Сraig. Dental Materials

are a combination of organic and inorganic phases, these materials are called resin composites. 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, although several newer systems seem to have minimal toxicity. The cytotoxicity is significantly reduced 24 to 48 hours after setting and by the presence of a dentin barrier. Several studies have shown that some materials are persistently cytotoxic in vitro even up to 4 weeks, whereas others gradually improve, and a few newer systems show little toxicity even initially. In all cases, cytotoxicity is thought to be mediated by resin components released from the materials. Evidence indicates that the light-cured resins are less cytotoxic than chemically cured systems, but this effect is highly dependent on the curing efficiency of the light and the type of resin system. In vivo, usage tests have been used to assess the biological response to resin composites. The pulpal inflammatory response to hemically cured and light-cured resin composites

is low to moderate after 3 days when they were placed in cavities with approximately 0.5 mm of remaining dentin. Any reaction diminished as the postoperative periods increased to 5 to 8 weeks and was accompanied by an increase in reparative dentin (Fig. 5-16). With a protective liner or a bonding agent, the reaction of the pulp to resin composite materials is minimal. The longer-term effects of resins placed directly on pulpal tissue are not known, but are suspected to be less favorable.

Amalgams and Casting Alloys Amalgams have been used extensively for dental restorations. 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 y, phase, and the composition of the amalgam. In cell-culture screening tests, free or unreacted mercury from amalgam is toxic, but low-copper amalgam that has set for 24 hours does not inhibit cell growth. With the addition of copper, amal-

152 Chapter 5 BIOCOMPATIBILITYOF DENTAL MATERIALS

gams become toxic to cells in culture. Implantation tests show that low-copper amalgams are well tolerated, but high-copper amalgams cause severe reactions when in direct contact with tissue. 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 relnaining 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 products.

A number of high-copper amalgams are in clinical use. Usage tests reported that at 3 days the pulpal responses elicited by these materials appear similar to those elicited by low-copper amalgams in deep, unlined cavities. At 5 weeks they elicited only slight pulpal response. At 8 weeks the inflammatory response was reduced. Bacterial tests on the high-copper amalgam pellets have revealed little inhibitory effect on serotypes of Streptococcus mutans, thus suggesting that the elements were not released in amounts necessary to kill these microorganisms. Although the high-copper amalgams seem biologically acceptable in usage tests, liners are suggested for all deep cavities.

Amalgams based on gallium rather than mercury have been developed to provide direct restorative materials that are free of mercury, although their use is not common. In cell culture, these alloys appear to be no more cytotoxic than traditional high-copper amalgams. These restorations release significant amounts of gallium

in vitro, but the importance of this release is not known. In implantation tests, gallium alloys caused a significant foreign body reaction. 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.

Cast alloys have been used for single restorations, bridges, porcelain-fused-to-metal crowns, and partial dentures. The gold content in these alloys ranges from 0% to 85% (percentage by weight). These alloys contain several other noble and non-noble metals that may have an adverse effect on cells if they are released from the alloys. However, released metals are most likely to contact gingival and mucosal tissues, and the pulp is more likely to be affected by the cement retaining the restoration.

Glass Ionomers Glass ionomers are another type of material that have been used both as a cement (luting agent) and as a restorative material. 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. Some researchers have reported that some systems are more cytotoxic than others, but the reasons for this observation are not clear. 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. Histological studies in usage tests show that any inflammatory infiltrate from ionomer is minimal or absent after 1 month. 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.

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. Resin-containing preparations can be polymerized chemically, but lightactivated systems have also been introduced. 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. When resins are incorporated into the formulae, these calcium hydroxide compounds become less irritating and are able to stimulate reparative dentin bridge formation more quickly than the Ca(OH), suspensions, and with no zone of necrosis. Therefore reparative dentin is laid down adjacent to the liner (Fig. 5-17), an indication that the replacement odontoblasts form the dentin bridge in contact with the liner. However, some of these materials evidently break down with time and create a gap between the restoration and the cavity wall. Resin-containing calcium hydroxide pulp capping agents are the most effective liners now available for treating pulp exposures. After pulp exposure, the uninfected pulp undergoes a relatively uncomplicated wound-healing process.

Numerous investigators have analyzed the ef-

Fig. 5-17 Light micrograph of a dentin bridge that has formed between a material and the pulp in a monkey. Initially, the pulp of the tooth was purposely exposed (top right) with a bur. The exposure was covered with a calcium hydroxide pulp-capping agent for 5 weeks before histological evaluation. A layer of secondary dentin has formed at the site of the pulp exposure, forming a dentin bridge. Some inflammatory cells are evident under the bridge, but the pulpal response is generally favorable.

(Courtesy Heys DR: Ann Arbor, 1987, University of Michigan School of Dentistry.)

fects of applying thin liners such as copal varnishes and polystyrenes under restorations. These materials are not generally used under resin-based materials, because resin components dissolve the thin film of varnish. Because liners are used in such thin layers, they do not provide thermal insulation, but they initially isolate the

dentinal tubule contents from the cavity preparation. 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.

Zincphosphate has been a widely used dental cement for castings, orthodontic bands and to base cavity preparations. Because the thermal conductivity of this cement is approximately equal to that of enamel and considerably less than that of metals, it has also been used to build up the remaining tooth structure. In vitro screening tests indicate that zinc-phosphate cement elicits strong-to-moderate cytotoxic reactions that decrease with increased time after setting. 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, confirm the cytotoxic effects of this cement where it contacts tissue. In usage tests in deep cavity preparations, moderate-to-severe localized pulpal damage is produced within the first 3 days, probably because of the initial low pH on setting (4.2 at 3 minutes). 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 of 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 under the cement. Other formulae have included calcium hydroxide in the powder, lowering the concentrations of phosphoric acid in the liquid, or included materials such as copper and fluoride ions that may function as antimicrobial agents. However, copper ions have proven extremely toxic to cells in culture and in implantation tests.

Zinc polyac ylate cements (polycarboxylate cements) were developed to combine the strength of zinc phosphate cements with the adhesiveness and biocompatibility of zinc oxide

eugenol (ZOE). In short-term tissue culture tests, cytotoxicity of freshly set and completely set cements has correlated both with the release of zinc and fluorides 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 encourage zinc ions to leach 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. In addition, 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-yearperiod 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. Polyacrylic cements cause 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.

Zinc oxide eugenol cements (ZOE) 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 in 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 M (bactericidal), the concentration on the pulpal side of the dentin may be lo4 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 inflamma-

tory reaction within the first week, and this was reduced to a mild, chronic inflammatory reaction, with some reparative dentin formation when cavities were deep, within 5 to 8 weeks. For this reason, it has been used as a negative control substance for comparison with restorative procedures in usage tests.

Bleaching Agents Bleaching agents have been used on nonvital and vital teeth for many years, but their use on vital teeth has increased astronomically in recent years. These agents usually contain some form of peroxide (generally carbamide peroxide) in a gel that can be applied to the teeth either by the dentist or at home by the patient. The agents may be in contact with teeth for several minutes to several hours depending on the formulation of the material. Home bleaching agents may be applied for weeks to even months in some cases. 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 further shown that the peroxides rapidly even 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 are not known. Bleaching agents will also chemically burn the gingiva if the agent is not sequestered adequately in the bleaching tray, although this is not a problem with a properly constructed tray. Long-term, low-dose effects of peroxides on the gingival and periodontal tissues are not currently known.

REACTION OF OTHER ORAL SOFT TISSUES TO RESTORATIVE MATERIALS

Restorative materials may cause reactions in the oral soft tissues such as gingiva. At present, it is not clear how much of the in vivo cytotoxicity

observed is caused by the restorative materials and how much is caused by products of bacterial plaque that accumulate on teeth and restorations. In general, conditions that promote retention of plaque, such as rough surfaces or open margins, increase inflammatory reactions in gingiva around these materials. However, released products of restorative materials also contribute either directly or indirectly to this inflammation, particularly in areas where the washing effects of saliva are less, such as in interproximal areas, in deep gingival pockets, or under removable appliances. Several studies have documented increased inflammation or recession of gingiva adjacent to restorations where plaque indexes are low. In these studies, released products from materials could cause inflammation in the absence of plaque or could inhibit formation of plaque and cause inflammation in gingiva. Some basic research has been done in vitro that shows, in principle, that components from dental materials and plaque may synergize to enhance inflammatory reactions.

Cements exhibit some cytotoxicity in the freshly set state, but this decreases substantially with time. The buffering and protein-binding effects of saliva appear to mitigate against the cytotoxic effects.

Composites are initially very cytotoxic in in vitro tests of direct contact with fibroblasts. The cytotoxicity is most probably primarily from unpolymerized components in the air-inhibited layer that leach out from the materials. Other in vitro studies, which have 'aged' the composites in artificial saliva for up to six weeks, have shown that the toxicity diminishes in some materials but remains high for others. Some of the newer composites with non-BisGMA non-UDMA matrices have significantly lower cytotoxicity in vitro, presumably because of lower amounts of leached components. Polished composites show markedly less cytotoxicity in vitro, although some materials are persistently toxic even in the polished state. Recently, there has been significant controversy about the ability of bis-phenol A and bis-phenol A dimethacrylate to cause estrogen-like responses in vitro. These compounds are basic components of many commer-

cia1 composites. However, there is no evidence that xenoestrogenic effects are a concern in vivo from any commercially available resin. Relatively little is known about other in vivo effects of released components of composites on the soft tissues, although the concerns are similar to those regarding denture base resin and soft liners (see later discussion, this section). There is some evidence that methacrylate-based composite components may cause significant rates of hypersensitivity, although few clinical trials exist.

Amalgam restorations carried into the gingival crevice may cause inflammation of the gingiva because of products of corrosion or bacterial plaque. Seven days after placing an amalgam, a few inflammatory cells appear in the gingival connective tissue, and hydropic degeneration of some epithelial cells may be seen. Some proliferation of epithelial cells into the connective tissue may also occur by 30 days, and chronic mononuclear cell infiltration of connective tissue is evident. Increased vascularity persists, with more epithelial cells invaginating into the connective tissue. Some of these changes may be a chronic response of gingiva to plaque on the margins of the amalgams. Nevertheless, corrosion products from amalgam cannot be ruled out at this time because implanted amalgams produce similar responses in connective tissues in animals. In addition, although copper enhances the physical properties of amalgam and is bactericidal, it is also toxic to host cells and causes severe tissue reactions in implantation tests. Animal implantation studies have also shown severe reactions to gallium-based alloys that have been used as amalgam replacements.

There is a report in the literature in which amalgam and resin composite restorations were placed in cavity preparations in monkey central incisors that had been extracted for less than one hour. The cavities, with depths of about 2 mm, were placed halfway between the cementoenamel junction and the root tip. The teeth were immediately reimplanted after restoration, and the animals were sacrificed at intervals up to 6 months. Repair of the PDL took place in a normal fashion except for the presence of an intense inflammatory infiltrate in the PDL adja-

cent to the amalgams through 2 weeks, but to the resin composites through 3 to 6 months. This result suggests that resin composites and amalgam release cytotoxic materials that cause tissue responses, at least at sites of implantation. For materials placed where they are rinsed in saliva, these cytotoxic agents are probably washed away before they harm the gingiva. However, rough surfaces on these types of restorations have been associated with increased inflammation in vivo. Usage tests in which restorations were extended into the gingival crevice have shown that finished materials gave a much milder inflammatory response than unfinished materials. The detrimental effect of surface roughness has been attributed to the increased plaque retention on these surfaces. However, rough surfaces on alloy restorations also caused increased cytotoxic effects in in vitro experiments where plaque was absent. This and other in vitro studies again would suggest that the cytotoxic response to alloys may be associated with release of elements from the alloys. The increased surface area of a rough surface may enhance release of these elements.

Casting alloys have a long history of in vivo use with a generally good record of biocompatibility. Some questions about the biological liability of elemental release from many of the formulations developed in the past 10 years have arisen, but there is no clinical evidence that elemental release is a problem, aside from hypersensitivity. Nickel allergy is a relatively common problem, occurring in 10% to 20% of females, and is a significant risk from nickel-based alloys because release of nickel ions from these alloys is generally higher than for high-noble or noble alloys. Palladium sensitivity has also been a concern in some countries, although the incidence of true palladium allergy is one third that of nickel allergy. However, it has been clinically documented that patients with palladium allergy are virtually always allergic to nickel. The converse is not true however. In vitro, there have been numerous articles published on the effects of metal ions on cells in the gingival tissues, such as epithelial cells, fibroblasts, and macrophages. For the most part, the concentrations of metal

ions required to cause problems with these cells in vitro are greater than those released from most casting alloys. However, the most recent research has shown that extended exposures to low doses of metal ions may also have biological liabilities. These new data are noteworthy because the low-dose concentrations approach those known to be released from some alloys. The clinical significance of this research is not known, however.

Denture base materials, especially methacrylates, have been associated with immune hypersensitivity reactions of gingiva and mucosa probably more than any other dental material. The greatest potential for hypersensitization is for dental and laboratory personnel who are exposed repeatedly to a variety of unreacted components. Hypersensitivity has been documented to the acrylic and diacrylic monomers, certain curing agents, antioxidants, amines, and formaldehyde. For the patient, however, most of these materials have reacted in the polymerization reaction, and the incidence of hypersensitization is quite low. Screening tests for sensitization potential include testing of unreacted ingredients, the polymeric substance after reaction, and oil, saline, or aqueous extracts of the polymer in in vitro tests described previously and in skin tests in animals. In addition to hypersensitivity, visible light-cured denture base resins and denture base resin sealants have been shown to be cytotoxic to epithelial cells in culture.

Soft tissue responses to soft denture liners and adhesives are of concern because of the intimate contact between these materials and the gingiva. Plasticizers, which are incorporated into some materials to make them soft and flexible, are released in vivo and in vitro. Cell culture tests have shown that some of these materials are extremely cytotoxic and affect a number cellular metabolic reactions. In animal tests, several of these materials have caused significant epithelial changes, presumably from the released plasticizers. In usage, the effects of the released plasticizers are probably often masked by the inflammation already present in the tissues onto which these materials are placed. Denture adhesives have been evaluated in vitro and show severe

cytotoxic reactions. Several had substantial formaldehyde content. The adhesives also allowed significant microbial growth. Newer formulations that add antifungal or antibacterial agents have not yet been shown to be clinically efficacious.

REACTION OF BONE AND SOFT TISSUES TO IMPLANT MATERIALS

There are four basic materials used in implant fabrication: ceramics, carbon, metals, and polymers (and combinations of the above). Interest in the biocompatibility of implant materials has grown as the use of implants in clinical practice has increased dramatically in the past 10 years. Most successful dental implant materials either promote osseointegration (an approximation of bone on the implant within 100 A of the implant) or biointegration (a continuous fusion of bone with the implant).

Reactions to Ceramic Implant Materials

Most ceramic implant materials have very low toxic effects on tissues, either because they are in an oxidized state or are corrosion resistant. As a group, they have low toxicity, and are nonimmunogenic and noncarcinogenic. However, they are brittle and lack impact and shear strength, and therefore have been used as porous or dense coatings on metals or other materials. If the root surface porosities are more than 150 pm in diameter, the implants often become firmly bound to bone (through ankylosis or biointegration), especially if they are taken out of occlusion for a time. If the porosities are smaller, the tissue usually forms only fibrous ingrowth. Dense ceramics are also used as root replicas or bone screws. Made of either single crystal (sapphire) or polycrystalline aluminum oxide, they become biointegrated and provide excellent stability if left unloaded for a time. In one study, 60% of the restorations still performed adequately after 6 years in place.

Hydroxyapatite, a relatively nonresorbable form of calcium phosphate, has been used with some success as a coating material for titanium implants and as a ridge augmentation material. Studies indicate that the hydroxyapatite increases

the rate of bony ingrowth toward the implant. However, the long-term corrosion of these coatings and the stability of the bond of the coating to the substrate are still controversial. Retrieval evidence indicates that even these "nonresorbable" coatings are resorbed over the long-term. Beta-tricalcium phosphate, another form of calcium phosphate, has been used in situations where resorption of the material is desirable, such as repair of bony defects. Carbon has been used as a coating and in bulk forms for implants. Although the biologic response to carbon coatings can be favorable, they have been supplanted by titanium, aluminum oxide bulk materials, and hydroxyapatite coatings. Finally, Bioglass forms a surface gel that reacts favorably with connective tissue, allowing bone formation adjacent to it.

Reactions to Pure Metals and Alloys

Pure metals and alloys are the oldest type of oral implant materials. All metal implants share the quality of strength. Initially, metallic materials were selected on the basis of ease of fabrication. Over time, however, biocompatibility with bone and soft tissue and the longevity of the implant have become more important. A variety of implant materials has been used, including stainless steel, chromium-cobalt-molybdenum, and titanium and its alloys. These materials have been used in a variety of forms, including root forms and subperiosteal and transosteal implants. In dentistry, the only metallic implant materials in common use today are titanium alloys.

Titanium is a pure metal, at least when first cast. In less than a second the surface forms a thin film of various titanium oxides, which is corrosion resistant and allows bone to osseointegrate. The major disadvantage of this metal is that it is difficult to cast. It has been wrought into endosteal blades and root forms, but this process introduces metallic impurities into the surface that may adversely effect bony response unless extreme care is taken during manufacturing. Titanium implants have been used with success as root forms that are left unloaded under the mucosa for several months before they are used to

support a prosthesis. With frequent recall and good oral hygiene, the implants have been maintained in healthy tissue for up to 2 decades. Titanium-aluminum-vanadium alloys (Ti6A14V) have been used successf~~llyin this regard as well, but questions remain about the liability of released aluminum and vanadium. Clinical studies have been positive. Although titanium and titanium alloy implants have corrosion rates that are markedly less than other metallic implants, they do release titanium into the body. Currently there is no evidence that these released elements are a problem locally or systemically. The issue remains unresolved.

In the soft tissue, the bond epithelium forms with titanium is morphologically similar to that formed with the tooth, but this interface has not been fully characterized. Connective tissue apparently does not bond to the titanium, but does form a tight seal that seems to limit ingress of bacteria and bacterial products. Techniques are being developed to limit downgrowth of the epithelium and loss of bone height around the implant, which ultimately cause implant failure. Peri-implantitis is now a documented disease around implants and involves many of the same bacteria as periodontitis. The role of the implant material or its released components in the progression of peri-implantitis is not known.

SUMMARY

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 location of a material in the oral cavity partially determines its biocompatibility. Materials that appear biocompatible when in contact with the oral mucosal surface may cause adverse reactions if they are

implanted beneath it. Materials that are toxic when in direct contact with the pulp may be essentially innocuous if placed on dentin or enamel. Finally, interactions between the material and the body influence the biocompatibility of the material. The material's response to changes in pH, the application of force, or the

degradative effects of biological fluids can alter its biocompatibility. Features of a material's surface that promote or discourage the attachment of bacteria, host cells, or biological molecules determine whether the material will promote plaque retention, integrate with bone, or adhere to dentin.

I SELECTED PROBLEMS

Problem 1

You have a group of six materials, all of which can be classified as posterior composites, but each of which has a slightly different formula. How would you determine which of these freshly set materials is the least toxic using the least expensive, least time-consuming tests?

Solution

You may choose a direct in vitro cell-culture test. The materials would be formed into disks of equal dimensions. The disks should be stabilized on the bottoms of cell-culturewells. Cells are then placed into the wells with the materials and are incubated in an appropriate incubation medium and environment for 24 hours or more. Then the disks and wells are observed and photographed under phasecontrast microscopy, taking special note of cytopathic effects around each disk for semiquantitative results.

Problem 2

If you have the same situation as in Problem 1, but you want to quantify your results, what are your options?

Solution

You may choose the same test model as in the solution for Problem 1, but you would measure the cellular response quantitatively rather

then by visual observation. There are two primary options: (1) measure the membrane permeability of the cells that remain around the disks using neutral red or jlcr (see Fig. 5-10); or (2) measure some aspect of cellular biosynthesis or metabolism (MTT test, DNA synthesis, protein synthesis, total protein). You might also choose another model, such as extracting the sample for 1 to 3 days in given volumes of solvent and then treating the cells with serial dilutions of the elutants.

Problem 3

With the same situation as in Problem 1,you want to know the effect of the setting reaction or time on the toxicity of the material. What would you do?

Solution

Usually the material is made into disks and allowed to set for varying periods (from 1 to several days). Then, for each period, the set disks are placed into tissue culture wells or are extracted with a solvent that is placed into tissue culture wells. Then your options are the same as those in the solutions for Problems 1 or 2.

Problem 4

You read a research paper on the in vitro cytotoxicity of composites that shows that

some composites are initially very cytotoxic but improve with time of elution into artificial saliva, whereas others continue to be toxic. How can you interpret these results in terms of clinical use of the composites?

Solution

Extrapolation of in vitro tests to clinical situations is always difficult, but one interpretation could be that the composites that improve with time in vitro pose lower long-term risks clinically than those which continue to be cytotoxic. This line of reasoning may be dangerous, however, if the material is designed to release a substance that is cytotoxic in vitro but therapeutic in vivo. Such is the case for fluoride release from some composites and glass ionomer cements.

Problem 5

If you have a series of composites and you want to rank them according to the toxicity they may have specifically for pulpal tissues, what would you do?

Solution

You should choose a test model in which dentin is interspaced between material and cell test system. The dentin barrier may alter the effects of released components from the material and change the response of pulpal cells. Without such a barrier, the composites may appear more cytotoxic in vitro than they would be clinically.

Problem 6

One of your six composite materials is associated fairly consistently with pulpitis when used by you and your fellow clinicians. The cytotoxicity test that you chose indicates that this material is not much more toxic than the other materials. How can you better understand what is causing the pulpitis?

Solution

The pulpitis may be caused by microleakage of bacteria or by the material's ability to cause a chronic inflammatory (vs, toxicity) response.

If the facilities are available, you may do in vitro chemotaxis tests, using human peripheral leukocytes with the test sample to determine which materials might be responsible for the inflammation. Tests on monocytes could also be done, using materials or material components at nontoxic concentrations. If the material does not show an inflammatory response in these tests, then usage tests in animals could be used to verify the pulpitis in vivo in a more controlled environment. Then, if usage tests in animals substantiate the pulpitis, you should be highly suspicious of microleakage of environmental materials or bacteria products. To confirm this suspicion, additional usage tests could be used where the margins of the restoration are surface-sealed to prevent microleakage.

Problem 7

You have a new polymeric substance that you believe might function well as a root cylinder implant. What kinds of tests should you conduct to answer questions of safety according to FDA standards, and how long will this take?

Solution

Initial tests should include cytotoxicity tests, a hemolysis test, some test for mutagenesis or gene toxicity, and probably an oral LD50test. (All tests except mutagenesis can be performed and analyzed in about 2 to 3 weeks if run concurrently. The mutagenesis tests may require between 1 and 3 months for performance analysis.) Secondary tests might include implantation into bone and soft tissue of small animals, tests for mucous membrane irritation, and hypersensitivity tests. If done concurrently, this will take about 1 month before histological preparation of tissue is begun. Time required for histology and reading of slides may vary, depending on the size of the project. Usage tests (percutaneous implants) in jaws of larger animals may require that the implants remain in place for 1 to 2 years, followed by histological processing and evaluation.