 (wt.) 50 (C) 3.5–6×10 3 141 Human cortical bone 50–150 (T) 130–180 (C) 12–18×10 3 28,43–46 Porous composites Amorphous CaP PLGA 28–75 (wt.) 75 >100 65 142,143 β-TCP Chitosan–Gelatin 10–70 (wt.) 322–355 0.32–0.88 (C) 3.94–10.88 144 HA PLLA 50 (wt.) 85–96 100×300 0.39 (C) 10–14 145 PLGA 60–75 (wt.) 81–91 800–1800 0.07–0.22 (C) 2–7.5 146 PLGA 30–40 110–150 337–1459 147 Bioglass PLGA 75 (wt.) 43 89 0.42 (C) 51 64,148,149 PLLA 20–50 (wt.) 77–80 ∼100 ∼10 1.5–3.9 (T) 137–260 1.1–13.7 150 PLGA 0.1–1 (wt.) 50–300 151 PDLLA 5–29 (wt.) 94 ∼100 10–50 0.07–0.08 (C) 0.65–1.2 7.21–13.3 111,112,152 Phosphate glass A/W PLA–PDLLA PDLLA 40 (wt.) 20–40 (wt.) 93–97 85.5–95.2 98–154 0.017–0.020 (C) 0.075–0.12 153 154 Human cancellous bone 4–12 (C) 100–500 1.65–2.11 155,156 CRC_7973_Ch001.indd 17 CRC_7973_Ch001.indd 17 1/25/2008 7:14:34 PM 1/25/2008 7:14:34 PM
- 39. 18 Biomaterials Fabrication and Processing Handbook [147,157,158]. Similarly, composites of bioactive glass and PLA were observed to form calcium phosphate layers on their surfaces and support rapid and abundant growth of human osteoblasts and osteoblast-like cells when cultured in vitro [109–112,148–154]. A comparison between the dense composites and cortical bone indicates that the most promis- ing synthetic composite seems to be hydroxyapatite fiber–reinforced PLA composite [134], which, however, exhibit mechanical property values close to the lower values of the cortical bone. Other promising composite scaffolds reported in literature are those from Bioglass and PLLA or PDLLA [149–152]. They have a well-defined porous structure, for example obtained by thermally induced phase separation [151], at the same time their mechanical properties are close to (but lower than) those of cancellous bone. 1.3.7 SUMMARY To design an ideal scaffold, which is bioresorbable, biocompatible, provides for cell attachment, proliferation, and maturation, and which disappears whenever a new bone forms allowing the new bone to undergo remodeling, it is necessary to weight up the pros and cons of the potential precursor materials, as summarized in Table 1.8. Among the bioactive ceramics and glasses listed in Table 1.8, bioactive (silicate) glasses have remarkable advantages. The ability to enhance vascularization, the role of silicon in upregulating TABLE 1.8 Advantages and Disadvantages of Synthetic Scaffold Biomaterials in Bone Tissue Engineering Biomaterials Positive Negative Calcium phosphates (e.g., HA, TCP, and BPCP) 1. Excellent biocompatibility 2. Supporting cell activity 3. Good osteoconductivity 1. Too fragile in amorphous structure 2. Nearly bioinert in crystalline phase Bioactive glasses and glass-ceramics 1. Excellent biocompatibility 2. Supporting cell activity 3. Good osteconductivity 4. Vascularization 5. Upregulation of gene expression 6. Tailorable degradation rate 1. Mechanically brittle and weak in the glass state 2. Degrade slowly in crystalline structures, except for 45S5 Bioglass-derived glass-ceramics Bulk biodegradable polymers (e.g., PLA, PGA, PLGA, PPF) 1. Good biocompatibility 2. Biodegradable with a wide range of degradation rates 3. Bioresorbable 4. Good processability 5. Good ductility 1. Inflammation caused by acid degradation products 2. Accelerated degradation rates cause collapse of scaffolds Surface bioerodible polymers (e.g., POE, poly(anhydrides), poly(phosphazene)) 1. Good biocompatibility 2. Retention of mechanical integrity over the degradative life of the device 3. Significantly enhanced bone ingrowth into the porous scaffolds, owing to the increment in pore size 1. They cannot be completely replaced by new bone tissue Composites 1. Excellent biocompatibility 2. Supporting cell activity 3. Good osteconductivity 4. Tailorable degradation rate 5. Improved mechanical properties 1. Fabrication techniques can be complex CRC_7973_Ch001.indd 18 CRC_7973_Ch001.indd 18 1/25/2008 7:14:35 PM 1/25/2008 7:14:35 PM
- 40. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 19 gene expression, and the tailorable degradation rate make bioactive glasses promising scaffold materials over others, and so they could be the material of choice as the inorganic component of composite scaffolds. Although bioactive glasses are brittle with low fracture toughness (Table 1.4), they can be used in combination with polymers to form composite materials. The ability to couple mechanical strength with tailorable biodegradability makes 45S5 Bioglass-derived glass-ceramics advantageous over calcium phosphates (including hydroxyapatite), as well as other bioactive glasses and related glass-ceramics. Between the two types of polymers, the bulk degradable type is more promising than the surface-erosive group, considering that being replaced by new bone tissue is one of the important criteria of an ideal scaffold material (Table 1.1). Finally, it is obvious that composites can be consid- ered ideal scaffolding materials for bone tissue engineering if fabrication processes suitable for the production of 3-D structures of the required size and shape and amenable to commercialization are further developed and optimised. 1.4 FABRICATION OF TISSUE-ENGINEERING SCAFFOLDS 1.4.1 FABRICATION OF INORGANIC SCAFFOLDS Porous ceramics can be produced by a variety of different processes [2,159], which may be classi- fied into two main categories: (1) manual-based processing techniques and (2) computer-controlled fabrication processes, such as solid free-form (SFF) technology, which is also commonly known as rapid prototyping (RP) [160]. Most manual-based processing techniques can further be divided into two groups: conventional powder-forming processes and sol–gel techniques [161]. 1.4.1.1 Powder-Forming Processes A flowchart that is common to all powder-forming processes is shown in Figure 1.2, and the differ- ent steps involved in these processes are discussed in this section. 1.4.1.1.1 Preparation of Slurries Slurry is a suspension of ceramic particles in a suitable liquid (e.g., water or ethanol) used to prepare green bodies. The inherent mechanism of pore formation in a powder compact is illustrated in Figure 1.3. Attractive forces that consist of hydrogen bonds, van der Waals forces, Coulomb’s forces, and physical friction between particles cause agglomeration of particles. Addition of fillers to the FIGURE 1.2 Flowchart of the powder-sintering method to produce porous ceramic scaffolds. Heat treatment of the green body to sinter the ceramic structure Prepare slurry from the powder Form a green body from the slurry Start with a ceramic powder Porous ceramic Add Additives (e.g., porogen, binder) CRC_7973_Ch001.indd 19 CRC_7973_Ch001.indd 19 1/25/2008 7:14:35 PM 1/25/2008 7:14:35 PM
- 41. 20 Biomaterials Fabrication and Processing Handbook slurry, such as sucrose, gelatine, and PMMA microbeads, and a wetting agent (i.e., a surfactant) can increase porosity. These chemicals, which are called porogens, are evaporated or burned out during sintering, and as a result pores are formed [2,159]. One successful formulation has been the use of hydroxyapatite powder slurries (dispersed with vegetable oil) added with gelatine solution [162], which has led to porous scaffolds with interconnected pore structure with pore diameters of ∼100 µm. A similar process has been used to prepare melt-derived Bioglass scaffolds using cam- phor (C10H16O) as the porogen [163]. Binders are also added to slurries. The most important function of a binder is to improve the strength of the green body in order to provide structural integrity for handling (green strength) before the product is sintered [164]. Polysaccharides [165], polyvinyl alcohol (PVA) [166], and poly- vinyl butyl (PVB) [167] are the frequently added binders in bioceramic slurries. 1.4.1.1.2 Formation of Green Bodies In ceramic production, a green body is always porous, and its structure largely determines that of the sintered product. Table 1.9 lists different methods of obtaining green bodies for 3-D porous ceramics. These methods can be classified into two categories: dry and wet processes [159]. They lead to different porous structures and pore volume fractions. Certain techniques, such as tape casting, extrusion, slurry dipping, and spraying, are not included here; because they aim at achieving a predetermined geometric shape of ceramic parts (such as rods, tubes, sheets, and coating on films), instead of a given porous structure. Except injection molding, all conventional processes listed in Table 1.9 have been applied to synthesize ceramic scaffolds for tissue engi- neering as discussed below. FIGURE 1.3 Schematic illustration of pores among agglomerates and particles [159]. Primary particle Agglomerate of primary particles Pore among agglomerates Pore among primary particles TABLE 1.9 Methods for Obtaining Ceramic Bodies for 3-D Porous Ceramics [159] Dry processes Loose packing Compaction Uniaxial pressing Cold isostatic pressing (CIPing) Wet processes Slip casting Injection molding Phase separation/freeze-drying Polymer replication Gelcasting CRC_7973_Ch001.indd 20 CRC_7973_Ch001.indd 20 1/25/2008 7:14:35 PM 1/25/2008 7:14:35 PM
- 42. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 21 Dry methods. The simplest way to prepare ceramic green bodies is the dry powder method where powders are directly compressed by pressing (uniaxially or isostatically) into molds, thereby forming green bodies. Pore diameters decrease and mechanical properties increase as the packing density of the particles in the green bodies increases. A densification step by sintering at high tem- perature is required (see Section 1.4.1.1.3). Mechanical properties can be increased further by hot- isostatic pressing (HIP) [168] or by uniaxial hot pressing. These pressure-assisted methods decrease the pore diameter as well. The addition of porogens, such as sucrose and camphor, enhances the formation of pores [159]. Slip casting. Slip is a creamy (relatively thick) slurry. In this method, the slurry is cast into a porous mold. The liquid of the slurry is absorbed into the porous mold, and as a result the particles in the slurry are filtered, which adhere to the mold surface. After this process, a porous green body is obtained through further drying [161,169]. Phase separation/freeze-drying. In this method, a ceramic slurry is poured into a container, which is immersed in a freezing bath. Thus, ice is stimulated to grow and ceramic particles are piled up between the columns of the growing ice. After the slurry is completely frozen, the container is dried in a drying vessel, usually under vacuum [170]. The pores are created by the ice crystals that sublimate at a reduced pressure. Freeze-drying removal of ice crystals creates 3-D interconnected pore channels with complex structures. The porous structure can be customized by the variation of the slurry concentration, freezing temperature, and pressure. Replication technique. This method, which is also called the polymer-sponge method, was patented for the manufacturing of ceramic foams [171]. In the polymer-replication process, the green bodies of ceramic foams are prepared by coating a polymer (e.g., polyurethane) foam with a ceramic slurry. The polymer foam, which already has the desired macrostructure, simply serves as a sacrificial template for the ceramic coating. The polymer template is immersed in the slurry, which subsequently infiltrates the structure, and so the ceramic particles adhere to the surface of the polymer substrate. Excess slurry is squeezed out leaving a ceramic coating on the foam struts. After it is dried, the polymer is slowly burned out in order to minimize dam- age to the porous ceramic coating. After the removal of the polymer, the ceramic is sintered to the desired density. The process replicates the macroporous structure of the polymer foam and results in a rather distinctive microstructure within the struts. A flowchart of the process is given in Figure 1.4 [172]. This method has been applied for the preparation of foam-like scaffolds for tissue engineering, including porous calcium phosphates [173], Bioglass [80], and other inert bioceramics [172,174]. FIGURE 1.4 Flowchart of the replication process to produce a ceramic foam. Dry, burn out the polymer substrate, and sinter the green body Coat a polymer foam with the slurry Prepare slurry from the powder Ceramic powder Ceramic foam Add Binder CRC_7973_Ch001.indd 21 CRC_7973_Ch001.indd 21 1/25/2008 7:14:35 PM 1/25/2008 7:14:35 PM
- 43. 22 Biomaterials Fabrication and Processing Handbook Apart from the slurry-immersion coating, electrospray coating techniques have also been applied together with the polymer-sponge process to produce ceramic foams, for example, Al2O3 [175] and ZrO2 foams [176]. Unlike the foams produced by the slurry-immersion method, the struts of the ceramic foams produced by the process of electrospray coating contain fewer holes and cracks. This microstructure can lead to improved mechanical properties of the foams [176]. Another possibility investigated to improve the mechanical properties of foams made by the rep- lication method is to apply a thin polymer coating on the porous structure. For example, to improve the mechanical stability of highly porous Bioglass-derived scaffolds produced by the replication technique [80], a polymer coating, such as poly(d,l-lactic acid) (PDLLA), was applied [177]. The coating thickness was approximately 3 µm on an average. Although the thin coating layer did not increase the mechanical strength of the foams considerably, it significantly improved the mechani- cal stability of the structure. The fracture energy of the coated foams was ∼20 times higher than that of uncoated foams. More importantly, upon immersion in SBF, nanofibers of hydroxyapatite deposited within the PDLLA coating layer, eventually a nanocomposite layer, formed biomimeti- cally on the strut surfaces. This method has remarkably improved the mechanical performance of the scaffolds in a biological environment [177]. Gelcasting. This method adopts one of the direct-foaming techniques mentioned in Table 1.10 to achieve highly porous green bodies. The foamed suspension is set through a direct-consolidation technique, listed in Table 1.10, that is, polymerization of organic monomers (i.e., gelation), in which the particles of the slurry are consolidated through polymerization reaction. A green body is formed after the gel is cast in a mold [178–180]. Figure 1.5 gives the flowchart of the gelcasting process. Two factors are critical in the gelcasting process: (1) the gelation speed must be fast enough to prevent foam collapse, and (2) the gel rheology is important because the process involves casting. Systems of high fluidity are required in order to enable easy filling of small details in molds to allow production of high-complexity shapes. Gelcasting techniques have been applied to produce hydroxy- apatite foams [181–183]. Gelcasting has also been combined with the replication process (described above in this section) to produce hydroxyapatite scaffolds with interconnected pores [184]. 1.4.1.1.3 Sintering The final step in the production of a ceramic foam is the densification of the green bodies by con- ducting a high temperature sintering process. Foams are normally dried at room temperature for at least 24 h prior to sintering. In this step, controlled heating is important to prevent collapse of the ceramic network. The heating rate, sintering temperature, and holding time depend on the ceramic starting materials. For example, values are in the range of 0.5–2°C/min, 1200–1350°C, and 2–5 h, TABLE 1.10 Techniques of Direct Foaming and Direct Consolidation Techniques References Direct foaming 1. Injection of gases through the fluid medium 2. Mechanically agitating particulate suspension 3. Blowing agents 4. Evaporation of compounds 5. Evaporation of gas by in situ chemical reaction 20 Direct consolidation 1. Gelcasting 2. Direct coagulation consolidation (DCC) 3. Hydrolysis-assisted solidification (HAS) 4. Freezing (quick set) 178 CRC_7973_Ch001.indd 22 CRC_7973_Ch001.indd 22 1/25/2008 7:14:36 PM 1/25/2008 7:14:36 PM
- 44. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 23 respectively, in the case of porous hydroxyapatite [173,181,183,185]. It is worthwhile noticing that there is a narrow time–temperature window for densification of foams made from bioactive glasses, which are prone to crystallize while sintering by viscous flow. Hence the production of bioactive glass foams by powder-based methods presents difficulties [80]. 1.4.1.2 Sol–Gel Techniques 1.4.1.2.1 Sol–Gel Process and Synthesis of Aerogel Ceramics The sol–gel process is a well-developed, robust, and versatile “wet” technique for the synthesis of ceramics and glasses. By applying the sol–gel process, it is possible to fabricate inorganic mate- rials in various forms: ultrafine or spherical shaped powders, thin film coatings, ceramic fibers, microporous inorganic membranes, monolithic ceramics and glasses, and extremely porous aerogel materials [186]. The processing path of aerogel ceramics starts with an alkoxide precursor. Alkoxide precursors, such as tetraethyl orthosilicate (TEOS) and triethoxyl orthophosphate (TEP), undergo hydrolysis and condensation reactions to form a sol. In case of silicate precursors, polymerization of –Si–OH groups continues after hydrolysis is complete, beginning the formation of the silicate (–Si–O–Si–) network. The network connectivity increases until it spans throughout the solvent medium. Eventually a wet gel forms. The wet gel is then subjected to controlled thermal processes of aging to strengthen the gel, drying to remove the liquid by-product of the polycondensation reaction, and thermal sta- bilization (or sintering) to remove organic species from the surface of the material; and as a result, a porous aerogel forms [2,187]. 1.4.1.2.2 Production of Highly Porous Glasses Highly porous glasses (or glass foams) have been developed by a slightly modified sol–gel pro- cess [188]. The sol–gel process is based on the polymerization reactions of metal alkoxide precur- sors (usually TEOS and TEP). These precursors are dissolved in a solvent, and a gel is formed by hydrolysis and condensation reactions. The gel is then subjected to controlled thermal processes of aging to strengthen the gel, drying to remove the liquid by-product of the polycondensation reac- tion, and thermal stabilization/sintering to remove organic species from the surface of the material (500–800°C). Sol–gel derived glass scaffolds are obtained by directly foaming the sol with the use Prepare suspension from the powder Ceramic powder Add Dispersant, surfactant, monomer, cross-linker Foam the suspension using one of the foaming techniques in Table 2.16 While the foamed suspension is poly merized to form a gel, cast the gel Ceramic foam Dry and sinter the green body Initiator, catalyst Add FIGURE 1.5 Flowchart of the gelcasting method to produce a ceramic foam. CRC_7973_Ch001.indd 23 CRC_7973_Ch001.indd 23 1/25/2008 7:14:36 PM 1/25/2008 7:14:36 PM
- 45. 24 Biomaterials Fabrication and Processing Handbook of a surfactant and catalysts [188–190]. Therefore, after sol hydrolysis, the surfactant (e.g., Teepol, a detergent containing a low-concentration mixture of anionic and nonionic surfactants), water (improves foamability of surfactant), and the catalyst for polycondensation (e.g., HF) are added by vigorous agitation. A flowchart of the process is given in Figure 1.6. Porosity of the foam scaffolds is influenced by the foaming temperature, water content, and catalyst content. Sol–gel derived bio- active glass foams [191,192] and gelcast hydroxyapatite scaffolds [181,183] have shown favorable results in both in vitro and in vivo tests for bone regeneration. 1.4.1.3 Solid Free-Form Techniques SFF techniques, also known as RP, are computer-controlled fabrication processes. They can rapidly produce highly complex 3-D objects using data generated by computer-aided design (CAD) systems. In a typical case, an image of a bone defect in a patient can be taken, which is used to develop a 3-D CAD model. The computer can then reduce the model to slices or layers. The 3-D objects are constructed layer-by-layer using RP techniques such as fused deposition modeling (FDM), selective laser sintering (SLS), 3-D printing (3-DP), or stereolithography [160]. Calcium phosphate scaffolds have been produced using the FDM process [193,194], SLS, 3-DP processes [160], stereolithogra- phy [195,196], and RP combined with replication technique [197]. The typical process chain for all SFF techniques is presented in Figure 1.7. To date, only a small number of SFF techniques, such as 3-DP, FDM, and SLS, have been adopted for tissue-engineering scaffolds. The following paragraphs give brief descriptions of the principles on which these three techniques are based. Comprehensive technical details can be found in previous detailed reviews [160,198–201]. 1.4.1.3.1 Three-Dimensional Printing Three-dimensional printing employs ink-jet printing technology for processing materials from pow- ders. Therefore, this technique is a combination of SFF and powder sintering. During fabrication, a printer head is used to print a liquid binder onto thin layers of powder following the object’s profile being generated by the system computer. The subsequent stacking and printing layer recreates the full structure of the desired object. Alkoxides: TEOS and TEP Prepare a sol from the alkoxides and Ca(NO3)2 in deionized water solvent Add Catalysis (HNO3) to speed up hydrolysis Foam the sol by vigorous agitation When the gelation of the foamed sol is nearly completed, cast the gel in molds Glass foam Age, dry, and sinter the gel Surfactant for foaming, catalyst (HF) for gelation Add FIGURE 1.6 Flowchart of the production of bioactive glass foams using sol–gel technology. CRC_7973_Ch001.indd 24 CRC_7973_Ch001.indd 24 1/25/2008 7:14:36 PM 1/25/2008 7:14:36 PM
- 46. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 25 1.4.1.3.2 Fused Deposition Modeling FDM employs the concept of melt extrusion to deposit a parallel series of material rods that forms a material layer. In FDM, filament material stock (generally thermoplastic) is fed and melted inside a heated liquefier head before being extruded through a nozzle with a small orifice. Indirect fabrication methods involving FDM have been applied for producing porous bioc- eramic implants. In this method, FDM was employed to fabricate wax molds containing the nega- tive profiles of the desired scaffold microstructure. Ceramic scaffolds were then cast from the mold through a lost mold technique [193,194]. 1.4.1.3.3 Selective Laser Sintering SLS employs a CO2 laser beam to selectively sinter polymer, ceramic, or polymer-ceramic com- posite powders to form material layers. The laser beam is directed onto the powder bed by a high precision laser scanning system. The fusion of material layers that are stacked on top of one another replicates the object’s height [202,203]. 1.4.1.4 Comparison of Fabrication Techniques for Ceramic or Glass Scaffolds Table 1.11 lists the porosity, pore size, and mechanical properties of several porous ceramics pro- duced by different techniques. Figure 1.8 shows typical pore structures produced by different tech- niques. Comparing the pore structures of ceramic scaffolds shown in Figure 1.8 with the structure of cancellous bone, it is evident that the pore morphology produced by the replication technique is the most similar one, containing completely interconnecting pores and solid material forming only the struts. The ceramic foams synthesized by gelcasting and sol–gel techniques come next in terms of structural similarity to cancellous bone, however, it is expected that these foams exhibit lower pore interconnectivity than foams made by the replication method. The advantages of replication method over other ceramic foaming techniques are summarized in Table 1.12. In brief, the replication technique meets all criteria posed on the fabrication process of tissue-engineering scaffolds: suitable for commercialization, reproducible, cost-effective, safe, and capable of producing irregular or complex shapes. Contemporary authors consider the replication technique as the optimal technique for production of novel bioactive glass-ceramic scaffolds for bone tissue engineering [204]. FIGURE 1.7 Flowchart of the typical rapid prototyping (RP) process [160]. Medical imaging • CT, MRI, etc. 3-D solid model creation in CAD • Pro/engineer (PTC) SFF system computer • Generation of slice data, etc. SFF fabrication • SLS, FDM, etc. Post-processing • Finishing and cleaning CRC_7973_Ch001.indd 25 CRC_7973_Ch001.indd 25 1/25/2008 7:14:37 PM 1/25/2008 7:14:37 PM
- 47. 26 Biomaterials Fabrication and Processing Handbook TABLE 1.11 Porous Structures and Mechanical Properties of Porous Bioceramics Produced by Different Techniques Technique Materials Porosity (%) Pore Size (µm) Closed (C) or Open (O) Compressive/Flexural Strength (MPa) References Powder forming–sintering Dry process with porogens Hydroxyapatite NA Varying between 40 and 100 C NA 166 Hydroxyapatite 67 250–400 O 205 45S5 Bioglass 21 42 200–300 80 C C 163 206 69 Phase separation/ freeze-drying Al 2 O 3 30–60 ∼50 in width, 300–500 in length O NA 170 Replication technique/coated by Slurry-immersion Al 2 O 3 TiO 2 Glass-reinforced HA Hydroxyapatite HA coated by PLGA Bioglass 87 74 85–97.5 69–86 69–86 >90 Up to 800 385–700 Average size 420–560 490–1130 490–1130 400–800 O O O O O O NA 0.01–0.175 0.03–0.29 0.31–4.03 0.4–0.5 169 165 185 173 173 80 Electrospray Al 2 O 3 96 ∼800 O 175 Gelcasting/foamed by Starch Al 2 O 3 23–70 10–80 C NA 178 CRC_7973_Ch001.indd 26 CRC_7973_Ch001.indd 26 1/25/2008 7:14:37 PM 1/25/2008 7:14:37 PM
- 48. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 27 Vigorous stirring Al 2 O 3 Al 2 O 3 Hydroxyapatite Hydroxyapatite Hydroxyapatite 70–92 NA 76.7–80.2 48 NA Average size: 260–700 Range in 50–2000 NA 20–1000 50–300 Cell: 100–500 Window: 30–120 Partly O/C NA Partly O/C Partly O/C Partly O/C 2–26 3–20 179 180 Replication technique Hydroxyapatite β-TCP+HA 70–77 73 200–400 NA O O 4.4–7.4 8 1.6–5.8 0.55–5 9.8 181 183 182 184 207 Sol–gel/foamed by Burning PMMA beads Decomposition of H 2 O 2 Burning EO-PO-EO blocks Vigorous stirring CaO–SiO 2 glass (CH 3 O) 4 Si SiO 2 glass Bioactive glasses 70–95 ∼0.5 <0.7 1–10 Up to 600, size of cell windows mostly in 80–120 Partially O/C Partially O/C Partially O/C Partially O/C 208 209 210 189 Solid free-form (SFF) FDM Al 2 O 3 β-TCP CaO-Al 2 O 3 PP-TCP composite 29–44 29–44 29–44 36–52 305–480 305–480 300 160 O O O O 62–128 0.25–1.45 2–24 12.7–10 211 211 212 213 SLS Calcium phosphates 30 200 O 13.8 214 CRC_7973_Ch001.indd 27 CRC_7973_Ch001.indd 27 1/25/2008 7:14:37 PM 1/25/2008 7:14:37 PM
- 49. 28 Biomaterials Fabrication and Processing Handbook e VFP = 0.35 a b c d f g h 500 µm 100 µm FIGURE 1.8 Typical structures of porous ceramics produced by different techniques: (a) porous hydroxya- patite produced by the powder method combined with PVA as a porogen additive [205], (b) porous alumina by freeze-drying method [170], (c) porous hydroxyapatite by gelcasting method [181], (d) bioactive glass foams by sol–gel technique [188], (e) porous β-TCP by solid free-form technique [211], (f) β-TCP+hydroxyapatite foam produced by the replication technique [207], (g) porous bioglass-based glass-ceramic foam by the replication technique [80], and (h) porous structure of cancellous bone [215]. TABLE 1.12 Advantages of the Replication Technique over Other Methods 1. Cancellous bone-like macroporous structure The porous structure produced by the replication technique is very similar to cancellous bone: highly porous network with open and highly interconnected porosity, compared with the rest of the techniques (see Figures 1.8 (f, g, h)). 2. High commercialization potential This technique is the simplest and most cost-effective method, and thus most suitable for commercialization, for example, compared with SFF. SFF-RP, which are expensive processes, it may be a method for producing specific and complex scaffold architectures. 3. Safety It does not involve any toxic chemicals, compared with sol–gel and gelcasting techniques, which use HF to accelerate polymerization. 4. Irregular or complex shape production ability It can produce scaffolds of irregular or complex shapes, compared with standard dry powder processing or sol–gel-based methods. 1.4.2 FABRICATION OF COMPOSITE SCAFFOLDS The fabrication of polymer–ceramic composite scaffolds is based on conventional processes used for neat polymeric scaffolds. Numerous techniques have been developed to process porous poly- mer scaffolds for use in tissue engineering. Table 1.13 lists currently applied 3-D polymer scaf- fold fabrication technologies, based on Ref. 1. Excellent reviews can be found in the literature [1,216–219]. While intensive efforts have been made to develop the processing technologies of polymer scaf- folds, relatively less attention has been paid to the fabrication of porous composite scaffolds. Among the technologies in Table 1.13, solvent casting with or without particle leaching [146,150,153,154] and TIPS combined with freeze-drying [109–112,144,145] seem to be the most applied method to the fabrication of polymer–ceramic composite scaffolds. In addition to these methods, there are CRC_7973_Ch001.indd 28 CRC_7973_Ch001.indd 28 1/25/2008 7:14:37 PM 1/25/2008 7:14:37 PM
- 50. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 29 TABLE 1.13 3-D Fabrication Technologies of Polymer Scaffolds [1] Fabrication Technology Required Properties of Materials Reproducibility Available Pore Size (µm) Porosity (%) Architecture Solvent casting/particle leaching Soluble User, material and technique sensitive 30–300 20–50 Spherical pores Membrane lamination Soluble User, material and technique sensitive 30–300 <85 Irregular pores Textile technology Fibers Machine controlled 20–100 <95 Melt molding Thermoplastic Machine controlled 50–500 <80 Extrusion/particle leaching Thermoplastic Machine controlled <100 <84 Spherical pores Emulsion freeze-drying Soluble User, material and technique sensitive <200 <97 High volume of interconnected micropores Thermally induced phase separation Soluble User, material and technique sensitive <200 <97 High volume of interconnected micropores Gas foaming Amorphous Material and technique sensitive <100 10–30 High volume of noninterconnected micropores Gas foaming/particle leaching Amorphous Material and technique sensitive Micropores <50 Macropores <400 <97 Low volume of noninterconnected micropores combined with high volume of interconnected macropores Three-dimensional printing Soluble Machine and computer controlled 45–150 <60 100% interconnected macrospores Fused deposition modeling Thermoplastic Machine and computer controlled >150 <80 100% interconnected macrospores CRC_7973_Ch001.indd 29 CRC_7973_Ch001.indd 29 1/25/2008 7:14:39 PM 1/25/2008 7:14:39 PM
- 51. 30 Biomaterials Fabrication and Processing Handbook two other methods: microsphere sintering and foam coating, which have been considered exten- sively for the combination of ceramic and polymeric materials, as listed in Table 1.14 and shown in Figure 1.9. This section will briefly introduce the processing techniques for polymer-ceramic composite scaffolds. 1.4.2.1 Solvent Casting Solvent casting of biocomposite scaffolds involves the dissolution of the polymer in an organic solvent, mixing it with ceramic granules, and casting the solution into a predefined 3-D mold. The solvent is subsequently allowed to evaporate. The main advantage of this processing technique is the ease of fabrication without the need of specialized equipment. The primary disadvantages of solvent cast- ing are (1) the limitation in the shapes (typically flat sheets and tubes are the only shapes that can be formed), (2) the possible retention of toxic solvent within the polymer, and (3) the denaturation of the proteins and other molecules incorporated into the polymer by the use of solvents. The use of organic solvents to cast the polymer may decrease the activity of bioinductive molecules (e.g., protein). The detailed processing steps can be found in Ref. 65. 1.4.2.2 Solvent Casting or Particle Leaching and Microsphere Packing Polymer-ceramic constructs can be fabricated by the solvent aggregation method. The polymer microspheres are first formed from traditional water oil/water emulsions. Solvent-aggregated polymer-ceramic scaffolds can then be constructed by mixing solvent, salt or sugar particles, ceramic granules, and prehardened microspheres [220]. A 3-D structure of controlled porosity TABLE 1.14 Fabrication Methods of Three-Dimensonal Porous Composite Scaffolds Fabrication Technique Biocomposites Percentage of Ceramic (%) Porosity (%) Pore Size (μm) References Ceramic Polymer Solvent casting/particle leaching HA PLGA 60–75 (wt.) 81–91 800–1800 146 Bioglass PLLA 20–50 (wt.) 77–80 ∼100 (macro) ∼10 (micro) 150 Phosphate glass PLA–PDLLA 40 (wt.) 93–97 153 A/W PDLLA 20–40 (wt.) 85.5–95.2 98–154 154 Thermally induced phase separation/freeze-drying β-TCP Chitosan– gelatin 10–70 (wt.) 322–355 144 HA PLLA 50 (wt.) 85–95 100×300 145 Bioglass PDLLA 5–29 (wt.) 94 ∼100 (macro) 10–50 (micro) 109–112, 152, 221 Microsphere/sintering Amorphous CaP PLGA 28–75 (wt.) 75 >100 142, 143 Bioglass PLGA 75 (wt.) 43 89 149 Polymer foam/ceramic coating HA Bioglass PLGA PDLLA 40–85 (vol.) 135, 136 109, 110, 222, 223 Ceramic foam/polymer coating HA foam PDLLA 173, 221–223 CRC_7973_Ch001.indd 30 CRC_7973_Ch001.indd 30 1/25/2008 7:14:39 PM 1/25/2008 7:14:39 PM
- 52. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 31 is formed based on this method combined with particle leaching and microsphere packing. This method shares similar advantages and disadvantages with the solvent casting technique. Details of the method are presented in Ref. 65. 1.4.2.3 Thermally Induced Phase Separation or Freeze-Drying Three-dimensional porous structures can also be achieved through phase separation and evapora- tion. An approach to induce phase separation is to lower the temperature of the suspension of poly- mer and ceramic materials. The solvent is solidified first, forcing the polymer and ceramic mixture into the interstitial spaces. The frozen mixture is then lyophilized using a freeze dryer in which the ice solvent evaporates [109,144,145]. 1.4.2.4 Microsphere Sintering In this process, microspheres of a ceramic and polymer composite are synthesized first, using emul- sion or solvent evaporation technique. Sintering the composite microspheres yields a 3-D porous scaffold [142,143]. 1.4.2.5 Foam Coating An alternative approach to address the combination of polymeric and ceramic materials is to coat bioactive ceramics onto polymeric foams [220–222]. The inverse method, known as polymer-coated ceramic scaffolds, has also been investigated [173,177,223], as discussed in Section 1.4.1.1.2. FIGURE 1.9 Typical structures of porous biocomposites by various techniques: (a) solvent casting or parti- cle leaching [146,150], (b) phase separation or freeze-drying [145], (c) microsphere sintering [143], (d) polymer coating of Bioglass foam [177]. c a b d 200 µm CRC_7973_Ch001.indd 31 CRC_7973_Ch001.indd 31 1/25/2008 7:14:39 PM 1/25/2008 7:14:39 PM
- 53. 32 Biomaterials Fabrication and Processing Handbook 1.5 SURFACE FUNCTIONALIZATION The biocompatibility of biomaterials is related to cell behavior in contact with the biomaterial surface and particularly to cell adhesion to the surface. Surface characteristics of materials, such as topography (roughness), chemistry, or surface energy, play a key role in the cell adhesion behavior on biomaterials. The first stage in cell or material interactions involves cell attachment, adhesion, and spreading. The quality of this first phase will influence the cell’s capacity to proliferate and to differentiate (second stage) in contact with the scaffold [224–228]. The process of bone tissue regeneration involves expression of genes and synthesis of proteins known to be important in the mineralization process. To ensure the in vivo biocompatibility of an implant, it is often necessary to modify the material surface, either physically by surface roughen- ing, for example, or chemically, such as by attachment of chemically active species [228,229]. To improve the cell–substrate interaction, different strategies including surface modification have been developed. The functionalized surfaces can control not only the initial protein adsorp- tion and production, but also the differentiation potential of different cells (such as human stem cells) [229]. The main strategies developed for surface modification are presented in Sections 1.5.1 through 1.5.4. 1.5.1 PROTEIN ADSORPTION This first phase of cell–implant interaction depends on protein adhesion. The proteins currently used for chemical surface modification of biomaterials are growth factors (or related proteins) and adhesion proteins (or related peptides). Members of the transforming growth factor-β family are widely studied: TGF-β1, bone morphogenetic proteins BMP-2, BMP-7, or osteogenic protein OP-1 [230–233]. Among adhesion proteins, RGD-peptides (contain a sequence of the amino acids: argi- nine R, glycine G, and aspartic acid D) have a high efficacy in promoting osteoblast adhesion [226,234,235]. All these proteins can be adsorbed in vitro from the serum containing media or in vivo from biological fluids. The pH, ionic composition of biological solution, temperature, and the functional group of proteins are the main factors determining protein adsorption on a specific substrate [226]. Another important factor that influences the protein adsorption is the surface energy. Positively and negatively charged substrates adsorb different proteins [226]. In general, proteins that have a number of positively charged residues are expected to show a high affinity for the anionic surfaces, due to electrostatic attractions. By an appropriate pretreatment of the ceramic substrate (e.g., in phosphate solution), the ionic charges from the surface can be modified, providing an increasing affinity of the proteins or other biomolecules [236]. 1.5.2 SILANE-MODIFIED SURFACES (SILANIZATION TECHNIQUE) The ability of organosilanes to bond to surfaces arises from the fact that the ethoxy groups (–Si– (OCH2CH3)3) of aminosilanes, such as aminopropyltriethoxysilane (APTES), form silanols (–Si– (OH)3) in aqueous solution. These silanol groups can then bond covalently to a suitable substrate, usually inorganic solids displaying appropriate surface chemical groups (such as –OH), thus leaving terminal (nonbonded) amino groups free to serve as attachment sites for biological modifiers like peptides and proteins. Attachment of biological modifiers to the terminal amino groups can be accomplished by either of the two processes: physisorption or chemisorption [226,227]. The functional groups such as methyl (–CH3), hydroxyl (–OH), carboxyl (–COOH), and amino (–NH2) groups are present in many biological molecules and have specific physical and chemical properties that influence the cellular process. By silanization, the substrate surface can be modified in order to immobilize specific biomolecules. The inclusion of all these functional groups, using silane modification techniques, especially on glass surfaces, provides a method that CRC_7973_Ch001.indd 32 CRC_7973_Ch001.indd 32 1/25/2008 7:14:41 PM 1/25/2008 7:14:41 PM
- 54. Inorganic and Composite Bioactive Scaffolds for Bone Tissue Engineering 33 TABLE 1.15 Functional Group Abbreviations, Chemical Formulae, and Silane Precursors for Silane-Modified Surfaces [235] Functional Group Formula Precursor NH2 COOH OH C6H5 CH3 –(CH2)3NH3 + –COO− –CH2CH2OH –C6H5 –CH3 3-Aminopropyltrimethoxysilane Trichlorovinylsilanea Trichlorovinylsilanea Phenyltrichlorosilane Dimethyldichlorosilane a The COOH and OH surfaces were produced by a postsilanization modification of the vinyl surfaces. produces well-defined and organized substrates with different surface chemistries and energies [225–228,235,236]. Table 1.15 presents the typical silane precursors for silane-modified surfaces. 1.5.3 TOPOGRAPHY (ROUGHNESS) MODIFICATION The cell adhesion on ceramic substrates can be enhanced by modifying the surface roughness (e.g., by sandblasting, heat treatment, acid etching, etc.). Increased surface wettability or hydrophilicity has been associated with enhanced protein adsorption and, consequently, cell adhesion on bioma- terials [237]. There has been, however, only limited work on developing techniques to modify the surface topography of 3-D ceramic or composite scaffolds [238–240], these techniques being devel- oped especially for metallic implant for orthopedics. Another strategy consists of immersing the ceramic substrate in an SBF to mimic the first stage of bone tissue integration on the in vivo implants. The integration of bone tissue on the bioactive ceramic or composite surfaces takes place by biomineralization of a thin layer of calcium phos- phate at the interface between the implant and the bone tissue. Therefore, by soaking the bioactive biomaterial in SBF, a uniform thick-film composed of nanocrystallites of biologically active cal- cium phosphate is produced [241]. During this biomimetic process, the topography of the ceramic implant is modified by the precipitation of nanocrystals of calcium phosphate on its surface. The new nanoscaled texture of the bioactive ceramic implant can improve the cellular adhesion. 1.5.4 POLYMER COATINGS Another strategy to improve the cell–scaffold interaction is coating the substrate with an organic phase, usually a biodegradable polymer [177]. Moreover, the strong interfacial adhesion between the ceramic biomaterial and the organic polymer is a key parameter in generating composites with good mechanical properties. A wide variety of polymers have been investigated for this application, including PLA, PGA, PLGA, PDLLA, PHA, and PCL [173,177,223,242–244]. It has been antici- pated that the combination of ceramic scaffolds and appropriate biodegradable polymer coatings can enhance the interfacial adhesion of proteins. 1.6 CONCLUSIONS Significant developments have been achieved in the design and fabrication of a variety of bioceramic and composite scaffolds, which have demonstrated outstanding properties for applications in bone tissue engineering. However, there are several challenges ahead for material scientists and tissue engi- neers, associated with, in particular, the improvement of biological functions and mechanical integrity of synthetic scaffolds. Vascularization is the single most important issue to be addressed prior to CRC_7973_Ch001.indd 33 CRC_7973_Ch001.indd 33 1/25/2008 7:14:42 PM 1/25/2008 7:14:42 PM
- 55. Exploring the Variety of Random Documents with Different Content
- 56. CHAPTER XII While Delamere, in the deepest despondence, which he could neither conquer or conceal, made a vain effort to divert his mind with those amusements for which he no longer had any relish, Emmeline, at her new residence, attracted the attention of many of Mrs. Ashwood's visitors. A widow, in possession of an handsome jointure, and her children amply provided for, Mrs. Ashwood was believed to entertain no aversion to a second marriage: and her house being so near London, was frequented by a great number of single men; many of whom came there because it was a pleasant jaunt from the city, where most of them resided; and others, with hopes of amending their fortunes by an alliance with the lady herself. These latter, however, were chiefly the younger sons of merchants; and though pleased with their flattery and assiduity, Mrs. Ashwood, who had an almost equal share of vanity and ambition, had yet given no very decided preference to any; for she imagined her personal attractions, of which she had a very high idea, added to the advantages of a good income, good expectations, and opulent connections, entitled her to marry into an higher line of life than that in which her father had first engaged her. Her acquaintance, however, was yet very limited among persons of fashion; and it was not wholly without hopes of encreasing it that she had consented to receive Miss Mowbray, whose relationship to Lord Montreville would, she imagined, be the means of introducing her to his Lordship's notice and to that of his family. Her civility and kindness to Emmeline were unbounded for some time. And as she was not easily convinced of her own want of beauty, she never apprehended that she ran some risk of becoming a foil, instead of the first figure, as she expected generally to be.
- 57. The extreme simplicity of Emmeline's appearance, who notwithstanding the remonstrances of Mrs. Ashwood continued to dress nearly as she did in Wales; and her perfect ignorance of fashionable life and fashionable accomplishments, gave her, in the eyes of many of Mrs. Ashwood's visitors, the air of a dependant; and those who visited with a view to the fortune of the latter, carefully avoided every appearance of preference to Emmeline, and kept her friend in good humour with herself. But there were, among those who frequented her house, some men of business; who being rather in middle life, and immensely rich, had no other views in going thither than to pass a few hours in the country, when their mercantile engagements prevented their leaving London entirely; and who loved pleasure better than any thing but money. With one or two of these, Mrs. Ashwood and her father had at different times encouraged overtures of marriage. But they knew and enjoyed the pleasure their fortune and single state afforded them too well to give those indulgences up for the advantage of increasing their incomes, unless the object had possessed greater attractions than fell to the share of Mrs. Ashwood; and her father could not be prevailed upon to give her (at least while he lived) a sum of money large enough to tempt their avarice. These overtures therefore had ended in nothing more than an intercourse of civility. But Emmeline no sooner appeared, than one of these gentlemen renewed his visits with more than his original assiduity. The extreme beauty of her person, and the naivetè of her manners, gave her, to him, the attractive charms of novelty; while the mystery there seemed to be about her, piqued his curiosity. It was known that she was related to a noble family; but Mrs. Ashwood had been so earnestly entreated to conceal as much as possible her real history, lest Delamere should hear of and discover her, that she only told it to a few friends, and it had not yet reached
- 58. the knowledge of Mr. Rochely, who had become the attendant of Mrs. Ashwood's tea table from the first introduction of Emmeline. Mr. Rochely was nearer fifty than forty. His person, heavy and badly proportioned, was not relieved by his countenance, which was dull and ill-formed. His voice, monotonous and guttural, was fatiguing to the ear; and the singularity of his manners, as well as the oddness of his figure, often excited a degree of ridicule, which the respect his riches demanded could not always stifle. With a person so ill calculated to inspire affection, he was very desirous of being a favourite with the ladies; and extremely sensible of their attractions. In the inferior ranks of life, his money had procured him many conquests, tho' he was by no means lavish of it; and much of the early part of his time had been passed in low amours; which did not, however, impede his progress to the great wealth he possessed. He had always intended to marry: but as he required many qualifications in a wife which are hardly ever united, he had hesitated till he had long been looked upon as an old bachelor. He was determined to chuse beauty, but expected also fortune. He desired to marry a woman of family, yet feared the expensive turn of those brought up in high life; and had a great veneration for wit and accomplishments, but dreaded, lest in marrying a woman who possessed them, he should be liable to be governed by superior abilities, or be despised for the mediocrity of his own understanding. With such ideas, his relations saw him perpetually pursuing some matrimonial project; but so easily frightened from his pursuit, that they relied on his succession with the most perfect confidence. When first he beheld Emmeline, he was charmed with her person; her conversation, at once innocent and lively, impressed him with the most favourable ideas of her heart and understanding; and, brought up at a great distance from London, she had acquired no taste for expences, no rage for those amusements and dissipations which he so much apprehended in a wife.
- 59. When he came to Mrs. Ashwood's, (which was almost every afternoon) Emmeline, who was generally at work, or drawing in the dressing-room, never discomposed herself; but sat quietly to what she was doing; listening with the most patient complaisance to the long and uninteresting stories with which he endeavoured to entertain her; an attention which greatly contributed to win the heart of Rochely; and he was as much in love as so prudent a man could be, before he ventured to ask himself what he intended? or what was the family and what the fortune of the person who now occupied most of his time and a great portion of his thoughts? Mrs. Ashwood, frequently engaged at the neighbouring card-tables, from which Emmeline almost always excused herself, often left her and Mr. Rochely to drink tea together; and when she was at home, would sometimes make her party in another room, where the subject of laughter with her own admirers, was the growing passion of the rich banker for the fair stranger. Emmeline did not, when present, escape ridicule on this subject: but as she had not the least idea that a man so much older than herself had any intention of offering himself as an husband, she bore it with great tranquillity, and continued to behave to Mr. Rochely with the attentive civility dictated by natural good breeding; while she heard, without any concern but on his account, the perpetual mirth and loud bursts of laughter which followed his compliments and attentions to her. If he was absent a few days, the door of Mrs. Ashwood was crouded with servants and porters with game from Mr. Rochely. And his assiduities became at every visit more marked. As it was now late in the autumn, Mrs. Ashwood was desirous of shewing Miss Mowbray some of those public places she had not yet seen; and Emmeline (not apprehending there was any reason to fear meeting Mr. Delamere at a season when she knew field sports kept him altogether in the country) made no difficulty to accompany her.
- 60. Mr. Rochely no sooner heard a party to the play proposed, than he desired to join it; and Mrs. Ashwood, Miss Galton, (an intimate friend of her's), with Miss Mowbray, Mr. Hanbury, (one of Mrs. Ashwood's admirers), and Mr. Rochely, met at Drury-Lane Theatre; where Emmeline was extremely well entertained. When the play was over, the box was filled with several of Mrs. Ashwood's acquaintance, who talked to her, while their eyes were fixed on her young friend; an observation that did not greatly lighten up her countenance. The most conspicuous among these was a tall, thin, but extremely awkward figure, which in a most fashionable undress, and with a glass held to his eye, strided into the box, and bowing with a strange gesture to Mrs. Ashwood, exclaimed—'Oh! my dear Mrs. A! —here I am!—returned from Spa only last night; and already at your feet. So here you are? and not yet enchained by that villainous fellow Hymen? You are a good soul, not to give yourself away while I was at Spa. I was horridly afraid, my dear widow! you would not have waited even to have given me a wedding favour.' To this speech, as it required no answer, Mrs. Ashwood gave very little; for besides that she was not pleased with the matter, the manner delighted her still less. The speaker had, during the whole of it, leaned almost across the person who was next to him, to bring his glass nearly close to Emmeline's face. Emmeline, extremely discomposed, drew back; and Mr. Rochely, who sat near her, putting away the glass softly with his hand, said very calmly to the leaning beau—'Sir, is there any occasion to take an account of this lady's features?' 'Ah! my friend Rochely!' answered he familiarly, 'what are you the lady's Cicisbeo? as we say in Italy. Here is indeed beauty enough to draw you from the contemplation of three per cent. consols, India bonds, omnium, scrip, and douceurs. But prithee, my old friend, is this young lady your ward?' 'My ward! no,' answered Rochely, 'how came you to think she was?'
- 61. Mr. Elkerton, who fancied he had vastly the advantage in point of wit, as well as of figure, over his antagonist, now desired to know, 'whether the lady was his niece? though if I had not recollected' said he, 'that you never was married, I should have taken her for your grand daughter.' This sarcasm had, on the features of Rochely, all the effect the travelled man expected. But while he was preparing an answer, at which he was never very prompt, the coach was announced to be ready, and Emmeline, extremely weary of her situation, and disgusted even to impatience with her new acquaintance, hastily arose to go. Elkerton offered to take her hand; which she drew from him without attempting to conceal her dislike; and accepting the arm of Rochely, followed Mrs. Ashwood; while Elkerton, determined not to lose sight of her, seized the hand of Miss Galton, who being neither young, handsome, or rich, had been left to go out alone: they followed the rest of the party to the coach, where Mrs. Ashwood and Miss Mowbray were already seated, with Mr. Hanbury; who, as he resided with his mother in the village where Mrs. Ashwood lived, was to accompany them home. The coach being full, seemed to preclude all possibility of Elkerton's admittance. But he was not so easily put off: and telling Mrs. Ashwood he intended to go home to sup with her, he stepped immediately in, and ordered his servant, who waited at the coach door with a flambeau, to direct his vis-a-vis to follow. Rochely, who meant to have wished them a good night after seeing them to their carriage, was too much hurt by this happy essay of assurance not to resolve to counteract **it's consequences. Elkerton, though not a very young man, was near twenty years younger than Rochely; besides the income of his business (for he was in trade) he had a large independent fortune, of which he was extremely lavish; his equipages were splendid; his house most magnificently furnished; and his cloaths the most expensive that could be bought.
- 62. Rochely, whose ideas of elegance, manners, or taste, were not very refined, had no notion that the absurdity of Elkerton, or his disagreeable person, would prevent his being a very formidable rival. He therefore saw him with great pain accompany Emmeline home; and though he had formed no positive designs himself, he could not bear to suppose that another might form them with success. Directing therefore his chariot to follow the coach, he was set down at the door a few minutes after Mrs. Ashwood and her party; where Emmeline, still more displeased with Elkerton, and having been teized by his impertinent admiration the whole way, looked as if she could have burst into tears. Mrs. Ashwood, in a very ill humour, hardly attended to his flourishing speeches with common civility; he had therefore recourse to Miss Galton, to whom he was giving the history of his travels, which seemed to take up much of his thoughts. Miss Galton, who by long dependance and repeated disappointments had acquired the qualifications necessary for a patient hearer, acquiesced in smiling silence to all his assertions; looked amazed in the right place; and heard, with great complacency, his wonderful success at cards, and the favour he was in with women of the first fashion at Spa. The entrance of Mr. Rochely gave no interruption to his discourse. He bowed slightly to him without rising, and then went on, observing that he had now seen every part of Europe worth seeing, and meant, at least for some years, to remain in England; the ladies of which country he preferred to every other, and therefore intended taking a wife among them. Fortune was, he declared, to him no object; but he was determined to marry the handsomest woman he could meet with, for whom he was now looking out. As he said this, he turned his eyes towards Emmeline; who affecting not to hear him, tho' he spoke in so loud a tone as to make it unavoidable, was talking in a low voice to Mr. Rochely.
- 63. Rochely placing himself close to her, had thrown his arm over the back of her chair; and leaning forward, attended to her with an expression in his countenance of something between apprehension and hope, that gave it the most grotesque look imaginable. Mrs. Ashwood, who had been entertained apart by Mr. Hanbury, now hurried over the supper; during which Elkerton, still full of himself, engrossed almost all the conversation; gave a detail of the purchases he had made abroad, and the trouble he had to land them; interspersed with bon mots of French Marquises and German Barons, and witty remarks of an English Duke with whom he had crossed the water on his return. But whatever story he told, himself was still forwardest in the picture; his project of marrying an handsome wife was again repeated; and he told the party how charming a house he had bought in Kent, and how he had furnished his library. Rochely, who lay in wait to revenge himself for all the mortifications he had suffered from him during the evening, took occasion to say, in his grave, cold manner, 'to be sure a man of your taste and erudition, Mr. Elkerton, cannot do without a library; but for my part, I think you will find no books can say so much to the purpose as those kept by your late father in Milk-Street, Cheapside.' Elkerton turned pale at this sneer; but forcing a smile of contempt, answered, 'You bankers have no ideas out of your compting-houses; and rich as ye are, will never be any thing but des bourgeois les plus grossieres! For my part I see no reason why—why a man's being in business, should prevent his enjoying the elegancies and agréments of life, especially if he can afford it; as it is well known, I believe, even to you, Sir, that I can." 'Oh! Sir,' replied Rochely, 'I know your late father was reputed to have died rich, and that no body has made a better figure about town than you have, ever since.' 'As to figure, Sir,' returned the other, 'it is true I like to have every thing about me comme il faut. And though I don't make fifty per
- 64. cent. of money, as some gentlemen do in your way of business, I assure you, Sir, I do nothing that I cannot very well afford.' Mrs. Ashwood, who thought it very likely a quarrel might ensue, here endeavoured to put an end to such very unpleasant discourse; and prevented Mr. Hanbury, who equally hated them both, from trying to irritate them farther, to which he maliciously inclined. The hints, however, of fatigue, given by her and Miss Mowbray, obliged Mr. Rochely to ring that his chariot might be called, which had waited at the door; while Elkerton, who had a pair of beautiful pied horses in his vis-à-vis, desired to have them sent for from a neighbouring inn—'for I' said he, rising and strutting round the room, 'never suffer my people or my horses to wait in the streets.' He then leant over Emmeline's chair, and began in a court tone to renew his compliments. But she suddenly arose; and begging Mrs. Ashwood would give her leave to retire, wished Mr. Rochely and ladies a good night; and slightly curtseying to Elkerton, who was putting himself into the attitude for a speech and a bow, she tripped away. Rochely, as soon as she was gone, hastened to his chariot; and Elkerton, whose people were in no haste to leave the ale-house, begged to sit down 'till they came. Mrs. Ashwood had been the whole evening particularly out of humour, and being no longer able to command it, answered peevishly, 'that her house was much at his service, but that she was really so much fatigued she must retire—however,' said she, 'Miss Galton, you will be so good as to stay with Mr. Elkerton—good night to you, Sir!' He was no sooner alone with Miss Galton, than he desired her, after a speech (which he endeavoured to season with as much flattery as it would bear) to tell him who Emmeline was? 'Upon my word, Sir,' answered she, 'it is more than I know. Her name is Mowbray; and she is somehow connected with the family of
- 65. Lord Montreville; but what relation,' (sneeringly answered she) 'I really cannot pretend even to guess.' 'A relation of Lord Montreville!' cried Elkerton; 'why I knew his Lordship intimately when I was abroad three or four years ago. He was at Naples with his son, his lady, and two daughters; and I was domesticated, absolutely domesticated, among them. But pray what relation to them can this Miss Mowbray be?' 'Probably,' said Miss Galton, 'as you know his Lordship, you may know what connections and family he has. I suppose she may be his cousin—or his niece—or his——.' Here she hesitated and smiled; and Elkerton, whose carriage was now at the door, and who had a clue which he thought would procure him all the information he wanted, took leave of Miss Galton; desiring her to tell Mrs. Ashwood that he should wait upon her again in a few days. CHAPTER XIII Delamere continued in Norfolk only a few weeks after his father and the family came thither. During that time, he appeared restless and dissatisfied; his former vivacity was quite lost; he shunned society; and passed almost all his time in the fields, under pretence of hunting or shooting, tho' the greatest satisfaction those amusements now afforded him was the opportunity they gave him of absenting himself from home. He seldom returned thither 'till six or seven o'clock; dined alone in his own apartment; and affected to be too much fatigued to be able to meet the party who assembled to cards in the evening. Lady Mary Otley and her daughter, a widow lady of small fortune in the neighbourhood, with Lord and Lady Montreville and their eldest daughter, made up a party without him. Augusta Delamere had been
- 66. left in their way from the North, with a relation of his Lordship's who lived near Scarborough, with whom she was to remain two months. The party at Audley-Hall was soon encreased by Sir Richard Crofts and his eldest son, who came every autumn on a visit to Lord Montreville, and who was his most intimate friend. Lord Montreville, during the short time he studied at the Temple, became acquainted with Sir Richard, then clerk to an attorney in the city; who, tho' there was a great difference in their rank, had contrived to gain the regard and esteem of his Lordship (then Mr. Frederic Mowbray) and was, when he came to his estate, entrusted with it's management; a trust which he appeared to execute with such diligence and integrity, that he soon obtained the entire confidence of his patron; and by possessing great ductility and great activity, he was soon introduced into a higher line of life, and saw himself the companion and friend of those, to whom, at his setting out, he appeared only an humble retainer. Born in Scotland, he boasted of his ancestry, tho' his immediate predecessors were known to be indigent and obscure; and tho' he had neither eminent talents, nor any other education than what he had acquired at a free-school in his native town, he had, by dint of a very common understanding, steadily applied to the pursuit of one point; and assisted by the friendship of Lord Montreville, acquired not only a considerable fortune, but a seat in Parliament and a great deal of political interest, together with the title of a Baronet. He had less understanding than cunning; less honesty than industry; and tho' he knew how to talk warmly and plausibly of honour, justice, and integrity, he was generally contented only to talk of them, seldom so imprudent as to practice them when he could get place or profit by their sacrifice. He had that sort of sagacity which enabled him to enter into the characters of those with whom he conversed: he knew how to humour their prejudices, and lay in wait for their foibles to turn them to his own advantage.
- 67. To his superiors, the cringing parasite; to those whom he thought his inferiors, proud, supercilious, and insulting; and his heart hardening as his prosperity encreased, he threw off, as much as he could, every connection that reminded him of the transactions of his early life, and affected to live only among the great, whose luxuries he could now reach, and whose manners he tried to imitate. He had two sons by an early marriage with a woman of small fortune, who was fortunately dead; for had she lived, she would probably have been concealed, lest she should disgrace him. To his sons, however, he had given that sort of education which was likely to fit them for places under government; and he had long secretly intended the eldest for one of the Miss Delameres. Delamere, all warmth and openness himself, detested the narrow- minded and selfish father; and had shewn so much coolness towards the sons, that Sir Richard foresaw he would be a great impediment to his designs, and had therefore the strongest motive for trying to persuade Lord Montreville, that to send him on another tour to the Continent, would be the best means of curing him of what this deep politician termed 'a ridiculous and boyish whim, which his Lordship ought at all events to put an end to before it grew of a more dangerous consequence.' Mr. Crofts, as he was no sportsman, passed his mornings in riding out with Miss Delamere and Miss Otley, or attending on the elder ladies in their airings: while Delamere, who wished equally to shun Miss Otley, whom he determined never to marry, and Crofts, whom he despised and hated, lived almost alone, notwithstanding the entreaties of his father and the anger of his mother. Her Ladyship, who had never any command over her passions, harrassed him, whenever they met, with sarcasms and reflections. Lady Mary, scorning to talk to a young man who was blind to the merits of her daughter, talked at him whenever she found an opportunity; and exclaimed against the disobedience, dissipation, and ill-breeding of modern young men: while Miss Otley affected a
- 68. pretty disdain; and flirted violently with Mr. Crofts, as if to shew him that she was totally indifferent to his neglect. The temper of Delamere was eager and irritable; and he bore the unpleasantness of this society, whenever he was forced to mix in it, with a sort of impatient contempt. But as he hourly found it more irksome, and the idea of Emmeline press every day more intensely on his heart, he determined, at the end of the third week, to go to London. Not chusing to have any altercation with either Lord or Lady Montreville, he one evening ordered his man to have his horses ready at five o'clock the next day, saying he was to meet the foxhounds at some distance from home; and having written a letter to his Lordship, in which he told him he was going to London for a fortnight, (which letter he left on the table in his dressing-room) he mounted his horse, and was soon in town; but instead of going to the house of his father in Berkley-Square, he took lodgings in Pall- Mall. Every night he frequented those public places which were yet open, in hopes of finding Emmeline; and his servant was constantly employed for the same purpose; but as he had no trace of her, all his enquiries were fruitless. On the night that Emmeline was at the play, he had been at Covent- garden Theatre, and meant to have looked into the other house; but was detained by meeting a young foreigner from whom he had received civilities at Turin, 'till the house was empty. So narrowly did he miss finding her he so anxiously sought. Elkerton, in looking about for the happy woman who was worthy the exalted situation of being his wife, had yet seen none whom he thought so likely to succeed to that honour as Miss Mowbray; and if she was, on enquiry, found to be as she was represented, (related to Lord Montreville) it would be so great an additional advantage, that he determined in that case to lay himself and his pied horses, his house in Kent, his library, and his fortune, all at her feet immediately.
- 69. Nor did he once suffer himself to suspect that there was a woman on earth who could withstand such a torrent of good fortune. In pursuance therefore of this resolution, he determined to make enquiry of Lord Montreville himself; of whom he had just known so much at Naples as to receive cards of invitation to Lady Montreville's conversationes. There, he mingled with the croud; and was slightly noticed as an Englishman of fortune; smiled at for his affectation of company and manners, which seemed foreign to his original line of life; and then forgotten. But Elkerton conceived this to be more than introduction enough; and dressing himself in what he thought un disabille la plus imposante, and with his servants in their morning liveries, he stopped at the door of Lord Montreville. 'Lord Montreville was not at home.' 'When was he expected?' 'It was uncertain: his Lordship was at Audley-hall, and might be in town in a fortnight; or might not come up till the meeting of Parliament.' 'And are all the family there?' enquired Elkerton of the porter. 'No, Sir; Mr. Delamere is in town.' 'And when can I see Mr. Delamere?' The porter could not tell, as he did not live in Berkley-Square. 'Where, then, is he?' 'At lodgings in Pall-Mall:' (for Delamere had left his direction with his father's servants.) Elkerton therefore took the address with a pencil; and determined, without farther reflection, to drive thither. It was about four o'clock, and in the middle of November, when Delamere had just returned to his lodgings, to dress before he met
- 70. his foreign friend, and some other young men, to dine at a tavern in St. James's-Street, when a loud rap at the door announced a visitor. Millefleur having no orders to the contrary, and being dazzled with the splendour of Elkerton's equipage, let him in; and he was humming an Italian air out of tune, in Delamere's drawing-room, when the latter came out in his dressing-gown and slippers to receive him. Delamere, on seeing the very odd figure and baboonish face of Elkerton, instead of that of somebody he knew, stopped short and made a grave bow. Elkerton advancing towards him, bowed also profoundly, and said, 'I am charmed, Sir, with being permitted the honour of paying you my devoirs.' Delamere concluded from his look and bow, as well as from a foreign accent, (which Elkerton had affected 'till it was become habitual) that the man was either a dancing master or a quack doctor, sent to him by some of his companions, who frequently exercised on each other such efforts of practical wit. He therefore being not without humour, bowed again more profoundly than before; and answered, 'that the honour was entirely his, tho' he did not know how he had deserved it.' 'I was so fortunate, Sir,' resumed Elkerton, 'so fortunate as to—have the honour—the happiness—of knowing Lord Montreville and Lady Montreville a few years ago at Naples.' Delamere, still confirmed in his first idea, answered, 'very probably, Sir.' 'And, Sir,' continued Elkerton, 'I now waited upon you, as his Lordship is not in town.' 'Indeed, Sir, you are too obliging.' 'To ask, Sir, a question, which I hope will not be deemed—be deemed—' (a word did not immediately occur) 'be deemed— improper—intrusive—impertinent—inquisitive—presuming—— '
- 71. 'I dare say, Sir, nothing improper, intrusive, impertinent, inquisitive, or presuming, is to be apprehended from a gentleman of your appearance.' Delamere expected something very ridiculous to follow this ridiculous introduction, and with some difficulty forbore laughing. Elkerton went on—— 'It relates, Sir, to a Lady.' 'Pray, Sir, proceed. I am really impatient where a lady is concerned.' 'You are acquainted, Sir, with a lady of the name of Ashwood, who lives at Clapham?' 'No, really Sir, I am not so happy.' 'I fancy then, Sir, I have been misinformed, and beg pardon for the trouble I have presumed to give: but I understood that the young lady who lives with her was a relation of Lord Montreville.' A ray of fire seemed to flash across the imagination of Delamere, and to inflame all his hopes. He blushed deeply, and his voice faultering with anxiety, he cried— 'What?—who, Sir?—a young lady?—what young lady?' 'Miss Mowbray, they tell me, is her name; and I understand, Sir—but I dare say from mistake—that she is of your family.' Delamere could hardly breathe. He seemed as if he was in a dream, and dared not speak for fear of awaking. Elkerton, led on by the questions Delamere at length summoned resolution to ask, proceeded to inform him of all he knew; how, where, and how often, he had seen Emmeline, and of his intentions to offer himself a candidate for her favour—'for notwithstanding, Sir,' said he, 'that Mr. Rochely seems to be fort avant en ses bon graces, I think—I hope—I believe, that his fortune—(and yet his fortune does not perhaps so much exceed mine as many suppose)—his fortune will hardly turn the balance against me; especially if I have
- 72. the sanction of Lord Montreville; to whom I suppose (as you seem to acknowledge some affinity between Miss Mowbray and his Lordship) it will be no harm if I apply.' Thro' the mind of Delamere, a thousand confused ideas rapidly passed. He was divided between his joy at having found Emmeline, his vexation at knowing she was surrounded by rivals, and his fear that his father might, by the application of Elkerton to him, know that Emmeline's abode was no longer a secret: and amidst these various sensations, he was able only to express his dislike of Elkerton, whose presumption in thinking of Emmeline appeared to cancel the casual obligation he owed to him for discovering her. 'Sir,' said he haughtily, as soon as he could a little recover his recollection, 'I am very well assured that Lord Montreville will not hear any proposals for Miss Mowbray. His Lordship has, in fact, no authority over her; and besides he is at present about to leave his house in Norfolk, and I know not when he will be in town; perhaps not the whole winter; he is now going to visit some friends, and it will be impossible you can have any access to him for some months. As to myself, you will excuse me; I am engaged to dine out.' He rang the bell, and ordered the servant who entered to enquire for the gentleman's carriage. Then bowing coolly to him, he went into his dressing room, and left the mortified Elkerton to regret the little success of an attempt which he doubted not would have excited, in the hearts of all those related to Miss Mowbray, admiration at his generosity, and joy for the good fortune of Emmeline: for he concluded, by her being a companion to Mrs. Ashwood, that she had no fortune, or any dependance but on the bounty of Lord Montreville. Delamere, whose ardent inclinations, whatever turn they took, were never to be a moment restrained, rang for his servants; and dispatching one of them with an excuse to his friends, he sent a second for an hackney-coach. Then ordering up a cold dinner, which he hardly staid to eat, he got into the coach, and directed it to be
- 73. driven as fast as possible to Clapham Common; where he asked for the house of Mrs. Ashwood, and was presently at the door. The servant had that moment opened the iron gate, to let out a person who had been to his mistress upon business. Delamere therefore enquiring if Miss Mowbray was at home, entered without ringing, and telling the servant that he had occasion to speak to Miss Mowbray only, the man answered, 'that she was alone in the dressing room.' Thither therefore he desired to be shewn; and without being announced, he entered the room. Instead of finding her alone, he saw her sit at work by a little table, on which were two wax candles; and by her side, with his arm, as usual, over the back of her chair, and gazing earnestly on her face, sat Mr. Rochely. Emmeline did not look up when he came in, supposing it was the servant with tea. Delamere therefore was close to the table when she saw him. The work dropped from her hands; she grew pale, and trembled; but not being able to rise, she only clasped her hands together, and said faintly, 'Oh! heaven!—Mr. Delamere!' 'Yes, Emmeline, it is Mr. Delamere! and what is there so extraordinary in that? I was told you were alone: may I beg the favour of a few minutes conversation?' Emmeline knew not what to reply. She saw him dart an angry and disdainful look at poor Rochely; who, alarmed by the entrance of a stranger that appeared on such a footing of familiarity, and who possessed the advantages of youth and a handsome person, had retreated slowly towards the fire, and now surveyed Delamere with scrutinizing and displeased looks; while Delamere said to Emmeline —'if you have no particular business with this gentleman, will you go into some other room, that I may speak to you on an affair of consequence?' 'Sit down' said Emmeline, recovering her surprize; 'sit down, and I will attend you presently. Tell me, how is your sister Augusta?' 'I know not. She is in Yorkshire.'
- 74. 'And Lord Montreville?' 'Well, I believe. But what is all this to the purpose? can I not speak to you, but in the presence of a third person?' Unequivocal as this hint was, Rochely seemed determined not to go, and Delamere as resolutely bent to affront him, if he did not. Emmeline therefore, who knew not what else to do, was going to comply with his request of a private audience, when she was luckily relieved by the entrance of Mrs. Ashwood and the tea table. Mrs. Ashwood, surprized at seeing a stranger, and a stranger whose appearance had more fashion than the generality of her visitors, was introduced to Mr. Delamere; a ceremony he would willingly have dispensed with; and having made his bow, and muttered something about having taken the liberty to call on his relation, he sat down by Emmeline, and in a whisper told her he must and would speak to her alone before he went. Emmeline, to whose care the tea table was allotted when Miss Galton happened not to be at Mrs. Ashwood's, now excused herself under pretence of being obliged to make tea; and while it was passing, Mrs. Ashwood made two or three attempts to introduce general conversation; but it went no farther than a few insignificant sentences between her and Mr. Rochely. Delamere, wholly engrossed by the tumultuous delight of having recovered Emmeline, and by contriving how to speak to her alone, thought nothing else worthy his attention; and sat looking at her with eyes so expressive of his love, that Rochely, who anxiously watched him, was convinced his solicitude was infinitely stronger than his relationship only would have produced. He had at length learned, by constant attention to every hint and every circumstance that related to Emmeline, who she was; and had even got from Mrs. Ashwood a confused idea of Delamere's attachment to her, which the present scene at once elucidated.
- 75. Rochely saw in him not only a rival, but a rival so dangerous that all his hopes seemed to vanish at once. Unconscious, 'till then, how very indiscreetly he was in love, he was amazed at the pain he felt from this discovery; and with a most rueful countenance, sat silent and disconcerted. Mrs. Ashwood, used to be flattered and attended to, was in no good humour with Mr. Delamere, who gave her so little of his notice: and never perhaps were a party more uncomfortable, 'till they were enlivened by the entrance of Miss Galton and Mr. Hanbury, with another gentleman. They were hardly placed, and had their tea sent round, before a loud ring was heard, and the servant announced 'Mr. Elkerton.' Mr. Elkerton came dancing into the room; and having spoken to Mrs. Ashwood and Emmeline, he slightly surveyed the company, and sat down. He was very near sighted, and affected to be still more so; and Delamere having drawn his chair out of the circle, sat almost behind Emmeline; while the portly citizen who had accompanied Mr. Hanbury sat forward, near the table; Delamere was therefore hardly seen. Elkerton began to tell them how immoderately he was fatigued. 'I have been over the whole town,' said he, 'to-day. In the morning I was obliged to attend a boring appointment upon business relative to my estate in Kent; and to meet my tenants, who disagreed with my steward; and then, I went to call upon my old friend Delamere, Lord Montreville's son, in Pall-Mall; we passed a very chearful hour discoursing of former occurrences when we were together at Turin. Upon my word, he is a good sensible young man. We have renewed our intimacy; and he has insisted upon my going down with him to his father's house in Norfolk.' Emmeline suspended her tea making, and looked astonished. Mrs. Ashwood seemed surprized.
- 76. But Delamere, who had at first felt inclined to be angry at the folly and forwardness of Elkerton, was now so struck with the ridicule of the circumstance, that he broke into a loud laugh. The eyes of the company were turned towards him, and Elkerton with great indignation took his glass to survey who it was that had thus violated the rules of good breeding; but great was his dismay and astonishment, when he beheld the very Delamere, of whom he had spoken with so much assurance, rise up, and advancing towards him, make a grave bow.— 'Sir,' said Delamere, very solemnly, 'I cannot sufficiently express my gratitude for your good opinion of me; nor my happiness to hear you intend to honour me with a visit at Audley Hall. Upon my word you are too obliging, and I know not how I shall shew my gratitude!' The ironical tone in which this was delivered, and the discomposed looks of the distressed Elkerton, explained the matter to the whole company; and the laugh became general. Elkerton, tho' not easily disconcerted, could not stand it. After a sort of apology to Delamere, he endeavoured to reassume his consequence. But he had been too severely mortified; and in a few minutes arose, and under pretence of being engaged to a rout in town, went away, nobody attempting to stop him. Rochely, who hated Elkerton, could not forbear to triumph in this discomfiture. He spoke very severely of him as a forward, impertinent, silly fellow, who was dissipating his fortune. The old citizen heartily joined in exclaiming against such apostates from the frugality of their ancestors. 'Sir,' said he to Rochely, 'we all know that you are a prudent man; and that cash at your house is, as it were, in the Bank. Sir, you do honour to the city; but as to that there Mr. Elkerton, one must be cautious; but for my part, I wonder how some people go on. To my certain knowledge his father didn't die so rich as was supposed—no—not by a many thousands. Sir, I remember him—(and I am not ashamed to say it, for every body knows I have got my money honestly, and that it's all of my own
- 77. getting)—but, Sir, I remember that man's father, and not a many years ago neither, carrying out parcels, and sweeping the shop for old Jonathan Huggins. You knew old Jonathan Huggins: he did not die, I think, 'till about the year forty-one or two. You remember him, to be sure?' Rochely, ever tremblingly alive when his age was called in question, yet fearing to deny a fact which he apprehended the other would enter into a convincing detail to prove, answered that 'he slightly remembered him when he was quite a boy.' But his evasion availed him nothing. The old citizen, Mr. Rugby, was now got upon his own ground; and most inhumanly for the feelings of poor Rochely, began to relate in whose mayoralty old Jonathan Huggins was sheriff, and when he was mayor; who he married; who married his daughters; and how he acquired an immense fortune, all by frugality at setting out; and how one of his daughters, who had married a Lord against the old man's will, had spent more in one night than his father did in a twelvemonth. Delamere, who sat execrating both Jonathan Huggins and his historian, at length lost all patience; and said to Emmeline, in an half whisper, 'I can bear this no longer: leave these tedious old fools, and let me speak to you for two minutes only.' Emmeline knew not how to refuse, without hazarding some extravagance on the part of Delamere. But as she did not like the appearance of leaving the room abruptly, she desired Mrs. Ashwood would give her permission to order candles in the parlour, as Mr. Delamere wished to speak with her alone. As soon as the servant informed her they were ready, she went down: and Delamere followed her, having first wished Mrs. Ashwood a good night; who was too much displeased with the little attention he had shewn her, to ask him to supper, tho' she was very desirous of having a man of his fashion in the list of her acquaintance. Delamere and Emmeline were no sooner alone, than he began to renew, with every argument he thought likely to move her, his
- 78. entreaties for a private marriage. He swore that he neither could or would live without her, and that her refusal would drive him to some act of desperation. Emmeline feared her resolution would give way; for the comparison between the people she had lately been among, and Delamere, was infinitely favourable to him. Such unabated love, in a man who might chuse among the fairest and most fortunate of women, was very seducing; and the advantages of being his wife, instead of continuing in the precarious situation she was now in, would have determined at once a mind more attentive to pecuniary or selfish motives. But Emmeline, unshaken by such considerations, was liable to err only from the softness of her heart. Delamere unhappy—Delamere wearing out in hopeless solicitude the bloom of life, was the object she found it most difficult to contend with: and feeble would have been her defence, had she not considered herself as engaged in honour to Lord Montreville to refuse his son, and still more engaged to respect the peace of the family of her dear Augusta. Strengthened by these reflections, she refused, tho' in the gentlest manner, to listen to such proposals; reproached him, tho' with more tenderness in her voice and manner than she had yet shewn, for having left Audley Hall without the concurrence of Lord Montreville; and entreated him to return, and try to forget her. 'Let me perish if I do!' eagerly answered Delamere. 'No, Emmeline; if you determine to push me to extremities, to you only will be the misery imputable, when my mistaken parents, in vain repentance, hang over the tomb of their only son, and see the last of his family in an early grave. It is in your power only to save me—You refuse— farewel, then—I wish no future regret may embitter your life, and that you may find consolation in being the wife of some one of those persons who are, I see, offering you all that riches can bestow. Farewel, lovely, inhuman girl! be happy if you can—after having
- 79. sacrificed to a mistaken point of honour, the repose and the life of him who lived only to adore you.' So saying, he suddenly opened the door, and was leaving the room. But Emmeline, who shuddered at the picture he had drawn of his despair, and saw such traces of its reality on his countenance, caught his arm. 'Stay! Mr. Delamere,' cried she, 'stay yet a moment!' 'For what purpose?' answered he, 'since you refuse to hear me?' He turned back, however, into the room; and Emmeline, who fancied she saw him the victim of his unfortunate love, could no longer command her tears. Delamere threw himself at her feet, and embraced her knees. 'Oh Emmeline!' cried he, weeping also, 'hear me for the last time. Either consent to be mine, or let me take an eternal adieu!' 'What would you have me do? good God! what is it you expect of me?' 'To go with me to Scotland to-morrow—to night—directly!' 'Oh, no! no!—Does not Lord Montreville depend upon my honour?— can I betray a trust reposed in me?' 'Chimeras all; founded in tyranny on his part, and weakness on yours. He had no right to exact such a promise; you had no right to give it. But however, send to him again to say I have seen you— summons him hither to divide us—you may certainly do so if you please; but Lord Montreville will no longer have a son; at least England, nor Europe, will contain him no longer—I will go where my father shall hear no more of me.' 'Will it content you if I promise you not to write to Lord Montreville, nor to cause him to be written to; and to see you again?' 'When?' 'To-morrow—whenever you please.'
- 80. Delamere, catching at this faint ray of hope, promised, if she would allow him to come thither when he would, he would endeavour to be calm. He made her solemnly protest that she would neither write to Lord Montreville, or procure another to do it; and that she would not leave Mrs. Ashwood without letting him know when and whither she went; and if by any accident his father heard of his having found her, that she would enter into no new engagements to conceal herself from him. Having procured from her these assurances, which he knew she would not violate, and having obtained her consent to see him early the next morning, he at her request agreed to take his leave; which he did with less pain than he had ever before felt at quitting her; carrying with him the delightful hope that he had made an impression on her heart, and secure of seeing her the next day, he went home comparatively happy. Emmeline, who had wept excessively, was very unfit to return to the company; but she thought her not appearing again among them would be yet more singular. She therefore composed herself as well as she could; and after staying a few minutes to recollect her scattered spirits, she entered the room where they were at cards. Rochely, who was playing at whist with Mrs. Ashwood, Mr. Rugby, and Mr. Hanbury, looked anxiously at her eyes; and presently losing all attention to what he was about, and forgetting his game, he played so extremely ill, that he lost the rubber. The old cit, who had three half crowns depending, and who was a determined grumbler at cards, fell upon him without mercy; and said so many rude things, that Rochely could not help retorting; and it was with some difficulty Mrs. Ashwood prevented the grossest abuse being lavished from the enraged Rugby on the enamoured banker; who desiring to give his cards to Miss Galton, got up and ordered his carriage. Emmeline sat near the fire, with her handkerchief in her hand, which was yet wet with tears.
- 81. Rochely, with a privilege he had been used to, and which Emmeline, from a man old enough to be her father, thought very inconsequential, took her hand and the handkerchief it held. 'So, Miss Mowbray,' said he, 'Mr. Delamere is your near relation?' 'Yes, Sir.' 'And he has brought you, I fear, some ill news of your family?' 'No, Sir,' sighed Emmeline. 'No death, I hope?' 'No, Sir.' 'Whence then, these tears?' Emmeline drew her hand away. 'What a strange young man this is, to make you cry. What has he been saying to you?' 'Nothing, Sir.' 'Ah! Miss Mowbray; such a lad as that is but an indifferent guardian; pray where does his father live?' Miss Mowbray, not aware of the purpose of this enquiry, and glad of any thing that looked like common conversation, answered 'at Audley Hall, in Norfolk; and in Berkley-Square.' Some other questions, which seemed of no consequence, Rochely asked, and Emmeline answered; 'till hearing his carriage was at the door, he went away. 'I don't like your Mr. Delamere at all, Miss Mowbray,' said Mrs. Ashwood, as soon as the game ended. 'I never saw a prouder, more disagreeable young man in my life.' Emmeline smiled faintly, and said she was sorry he did not please her. 'No, nor me neither,' said Miss Galton. 'Such haughtiness indeed!— yet I was glad he mortified that puppy Elkerton.'
- 82. Emmeline, who found the two friends disposed to indulge their good nature at the expence of the company of the evening, complained of being fatigued, and asked for a glass of wine and water: which having drank, she retired to bed, leaving the lady of the house, who had invited Mr. Hanbury and his friend to supper, to enjoy more stories of Jonathan Huggins, and the pretty satyrical efforts of Miss Galton, who made her court most effectually by ridiculing and villifying all their acquaintance whenever it was in her power. CHAPTER XIV When Rochely got home, he set about examining the state of his heart exactly as he would have examined the check book of one of his customers. He found himself most miserably in love. But avarice said, Miss Mowbray had no fortune. By what had passed in his bosom that evening, he had discovered that he should be wretched to see her married to another. But avarice enquired how he could offer to marry a woman without a shilling? Love, represented that her modest, reserved, and unambitious turn, would perhaps make her, in the end, a more profitable match than a woman educated in expence, who might dissipate more than she brought. Avarice asked whether he could depend on modesty, reserve, and a retired turn, in a girl not yet eighteen? After a long discussion, Love very unexpectedly put to flight the agent of Plutus, who had, with very little interruption, reigned despoticly over all his thoughts and actions for many years; and Rochely determined to write to Lord Montreville, to lay his
- 83. circumstances before him, and make a formal proposal to marry Miss Mowbray. In pursuance of this resolution, he composed, with great pains, (for he was remarkably slow in whatever he undertook) the following epistle. — 'My Lord, 'This serves to inform your Lordship, that I have seen Miss Mowbray, and like her well enough to be willing to marry her, if you, my Lord, have not any other views for her; and as to fortune, I will just give your Lordship a memorandum of mine. 'I have sixty thousand pounds in the stocks; viz. eighteen in the three per cent. consols. twenty in Bank stock: ten in East India stock; and twelve in South Sea annuities. 'I have about forty thousand on different mortgages; all good, as I will be ready at any time to shew you. I have houses worth about five more. And after the death of my mother, who is near eighty, I shall have an estate in Middlesex worth ten more. The income of my business is near three thousand pounds a year; and my whole income near ten thousand. 'My character, my Lord, is well known: and you will find, if we agree, that I shall not limit Miss Mowbray's settlement to the proportion of what your Lordship may please to give her, (for I suppose you will give her something) but to what she ought to have as my widow, if it should so happen that she survives me. 'I have reason to believe Miss Mowbray has no dislike to this proposal; and hope to hear from your Lordship thereon by return of post. I am, my Lord, your Lordship's very humble servant, Humphrey Rochely.'
- 84. Lombard-Street, Nov. 20th. 17—. This was going to the point at once. The letter arrived in due time at Audley-Hall; and was received by Lord Montreville with surprise and satisfaction. The hint of Miss Mowbray's approbation made him hope she was yet concealed from Delamere; and as he determined to give the earliest and strongest encouragement to this overture, from a man worth above an hundred thousand pounds, he called a council with Sir Richard Crofts, who knew Rochely, and who kept cash with him; and it was determined that Lord Montreville should go to town, not only to close at once with the opulent banker, but to get Delamere out of the way while the marriage was in agitation, which it would otherwise be impossible to conceal from him. To persuade him to another continental tour was what Sir Richard advised: and agreed to go to town with his Lordship, in order to assist in this arduous undertaking. Lord Montreville, however, failed not immediately to answer the letter he had received from Mr. Rochely, in these terms— 'Sir, 'This day's post brought me the honour of your letter. 'If Miss Mowbray is as sensible as she ought to be, of so flattering a distinction, be assured it will be one of the most satisfactory events of my life to see her form a connection with a gentleman truly worthy and respectable. 'To hasten the completion of an event so desirable, I fully intend being in town in a very few days; when I will, with your permission, wait on you in Lombard-Street. 'I have the honour to be, with great esteem, Sir, your most devoted,
- 85. and most obedient servant, Montreville.' Audley-Hall, Nov. 23. The haughty Peer, who derived his blood from the most antient of the British Nobility, thus condescended to flatter opulence and to court the alliance of riches. Nor did he think any advances he could make, beneath him, when he hoped at once to marry his niece to advantage, and what was yet more material, put an invincible bar between her and his son. While this correspondence, so inimical to Delamere's hopes, was passing between his father and Mr. Rochely, he was every hour with Emmeline; intoxicated with his passion, indulging the most delightful hopes, and forgetting every thing else in the world. He had found it his interest to gain (by a little more attention, and some fine speeches about elegance and grace,) the good opinion of Mrs. Ashwood; who now declared she had been mistaken in her first idea of him, and that he was not only quite a man of fashion, but possessed an excellent understanding and very refined sentiments. The sudden death of her father had obliged her to leave home some days before: but as soon as she was gone, Emmeline, who foresaw that Delamere would be constantly with her, sent for Miss Galton. No remonstrance of her's could prevent his passing every day at the house, from breakfast 'till a late hour in the evening. On the last of these days, he was there as usual; and it was past eight at night, when Emmeline, who had learned to play on the harp, by being present when Mrs. Ashwood received lessons on that instrument, was singing to Delamere a little simple air of which he was particularly fond, and into which she threw so much pathos, that lost in fond admiration, he 'hung over her, enamoured,' when she was interrupted by the entrance of a servant, who said that a Lord, but he forgot the name, was below, and desired to speak with Miss Mowbray.
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