![• Fig. 2. Illustration of various
features of polymer important
from bioprinting perspective: (a)
hydrophilic and hydrophobic
properties, (b) cross-linking
potential, (c) viscosity,(d)
mechanical features, (e) cell
adhesion and biocompatibility,
and (f) biodegradation ability.The
figure has been reproduced from
[20] with permission from
Academic Press.](https://image.slidesharecdn.com/tissueengineering-240309212224-ac5c6ad0/85/tissue-engineering-basics-and-challenges-pdf-78-320.jpg)
tissue engineering ; basics and challenges.pdf
- 2. Tissue engineering • By • Romissaa Aly Esmail • Assistant lecturer of Oral Medicine, Periodontology, Diagnosis and Dental Radiology (Al-Azhar University)
- 3. • Contents: • Introduction • TYPES OF CELLS (PROLIFERATION AND DIFFERENTIATION) • Scaffold • THE CHALLENGES OF TISSUE ENGINEERING
- 4. • The definition of tissue engineering, according to International Union of Pure and Applied Chemistry (IUPAC), is “to use of a combination of cells, engineering and materials, and suitable biochemical and physiochemical factors to improve or replace biological functions” • (Griffin et al., 2009).
- 14. • The main approaches of tissue engineering can be juxtaposed as: • I. Use of an instructive environment (e.g., bioactive material) to recruit and guide host cells to regenerate a tissue; • II. Delivery of repair cells and/or bioactive factors into the damaged area; and
- 15. • III. Cultivation of cells on a biomaterial scaffold in a culture system (bioreactor), under conditions designed to engineer a functional tissue for implantation • (
- 24. Tissue engineering approaches with a combination of cells, biofactors and scaffolds.
- 26. TYPES OF CELLS (PROLIFERATION AND DIFFERENTIATION
- 27. Stemcells
- 28. Whether it is expected to start tissue cellularization with fully differentiated cells, it is better to obtain a cell biopsy from the patient.
- 29. • As another option, multipotent stem cells (e.g., mesenchymal stromal cells and endothelial progenitor cells; EPCs) can be directed into desired differentiation. • Lastly, autologous induced pluripotent stem (iPS) cells are a good alternative choice for the source of required cell types.
- 30. Embryonic-like stem cells, like the newly derived iPS, have essentially unlimited potential for expansion in vitro which might open the possibility of creating autologous embryonic-like cells that could solve the matter of assembling more complex tissues including cardiac regeneration. (Jakab et al., 2010).
- 31. One obvious requirement is that of immune tolerance of the repaired cells. It is essential to use patient- specific autologous cells, in the transplantation to avoid disease transmission, life-long immunosuppression, or the possibility of rejection of transplanted organ
- 33. Reprogrammed cells are characterized by a high plasticity, being able to differentiate in cells of the three embryonal germ layers and thus potentially having the capacity to differentiate in most of the human body's cells. Induced pluripotent stem cells (iPS) are pluripotent cell lines obtained from the ex vivo reprogramming of fetal or adult somatic cells, like fibroblasts.
- 34. To date, the limitation to the clinical use of these cells is given by the risk of tumorigenesis deriving from the genomic integration of the viral vectors. Umbilical cord is collected at the time of birth; umbilical cord blood mononuclear cells (UCBMNCs) can be isolated from the blood, while mesenchymal stem cells are extracted from the Wharton's jelly. Cord stem cells are able to differentiate in cardiomyocyte-like cells and endothelial cells (ECs)
- 35. Foetal-derived stem cells can be isolated from the amniotic fluid and include both pluripotent stem cells and more committed cells (Klemmt et al., 2011).
- 36. • Endothelial cells and progenitor cells • The first can be isolated from peripheral, easy accessible veins, such as the saphenous or forearm veins, while the second ones can be purified from the peripheral blood or bone marrow. • EPCs can circulate in peripheral blood and can be incorporated in regions of active neovascularization, (Xin et al., 2008).
- 37. Experimental evidence suggests that EPCs participate not only in the process of vasculogenesis substituting the lost ECs but also in the endothelialization of grafts (Young et al., 2007). The significance of EPCs in cardiovascular disease has been reviewed in Madonna and De Caterina (2015).
- 38. Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues, including the heart, skeletal muscle, bone marrow and adipose tissue. (Uccelli et al., 2008).
- 39. • MSCs stand out as an encouraging option for cell therapy due to their accessible isolation, great expansion potential, immunoregulatory activity and angiogenic properties. • (Dimarino et al., 2013)
- 40. Not less relevant, MSCs possess a multipotential differentiation capacity, being able to differentiate, in vitro, into cells of the mesodermal lineage ,and possibly toward cells of endoderm and ectoderm derivation. The immune compatibility of the MSCs is a remarkable advantage for the translation of their use in clinics (Castro- Manrreza and Montesinos, 2015).
- 41. Bone marrow-derived progenitor cells The advantage of using these cells is the easy accessibility with low risks for the patients, and the possibility to harvest high numbers of cells without requirement of long-time in vitro expansion.
- 43. SCAFFOLDS
- 44. “Scaffolding” term is first introduced by Barth in 1893 (Barth, 1893) as to use this notion like a porous matrix or an implant allowing cells to infiltrate and regenerate the local tissue. The term, eventually, became possessing alternative concepts (Jakab et al., 2010)
- 45. such as using natural and synthetic substrates, nanocomposite materials, or decellularized extracellular matrix (ECM), and additionally, maturing the concept towards cell material interactions, release of biological factors, and design of shape and functionality for specific purposes. (Gloria et al., 2010).
- 46. • Scaffolds should also establish a tissue-specific microenvironment to maintain and regulate cell behavior and function (Khademhosseini et al., 2009). • The scaffold, onto which cells are seeded, enabling them to attach and colonize, is therefore a key element for tissue engineering. •
- 47. Scaffolds can cause problems owing to their degradation, evoking immunogenic reactions and other unforeseen complications, which arouse the importance of further development and research about scaffolds. The properties of scaffolds consist of several parameters, including biological substances used, porosity, elasticity, stiffness, and specific anatomical shapes. It is also needed to reinstate the tissue-specific structure, activity, and physical behavior.
- 48. • Ultimately, scaffold should also provide repopulated cell- specific topological features (nano- or micro- and macroscale), mechanical environment, surface ligands, and facility to release chemical compounds (angiogenic factors or cytokines) (Freytes et al., 2009).
- 49. some examples of the diverse scaffolding strategies PRE-MADE POROUS SCAFFOLD CAN BE PROCESSED AND THEN CELL-SEEDED (E.G. FIBER SCAFFOLD, FOAMS, FILMS). HUMAN TISSUE CAN BE DECELLULARIZED USING CHEMICAL AGENTS AND THEN CELL-SEEDED WITH TREATED CELLS. CELLS CAN BE CULTURED TO PRODUCE ECM AND CELL SHEETS. SHEETS ARE HARVESTED AND ASSEMBLED LAYER-BY-LAYER TO PRODUCE TISSUES AND ORGANS. POLYMER SOLUTION CAN BE DIRECTLY MIXED WITH CELLS AND CROSSLINK EITHER PHYSICALLY OR CHEMICALLY AND CAN BE USED AS 3D CULTURE ENVIRONMENT.
- 53. • FIGURE 20.2 The self-assembly approach depends on the natural ability of cells to unite and generate new natural environment by secreting extracellular matrix components. (A) The basic construct of vascular graft to provide nutrient support to engineered tissues. (B) Prepared multicellular blocks are seeded into the desired position in the tissue. (C) The blood flow is conducted through cellular blocks, while the conditions of incubation for the cells have been prepared. (D) Self- assembling cells proliferate and generate new ways to provide maximum perfusion among them. These unnatural spaces between the cells become the new coronary bed for the engineered tissue.
- 54. Figure 2. Blood vessel tissue engineering. Source: Seifu DG et al. Nat Rev Cardiol, 2013; 10(7), 410–21.
- 55. In In vivo TE, the scaffold is implanted usually with cells and an animal is used as an incubator to grow the tissue or the organ before being re-implanted in the same or another patient. Figure 3 shows the impressive example of a human ear scaffold seeded with cow cells implanted in an immune deprived mouse. Figure 3. The Vacanti mouse. Original article: Y. Cao et al. Plast Reconstr Surg, 1997; 100(2), 297-302.
- 56. • In in situ TE, the scaffold is implanted or injected with or without cells into the patient’s (or animal’s) body. The tissue is expected to self-repair due to cell migration and cells growing directly in the body’s environment, such as in the example illustrated in Figure 4. •
- 57. Figure 3. Cartoon illustrating the promising strategy of tissue engineering based on which grafts and materials are combined with patient autologous cells and grown in vitro or in a bioreactor, in order to obtain an optimized cellularized graft that lacks of immunogenicity, thrombogenicity, and risk of calcification, while having the potential to grow in parallel with the child growth.
- 59. ECM Scaffolds
- 60. Furthermore, ECM is considered as a dynamic interchangeable media with the resident cell population which turns out that ECM can affect genetic profile, proteome, and protein functionality of cells depending on the parameters of pH, oxygen concentration, mechanical forces, and its biochemical milieu.
- 61. Thus, native ECM is a logical and ideal scaffold for organ and tissue reconstruction. (Tottey et al., 2011). To establish intact 3D ECM scaffolds, varying allogeneic, xenogeneic tissues and organs have been manufactured. They are prepared by the process of decellularization.
- 62. The strategy contains the principles of decellularizing a site-specific ECM and repopulating it with autologous fully differentiated, progenitor or stem cells of the current patient to prevent adverse effects of implantation or immune responses. It is shown that ECM successfully directed cells to their target region and supported growth and differentiation of local stem and progenitor cells in skin graft that are potential cure for scar-free healing (Guenou et al., 2009).
- 63. One additional advantage of using decellularized ECM scaffolds is its ability to serve 3D architecture of the capillary bed, besides its biomechanical properties of the constituent fibers. To empower the repopulation and differentiation processes of implanted cells, and, to maintain life-long restoration of tissue, enough oxygen and nutrient support must be conducted, as well as the transfer of bioactive molecules throughout the tissue
- 64. • In a recent study, it is stated that after the decellularization process, the first three four branches of capillary vasculature are preserved, which allows enough perfusion and nutrition conveyance to the tissue • (Sarig et al., 2012).
- 65. DECM SCAFFOLDS
- 66. • Figure 1. Timeline of key events leading to whole organ decellularization methodologies and major milestones using hPSC- derived cells to repopulate organ-derived dECM scaffolds (*).
- 76. hydrogels made from decellularized tissues including urinary bladder heart, liver, dermis , adipose tissue bone and lung among others, were developed . Nowadays, one of the main hurdles when using dECM hydrogels as bioinks relies on their low viscosity, which inevitably compromise shape fidelity of the bioprinted 3D construct, worsening printing resolution.
- 77. Fig. 1. Chart diagramming natural (red) and synthetic (blue) polymer distributions for use as bioinks. (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)
- 78. • Fig. 2. Illustration of various features of polymer important from bioprinting perspective: (a) hydrophilic and hydrophobic properties, (b) cross-linking potential, (c) viscosity,(d) mechanical features, (e) cell adhesion and biocompatibility, and (f) biodegradation ability.The figure has been reproduced from [20] with permission from Academic Press.
- 80. • FIGURE 3 Schematic representation of a 3D bioprinting system consisting of a computer aided 3-axis stage controller and a deposition module including three different print heads connected to a pressure controller (a). Computer aided design and computer aided manufacturing (CAD/CAM) process for 3D bioprinting of a human size kidney. A 3D CAD model generated from medical imaging data (CT: computed tomography; MRI: magnetic resonance imaging) produces a visualized motion program which dictates the XYZ stage movements to generate the 3D bioprinted kidney prototype (b).
- 82. NATURAL SCAFFOLD
- 83. Natural polymers used in tissue engineering consist of collagen, alginate, agarose, chitosan, chitin, fibrin, silk fibroin, and hyaluronic acid (or hyaluronan). Collagen is a natural protein material that is commonly used in tissue engineering because environment of the cell metabolism by collagen scaffold is close to physiological conditions.
- 86. Three major classes of biomaterials, ceramics, biopolymers and synthetic polymers are widely employed for fabrication of various scaffolds structures. Biomaterials such as hydroxyapatite (HAP) calcium phosphate and magnesium phosphate-based ceramics are extensively used for making porous scaffolds for bone tissue regeneration. J.A. Kim, J. Lim, R (2016).
- 87. Recently, nano- and bio- glass based ceramics have also been employed in bone tissue engineering . Biopolymers such as collagen, chitosan (CS) and hyaluronic acid (HA) find potential applications in various tissue engineering fields. E. Entekhabi, M. (2016)
- 89. However, non-conductive biomaterials cannot respond to the electrical stimuli and hence they cannot mimic the cellular properties. On the other hand, the invention of synthetic conductive materials overwhelms the drawbacks of non-conductive materials .
- 113. The obstacles of EMC scaffolding are the need of the identification of the optimal cell source for different organs, the loss of an effective method for recellularization of denuded vascular structure in whole-organ scaffolds, and problems on the identification of the appropriate population of patients (Badylak et al., 2012).
- 114. In addition, scaffold-based tissue engineering still faces the problems of immunogenicity, acute and chronic inflammatory response resulting from the host response to the scaffolds,
- 115. • and its biodegradation products, mechanical mismatch with the surrounding tissue, difficulties in incorporating high numbers of cells uniformly distributed within the scaffold, and the limitation in introducing multiple cell types with positional specificity. • (Langer, 2007).
- 116. • Another critical problem present in the field is to provide sufficient vascular supply to thick constructs, as molecular diffusion can assure the exchange of nutrients and oxygen within limited ranges (Ko et al., 2008). • This could only be done by conducting proper capillary bed within the scaffold in nanoscale.
- 117. Another problem waiting for being addressed is the transition and the integration of a tissue from an in vitro to an in vivo setting. According to the surgical point of view, macro- and microvascular tree must be connected successfully to provide enough perfusion among the tissue.
- 118. Three main components of vasculature, capillary, intermediate microvessels, and microvasculature, must work together. Bioprinting also faces some difficulties. Aside from the expense of the printers, it is hard to assure high cell density to build up solid organs.
- 119. Sometimes, high-speed deposition of cells may damage the construction. Additionally, the success of printing depends on the control of the gelation state of the collagen layers. To integrate the final structure of tissue, collagen must be removed, which is very difficult.
- 120. Also, printing larger and morecomplex patterns like branching tubes limits the use of printing, and it is excessively time consuming. Establishing compatible bioreactors is also difficult. The maturation of the tissue in bioreactor takes really long time and new designs of bioreactors are certainly needed
- 121. Economical and financial aspects of the field are in their infancy. CTI, in which tissue engineering is one of the fields, has some problems to market its products and to make them approved by FDA. It is also stated that the necessary infrastructure, including appropriate regulation, reimbursement regimes, scalable manufacturing, robust business models, and clinical outlets are not yet in place (Mason et al., 2011).