Advances in Dental Research

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In Vitro Models of Biological Responses to Implants D.M. Brunette ADR 1999 13: 35 DOI: 10.1177/08959374990130011201 The online version of this article can be found at: http://adr.sagepub.com/content/13/1/35.citation

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IN VITRO MODELS OF BIOLOGICAL RESPONSES TO IMPLANTS D.M. BRUNETTE

Faculty of Dentistry University of British Columbia 2199 Wesbrook Mall Vancouver, BC, Canada V6T 1Z3 Adv Dent Res 13:35-37, June, 1999

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n vitro models have been widely used to investigate biological responses to biomaterials, and the materials adopted for implantology have been no exception. Diverse populations of cells—including epithelium (Brunette, 1986b), fibroblasts (Brunette, 1986a), osteogenic {e.g., Chehroudi et aL, 1992; Martin et aL, 1995; Morgan et aL, 1996; Layman and Ardoin, 1998), and osteoclastic populations (Gomi et aL, 1993)—have been cultured on materials used in implantology, such as titanium and hydroxyapatite. Studies of implant failure have indicated the necessity of proper surface preparation for clinical success (Lemons, 1988). Similarly, commercially available dental implants differ in their topographies (Brunette, 1988), from machined surfaces to plasma-sprayed or porous coatings, with each manufacturer providing a rationale for its choice of surface. Since surface chemistry and surface topography are important determinants of implant performance, the effects of topography {e.g., Martin et aL, 1995; Qu et aL, 1996) or surface preparation (Baier et aL, 1984; Stanford et aL, 1994) have been studied in vitro by a wide variety of outcome variables. Given their relative ease of use and lack of expense, in vitro models would appear to provide an attractive approach to investigating biological responses to implant materials. There are two broad categories of use for in vitro systems in investigating cell responses to biomaterials: (1) fundamental studies on mechanisms of cell response, and (2) modeling physiological function in vivo. As an example of a mechanistic study, consider experiments on the cytoskeletal response of fibroblasts to the topography of titanium surfaces (Oakley et aL, 1997). This study used precisely defined topographies produced by microfabrication techniques that were subsequently coated with titanium. The cytoskeleton was observed by means of immunostaining techniques and confocal laser scan microscopy, and the media in Key words: In vitro models, implants. Presented at the 15th International Conference on Oral Biology (1COB), "Oral Biology and Dental Implants", held in Baveno, Italy, June 28-July 1, 1998, sponsored by the International Association for Dental Research and supported by Unilever Dental Research

some experiments included inhibitors of microtubule and actin filament formation, conditions not normally encountered in clinical implantology. The conclusion of the paper was that microtubules were the primary cytoskeletal component involved in topographic guidance. There was no claim that the results applied to any aspect of implantology, and the paper was published in a journal devoted to cell biology and not biomaterials. The use of cell culture for this kind of mechanistic study is common and not controversial. The value of using cell cultures to model physiological function in vivo, however, is controversial. Models are supposed to portray the fundamental properties of the system of interest. Models are grounded in the logic of analogy, and all analogies will at some point fail, because similarity is not identity. Typically, critics of analogies point out relevant differences between the systems being compared. Because cells must surely experience different environments in vitro and in vivo, studies using culture models are theoretically open to the criticism that they may well be irrelevant to cell responses in vivo. Debates over what conditions are relevant or irrelevant, however, are sterile, and eventually the question of the value of a model must be decided on how well the in vitro model predicts cell responses in vivo. For example, an early study on cell response to microfabricated titanium surfaces noted that the direction of epithelial outgrowths from gingival explants could be controlled by a grooved surface topography and suggested that grooves could be used to inhibit epithelial downgrowth on implants (Brunette et aL, 1983). That suggestion appeared to be correct when some grooved surfaces were placed on percutaneous implants in rats. But when it was examined in detail (Chehroudi et aL, 1990), it was found that the deeper grooved surfaces inhibited epithelial downgrowth, but that the interaction of the epithelium with the implant was markedly different from that observed in vitro. Thus, the in vitro experiments had predicted the response of shallow grooved surfaces in vivo, but for deeper grooves, the model had failed. Failures of in vitro models must be expected because, to a large degree, many of the questions raised about the validity of cell cultures as models of cell function in vivo have never been answered. Harris (1964), in his seminal book Cell Culture and Somatic Variation, reviewed what we might now call the early days of cell and tissue culture. Many of the problems of interpreting culture data were evident even then, and Harris pointed out that: (1) cultured cells underwent morphological simplification; (2) specialized features such as glandular tubules tended to disappear in vitro', (3) growth often produced simplified unorganized patterns; (4) some cells became transformed during culture so that they could proliferate indefinitely, and these deviant cells had chromosomal aberrations and differences in morphology; and (5) with increasing time of culture, initially complex mixtures of cells became dominated by a few cell types that thrive under culture


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conditions. Twenty-three years after Harris' book, a widely used text on cell culture asked the question, "What is a cultured cell?", and concluded that the exact nature of the cells that grow in culture is unknown (Freshney, 1987). Against the pessimistic viewpoint that using cultured cells to investigate possible biological responses to materials represents the dubious practice of using an unknown to investigate an unknown is the observation, repeated for many cell types, that even morphologically similar cells differ in their functional properties, depending on their tissue of origin. Thus, in dental research, for example, it has been found that fibroblasts cultured from the periodontal ligament differ from those of skin or gingiva when several culture parameters were measured (Marmary et ah, 1976). Moreover, complex mixtures of cells can differentiate in vitro to form structures typical of their tissue of origin, so that, for example, cells dissociated from fetal calvaria can produce bone-like nodules in vitro when given the appropriate conditions (Bellows et al., 1986). Thus, in vitro systems can model in vivo functions, but the validity of the model must be considered on a case-by-case basis. In my view, in vitro models are often poorly justified, because decisions are made on the basis of experimental convenience and potential for rapid publication. In the publish-or-perish world that most readers of this journal inhabit, there is no reason to belabor the need for investigators to maximize their numbers of publications. In my view, this need has led many investigators who use culture systems into following two simple rules for optimizing publication performance: (1) They choose an in vitro system with which they have experience. (2) They then practice the "transfer method" of investigation, which consists of applying or transferring a new principle or technique discovered in another field to one's own research (Beveridge, 1957). I should point out that there is no shame in practicing the transfer method. I have used it myself on occasion, and in doing so I am in good company. Beveridge (1957) notes that, for example, Lister's development of antiseptic surgery was largely a transfer of Pasteur's work showing that decomposition was caused by bacteria. So the transfer method is not to be despised, and Beveridge (1957) rates it as the most fruitful and easiest method in research. In practice, it works out to a number of simple steps: (1) Choose a novel technique that has been proven successful in some related field of investigation at the current time. (In the study of in vitro models of biological responses to implants, almost any technique of molecular biology or cell signaling will do.) (2) Arrange a collaboration with competent practitioners of the technique. (3) Apply the technique to your in vitro system and choose an advantageous outcome variable. (4) Adjust the system to produce a statistically significant effect. Adjustments might include altering concentrations of growth factors to extreme levels or, if

you are dealing with surfaces, producing substrata that vary widely in surface energy or density of topographic features. (5) Publish the results, taking care to include the collaborators and their grants in the list of authors and acknowledgments, respectively. For a paper to be publishable, it is often the case that there must be a statistically significant difference between groups. In practice, obtaining novel and publishable results with the transfer method is not difficult, because the investigator has so many options for making groups differ. Consider osteogenesis as an example. First, a virtual host of factors can be added to inhibit or enhance osteogenesis, including vitamin C, dexamethasone, beta glycerophosphate, bone morphogenic proteins, cytokines, proteoglycans, collagen, drugs, bone cements, and extracts that remind one of the witches' brew in Macbeth, and all of these can be delivered in various forms and concentrations. Second, the substrata for culture can be varied, from standard tissue culture through surfaces currently used for implants to experimental surfaces produced by novel fabrication or surface-treatment methods. Third, an investigator can choose from an abundance of possible outcome variables, such as mineralized matrix production (e.g., bone nodules), cell morphology, histochemical or immunochemical identification of markers (e.g., APase, osteocalcin, BSP), gene expression for various markers, receptors, and signals. It is evident from even this brief examination that there are many options available to investigators to ensure that at least some groups differ in some parameter, so an investigator, if sufficiently determined, is almost guaranteed a publishable result. The result of the transfer method is that it does generate novel, sometimes interesting, and sometimes unexpected results. The problem for readers of the literature is that, since systems are chosen on the basis of experimental convenience, they can be irrelevant to the actual biological phenomenon that is being modeled. The sequelae of the transfer method are that the literature contains isolated results on many systems, bandwagons form and roll along with their own momentum, few studies are ever fully replicated, and conflicting data make it difficult for conclusions to be generalized. What, then, is the reader to make of the relevance of the rapidly expanding literature of cell culture studies on biological responses to implant materials? First, it should be recognized that the greater part of the literature will give information on possible mechanisms underlying responses to biomaterials. This literature can be appreciated for the insight it gives into cell biology, but much of it will not be of relevance to implants in vivo, because the conditions used, chosen as they most likely were on the basis of experimental convenience, are unlikely to model what happens in vivo. It would be useful to distinguish the hypothesis-driven studies from those generated by a "shotgun" approach, but in practice this is difficult, for, as stated by Medawar (1964), "the scientific paper is a fraud in the sense that it does give a totally misleading narrative of the process of thought that goes into the making of scientific discoveries". After the data have been produced, it is not difficult to generate the hypothesis that might have been tested. I think that the most important criterion for distinguishing the


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useful from the irrelevant is fruitfulness. That is, in this instance, can one imagine an in vivo system where the observed effect or conclusion can be tested? If the line of inquiry begins and can end only in vitro, it cannot provide much insight into how implants interact with cell populations in vivo. A second criterion concerns the investigative team, and briefly it involves the consideration of whether the studies being reported are part of a program of research rather than a "one-off opportunistic commando raid on an unsuspecting population of cultured cells. In this regard, the speakers on in vitro models of response to implants at the Baveno ICOB meeting are exemplary: Each has made a continuing contribution to our understanding of biological responses to implants, and the meeting provided an opportunity to review their ongoing studies.

REFERENCES Baier RE, Meyer AE, Natiella JR, Natiella RR, Carter JM (1984). Surface properties determine bioadhesive outcomes: methods and results. J Biomed Mater Res 18:337-355. Bellows CG, Aubin JE, Heersche JN, Antosz ME (1986). Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif 7W.SK* 7/tf 38:143-154. Beveridge WIB (1957). The art of scientific investigation. New York: Vintage Books, pp. 172-175. Brunette DM (1986a). Fibroblasts on micromachined substrata orient hierarchically to grooves of different dimensions. Exp Cell Res 164:11-26. Brunette DM (1986b). Spreading and orientation of epithelial cells on grooved substrata. Exp Cell Res 167:203-217. Brunette DM (1988). The effects of implant surface topography on the behavior of cells. Int J Oral Maxillofac Implants 3:231-246. Brunette DM, Kenner GS, Gould TR (1983). Grooved titanium surfaces orient growth and migration of cells from human gingival explants. J Dent Res 62:1045-1048. Chehroudi B, Gould TR, Brunette DM (1990). Titanium-coated micromachined grooves of different dimensions affect epithelial and connective-tissue cells differently in vivo. J Biomed Mater Res 24:1203-1219.

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Chehroudi B, Ratkay J, Brunette DM (1992). The role of implant surface geometry on mineralization in vivo and in vitro; a transmission and scanning electron microscopic study. Cells Mater 2:89-104. Freshney RI (1987). Culture of animal cells: a manual of basic technique. New York: A.R. Liss, Inc., p. 12. Gomi K, Lowenberg B, Shapiro G, Davies JE (1993). Resorption of sintered synthetic hydroxyapatite by osteoclasts in vitro. Biomaterials 14:91-96. Harris M (1964). Cell culture and somatic variation. New York: Holt-Rinehart Winston, pp. 123-195. Layman DL, Ardoin RC (1998). An in vitro system for studying osteointegration of dental implants utilizing cells grown on dense hydroxyapatite disks. J Biomed Mater Res 40:282-290. Lemons JE (1988). Dental implant retrieval analyses. J Dent Educ 52:748-756. Marmary Y, Brunette DM, Heersche JN (1976). Differences in vitro between cells derived from periodontal ligament and skin of Macaca irus. Arch Oral Biol 21:709-716. Martin JY, Schwartz Z, Hummert TW, Schraub DM, Simpson J, Lankford J Jr, et al. (1995). Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). / Biomed Mater Res 29:389-401. Medawar PW (1964). Is the scientific paper fraudulent? Saturday Review, August 1, pp. 42-43. Morgan J, Holtman KR, Keller JC, Stanford CM (1996). In vitro mineralization and implant calcium phosphatehydroxyapatite crystallinity. Implant Dent 5:264-271. Oakley C, Jaeger NA, Brunette DM (1997). Sensitivity of fibroblasts and their cytoskeletons to substratum topographies: topographic guidance and topographic compensation by micromachined grooves of different dimensions. Exp Cell Res 234:413-424. Qu J, Chehroudi B, Brunette DM (1996). The use of micromachined surfaces to investigate the cell behavioural factors essential to osseointegration. Oral Dis 2:102-115. Stanford CM, Keller JC, Solursh M (1994). Bone cell expression on titanium surfaces is altered by sterilization treatments. J Dent Res 73:1061-1071.
