BIOSTATISTICS hypothesis testings ,sampling

CONTENTS
 INTRODUCTION
 USES OF BIOSTATISTICS
 COLLECTION OF DATA
 SAMPLING AND SAMPLE DESIGN
 MEASURES OF CENTRAL TENDENCY
 MEASURES OF DISPERSION
 THE NORMAL CURVE
 TEST OF SIGNIFICANCE
 CORRELATION AND REGRESSION

INRODUCTION
Analysis and interpretation is done using biostatistics. The word ‘statistics’comes
from the Italian word ‘statica’meaning ‘statesman’ or German word ‘statistik’which
means a political state. The science of statistics is said to have developed from
registration of heads of families in ancient Egypt to the roman census on military
strength, births and deaths , etc.and found its application gradually in the field of
health and medicine. John Graunt(1620-1674),who was neither a physician nor a
mathematician is considered the father of health statistics.
STATISTICS;
It is the science of compiling, classifying and tabulating numerical data and
expressing the results in a mathematical or graphical form
Types
A. Descriptive statistics ; enumeration, organization and graphical representation of
data from a sample by tabulation and graphical presentation.
B. Inferential statistics; Methods of making generalizations about a larger group
based on information about a sample of that group, use data from sample to make
inferences about a population.
BIOSTATISTICS;

Biostatistics is that branch of statistics concerned with mathematical facts and data
related to biological events
USES OF BIOSTATISTICS
 To test whether the difference between two populations is real or a chance
occurrence
 To study the correlation between attributes in the same population
 To evaluate the efficacy of vaccines ,sera etc
 To measure mortality and morbidity
 To evaluate achievements of public health programs
 To help promote health legislation and create administrative standards for oral
health
BASICS FOR STATISTICAL ANALYSIS
Statistical analyses are based on three primary entities;
 The population(U)that is of interest
 The sets of characteristics(variables)of the units of this population(V)
 The probability distribution(P) of these characteristics in the population
The population(U)
The population is a collection of units of observation that are of interest e.g; In
determining the effectiveness of a particular drug for a disease, the population would
consist of all possible patients with this disease. It is essential, in any research study,
to identify the population clearly and precisely. The success of the investigation will
depend to a large extent on the identification of the population of interest.

The variables(V)
A variable is a state ,condition ,concept or event whose value is free to vary within
the population.
Once the population is identified, we should clearly define what characteristics of the
units of this population (subjects of the study) we are planning to investigate.For
example, in the case of the HIV study above, one needs to define HIV (reliable and
valid method of identifying HIV in people), and what other characteristics of the
people (e.g. age, sex, education, etc.) one intends to study. Clear and precise
definitions and methods for measuring these characteristics (a simple observation, a
laboratory measurement, or a battery of tests using a questionnaire) are essential for
the success of the research study.
The variables are characterized in many ways; for statistical considerations, the
variables are usually classified as discrete or continuous. Discrete variables are
those in which only a small number of values is possible (e.g. sex: male, female),
incidence of a disease (yes, no)). Continuous variables are those which, theoretically,
can take any value within a specified range of minimum and maximum value (e.g.
age, blood pressure). There are some variables that are discrete in nature, but the
number of categories make them similar to continuous variables, and these are
considered as continuous in most statistical calculations (e.g. number of years of
schooling, number of people in a household).
Types of variables
 Independent variable;
variables that are manipulated or treated in a study in order to see what effect,
difference in them will have on those variables proposed as being dependent on them.
 Dependent variable
Variables in which changes are results of the level or amount of the independent
variable or variables.

 Confounding variable or intervening variable
Variables that should be studied because they may influence or confound the effect
of the independent variable on the dependent variable.
Example; In a study of the effect of tobacco(independent variable) on oral cancer
(dependent variable), the nutritional status of the individual may play an intervening
role.
 Background variable
Variables that are so often of relevance in investigations of groups or populations that
they should be considered for possible inclusion in the study
The probability distribution (P)
The most crucial link between the population and its characteristics, which allows us
to draw inferences on the population based on sample observations, depends on this
probability distribution. The probability distribution is a way to enumerate the
different values the variable can have, and how frequently each value appears in the
population. The actual frequency distribution is approximated to a theoretical curve
that is used as the probability distribution. Common examples of probability

distributions are the binomial, Poisson and normal. Most statistical analyses in health
research use one of these three common probability distributions. For example, the
incidence of a relatively common illness may be approximated by a binomial
distribution, whereas the incidence of a rare condition (e.g. number of deaths from
motor vehicle accidents) may be considered to have a Poisson distribution.
Distributions of continuous variables (blood pressure, heart rate) are often considered
to be normally distributed.
Probability distributions are characterized by ‘parameters’: quantities that allow us to
calculate probabilities of various events concerning the variable, or that allow us to
determine the value of probability for a particular value. For example, the binomial
distribution has two parameters: n and p. The binomial distribution occurs when a
fixed number (n) of subjects is observed, the characteristic is dichotomous in nature
(only two possible values), and each subject has the same probability (p) of having
one value and (1-p) of the other value. The statistical inference then involves finding
out the value of p in the population, based on an observation of a carefully selected
sample.
The normal distribution, on the other hand, is a mathematical curve represented by
two quantities, m and s. The former represents the mean of the values of the
variables, and the latter, the standard deviation. The type of statistical analysis done
depends very much on the design of the study: in particular, whether the study was
descriptive, and what sampling design was used to draw the sample from the
population.
COLLECTION OF DATA
Collective recording of observations either numerical or otherwise is called data.
Demographic data comprise details of population size, geographic distribution, ethnic
groups, socioeconomic factors and their trends over time. Such data are obtained
from census/surveys, experiments, hospital records and other public service reports
and are important determinants for oral health care programs.
Depending on the nature of the variable, data is classified into two broad categories,
1. Qualitative data; when the data is collected on the basis of attributes or qualities
like sex, malocclusion, cavity etc.

2. Quantitative data; when the data is collected through measurement using
calipers, like arch length, arch width, fluoride concentration in water supply etc.,
Quantitative data can be classified into two, Discrete and Continuous
 Discrete
When the variable under observation takes only fixed values like whole numbers,
the data is discrete
Example; DMF teeth
 Continuous
If the variable can take any value in a given range, decimal or fractional, it is called
as continuous data
Example; arch length, mesiodistal width of erupted teeth.
 Data can be collected through,

I. Primary source
Here the data is obtained by the investigator himself. This is first hand information.
II. Secondary source
The data already recorded is utilized to serve the purpose of the objective of the
study.
Presentation of data
2 main methods of presenting data
 Tabulation
 Charts and diagrams
Tabulation
Tables are simple device used for the presentation of statistical data
Principles
 Tables should be as simple as possible(2-3 small tables)
 Data should be presented according to size or importance, chronologically or
alphabetically.
 Should be self explanatory.
 Each row and each column should be labeled concisely and clearly.
 Specific unit of measure for the data should be given. Title should be clear,
concise and to the point.
 Total should be shown.
 Every table should contain a title as to what is depicted in the table.
 In small table, vertical lines separating the columns may not be necessary.
 If the data are not original, their source should be given in a footnote

Types of tables
 Master table
They are tables, which contain all the data obtained from a survey
 Simple table
They are one way tables which supply answers to questions about one
characteristic of data only.
Frequency distribution table
The simplest table is a two-column frequency table.
 The 1st
column lists the classes into which the data are grouped.

 The 2nd
column lists the frequencies for each classification
CHARTS ANDDIAGRAM
Most convincing and appealing ways of depicting statistical results.
Principles
— Every diagram must be given a title that is self explanatory
— Simple and consistent with the data
— The values of the variable are presented on the horizontal or x-axis and frequency
on the vertical line or y -axis
— The number of lines drawn in any graph should not be many.
— The scale of presentation for the x-axis and Y-axis should be mentioned.
— The scale of division of 2 axes should be proportional and the divisions should be
marked along with the details of the variables and frequencies presented on the
axis
Barchart
— Represents qualitative data
— Bars can be either vertical or horizontal.
They are of 3 types,
— Simple bar chart
— Multiple bar chart

— Component/proportional bar chart
 SIMPLEBARCHART
Represents only one variable
 MULTIPLEBARCHART
Each category of a variable there are set of bar
 COMPONENT/PROPORTIONALBARCHART
Individual bar is divided into 2 or more parts

 PIEDIAGRAM
Entire graph look like a pie
— It is divide into different sectors corresponding to the frequencies
 LINEDIAGRAM

— Useful to study changes of values in the variable over time and is the simplest
type of diagram
— X-axis-time such as hours, days, weeks, months or years
— Y-axis-value of any quantity
 HISTOGRAM
Pictorial presentation of frequency distribution
— No space between the cells on a histogram

— Area of rectangle is proportional to the frequency
 FREQUENCY POLYGON
Obtained by joining midpoints of histogram blocks at the height of frequency by
straight lines usually forming a polygon
 FREQUENCY CURVE
When number of observations is very large and class interval is reduced the frequency
polygon losses its angulation becoming a smooth curve known as frequency curve.

 PICTOGRAM
Popular method of presenting data to the common man through small pictures or
symbols
 SPOT MAP/ SHADED MAP/ CARTOGRAM
These maps are prepared to show geographic distribution of frequencies of
characteristics.

SAMPLING AND SAMPLE DESIGN
A sample is a part of a population, called the ‘universe’, reference’ or ‘parent’
population. sampling is the process or technique of selecting a sample of appropriate
characteristics and adequate size.
 Probability sampling ; subject of population get an equal opportunity to be
selected as a representative sample
 Non –probability sampling ; it is not known that which individual from the
population will be selected as sample
Types

PROBABILITYSAMPLING
a) Simple random sample
This is the most common and the simplest of the sampling methods. In this method,
the subjects are chosen from the population with equal probability of selection. One
may use a random number table, or use techniques such as putting the names of the
people into a hat and selecting the appropriate number of names blindly. Recently,
computer programs have been developed to draw simple random samples from a
given population. The simple random sample has the advantages that it is easy to
administer, is representative of the population in the long run, and the analysis of data
using such a sampling scheme is straightforward. The disadvantage is that the
selected sample may not be truly representative of the population, especially ifthe
sample size is small.
b) Stratified sampling
Population is divided into 2 or more groups called strata. Sub samples are randomly
selected from each strata. When the size of the sample is small and we have some
information about the distribution of a particular variable (e.g. gender: 50% male/50%
female), it may be advantageous to select simple random samples from within each of
the subgroups defined by that variable. By choosing half the sample from males and
half from females, we assure that the sample is representative of the population with
respect to gender. When confounding is an important issue (such as in case-control

studies), stratified sampling will reduce potential confounding by selecting
homogeneous subgroups.
c) Cluster sampling
The population is divided into subgroups (clusters) like families. A simple random
sample is taken from each cluster. In many administrative surveys, studies are done on
large populations which may be geographically quite dispersed. To obtain the
required number of subjects for the study by a simple random sample method will
require large costs and will be inconvenient. In such cases, clusters may be identified
(e.g. households) and random samples of clusters will be included in the study; then
every member of the cluster will also be part of the study.
This introduces two types of variations in the data – between clusters and within
clusters – and this will have to be taken into account when analyzing data.
d) systematicsampling
Selecting one unit at random and then selecting additional units at evenly spaced
interval till the sample of required size has been got

NONPROBABILITYSAMPLING
A. QUOTASAMPLING
General composition of the sample is decided in advance.
— The only requirement is that the right number of people be somehow found to fill
these quotas
B.PURPOSIVESAMPLING
— Sample is constructed to serve a very specific need or purpose
— A researcher may have a specific group in mind, such as high level business
executives.

C.SNOWBALLSAMPLE(CHAINREFERRALSAMPLING)
It is a subset of purposive sampling.
— So named because one picks up the sample along the way, analogous to a snowball
accumulating snow.
A snowball sample is achieved by asking a participant to suggest someone else who
might be willing or appropriate for the study.
— Useful in hard to crack population such as drug users and homeless people.
D.CONVENIENCESAMPLING

 Multi-stage sampling
Many studies, especially large nationwide surveys, will incorporate different
sampling methods for different groups, and may be done in several stages. In
experiments, or common epidemiological studies such as case-control or cohort
studies, this is not a common practice.
DESCRIPTIVESTATISTICS
MEASURES OF CENTRAL TENDENCY/STATISTICAL
AVERAGES
It is the central value around which the other values are distributed. The main
objective of measure of central tendency is to condense the entire mass of data and to
facilitate comparison. A good measure of central tendency should satisfy the
following properties,
 It should be easy to understand and compute.
 It should be based on each and every item in the series.
 It should not be affected by extreme observations(either too small or too large
values)

 It should have sampling stability , i.e, if different samples of same size say 10,are
picked up from the same population, and the measure of central tendency is
calculated ,they should not differ from each other markedly
Most common measures of central tendency that are used in dental science are,
 Arithmetic mean-mathematical estimate
 Median-positional estimate
 Mode-based on frequency.
A) Arithmetic mean
It is the simplest measure of central tendency. it is obtained by adding the individual
observations and then divided by the total number of observations.
Mean is calculated using the formula,
∑Xi / n
Where, ∑ (sigma), means the sum of, Xi is the value of each observation in the data,
n is the number of observations in the data.
Example; The number of decayed teeth in a group of 10 children aged 5 years are as
follows; 2,2,4,1,3,0,5,2,3,4.
Then the mean number of decayed teeth for this group is calculated as;
n =10
∑Xi =2+2+4+1+3+0+5+2+3+4=26
Mean number of decayed teeth = 26/10 =2.6 teeth.
Advantages;

 Easy to calculate and understand
 It is the most useful of all the averages
Disadvantages;
 It may be unduly influenced by abnormal values
 Sometime it might look ridiculous
B) Median
The median is the middle value in a distribution such that one half of the units in the
distribution have a value smaller than or equal to the median and one half has a value
higher than or equal to the median. To calculate the median, all the observations are
arranged in either ascending or descending order of their magnitude and then the
middle value of the observations is selected as the median. when the number of
observations is even, the mean of the two middle values may be taken as the median.
Example; the following are the numbers of visits to a dentist by 10 patients in one
year
13,8,4,3,5,2,8,1,7,4
For calculating the median, the numbers are arranged in order of magnitude as
1,2,3,4,4,5,7,8,8 and 13.since there are 10 patients , the average of the 5th
and 6th
patient is calculated as the median ,which is (4+5)/2=4.5 visits. Thus, it is seen that
median is a positional average. It is not capable of future treatment.
Advantage
It is not affected by abnormal values

C) Mode
The mode or the modal value is that value in a series of observations that occurs with
the greatest frequency.
For example ,if the age at eruption of the canine is 6,6,5,7,8,6,7,5 for 8 children, the
mode will be 6,since it occurs more often than any of others. The mode is located
from the frequency distribution table, taking the value of variable with the maximum
frequency. There can be more than one mode for a series. When mode is ill defined,
it can be calculated using the relation
Mode=3 median-2 mean
Depending on the nature of data and the objective of the study, the appropriate
measure of central tendency may be used.
The most commonly used measure is the arithmetic mean
If there are extreme values in the series of data, median may be used. If it is required
to know the value that has high influence in the series, mode may be computed.
 The most commonly used measure is the arithmetic mean
 If there are extreme values in the series of data, median may be used.
 If it is required to know the value that has high influence in the series, mode may
be computed.

MEASURES OF DISPERSION
Measures of dispersion helps to know how widely the observations are spread on
either side of the average. dispersion is the degree of spread or variation of the
variable about a central value. The most common measures of dispersion used in
dental science are,
— Range
— Mean deviation
— Standard deviation
i. Range;
It is the simplest method, defined as the difference between the value of the largest
item and the value of the smallest item.
ii. Mean deviation
It is the average of the deviations from the arithmetic mean. It is given by,
M.D. =∑(x-xi)
n
Where, ∑(sigma), is the sum of ,X is the arithmetic mean, xi is the value of each
observation in the data , n is the number of observation in the data.

iii. Standard deviation
The standard deviation is the most important and widely used measure of studying
dispersion. It is also known as root mean square deviation because it is the square root
of the mean of the squared deviations from arithmetic mean.
Greater the standard deviation, greater will be the magnitude of dispersion from the
mean. A small standard deviation means a higher degree of uniformity of the
observations.
S.D. =√(x-x1)2
n
Steps,
a) calculate the mean of the series, X

b) Take the deviations of the items from the mean ,x-xi
c) Square these deviations and add them up,∑(x-xi)2
d) Divide the result by the total number of observations, n(or n-1 if sample size is
less than 30)
e) Then obtain the square root. this gives the standard deviation
EXAMPLE
— Suppose you're given the data set,
STEP- 1
— Calculate the mean of your data set.
— The mean of the data is (1+2+2+4+6)/5 = 15/5 = 3
STEP -2
— Subtract the mean from each of the data values and list the differences.
— Subtract 3 from each of the values 1, 2, 2, 4, 6
1-3 = -2
2-3 = -1
2-3 = -1
4-3 = 1
6-3 = 3
— Your list of differences is -2, -1, -1, 1, 3
STEP-3
Square each of the differences from the previous step and make a list of the squares.
— You need to square each of the differences -2, -1, -1, 1, 3

— Your list of squares is 4, 1, 1, 1, 9
STEP-4
— Add the squares from the previous step together.
4+1+1+1+9 = 16
— Subtract one from the number of data values you started with. One less than this is
5-1 = 4
STEP-5
Divide the sum of square values with number of observations.
16/4 = 4
STEP-6
Take the square root of the number from the previous step.
— This is the standard deviation.
— Standard deviation=√4=2
THENORMALCURVE/NORMALDISTRIBUTION/GAUSSIAN
DISTRIBUTION
when data collected from a very large number of people and a frequency distribution
is made with narrow class intervals, the resulting curve is smooth and symmetrical
and is called a normal curve.
In a normal curve,
i. The area between one standard deviation on either side of the mean will include
approximately 68% of values.
ii. The area between two standard deviations on either side of the mean will include
approximately 95% of the values.

iii. The area between 3 standard deviations on either side of the mean will include
approximately 99.7% of the values.
The limits on either side of the mean are called ‘confidence limits’
Standard normal curve
There might be many normal curves but there is only one standard normal curve.
 The standard normal curve is bell shaped.
 The curve is perfectly symmetrical based on an infinitely large number of
observations. The maximum number of observations is at the mean and number
of observations is gradually decreased on either side with few observations at the
extreme points.
 The total area of the curve is one, its mean is zero and standard deviation one.
 All the 3 measures of central tendency, the mean, median and mode coincide.
Hypothesis;
a theory or statement of belief about the population of interest example; there is a
difference in the mean caries experience between all 5 yr old children living in urban
and rural area.
Null hypothesis
H0 ; There is no difference between the groups

Alternative hypothesis
H 1; there is difference between the groups. Alternative when null hypothesis is
rejected.
A test of hypothesis has several steps:
Step 0
Identify the null hypothesis This is a re-statement of the research hypothesis in the
‘null’ form, i.e. ‘no effect of treatment’, ‘no difference in survival rates’, ‘no
difference in prevalence rates’, ‘relative risk is one’, etc. The null hypothesis is often
stated with the research objectives. The null hypothesis should be ‘testable’, i.e. it
should be possible to identify which parameters need to be estimated, and it should
be possible to estimate the parameter, its standard error and the sampling distribution,
given the study design.
Step 1.
Determine the levels, a and b of errors acceptable in the inference since the inference
is based on a sample of the population, one will never be absolutely sure if the
hypothesis is true or not in the population. The decision is a dichotomous one: to
accept the null hypothesis H, or to reject H0. Two types of errors in inference are
possible. The type I error (a) is the probability of falsely rejecting the null hypothesis,
and the type II error (b) is the probability of falsely accepting the null hypothesis.
These are summarized in the table below:
‘Truth’ (in the population)
Decision (based
On sample results) H0 is true H0 is false
Accept H0 No error Type II or β
Reject H0 Type I or α No error

a) Standard error of mean the standard error of mean gives the standard
deviation of the means of several samples from the same population. Standard error
can be estimated from a single sample.
Standard error(S .E) of mean =S.D/√n
b) Standard error of proportion
Standard error of proportion =√ pq/n
Where p and q are the proportion of occurrence of an event in two groups of the
sample and n is the sample size.
c) Standard error of difference between 2 means
It is used to find out whether the difference between the means of 2 groups is
significant to indicate that the samples represent 2 different universe.
Standard error between means=√ σ 1
2
+σ 2
2
n1 n2
D) Standard error of difference between proportions
It is used to find out whether the difference between the proportions of two groups is
significant or has occurred by chance.
CONFIDENCELIMIT
Confidence limit is the range within which all the possible sample mean will lie.
— A population mean ±1 std. Error limit correspond to 68.27 percent of sample mean
value
— A population mean ±1.96 std. error corresponds to 95% of the sample mean value.
— Population mean±2.58 std. error corresponds to 99% of the sample mean value.
— Population mean ±3.29 correspond to 99.9% of the sample mean value
— Range between the two limits is called confidence interval.
P-V
ALUE

— P value provide significant departure or some degree of evidence against null
hypothesis
— p‹0.05 = significant , P‹0.01 or p‹0.001=highly significant

Tests of significance/Hypothesistesting
When different samples are drawn from the same population, the estimates might
differ. This difference in the estimates is called sampling variability.
Tests of significance deals with techniques to know how far the difference between
the estimates of different samples is due to sampling variation.
PARAMETRIC TESTS
1. LARGE SAMPLE
F- Test(ANOVA)
Z-Test
2. SMALL SAMPLE
T-test
NON PARAMETRIC TESTS:
 Chi square test
 Wilicoxon signed rank test
 Mann-Whitney U test
 Spearman’s correlation test
 Mc Nemar’s test
 Fisher’s exact probability test

Z test
It is used to test the significance of difference in means of large samples (>30)
The pre-requisites to apply Z test for means are,
a. The sample must be randomly selected.
b. The data must be quantitative.
c. The variable is assumed to follow a normal distribution in the population.
d. Sample should be larger than 30
Observation – mean = x- x
Standard deviation SD
t test
When sample size is small, t test is used to test the hypothesis. This test was designed
by W.S.Gossett, whose pen name was ‘student’. Hence this test is also called
‘student’s t-test.
T=ratio of observed difference between two means of small samples to the standard
error of difference in the same.
It is applied to find the significance of difference between two proportions as,
 Unpaired t test(independent t test)
 Paired t test(dependent t test)
Criteria for applying t test,
 The sample must be randomly selected.
 The data must be quantitative.
 The variable is assumed to follow the normal distribution in population.
 The sample should be less than 30.

Unpaired t test
This test is applied to unpaired data of independent observations made on individuals
of two different of separate group or samples drawn from two populations, to test if
the difference between the means is real or it can be attributed to sampling
variability.
Paired t test
It is applied to paired data of independent observations of one sample only when each
individual gives a pair of observations.
ANOVA (Analysis of Variance)
Many situations involve collecting data on three or more group of individuals, with
the objective of determining whether any true differences in mean performances exist
among the condition under the study. This often happens in experimental situations
where several different treatment ( for example , various therapeutic approaches to a
specific problem or, various dosage levels of a particular drug) may be under
comparison. In the above situation, ANOVA is a way to test the quality of three or
more means of more than two groups.
 One way ANOVA
Where only one factor will affect the result between 2 groups
 Two way ANOVA
Where we have 2 factors that affect the result or outcome

 Multi way ANOVA
Three or more factors affect the outcomes between groups
NON PARAMETRIC TEST
chi square test
It is used to test the significance of difference between two proportions and can be
used when there are two groups to be compared
Example: if there are two groups, one of which has received oral hygiene instructions
and other has not received any instruction and if it is desired to test if the occurrence
of new cavities is associated with the instructions.
Mann-Whitney u test
 Non parametric equivalents of the unpaired t-test. They can be viewed as tests for
equality of medians
 Example ;is there any evidence that patients who visit the dentist at least
annually are more or less satisfied with their dental care than patients who
visit less often(where satisfaction is scored on a 5 point scale such that,
 1=completely dissatisfied ,2=moderately dissatisfied , 3=indifferent ,
4=moderately satisfied , 5=completely satisfied
 Null hypothesis; “there is no difference in the median level of satisfaction in the
population of patients who visit the dentist at least annually and in the population
of patients who visit less.
CORRELATION AND REGRESSION
Correlation;
When dealing with measurement on 2 sets of variable in a same person, one variable
may be related to the other in same way.(i.e. change in one variable may result in
change in the value of other variable)
Correlation is the relationship between two sets of variables.

Correlation coefficient is the magnitude or degree of relationship between 2 variables.
(varies from -1 to +1).
Obtained by plotting scatter diagram (I .e one variable on x axis and other on y-axis)
 Perfect positive correlation
In this, the two variables denoted by letter x and y are directly proportional and fully
correlated with each other.
The correlation coefficient(r)=+1 ,i.e. both variables rise or fall in the same
proportion.
 Perfect negative correlation
Values are inversely proportional to each other ,i.e, when one rises, the other falls in
the same proportion ,Correlation coefficient(r) =-1
Regression
To know in an individual case the value of one variable, knowing the value of the
other, we calculate what is known as the regression coefficient of one measurement to
the other.
It is customary to denote the independent variate by x and dependent variate by y.

REFERENCES
 Parks textbook of preventive and social medicine.18th
edition
 Mahajan’s methods in biostatistics for medical students and research workers-8th
edition
 Essentials of preventive community dentistry-Dr.soben peter-3rd
and 4th
edition

INTRODUCTION
The primary use of waxes in dentistry is to make a pattern of appliances prior to
casting as many dental restorations are made by lost-wax technique, in which a
pattern is made in wax and put in the mold (investment materials). After setting, the
wax is burnt out and the space is filled with molten metal or plastic acrylic.
Chemically waxes are polymers consisting of hydrocarbon and their derivatives like
ester and alcohol. Dental waxes are mixture of natural and synthetic waxes gums, fat,
oils, natural and synthetic resins and coloring agents.
HISTORY
 The oldest wax used by people were the beeswax.
 Over 60 million years ago, the insects wax production was already accepted by
people as a diet source.
 First inlay in dentistry is credited to “ john murphy” of london, who was
fabricating porcelain inlay in 1855.
 In 1880 ,Ames used a burnished –foil technique for fabrication of inlays.
 First cast inlay is attributed to “philbrook” -1897
DEFINITION
According to ANUSAVICE ;-
A low molecular weight ester of fatty acids with monohydrate alcohol derived from
natural and synthetic components such as petroleum derivatives that softens to a
plastic satate at a relatively low temperature.

Classification of waxes:
According to origin
1. Mineral:
a. Paraffin: Refined from crude oil, has relatively low melting point (50-70°C) and
relatively brittle.
b. Ceresin: Refined from petroleum, has medium melting range (60°C).

2. Plants:
a. Carnauba: Obtained from palm trees, it is hard, tough, and has high melting point
(80-85°C).
b. Candelilla: It is hard, tough, and has high melting point (80-85°C), used to increase
the melting point and reduce flow at mouth temperature.
3. Animal:
a. Stearin: Obtained from beef fat, has low melting point (50 C)
b. Bees: Obtained from honey-comb, consist of partially crystalline natural polyester.
It is brittle, has medium melting temperature (60-70°C).
4. Synthetic:
They are used to modify some properties of natural waxes like polyethylene

According to use:
1. Pattern wax
a. Inlay wax: It should be hard and brittle in order to fracture rather than to distort
when removal from undercut areas. The wax is blue in color. They are used to make
inlays, crowns and pontic replicas.
They are mostly paraffin with carnauba wax. There are two types:
Type 1: for direct technique.
Type 2: for indirect technique.
b. Denture casting wax:
It is used to produce the metal components of cobalt/chromium partial denture. It is
based on paraffin wax with bees wax to give softness necessary for molding and
stickiness necessary to ensure adhering to an investment cast material of refractory
cast. It is green in color.

c. Denture base plate wax: It is used to form the base of the denture and in setting of
teeth. It is pink in color.
2. Processing wax
Waxes are used during processing of the appliance.
a. Beading: It is used to make beading around the impression before pouring gypsum
to protect the margins of the cast.

b. Boxing: It is used to make box around the impression to make pouring gypsum into
the impression easier and more perfect.
c. Block out: It is used to block out undercut areas on cast during processing of co/cr
metal framework.
d. White: It is used to make pattern simulate veneer facing in crowns.

e. Sticky: It is used to join and stabilize temporary broken pieces of the broken
denture before repair.
3. Impression wax
They are previously used to make impression, but they distort when removal from
undercut areas, they have high flow.
a. Impression wax: It is used to make the impression.
b. Corrective wax: It is used to record selected areas of soft tissues in edentulous
arches.

REQUIREMENTS OF DENTAL WAXES
1. Must conform to the exact size and shape and contour of the appliance which is to
be made.
2. Should have enough flow when melted to reproduce the fine details.
3. No dimensional changes should takes place once it is formed.
4. Boiling out of the wax without any residue.
5. Easily carved and smooth surface can be produce.
6. Definite contrast in color to facilitate proper finishing of the margins.
PROPERTIES OF DENTAL WAXES
1. They are thermoplastic materials that are soft when heated and are solid at room
temperature.
2. They have high coefficient of thermal expansion and contraction. They are the
highest of dental materials; it is about 300*10-6 to 1000*10-6 cm/cm C. The
shrinkage of wax from liquid to solid at room temperature is 0.4 %.
Thermal contraction of wax is compensated by expansion of investment.

3. They are poor thermal conductivity. After softening of the wax, it is allowed to
cool, which accompanied by contraction because of poor thermal conductivity only
the
outer layer solidify and the inner solidify later which will produce internal stress.
Relief of the stresses accrues later especially when temperature increases, greater
stresses may be incorporated if the wax is not properly softened.
The best way to soften the wax is to be held in the warm raising air above the flame
and not in the flame itself.
4. They should have high flow when softened, but should little or no flow at room
temperature or mouth temperature in order not to distort.
5. Inlay should be brittle in order to fracture rather than distort when removed from
undercut of the cavity.
Thermal Properties of Dental Waxes
MELTING RANGE
Waxes have a melting range rather than a melting point.
Example: paraffin 44-62 °C , carnauba 50-90 °C
COEFFICENT OF THERMAL EXPANSION
Waxes expand when there is increase in temperature and Contract when there is
decrease in temperature
Dental waxes have the greatest co-efficient of thermal expansion than any other
restorative materials in dentistry.

THERMAL CONDUCTIVITY
The thermal conductivity of the waxes is low . So that sufficient time must be allowed
both to heat them uniformly throughout and to cool them to body or room
temperature.
FLOW OF DENTAL WAX
 One of the desirable properties of type I inlay wax is that it should exhibit a
marked plasticity or flow at a temperature slightly above that of the mouth.
 The temperatures at which the wax is plastic are indicated by the time-
temperature cooling curve for a typical type I wax.
Time-temperature cooling curve for type I inlay wax.
Each wax exhibits a sharp transition temperature at which it loses its plasticity. Soft
wax exhibits a transition point at a lower temperature than hard wax.
DESIRABLE PROPERTIES OF WAX
 The wax should be uniform when softened.
 The color should contrast with die materials or prepared teeth.
 The wax should not fragment into flakes or similar surface particles when it is
molded after softening.

 The wax must not be pulled away by the carving instrument or chip as it is carved
or such precision cannot be achieved.
 Expansion and shrinkage of casting wax are extremely sensitive to temperature.
 Normally soft wax shrinks more than hard wax. High-shrinkage wax may cause
significant distortion when it solidifies.
Distortion of wax pattern
Most serious problem to be experience In forming and removing the pattern from a
tooth or die.Distortion of a wax pattern results from occluded air in the pattern,
physical deformation (during molding, carving, or removal),release of stresses
“trapped” during previous cooling excessive storage time, and extreme temperature
changes during storage.
Like other thermoplastics, waxes tend to return partially to their original shape after
manipulation. This is known as elastic memory.This can be depicted by opening of a
horse –shoe shape molded inlay wax kept in water after manipulation. So to
counteract the property of distortion, the pattern should be invested immediately on
removal so as for best fitting of the casting.
A, A stick of inlay wax is bent into the shape of a horseshoe and floated on water at
room temperature.

B, After 24 hours the same stick of wax tends to relax and distortion occurs.
BIOCOMPATIBILITY
OF
DENTAL MATERIALS

CONTENTS
 INTRODUCTION
 DEFINITION
 HISTORICAL BACKGROUND
 RELEVANCE OF BIOCOMPATIBILITY IN DENTISTRY
 ADVERSE EFFECTS FROM DENTAL MATERIALS
 BIOCOMPATIBILITY TESTS
 BIOCOMPATIBILITY OF DENTAL MATERIALS
 OCCUPATIONAL HAZARDS FOR DENTAL PERSONNEL
 CLINICAL GUIDELINES
 CONCLUSION
 REFERENCES

INTRODUCTION
The three major factors that are linked to the success of dental materials are
material properties, the design of the dental device and the biocompatibility of
component materials. The biocompatibility of dental restorative materials is evaluated
using compositional analysis, surface degradation tests, cell culture tests, clinical
testing in humans, and animal model tests.
Since no dental biomaterial is absolutely free from the potential risk of adverse
reactions, the testing of biocompatibility is related to risk assessment. Thus, the
challenge for the users of dental biomaterials is to select those products for which the
known benefits far outweigh the known risks.
Specific tests have been developed to screen restorative and implant materials
for their biocompatibility. For materials, models have been developed to analyze the
uptake, distribution, biotransformation, and excretion of metal ions or metal
complexes in the body.
Adverse reactions to dental restorative materials and auxiliary materials
include one or more of the following effects: allergic reaction, chemical burn, pulp
irritation, pulp damage, thermal injury, tissue irritation, and toxic reaction. The
specific causes of these effects are difficult to diagnose because of the multifactorial
nature of dental treatment and the subjective nature of patients’ complaints or
descriptions of their symptoms. Furthermore, there are no perfect tests for the
confirmation or validation of diagnoses.
DEFINITION: The ability of a biomaterial to perform its desired function with
respect to a medical (or dental) therapy, without eliciting any undesirable local or
systemic effects in the recipient or beneficiary of that therapy, but generating the most
appropriate beneficial cellular or tissue response in that specific situation, and
optimizing the clinically relevant performance of that therapy (Williams, 2008)

In simple words, it is defined as the ability of a material to elicit an appropriate
biological response to a given application in the body.
The biocompatibility of a material depends on several factors:
1. The chemical nature of its components
2. The physical nature of the components
3. The types and locations of patient tissues that will be exposed to the device
4. The duration of the exposure
5. The surface characteristics of the material
6. The amount and nature of substances eluted from the material
This implies there is an interaction between the body and the material. The
placement of the material creates an interface that is otherwise absent in the body. The
interface is a site of many dynamic reactions, that is, the material may alter the body
or the body may alter the material. These dynamics determine both the biological
response of the body to the material (its biocompatibility) and ability of the material
to survive or resist or resist degradation or corrosion in the body.
Every biological surface is active therefore it is not possible to have a material
that is inert. The activity of this interface depends on the location of the material, its
duration in the body, the properties of the material, and health of the host. There is an
expectation for the biological performance of every material. In a bone implant, the
expectation is that the material will allow the bone to integrate with the implant. Thus,
an appropriate biological response for the implant is osseointegration. Whereas in a
full cast crown, the expectation is that the material will not cause inflammation of the
pulpal or periodontal tissues, but osseointegration is not an expectation. Whether or

not a material is biocompatible therefore depends on the physical function for which
the material will be used and the biological response that will be required from it.
The biocompatibility requirements of a material include the following:
 They should not sensitize and produce allergic reactions
 They should not undergo degradations
 They should not be carcinogenic
 They should not contain any toxic diffusible substances which get released
and enter into the circulatory system
 They should not be harmful to soft and hard tissues of the oral cavity in
particular, and the whole body, in general
HISTORICAL BACKGROUND
Since ancient times, a wide variety of relatively inert materials have been
placed or implanted in humans to replace or repair missing, damaged, or defective
body tissues. Bone, seashells, animal teeth, human teeth, metals, resin materials,
inorganic compounds, and other tooth replacement materials have been used for
replacement of missing teeth. For the restoration of damaged or decayed teeth, metals
and nonmetals have also been used, with outcomes that have varied from short-term
failure to limited success in certain individuals. Some of these materials have caused
immediate or delayed adverse reactions because of their allergenic or toxic potentials.
Although the concept of ethical treatment of patients extend back to the time
of Hippocrates (460-377 B.C.), the idea that new dental materials must be tested for
safety and efficacy before clinical use is much more recent. As late as the mid-1800s,
dentists tried new materials for the first time by putting them into patients’ mouths.
Many exotic formulations were used. For example, Fox developed a “fusible metal”
that consisted of bismuth, lead, and tin, which he melted and poured into the cavity
preparation at a temperature of approximately 100-degree Celsius. Even G.V. Black

used patients to test many of his new ideas for restorative materials, such as early
amalgams.
The current philosophy about testing the biological properties of dental
materials in a systematic way evolved in the 1960s. Using humans as research
subjects today without some previous testing or knowledge of the biological
properties of a material is unethical and illegal. Still, every new material must be
inserted into a human for the first time at some point. In most cases, a committee of
clinicians, basic scientists, and laypersons regulate and oversee the testing of new
materials in humans. Therefore, many alternate tests have been developed to try to
minimize the risks to humans. Despite good clinical research, materials are still used
before their biological properties can be fully ascertained.
Tests for the safety of restorative dental materials must ensure that a candidate
material is nontoxic and unlikely to cause adverse immunological effects. Evaluations
of toxicity are designed to identify adverse health events caused by physical agents,
chemical agents, or both. Paracelsus (1493−1541) correctly proposed that only the
dose of a substance differentiates a toxic agent from a remedy. No test can produce
results that can guarantee that a substance will not cause adverse effects in all
individuals who are treated with the substance. The allowable percentage of adverse
effects in a population is based on the risks to the health and life expectancy of the
individuals who will be exposed to the product under the indicated conditions and the
corresponding exposure doses for its components.
Initially, most biological reactions to materials were categorized empirically
and relied on animal models. Many studies between the 1950s and the 1970s involved
the use of premolar teeth that were scheduled for orthodontic extraction. Since the
1980s, testing has focused on primary tests for cytotoxicity, hemolysis, Styles’ cell
transformation, the Ames test, the dominant lethal response, oral LD50,
intraperitoneal (IP) LD50, and the acute inhalation test. Secondary tests are also used.
These include the mucous membrane irritation test (in hamsters’ cheek pouches),
dermal toxicity from repeated exposures, responses to subcutaneous implantation
(e.g., in rats), and sensitization (of guinea pigs). Testing of dental materials also
includes tests for pulp irritation responses, pulp capping effects, endodontic
applications, and dental implant performance.

As cell culture techniques developed, research focused on the mechanisms that
affected biological responses to materials. In the past decade, new molecular
biological and imaging techniques have been applied to assist our understanding of
the biological response to materials. Today, the field of biocompatibility testing has
reached a point where some prediction of biological properties is possible and the
future will likely provide the ability to design materials that elicit customized
biological responses.
DEFNITION
BIOCOMPATIBILITY- The ability of a biomaterial to perform its desired function
with respect to a medical (or dental) therapy, without eliciting any undesirable local or
systemic effects in the recipient or beneficiary of that therapy, but generating the most
appropriate beneficial cellular or tissue response in that specific situation, and
optimizing the clinically relevant performance of that therapy (Williams, 2008)
Biocompatibility of a material cannot be evaluated by using a single test rather than a
group of various techniques
The major requirements of a biocompatibility test are:
1. The test should be performed under conditions that simulate the actual use of the
material in the body

2. The test conditions should reflect the effects of the material’s time in the body on
the biological response
3. The stresses induced in the material under its intended function should be
considered in the interpretation of the biological response
BIOCOMPATIBILITY TESTS
Measuring biocompatibility continues to evolve as more is known about the
interactions between dental materials and oral tissues and as technologies for testing
improve. New materials must be extensively screened to ensure that they are
biologically acceptable before they are used in humans. Several varieties of tests are
used for this purpose, and are classified as in vitro, animal, and usage tests, the latter
including clinical trials.
Test Requirements:
The specific use of a material in the body has a direct bearing on the biological
response it produces. There are some major requirements of any test for
biocompatibility. Three of the most significant ones are that: (1) the test should be
performed under conditions that simulate the actual use of the material in the body;
(2) the test conditions should reflect the effects of the material’s time in the body on
the biological response; and (3) the stresses induced in the material under its intended
function should be considered in the interpretation of the biological response.
The test conditions should reflect whether or not the material will (1) contact
soft tissue or mineralized tissue; (2) be external to the oral epithelium; (3) serve as an
endosseous implant; (4) be exposed directly to bone, tissue fluid, blood, and saliva;

and (5) be separated by some barrier such as dentin between the material and living
cells. Special attention must be paid to materials that communicate through the
epithelium or lie completely beneath the epithelium.
Short-term exposures such as those of impression materials or temporary
cements are used only for a few minutes to a few weeks in the mouth. Their biological
responses are likely to differ from those that occur after 10 years of exposure. The
short-term responses are likely to be allergic reactions, but they are unlikely to be
toxic or mutagenic effects. In general, the most demanding tests are designed to
evaluate materials that are expected to remain present for the longest times.
Types of Biocompatibility Tests:
Autian (1970) was the first to propose a structured approach in biocompatibility
testing:
1. Non specific toxicity (Cell culture or small laboratory animals):
These tests are carried out on models which do not simulate clinical situation
2. Specific toxicity (Usage tests e.g. in subhuman primates):
Tests are conducted on models which simulate the clinical situation
3. Clinical testing in humans
Currently, three types of tests are used to analyze the biocompatibility of dental
materials: (1) an in vitro test, (2) an animal test, and (3) a usage test performed
clinically in animals or humans. No single test can accurately estimate the biological

response to a material. In addition, there is no clear consensus on the optimal
combinations of tests that must be performed for each type of material.
In Vitro Tests:
In vitro tests for biocompatibility require placement of a material or a
component of a material in contact with a cell, enzyme, or some other isolated
biological system. The contact can be either direct, when the material contacts the cell
system without barriers, or indirect, when there is a barrier of some sort between the
material and the cell system. Direct tests can be further subdivided into those in which
the material is physically present with the cells and those in which some extract from
the material contacts the cell system. In vitro tests can be roughly subdivided into
those that measure cytotoxicity or cell growth, those that measure some metabolic or
other cell function, and those that measure an effect on the genetic material in a cell
(mutagenesis assays). Often there is overlap in what a test measure.
In vitro tests have a number of significant advantages over other types of
biocompatibility tests. They are relatively quick, generally cost less than animal or
usage tests, can be standardized, are well suited to large-scale screening, and can be
tightly controlled to address specific scientific questions. The overriding disadvantage
of in vitro tests is their questionable relevance to the final in vivo use of the material.
Other significant disadvantages include the lack of inflammatory and other tissue-
protective mechanisms in the in vitro environment.
Standardization of in vitro tests is a primary concern. Two types of cells can
be used for in vitro assays. Primary cells are those cells taken directly from an animal
and cultured. These cells will grow for only a limited time in culture but usually retain
many of the characteristics of cells in vivo. Continuously grown cells or cell lines are
cells that have been transformed previously to allow them to grow more or less
indefinitely in culture. Because of this transformation, these cells do not retain all in
vivo characteristics, but they do consistently exhibit those features that they do retain.
Primary cell cultures seem to be more relevant than continuous cell lines for

measuring cytotoxicity of materials. However, primary cells, being from a single
individual, have limited genetic variability, may harbor viral or bacterial agents that
alter their behavior, and often rapidly lose their in vivo functionality once placed in
culture. Furthermore, the genetic and metabolic stability of continuous cell lines
contributes significantly toward standardizing assay methods.
Cytotoxicity Tests
Cytotoxicity tests assess cell death caused by a material by measuring cell
number or growth before and after exposure to that material. Control materials should
be well defined and commercially available to facilitate comparisons among other
testing laboratories. Membrane permeability tests are used to measure cytotoxicity by
the ease with which a dye can pass through a cell membrane, because membrane
permeability is equivalent to or very nearly equivalent to cell death.
Tests for Cell Metabolism or Cell Function
Some in vitro tests for biocompatibility use the biosynthetic or enzymatic activity of
cells to assess cytotoxic response. Tests that measure DNA synthesis or protein
synthesis are common examples of this type of test. A commonly used enzymatic test
for cytotoxicity is the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) test; other tests include the nitroblue tetrazolium (NBT), 2,3-Bis-(2-methoxy-
4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), and a water-
soluble tetrazolium (WST) assays, all being colorimetric assays based on different
tetrazolium salts. alamarBlue tests quantitatively measure cell proliferation using a
fluorescent indicator that allows continuous monitoring of cells over time.
Tests That Use Barriers (Indirect Tests)

Because direct contact often does not exist between cells and materials during in vivo
use, several in vitro barrier tests have been developed to mimic in vivo conditions.
These tests include an agar overlay method, which uses agar to form a barrier between
the cells and the material, and the Millipore filter assay, in which a monolayer of cells
is grown on a filter that is turned over so that test materials are placed on the filter and
leachable diffusion products are allowed to interact with the cells. The agar diffusion
and Millipore filter tests can provide, at best, a qualitative cytotoxic ranking among
materials.
For many materials, dentin is a barrier through which toxic components must
diffuse to reach pulp tissue, with the thickness of the dentin directly correlating with
the protection offered to the pulp. These assays, which incorporate dentin disks
between the test sample and the cell assay system, have the added advantage of
directional diffusion between the restorative material and the culture medium.
Other Assays for Cell Function
In vitro assays to measure immune function or other tissue reactions have also
been used. These assays measure cytokine production by lymphocytes and
macrophages, lymphocyte proliferation, chemotaxis, or T-cell rosetting to sheep red
blood cells. Other tests measure the ability of a material to alter the cell cycle or
activate complement. The in vivo significance of these assays is yet to be ascertained,
but many are promising for being able to reduce the number of animal tests required
to assess the biocompatibility of a material.
Mutagenic Assays
Mutagenesis assays assess the effect of a biomaterial on a cell’s genetic material.
Genotoxic mutagens directly alter cell DNA through various types of mutations. Each
chemical may be associated with a specific type of DNA mutation. Genotoxic

chemicals may be mutagens in their native states, or may require activation or
biotransformation to be mutagens, in which case they are called promutagens.
Epigenetic mutagens do not alter the DNA themselves, but support tumor growth by
altering the cell’s biochemistry, altering the immune system, acting as hormones, or
by other mechanisms. Carcinogenesis is the ability to cause cancer in vivo. Mutagens
may or may not be carcinogens, and carcinogens may or may not be mutagens. Thus,
quantitation and relevance of tests that measure mutagenesis and carcinogenesis are
extremely complex.
The Ames test is the most widely used short-term mutagenesis test and the
only one that is considered thoroughly validated. It looks at the conversion of a
mutant stock of Salmonella typhimurium back to a native strain, because chemicals
that increase the frequency of reversion back to the native state have a high
probability of being carcinogenic in mammals. A second test for mutagenesis is the
Styles’ cell transformation test. This test on mammalian cells offers an alternative to
bacterial tests (Ames test), which may not be relevant to mammalian systems.
However, because the Ames test is widely used, extensively described in the
literature, and technically easier to conduct, it is most often conducted in a screening
program.
Animal Tests:
Animal tests for biocompatibility, usually involving mammals such as mice,
rats, hamsters, or guinea pigs, are distinct from usage tests (which are also often done
in animals) in that the material is not placed in the animal with regard to its final use.
The use of an animal allows for the complex interactions between the material and a
functioning, complete biological system to occur. This is extremely difficult to mimic
in a cell-culture system. Thus, the biological responses in animal tests are more
comprehensive and may be more relevant than in vitro tests, and these features are the
major advantages of these tests. The main disadvantages of animal tests are that they
can be difficult to interpret and control, are expensive, time consuming, and often
involve significant ethical concerns and oversight. Furthermore, the relevance of the

test to the in vivo use of a material is often unclear, especially in estimating the
appropriateness of an animal species to represent a human. A variety of animal tests
have been used to assess biocompatibility.
Usage Tests
Usage tests may be done in animals or in human study participants. They are distinct
from other animal tests because they require that the material be placed in a situation
identical to its intended clinical use. The usefulness for predicting biocompatibility is
directly proportional to the fidelity with which the test mimics the clinical use of the
material, including time, location, environment, and placement technique. For this
reason, usage tests in animals usually employ larger animals that have similar oral
environments to humans, such as dogs, mini-swine, or monkeys.
When humans are used, the usage test is termed a clinical trial. The
overwhelming advantage for usage tests is their relevance. These tests are the gold
standard, in that they give the ultimate answer to whether or not a material will be
biocompatible and clinically useful. One might ask, then, why bother with in vitro or
animal tests at all. The answer is in the significant disadvantages of the usage test.
These tests are extremely expensive, last for long periods, involve many ethical and
often legal concerns, are exceptionally difficult to control and interpret accurately,
and may harm the test participants. In addition, statistical analysis of these tests is
often a daunting process. In dentistry, dental pulp, periodontium, and gingival or
mucosal tissues are the main targets of usage tests.
Dental Pulp Irritation Tests
In general, materials to be tested on the dental pulp are placed in class 5 cavity
preparations in intact teeth without caries. At the conclusion of the study, the teeth are
removed and sectioned for microscopic examination, with tissue necrotic and

inflammatory reactions classified according to the intensity of the response. Although
most dental-pulp irritation tests have involved teeth without inflamed pulps, there has
been increased concern that inflamed dental pulp tissue may respond differently than
healthy pulps to liners, cements, and restorative agents. Thus, usage tests on teeth
with induced pulpitis, which allow evaluation of the type and amount of reparative
dentin formed, will likely continue to be developed and refined.
Dental Implants in Bone
At present, the best predictors for success of implants are careful patient selection and
ideal clinical conditions. The following terms are used to define various degrees of
success: early implant success for implants surviving 1 to 3 years, intermediate
implant success for implants surviving 3 to 7 years, and long-term success for
implants surviving more than 7 years. As such, there are three commonly used tests to
predict implant success: (1) penetration of a periodontal probe along the side of the
implant, (2) mobility of the implant, and (3) radiographs indicating either osseous
integration or radiolucency around the implant. A bone implant is considered
successful if it exhibits no mobility and no radiographic evidence of periimplant
radiolucency, has minimal vertical bone loss and is completely encased in bone, and
has an absence of persistent periimplant soft-tissue complications. Any fibrous
capsule formation is a sign of irritation and chronic inflammation, which is likely to
lead to micromotion of the implant and ultimately to loosening and failure.
Mucosa and Gingival Usage Tests
Tissue response to materials with direct contact of gingival and mucosal tissues is
assessed by placement in cavity preparations with subgingival extensions. The
material’s effect on gingival tissues is observed and responses are categorized as
slight, moderate, or severe, depending on the number of mononuclear inflammatory

cells (mainly lymphocytes and neutrophils) in the epithelium and adjacent connective
tissues. A difficulty with this type of study is the frequent presence of some degree of
preexisting inflammation in gingival tissue due to the presence of bacterial plaque,
surface roughness of the restorative material, open or overhanging margins, and over-
contouring or under-contouring of the restoration.
Correlation Among In Vitro, Animal and Usage Tests
In the field of biocompatibility, some scientists question the usefulness of in vitro and
animal tests in light of the apparent lack of correlation with usage tests and the
clinical history of materials. However, lack of correlation is not surprising in light of
differences among these tests, in that in vitro and animal tests often measure aspects
of biological response that are more subtle or less prominent than those observed
during a material’s clinical use. Furthermore, barriers between the material and tissues
may exist in usage tests or clinical use, but may not exist in in vitro or animal tests.
Thus, it is important to remember that each type of test has been designed to measure
different aspects of biological response to a material, and correlation is not always to
be expected.
The best example of a barrier that occurs in use but not during in vitro testing
is the dentin barrier. When restorative materials are placed in teeth, dentin will
generally be interposed between the material and the pulp. The dentin barrier,
although possibly only a fraction of a millimeter thick, is effective in modulating the
toxic effect of a dental material.
Three methods were used to evaluate the following materials: zinc oxide–
eugenol (ZOE) cement, resin composite, and silicate cement. The evaluation methods
included (1) four different cell culture tests, (2) an implantation test, and (3) a usage
test in class 5 cavity preparations in monkey teeth.
The results of the four cell culture tests were relatively consistent, with silicate
having only a slight effect on cultured cells, composite a moderate effect, and ZOE a
severe effect.

These three materials were also embedded subcutaneously in connective tissue
in polyethylene tubes (secondary test), and observations were made at 7, 30, and 90
days. Reactions at 7 days could not be determined because of inflammation caused by
the operative procedure. At 30 days, ZOE caused a more severe reaction than silicate
cement. The inflammatory reactions at 90 days caused by ZOE and silicate were
slight, whereas the reaction to resin composites was moderate.
When the three materials were evaluated in class 5 cavity preparations under
prescribed conditions of cavity size and depth (usage test), the results were quite
different from those obtained by the other methods. The silicate was found to have the
most severe inflammatory reaction, the composite had a moderate-to-slight reaction,
and the ZOE had little or no effect.
Apparent contradictions in this study are explained by considering the
components that were released from the materials and the environments into which
they were released. The silicate cement released hydrogen ions that were probably
buffered in the cell culture and implantation tests but were not adequately buffered by
the dentin in the usage tests. Microleakage of bacteria or bacterial products may have
added to the inflammatory reaction in those usage tests. Thus, this material appeared
to be the most toxic in the usage test. The composites released low-molecular-weight
resins, and the ZOE released eugenol and zinc ions. In the cell culture tests, these
compounds had direct access to cells and probably caused the moderate-to-severe
cytotoxicity. In the implantation tests, the released components may have caused
some cytotoxicity, but the severity may have been reduced because of the capacity of
the surrounding tissue to disperse the toxins. In usage tests, these materials probably
were less toxic because the diffusion gradient of the dentin barrier reduced
concentrations of the released molecules to low levels. The slight reaction observed
with the composites may also have been caused in part by microleakage around these
restorations. The ZOE did not show this reaction, however, because the eugenol and
zinc probably killed bacteria in the cavity, and the ZOE may have reduced
microleakage.
Another example of the lack of correlation of usage tests with implantation
tests is the inflammatory response of the gingiva at the gingival and proximal margins
of restorations that accumulate bacterial plaque and calculus. Plaque and calculus

cannot accumulate on implanted materials and therefore the implantation test cannot
hope to duplicate the usage test. However, connective tissue implantation tests are of
great value in demonstrating the cytotoxic effects of materials and evaluating
materials that will be used in contact with alveolar bone and apical periodontal
connective tissues. In these cases, the implant site and the usage sites are sufficiently
similar to compare the test results of the two sites.
Using In Vitro, Animal and Usage Tests Together
For about 25 years, scientists, industry, and the government have recognized that the
most accurate and cost-effective means to assess biocompatibility of a new material is
a combination of in vitro, animal, and usage tests. Implicit in this philosophy is the
concept that no single test will be adequate to completely characterize
biocompatibility of a material.
Early combination schemes proposed a pyramid testing protocol, in which all
materials were tested at the bottom of the pyramid and materials were “weeded out”
as the testing continued toward the top of the pyramid. Tests at the bottom of the
pyramid were “unspecific toxicity” tests of any type (in vitro or animal) with
conditions that did not necessarily reflect those of the material’s use. The next tier
shows specific toxicity tests that presumably dealt with conditions more relevant to
the use of the material. The final tier was a clinical trial of the material.
Later, another pyramid scheme was proposed that divided tests into initial,
secondary, and usage tests. The philosophy was similar to that used in the first
scheme, except that the types of tests were broadened to encompass biological
reactions other than toxicity, such as immunogenicity and mutagenicity. The concept
of a usage test in an animal was also added (versus a clinical trial in a human).
The features of this type of testing encompass the following. Firstly, only
materials that “passed” the first tier of tests were graduated to the second tier, and
only those that passed the second tier were graduated to the clinical trials. This
scheme funneled safer materials into the clinical trials area and eliminated unsafe

materials. This strategy was appreciated because clinical trials are the most expensive
and time-consuming aspect of biocompatibility testing. Second, any material that
survived all three tiers of tests was deemed acceptable for clinical use. Third, each tier
of the system put a great deal of weight on the tests used to accurately screen in or out
a material. Although still used in principle today, the inability of in vitro and animal
tests to unequivocally screen materials in or out has led to development of newer
schemes in biocompatibility testing.
Two newer testing schemes have evolved in the past 5 years with regard to
using combinations of biocompatibility tests to evaluate materials. In both of these
schemes, all tests (in vitro, animal, and usage) continue to be of value in assessing
biocompatibility of a material during its development and even in its clinical service.
For example, tests of inflammatory response in animals may be useful not only during
the development of a material, but also if a problem is noted with the material after it
has been on the market for a time. These new schemes also recognize the inability of
current testing methods to accurately and absolutely screen in or out a material. In
addition, both incorporate the philosophy that assessing the biocompatibility of a
material is an ongoing process.
Diagnostic tests on patients
Diagnostic tests on patients are used to more deeply analyze claimed or real
unwanted side effects in individual subjects (individual compatibility). This branch of
biocompatibility studies has become very important during recent years, since many
materials do not cause clinically manifest reactions in the vast majority of the
population but may generate claimed or real disease symptoms linked to materials in
single cases. The assumption of an individual compatibility for dental materials is
based on these observations. Thus, examination of the individual compatibility of
various materials has been attempted by means of one or more test methods in order
to find a feasible explanation for certain symptoms, to perform a causal treatment, or,
if possible, to avoid such symptoms by a preceding examination.

Allergy tests:
The patch test, originally developed and described by Jadassohn, is the most
important allergy test regarding dental materials. This test can be applied to identify
delayed type hypersensitivity (type IV reactions) as the cause for an allergic contact
dermatitis. Immediate reactions (type I reaction, such as asthma) can be diagnosed by
the prick test. The Radioallergosorbent test (RAST) may be used as an alternative or
supplement to the prick test.
Measurement of intraoral voltage:
All metals in the oral cavity are exposed to an aqueous environment. They
corrode (more or less) and at the same time release different positively charged ions.
The metal surface thereby becomes negatively charged, which will then cause the
attachment of positively charged ions from saliva (Ca2+, Na+, or K+). Voltage
differences can be found against a reference electrode or between two metals in the
oral cavity (e.g., between two equivalent gold alloys). If there is a conductive contact
between the two metals (e.g., direct contact or through wires), then ionic electricity
can circulate (ion shift) in the tissue/saliva. The electric current or the current density
per cross-sectional tissue area cannot be directly measured.
A number of measurement devices are available on the market that can be
used for determining intraoral voltages between different restorations. These devices
require a high internal resistance (at least 20 megaohms). Certain techniques measure
a “current.” But it should be kept in mind that this does not represent a current
(electricity) in tissue or in saliva, but a discharge via an instrument-specific internal
resistance, although it is sometimes referred to as measurement of intraoral current.
Change of electric current by time (e.g., per second) can be measured by means of
appropriate computer programs. Some techniques even claim to be able to measure
currents between resins.
Evaluation of pulp sensitivity:

The sensibility test of the pulp may demonstrate functional nerval structures.
This method is used for pulp diagnosis and is mainly based on the application of cold
and of electric current. The threshold of pulp nerves regarding electric current varies
between 20 and 100 μA, whereas this value for periodontal structures ranges between
176 and 250 μA. Thus, it is possible to differentiate between an irritation of nerves in
the pulp and in the periodontium. Thermal examination is performed with sticks of
ice, CO2-snow (– 78.5 oC), or cold sprays, which, for instance, contain propane,
butane or similar substances (– 22 to – 50 oC). Dichlorine–difluorine–methane, which
has been used previously, has been discontinued for environmental reasons. All
substances will cause a similar temperature decrease in the pulp.
Analysis of intraoral alloys:
Knowledge about the exact composition of materials in patients’ oral cavities
is an important prerequisite for subsequent clinical tests, such as allergy tests. But so
far, appropriate techniques are available only for the routine analysis of metals. For
removable restorations and dentures, processing and corrosive alterations can be
examined by means of modern analytical methods (polished metallic micrograph
sections in combination with energy dispersive x-ray analysis, or EDX). However,
clinical evaluation is much more difficult if restorations such as crowns, inlays, or
bridges are fixed in the oral cavity and thus cannot be removed for identification of
the alloy and the structure in the laboratory. In these cases, the composition of an
intraoral alloy can be identified using the chip test. A small amount of alloy particles
(chips) is produced intraorally using a silicon carbide stone or a tungsten carbide bur.
The alloy particles are collected on a small, circular, self-adhesive graphite plate. This
self-adhesive carrier conducts electricity. Subsequently, the collected alloy particles
can be identified quantitatively and qualitatively by means of EDX analysis.
Analysis of metals in saliva and biopsies:
So far, examination of saliva to diagnose material-linked side effects
concentrates on the detection of metals, although most recently, resin components

were also identified in saliva. A defined amount of “morning saliva” (before any food
or drink intake or oral hygiene measures) is collected and, after chemical pulping, is
analyzed, such as by atomic absorption spectrometry (AAS). Biopsies, for instance
from the gingiva adjacent to metal restorations, were also used to determine the metal
content. Metal concentrations in biopsies are usually analyzed by AAS after chemical
pulping.
STANDARDS THAT REGULATE THE TESTING OF
BIOCOMPATIBILITY
Standardization is a difficult and lengthy process, made more difficult by
disagreement on the appropriateness and significance of particular tests. In early
attempts to develop a uniform test for toxicity of dental materials, small, standard-
sized pieces of gold, amalgam, gutta-percha, silicates, and copper amalgam were
sterilized and placed in uniformly sized pockets within skeletal muscle tissue. Biopsy
specimens were evaluated microscopically after 6 months. Somewhat later came
attempts to standardize techniques by placing materials within connective tissue and
tooth pulp. Not until the passage of the Medical Device Bill by Congress in 1976 was
biological testing for all medical devices (including dental materials) given a high
priority. In 1972 the ADA Council on Dental Materials, Instruments, and Equipment
(now the Council on Scientific Affairs) approved specification No. 41 for
Recommended Standard Practices for Biological Evaluation of Dental Materials. The
committee that developed this document recognized the need for standardized
methods of testing and for sequential testing of materials to reduce the number of
compounds that would need to be tested clinically. In 1982, an addendum was made
to this document to include tests for mutagenicity, and it was further updated in 2005.
Based on a thorough risk assessment according to relevant ISO standards and an
evaluation of the existing literature, the manufacturers have the responsibility of
selecting the appropriate and necessary biological tests for their products. Finally, the

manufacturer is fully responsible legally for any adverse effects arising from the use
of the products, which may have been prevented by performing state-of-the-art tests.
ANSI/ADA Specification 41
Three categories of tests are described in the 2005 American National
Standards Institute (ANSI)/ ADA specification: initial, secondary, and usage tests.
The standard was most recently revised to conform to International Organization for
Standardization (ISO) 10993, and was released as ANSI/ADA specification No. 41,
Recommended Standard Practices for Biological Evaluation of Dental Materials
(2005).
ISO 10993
In the 1980s, international efforts were initiated by several organizations to
develop international standards for biomedical materials and devices. Several
multinational working groups, including scientists from ANSI and the ISO, were
formed to develop standard ISO 10993, published in 1992. Revision of the dental
components of this document resulted in ISO 7405:2008 “Preclinical evaluation of
biocompatibility of medical devices used in dentistry—Test methods for dental
materials.” This is the most recent ISO standard available for biocompatibility testing
of dental materials.
The standard divides tests into initial and supplementary tests to assess the
biological reaction to materials. Initial tests are tests for cytotoxicity, sensitization,
and systemic toxicity. Some of these tests are done in vitro, others in animals in non-
usage situations. Most of the supplementary tests for assessing chronic toxicity,
carcinogenicity, and biodegradation are done in animal systems, many in usage
situations. Significantly, although guidelines for the selection of tests are given in part
1 of the standard and are based on how long the material will be present; whether it

will contact body surface only, blood, or bone; and whether the device communicates
externally from the body, the ultimate selection of tests for a specific material is left
up to the manufacturer, who must present and defend the testing results.
MATERIAL SAFETY DATA SHEETS
Each dental product is supplied with a Material Safety Data Sheet (MSDS), also
known as a Product Safety Data Sheet (PSDS). This is a report on the properties of a
particular substance, for example, melting point, boiling point, and flash point. This
report is intended for occupational settings and is essential for workers and emergency
personnel because it describes procedures for handling or working with the material
safely. In addition to physical data, it identifies toxicity risks, health effects, first-aid
procedures, reactivity, storage and disposal conditions, and, where applicable,
procedures for firefighting, the types of protective equipment that should be used and
procedures that must be followed for accidental releases and handling of spills.
Potentially harmful substances must be properly labeled to minimize the risk
of injuries to personnel and others who may come in contact with the material or
substance, risks to the health of individuals, and risks of environmental exposure.
Labels may include hazard symbols, for example, the European Union label, e.g., with
a black diagonal cross on an orange background.
Labeling on packages or delivery devices that contain potentially hazardous
substances exhibits symbols indicating the types of hazards that may be encountered.
The colour coded markings fall into one of four categories: (1) blue for the level of
health hazard, (2) red for flammability, (3) yellow for (chemical) reactivity, and (4)
white for unique hazards. Each of the health, flammability, and reactivity categories is
rated on a scale from 0 (no hazard or normal substance) to 4 (severe risk).
CURRENT BIOCOMPATIBILITY ISSUES IN DENTISTRY
Mercury and Amalgam:

The controversy over the biocompatibility of amalgam has waxed and waned
several times in the 170-plus year history of its dental use. Most of the controversy
stems from the known toxicity of mercury and the question of whether mercury from
amalgam restorations has toxic effects. Mercury occurs in four forms: as the metal
(Hg0), as an inorganic ion (Hg2+), as a component of the silver-mercury phase, or in
one of several organic forms such as methyl or ethyl mercury. Metallic mercury gains
access to the body via the skin or as a vapor through the lungs. Ingested metallic
mercury is poorly absorbed from the gut (0.01%), so the primary portal into the body
is through inhalation of mercury vapor. Mercury vapor is readily absorbed after
inhalation. Dissolved mercury can be transported through blood and distributed to the
brain and other organs and excreted by exhalation and in urine. Elemental mercury is
transported to blood cells and tissues, where it is oxidized rapidly to mercuric mercury
(Hg2+).
Mercury accumulates in the kidneys. In the brain, metallic mercury can be
converted to an inorganic form that is retained in the brain. Elemental mercury and
mercury vapor have a half-life of 1 to 3 months. Mercury leaves the body by excretion
through urine and feces. Chronic mercury toxicity may be manifested as tremors;
memory loss; and changes in personality, vision, and hearing. Children and fetuses
are most sensitive to the effects of mercury on the nervous system. Selenium, an
essential element, is claimed to be protective against the toxic effects of mercury.
Several studies have shown that amalgams release sufficient vapor to cause
absorption of between 1 and 3 μg/day of mercury, depending on the number and size
of amalgam restorations present (Langworth et al., 1988; Berglund, 1990; Mackert
and Berglund, 1997; Ekstrand et al., 1998). The inhaled mercury gains access to the
bloodstream via the alveoli of the lungs. From the blood, mercury is distributed
throughout the body, with a preference for fat and nerve tissues. Mercury is also
ingested as particles produced by wear, and about 45 μg/day of mercury may reach
the gut either as the amalgam form or as dissolved and released Hg2+ ions. The
absorption of ionic mercury is also poor (approximately 1% to 7%). Mercury trapped
in amalgam particles is also poorly resorbed. Methyl mercury is not produced from
amalgams but is generally a product of bacteria or other biological systems acting on
metallic mercury. Methyl mercury is the most toxic form of mercury and is also very
efficiently absorbed from the gut (90% to 95%). Methyl mercury is absorbed mainly

from the diet, particularly from fish (especially shark, swordfish, and tuna), which
contribute a significant portion.
Concerns about mercury stem from its toxicity and its relatively long half-life
in the body. The toxicity of mercury is well known; the symptoms depend somewhat
on the form. Acute symptoms are neurologically based or kidney based, ranging from
paresthesia (at 500 μg/kg or above) to ataxia (at 1000 μg/kg or above), joint pain (at
2000 μg/kg or above), and death (at 4000 μg/kg or above). The lowest known level
for any observable toxic effect is 3 μg/kg. This level translates to about 30 μg of
mercury per gram of creatinine clearance in the urine. At chronic exposure levels, the
symptoms are more subtle and include weakness, fatigue, anorexia, weight loss,
insomnia, irritability, shyness, dizziness, and tremors in the extremities or the eyelids.
Although amalgams do not release anywhere near toxic levels of mercury, the long
half- life of mercury in the body raises concerns among some individuals. The half-
life ranges from 20 to 90 days, depending on the form, with methyl mercury
exhibiting the longest half-life and inorganic forms the shortest. Numerous tests for
the body burden of mercury have been developed, including those based on the
analysis of blood, urine, and hair. Of these test parameters, measurement of mercury
in the urine after 24 hours may be the best long-term indicator of the total metallic
mercury body burden, normalized to grams of creatinine clearance from the kidneys.
Numerous studies have attempted to determine whether mercury exposure
from dental restorations or other sources contributes to any documentable health
problem. Several studies have estimated the number of amalgam surfaces needed to
expose an individual to mercury concentrations with a minimum observable effect
(slightly impaired psychomotor performance, detectable tremor, and impaired nerve
conduction velocity). Estimates are that several hundred amalgam surfaces would be
necessary to achieve these levels. Even if all 32 teeth were restored on all surfaces
with amalgam, the total number of surfaces (counting incisal edges) would be only
160. Other studies have measured renal function in patients in whom all of the
amalgam was removed at the same time (the worst possible case). Despite markedly
elevated blood, plasma, and urine levels of mercury, no renal impairment was noted.
Still other studies have attempted to look at blood cell types and cell numbers in
dentists, who are presumably exposed to higher levels of mercury because of their
daily occupational exposure. No effects of mercury have been noted. Other studies for

neurological symptoms in children populations occupationally exposed have shown
no effects (Bellinger et al., 2006, 2007; DeRouen et al., 2002, 2006). In summary,
there are simply no data to show that mercury released from dental amalgam is
harmful to the general population.
Base Metal and Noble Metal Alloys
Predominantly base metal alloys are classified by the ADA as those containing
less than 25% by weight of noble metals (gold, platinum, palladium, rhodium,
ruthenium, iridium, and osmium). Noble alloys are classified as those that contain
between 25% and 60% of these noble metal elements. High noble metal alloys are
classified by the ADA as those containing at least 40% gold and 60% of noble metal
elements. Stainless steel (Fe-C-Ni-Cr), cobalt–chromium (Co-Cr), nickel–chromium
(Ni-Cr), and cobalt–nickel–chromium (Co-Ni-Cr), classified as base metal alloys, and
commercially pure titanium (CP Ti) are used most often for removable fixed
restorations and orthodontic appliances. Some evidence suggests that metal appliances
can lead to gingivitis or periodontitis. The severity of these adverse effects varies as a
function of atomic or molecular characteristics (Schmalz and Garhammer, 2002).
Metallic components and microparticles from cast metal restorations have been found
in contiguous plaque and gingival tissues. Although high-gold-content noble alloys
are more resistant to corrosion than other alloys, most local adverse effects seem to
occur when noble and base alloys are used together.
Metal ions can be leached from cast metal restorations or wrought appliances
into the oral cavity. Since high-noble (HN) and noble (N) alloys are corrosion
resistant, one might expect a negligible level of leaching. However, allegations of
adverse effects caused by leaching of palladium have raised concerns on the
biocompatibility of these alloys (Wataha and Hanks, 1996). Biocompatibility studies
of a high-gold alloy (Iropal W), two low-gold alloys (Argenco 9 and Gold EWL-G), a
high-palladium alloy (Argipal), two palladium–silver (Pd-Ag) alloys (Argenco 23 and
EWL-G), one Ni-Cr alloy (Wiron-88), two Co-Cr alloys (Wironium and Wirocast),
and a 22k gold alloy revealed that the strongest responses were derived from the Ni-
Cr alloy and the weakest response was from the 22k gold alloy. These analyses were

based on use of the subcutaneous implantation (histopathological) method. Cast metal
discs were implanted for 15, 30, or 60 days in rats. The high-gold alloy and the high-
palladium group showed reactions similar to those of the 22k gold alloy. However,
the low-gold alloy and the Pd-Ag alloys ranked between the base metal alloy and the
precious metal alloys.
Metallic ions released through corrosion processes are responsible for much of
the metal–protein or metal–cell interaction behavior of dental metals and alloys.
However, the surface structure of the metal, its composition, and its electrochemical
properties also contribute to local interactions. Increased plaque accumulation can
cause adverse inflammatory reactions in the adjacent soft tissues. Ions released from
the superficial layers of cast alloys may be quite cytotoxic.
No correlation has been found between the noble metal content of alloys and
the severity of corrosion. However, some base metals, such as nickel-chromium-
beryllium (Ni-Cr-Be) alloys, exhibit increased corrosion in low-pH environments.
Also, microscopic particles can be abraded from metallic restorations during wear
processes. In sufficient quantities, metal ions such as copper, nickel, and beryllium
can be released and subsequently induce inflammation of the adjacent periodontal
tissues and the oral mucosa.
No evidence exists to prove that dental metallic materials are mutagenic,
genotoxic, or carcinogenic. Although in vitro evidence suggests that the immune
response can be altered by various metal ions, their role in oral inflammatory diseases
such as gingivitis and periodontitis is unknown. Nickel is known to be highly
allergenic, especially in females. It has been reported that 34% to 65% of patients who
are allergic to nickel are also allergic to palladium. Further, palladium allergy seems
to occur when individuals have been sensitized to nickel.
Few studies have measured the release rate of metal ions via in vivo corrosion.
The amount and nature of released cations varies depending on the type of alloy, the
environment, and the corrosion mechanism, including the concentration cell type,
crevice corrosion, galvanic cell corrosion, stress corrosion, and pitting corrosion. The
chemical composition of the corrosive solution, the pH, ion composition, artificial
saliva characteristics, cell culture medium, and serum are significant variables as well.

Some evidence indicates that multiphase alloys tend to release metal ions in
proportion to their ion compositions.
Nickel and Beryllium
Of many metals used in dentistry, nickel is a common chemical element in
many base metal dental alloys, such as those used for crowns, fixed dental prostheses,
removable partial dentures, and some orthodontic appliances. Nickel is also used in
many types of endodontic files, although the duration of exposure through the use of
files is far shorter. The use of nickel in dental alloys has been controversial for many
years because of the allergenic potential of nickel ions and nickel compounds.
Nickel is the most allergenic metal known, with an incidence of allergic
reactions between 10% and 20%. Reactions to nickel are more common among
women, presumably because of the chronic exposure to nickel through jewelry,
although the incidence among men is increasing. Reactions to nickel-containing
dental alloys are well documented, and these can be quite severe in sensitized
individuals. These reactions are probably under-reported because they are often subtle
and can resemble periodontal inflammation or the erythema that results from
excessive pressure on the palatal mucosa by metal frameworks. These reactions may
also occur only outside the mouth.
Not all individuals with nickel allergy will react to intraoral nickel, and it is
currently not possible to predict which individuals will react. Because the frequency
of nickel allergy is high, it is possible that individuals will become sensitized to nickel
after placement of nickel-containing alloys in the mouth.
Some studies in guinea pigs have suggested that oral exposure to nickel
induces immunological tolerance. As stated previously, there is a possible cross-
reactivity between nickel and palladium allergy. Almost all patients who are allergic
to palladium will be allergic to nickel, whereas only about 33% of those allergic to
nickel will be allergic to palladium. The mechanisms of the high allergy frequency to
nickel are not known, but there is probably a genetic component. In addition, the

tendency of nickel-containing alloys to release relatively large amounts of nickel ions
probably contributes to their allergenicity. This release is particularly high in acidic
conditions, especially for Ni-Cr alloys with less than 20% by weight of chromium.
Nickel has other adverse biological effects in addition to allergy. Nickel ions
(Ni2+) are a documented mutagen in humans, but there is no evidence that nickel ions
cause any carcinogenic response intraorally. Nickel ions, along with cobalt and
mercury, have also been shown to be nonspecific inducers of inflammatory reactions.
Specifically, nickel ions appear to induce intercellular adhesion molecules in the
endothelium, and they induce the release of cytokines from monocytes and other cells.
It is not known to what extent these mechanisms contribute to any intraoral
inflammation around nickel-containing crowns.
It is well known that nickel-based alloys can exhibit significant corrosion and
release of nickel ions in a low-pH environment. Beryllium-free Ni-Cr alloys are more
corrosion-resistant than beryllium-containing alloys. Base metal alloys containing
both beryllium and nickel exhibit high beryllium release rates, which may pose a
health risk. Many biological factors—including the biofilm characteristics, organic
acid composition, and types of enzymes produced by oral microorganisms or those
present in food—may contribute to alloy corrosion in vivo. Interactions between
metallic restorations and patient factors such as consumption frequency of acidic
foods and beverages and composition of saliva, can significantly affect intraoral
corrosion. Corrosion may also be accelerated by phagocytotic cells such as human
neutrophils.
Wear can also accelerate the corrosion processes in vivo because of the local
breakdown of the passivation layer (Khan et al., 1999). The dual action of corrosion
and wear may accelerate breakdown in the oral environment.
Although no general correlation has been demonstrated between alloy
composition and cytocompatibility, severely cytotoxic alloys generally have
contained higher amounts of nickel than biocompatible products.
Although beryllium is known to be highly toxic, it is used in some Ni-Cr
alloys in concentrations of 1% to 2% by weight (approximately 5.5% to 11% atomic
content) to increase the castability of these alloys and lower their melting range. It

also tends to form thin adherent oxides that are required to promote atomic bonding of
porcelain. The use of beryllium in dental alloys is controversial because of its
biological effects. First, beryllium is a documented carcinogen in either the metallic
(Be0) or ionic (Be2+) state, although there are no studies showing that dental alloys
containing beryllium cause cancer in humans. Any reaction is most probably mediated
by beryllium released from the alloys, and although such release has been
documented intraorally and in vitro, it is not as prominent as for nickel. Acidic
environments enhance beryllium release from Ni-Cr alloys.
Furthermore, beryllium-containing particles that are inhaled and reach the
alveoli of the lungs may cause a chronic inflammatory condition called berylliosis. In
this condition, the alveoli of the lung are engorged with lymphocytes and
macrophages. T cells in susceptible individuals proliferate locally in the lung tissue,
presumably in a delayed hypersensitivity reaction to the beryllium metal. Berylliosis
occurs only in individuals with a hypersensitivity to beryllium and may occur from
inhalation of beryllium dusts (from grinding or polishing alloys), salts, or fumes such
as those encountered in casting beryllium-containing alloys. Thus, dental lab
technicians would presumably be at the highest risk of adverse effects from exposure
to beryllium dusts and vapors.
Titanium and Titanium Alloys
In vitro evaluation of titanium biocompatibility, percentage attachment
efficiency, and proliferation of human fetal fibroblasts and human gingival fibroblasts
reveals that a surface layer of titanium oxide (Ti2O3) has the ability to coexist with
living tissues and organisms. Based on these studies one can conclude that titanium is
relatively nontoxic, non-injurious, and not physiologically reactive. Titanium has a
light weight, high strength, and excellent durability when exposed to chemical agents.
However, it is susceptible to attack by acidic fluoride products.

Cytotoxicity of Metals and Dental Casting Alloys
Cytotoxicity is often reported by an IC50 value, which is the inhibitory
concentration (IC) that causes a 50% reduction in cell growth. A study of 43 metal
salts, using the colony formation method and two types of cells (fibroblasts and
osteoblast-like cells), revealed that IC50 depends on the types of metallic elements,
their chemical states, and their elemental concentrations (Yamamoto et al., 1998).
Another study reported the metabolic as well as the morphological response of
cultured human gingival fibroblasts to salt solutions of beryllium (Be2+), chromium
(Cr6+ and Cr3+), nickel (Ni2+), and molybdenum (Mo6+) ions that may be released
from dental alloys (Messer et al., 1999). The evaluation parameters included viability,
lysosomal activity, oxygen consumption, membrane integrity, DNA synthesis,
ribonucleic acid (RNA) synthesis, protein synthesis, intracellular adenosine
triphosphate (ATP) levels, and glucose-6-phosphate dehydrogenase activity. Whereas
Ni2+ ion solutions altered metabolic functions at concentrations of 3 to 30 parts per
million (ppm) compared with Cr3+ and Mo6+ at concentrations of 10 and 100 ppm,
Cr6+ and Be2+ were the most toxic ions, which caused cellular alterations at
concentrations of 0.04 to 12 ppm. Heavy metal ions such as Ni2+ and Co2+, released
from an implanted alloy by corrosion, can be distributed systemically by proteins,
such as albumin. Cast CP Ti has been found to be highly biocompatible (Berstein et
al., 1992; Wang and Li, 1998). Further, it was observed that the corrosion resistance
of a noble or base metal alloy does not permit one to draw conclusions as to its
biocompatibility. It was also found that solid specimens of gold-based solders
combined with a substrate alloy were very often less cytotoxic than the solders alone.
Only three solder-substrate alloy combinations revealed more pronounced toxic
reactions than the single solders (Wataha et al., 1995).
A shortcoming of conventional cell-culture studies is that the results do not
usually reflect the long-term in vivo behavior of cast dental alloys. Thus, some alloys
are tested both immediately after being polished and up to a year after being
conditioned in a biological medium. Some alloys that were cytocompatible at baseline
were also not cytotoxic after 10 months, and highly cytotoxic alloys were
significantly less cytotoxic after 10 months. Thus, one could assume that restorations

that have been in place for a year or more may present a reduced risk of cytotoxic
effects.
Methacrylates and Resin-based Composites
The best screening substance for methacrylate allergy caused by dental
material products is HEMA. This result confirms previous findings (Goon et al.,
2006), which revealed that HEMA alone picked up 96.7% of the patients with
methacrylate allergy and 100% of the dental personnel with methacrylate allergy. The
frequency of positive responses to the common allergen test substances were reported
as follows: gold sodium thiosulfate, 14.0%; nickel sulfate, 13.2%; mercury, 9.9%;
palladium chloride, 7.4%; cobalt chloride, 5.0%; and HEMA 5.8% (Goon et al.,
2006).
The primary risk of these materials appears to be allergy related, and the risk
is highest for dental personnel because of frequent exposure to non-polymerized
materials. Allergy to other types of dental materials such as latex gloves and
monomeric substances also represents the greatest risks of an adverse biological effect
for dental office staff. The allergenicity of methylmethacrylate is well documented,
and the use of gloves is not effective in preventing contact because most resin
monomers pass easily through gloves. Also, allergic reactions to other methacrylates
have been reported. The allergic reactions occur primarily as contact dermatitis, with
the resins acting as haptens via delayed hypersensitivity (Type IV) mechanisms. In
rare cases, anaphylactic responses have been reported, and dermatitis may be so
severe as to be disabling. In the most severe cases, individuals may need to change
work activities or change to a different profession.
Resins also have significant toxic effects, which are clearly demonstrated
through the use of in vitro tests. The results of these tests are often comparable with,
and sometimes worse than, the effects resulting from the use of metals. There is ample
evidence that resins release non-polymerized components into biological
environments, although the release in vivo of specific substances is not well
documented for either resins or metals. Resin components have also been shown to
traverse dentin, and newer techniques that advocate direct pulp capping with resins

expose pulp directly to these materials. The long-term, low-dose effects of resin
components that are released are not well understood, and detecting adverse effects in
vivo is difficult. Limited clinical evidence has linked the use of resins to oral
inflammation. There is also limited in vivo evidence to show that resins may allow the
growth of some bacterial species. Other studies have advocated the use of special
resins as antimicrobial agents to be incorporated into dental restorative materials.
Dental Ceramics
Ceramic materials are known for their high levels of biocompatibility. Metal oxides
such as Al2O3, BaO, CaO, K2O, Li2O, Na2O, ZnO, and ZrO2 are components of
either dental core ceramics or dental veneering (layering) ceramics, and silicon
dioxide (SiO2) is the principal matrix phase component of all veneering ceramics.
These oxides and related compounds in dental ceramics exhibit minimal dissolution in
normal oral fluids and beverages. However, highly acidic environments can increase
the release rates of certain metal and silicon ions. For example, acidulated phosphate
fluoride (APF) is known to corrode the surfaces of veneering porcelains as well as
glaze and stain ceramics. This is an extreme case, which suggests that APF should not
be used in patients who have ceramic or metal–ceramic restorations.
Glass Ionomers
Glass ionomer has been used as a cement (luting agent), liner, base, and restorative
material. Light-cured ionomer systems use HEMA or other monomers or oligomers as
additives or as pendant chains on the polyacrylic acid main chain. In screening tests,
freshly prepared ionomer is mildly cytotoxic, but this effect is reduced over time. The
fluoride release from these materials, which may have some therapeutic value, has
been implicated in this cytotoxicity in vitro. Some researchers have reported that

certain systems are more cytotoxic than others, and though the reasons for this are not
clear, presumably it is related to the composition of the glasses used in the material,
which may contain aluminum, calcium, manganese, zinc, strontium, and other
metallic elements.
There have been several reports of pulpal hyperalgesia for short periods (days)
after placing glass ionomers in cervical cavities. This effect is probably the result of
increased dentin permeability after acid etching. In any case, glass ionomer has not
been shown to be well tolerated when placed directly upon living pulp tissue as a
direct pulp-capping agent.
Acidic dental materials have the capacity for demineralizing dentin and
therefore releasing bioactive molecules present within this tissue. Although this effect
has not been shown specifically for glass ionomers to date, it seems reasonable to
assume that it does occur clinically. In a recent study in nonhuman primates, dentin
matrix proteins were shown to enhance the formation of reactionary dentin over
exposed pulps, compared with calcium hydroxide or resin-modified glass ionomer.
Although the response to resin-modified glass ionomer was less consistent than
calcium hydroxide, in many cases it did result in new dentin formation, even when
directly exposed to the pulp. It is important to note that the natural tooth repair
process producing reactionary dentin does occur, following an initial inflammatory
reaction, under glass ionomer when the material is placed over an existing dentin
surface. Thus, it is possible that the repair process is again aided by the presence of
the bioactive molecules released from the dentin by the mild demineralization
produced by the material under these conditions.
Liner, Varnishes and Non-Resin Cements
Calcium hydroxide cavity liners come in many forms, typically as pastes with a very
alkaline pH (>12). Resin-containing preparations also exist and are capable of light-
activated polymerization. The high pH of calcium hydroxide in suspension leads to
extreme cytotoxicity in screening tests. Calcium hydroxide cements containing resins
cause mild to moderate cytotoxic effects in tissue culture in both the freshly set and

long-term set conditions. Inhibition of cell metabolism is reversible in tissue culture
by high levels of serum proteins, suggesting that protein binding or buffering in
inflamed pulp tissue may play an important role in detoxifying these materials in vivo.
The initial response after exposing pulp tissue to these highly alkaline aqueous pulp-
capping agents is necrosis to a depth of 1 mm or more. The alkaline pH also helps to
coagulate any hemorrhagic exudate of the superficial pulp.
Shortly after necrosis occurs, neutrophils infiltrate into the sub-necrotic zone.
After 5 to 8 weeks, only a slight inflammatory response remains. Within weeks to
months, however, the necrotic zone undergoes dystrophic calcification, which appears
to be a stimulus for dentin bridge formation.
When resins are incorporated into the compound, these calcium hydroxide
compounds become less irritating and are able to stimulate dentin bridge formation
more quickly than the Ca (OH)2 suspension alone. Significantly, this occurs with no
zone of necrosis, and reparative dentin is laid down adjacent to the liner.
Numerous investigators have analyzed the effects of applying thin liners such
as resin-based copal varnishes and polystyrenes under restorations. They may also
reduce penetration of bacteria or chemical substances for a time. However, because of
the thinness of the film and formation of pinpoint holes, the integrity of these
materials is not as reliable as that of other cavity liners applied in a greater thickness.
Zinc phosphate has been widely used as a cement for seating castings and
fixing orthodontic bands, and as a thermal insulating base under metallic dental
restorations, because the thermal conductivity of this cement is approximately equal
to that of enamel and is considerably less than that of metals. In vitro screening tests
indicate that zinc phosphate cement elicits strong-to-moderate cytotoxic reactions that
decrease with time. Leaching of zinc ions and a low pH may explain these effects.
The dilution of leached cement products by dentin filtration has been shown to protect
the pulp from most of these cytotoxic effects. Focal necrosis, observed in implantation
tests with zinc phosphate cements injected into rat pulp, confirms the cytotoxic effects
of this cement when it contacts pulp tissue. In usage tests in deep cavity preparations,
moderate-to-severe localized pulpal damage is produced within 3 days, probably
because of the initial low pH (4.2 at 3 minutes). However, the pH of the set cement
approaches neutrality after 48 hours. By 5 to 8 weeks, only mild chronic inflammation

is present, and reparative dentin has usually formed. Because of the initially painful
and damaging effects on the pulp by this cement when placed in deep cavity
preparations, the placement of a protective layer of a dentin-bonding agent, ZOE,
varnish, or calcium hydroxide, is recommended in preparations with minimal
remaining dentin covering the pulp.
Zinc polyacrylate cements (polycarboxylate cements) were developed as
biocompatible and cements chemically adhesive to tooth structure. In short-term
tissue culture tests, cytotoxicity of freshly set and completely set cements has
correlated with both the release of zinc and fluoride ions into the culture medium and
with a reduced pH. Some researchers suggest that this cytotoxicity is an artifact of
tissue culture because the phosphate buffers in the culture medium facilitate zinc ion
leaching from the cement. Supporting this theory, cell growth inhibition can be
reversed if EDTA, which chelates zinc, is added to the culture medium. Furthermore,
inhibition of cells decreases as the cement sets. The polymer component of the cement
may also be of concern, because concentrations of polyacrylic acid above 1% appear
to be cytotoxic in tissue culture tests. On the other hand, subcutaneous and bone
implant tests over a 1-year period have not indicated long-term cytotoxicity of these
cements. Thus, other mechanisms such as buffering and protein binding of these
materials may neutralize these effects in vivo over time. Polyacrylate cements evoke a
pulpal response similar to that caused by ZOE, with a slight-to-moderate response
after 3 days and only mild, chronic inflammation after 5 weeks. Reparative dentin
formation is minimal with these cements, and thus they are recommended only in
cavities with intact dentin in the floors of the cavity preparations.
ZOE cements have been used in dentistry for many years. In vitro, eugenol
from ZOE fixes cells, depresses cell respiration, and reduces nerve transmission with
direct contact. Surprisingly, it is relatively innocuous in usage tests with class 5 cavity
preparations. This is not contradictory for a number of reasons. The effects of eugenol
are dose dependent and diffusion through dentin dilutes eugenol by several orders of
magnitude. Thus, although the concentration of eugenol in the cavity preparations just
below the ZOE has been reported to be 10−2 M (bactericidal), the concentration on
the pulpal side of the dentin may be 10−4 M or less. This lower concentration
reportedly suppresses nerve transmission and inhibits synthesis of prostaglandins and
leukotrienes (anti-inflammatory). In addition, and as described before, ZOE may form

a temporary seal against bacterial invasion. In cavity preparations in primate teeth
(usage tests), ZOE caused only a slight to moderate inflammatory reaction within the
first week. This was reduced to a mild, chronic inflammatory reaction, with some
reparative dentin formation (within 5 to 8 weeks), when cavities were deep. For this
reason, it has been used as a negative control substance for comparison with
restorative procedures in usage tests.
Latex
Of particular interest in dentistry is the use of latex gloves and latex rubber
dams, which expose both patients and dental personnel to this potential allergen. In
the early 1980s, when HIV infection became a major safety issue, dental personnel
began to routinely wear gloves to reduce the risk of transmission. Since that time, the
incidence of latex hypersensitivity reactions has increased enormously. Natural latex
products are made from a white milky sap harvested from a tree growing in tropical
regions. Ammonia is added to the sap to preserve it, but at the same time, the
ammonia hydrolyzes and degrades the sap proteins to produce allergens.
Vulcanization is the process by which liquid latex is hardened into rubber through the
use of sulfur compounds and heat. These chemicals may be allergenic themselves and
are often present to some degree in the final product. The manufacturing process
leaches the allergens by soaking the rubber products in hot water. The leaching water
is changed repeatedly to decrease the concentration of the allergens, but leaching
brings other allergens to the surface and unfortunately places the highest
concentrations near the skin of the wearer. Thus, the allergenicity of a given batch of
latex will be dependent on how the latex was collected, preserved, and processed.
Synthetic latex is also available, but it produces the same problems except that
naturally occurring proteins and their degradation products are not present.
Oral Hygiene Products
Oral hygiene products are usually not classified as dental materials. Their use
is at least in part regulated by other laws, such as regulations regarding cosmetics. But

there is no clear borderline. Side effects due to oral hygiene products have been
observed that are similar to those caused by dental materials. Thus, such adverse
effects were often erroneously attributed to dental materials. In order to diagnose side
effects and identify their possible causes, information about adverse effects of oral
hygiene products can be very helpful.
Toothpastes and mouthwashes are the two most important oral hygiene
products available to patients. Because it has been shown that bacteria play a prime
role in the etiology of the major oral diseases, many of the so-called active ingredients
are directed toward the oral microflora or sequelae of their metabolism. Cleaning
stains from teeth requires an abrasive, and the overall sales success of a product may
depend on flavoring agents, which give a sensation of wellbeing, cleanliness, and
reduced fears of oral malodor. These substances influence the compatibility of a
product. A product’s biocompatibility may be altered by a change in any of these
substances – their addition or removal or merely an alteration in their concentration.
Other classes of substances contained in oral hygiene products include humectants,
homogenizers, preservatives, foaming agents, and so on. Nearly all ingredients are
potentially harmful, and the risk of harmful effects depends on concentration. Many
antimicrobial agents added to toothpastes and mouthwashes possess powerful
antimicrobial properties in concentrations that are used in other cosmetic products,
such as soap. But these concentrations may not be compatible with the oral
environment. At the same time, it is difficult to formulate products capable of
maintaining suitable concentrations of the active agents for a suitable period of time
because the fluids in the mouth are constantly replaced by new saliva and lost through
swallowing. This situation means that the effect can be sustained only by frequent use
of the product or by a high substantivity (adhesion to teeth and/or oral mucosa), which
in turn increases the risk of harmful effects of chronic exposure.
Topical fluoride applications with aqueous solutions with high fluoride
content have been used by health professionals for more than half a century. During
the last three decades, varnishes and gels containing fluoride have also been used for
topical fluoride application. The majority of studies on the effect of varnishes have
been done with Duraphat varnish, and studies on the caries-reducing effect of this
product have been subjected to a meta-analysis. Results of a meta-analysis of fluoride
gel studies have also recently been published. These meta-analyses do not suggest that

the effects of these two methods are superior or inferior to applications of aqueous
solutions of 2% sodium fluoride, which is in keeping with conclusions of earlier
reviews. Deleterious effects of these products have mainly been associated with the
fluoride content.
Thus, Dental health professionals should consider claims of health benefits of
oral hygiene products with sound scepticism and should continue to demand
documentation both of health benefits and adverse effects of these products. To avoid
unnecessary risks, potentially deleterious substances (e.g., alcohol) should not be
added at higher concentrations without a documented therapeutic effect.
Bleaching Agents
The use of bleaching agents on vital teeth has become commonplace. These
agents usually contain some form of peroxide (generally carbamide or hydrogen
peroxide) in a gel that can be applied to the teeth either by a dentist or at home by a
patient. In vitro studies have shown that peroxides can rapidly (within minutes)
traverse the dentin in sufficient concentrations to be cytotoxic. The cytotoxicity
depends to a large extent on the concentration of the peroxide in the bleaching agent.
Other studies have even shown that peroxides can rapidly penetrate intact enamel and
reach the pulp in a few minutes. In vivo studies have demonstrated adverse pulpal
effects from bleaching, and most reports agree that a legitimate concern exists about
the long-term use of these products on vital teeth. In clinical studies, the occurrence of
tooth sensitivity is very common with the use of these agents, although the cause of
these reactions is not known. Bleaching agents will also chemically burn the gingiva
if the agent is not sequestered adequately in the bleaching tray. This is not a problem
with a properly constructed tray, but long-term, low-dose effects of peroxides on the
gingival and periodontal tissues have not been completely elucidated.

CONCLUSION
During the past few years, the biocompatibility of dental materials has evolved into a
comprehensive, complex, and independent discipline of dental materials science. The
topic draws on knowledge from biology, patient risk factors, clinical experience, and
engineering. It is mandatory for the clinician to know and understand the

biocompatibility of dental materials, so as to provide maximum advantage and
minimum risk to the patient

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