Regulation of connexin 43 expression in human gingival fibroblasts.

AIMS: Abundance of connexin 43 (Cx43), a transmembrane protein that forms hemichannels (HCs) and gap junctions (GJs), is dynamically regulated in human gingival fibroblasts (GFBLs) during wound healing. This may be important for fast and scarless gingival wound healing as Cx43 is involved in key cell functions important during this process. Our aim was to uncover the factors that regulate Cx43 expression and abundance in GFBLs. We hypothesized that cytokines and growth factors released during wound healing coordinately regulate Cx43 abundance in GFBLs. RESULTS: TGF-ß1, -ß2, -ß3, PGE2 and IL-1ß significantly upregulated, while TNF-α and IFN-γ downregulated Cx43 in cultured GFBLs. TGF-ß1, -ß2, -ß3, IL-1ß and IFN-γ modulated Cx43 abundance at both mRNA and protein levels, while TNF-α and PGE2 regulated only Cx43 protein abundance, suggesting involvement of distinct transcriptional/post-transcriptional and translational/post-translational mechanisms, respectively. TGF-ß1-induced upregulation of Cx43 was mediated by TGFßRI (ALK5) and SMAD2/3 signaling, and this was potently suppressed by PGE2, IL-1ß, TNF-α and IFN-γ that inhibited SMAD2/3 phosphorylation. CONCLUSION: Regulation of Cx43 abundance in GFBLs involves transcriptional/post-transcriptional and translational/post-translational mechanisms that are distinctly modulated by an interplay between TGF-ß isoforms and PGE2, IL-1ß, TNF-α and IFN-γ.

Matrix metalloproteinase-7 in periodontitis with type 2 diabetes mellitus.

BACKGROUND AND OBJECTIVE: Elevated levels of matrix metalloproteinase-7 (MMP7) have been observed in serum samples of subjects with type 2 diabetes mellitus (T2DM) and in gingival tissues of subjects with periodontitis. The aim of the present study was to collect in vivo and in silico evidence on the role of MMP7 in the interplay between T2DM and generalized periodontitis (GP). MATERIAL AND METHODS: The extent of MMP7 expression and localization were immunohistochemically analyzed in gingival tissues of patients with GP with T2DM (T2DM/GP, n = 11), systemically healthy patients with GP (n = 7), and systemically and periodontally healthy controls (n = 11). An in silico network model was built to determine the interactions between MMP7 and T2DM pathways. Regulation of neutrophil transmigration by MMP7 was analyzed in a knock-out mice model. RESULTS: In human gingival tissues, the proportion of cells with robust MMP7 expression was elevated in patients with T2DM/GP in comparison to controls (P = .014). According to the in silico analysis, "hydroxyl radical" and "hydrogen peroxide" compounds were among the most central nodes of the network, and were within the shortest paths connecting "glucose" to "MMP7." In MMP7 knock-out mice, an intense accumulation of neutrophils was observed in the gingival epithelium as compared to wild-type mice (P = .0001). CONCLUSION: Elevated MMP7 expression in gingival tissues of patients with T2DM/GP is related to the activation of reactive oxygen species by hyperglycemia. Suppression of MMP7 expression results in impaired neutrophil transmigration in gingiva.

Platelet factor 4 upregulates matrix metalloproteinase-1 production in gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Periodontitis is a highly prevalent chronic inflammatory disease that causes tooth loss, morbidity and confers an increased risk for systemic disease. Tissue destruction during periodontitis is due in large part to collagen-degrading matrix metalloproteinases (MMPs) released by resident cells of the periodontium in response to proinflammatory cytokines. Platelets are immune-competent blood cells with a newly recognized role in chronic inflammation; however, their role in the pathogenesis of periodontitis is undefined. Consequently, the objective of this study was to assess the effect of platelet factor 4 (PF4), a major platelet-derived cytokine, on MMP-1 (collagenase) expression in human gingival fibroblasts (HGFs). MATERIAL AND METHODS: HGFs were cultured in the presence or absence of recombinant PF4. Pro-MMP-1 secretion was quantified by enzyme-linked immunosorbent assay analysis of the cell culture supernatants. MMP-1 transcription was quantified by real-time polymerase chain reaction. Regulation of MMP-1 production by the p44/42 MAP kinase (MAPK) signaling pathway was examined in the presence or absence of PF4. RESULTS: Exposure to PF4 caused a ~ 2-3-fold increase in MMP-1 transcription and secretion from cultured HGFs. PF4 treatment also enhanced phosphorylation of p44/42 MAPK, which has been previously shown to induce MMP-1 expression in fibroblasts. Blockade of p44/42 MAPK signaling with the cell-permeant inhibitors PD98059 and PD184352 abrogated PF4-induced pro-MMP-1 transcription upregulation and release from cultured HGFs. CONCLUSION: We conclude that PF4 upregulates MMP-1 expression in HGFs in a p44/42 MAPK-dependent manner. These findings point to a previously unidentified role for platelets in the pathogenesis of periodontal diseases.

Characterisation of human gingival neural crest-derived stem cells in monolayer and neurosphere cultures.

Neural crest (NC)-derived stem cells (NCSC) have an exceptionally wide differentiation potential, but their use in regenerative therapy has been hampered by their scarcity in adult tissues and complex isolation protocols. Human oral mucosal gingiva may provide an attractive source of these cells as it contains NC-derived cells, the tissue is easily accessible and wound healing is fast and scarless with very little morbidity. To this end, we first investigated whether NC-derived cells are retained in adult gingiva by examining 8-months-old NC-reporter Wnt1-Cre/R26RYFP mice. We then hypothesised that gingival cell NC-like phenotype can be further enhanced by floating neurosphere cultures generated from standard human gingival fibroblast (GF) and pooled CFU-F (GSC) cultures. Findings showed that NC-derived cells are retained in the gingival connective tissue of aged mice. Human GFs and GSCs expressed NC-related genes nestin, Snai1, Twist1, Pax3, Sox9 and FoxD3, and generated neurospheres. This was mediated via calcium- and connexin 43-dependent cell communication, which is similar to neurospheres formed by neural progenitors. Cells in the spheres showed significantly increased expression of NC-related genes, and down regulation of fibroblast-related type I collagen. Structurally, the neurospheres were polarised with nestin positive cells located on the outer layers underlined with an extracellular matrix rich in molecules typical to embryonic NC. Sphere-derived cells expressed significantly elevated levels of neural markers, and differentiated into Tau, neurofilament-M and GFAP-positive cells suggesting neural differentiation potential. Thus, human GF and GSC cultures may provide an efficient source of NC-derived cells via enrichment by floating sphere cultures.

Hereditary gingival fibromatosis: characteristics and novel putative pathogenic mechanisms.

Hereditary gingival fibromatosis (HGF) is a rare condition that can occur as an isolated disease or as part of a syndrome or chromosomal abnormality. In severe cases, the gingival enlargement may cover the crowns of teeth and cause severe functional and esthetic concerns. Histological and cell culture studies have uncovered some of the molecular and cellular changes associated with HGF. However, the pathogenesis of the disease is still largely unknown. Recent studies about the genetic characteristics of HGF have provided novel clues about the potential pathogenic mechanisms. In particular, mutation in the son-of-sevenless (SOS-1) gene has been associated with one form of the disease. However, HGF displays genetic heterogeneity, and mutations in other genes are also likely involved. This review outlines the current knowledge about the histological, cellular, and genetic characteristics of HGF. In addition, the potential role of the SOS-1 molecule and related novel intracellular signaling pathways in the pathogenesis of HGF will be discussed.

Epithelial Microvesicles Promote an Inflammatory Phenotype in Fibroblasts.

Microvesicles (MVs) are extracellular vesicles secreted by various cell types that are involved in intercellular communication. We hypothesized that in human periodontal disease, the pocket epithelium releases MVs, which then modulate gene expression in the underlying fibroblasts to control periodontal inflammation. MVs were isolated from culture medium of gingival epithelial cells (GECs) treated with oral bacterial biofilm extract or left untreated. Biofilm treatment significantly increased MV release from the GECs. Mass spectrometry of GEC-MVs identified a total of 2,173 proteins, of which about 80% were detected in MVs from both control and biofilm-treated GECs. Among 80 signature genes of human gingival fibroblasts, 20 were significantly regulated (P < 0.05) by MVs from control and biofilm-treated GECs in a similar manner. Matrix metalloproteinase 1 and 3 and interleukin 6 and 8 showed the strongest regulation at the mRNA and protein levels. Several cellular signaling pathways were activated by GEC-MVs in human gingival fibroblasts, including Smad and mitogen-activated protein kinase-associated pathways ERK1/2, JNK, and p38. However, ERK1/2 signaling dominated in the MV-induced gene expression changes. The results demonstrate that GEC-MVs have a strong regulatory effect on the expression of fibroblast genes associated with inflammation and matrix degradation and that bacterial biofilm stimulates the generation of GEC-MVs. This suggests that bacterial biofilms can contribute to the initiation and progression of periodontal disease by promoting a tissue-destructive phenotype in gingival fibroblasts via the enhanced secretion of epithelial MVs.

Immunohistochemical localization of proteoglycans in human periodontium.

Proteoglycans (PGs) are extracellular and cell surface-associated macromolecules that regulate cell adhesion, cell growth, matrix formation, and bind growth factors. In this work we studied the distribution of core proteins of four PGs (decorin, biglycan, a large molecular weight PG, and CD44) in human gingiva and periodontal ligament by immunohistochemical staining of frozen tissue sections with specific antibodies. Decorin, a major PG of this tissue, was localized on collagen fiber bundles in the gingival and periodontal connective tissues. Staining for decorin was most intense at the subepithelial region. Biglycan was a minor PG component of the human periodontium, showing some accumulation in connective tissue under the oral epithelium. At the immunohistochemical level, biglycan appeared to form fine filament-like structures on extracellular matrix fibers. Localization of large molecular weight PG differed from that of decorin and biglycan. It was concentrated in deep connective tissue areas of the gingiva and in the periodontal ligament, and was only weakly present at the subepithelial region. CD44 was mainly concentrated in cell-cell contact areas of basal and spinous layers of oral epithelium. In the connective tissue of gingiva and periodontal ligament, CD44 was localized on fibroblast cell surfaces. Connective tissue area under the junctional epithelium contained relatively small amounts of PGs. The results indicate that different parts of human periodontium contain a typical variety of PGs, suggesting a specific function for each PG species in the location at which they accumulate.

Characterization of fibroblast clones from periodontal granulation tissue in vitro.

Connective tissues are known to be composed of heterogeneous fibroblast subpopulations. The significance of this heterogeneity in different physiological and pathological conditions is poorly understood. Granulation tissue is formed in connective tissue during wound healing, chronic inflammation, and certain pathological conditions. In this study, heterogeneity of fibroblasts from granulation tissue was investigated by cell-cloning techniques. Granulation-tissue fibroblasts (GTFs) from both chronically inflamed periodontal lesions and healing wound granulation tissue behaved similarly. GTFs showed a more pronounced decrease in proliferative capacity with increasing cumulative population doubling levels (CPDLs) and 30-40% lower cloning efficiency compared with normal gingival fibroblasts (HGFs). Morphologically, cells in GTF cultures were mainly large, whereas HGFs were mainly small in size. Both cell-line types showed heterogeneity in cell morphology. Clones composed of large stellate-like cells predominated in GTF cultures, whereas clones composed of small spindle-shaped or epithelioid cells predominated in HGF cultures. In both cell-line type the proportion of clones composed of large cells increased without increasing CPDL. These findings show that the properties of the fibroblasts changed during their in vitro life spans. The finding that normal connective tissue and granulation tissues contain morphologically distinct fibroblast clones in different proportions suggests that local factors could stimulate local fibroblasts to differentiate into GTFs. Alternatively, local factors could select some fibroblast subpopulations to overgrow the others to form granulation tissue.

Expression of heparan sulphate and small dermatan/chondroitin sulphate proteoglycans in chronically inflamed human periodontium.

Proteoglycans (PGs) function in regulating aspects of cell behavior, such as proliferation, adhesion, and migration. In this report, we investigated the localization of three heparan sulphate PGs (basement membrane [BM] heparan sulphate PG, CD44, and syndecan-1) and two small dermatan/chondroitin sulphate PGs (decorin and biglycan) in chronically inflamed human periodontium. Frozen sections were analyzed by immunofluorescence microscopy. In inflamed tissue, BM heparan sulphate PG showed reduced immunostaining in subepithelial and subendothelial basement membrane. Loss of CD44 and syndecan-1 was common in epithelial cells of inflamed periodontal tissue. Suprabasal keratinocytes of epithelium expressed involucrin, a cornified envelope protein and marker for epithelial differentiation, while the expression of syndecan-1 was weak or absent. In contrast, expression of the mesenchymal variant of CD44 and syndecan-1 was strong in infiltrating lymphocytes. Small dermatan/chondroitin sulphate PGs, decorin and biglycan, were also present in markedly reduced amounts in the periodontal connective tissue in chronic inflammation. In addition, decorin localized in the connective tissue along short rod-like structures. The results suggest that proteoglycan-dependent intercellular adhesion of keratinocytes is decreased and that adhesion of lymphocytes to matrix molecules via cell surface PGs increased in chronic inflammation. Disappearance of adhesion-modulating small dermatan/chondroitin sulphate PGs may further regulate cell migration in inflamed periodontium.

Human granulation-tissue fibroblasts show enhanced proteoglycan gene expression and altered response to TGF-beta 1.

Granulation-tissue fibroblasts are phenotypically unique cells that play an important role in wound repair and the development of chronic inflammatory lesions in connective tissue. In the present study, we compared proteoglycan, type I, and type III procollagen gene expression by granulation-tissue fibroblasts from wound and chronically inflamed tissues with normal gingival fibroblasts. We also analyzed the effect of TGF-beta 1 on proteoglycan mRNA levels and macromolecule production by these cells. One granulation-tissue fibroblast strain that was composed exclusively of alpha-smooth-muscle actin-positive cells (myofibroblasts) expressed strongly elevated basal levels of biglycan, fibromodulin, and versican (the large chondroitin sulphate proteoglycan), as well as type I and III procollagen mRNA. TGF-beta 1 enhanced more potently the expression of types I and III procollagen, biglycan, and versican mRNA by these cells as compared with normal fibroblasts. Other granulation-tissue fibroblast strains, in which about half of the cells expressed alpha-smooth-muscle actin, also showed enhanced proteoglycan and types I and III procollagen expression as compared with normal fibroblasts. These results suggest that alterations in matrix composition during inflammation and wound healing are regulated partly by altered phenotypes of the cells that produce the matrix, and partly by altered responses of these cells to TGF-beta 1.

Effect of citric acid treatment on the migration of epithelium on root surfaces in vitro.

Explants of bovine gingival mucosa were cultured for four days on scaled and citric acid-conditioned root surfaces. Demineralization of the hard tissue with citric acid exposed the collagenous matrix of the root. Undemineralized islands were frequently seen among the collagen fibers of the treated roots. When cultured on scaled, control root surfaces, the epithelium migrated inwards between the connective tissue of the explant and the root surface. On citric acid-treated roots, epithelial migration in this direction was rare although it was possible. Citric acid treatment of the substratum directed the epithelium to migrate outwards from the explant. Only a few of the controls showed epithelial migration to the outward direction. The findings indicate that demineralization of the root surface has an influence on the direction in which the epithelium initially starts to migrate. An induced delay of epithelial migration between the gingival connective tissue and the hard tissue may be favorable for connective tissue attachment to the root surface.

Successful periodontal maintenance of a case with Papillon-Lefèvre syndrome: 12-year follow-up and review of the literature.

BACKGROUND: Various approaches to treating the periodontal condition associated with Papillon-Lefèvre syndrome have been reported. These include oral hygiene instruction, use of mouthrinses, frequent debridement, multiple antibiotic regimens, periodontal surgery, extraction of hopeless teeth, and extraction of all deciduous teeth. Because Papillon-Lefèvre syndrome is rare, most publications are case reports, and very few document long-term successful treatment of the periodontal condition. METHODS: In 1986, a 3.5-year-old Indo-Canadian male was diagnosed with Papillon-Lefèvre syndrome and began periodontal treatment. Initial therapy consisted of debridement every 3 weeks, a 0.12% chlorhexidine mouthrinse, 2 regimens of metronidazole, and oral hygiene instruction for his parents. After 10 months it became apparent that the treatment was having little beneficial effect, since the periodontal destruction continued and teeth 51 and 61 exfoliated. At age 4, all remaining deciduous teeth were extracted and complete dentures inserted for the following 2-year edentulous period; then a 3-month maintenance schedule was maintained. RESULTS: The patient is now 17 years old and all his adult teeth are present with the exception of the third molars. His oral hygiene varies between moderate and good, with his most recent plaque score at 80% effectiveness. There are no probing depths greater than 4 mm, with the exception of the distal of the lower second molars where opercula are present. CONCLUSIONS: Extraction of all the deciduous teeth followed by a period of edentulousness may partially explain the fact that there has been no recurrent attachment loss in the permanent teeth up to age 17. Other explanations are discussed as part of the literature review of Papillon-Lefèvre syndrome.

Naturally occurring periodontal bone loss in the wild deer mouse, genus Peromyscus.

BACKGROUND: Historically, animal models for the study of periodontal diseases have incorporated surgically created defects, plaque retentive ligatures, as well as soft and high-sucrose diets which may not accurately reflect progression of the natural disease. Spontaneous periodontal disease is seen in a few animal species, but these are often expensive to maintain and are unsuitable for manipulation using advanced molecular biology techniques. Mice are inexpensive, easy to maintain, and are routinely used for transgenic experiments and are therefore an optimal animal for research purposes. However, it is commonly accepted that mice do not spontaneously develop periodontal disease. The purpose of this study was to determine if a mouse population that exhibits periodontal breakdown in the wild could be found, allowing for genetic manipulation of naturally occurring periodontal disease. METHODS: We examined over 2,500 dry skulls of several Peromyscus species from various locations and habitats on the west coast of North America for periodontal bone loss in the molars, using furcation involvement as an indicator of disease severity. Alveolar bone loss was classified as Grade I) horizontal component of bone loss in the furcations; II) through-and-through furcations; and III) through-and-through furcations with alveolar bone loss into the apical third of the root. RESULTS: The proportions of individual mice experiencing bone loss were 3.8% for Class I-III involvement, 1.3% for Class II-III involvement, and 0.5% for Class III alone. Three subspecies of P. keeni and one subspecies of P. maniculatus had periodontal disease prevalences in 7% to 13.5% of their samples. Mice from isolated islands had 1.8- to 4.7-fold higher disease prevalence than those located on the mainland, with even greater prevalence on small islands. No statistically significant differences between genders were found. CONCLUSIONS: It appears that periodontal disease is far more common in this mouse genus than previously believed. Some of the subspecies demonstrated severe periodontal disease at a prevalence comparable to that found in humans.

Histochemical localization of hyaluronate in human oral epithelium using a specific hyaluronate-binding probe.

Biochemical data suggest that gingival epithelium contains hyaluronate, but there is little histochemical information about its localization. Hyaluronate was here visualized in gingival and buccal mucosa using a specific probe derived from the hyaluronate binding region of cartilage proteoglycan. Hyaluronate was found both in the gingival and buccal epithelium, but its localization was correlated with the type of keratinization. In the keratinized epithelium of gingiva, whether ortho- or parakeratotic, the intercellular spaces from basal to upper spinous layers displayed strong staining, most intense in the middle spinous cell layer. The uppermost vital cell layers as well as the cornified cell layer remained unstained. In the non-keratinized epithelium of buccal mucosa and the local non-keratinized areas of gingiva, only the basal cells and the lowermost spinous cell layers stained for hyaluronate, whereas the majority of the upper epithelium was negative. Electron microscopic examination of the basal and spinous cell layers displayed hyaluronate, both associated with the cell surface and free in the intercellular space. The subepithelial connective tissue showed positive but diffuse staining in all specimens.

Epithelial integrins with special reference to oral epithelia.

Adhesion of epithelium to the extracellular matrix is crucial for the maintenance of systemic and oral health. In the oral cavity, teeth or artificial dental implants penetrate the soft tissue of the gingiva. In this interface, gingival soft tissue needs to be well attached via the epithelial seal to the tooth or implant surface to maintain health. After injury or wounding, epithelial tissue rapidly migrates to form the initial epithelial cover to restore the barrier against infection. These events are crucially dependent on deposition of extracellular matrix and proper activation and function of integrin receptors in the epithelial cells. Recent experimental evidence suggests that epithelial integrins also participate in the regulation of periodontal inflammation. In this review, we will discuss the structure and function of epithelial integrins and their extracellular ligands and elaborate on their potential role in disease and repair processes in the oral cavity.

Expression of fibronectin-binding integrins in gingival epithelium in drug-induced gingival overgrowth.

BACKGROUND AND OBJECTIVE: Gingival overgrowth is a side-effect of nifedipine and cyclosporin medications. Integrins are transmembrane glycoproteins that mediate cell adhesion, regulate cell proliferation and participate in the regulation of tissue fibrosis. The aim of this study was to investigate whether expression of epithelial cell integrins is linked to the development of drug-induced gingival overgrowth. MATERIAL AND METHODS: Human gingival biopsies of patients taking nifedipine, cyclosporin, or a combination of both medications, were used. Expression of the alpha5beta1, alphavbeta1 and alphavbeta6 integrins, and of cellular extra domain A of fibronectin, was localized in frozen sections using immunohistochemistry. RESULTS: The activated conformation of the beta1, alpha5beta1 and alphavbeta6 integrins were more frequently expressed in distinct locations in the oral epithelium in the combined drug group. Cellular extra domain A of fibronectin, a ligand for both alpha5beta1 and alphavbeta6 integrins, was expressed within the connective tissue of all groups. It was also expressed around the basal keratinocytes of the control, nifedipine and cyclosporin-induced gingival overgrowth groups, but not in the combined medication group. No relationship between the presence of inflammation and integrin expression was found. CONCLUSION: The results indicate that expression of certain integrins is up-regulated in the epithelium of drug-induced gingival overgrowth where they could participate in controlling the formation of elongated rete ridges and tissue fibrosis.

Expression of Endo180 is spatially and temporally regulated during wound healing.

AIMS: Interactions of cells with the extracellular matrix are important for normal wound healing and may play a role in scar formation. Remarkably, wound healing in human gingiva does not result in scar formation and serves as a model for wound regeneration. Endo180 (CD280) is a cell surface receptor that has novel functions to regulate cell migration and bind and internalize collagens that are key processes in wound healing. The aim of this study was to examine the expression of Endo180 during gingival wound regeneration. METHODS AND RESULTS: Biopsies were collected from normal human gingiva and 1-60 days after wounding and expression of Endo180 was analysed by immunostaining. Expression of Endo180 by cultured fibroblasts and keratinocytes was studied by immunoblotting and semiquantitative reverse transcriptase-polymerase chain reaction. In normal gingiva, Endo180 was expressed by basal epithelial cells, fibroblasts, myofibroblasts, pericytes, macrophages and endothelial cells. In wounds, Endo180 expression was spatiotemporally increased in the migrating and differentiating wound epithelium, in subsets of myofibroblasts, pericytes, macrophages and endothelial cells. Growth factors involved in wound healing up-regulated the expression of Endo180 in keratinocytes and fibroblasts. CONCLUSIONS: The findings suggest that Endo180 plays a role in re-epithelialization and connective tissue remodelling during wound regeneration.

Distinctive localization and function for lumican, fibromodulin and decorin to regulate collagen fibril organization in periodontal tissues.

BACKGROUND: Small leucine-rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are secreted extracellular matrix molecules that associate with fibrillar collagens and regulate collagen fibrillogenesis. Collagens are the major extracellular matrix components of periodontal connective tissues where they provide mechanical attachment of the tooth to the bone and gingiva and mediate signals that regulate cell functions, including remodeling of the periodontal ligament and bone. Structural organization of collagen may also be important for the defense against periodontal disease, because in certain conditions abnormal collagen fibrils associate with increased susceptibility to periodontal disease. OBJECTIVES: The purpose of this study was to find out the role of SLRPs to regulate collagen fibril and fibril bundle formation in periodontal tissues. METHODS: The localization of SLRPs in human and mouse periodontal tissues was studied using immunohistochemical methods. To assess the function of SLRPs we studied periodontal tissues of mice harboring targeted deletions of decorin, fibromodulin or lumican genes and lumican and fibromodulin double knockout mice using histological and electronmicroscopical methods. RESULTS: The SLRPs were coexpressed in human and mouse gingival and periodontal ligament connective tissues where they colocalized with collagen fibril bundles. Teeth in the knockout animals were fully erupted and showed normal gross morphology. Targeted deletion of decorin, fibromodulin, lumican or both lumican and fibromodulin resulted in abnormal collagen fibril and fibril bundle morphology that was most evident in the periodontal ligament. Each of the gene deletions resulted in a unique fibril and fibril bundle phenotype. CONCLUSIONS: These findings indicate that decorin, fibromodulin and lumican coordinately regulate the fibrillar and suprafibrillar organization of collagen in the periodontal ligament.

Colocalization of the collagen-binding proteoglycans decorin, biglycan, fibromodulin and lumican with different cells in human gingiva.

BACKGROUND AND OBJECTIVE: Decorin, biglycan, fibromodulin and lumican are structurally related molecules that belong to the family of small leucine-rich proteoglycans (SLRPs). These SLRPs are secreted extracellular matrix molecules that interact with type I collagen and regulate collagen fibrillogenesis. They may also modulate cell functions that are important in maintenance of connective tissue structure. The aim of this study was to localize decorin, biglycan, fibromodulin and lumican in human gingiva. METHODS: Localization of decorin and its proform (prodecorin), biglycan, fibromodulin and lumican and mature and proform of type I collagen was studied by immunohistochemical staining of frozen tissue sections from healthy human attached gingiva. Double immunostaining with anti-SLRP or anti-type I procollagen antibodies and specific markers for different connective tissue cells was used to study association of these molecules with cells. RESULTS: The mature and proforms of decorin and collagen and biglycan, fibromodulin and lumican showed distinct localization in the extracellular matrix, where they associated with type I collagen fiber bundles. Prodecorin also localized to the epithelial basement membrane zone. Fibroblasts, myofibroblasts, endothelial cells and pericytes showed immunoreactivity for procollagen, prodecorin, biglycan and fibromodulin, whereas lumican associated with fibroblasts and myofibroblasts only. Biglycan and fibromodulin were also associated with macrophages. Basal epithelial cells of the gingival epithelium showed immunoreactivity for biglycan, fibromodulin and lumican. CONCLUSIONS: Decorin, biglycan, fibromodulin and lumican associate with type I collagen and may collaborate to regulate collagen fibrillogenesis in human gingiva. Each of the SLRPs showed a distinct association with different connective tissue cells, suggesting that the cells produce these molecules and/or that the cells interact with them. Localization of biglycan, fibromodulin and lumican at the epithelial cells suggests novel functions for these SLRPs in human gingival epithelium.

A biochemical analysis of human periodontal tissue proteoglycans.

Proteoglycans synthesized by periodontal (gingival, periodontal ligament, dental follicle) fibroblasts were analysed by SDS/polyacrylamide and agarose gel electrophoresis after being labelled with radioactive sulphate. Medium, cell membrane and extracellular matrix fractions were analysed separately. Samples were treated with chondroitinase AC, chondroitinase ABC, heparitinase or a combination of chondroitinase ABC and heparitinase before electrophoretic separation of proteoglycans. Antibodies to versican and decorin were used to identify these molecules by Western immunoblots. For steady-state metabolic radiolabelling of fibroblasts, medium and cell membrane fractions contained about equal proportions of radiolabelled proteoglycans (about 43%), whereas less radioactivity (about 14%) was found in proteoglycans of the matrix fraction. Periodontal fibroblasts produce six major proteoglycans: versican, a high-molecular-mass chondroitin sulphate proteoglycan (CSPG); decorin, a dermatan sulphate proteoglycan (DSPG); a membrane-associated heparan sulphate proteoglycan (HSPG); two medium- or matrix-associated HSPGs; and a 91 kDa membrane-associated CSPG. Variation in decorin molecular size was observed in mass cultures of fibroblasts. Similar polydispersity in molecular size of decorin was seen in several clones established from one mass culture.