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Tissue and cell type-specific cell-free extracellular matrices generated by various decellularization protocols can be important tools to study cell interactions with their distinct extracellular niche. Our recent findings have shown that the widely used NH4OH/Triton X-100/DNase decellularization method is not able to completely remove cytoskeletal proteins from long-term three-dimensional human gingival fibroblast cultures. Therefore, we developed a novel decellularization protocol that involves sequential incubation of the cultures with latrunculin B, an actin destabilizing agent, deoxycholate, a mild detergent, and DNase. This protocol efficiently removes cytoskeletal proteins, including actin and beta-tubulin, and DNA from the cultures, which could interfere with the cell-matrix interactions of cells seeded on the decellularized matrices.

Background Leucocyte- and platelet-rich fibrin (L-PRF) is a blood-derived biomaterial rich in leucocytes and platelets embedded in a high-density fibrin network that can be compressed into a membrane and used in surgical applications to stimulate tissue regeneration and wound healing, especially in oral cavity. This study aimed to determine the combined effects of the growth factors and cells present in L-PRF on fibroblasts that directly face the L-PRF membranes placed during surgical procedures. Methods The effect of L-PRF from six donors on the expression of 84 key wound healing genes in normal human gingival fibroblasts was tested by RT-qPCR. Results L-PRF significantly regulated the expression of 33 fibroblast genes (39%), including interleukins, myofibroblast-, extracellular matrix- and angiogenesis-associated genes, and matrix metalloproteinase-1 and -3. L-PRF regulated fibroblast gene expression both time- and dose-dependently, and the effects were mediated by mitogen-activated protein kinases ERK1/2, JNK and p38. L-PRF also stimulated fibroblast wound closure and promoted the ability of fibroblasts to induce endothelial tube formation. L-PRF-induced gene expression changes in fibroblast were similar to those observed in early human and pig wounds. Conclusions This study provides new insights into the biological mechanism by which L-PRF regulates key gingival fibroblast functions important in wound healing.

Soft tissue calcification occurs in many parts of the body, including the gingival tissue. Epithelial cell-derived MVs can control many functions in fibroblasts but their role in regulating mineralization has not been explored. We hypothesized that microvesicles (MVs) derived from gingival epithelial cells could regulate calcification of gingival fibroblast cultures in osteogenic environment. Human gingival fibroblasts (HGFs) were cultured in osteogenic differentiation medium with or without human gingival epithelial cell-derived MV stimulation. Mineralization of the cultures, localization of the MVs and mineral deposits in the HGF cultures were assessed. Gene expression changes associated with MV exposure were analyzed using gene expression profiling and real-time qPCR. Within a week of exposure, epithelial MVs stimulated robust mineralization of HGF cultures that was further enhanced by four weeks. The MVs taken up by the HGF's did not calcify themselves but induced intracellular accumulation of minerals. HGF gene expression profiling after short exposure to MVs demonstrated relative dominance of inflammation-related genes that showed increases in gene expression. In later cultures, OSX, BSP and MMPs were significantly upregulated by the MVs. These results suggest for the first time that epithelial cells maybe associated with the ectopic mineralization process often observed in the soft tissues.

Background Decontamination of biofilm-infected rough implant surfaces is challenging. Platelet rich blood products have been shown to have anti-microbial properties against periodontal pathogens. Our aim was to investigate the effect of a potential biological implant surface disinfectant, leukocyte- and platelet-rich fibrin (L-PRF), on a mature oral multispecies biofilm on a rough titanium surface. Methods Sandblasted, large grit, acid-etched (SLA) titanium disks were inoculated with subgingival dental plaque and cultured anaerobically for 21 days. The L-PRF membranes were collected from 12 donors in three trials (four donors in each trial). The disks were rinsed with 0.9% NaCl and exposed to the cell-rich portion of the L-PRF membranes for 48 hours followed by scanning electron microscope (SEM) analysis immediately or after rinsing with 0.9% NaCl prior to fixation. The presence of platelet factor-4 in the rinse samples was analyzed by Western blotting. Remaining bacteria were quantified from SEM images of the implant surfaces and their numbers statistically compared. Results The L-PRF-treated samples without rinsing displayed numerous cells with multiple pseudopodia in immediate contact with bacteria that appeared perforated and increased in size. The cells were identified as platelets based on morphological criteria and by positive reaction for platelet factor-4 by Western blotting. After post-treatment rinsing, the L-PRF-treated disks displayed a significant reduction in bacterial counts (in average 92% reduction). Conclusion Application of L-PRF significantly reduced bacterial counts on contaminated SLA titanium surface, most likely through anti-microbial action by platelets.

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-β1, -β2, -β3, PGE2 and IL-1β significantly upregulated, while TNF-α and IFN-γ downregulated Cx43 in cultured GFBLs. TGF-β1, -β2, -β3, IL-1β 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-β1-induced upregulation of Cx43 was mediated by TGFβRI (ALK5) and SMAD2/3 signaling, and this was potently suppressed by PGE2, IL-1β, 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-β isoforms and PGE2, IL-1β, TNF-α and IFN-γ.

Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo-like 3D culture model, we further show that the GJ, HC, and channel-independent functions of Cx43 distinctly regulated wound healing-related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts.

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. Sin financiación 2.613 JCR (2018) Q1, 18/91 Dentistry, Oral Surgery & Medicine 1.061 SJR (2018) Q2, 4/12 Periodontics No data IDR 2018 UEM

Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-β signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring.

In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvβ6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-β1 (TGF-β1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress β6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-β1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-β1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the β6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD. This article has an associated First Person interview with the first author of the paper.

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.

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.

Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial–stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing–associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P

Integrins are cell surface receptors that traditionally mediate cell-to-extracellular matrix and cell-to-cell adhesion. They can, however, also bind a large repertoire of other molecules. Integrin αvβ6 is exclusively expressed in epithelial cells where it can, for example, serve as a fibronectin receptor. However, its hallmark function is to activate transforming growth factor-β1 (TGF-β1) to modulate innate immune surveillance in lungs and skin and along the gastrointestinal tract, and to maintain epithelial stem cell quiescence. The loss of αvβ6 integrin function in mice and humans leads to an altered immune response in lungs and skin, amelogenesis imperfecta, periodontal disease and, in some cases, alopecia. Elevated αvβ6 integrin expression and aberrant TGF-β1 activation and function are associated with organ fibrosis and cancer. Therefore, αvβ6 integrin serves as an attractive target for cancer imaging and for fibrosis and cancer therapy.

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.

Connexin 43 (Cx43) is the most ubiquitous connexin in various cells, and presents as hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We have recently shown that Cx43 abundance was strongly reduced in fibroblasts of human gingival wounds, and blocking Cx43 function in cultured human gingival fibroblasts (GFBLs) strongly regulated the expression of wound healing-related genes. However, it is not known whether these responses involved Cx43 HCs or GJs. Here we show that Cx43 assembled into distinct GJ and HC plaques in GFBLs both in vivo and in vitro. Specific blockage of Cx43 HC function by TAT-Gap19, a Cx43 mimetic peptide, significantly upregulated the expression of several MMPs, TGF-β signaling molecules, Tenascin- C, and VEGF-A, while pro-fibrotic molecules, including several extracellular matrix proteins and myofibroblast and cell contractility-related molecules, were significantly downregulated. These changes were linked with TAT-Gap19-induced suppression of ATP signaling and activation of the ERK1/2 signaling pathway. Collectively, our data suggest that reduced Cx43 HC function could promote fast and scarless gingival wound healing. Thus, selective suppression of Cx43 HCs may provide a novel target to modulate wound healing.

Periodontal diseases manifest by the formation of deep pockets between the gingiva and teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Epithelial cell receptor αvβ6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-β1, is highly expressed in healthy junctional epithelium that connects the gingiva to the tooth enamel. However, its expression is attenuated in human periodontal disease. Moreover, Itgb6−/− mice display increased periodontal inflammation compared to wild-type mice. We hypothesized that bacterial biofilms present in the periodontal pockets suppress αvβ6 integrin levels in periodontal disease and that this change aggravates inflammation. To this end, we generated three-week-old multi-species oral biofilms in vitro and treated cultured gingival epithelial cells (GECs) with their extracts. The biofilm extracts caused suppression of β6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1β and -6. Furthermore, GECs with β6 integrin siRNA knockdown showed increased interleukin-1β expression, indicating that αvβ6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed β6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvβ6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.

Gingiva of the oral mucosa provides a practical source to isolate fibroblasts for therapeutic purposes because the tissue is easily accessible, tissue discards are common during routine clinical procedures and wound healing after biopsy is fast and results in complete wound regeneration with very little morbidity or scarring. In addition, gingival fibroblasts have unique traits, including neural crest origin, distinct gene expression and synthetic properties and potent immunomodulatory functions. These characteristics may provide advantages for certain therapeutic approaches over other more commonly used cells, including skin fibroblasts, both in intraoral and extra-oral sites. However, identity and phenotype of gingival fibroblasts, like other fibroblasts, are still not completely understood. Gingival fibroblasts are phenotypically heterogeneous, and these…fibroblast subpopulations may play different roles in tissue maintenance, regeneration and pathologies. The purpose of this review is to summarize what is currently known about gingival fibroblasts, their distinct potential for tissue regeneration and their potential therapeutic uses in the future.

Cell surface integrin receptors mediate cell adhesion, migration and cellular signaling in all nucleated cells. They are activated by binding to extracellular ligands or by intracellular proteins, such as kindlins that engage with their cytoplasmic tails. Cells in the periodontal tissues express several integrins with overlapping ligand-binding capabilities. A distinct phenotype in the periodontium has only been described for knockouts or mutations of three integrin subunits, α11, β6 and β2. Integrin α11β1 appears to have some regulatory function in the periodontal ligament of continuously erupting incisors in mice. Integrin αvβ6 is expressed in the junctional epithelium (JE) of the gingiva. Animals deficient in this receptor develop classical signs of periodontal disease, including inflammation, apical migration of the JE and bone loss, suggesting that it plays a role in the regulation of periodontal inflmmation, likely through activation of transforming growth factor-β1. Lack of integrin activation in the JE is also associated with periodontitis. Patients with kindlin-1 mutations have severe early-onset periodontal disease. Finally, patients with mutations in the leukocyte-specific β2 integrin subunit have severe periodontal problems due to lack of transiting neutrophils in the periodontal tissues.

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.

This study examined the effect of a new bioactive dentin substitute material (Biodentine) on the viability of human gingival fibroblasts.Biodentine, White ProRoot mineral trioxide aggregate (MTA), and glass ionomer cement were evaluated. Human gingival fibroblasts were incubated for 1, 3, and 7 days both in the extracts from immersion of set materials in culture medium and directly on the surface of the set materials immersed in culture medium. Fibroblasts cultured in Dulbecco modified Eagle medium were used as a control group. Cytotoxicity was evaluated by flow cytometry, and the adhesion of human gingival fibroblasts to the surface of the set materials was assessed by using scanning electron microscopy. The data of cell cytotoxicity were analyzed statistically by using a one-way analysis of variance test at a significance level of P.05.Cells exposed to extracts from Biodentine and MTA showed the highest viabilities at all extract concentrations, whereas cells exposed to glass ionomer cement extracts displayed the lowest viabilities (P.05). There was no significant difference in cell viabilities between Biodentine and MTA during the entire experimental period (P.05). Human gingival fibroblasts in contact with Biodentine and MTA attached to and spread over the material surface after an overnight culture and increased in numbers after 3 and 7 days of culture.Biodentine caused gingival fibroblast reaction similar to that by MTA. Both materials were less cytotoxic than glass ionomer cement.

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.

Shortly after wounding, epithelial keratinocytes become activated through the combined effects of the exposure to pro-migratory matrix molecules within the wound site and to growth factors that are released by other wound cells and from the blood clot as well as by wound-generated electrical fields. Within 24 hours, they start migrating from the wound edges into the fibrin–fibronectin-rich blood clot. They also deposit their own matrix molecules that facilitate their motility, including EDA fibronectin, laminin-332, and tenascin-C as well as express their receptors (mainly integrins). Basal keratinocytes adjacent to the wound site start proliferating 48–72 hours after the injury, contributing to the migrating cell pool. Re-epithelialization is stimulated by a number of cytokines and growth factors such as members of the epidermal growth factor, transforming growth factor-β, and keratinocyte growth factor families that promote keratinocyte migration and proliferation. Furthermore, re-epithelialization is dependent on regulated expression of proteases, including plasmin and matrix metalloproteinases, which break down extracellular matrix to allow keratinocyte invasion into wound provisional matrix as well as release and activate matrix-bound growth factors. Thus, wound re-epithelialization is a complex process that requires coordinated expression of several new extracellular matrix molecules, their receptors, and proteinases, and when dysregulated, can result in failures to re-epithelialize and formation of chronic wounds.

Integrin αvβ6 is a heterodimeric cell surface receptor, which is absent from the normal epithelium, but is expressed in wound-edge keratinocytes during re-epithelialization. However, the function of the αvβ6 integrin in wound repair remains unclear. Impaired wound healing in patients with diabetes constitutes a major clinical problem worldwide and has been associated with the accumulation of advanced glycated endproducts (AGEs) in the tissues. AGEs may account for aberrant interactions between integrin receptors and their extracellular matrix ligands such as fibronectin (FN). In this study, we compared healing of experimental excisional skin wounds in wild-type (WT) and β6-knockout (β6(-/-) ) mice with streptozotocin-induced diabetes. Results showed that diabetic β6(-/-) mice had a significant delay in early wound closure rate compared with diabetic WT mice, suggesting that αvβ6 integrin may serve as a protective role in re-epithelialization of diabetic wounds. To mimic the glycosylated wound matrix, we generated a methylglyoxal (MG)-glycated variant of FN. Keratinocytes utilized αvβ6 and β1 integrins for spreading on both non-glycated and FN-MG, but their spreading was reduced on FN-MG. These findings indicated that glycation of FN and possibly other integrin ligands could hamper keratinocyte interactions with the provisional matrix proteins during re-epithelialization of diabetic wounds.

Background Scar formation following skin trauma can have devastating consequences causing physiological and psychosocial concerns. Currently, there are no accepted predictable treatments to prevent scarring which emphasizes a need for a better understanding of the wound healing and scar formation process. Objectives Previously it was shown that healing of small experimental wounds in the oral mucosa of red Duroc pigs results in significantly reduced scar formation as compared with equivalent full-thickness skin wounds. In the present study, scar formation was assessed in 17 times larger experimental wounds in both oral mucosa and skin of the red Duroc pigs. Methods Equivalent experimental wounds were created in the oral mucosa and dorsal skin of red Duroc pigs, and scar formation, localization and abundance of key wound healing cells, transforming growth factor-β (TGF-β) and phosphorylated Smad3 (pSmad3) were assessed. Results Oral mucosal wounds displayed significantly less clinical and histological scar formation than did the corresponding skin wounds. The number of macrophages, mast cells, TGF-β and pSmad3 immunopositive cells was significantly reduced in the oral mucosal wounds as compared with skin wounds during the maturation stage of the healing process. Although the number of myofibroblasts was significantly elevated, the oral mucosal wounds showed significantly less contraction than did the skin wounds over time. Conclusions Earlier resolution of the inflammatory reaction and reduced wound contraction may promote scarless oral mucosal wound healing. In addition, scar formation likely depends not only on the number of myofibroblasts but also on the extracellular environment which regulates their function.

Kindlin-1 is an intracellular focal adhesion protein that regulates the actin cytoskeleton. Patients suffering from Kindler syndrome have a homologous mutation of the kindlin-1 gene and develop skin blisters, periodontal disease, and intestinal complications because of deficient adhesion of the basal epithelial cells. We investigated kindlin-1 localization in periodontal tissue and its functions in cultured keratinocytes and showed that kindlin-1 co-localizes with migfilin and paxillin in the basal epithelial cells of oral mucosa and in cultured keratinocytes. The kindlin-1-deficient oral mucosal tissue from a patient with Kindler syndrome showed a complete lack of paxillin and reduced migfilin immunostaining in the basal keratinocytes. Co-immunoprecipitation showed that migfilin directly interacted with kindlin-1. RNA interference-induced kindlin-1 deficiency in keratinocytes led to an altered distribution of migfilin-containing focal adhesions, reduced cell spreading, decreased cell proliferation, and decelerated cell migration. Disruption of microtubules in the kindlin-1-deficient cells further reduced cell spreading, suggesting that microtubules can partially compensate for kindlin-1 deficiency. Kindlin-1 supported mature cell-extracellular matrix adhesions of keratinocytes, as downregulation of kindlin-1 expression significantly reduced the cell-adhesion strength. In summary, kindlin-1 interacts with migfilin and plays a crucial role in actin-dependent keratinocyte cell adhesion essential for epidermal and periodontal health.

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin αvβ6 is induced during wound healing, and it can activate fibrogenic transforming growth factor β1 (TGF-β1) and anti-fibrogenic TGF-β3 that play key roles in scar formation. In this study, expression of β6 integrin and members of the TGF-β pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of β6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The αvβ6 integrin was colocalized with both TGF-β isoforms in the wound epithelium. Significantly higher expression levels of β6 integrin and TGF-β1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-β3 compared with skin. The spatio-temporal colocalization of αvβ6 integrin with TGF-β1 and TGF-β3 in the wound epithelium suggests that αvβ6 integrin may activate both isoforms during wound healing. Prolonged expression of αvβ6 integrin along with TGF-β3 in the gingival wound epithelium may be important in protection of gingiva from scar formation. (J Histochem Cytochem 57:543–557, 2009)

Wound healing in oral mucosa is fast and results in little scar formation as compared with skin. The biological mechanisms underlying this property are poorly understood but may provide valuable information about the factors that promote wound regeneration. Small leucine-rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are extracellular matrix molecules that regulate collagen fibrillogenesis, inhibit transforming growth factor-beta (TGF-beta) activity and reduce scarring. In the present study, we analyzed accumulation of SLRPs and TGF-beta during non-scarring human oral mucosal wound healing. Biopsies were collected from healthy volunteers from unwounded tissue and from standardized experimental wounds 3-60 days postwounding. Localization of SLRPs, TGF-beta1 and TGF-beta3 was analyzed by immunohistochemical staining and quantitated by image analysis. Double immunostaining was used to study localization of SLRPs or active TGF-beta in distinct cells. Decorin, biglycan, fibromodulin, and TGF-beta isoforms showed significantly increased accumulation in the wound extracellular matrix and distinct wound cells while the abundance of lumican in the extracellular matrix was strongly reduced during wound healing. Localization and abundance of fibromodulin, lumican, and TGF-beta isoforms was also spatiotemporally regulated in the wound epithelium. The findings suggest that SLRPs regulate wound reepithelialization and connective tissue regeneration during oral mucosal wound healing.

The association of aggressive periodontitis with Kindler syndrome was based on a single case in 1996 and later confirmed with a larger population. Since then, significant research has greatly increased our understanding of the molecular pathology of this disorder. We review recent advances in the molecular mechanisms of the syndrome and present a maintenance case report of a patient who has been followed in our clinic.A female patient who was diagnosed with Kindler syndrome and aggressive periodontitis at the age of 16 years has been followed and treated in our clinic for 12 years. Her main treatment has been maintenance therapy following her initial treatment and restorative work previously documented. Gingival biopsies obtained during the recent extraction of hopeless maxillary molars were used for histologic assessment of gingival tissue attachment apparatus and to isolate gingival fibroblasts. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using these cells to confirm the lack of expression of kindlin-1.RT-PCR showed the total loss of kindlin-1 mRNA in cultured gingival fibroblasts, supporting the clinical diagnosis of Kindler syndrome. Tissue biopsies revealed atypical pocket epithelium. Maintenance therapy has been moderately successful. Teeth that were recently lost had a poor prognosis at the initial assessment. The patient's gingiva and oral mucosa continue to be fragile with episodes of sloughing and inflammation.Periodontitis in Kindler syndrome responds to maintenance therapy, but the gingiva and oral mucosa continue to display an abnormal appearance with white patches. Histologic findings suggest that the junctional epithelium in Kindler syndrome may be abnormal and could explain why these patients have periodontal disease. Attachment loss progressed around teeth with an initial guarded or poor prognosis. Teeth that started with a good or fair prognosis continue to have a fair prognosis. Limited dental implant treatment is being considered.

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.

Background: Gingiva is composed of attached and marginal (free) gingiva and interdental papilla. Increasing esthetic demands in dentistry have created a need to restore all parts of the gingiva. However, the interdental papilla has limited regeneration potential compared to other parts of the gingiva. It also is more susceptible to gingival overgrowth, suggesting that it has distinct cellular and molecular properties from other parts of the gingiva. Very little is known about the possible differences in the molecular composition of different parts of the gingiva. Methods: We compared the expression of a set of key molecules in interdental papilla and marginal gingiva from seven healthy subjects by immunohistochemical staining. Results: In the interdental papilla, immunoreactivity for integrin αvβ6 and cytokeratin 19 in the oral epithelium was significantly higher than in marginal gingiva. Expression of type I procollagen, extra domain A (EDA) and extra domain B (EDB) fibronectin isoforms, tenascin-C, transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), and the signaling molecule son-of-sevenless (SOS)-1 also were increased in the interdental papilla. The expression of small leucine-rich proteoglycans decorin, biglycan, fibromodulin, and lumican in the interdental papilla was partially different from the marginal gingiva. Conclusions: Molecular composition of the interdental papilla is distinct from marginal gingiva. Increased expression of molecules normally induced in wound healing (αvβ6 integrin, fibronectin-EDB and -EDA, tenascin-C, type I procollagen, TGF-β, CTGF, and SOS-1) suggests that the cells in the interdental papilla are in an activated state and/or inherently display a specific phenotype resembling wound healing.

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.

Wound healing is regulated by a complex, spatiotemporally controlled interplay between resident and circulating cells, extracellular ­matrix (ECM), chemical, biochemical, electrochemical, and mechanical signals from the ECM and soluble mediators. Wound healing starts with coagulation and haemostasis that stops the bleeding. Platelets in the clot are activated and release growth factors and cytokines that stimulate resident cells to start re-epithelialization, angiogenesis, and connective tissue repair. In addition, platelet-derived factors, activated complement, and bacterial products trigger an inflammatory reaction with neutrophil and macrophage influx to clear the ­bacteria. How effectively the inflammation is resolved will play a crucial role in determining the healing outcome. Fast re-­epithelialization that establishes the barrier function and neutrophil-macrophage interactions are important for this process. Initial influx of macrophages contains mainly cells that produce proinflammatory cytokines while the reparative macrophages that support granulation tissue formation, angiogenesis, and remodeling dominate the later stages of wound healing. Macrophages also stimulate the differentiation of resident cells, possibly fibroblasts and/or pericytes, to myofibroblasts, which produce matrix that, after remodeling, turns to either scar in skin or normal mucosal tissue in palatal oral mucosa. Current understanding about wound healing is largely based on data obtained from skin that does not always replicate oral mucosal wound healing. In general, however, the wound healing stages and key principles are shared among different tissues. Therefore, in this chapter we will discuss the general principles of wound healing and include information that is distinct to oral mucosal wound healing when relevant.

Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/β and TGF-β signaling pathways, and may promote fast and scarless wound healing in human gingiva.

After epithelial disruption by tissue injury, keratinocytes migrate from the wound edge into a provisional matrix. This process is stimulated by growth factors that signal through epidermal growth factor (EGF) receptor, including EGF, heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha), and by for example keratinocyte growth factor (KGF) and TGF-beta1 that function through different receptors. We have previously shown that keratinocyte migration induced by EGF or staurosporine is dependent on the activity of glycogen synthase kinase-3 (GSK-3). In the present study, we show that keratinocyte migration induced by TGF-beta1, KGF, EGF, TGF-alpha and staurosporine depends on EGFR signaling, involves autocrine HB-EGF expression and is potently blocked by GSK-3 inhibitors SB-415286 and LiCl. Inhibition of GSK-3 also retards wound reepithelialization in vivo in mice. Moreover, inhibition of GSK-3 activity prevented cell rounding that is an early event in EGFR-mediated keratinocyte migration. Isoform-specific GSK-3alpha and GSK-3beta knockdown and overexpression experiments with siRNAs and adenoviral constructs, respectively, revealed that GSK-3alpha is required for keratinocyte migration, whereas excessive activity of GSK-3beta is inhibitory. Thus, induction of keratinocyte migration is conveyed through EGFR, promoted by endogenous HB-EGF and requires GSK-3alpha activity.

The alphavbeta6 integrin is an exclusively epithelial integrin that is highly expressed during fetal development. In adult tissue, alphavbeta6 integrin is expressed during inflammation, carcinogenesis, and in wound healing. We previously reported that alphavbeta6 integrin is highly expressed in poorly healing human wounds and its over-expression is associated with chronic wounds in a mouse model. The objective of this study was to investigate the role of alphavbeta6 integrin in compromised wound healing induced by hydrocortisone treatment or aging by using young and old mice deficient in or overexpressing the beta6 integrin subunit in the epidermis. Untreated aged beta6 integrin-deficient (beta6-/-) animals showed a significant delay in wound healing when compared to their age-matched controls or younger beta6-/- mice. The most significant delay was observed at the stages where granulation tissue deposition was occurring. Hydrocortisone treatment significantly delayed wound healing in wild-type and beta6 integrin-deficient mice in comparison with the untreated controls. However, hydrocortisone treatment in beta6 integrin overexpressing animals did not cause a significant delay in wound healing. The results of this study suggest that alphavbeta6 integrin plays an important role in wound healing in animals compromised by either age or stress mimicked by hydrocortisone.

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.

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts. Collagen beads bound to gingival cells by alpha2beta1 integrins. Binding and internalization of decorin/collagen-coated beads decreased dose-dependently with increasing decorin concentration (p < 0.001). Inhibition of binding was sustained over 5 h (p < 0.001) and was attributed to interactions between decorin and collagen and not to decorin-collagen receptor interactions. Both the non-glycosylated decorin core protein and the thermally denatured decorin significantly inhibited collagen bead binding (approximately 50 and 89%, respectively; p < 0.05). Mimetic peptides corresponding to leucine-rich repeats 1-3, encompassed by a collagen-binding approximately 11-kDa cyanogen bromide fragment of decorin and leucine-rich repeats 4 and 5, previously shown to bind to collagen, were tested for their ability to inhibit collagen bead binding. Although the synthetic peptide 3 alone exhibited saturable binding to collagen, neither peptides 3 nor 1 and 2 markedly inhibited phagocytosis. Leucine-rich repeat 3 bound to a triple helical peptide containing the alpha2 integrin-binding site of collagen. When collagen beads were co-incubated with peptides 3 and 4, inhibition of collagen phagocytosis (55%) was equivalent to intact native/recombinant core protein. Thus a novel collagen binding domain in decorin acts cooperatively with leucine-rich repeat 4 to mask the alpha2beta1 integrin-binding site on collagen, an important sequence for the phagocytosis of collagen fibrils.

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.

Integrin alphavbeta6 is an epithelial cell-specific receptor that is not normally expressed by resting epithelium but its expression is induced during wound healing. The function of alphavbeta6-integrin in wound repair is not clear. In the present study, we showed that beta6-integrin expression was strongly up-regulated in the epidermis in human chronic wounds but not in different forms of skin fibrosis. To test whether increased beta6-integrin expression plays a role in abnormal wound healing we developed four homozygous transgenic mouse lines that constitutively expressed human beta6-integrin in the epithelium. The mice developed normally and did not show any histological abnormalities in the skin. The rate of experimental skin wound closure was unaltered and the wounds healed without significant scar formation. However, during breeding program 16.1 to 27.0% of transgenic mice developed spontaneous, progressing fibrotic chronic ulcers. None of the wild-type animals developed these lesions. The chronic lesions had areas with severe fibrosis and numerous activated macrophages and fibroblasts expressing transforming growth factor (TGF)-beta. The level of TGF-beta1 was significantly increased in the lesions as compared with normal skin. The findings suggest that increased alphavbeta6-integrin in keratinocytes plays an active part in abnormal wound healing possibly through a mechanism involving increased activation of TGF-beta.

Squamous cell carcinoma (SCC) of the oral cavity is a highly invasive tumour of stratified squamous epithelium that spreads through degradation of the basement membrane (BM) and extracellular matrix (ECM). There are currently no reliable tissue or serum markers to predict whether the tumour has metastasized at the time of diagnosis. Verrucous carcinoma (VC) of the oral cavity is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. Many matrix metalloproteinases (MMPs), such as MMP-1, -2, -7, -9, -13, and -14, as well as integrin receptors have been implicated in cancer invasion. Integrin alphavbeta6 is induced in SCC and appears to be involved in up-regulation of MMP-9 expression by oral keratinocytes and promotion of their migration. The aim of this study was to investigate whether the pattern of MMP expression or that of alphavbeta6 integrin contributes to the differences in the biological behaviour of oral SCC and VC. The results show that the less aggressive nature of oral VC may be connected to its MMP expression profile. Typically, VCs were devoid of epithelial MMP-3, -7, -9, -12 and -13 expression, compared with SCCs. MMP-19 was expressed by epithelial keratinocytes in hyperproliferative areas of verrucous hyperplasia, VC, and SCC, but was absent in the invasive cancer cell nests of SCC. MMP-26 was expressed by hyperproliferative keratinocytes in VC as well as by invasive cancer cells in SCCs. MMP-10 was expressed widely in the epithelium of all SCC specimens. alphavbeta6 integrin expression was also detected in some cases of epithelial hyperplasia but was significantly more abundant in cancers at the invasive front. The absence of MMP-7, -9 and -12 from epithelial cells may serve as a good prognostic marker of non-invasive oral carcinoma. Blocking the activity of invasion-specific MMPs or alphavbeta6 integrin might offer novel therapeutic modalities in early-stage oral carcinoma.