Dent Mater 12:186-l 93, May, 1996

In vifro models of biocompatibility: A review

Carl T. Hanks’, John C. Watahaz, Zhilin Suni

‘Department of Oral Medicine, Pathology and Surgery School o~‘Lkntrstr~~~ liniversity of’Michig_an, Ann Arbor: Michig.an, I ISA

‘Department of’Ora1 Rehuhilitation, SC~IOO/ of’lk~rtistr\, Medical College of Georgia, Augusta. Cwr~rr~. 1’5%

ABSTRACT The objectives of this paper were to define in vitro biocompatibility of materials, to discuss some of the issues concerning why conclusions from tissue culture are sometimes different from in viva biocompatibility, to give highlights of the sequence of the development of these in vitro

assays from the early 1950s to their present state of development, and to discuss possible future trends for in vitro testing. In vitro

biocompatibility tests were developed to simulate and predict biological reactions to materials when placed into or on tissues in the body. Traditional assays have measured cytotoxicity by means of either an end-stage event, (Le., permeability of cytoplasmic membranes of dead and dying cells, or some metabolic parameter such as cell division or an enzymatic reaction). In vitro assays for initiation of inflammatory and immune reactions to materials have also begun to appear in the literature. More recently, the concept of dentin barrier tests has been introduced for dental restorative materials. Four models which measure both permeability and biological effects of materials are compared and discussed. Future efforts may be directed toward development of materials which will allow or promote function and differentiation of tissues associated with materials. New analytical procedures and understanding of optimal characteristics of materials should improve our ability to develop more biocompatible materials. Both molecular biology techniques, and altered design of material surfaces may make the materials either more or less reactive to the biological milieu. These trends suggest a greater future role of the biological sciences in the development of biomaterials.

INTRODUCTION

The objective of in vitro biocompatibility tests is to simulate biological reactions to materials when they are placed on or into tissues of the body. These methods offer less expensive ways to survey newly developed materials, reducing the probability of surprises when animal usage tests or clinical trials are performed. Without prior laboratory testing of materials, using animals to test materials could become very time-consuming and expensive.

There have been a number of problems m~:c)ivt:d Ivlth developing ill r)itro models of biocompatibility. The mayor problems are: 11 discerning significant ‘II !s;i*fi biological reactions for which simulation assays can be devised. and 2) designing 777 vitro assays which are relatively simple to perform and which produce consistent rclsults among laboratories. The earliest, in vitro tests of’ material+ cytotoxicity were relatively simple to design and oflered fewer complications than animal models. Whereas c,ytotoxicity test> are still kequently used to test biocompatibility of materials, there are many other types of assays which are capable of’ testing for activation of biological processes. These other assays require much longer periods of tissue reaction tc! materials than cytotoxic reactions (Hanks and Wataha. 1993 1. These other processes fall into the areas of 11 inflammation. 2) immune reactions, and 3) mutagenesis. In choosing an assay method. the question “what is the most usefL1 type 01. in vitro assay?“. should rather be phrased “what is ant: attempting to test?” Cytotoxicity assays measure only fmitc effects on cells during the first 12-24 h after exposure to toxic substances. The host cells either recover from or succumb to their chemical injury However, many biological reactions in uiuo are not simply cytotoxic and are propagated be.yonci 24 h. Examples are inflammatory and immune reactions. For this reason. it has been pointed out that there is pool’ correlation between cytotoxicity assays and pulp reactions 10 restorative materials in animals (Mjiir ef ni.. 1977 I

In order to understand the progress and limitations of U) vitro biocompatibility tests, the remainder of this paper will briefly review highlights of past and present investigations. and suggest areas of future research.

PREVIOUS INVESTIGATIONS From the earliest periods of cell culture, L~CYY 1~1s ken XII emphasis on establishing standards by: 1) gathering data which could be validated among several labs in the same way that materials properties could br validated 11~ chemical and

186 Hanks et a/.//n vitro models

physical testing, and 2) understanding of the general nature of the reactions which take place in response to materials, usually via direct contact. Use of in vitro techniques to study the toxicity of various synthetic materials began some 30 years after tissue culture was first established as a technique (Strangeways and Fell, 1926). Various investigators began to apply organ and tissue culture techniques to toxicological problems in the 1950s and 60s e.g., the toxicity of rubbers and plastics on chick embryonic tissue (Cruickshank ct c&1960), the effects of cyclic hydrocarbons on cultured rat tracheal epithelium and connective tissue (Crockeret aZ., 19651, and the toxicity of plastics and a variety of other materials on primary chick embryonic cells and mouse L cells (Guess et al., 1965). Kawahara’s laboratory began to use cell culture methods to investigate dental materials in the 1950s (Kawahara & al., 1968). Using the hanging drop method and monolayers of L-929 cells, these investigators reported on the cytotoxicity of pure metals, dental cements and medicaments, and plastics.

Ieirskar and Helgeland (1972) did the first study of the growth of human epithelial and L-929 cells on and around standard-sized disks of dental materials, including silver amalgam and copper amalgam, resins, silicate cement, and a gold alloy One of the earliest quantitative in. vitro tests was a membrane permeability assay for “lC!r-release using L-929 cells or HeLa cells (Spangberg, 1973). The specific purpose of this assay was to measure the number of dead and dying cells in a culture in the presence of a synthetic material. Because chromium is probably not a metabolic substrate, it is thought that there are probably no biological effects of the radioisotope itself at low concentrations. However, its use is limited to measurement of final events, i.e., cell membrane permeability, following cell death, and thus, sublethal changes are not measured.

Schmalz (1982; 1988) was one of the first dental investiga- tors to apply the agar overlay technique to test the cytotoxicity of dental materials. In this technique, L-929 cells are grown in plastic culture dishes, and the cells are overlaid by agar. Toxic test material which is placed on top of the agar diffuses through the agar to cause cell membrane permeabilization and release of the vital dye, neutral red. Using this technique to study a number of dental materials, he showed results similar to the “‘Cr-release assay at lower costs and with no radioactivity Wennberget ul. ( 1979) introduced the‘Millipore”6lter method, in which either human epithelial cells (HeLa) or mouse L cells (L-929) are grown on filter paper. Either membrane permeability (neutral red release) or succinyl dehydrogenase activity is used to measure cell viability The eluate Tom the dentin disks diffuses through the f&r membrane and affects the cell layer and, like the agar overlay test, this process results in artifactual variability in diffusion and is thus difficult to interpret.

There is still a significant effort being made to standardize in vitro tests. However, technology is moving much faster than standardization, which makes the development of standards a difficult and almost continuous process. There are a number of standards or guidelines which govern the testing of materials. For example, the American Society for Testing and Materials (ASTM, 1983) formulated several standards, including standard F813-83: Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices. This test uses monolayers of L-929 cells and tests for cytopathic effect

and viability of cells remaining on the substrate. A second ASTM standard is F895-84: Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity, which is the agar overlay technique using L-929 cells. The other two testing organizations, the International Organization for Standardization (ISO, 1993) and the Council on Dental Materials, Instruments and Equipment of the American Dental Assocation (1982) have recommended the use of batteries of in vitro and in uiuo tests to study the biocompatibility of materials since they believe no single test is capable of defining biocompatibility Technical report 7405 published by the IS0 (1984) includes: 1) Section 5.7: In vitro cytotoxicity tests (chromium-release method); 2) Section 5.8: Cytotoxicity test (Millipore filter method); and 3) Section 5.9: Cytotoxicity test (agar overlay). The ANSVADADoc. 41,1982 Addendum includes only the Section 4.4.1: Cytotoxicity:1~ vitro Test for Cytotoxicity (chromium-release assay). Both the IS0 and the ANSI/ADA documents are in the process of revision.

PRESENT INVESTIGATIONS Cytotoxicity. According to the above standards, the major category of tests for the initial evaluation of materials is the cytotoxicitytest. In addition to the previously mentioned tests, this laboratory has investigated the cytotoxlcity of resins and metals over the last ten years, mainly by utilizing metabolic assays(RathbunetaZ.,1991;HanksetuZ., 1991;WatahaetaZ., 1992b; 1994; 199513). Attempts have been. made to define and control variables which affect these cytotoxicity tests. The study of conditions for release of metal ions and measurement by atomic absorption spectrometry (Watahact al., 1992a),uptake of metal ions by target cells (Wataha et al., 1993) the variation in response by various cell lines of target cells (Wataha et al., 19941, and cytotoxic effects of metal ions on macrophages as a model for characterizing the response of another cell type to materials (Wataha et al., 1995a) have been facilitated by cytotoxicity tests.

Cell culture methods have also been used to study the effects of concentrations of resin components on connective tissue cell metabolism. There have been a number of reports of pulp”irritation” following resin composite restorations (Stanley et al., 1967; Baume and Fiore-Donno, 1968;Tobias et al., 1973; Stanleyet al., 1975; Block et al., 1977; Stanley, 1980; Qvist and Thylstrup, 1988; Qvist et al., 1989). Although there is even more evidence of bacterial microleakage around the margins of restorations (Br%innstrom and Nyborg, 1972; Bergenholtz et al., 1982; Cox et al., 1987; Bergenholtz, 19901, it is the authors’ opinion that pulpal irritation is not caused exclusively by bacterial products. Some in uiclo m.ethodologies which exclude bacteria suggest that resins may also contribute to tissue inflammation (Nasjleti et al., 1983‘). The cytotoxicity of polymerized resins in vitro has been reported by several investigators (Meryon and Brown, 1983; Hanks et aZ., 1988; Hensten-Pettersen and Jacobsen, 1991). 1 t was decided by the current authors that, as a first step, that toxic ranges of concentrations of resin components as well as the magnitude of the biological responses should be determined more systematically Standard toxicological measures, the TC,, (concentration at which halfofthe biological activity remains), allow comparison of a group of substances within an experiment, or if the experimental parameters are similar between experiments. Hanks et al. ( 1991) reported on the

Dental Materials/May 7996 187

metabolic cytotoxicity of 11 contemporary resin components, solubilized in DMSO and diluted 1:lOOO in DMSO before treating cultures of Balb/c 3T3 cells. Standard curves were drawn for concentrations of resins vs. either ‘IH-TdR uptake (DNA synthetic rate),“H-leucine uptake (protein synthetic rate\ or total protein per culture. It was found that Balb/c 3T3 cell responses for all three biological parameters were at the same level of magnitude for most resin components. Ethyoxylated b&phenol A dimethacrylate (E-BPA) was the most toxic with a 24 h TC,,, below 10 ymol/L. The TC,,, concentrations for 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy) phenyllpropane (BisGMA), urethane dimethacrylate KJDMA), 1,6 hexane diol dimethaclylate (HDDM) and bis glycidyl ether ofbis-phenol A (BGE-BPA) were between lo-20 pmol/L. TC,, values for triethylene-glycol-dimethacrylate (TEGDMA), glycidyl methacrylate (GMA) and bis phenol A (BPA) ranged between 30-100 ymoVL. The TC,, values for N,N dihyroxy-ethyl-p- toludidine (DHEpT), camphoroquinone (CAMP) and N,N dimethylaminoethyl methacrylate (DMAEM) were above 100 pmol/L, making them less toxic than the other groups. These comparative data suggested that these materials should !.x grouped according to their relative hazard levels for various biological parameters. Using flow cytometry, Nassiri et al. ( 19941, reported that at both lethal and sublethal doses, UDMA has the effect of allowing some of the test cells to synthesize DNA, but later blocking these cells in the G2 phase of the cell cycle.

To this point, most studies on component c.ptoxicity have concentrated on the effects of single matenals. However, clinical materials consist ofcombinations ofcomponents. Thus, in a study of combinations of two cations used at different concentrations on Balb/c 3T3 metabolism, Wataha et al. (199213) showed that the combinations had three kinds of effects: additive (the same as adding the effects of each cation i. synergistic (the effect was more toxic than the additive effects of each cation), or antagonistic (the effect was less toxic than the additive effects). Ratanasathien et al.(1995) used a similar approach to investigate the cytotoxic metabolic effects of binary combinations of components of some contemporary dentin bonding systems, i.e., 2-hydroxyethyl methacrylate (HEMA), BisGMA, TEGDMA and UDMA. Individually. TC. values for these four resins could be ranked from most t&l to least toxic as BisGMA > UDMA > TEGDMA >>> HEMA after exposure of 24 and 72 h. A synergistic effect was found when BisGMA was present at 25 ymolA+ and HEMA was present at any concentration. Synergistic effects are particularly important because unpolymerized components of dentin bonding agents may have the potential to cause toxic reactions in the pulp at levels lower than the individual component by itself In addition, low molecular-weight resins such as HEMA, 4-META and TEGDMA may also act as solvents for more viscous resins (BisGMA and UDMA) and make them more accessible to cells and tissues.

Dentin barrier tests. A recent adjunct to the cytotoxicity tests has been the development of dentin barrier tests. Since Outhwaite et 01. (1974) published a short paper on the fabrication of a split-chamber device, Pashley and colleagues have defined factors which affect diffusion through dentin tubules, including size and concentration of molecules, density of dentin tubules, length of dentin tubules, diameter of tubules, and effect of temperature on this process (Outhwaite rt al..

1976; Pashley and Livingston, 1978;Pashley, 1989; Pashiey anti Matthews: 1993). Tyas t 1977) probably introduced the concept of measurement ofa biological effect in the presence of a dentin barrier. The device, a simulated dental cavity consisting of a Pyrex cylinder projecting through the lid of :I 30 mm petri dish, later became a British standard (British Standards Institution. 1989) for testing of restorative materials. Using this model. Meryon (19841 showed that the thicker the dentin powder in the Pyrex cylinder. the less the cytotoxic effert ofzinc oxideeugenol. Later, Meryon and Brook ( 1989) compared the ability of 100 pm and 500 pm slices of dentin to alter the cytotoxic effects of three bonding agents using this device.

Hume ( 1985) fabricated a second dentin harrier devicct: 13 human molar tooth with a Class I cavity preparation in thtt occlusal surface and a wax trough replacing the root. With this device, he demonstrated that dentin indeed had the effect of’ reducing toxicity of various materials (zinc oxide-eugenol. ;:I glass ionomer cement, and an unfilled resin J placed in thcb cavity preparation as compared to direct contact with cell monolayers. Problems with this model lvere that tb(s 1.6-2.0 mm of dentin was too thick and could not be controlled. that the surf& area was not consistent. and that thr: concentrations ofthe tisates were unknown. Stanley ( 1980 1 has suggested that 0.5 mm dentin thickness was optimal to measure a range of cytotoxic concentration:: Hume’:: laboratory has advanced their in uitro model by using HPL( ’ to measure dif&sion ofresins across dentin (&r&a and Hume. 1994).They reported thatTEGDMAleaches readily from resin composite, diffuses readily through 1.6-2.0 mm t,hick dentill into tissue culture fluid, and is a major contributor to the’ cytotoxicity ofthat fluid to a cell test system. In a similar study (Gerzina and Hurne, 19951, they detected HEMA within 4 rntil of elution and TEGDMA for up to 30 cl of sampling? but a1 lower levels than HEMA. The hydrostatic pressure moving from pulp to cavity preparation made a significant difference in the diffusion of TEGDlVA, but not of HEMA. Hydrostatic- pressure did not prevent diffiision of either molecule.

The current authors have published several studies usm,g both single- and multiple-well split-chambered devices. At fist. multiple chamber devices were developed, using 12 humti-u molar dentin disks with similar thickness and hydraulic conductance values, to me‘asure both diffusion of molecules ar~i cytotoxicity of these molecules at the same time (Hanks ct cri.. 1988). Radioact,ive phenol was used to characterize tht. multiple-chambered device and as a positive control (Hanks cJt al., 19891. However, this multiple split-chambered de+<’ proved to he too cumbersome, Thus. it was decided that ;I two-stage process would be more accurat,e. The stages wen;: I) detemination of the range of concentrations causing tht-L biological eflect. and 2 I determination of the diffusibility of thf L material across dentin under various circumstances. c’.,~:, presence or absence of smear layers, positive or negative, pulpal pressures.

Following this strategy, Bouillaguet cd (11. ( 1996 I found that. the 24 hTC!_, concentration for HEMAfor Balb/c 3T3 cells wa.i; about 4 mmol/L, which means that it is about 1000 times less toxic than BisGMA. Subsequently, they used a split-chamber device connected to a flow-through cuvetlcl in a Ii\’ spectrophotometer. The diffusate chamber vol time plus t,ubing was only 70 III,. With this arrangement, HEMA

188 Hanks et a/.//n vitro models

diffusion was monitored across etched human molar dentin disks with thicknesses of 0.5 and 1.0 mm, and with either 0 or 10.0 cm H,O back pressure. HEMA diffusion was evident in the lower chamber within 3 min of application. The permeability of HEMA was directly proportional to the driving concentration and inversely proportional both to the dentin thickness and convection pressure of fluid moving in the opposite direction. Earlier reports by our laboratory (Hanks ct al., 1994b) showed that permeability coefficients of water-soluble (e.g., phenol, albumin, gamma globulin, and fibrinogen) and non-water-soluble (e.g., BisGMA, TEGDMA, bisphenol A, glycidyl methacrylate) molecules tend to correlate inversely with their molecular weight (molecular size) as well as dentin thickness (length of dentin tubules) Neither of these studies tested for the possibility that differences in diffusion permeability may have resulted in part because of either dentin adsorption, adsorption to other materials in the dentin tubules, or steric hindrance because of molecular shape.

Finally, Schmalz and Gymnick (1992) reported another dentin barrier model which utilizes L-929 mouse fibroblasts growing on bovine dentin slices. Fluorescent dyes were used to visualize the remaining viable cells on the “pulp,,, dentin surface following the incubation of the test material on the opposite dentin surface for 24 h. Using various phenol concentrations and thicknesses of dentin, this research group has also confirmed that the cytotoxic effect of phenol is proportional to the dentin thickness and the concentration of phenol.

At this stage of development, there are still several problems associated with the dentin barrier tests. The problems include: 1) the variability among different models, 2) the reduction of the volume of *sate on the “pulpal” side of the dentin disk so that it is more nearly approximates the volume of extracellular fluid in and around the pulpal cells adjacent to the dentin, and 3) development of relevant biological responses of many different types of cells in the pulp (e.g., odontoblasts and preodontoblasts, fibroblasts and less differentiated mesenchymal cells, endothelial cells and supportive cells for blood vessels and nerves, nerve axons themselves, macrophages, etc.). In the most simple model (British Standards Institution, 1989), it is impossible to quantitate the difisate, so that one can still only rank order materials &r diffusion through dentin. Although Meryon and Brook (1989) have estimated the total volume of pulpal fluid to be about 200 FL, this has not been studied extensively. Hume’s laboratory has recently been able to quantitate the absolute amounts of TEGDMA and HEMA crossing the dentin after HPLC, but have not related it to fluid volume, i.e., concentration (Gerzina and Hume, 1995). The authors’ laboratory has been able to reduce the diffusate chamber volume in the split-chamber device down to about 40 & so that the concentration of the difFusate is more realistic (Hanks et al., 199413). However, both of the latter two models have obtained more accurate readings by sacrificing simplicity of design. Finally it would seem that cells growing directly on the underside of a slice of dentin (Schmalz and Gymnick, 1992) would be an optimum arrangement because the data would not have to take into account the &ate volume. However, the authors reported that although fluorescein diacetate can indeed allow distinction between living and dead cells, variability in plating efficiency on dentin is so great that it is

impossible to distinguish between cytotoxicity and artifacts in plating density (Hanks et al., 1989). Thus, lack of information on the actual concentration of diffusate and presentation to the cell test system makes it difficult to fabricate a simple, reproducible standard test.

Much of the controversy about the effects of materials and bacterial products could be resolved if there were a good clinical model for measuring diffusion products in the human pulp. While such clinical studies have not been reported, several laboratory studies suggest that monomers do indeed leach out of polymerized resin composites and diffise across dentin. Thus, Tanaka et al. (1991) identified large amounts of eluted TEGDMA and smaller amounts of BisGMA from several dental composites by gas-liquid chromatography and mass spectroscopy, Ferracane ( 1994) estimated that approximately 5-10s of unbound monomer, which equals approximately 2% ofthe weight of the resin component in most composites, elutes into an aqueous solution. In other studies, Gerzina and Hume (1994) reported that TEGDMA leaches readily from resin composite. RecentI>; Rueggeberg et al. (1995) showed that for polymerized copolymers of BisGMA and TEGDMA ranging from 20%lOO’r(l BisGMA with the remainder being TEGDMA. BisGMA was the major component which leached into tetrahydrofuran from the copolymers, indicating that the leached component was not converted and was not present as pendant chain moieties.

Newer in vitro tests. Synthetic materials probably have significant influence on other biolo_gicall processes such as tiammation and the immune responses, although studies of this interaction are in their infancy There have been very few reports in the dental literature on the development of biocompatibility assays to predict inflammatory or immune reactions, e.g., Downing et al. (1989). Other “biocompatibility’ laboratories, especially those associated with the development of medical devices contacting blood, have published papers on macrophage/monocyte activation by polymers and other materials (Herzlinger et aZ., 1981; Chenoweth, 1986; Miller andAnderson, 1988; Johnson, 1990;Remes andwilliams, 1991). The authors’ (Hanks et al., 1994a) have also reported complement activation (C3a generation t in the presence of 1) polymerized resin formulae containing 70% or more BisGMA, 2) PBS extracts of polymerized resins, and 3) individual resin components, t’.~.: BisGMA or N,N dihydroxyethyl-p-toluidine (DHEpT) solubilized in DMSO or ethanol solvents.

The effects of resin components upon the function of lymphocytes and accessory pulp cells have also been reported recently (Jontell et al., 19951, using the same resin materials as were tested in the previous cytotoxicity study (Hankset al., 1991). These resins were tested in two different systems: 1) concanavalin-A induced spleen cells of Lewis strain rats as one test system, and 2) T-lymphocytes from cervical lymph nodes with a mixed incisor pulpal cell population from the same species. UDMA, BisGMA,TEGDMA and E!PA increased spleen cell proliferation in response to con A at low concentrations, while other resins, except CAMP, caused only inhibition of “H-TdR uptake (cytotoxicity). In the other assay, purified T-lymphocytes stimulated by pulpal cells (Class II expressing cells) did not show an enhanced response to any materials tested except CAMP. CAMP enhanced proliferation of splenic cells to over 400% of the con A controls at high doses (64-250 ymol/L),

Dental Materials/May 1996 189

and enhanced proliferation of T-lymphocytes in the presence of pulpal cells to more than 120% ofconcanavalinA controls at. these concentrations.

Finally, because cytokines are expressed in higher concentrations than normal during inflammatory and immune reactions, and are often integrally involved in the mediation of these reactions, they are good prospects for developing biocompatibility assays. Tumor necrosis factor-cc (TNF-c() in combination with interleukin-1 (IL-l) and?-interferon (y-IFN) are implicated in bone resorption (Wang and Stashenko, 1993). granuloma formation (Shikama et al., 1989; R&in and Gay, 19921, and other biological activities associated with inflammation. Macrophage release of TNF-ct has been intentionally used to develop a biocompatibility assay to study dentin adhesives by Yourtee and colleagues (Zhuang ct al.. 1994a; 199413 1.

FUTURE DEVELOPMENTS Future efforts in the development of biocompatibility methodology will probably be directed toward the development of materials which will allow normal differentiation and function of tissues into which the materials are placed. Advances in technology and understanding of biology, chemistry and related fields will inevitably lead to advances of the present models as well as to new models. In predicting areas in which new research will probably occur, two areas should be mentioned: 1) molecular biology of host tissue in response to materials, and 2) molecular interactions between biological molecules and synthetic materials or tissue- material combinations.

Examples of molecular biological approaches which apply to dental materials are as follows. Osteoblast-like cells (the ROS 17/2.8 cell line) can be made to differentiate in culture by various additives to the culture medium. At sublethal concentrations, several metal ions are able to alter gene expression of these cells (Sun et al., 1994; 1995 1. Al-:‘, Ti+,’ and v’- were shown to suppress transcription of alkaline phosphatase (ALP) and osteocalcin (OCN), but CO+~, Cr’:’ and N? did not. The transcription of ALP and OCN are thought to be an important part of bone matrix synthesis and mineralization. Thus, this approach may give additional information concerning the ability of metal alloys to integrate into osseous tissues.

One impediment to testing biocompatibility of restorative materials is that the only reliable model of the odontoblast has been the in viva model. Several interpretative problems occur because tissue reactions to synthetic materials as well as bacterial products are very complex. Thus, it is difficult to separate cytotoxicity and differentiation of replacement odontoblasts from inflammatory and immune reactions of the surrounding connective tissue in uino. Therefore, a reliable itz. vitro assay for restorative materials should include cell lines of odontoblasts and their precursors which have stable phenotypes so that the results of the assays can be verified in several laboratories. At present, several laboratories are interested in expression of genetic markers associated with precursor and mature odontoblasts during fetal and neonatal development (MacDougaU et al., 1985; Butler et al., 1992; D’Souzaet al., 1992; Bronckers et al., 1993; Ritchie et al., 1994: 1995; Butler and Ritchie, 1995; D’Souza et al., 1995). The technology is now available to investigate whether these

materials can alter the genetic mechanisms ot oduntoblast:; and thus alter dentin regeneration. Several labordtorics. including those of Drum ct rrl. i1985), Kawase VI cl/. (19901. Nakashima ( 1991), Tsukamoto et n/. (1992 I. MacDougall tjt oi. (19921, Andrews et al. (1993). and Takeda rf tri. f 19941, have reported cultured odontoblast-like cell lines. The present authors’ laboratory has developed :l Jinc, of’ mous:1” odontoblast-like cells (MDPC-23). parallel to the osteohlasl system, expressly to assay restorative materials t Hanks & (I/.

1995). This established cell line has iwl? ~*loned ;mri characterized in experiments parallel to those p&ormed to1 the ROS 17/2.8 cells. This MDPC-23 cell line is considered odontoblast-like because it expresses dentin siaioprotein ( DST’ b and several phosphate-rich proteins which ;lre specificall!. synthesized bv odontoblasts, as well as other markers specific for mineralizing tissues, r.(‘. , alkaline phosphatase / AlA’ 1 osteopontin (OPN). osteocalcin (OCN) and Type I cacrllagen, as shown by Northern and Western blotting techniques. Both kglycerophosphate (P-GP) and dexamethasone ( DEX) depress cell proliferation and DNA s_ynthesis. p-GP enhances AI,J’ expression as well as mineralization of these cultures. but inhibits cellular metabolic activity (M’lTl. DEX inhibits I>N,\ s_ynthesis. ALP activity and m&eralizat.ion. In :jddition contrary to its effect on ROS 17pL.8 osteoblast-like cells and primary fetal rat calvarium osteoblast-like cells. DEX does no1 appear to havtl an inductive effect on differentiation of thrsc* odontoblast-like cells. Data on the effects of’dental materialh on this system have not been reported. but arc bring gathered

The second area of interest is biological tissue-synthetic’ material interactions, which may be descrihcd ;is “ti;;snt’ engineering.” The term “tissue engintlrring” ~+;uggest:- replacing body parts by either s_ynthetic mat&:& or norm:) 1 body tissue. cells or molecules which have been reorganized il/ dome way before being replaced in the body. Or+> ex‘amplt? 4 11 this is in “rt:-engineering” synthetic molecll Et-;. Sfx\‘e ri! 1 laboratories have suggested that, salivary. rnlcrobial ani{ tissue hydrolytic enzymes may play significant roles in both: the breakdown of dental resins and the production {.it degradation products (Van Groeningen V! rri.. I%%. Munksgaard and Freund, 1990: Freund and Munksg~ard, I 99U; Bean et r/f.. 19941 These degradation product.s may br %I potential sourct’ 01‘ biologically activr molrc1lli?.-; .;uch ;ib antigens, toxicants, growth factors, or gene-modu lating agent> Computer molecular modeling of enzymes and terminal group+ on polymers allows investigators to company kinetic artci potential energy values of enzymatic degradat,lon reactions oi terminal groups to determine which reaction i:: stcricall>- hindered, i.t>. most energy-consuming (You&m (‘I if/., 1995 1 Thus, an est.rr linkage which does not clasil,v fit into ,.u, -‘enzyme dock” should provide morth b~odut,~bjlil y illll: biocompatibilitlv.

Additionally, the telm “tissue engineer-rng t:r~compasst~~. such areas as: 1 I the fabrication of’vascular gyaii, materiais 2) materials which will provide structural replacement of born and connective t,issue which has attached to it an integral layer of protein containing an amino acid sequence’ ! the so-called RGD sequence) to which cells readily attach ! J’klnssia anti Hubbell, 1990; Anderson rf. trl.. 1994): and :Zi the US*! :ji biologically active molecules (Rutherford c>f ai., I 993a; 199% or the transplantation of cells which producp biologicalb, functional molecules (McGrath et rrl., 1992 i, What roLe might

190 Hanks ef a/.//n vitro models

tissue engineering play in the future of dental materials? Areas which readilv come to mind are: 1) the use of dental implants fabricated in such a way as to assure osteoinduction; 2) generation of new bone to replace bone lost because of disease or trauma; 3) a root surface treatment which will promote the regeneration of the periodontal membrane; and 4) treatment of the surfaces of exposed vital pulps to guarantee dentin bridging and maintenance of viability of the PdP.

ACKNOWLEDGMENTS We acknowledge the support of Research Grant No. DE09296 from the National Institute of Dental Research, Bethesda, MD 20892.

I’rewntc~d at thv 199.5 AI)M annual meeting; La Jolla, CA, USA; November 1995

Address correspondence and reprint requests to: (h-l T Hanks 5223 School of Dentistry LTniversity of Michigan 1011 N. Ibivewity Avenus Ann Arbor. MI 48109-1078 ITSA

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