Role of fibroblast subpopulations in periodontal physiology and pathology Christopher A. G. McCulloch’ and Sandra Bordin®® ‘Faculty of Dentistry, University of Toronto, Toronto, Canada and "Department of Periodontics and "Research Center in Oral Biology, University of Washington, Seattle, Washington, USA, McCulloch CAG, Bordin S: Role of fibroblast subpopulations in periodontal physiology and pathology. J Periodont Res 1991; 26. 144-154. Fibroblasts are the principal cell type in the soft connective tissues of the periodontium; they perform important functions in development, physiology, and disease. A growing number of reports have indicated site-specific pheno- typic variation of fibroblasts. Heterogeneity of metabolic traits has been demon- strated in cells from healthy and diseased tissues. The tissue distribution and relative proportions of fibroblast subpopulations have a significant impact on the regulation of connective tissue function in health and disease, Introduction The fibroblast is the predominant cell type in the soft connective tissues of the periodontium and consequently plays a central role in normal func- tion and in pathologic alterations, Fibroblasts, thesize and maintain a diverse group of connective tissue matrices throughout the periodontium, and exhibit motility and contractility, functions which help shape structural organization of the tissue during regeneration and development (1). Despite their ubiquity and their relative facility for propa- gation in vitro, fibroblasts are still an ill-defined group of cells and are usually identified by cigar- shaped or stellate morphology, presence of vimen- tin intermediate filaments, and synthesis of matrix collagens and fibronectin. These features suggest a similarity of form and function which may be misleading. Examination of the diversity of syn- thetic products, rates of product synthesis, re- sponses to regulatory molecules, cellular turnover rates, and morphological features indicates that fibroblasts of healthy and diseased tissues are het- erogeneous with respect to these and perhaps other unexamined properties. Fibroblast heterogeneity in oral tissues was last reviewed in 1983 (2). In the present review, some earlier data and recent e dence for generation of separate cell lineages and of functional heterogeneity of fibroblasts is sum- marized. The fundamental roles that fibroblast subsets may play in development, maintenance, pathological alteration, and wound healing in the periodontium are critically discussed. This study was supported by MRC (Canada) MA-8903 and by (USA) NIH /NIDR DE08229-03, key words: fibroblast — pathology ~ physiology ‘Accopted for publication 6 August 1990 Origin of cell lines during development of the periodontium Phenotypic heterogeneity within a cell population implies the existence of mechanisms for generation of cellular diversity and subsequent modulation At least some of this generation of diversity might be expected to occur during development. Develop- ‘mental processes proceed by means of discrete (and often overlapping) events involving synthesis and remodeling of new structures that ultimately result in the highly specialized states necessary for adult function. Caplan et al. (3) have hypothesized that, during embryogenesis, variants of individual mol- ecules and cells, called isoforms, arise and may exert a regulatory role in remodeling during devel- opment. In this context, fibroblast isoforms arising from mesenchyme could be responsible for the dif- ferent types of soft-tissue matrices formed in dental and periodontal structures. In embryonic and fetal tissues, mesenchyme ap- pears as a loose connective tissue with populations of stellate-shaped cells embedded in an extracellu- lar matrix. Mesenchymal cells are precursors for a diverse group of connective tissue cells including smooth muscle cells, endothelial cells, chondrobla- sts, pericytes, adipocytes, osteoblasts, odonto- blasts, and fibroblasts. In fetal jaws, mesenchymal cells are induced by oral epithelium to an odonto- genic specificity (4). In the layer of dental follicle that invests the tooth germ, some mesenchymal cells are capable of differentiating into cells of ce- mentogenic and osteogenic lineages, as well as into fibroblasts of the periodontal ligament (5-7) By comparison, little is known about the devel- opmental origin of gingival fibroblasts. Prior totooth eruption, the gingival ridge and later the gum pads mark the sites of tooth crown penetration through the mucosa. The fibroblasts located in the gum pads and in the fully developed lamina propria of the gingiva probably arise from cells in the peri- follicular mesenchyme (8). It is conceivable that some cells of the nascent periodontal ligament could contribute to gingival fibroblast populations. Currently it is not clear whether a single stem cell for fibroblasts, osteoblasts, and cementoblasts exists in fetal or adult periodontal tissues. Large changes in the level of expression of the c-myc protooncogene (9), the extracellular matrix mol- ecule tenascin (10), and cell-surface proteoglycan (11) in cells of dental mesenchyme during tooth development indicate considerable temporal, modulation of important regulatory molecules. These processes may reflect the earliest detectable sorting of cells along separate phenotypic path- ways. Wide variations in binding of "I-EGF (epi dermal growth factor) to dental mesenchyme cells, and dental follicle cells, both within the same tooth germ and at different developmental stages, have been interpreted to show that EGF-receptor ex- pression is specific to the developmental stage of the tooth, and is related to maintenance of prolif- erating mesenchymal cells (12). While the above reports may indicate tissue-spe- cific and within-tissue variation of expression of regulatory cellular molecules, they do not prove the existence of separate cell lines. The data could be interpreted equally to show site-specific modu- lation for that particular developmental stage and that these cell populations are of uniform compo- ion and are possibly totipotent. Indeed, the ca- pacity of follicle and papilla cells to produce teeth with cementum, periodontal ligament and bone upon recombination (6, 7) strongly supports the totipotency of these cells. At early times of tooth formation (which is synchronous with periodontal ligament formation), there is no compelling data to demonstrate the existence of phenotypically stable and heterogeneous populations of fibroblasts in periodontal ligament. Indication of sorting of cells into populations with different functions has been reported during later stages of root development and tooth erup- tion. Cell kinetic data indicated the presence of discrete populations of progenitor cells in the api cal portion of the periodontal ligament in 12-day- old mouse molars (13). These progenitor cells ex- ibited some of the characteristics of stem cells and gave rise to progeny which migrated coronally. A close temporal relationship between fibroblast proliferation and migration and molar tooth erup- tion was demonstrated, indicating that, in the st functional stage of periodontal ligament, Fibroblast subpopulations 145 discrete populations of fibroblasts arise both with and without the capacity for cell division. These findings suggest the existence of separate popula- tions of proliferating and differentiating cells, Such a hierarchy is observed in mammalian cell systems in which considerable diversity of cell functions is required (e.g., hemopoietic tissues). The existence of phenotypically stable cell lines in developing periodontal tissues has been exam- ined indirectly by Barrett and Reade (14), Unmi- neralized third-molar tooth germs, teeth with partly formed roots, and fully erupted teeth with developed roots were removed from mice at vari- ous ages and transplanted to recipients beneath the renal capsule. Only cells from incompletely formed teeth were capable of synthesizing all the peri- odontal tissues. Apparently, cells that had differen- tiated into fully functional cells of periodontium had lost the capacity for formation of bone and cementum, and were lineage-restricted. To demonstrate existence of separate fibroblast subpopulations in either early or late tooth and periodontal development, it would be necessary to provide unambiguous evidence of discrete cell types using a variety of different markers, and then to use these markers to separate and characterize individual cell types. Current challenges in these studies are the isolation and production of markers specific to a given phenotype, and the demon- stration that absence of expression of a particular phenotype will lead to inappropriate development of the tooth and periodontium. Such information on separate origins and lineages of periodontal ligament and gingival fibroblast cell populations would not only y the biology of tooth devel- opment, but could be important in rationalizing and improving the response to regenerative wound healing procedures. Boyko et al. (15) indicated that gingival fibroblasts grown in vitro fail to contribute to reformation of a periodontal ligament around teeth implanted in dogs, while periodontal ligament fibroblasts were observed to either contribute to regeneration or at least not to inhibit regeneration, These phenomenological findings suggest that dif- ferent functional properties between gingival and periodontal ligament fibroblasts may be important in developmental processes. More definitive studies of cellular lineages in developing and adult peri- odontium are needed. Generation of cell lines in adult tissues Adult tissues such as bone, muscle, dermis, and brain contain cell lines that are organ- or tissue- specific and are responsible for tissue function and homeostasis. In adult mammals, most renewing cell systems (¢.g., blood, gut epithelium) contain148 = McCulloch and Bordin subpopulations which can be discriminated on the basis of proliferative capacity and various special- ized cellular functions. For example, the hemopoie- tic stem cell can give rise to all types of blood cells ate numbers of ampli visions and differentiation steps while, in comparison, the tissue macrophage is an end-of-line, fully speci -d cell type that is usually incapable of further cell division. The search for such lineage characteristics and cellular hierarchies in fibroblast populations within mature periodontal tissues has been hampered by lack of clear-cut specialization markers and ob- scured by the occasional report showing that even cells with differentiated phenotypes are capable of cell division (16-18). Bayreuther and colleagues (19, 20) have shown the existence of mitotic and postmitotic skin fibroblast populations that can be further subdivided on the basis of certain morpho- logical features and “'S-methionine-labeled nuclear and cytoplasmic protein patterns on 2-dimensional gels. The data have been interpreted to show spon- taneous differentiation along a 7-stage cell lineage. In vivo correlates to these in vitro cell types have been proposed (19, 20). Taken together, these find- re Suggestive of a system of lineages in fibro- blast populations which are reminiscent of other renewal cell systems, and which include fibroblast stem cells (20; Fig. 1). Progenitor cells Are stem cells found in periodontal tissue, and if so, in what sites? In periodontal ligament, a num- ber of reports in both wounded (21, 17) and nor- mally functioning tissues (22, 23) have indicated that paravascular cells with small nuclei exhibited many characteristics of early progenitor cells, poss- Stromal Cell Lineages in Periodontium Tncreasing Differentiation Fig. I. Hypothetical diagram illustrating possible lineage re- lationships between synthetic cell types of periodontal tissues. Periodontal stem cells may be located in bone marrow spaces adjacent to the periodontal ligament, and/or around blood vessels in the periodontal ligament. ibly stem cells. These paravascular cells have a relatively high basal rate of proliferation, undergo increased proliferation during a repopulation re- sponse, and show certain kinetic characteristics similar to stem cells in renewal cell systems. On the basis of these properties, it has been proposed that the paravascular cell may be a separate cell type from fibroblasts located in the avascular part of the ligament, and may represent an early progeni- tor cell. Paravascular cells exhibit spatial cluster- ing, which suggests a possible clonal distribution of progenitors and their progeny (23). If separate lineages of fibroblasts start to emerge after tooth eruption, then clonal expansion of phenotype-re- stricted cell populations could be produced from these clusters. Cell kinetic methods have suggested that progenitor cells in endosteal spaces may mi- grate via communicating channels into the peri- odontal ligament and there augment fibroblast, cementoblast, or osteoblast cell populations (24; Fig. 2). This interpretation has received support from the work of Cho ef al. (25) showing that labeling of the epidermal growth factor receptor with “I-EGF is much more intense over bone and periodontal ligament cells than gingival cells. Little is known about progenitor cell popula- tions in gingiva. Pender ef al. (26) have identified two putative progenitor cell populations in rat gin- giva; one population is located in the midpapilla and another is found close to the epithelial attach- ment. The functional characteristics of these cells, are unknown, as is their capacity to repopulate wounded tissue, When gingival fibroblasts are propagated in vitro.they appear to be unable to participate in regeneration of periodontal ligament Paravascular Brogenior cells jood vessels _ Blood vessel Vascular channel>; Periodontal Progenitor 2 cel lning ‘vascular channels “Bone marrow space Fig. 2. Illustration to show connections between periodontal ligament and adjacent endosteal spaces. Separate populations of fibroblast progenitors are located on walls of vascular chan- nels and immediately around blood vessels in periodontal liga ‘ment, Some of the proliferating cells lining vascular channels contribute to periodontal ligament cell populations by mi- aration (24)(15). This may indicate lineage restriction if gin- gival progenitor cells are unable to switch from a gingival to a periodontal ligament phenotype. In vivo evidence of heterogeneity In periodontium supporting fully erupted teeth, fibroblast populations are in steady-state (22). No net growth of cells or increase of tissue volume occurs in physiological function in spite of rapid and extensive remodeling of periodontal tissues (27). Homeostasis is achieved in large part by the metabolic activities of fibroblasts. One of the major problems in studying fibroblast function is to ident- ify cell populations of a single type among a mix- ture of different cell types. Examination of peri- odontium by light microscope alone provies little reason to suspect the existence of discrete subsets of fibroblasts, each with a specific function in re- modeling and homeostatic processes (1). Detailed examinations by Roberts _ and Chamberlain (28) have identified four different morphological types of cells in periodontal liga- ment using scanning electron microscopy. One of these cell types exhibited numerous pseudopodia- like structures which were similar in morphology to those of migrating periodontal ligament cells observed in culture. Such in vivo morphology was interpreted as being indicative of a migratory cell type. This is not an unreasonable supposition in view of the large body of data showing migration of fibroblasts in periodontal tissues (17, 29-33), These reports do not provide any specific indi- cation that there is a separate subset of migrating fibroblasts. Roberts and coworkers (34, 35) have used light microscope nuclear morphometry of periodontal ligament fibroblasts to examine cel linages and the level of differentiation of fibroblast and osteogenic precursor cells, The data show that discrete popula- tions of cells with varying nuclear size are related to level of differentiation and ability to form osteo- blasts and fibroblasts. Both osteogenic and fibro- blastic precursors and differentiated cell types were identified, and some of these cell types were more capable than others of subsequently undergoing cell division. Cho and Garant (36) examined the morphology of fibroblasts in aged mice periodontal ligament and found that up to 17% of cells were multi- nucleated. These cells did not resemble osteoclasts or foreign body giant cells. They contained dense granules which could have been residues of pre- vious lysosomal activity, and exhibited collagen secretion granules and Golgi apparatus. The func- tion of these cells is unknown, but they are not found in other connective tissues such as tail ten- Fibroblast subpopulations 147 don. These findings suggest that the periodontal ligament has conditions favorable for cell fusion ‘or possibly requires multinucleated cells for homeostasis in aging animals. Electron microscopy studies have revealed sig- nificant morphological heterogeneity in actin content (37), amount of endoplasmic reticulum (38), phagocytosis of collagen fibers (39-41), and nuclear-cytoplasmic ratio (18), both within a tissue and between anatomically separate tissues, The existence of fibroblasts with such diverse morphologies is not in itself indicative of any functional heterogeneity. The data of Azuma er al. (37) showing cells with wide variation in num- bers of microfilaments and other cytological fea- tures associated with myofibroblasts (42) could be interpreted to show that periodontal ligament contains cells with a contractile or migratory cellular phenotype (43). This concept has been questioned by Shore and Berkovitz (44), who suggested that microfilaments are associated with endocytosis and exocytosis. More recent data using immunofluorescence and cytoskeletal markers have supported the view that the myofi- broblast is a separate cell type involved in tissue contraction (45) and is particularly common in granulation tissue Phagocytosis One of the most striking and widely cited features of periodontal tissues is the rapidity of collagen turnover, which is mediated at least in part by intracellular degradation (46). Therefore, phago- cytosis of collagen is likely to be an expression of collagen turnover and/or remodeling (39, 47, 48). Depending on experimental conditions, mem- brane-bound collagen profiles are found in varying proportions in fibroblasts of both periodontal iiga- ment and gingiva (38, 39, 41), Whether all fibro- blasts are capable of phagocytosis is unknown, but the finding of relatively high proportions of phagocytic cells in specific sites within the pe odontium indicates that discrete fibroblast subsets may be committed to phagocytosing collagen and pethaps other components of extracellular matrix. The observation that volume density of ingested collagen can be modulated by hypofunction (48) provides further support for the existence of a phagocytic phenotype, and may provide a mechan- ism by which collagen turnover rates are adjusted throughout tissue. Increasing the number of, phagocytic cells engaged in collagen remodeling could provide a means of regulating matrix degra- dation.448 = McCulloch and Bordin In vitro metabolic studies Current in vivo reports are consistent with, but do not prove, the existence of heterogeneous popula- tions of fibroblasts. In contrast, a growing body of literature indicates that when fibroblasts from normal and diseased tissues are grown in vitro, large variations in terms of matrix biosynthesis, enzyme activities, expression of surface proteins, response to inflammatory mediators, and prolifer- ative potential are observed. Hassell and Stanek (49) observed a considerable degree of stable vari- ations in synthetic properties and_ proliferative rates among six morphologically indistinguishable mass cultures of human diploid fibroblasts derived from a single biopsy of a normal gingival papilla tip. These data suggest that fibroblast subtypes displaying quite different phenotypic character- istics may coexist within a given tissue. Studies of, clones derived from single cells may aid in eludica- tion of this hypothesis; however, from a practical point of view such an approach could prove diffi- cult, Clonogenic studies of in vitro systems rely on the intrinsic ability of a cell to proliferate to form a colony of a size large enough for reliable bioassays Since an intrinsic property of normal diploid fibro- blasts is finite replicative life span, clone size is related to the number of divisions which the cell has undergone: the more divisions that have oc- curred, the smaller the clone size. Furthermore, biosynthetic changes occur as a culture ages: there- fore experiments designed to evaluate activities of, fibroblast classes must preclude evaluating culture age rather than clonal peculiarities (50). Reports from in vitro work are reviewed to describe a) the nature and extent of functional heterogeneity among resident fibroblasts; b) the mechanisms reg- ulating synthetic activities; and c) the involvement of fibroblast subtypes in pathogenesis of various disease states and in wound hea Collagen synthesis and degradation Gingival and periodontal connective tissues are composed of a number of structurally and fune- tionally distinct collagen types that serve to anchor teeth to supporting structures and to provide tone and form to the tissue (51). Normal, inflamed, and hyperplastic tssues contain types I and III collagen as the major collagens, and types IV and V as, minor species (see review, (2)). Qualitative and quantitative differences in relative proportions of type III collagen, and in rates of procollagen pro- cessing among fibroblasts cultured from different periodontal tissues have been described (52). Com- parisons in vitro of collagen production (as well as, protein synthesis and alkaline phosphatase levels) between human periodontal and gingival fibroblast populations derived from the same patient revealed different metabolic behavior, which may be indica- tive of separate cellular functions for maintenance and regeneration of periodontium (53). Clonal fibroblast populations from human gingiva have been shown to be heterogeneous with regard to production of different amounts and types of col- lagen (54-56). Similarly, clonal populations of fibroblasts from periodontal ligament synthesize different levels of collagens, and produce distinctly different ratios of collagen types (57). The content, synthesis, and distribution of col- lagen types are altered by pathological conditions. Modifications in distribution, ultrastructure and organization of types I and III collagen have been observed in fibrotic gingiva of patients with chronic periodontitis (58). Fibroblasts derived from oral fibrous papular lesions synthesize an increased ratio of type I to type III collagens (59). Types I and III collagen are both lost from foci of inflammation in human gingiva. At these sites type V collagen content is increased, and a new collagen, type I trimer, can be detected (60). Type I trimer is synthesized in vitro by fibroblasts obtained from inflamed but not from normal gingiva (61). Alter- ations in type I collagen production in diseased periodontium as measured in vivo by in situ hybrid- izaion with specific CDNA probes indicated re- gional variation of mRNA content (62). It has not, yet been established whether observed variations in collagen activities may be due to selection of specific fibroblast subtypes during progression of the disease, or to other factors such as modulation of fibroblast synthesis activities by environmental ligands (59, 62, 63). athologic alterations observed in hyperplastic gingiva have been interpreted to involve cell selec tion (64, 65). Excessive accumulation of collagens, especially type III, and other matrix components, has been described in fibroblasts cultured from drug-induced hyperplasia (64-66). In contrast, compared to normal cells, collagen synthesis was reduced by one-half in fibroblasts cultured from gingival biopsies of a patient with familial gingival hyperplasia. (67). Taken together, the reported ob- servations indicate that fibroblasts obtained from both spontaneous and drug-induced hyperplasias are abnormal, and that the abnormalities persist in culture (Table 1). Rapid turnover of collagens in the matrix of both gingiva (51) and periodontal ligament (46) is, essential for continuous attachment of the roots, to the alveolar bone. Two pathways of collagen degradation have been identified: an extracellular collagenase-dependent route, and an intracellular pathway independent of collagenase (68). LittleTable 1. Distribution of collagen types in cultured oral fibro- blasts from Collagen Types Type Firovie gingiva 38 Fibrous papules same] 39 Inflamed gingiva |} _ present 2,60, 62,63 Fibroblasts near periodontal pocket | a Fibroblasts near inflammatory infil- trates tf a Phenytoin-induced ot hyperplastic gingiva tf same detected 2, 64-66 Familial gingival not hyperplasia i detected 67 information is available on the relative utilization, of these routes in health and disease. Fibroblasts from periodontal tissues utilize both pathways; by extracellular release of metalloproteinases which degrade mature collagens (69, 70), by intracellular degradation of newly synthesized collagens after exposure to inflammatory agents like prostaglandi- (71), and by phagocytosis with subsequent lysosomal degradation of fragments of collagen fibrils (see above section on phagocytosis). Fibro- blasts of rodent periodontal ligament appear to be unique in that all degraded collagen passes through the phagolysosome pathway during physiological turnover and remodelling (72) Fibroblasts of periodontal granulation tissue ex- t in vitro altered ratios of mRNAs for type I to type III collagens, and a difference in glycosyla- tion of dermatan sulfate proteoglycan (73). These cells might be similar to myofibroblasts as orig- inally described by Gabbiani e al. (42, 74) in vari- ous models of granulation tissue formation. Peri- odontal ligament fibroblasts contracted collagen gels faster than did gingival fibroblasts, suggesting that these cells may reversibly modulate to a con- tractile state (75). Whether only cells of granulation tissue and certain periodontal fibroblast subsets. exhibit the contractile phenotype is unknown. So far, it has not been demonstrated that all peri- odontal ligament cells are capable of switching to 4 contractile phenotype, nor that such a switch is reversible or irreversible. Noncollagenous proteins A group of acid-soluble noncollagenous proteins ranging from 15 kDa to 75 kDa in size, and consist- ing of at least 45%-S6% keratins, appears to be present in moderate to large amounts in whole extracts of healthy gingival tissues and in traces in dental lamina (76, 77). The amounts of regenerated Fibroblast subpopulations 149 gingiva were much greater than in extracts of nor- mal gingiva. Only traces were found in extracts of gingiva from animals with spontaneous perio- dontitis. While these observations clearly indicate that pathological conditions affect gingival noncol- lageneous proteins, their locations, biochemical na- ture, and biological function are not well under- stood. Extracts of gingival noncollagenous pro- teins, partially purified by exclusion of the keratin fraction, have the ability to bind to native collagen (76). Such association with collagen supports the hypothesis that these proteins may play a structural role in gingiva, and their loss during the early stages of periodontitis may be an important event in progressive tissue destruction (77). Glycosaminoglycans (GAGs) and proteoglycans (PGs) GAGs and PGs play an important role in assembly of extracellular matrix in both health disease (see reviews, refs. 78, 79). Activities of PGs in normal and diseased gingival and periodontal tissues have been recently reviewed (80). Normal and inflamed oral tissues display measurable differences in secre- tion of chondroitin sulfate (CS) and dermatan sul- fate (DS). Hyaluronic acid (HA) from diseased gingiva has a smaller molecular size than HA from normal tissues, Whether these differences represent alterations in existing fibroblast populations. or selection of predominant subsets of cells within the tissue under inflammatory conditions, was not stablished. The possibility that granulation tissue ibroblasts may represent a distinct phenotype with regard to GAG production was supported by ob- servations that these cells contain shorter GAG. chains attached to the core. Whether they have a role in matrix assembly of granulation tissue re- mains to be shown (73). Differences in CS and HA production have been reported in uninjured and wounded rabbit oral mucosa, and have been inter- preted to indicate that different fibroblast sub- strains can populate a given oral site as a function of variables such as injury and/or healing status (81) Modul: ion of fibroblast populations As soon as separate lines of fibroblast subsets are established in various sites within the periodon- tium, then the size of the subpopulation and its metabolic regulation may be achieved by several different mechanisms. Regulation of function and population size may be mediated by: a) clonal ex- pansion of a subset of cells with high proliferative rate (23); b) clonal deletion caused by either selec- tive cell death and/or inhibition of proliferation by growth regulatory factors or cytotoxic substances150 McCulloch and Bordin from activated lymphoid cells; or c) directed mi- gration of subsets into a site by local release of chemoattractants. Evidence for these putative mechanisms in regulation of fibroblast populations is described below. Influence of extracellular ligand ‘A variety of substances derived from blood and inflammatory cells, including growth factors and cytokines, regulate fibroblast growth and synthesis. There is wide variation in the response of fibro- blasts from periodontal tissues to some of these factors. Bronson et al. (82) observed that exposure of rabbit oral mucosa cells to interleukin-1 (IL-1) altered the GAG profiles of uninjured cells, while GAG profiles of wounded cells remained un- altered. Early observations by Ko et al. (83) indi- cated that prostaglandin-E, inhibits growth and synthesis of gingival fibroblasts, and that the effect is total on a subpopulation of cells, while not af- fecting the remaining nonsensitive cells. Korotzer et al. (84) reported that normal human serum con- tains mitogen(s) capable of inducing DNA syn- thesis and growth in a subpopulation of human gingival fibroblasts, and that C1 complement com- ponent may be an active factor. Bordin and Page (85) reported that a fibroblast subtype with unique surface markers for Clq com- plement, and expressing proliferative and synthetic properties expected of wound-healing cells, could be generated from tissue only when factors from platelets were present in the medium, Platelet fac- tors are found in high concentration at sites of connective tissue injury and inflammation (review, 86). Not surprisingly, the proportion of this sub- type was greater in cultures obtained from chronic- ally inflamed gingiva (Fig. 3). Whether this subtype comprises a totally independent lineage, or whether it may be a relatively undifferentiated progenitor population that gives rise not only to like progeny but also to other more differentiated cell types, has not been resolved. Cell surtace heterogeneity: jeceptors The cell surface plays a vital role in transduction and modulation of external signals that initiate and regulate a variety of fibroblast functions, including cellular proliferation, migration, and. differen- tiation (87). Variations in expression and function of cell-surface components of different fibroblast subtypes could have profound effects on modu- lation of synthetic activities of healthy and diseased periodontal tissues. Fibroblasts from normal oral connective tissues express surface receptors for a great variety of substances including extracellular Normal Tissue Granulation Tissue » a o o Relative number of cells ° T do” 80 "120" 160200 7” 40 " 80 120160” 200 Relative C1q fluorescence intensity Fig. 3. Clq binding profiles determined by using a fluorescence- ‘activated cell sorter in cultures of human healthy gingival or ‘granulation tissue. Cells in panels B and E, which were isolated and grown in Dulbecco medium with 10% platelet-rich human serum (PRHS), displayed two major peaks of Clq binding activity, Cells in panels C and F, which were isolated and grown in medium with 10% platelet-poor human serum (PPS), and the control cells in panels A and B, which were isolated and grown in medium with 10% [etal bovine serum (FBS), displayed only one peak of Clq binding activity. Notice the higher pro- portion of cells with increased Clq binding activity in panels DEF matrix components such as fibronectin and vi- tronectin (88), jokines such as IL-1 (89), and complement proteins (90). Investigation of func- tional changes of cell-surface receptors could pro- vide a well-defined experimental approach to the study of fibroblast heterogeneity, although such investigation has been limited in part becau: tailed knowledge of the functional mechanisms of, many receptors is still unknown. There is heterogeneity in complement Clq recep- tor expression among individual cells of the same strain and among strains of fibroblasts derived from different donors (90). At least two popula- tions were identified and separated using a fluor- escence-activated cell sorter and fluorescent anti- Clq antibodies (90). One population was found to express receptors specifically binding the globular domain of Clq with a high affinity, while the other type expressed receptors of relatively lower affinity which bind the collagen-like domain of the mol- ecule (84, 91). The two different forms of Clq receptors appear to have different functions. Re- ceptors for the globular domain of Clq have the capacity to induce nonimmune activation of classi- cal complement cascade, as assessed by generation of C4a and C4d fragments in normal AB serum (91). Receptors for the collagen-like domain of Clq appear to play a role in adhesion of fibroblasts tomatrix components (92). Inflammatory metabo- lites and mediators, including complement and platelet factors, may regulate activities of the differ ent fibroblast subtypes (86, 93) by amplification of the subtype with receptor for the globular domain of Clq. It has been proposed that this particular subtype may participate in tissue regeneration by activating the classical complement pathway, gen- erating mitogenic complement fragments on-site and Clq. The generated Clq could provide anchor- age for fibroblasts that express the adherence re- ceptor for the collagen-like domain of Clq. These cells, which represent the predominant resident cell subpopulation of normal gingiva may then repopu- late and remodel the tissue (Fig. 4). In pathological disorders, this mechanism could be a contributing factor in chronic inflammatory fibrotic lesions (92). Cell attachment Normal diploid fibroblasts require attachment to their extracellular matrix (ECM) for maintenance of normal tissue functions, as well as for wound healing and tissue regeneration. The biology of fibroblast-matrix interaction has been the focus of keen interest in studies of periodontal regeneration, and the composition and functions of components, of extracellular matrix of the periodontium have been recently reviewed (94). Key effectors in inter- actions of fibroblasts with ECM are glycoprotein- attachment factors on the cell surface. Alterations in amount, distribution, and specific interaction of these factors have been observed in the phenotypes, of fibroblasts from diseased tissues. A decrease in relative amount of a 140 kDa cell-surface sialogly- coprotein, known to the main cell-surface compo- nent of fibroblasts, has been observed in granu- ADHESION AND, SYNTHESIS GROWTH Fig. 4. Scheme of the proposed functional interactions of CL and Clq with fibroblast subtypes expressing receptors for the globular and collagen-like domains of the molecule, (C1-inh= Cl-inkibitor), Fibroblast subpopulations 154 lation-tissue fibroblasts of the periodontium (73). Fibroblasts can attach in vitro to a variety of ECM components through specific cell-surface re- ceptors. Farsi e¢ al. (95) have documented hetero- geneity in attachment of gingival fibroblasts to collagen substrates. Jn vivo, itis likely that a major ECM component for fibroblast attachment is fib- ronectin (FN). FN coats collagen fibrils of peri- odontal ligament (96) and gingiva, and sometimes fills spaces between cell membrane and adj collagen fibrils (97). Immunocytochemical loc: tion of FN at specific fibroblast-to-matirx contact, sites in periodontal tissues of the Beagle dog re- vealed that, in healthy tissue, contact sites are small, and evenly distributed on the cell surface. In in- flamed tissue, contact sites become enlarged and fibronexi associated with stress fibers are found (98). The authors speculated that such increased cell-to-matrix contacts may result in changes in cell motility. Significance of fibroblast heterogeneity in health and disease The interrelationships between cell lineages and differentiation potential contribute to fibroblast di- versity in the adult. Understanding of the biology of the fibroblast cell system is complicated by the functional demands placed on different tissues within the periodontium, Schor and Schor (99) and MacKenzie (100) suggested that specificity of fibroblast phenotype may influence not only the metabolism of connective tissues but also of ad- acent epithelia. An intriguing concept in this re- gard is that regional specificity of epithelia in peri- odontal tissues may depend at least in part on the phenotype of underlying fibroblasts. Epithelial cells close to fibroblasts in the transseptal fiber zone of gingiva and in periodontal ligament may exhibit a junctional epithelium phenotype, while epithelial cells near the lamina propria of gingival connective tissue may exhibit an oral epithelial or sulcular epithelial phenotype. Studies of regulation of epithelial cell populations by direct cellular in- teractions or by regulatory products produced by fibroblasts subsets are at an early stage. Involvement of fibroblast subtypes in the patho- genesis of periodontal diseases has been discussed by Hassell er al. (66), Hassell and Stanek (49), Ko. et al, (83), and Narayanan and Page (2). They suggested that pathologic alterations of oral con- nective tissues may result from a clonal imbalance of resident fibroblast subtypes rather than the pres- ence of abnormal cells. Relative proportions of various subtypes and their functional activities in tissues may be regulated by specific cellular interac- mns with bioactive molecules present in the sys-182. McCulloch and Bordin temic circulation, as well as with cytokines pro- duced by fibroblasts themselves, neighboring epi- thelial cells, platelets, and infiltrating inflammatory cells. Once healing and tissue repair is completed, composition of tissue fluids and the proportions and numbers of fibroblast subtypes return to nor- mal, Any aberration in these processes may lead to pathological alterations. Recently, Cockey et al. (101) have modified and extended these concepts by proposing that, under optimal growth conditions, the genotype may be chiefly controlling proportions of the various fibro- blast subtypes in oral tissues, but environmental factors may be more important regulators under suboptimal conditions. Additional evidence for a potential but restricted environmental influence on cell metabolism has been provided by the studies of Bronson er al. (81) which indicate that range of behavior expressed by fibroblast substrains under changing environmental stimuli is intrinsic to the cells. Existing data in support of the heterogeneity hypothesis are largely indirect, and represent the average of whole cultures. Definitive proof for fibroblast heterogeneity rests on availability of bio- markers which could aid in unequivocal identifi- cation of subtypes, both i vitro and in vivo. Such biomarkers could be used to determine whether a phenotype 1) is retained in a heritable manner; 2) is unaffected by epigenetic factors; and 3) can be selected in vivo under specific conditions. 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Cockey GH, Boughman JA. control of variation in hum: ation rate, Jn Vitro 1989; 28: Harris EL, et al. Genetic tingival fibroblast prolifer- 5-258, Address: CAG. McCulloch Faculty of Dentistry University of Toronto 124 Edward Street Toronto, Ontario, Canada MSG 166This document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.