BIOCOMPATIBILITY OF DENTAL MATERIALS

Presented by

Sharique Alam

History

HISTORY

• Hippocrates (460-377B.C)-developed the concept of ethical treatment of patients

• Concept of protecting patient is only 30-40 years old

• IRB oversee testing of new materials in humans.

• The oversight of testing materials in a systematic way now rests largely with FDA

• Also regulated by ANSI ,ADA and ISO

• HIPPOCRATIC OATH: MODERN VERSION• I swear to fulfill, to the best of my ability and judgment, this covenant:• I will respect the hard-won scientific gains of those physicians in whose steps I

walk, and gladly share such knowledge as is mine with those who are to follow.• I will apply, for the benefit of the sick, all measures [that] are required, avoiding

those twin traps of overtreatment and therapeutic nihilism.• I will remember that there is art to medicine as well as science, and that warmth,

sympathy, and understanding may outweigh the surgeon's knife or the chemist's drug.

• I will not be ashamed to say "I know not," nor will I fail to call in my colleagues when the skills of another are needed for a patient's recovery.

• I will respect the privacy of my patients, for their problems are not disclosed to me that the world may know. Most especially must I tread with care in matters of life and death. If it is given me to save a life, all thanks. But it may also be within my power to take a life; this awesome responsibility must be faced with great humbleness and awareness of my own frailty. Above all, I must not play at God.

• I will remember that I do not treat a fever chart, a cancerous growth, but a sick human being, whose illness may affect the person's family and economic stability. My responsibility includes these related problems, if I am to care adequately for the sick.

• I will prevent disease whenever I can, for prevention is preferable to cure.• I will remember that I remain a member of society, with special obligations to all

my fellow human beings, those sound of mind and body as well as the infirm.• If I do not violate this oath, may I enjoy life and art, respected while I live and

remembered with affection thereafter. May I always act so as to preserve the finest traditions of my calling and may I long experience the joy of healing those who seek my help.

• —Written in 1964 by Louis Lasagna, Academic Dean of the School of Medicine at Tufts University, and used in many medical schools today.

Definition

Definition

• Biocompatibility is formally defined as the ability of a material to elicit an appropriate biological response in a given application in the body.

• Implicit in this definition is that biocompatibility is an interaction between body and material –depends on condition of host, properties of material and context of use

• Inherent in this definition also is the idea that a single material may not be biologically acceptable in all applications.

Anatomical and pathological aspects of oral tissues

ENAMEL

• Mature human enamel is highly mineralized (96% by weight),

with only 1% of its weight being organic molecules and 3% being water.

• The permeability of enamel to most oral molecules is quite low, and in this sense enamel "seals" the tooth to outside agents. However, recent evidence indicates that enamel is not impermeable. Peroxides in bleaching agents have been shown to permeate intact enamel in just a few seconds.

DENTIN AND PULP

• Because of their close anatomical relationship, most researchers consider the dentin and pulp to be a single organ.

• The dentinal matrix (both calcified and uncalcified) forms the greatest bulk of the tooth. Calcified dentin is about 20% organic, 70% inorganic, and 10% aqueous by weight. Collagen constitutes approximately 85% of the organic portion of dentin, and hydroxyapatite is the main inorganic compound.

• The dentinal matrix surrounds dentinal tubules that are filled with the odontoblastic processes.

• A serum-like fluid fills the dentinal tubules. This fluid has continuity with the extracellular fluid of the pulp tissue. The pulpal circulation maintains an intercellular hydraulic pressure of about 24 mm Hg (32.5 cm H20)

• Moderately deep cavity preparation will damage odontoblasts by severing the odontoblastic processes. Deep cavity preparation may destroy most of the dentin and kill the primary odontoblasts.

• A number of investigators think that the extracellular matrix (ECM) of dentin and pulp is largely responsible for the differentiation of secondary odontoblasts that form reparative dentin.

• The source of secondary odontoblasts is not known, but much of the proliferative activity of granulation tissue following pulpal insult is found in perivascular areas proximal to the core of the pulp.

• Nerves and blood vessels, which arborize from the core of the pulp as they approach the odontoblastic layer,may influence the extent of the inflammatory response and the amount of new dentinal matrix formed during dentin repair.

• When toxic bacterial or chemical products traverse the dentin, odontoblasts and the pulpal connective tissue usually respond first by focal necrosis (0 to 12 hours), which may be followed by an acute, but more widespread, pulpitis (12 hours to several days). This response may resolve naturally if the injurious agent is removed or the tubules are blocked. If the pulpitis does not resolve, it may spread to more completely involve the pulp in liquefaction necrosis (especially if the pulpitis results from bacterial products) or in chronic inflammation

DENTIN PERMEABILITY

• Two types of dentin permeability occur-Convection and diffusion

• Convection of fluids across dentin varies with the fourth power of the radius of the dentinal tubule(r4), and thus very sensitive to the diameter of the tubules.

• Diffusion is proportional to the length of the dentinal tubules and thus ,roughly, to the thickness of the dentin between cavity preparation and the pulp

• Smear layers created in cavity preparation are better than cavity liners and sealers at limiting diffusional permeability

.• the capillary beds and vascular dynamics in most healthy pulps

are probably capable of removing relatively large amounts of cytotoxic chemicals and bacterial products once they diffuse through the dentin. However, if the pulp is already damaged (inflamed because of caries or trauma), edema and sluggish circulation probably compromise the removal of these materials.

BONE

• Bone is an extracellular matrix (ECM) with accompanying cells and tissue. The ECM of bone is a mineralized tissue composed of about 23% organic substances and about 77% hydroxyapatite.

• Osteoblasts die if surgical manipulation destroys the vascular supply or heats the bone above 45' C for more than a few minutes

Osseointegration and Biointegration

• Osseointegration is defined as the close approximation

of bone to an implant material

• To achieve osseointegration, the bone must be viable, the space between the bone and implant must be less than 100 A and contain no fibrous tissue, and the bone-implant interface must be able to survive loading by a dental prosthesis. In current practice, osseointegration is an absolute requirement for the successful implant-supported dental prosthesis.

• In recent years there has been a trend to coat titanium alloys with a calcium-phosphate ceramic to better promote an implant bone connection. If successful, the ceramic coating becomes completely fused with the surrounding bone. In this case, the interface is called biointegration rather than osseointegration and there is no intervening space between the bone and the implant .

• A number of ceramic coatings have been used in this manner, including tricalcium phosphate, hydroxyapatite, and Bioglass. The long-term integrity of the ceramic coating in vivo is not known, but some evidence indicates that these coatings will resorb over time.

PERIODONTIUM

• The periodontium is a combination of tissues, including the periodontal ligament (PDL), cementum, and alveolar bone. The cementum and alveolar bone are mineralized extracellular matrices with the associated cells that are responsible for generating and maintaining them.

GINGIVA AND MUCOSA

• The gingiva is a connective tissue with an epithelial surface that covers the alveolar ridge, surrounds the cervices of the teeth, and fills interproximal spaces between teeth.

• The free gingiva fuses with the attachment epithelium, which surrounds the tooth at its cervix in the young, healthy tooth. Some of the crevicular epithelium and all the attachment epithelium, at least in the young individual, are derived embryologically from reduced enamel epithelium.

• The gingival response to injury may be complicated by its association with the tooth. Calculus deposition on the tooth, malocclusion, and faulty restorations may enhance the destructive effects of microorganisms. The crevicular epithelium then becomes vulnerable to endotoxin and various exogenous and endogenous chemicals.

• The resulting breakdown of tissue leads to an acute inflammatory response by the gingival connective tissue called acute gingivitis. This condition is often reversible if the injurious agent is removed and the reaction is limited to the connective tissue above the alveolar bony crest.

• If the insult continues, the inflammatory infiltrate becomes mixed and then predominantly mononuclear. Inflamed epithelial-lined granulation tissue gradually spreads apically to and below the alveolar bony crest. At this point the condition is described as chronic periodontal disease, a progressive disease process that is probably reinforced by immune mechanisms. The relationship between periodontal disease and dental materials that are in close contact with the gingiva is not known but is an active area of research.

• The oral mucosa is composed of a loose fibro elastic connective tissue with a well-vascularized and innervated lamina propria and submucosa and is covered mainly by a parakeratinized stratified squamous epithelium.

• The oral mucous membrane can be injured chemically or physically by dental materials. If the injury is short-term (acute) and leads to loss of tissue but does not involve infection by pathogenic microorganisms, the connective tissue defect is filled in with granulation tissue within 3 to 4 days and epithelium regenerates over the surface within a week. The tissue is remodeled to nearly normal by the end of 2 to 3 weeks.

MEASURING BIOCOMPATIBILTY

MEASURING BIOCOMPATIILITY

• Historically, new materials were simply tried in humans to see if they were biocompatible

• Current materials must be extensively screened for biocompatibility before they are ever used in humans.

• These tests are classified as in vitro, animal, and usage tests.

• These three testing types include the clinical trial, which is really a special case of a usage test in humans.

1)IN VITRO TESTS

• In vitro tests for biocompatibility are done in a test tube, cell-culture dish, or otherwise outside of a living organism.

• These tests 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, where the material contacts the cell system without barriers, or indirect, where 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 the effect on the genetic material in a cell (mutagenesis assays).

ADVANTAGES AND DISADVANTAGES OF BIOCOMPATIBILITY TESTS

• Standardization of in vitro tests is a primary concern of those trying to evaluate materials.

• Two types of cells can be used for in vitro assays.• Primary cells are cells taken directly from an animal into culture.

These cells will grow for only a limited time in culture but may retain many of the characteristics of cells in vivo.

• Continuous cells are primary cells that have been transformed to allow them to grow more or less indefinitely in culture. Because of their transformation, these cells may not retain all in vivo characteristics, but they consistently exhibit any features that they do retain.

• Primary cell cultures would seemingly be more relevant than continuous cell lines for measuring cytotoxicity of materials.

• However, primary cells have the problems of being from a

single individual, possibly harboring viral or bacterial agents that alter their behavior, and often rapidly losing their in vivo functionality once placed in a cell culture.

• Furthermore, the genetic and metabolic stability of continuous cell lines contributes significantly toward standardizing assay methods.

• In the end, both primary and continuous cells play an important role in in vitro testing; both should be used to assess a material

A)Cytotoxicity Tests

• Cytotoxicity tests assess the cytotoxicity of a material by measuring cell number or growth after exposure to a material.

• Cells are plated in a well of a cell-culture dish where they attach. The material is then placed in the test system. If the material is not cytotoxic, the cells will remain attached to the well and will proliferate with time. If the material is cytotoxic, the cells may stop growing, exhibit cytopathic features or detach from the well.

• If the material is a solid, then the density (number of cells per unit area) of cells may be assessed at different distances from the material, and a "zone" of inhibited cell growth may be described . Cell density can be assessed either qualitatively, semi-quantitatively, or quantitatively.

Substances such as Teflon can be used as negative (non-cytotoxic) controls, whereas materials such as plasticized polyvinyl chloride can be used as positive (cytotoxic) controls. Control materials should be well defined and commercially available to facilitate comparisons among testing laboratories.

• Another group of tests is used to measure cytotoxicity by a change in membrane permeability.

• Membrane permeability is the ease with which a dye can pass through a cell membrane. This test is used on the basis that a loss in membrane permeability is equivalent to or very nearly equivalent to cell death.

• The advantage of the membrane permeability test is that it identifies cells that are alive (or dead) under the microscope. This feature is important because it is possible for cells to be physically present, but dead (when materials fix the cells)

• There are two basic types of dyes used.

• Vital dyes are actively transported into viable cells, where they are retained unless cytotoxic effects increase the permeability of the membrane. It is important to establish that the dye itself does not exhibit cytoxicity during the time frame of the test.

• Nonvital dyes are not actively transported, and are only taken up if membrane permeability has been compromised by cytotoxicity.

• Many types of vital dyes have been used, including neutral red and Na2

51CrO4.The use of neutral red andNa251CrO4 are particularly

advantageous because they are neither synthesized nor metabolized by the cell.

• Examples of nonvital dyes include trypan blue and propidium iodide.

B)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 deoxyribonucleic acid (DNA) synthesis or protein synthesis are common examples of this type of test. The synthesis of DNA or protein by cells is usually analyzed by adding radioisotope labeled precursors to the medium and quantifying the radioisotope (e.g., 3thymidine or 3leucine) incorporated into DNA or protein.

• A commonly used enzymatic test for cytotoxicity is the MTT test

• Recently in vitro tests to measure gene activation, gene expression, cellular oxidative stress, and other specific cell functions have been proposed.

The production of formazan can be quantified by dissolving it and measuring the optical density of the resulting solution. Alternatively, the formazan can be localized around the test sample by light or electron microscopy. Other formazan-generating chemicals have been used, including NBT, XTT, and WST.

C)Tests That Use Barriers (Indirect Tests)

• Researchers have long recognized that in vivo, direct contact often does not exist between cells and the materials. Separation of cells and materials may occur from keratinized epithelium, dentin, or extracellular matrix.

• Several in vitro barrier tests have been developed to mimic in vivo conditions. One such test is the agar overlay method

This assay correlates positively with the direct-contact assays and the intramuscular implantation test in rabbits. However, the agar may not adequately represent barriers that occur in vivo. Furthermore, because of variability of the agar's diffusion properties, it is difficult to correlate the intensity of color or width of the zone around a material with the concentration of leachable toxic products.

• A second barrier assay is the Millipore filter assay.

• Dentin barrier tests have shown improved correlation with the cytotoxicity of dental materials in usage tests in teeth, and are gradually being developed for screening purposes

D)Other Assays for Cell Function

• In vitro assays to measure immune function or other tissue reactions have also been used. The in vivo significance of these assays is yet to be ascertained

• 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 activation of complement is of particular concern to researchers working on artificial or "engineered" blood vessels and other tissues in direct contact with blood. Materials that activate complement may generate inflammation or thrombi, and may propagate a chronic inflammatory response.

E)Mutagenesis Assays• Mutagenesis assays assess the effect of materials on a cell's

genetic material.

• Genotoxic mutagens directly alter the DNA of the cell 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 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 the quantification and relevance of tests that attempt to measure mutagenesis and carcinogenesis are extremely complex

• The Ames' test is the most widely used mutagenesis test and the only short-term test that is considered thoroughly validated.

• It uses mutant stocks of Salmonella typhimurium that require exogenous histidine. Native stocks of bacteria do not require exogenous histidine.

• Exclusion of histidine from the culture medium allows a chemical to be tested for its ability to convert the mutant strain to a native strain

Ames Test

• A second test for mutagenesis is the Styles' Cell Transformation test. This test on mammalian cells was developed to offer an alternative to bacterial tests (Ames test), which may not be relevant to mammalian systems.

• This assay quantifies the ability of potential carcinogens to transform standardized cell lines so they will grow in soft agar.

• Untransformed fibroblasts normally will not grow within an agar gel, whereas genetically transformed cells will grow below the gel surface. This characteristic of transformed fibroblasts is the only characteristic that correlates with the ability of cells to produce tumors in vivo. At least four different continuous cell lines (Chang, BHK, HeLa, WI-38) have been used.

COMPARISION OF MUTAGENIC TESTS

These studies suggest that not all carcinogens are genotoxic (mutagenic) and not all mutagens are carcinogenic.

11)ANIMAL TESTS• Animal tests for biocompatibility are usually used in

mammals such as mice, rats, hamsters, or guinea pigs

• Animal tests 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 many complex interactions between the material and a functioning, complete biological system to occur. For example, an immune response may occur or complement may be activated in an animal system in a way that would be 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

• The mucous membrane irritation test determines if a material causes inflammation to mucous membranes or abraded skin.

• This test is conducted by placing the test materials and positive and negative controls into contact with hamster cheek-pouch tissue or rabbit oral tissue.

• After several weeks of contact, the controls and test sites are examined, and the gross tissue reactions in the living animals are recorded and photographed in color. The animals are then sacrificed, and biopsy specimens are prepared for histological evaluation of inflammatory changes.

• In the skin sensitization test in guinea pigs(guinea pig maximization test), the materials are injected intradermally to test for development of skin hypersensitivity reactions. Freund's adjuvant can be used to augment the reaction. This injection is followed by secondary treatment with adhesive patches containing the test substance.

• If hypersensitivity developed from the initial injection, the patch will elicit an inflammatory response.

• The skin-patch test can result in a spectrum from no reaction to intense redness and swelling. The degree of reaction in the patch test and the percentage of animals that show a reaction are the bases for estimating the allergenicity of the material.

• Animal tests that measure the mutagenic and carcinogenic properties of materials have been developed by toxicologists.

• These tests are employed with a strategy called the decision-point approach.

• Using this strategy, tests are applied in a specific order, and testing is stopped when any one indicates mutagenic potential of the material or chemical.

• The validity of any of these tests may be affected by issues of species, tissue, gender, and other factors. Tests are generally divided into limited-term in vivo tests and long-term or lifetime tests.

• Limited-term in vivo tests measure altered liver function or increased tumor induction when animals are exposed to the chemicals for a fraction of their lifetimes. Long-term in vivo tests are performed by keeping the chemical in contact with the animal over the majority of its lifetime.

• Implantation tests are used to evaluate materials that will contact subcutaneous tissue or bone. The location of the implant site is determined by the use of the material, and may include connective tissue, bone, or muscle.

• Although amalgams and alloys are tested because the margins of the restorative materials contact the gingiva, most subcutaneous tests are used for materials that will directly contact soft tissue during implantation, endodontic, or periodontal treatment.

• Implantation tests of longer duration, for identification of either chronic inflammation or tumor formation, are performed in a manner similar to that of short-term tests except the materials remain in place for 1 to 2 years before examination.

111)USAGE TESTS

• Usage tests may be done in animals or in human volunteers. 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 of a usage test for predicting biocompatibility is directly proportional to the fidelity with which the test mimics the clinical use of the material in every regard, 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 or monkeys. If humans are used, the usage test is identical to a clinical trial.

• These tests are the gold standard of tests in that they give the ultimate answer to whether a material will be biocompatible

Dental Pulp Irritation Tests

• Generally, materials to be tested on the dental pulp are placed in class-5 cavity preparations in intact, noncarious teeth of monkeys or other suitable animals.

• The compounds are placed in an equal number of anterior and posterior teeth of the maxilla and mandible to ensure uniform distribution in all types of teeth. The materials are left in place from 1 to 8 weeks. Zinc oxide-eugenol and silicate cement have been used as negative and positive control materials, respectively.

• At the conclusion of the study, the teeth are removed and sectioned for microscopic examination.

• The thicknesses of the remaining dentin and reparative dentin for each histological specimen is measured with a photomicrometer and recorded. The response of the pulp is evaluated based on its appearance after treatment.

• Until recently, most dental-pulp irritation tests have involved intact, noncarious teeth, without inflamed pulps. There has been increased concern that inflamed dental pulp tissue may respond differently than normal pulps to liners, cements, and restorative agents.

• Usage tests that study teeth with induced pulpitis allows evaluation of types and amount of reparative dentin formed.

Dental Implants into Bone

At present, the best estimations of the success and failure of

implants are gained from three tests:

(1) penetration of a periodontal probe along the side of the implant

(2) mobility of the implant,

(3) radiographs indicating either osseous integration or radiolucency around the implant

Mucosa and Gingival Usage Tests

• Because various dental materials contact gingival and mucosal

tissues, the tissue response to these materials must be measured.

• Materials are placed in cavity preparations with subgingival extensions.

• The materials' effects on gingival tissues are observed at 7 days and again after 30 days.

• Responses are categorized as slight, moderate, or severe. A slight response is characterized by a few mononuclear inflammatory cells (mainly lymphocytes) in the epithelium and adjacent connective tissue.

• A moderate response is indicated by numerous mononuclear cells in the connective tissue and a few neutrophils in the epithelium.

• A severe reaction evokes a significant mononuclear and neutrophilic infiltrate and thinned or absent epithelium.

• A difficulty with this type of study is the frequent presence of some degree of preexisting inflammation in gingival tissue. Bacterial plaque is the most important factor in causing this inflammation.

• Secondary factors are the surface roughness of the restorative material, open or overhanging margins, and overcontouring or undercontouring of the restoration. One way to reduce the interference of inflammation caused by plaque is to perform dental prophylaxis before preparing the cavity and placing the material.

• However, the prophylaxis and cavity preparation will themselves cause some inflammation of the soft tissues. Thus, if margins are placed subgingivally, time for healing (typically 8 to 14 days) must be allowed before assessing the effects of the restorative agents.

CORRELATION BETWEEN TESTS

CORRELATION AMONG IN VITRO, ANIMAL, AND USAGE TESTS

• In vitro and animal tests often measure aspects of the biological response that are more subtle or less prominent than in a material's clinical usage.

• Barriers between the material and tissues may exist in usage tests or clinical use that may not exist in in vitro or animal tests.

• It is important to remember that each type of test has been designed to measure different aspects of the biological response to materials, and correlation may not always be expected.

• The best example of a barrier that occurs in use but not in vitro is the dentin barrier.

• The effect of the dentin barrier is illustrated by the following classic study

Comparision reactions of 3 materials by screening and usage tests

USING IN VITRO, ANIMAL, AND USAGE TESTS TOGETHER

• Scientists, industry, and the government have recognized that the most accurate and cost-effective means to assess the biocompatibility of a new material is a combination of in vitro, animal,and usage tests.

• Implicit in this philosophy is the idea that no single test will be adequate to completely characterize the biocompatibility of a material. The ways by which these tests are used together, however, are controversial and have evolved over the years as knowledge has increased and new technologies developed

• Two newer testing schemes have evolved in the past few years with regard to using combinations of biocompatibility tests to evaluate materials

• Both of these newer schemes accommodate several important ideas. • First, all tests (in vitro, animal, and usage) continue to be of value in

assessing the biocompatibility of a material during its development and even in its clinical service. For example, tests in animals for inflammation may be useful during the development of a material, but may also be useful after a problem is noted with the material after it has been on the market for a time.

• Second, the newer schemes recognize the inability of current testing methods to accurately and absolutely screen in or out a material.

• Third, these newer schemes incorporate the philosophy that assessing the biocompatibility of a material is an ongoing process.

STANDARDS THAT REGULATE THE MEASUREMENT OF BlOCOMPATlBlLlTY

• The first efforts of the ADA to establish guidelines for dental materials came in 1926 when scientists at the National Bureau of Standards, now the National Institute of Science and Technology, developed specifications for dental amalgam.

• One of the early attempts to develop a uniform test for all materials was the study by Dixon and Rickert in 1933, in which the toxicity of most dental materials in use at that time was investigated by implanting the materials into pockets in subdermal tissue.

• Other early attempts to standardize techniques were carried out by Mitchell (1959) on connective tissue and by Massler (1958) on 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 Council on Dental Materials, Instruments, and Equipment of ANSI/ADA approved Document No. 41 for Recommended Standard Practices for Biological Evaluation of Dental Materials

ANSI/ADA Document 41

• Three categories of tests are described in the 1982 ANSI/ ADA document: initial, secondary, and usage tests.

• The initial tests include in vitro assays for cytotoxicity, red blood cell membrane lysis (hemolysis), mutagenesis and carcinogenesis at the cellular level, and in vivo acute physiological distress and death at the level of the whole organism.

• Based on the results of these initial tests, promising materials are tested by one or more secondary tests in small animals (in vivo) for inflammatory or immunogenic potential (e.g., dermal irritation, subcutaneous and bony implantation, and hypersensitivity tests).

• Finally, materials that pass secondary tests and still hold potential are subjected to one or more in vivo usage tests

IS0 10993 • In the past decade, an international effort was initiated by several

standards organizations to develop international standards for biomedical materials and devices.

• Several multinational working groups, including scientists from ANSI and the International Standards Organization (ISO) were formed to develop these standards.

• The final document (IS0 10993) was published in 1992 and is the most recent standard available for biological testing. IS0 10993 contains 12 parts, each dealing with a different aspect of biological testing.

• 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 nonusage situations. Supplementary tests are tests such as chronic toxicity, carcinogenicity, and biodegradation. Most of the supplementary tests are done in animal systems, many in usage situations.

• Unlike the IS0 standard, which covers all biomedical devices, the ANSI/ADA document is limited to dental devices

BlOCOMPATlBlLlTY OF DENTAL MATERIALS

• REACTIONS OF PULP• Microleakage

• If a bond does not form or debonding occurs, bacteria, food debris, or saliva may be drawn into the gap between the restoration and the tooth by capillary action. This effect has been termed microleakage

• Recently, the new concept of nanoleakage hasbeen put forward.

• Nanoleakage refers to the leakage of saliva, bacteria, or material components through the interface between a material and tooth structure. However, nanoleakage refers specifically to dentin bonding, and may occur between mineralized dentin and a bonded material in the very small spaces of demineralized collagen matrix into which the bonded material did not penetrate.

• it is thought to play at least some role and it is suspected of contributing to the hydrolytic degradation of the dentin-material bond, leading ultimately much more serious microleakage.

Dentin Bonding• Because the dentinal tubules and their resident

odontoblasts are extensions of the pulp, bonding to dentin also involves biocompatibility issues.

• From the standpoint of biocompatibility, the removal of the smear layer may pose a threat to the pulpal tissues for three reasons.

• First, its removal juxtaposes resin materials and dentin without a barrier.

• Second, the removal of the smear layer makes any microleakage more significant because a significant barrier to the diffusion of bacteria or bacterial products toward the pulp is removed.

• Third, the acids used to remove the smear layer are a potential source of irritation themselves.

• The biocompatibility of acids used to remove the smear layer has been extensively studied. Numerous acids have been used to remove the smear layer, including phosphoric, hydrochloric, citric, and lactic acids. The effect of the acids on pulpal tissues depends on a number of factors, including the thickness of dentin between the restoration and the pulp, the strength of the acid, and the degree of etching. Most studies have shown that dentin is a very efficient buffer of protons, and most of the acid may never reach the pulp if sufficient dentin remains.

• A dentin thickness of 0.5 mm has proven adequate in this regard. Citric or lactic acids are less well buffered, probably because these weaker acids do not dissociate as efficiently.

• Usage tests that have studied the effects of acids have shown that phosphoric, pyruvic, and citric acids produce moderate pulpal inflammatory responses, which resolve after 8 weeks. Recent research has shown that in most cases the penetration of acids into the dentin is probably less than 100 µm.

• Dentin Bonding Agents

• Many of these reagents are cytotoxic to cells in vitro if tested alone.

• However, when placed on dentin and rinsed with tap water between applications of subsequent reagents as prescribed, cytotoxicity is often reduced.

• Longer-term in vitro studies suggest, however, that sufficient components of many bonding agents permeate up to0.5 mm of dentin to cause significant suppression of cellular metabolism for up to 4 weeks after their application. This suggests that residual unbound reagents may cause adverse reactions.

• In 1996 Olea et al claimed that dental sealants released estrogenic substances

• The concern about estrogen centred around a chemical called bisphenol A(BPA)

• One common test used to assess xenoestrogenic activity is called E Screen Assay

• This in vitro test relies on growth response of breast cancer cells that are estrogen sensitive to purported estrogenic compounds

Resin-Based Materials

• In vitro, freshly set chemically cured and light-cured resins often cause moderate cytotoxic reactions in cultured cells over 24 to 72 hours of exposure

• The cytotoxicity is significantly reduced 24 to 48 hours after setting and by the presence of a dentin barrier

• In all cases, cytotoxicity is thought to be mediated by resin components released from the materials

• The primary risks for these materials appear to be allergy

• The allergenicity of methylmethacrylate is well documented and the use of gloves is not effective in preventing contact because most monomers pass easily through gloves

• The allergic reactions are primarily contact dermatitis

Amalgams

• The biocompatibility of amalgams is thought determined largely by corrosion products released while in service. Corrosion depends on the type of amalgam, whether it contains the γ2, phase, and the composition of the amalgam.

• In usage tests, the response of the pulp to amalgam in shallow cavities or in deeper but lined cavities is minimal. In deep cavities (0.5 mm or less of remaining dentin), pain results from using amalgams in unlined cavity preparations.

• An inflammatory response is seen after 3 days and after 5 weeks. In cavities with 0.5 to 1.0 mm of dentin relining in the floor, the cavity preparation should be lined for two other reasons.

• First, thermal conductivity with amalgam is significant and can be a problem clinically.

• Second, margins of newly placed amalgam restorations show significant microleakage. Marginal leakage of corrosion and microbial products is probably enhanced by the daily natural thermal cycle in the oral cavity. The short-term pulpal response is significantly reduced when the cavity is lined, and amalgam rarely causes irreversible damage to the pulp. Long-term sealing of the margins occurs through the buildup of corrosion

• Amalgams based on gallium rather than mercury have been developed to provide direct restorative materials that are free of mercury

• Clinically, these materials show much higher corrosion rates than standard amalgams, leading to roughness and discoloration. There are few reports of pulpal responses to these materials

Glass Ionomers• Light-cured ionomer systems have also been introduced; these

systems use Bis-GMA or other oligomers as pendant chains on the polyacrylate main chain. In screening tests, freshly prepared ionomer is mildly cytotoxic, but this effect is reduced with increased times after setting.

• The fluoride release from these materials, which is probably of some therapeutic value, may cause cytotoxicity in in vitro tests

• The overall pulpal biocompatibility of these materials has been attributed to the weak nature of the polyacrylic acid, which is unable to diffuse through dentin because of its high molecular weight. In usage tests the pulp reaction to glass ionomer cements is mild.

Liners, Varnishes, and Nonresin Cements

• Calcium hydroxide cavity liners come in many forms, ranging from saline suspensions with a very alkaline pH (above 12) to modified forms containing zinc oxide, titanium dioxide, and resins.

• 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. The inhibition of cell metabolism is reversible in tissue culture by high levels of serum proteins, suggesting that protein binding or buffering in inflamed pulpal tissue may play an important role in detoxifying these materials in vivo.

• The initial response of exposed pulpal tissue to the 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 subnecrotic zone. Eventually, after 5 to 8 weeks, only a slight inflammatory response remains.

• Within weeks to months, the necrotic zone undergoes dystrophic calcification, which appears to be a stimulus for dentin bridge formation.

Conclusion

Conclusion• The biocompatibility of a dental material depends on its

composition, location, and interactions with the oral cavity. Metal, ceramic, and polymer materials elicit different biological responses because of their differences in composition.

• Furthermore, diverse biological responses to these materials depend on whether they release their components and whether those components are toxic, immunogenic, or mutagenic at the released concentrations.

• The paradigm of evaluating material biocompatibility needs to be meticulously assessed and informed consent and continual evaluation and reevaluation of materials must be done