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.

Global TLR2 and 4 deficiency in mice impacts bone resorption, inflammatory markers and atherosclerosis to polymicrobial infection.

Toll-like-receptors (TLRs) play a significant role in the generation of a specific innate immune response against invading pathogens. TLR2 and TLR4 signaling contributes to infection-induced inflammation in periodontal disease (PD) and atherosclerosis. Observational studies point towards a relationship between PD and atherosclerosis, but the role of TLR2 and TLR4 in the recognition of multiple oral pathogens and their modulation of host response leading to atherosclerosis are not clear. We evaluated the role of TLR2 and TLR4 signaling in the induction of both PD and atherosclerosis in TLR2-/- and TLR4-/- mice to polymicrobial infection with periodontal pathogens Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Polybacterial infections have established gingival colonization in TLR2-/- and TLR4-/- mice and induction of a pathogen-specific immunoglobulin G immune response. But TLR deficiency dampened accelerated alveolar bone resorption and intrabony defects, indicating a central role in infection-induced PD. Periodontal bacteria disseminated from gingival tissue to the heart and aorta through intravascular dissemination; however, there was no increase in atherosclerosis progression in the aortic arch. Polybacterial infection does not alter levels of serum risk factors such as oxidized low-density lipoprotein, nitric oxide, and lipid fractions in both mice. Polymicrobial-infected TLR2-/- mice demonstrated significant levels (P < 0.05 to P < 0.01) of T helper type 2 [transforming growth factor-ß1 , macrophage inflammatory protein-3α, interleukin-13 (IL-13)] and T helper type 17 (IL-17, IL-21, IL-22, IL-23) splenic T-cell cytokine responses. Increased heat-shock protein expression, hspa1a for Hsp 70, was observed for both TLR2-/- and TLR4-/- mice. This study supports a role for TLR2 and TLR4 in PD and atherosclerosis, corroborating an intricate association between two inflammatory diseases.

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.

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.

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.

Increased cytoplasmic level of migfilin is associated with higher grades of human leiomyosarcoma.

AIMS: Leiomyosarcomas (LMS) are malignant neoplasms composed of cells that exhibit distinct smooth muscle differentiation. The molecular and cytogenetic features of LMS are complex and no consistent aberrations have been reported to date. Mitogen inducible gene-2 (Mig-2), kindlin and migfilin are recently identified cell-matrix adhesion proteins. The aim was to determine the expression and distribution of these proteins in human smooth muscle tumours of somatic soft tissue. METHODS AND RESULTS: Immunohistochemistry was performed on a human LMS tissue microarray and on sections of human leiomyomas (LM) and normal smooth muscle. Migfilin was barely detectable in normal smooth muscle cells, whereas increased levels of migfilin were observed in the majority of LM and LMS. Furthermore, the cytoplasmic level of migfilin was strongly associated with higher tumour grades. Additionally, the cytoplasmic levels of migfilin and Mig-2 were correlated with each other, suggesting an association between the two in the cytoplasm. Kindlin was expressed in normal smooth muscle, LM and LMS, and its level did not correlate with tumour grade. CONCLUSIONS: Our results suggest a role for cytoplasmic migfilin in the progression of LMS and identify cytoplasmic migfilin as a potentially important biological marker for human LMS progression.

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.

Severe periodontitis in a 5-year-old girl with hyperimmunoglobulin E syndrome.

The hyperimmunoglobulin E syndrome (HIES) is a multisystem disorder that affects the: (1) dentition; (2) skeleton; (3) connective tissues; and (4) immune system. Little is known about periodontal manifestations of the syndrome. The purpose of this report was to describe a 5-year-old girl with suspected autosomal-recessive HIES syndrome who revealed profusely bleeding and painful gingiva and generalized aggressive periodontitis. A polymerase chain reaction (PCR)-based microbiological examination detected Porphyromonas gingivalis, Tannerella forsythia, Prevotella nigrescens, Treponema denticola, Eikenella corrodens, and Campylobacter rectus in the deep periodontitis lesions. The extraction of all deciduous teeth due to a poor prognosis and risk of systemic infection led to resolution of the oral inflammation. Long-term follow-up is required to determine the periodontal prognosis of the erupting permanent teeth.

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.

Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9), and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer.

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.

Characterization of organotypic keratinocyte cultures on de-epithelialized bovine tongue mucosa.

Organotypic cultures have been used to study epithelial cell behavior for many years. The aim of this study was to develop an organotypic culture method that better mimics the three-dimensional morphology of interdigitating rete ridges and connective tissue papillae and that also conserves the basement membrane zone (BMZ). Bovine tongue mucosa connective tissue, separated from epithelium after 1 M NaCl incubation, was used as organotypic culture substratum for different human keratinocyte cell lines. Organotypic cultures were characterized by electron and immunofluorescence microscopy for expression of integrin subunits and extracellular matrix components. While spontaneously immortalized mucosal keratinocytes produced highly irregular stratified organotypic cultures, the normal human epidermal keratinocytes (NHEK) demonstrated culture morphology that resembled in vivo epidermis. However, in this model, the histomorphology, expression of differentiation markers involucrin, keratin 10 and 14, and integrins varied significantly between the cell lines. Some cultures appeared to have an extended survival since they were maintained up to 40 days without histological signs of degeneration. The ultrastructure of the BMZ including hemidesmosomes was similar to the normal dermo-epidermal junction. Extracellular matrix molecules, including tenascin, laminin-1 and -5, were expressed in the cultures demonstrating their secretion solely by keratinocytes. Distribution and expression of integrins in NHEK cultures was similar to that seen in vivo skin with the exception of additional expression of alpha5beta1 and alpha(v)beta6 integrins. Organotypic NHEK cultures show similarities to normal stratified epithelium and are potentially useful for multiple applications for studies on epithelial cell behavior in vitro.

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.

Exogenous phospholipase C stimulates epithelial cell migration and integrin expression in vitro.

Phospholipase C secreted by bacterial pathogens has been identified as a virulence factor in several human diseases and has been implicated in impeding wound healing. The role of phospholipase C in the intracellular signal control of epithelial growth was studied in normal human skin keratinocytes cultured in conditions simulating aspects of wound healing. Bacillus cereus phospholipase C decreased cell-cell contact and increased cell migration resulting in disruption of the advancing epithelial sheet. Phospholipase C-induced migration was blocked by inhibitor of the phosphoinositol signal transduction pathway neomycin sulfate and protein kinase C inhibitor RO-31-8220. Induced migration was associated with elevated levels of matrix metalloproteinase-9 which, when blocked by tissue inhibitor of metalloproteinase-1, was accompanied by a loss of migration. Adhesion studies showed that phospholipase C treatment enhanced cell binding to fibronectin, vitronectin and collagen IV. Immunostained phospholipase C-stimulated cells cultured on fibronectin showed enhanced expression and relocation of the integrin subunits alpha(v), alpha5 and beta1. Confocal microscopy showed that phospholipase C-induced levels of integrin subunit beta1 were predominantly deposited on the basal surface of the cell apparently in focal contacts and associated with actin stress fibers. These results indicate that exogenous phospholipase C signaling from a bacterial source may play an important role in perturbing normal reepithelialization via altered expression of integrins and matrix metalloproteinase-9.

An improved method for culture of epidermal keratinocytes from newborn mouse skin.

Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture medium containing low Ca2+ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin alpha v beta 6. The expression level of alpha v beta 6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.

Proteolytic events of wound-healing--coordinated interactions among matrix metalloproteinases (MMPs), integrins, and extracellular matrix molecules.

During wound-healing, cells are required to migrate rapidly into the wound site via a proteolytically generated pathway in the provisional matrix, to produce new extracellular matrix, and, subsequently, to remodel the newly formed tissue matrix during the maturation phase. Two classes of molecules cooperate closely to achieve this goal, namely, the matrix adhesion and signaling receptors, the integrins, and matrix-degrading and -processing enzymes, the matrix metalloproteinases (MMPs). There is now substantial experimental evidence that blocking key molecules of either group will prevent or seriously delay wound-healing. It has been known for some time now that cell adhesion by means of the integrins regulates the expression of MMPs. In addition, certain MMPs can bind to integrins or other receptors on the cell surface involved in enzyme activation, thereby providing a mechanism for localized matrix degradation. By proteolytically modifying the existing matrix molecules, the MMPs can then induce changes in cell behavior and function from a state of rest to migration. During wound repair, the expression of integrins and MMPs is simultaneously up-regulated. This review will focus on those aspects of the extensive knowledge of fibroblast and keratinocyte MMPs and integrins in biological processes that relate to wound-healing.

Immunolocalization of tenascin-C, alpha9 integrin subunit, and alphavbeta6 integrin during wound healing in human oral mucosa.

Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal-epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-C(L)) and the putative TN-C-binding alpha9 and alphavbeta6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for alpha9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-C(L) were localized in the matrix between and underneath alpha9-expressing epithelial cells. In parallel with gradual downregulation of alpha9 integrin immunoreactivity in 7-day and older wounds, the expression of alphavbeta6 integrin was temporarily induced. Integrin alphavbeta6 co-localized in the same area as TN-C and TN-C(L) immunoreactivity at the cell-cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-C(L) were abundantly localized in the granulation tissue. The findings show that TN-C(L) is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of alpha9 integrin in migrating epithelial cells and of alphavbeta6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.