The influence of type I diabetes mellitus in periodontal disease induced changes of the gingival epithelium and connective tissue

Periodontal disease constitutes the most frequent chronic diseases in human dentition. Bacterial plaque is the main etiologic agent, although it is the host immune response that causes periodontal tissue destruction. Diabetes is considered an important risk factor, not only for the onset but also for progression of the disease. The aim of this study was to analyze structural changes in the rat gingival epithelium and connective tissue in response to the experimental periodontal disease induced by the ligature technique, under the influence of diabetes. The results showed that experimental periodontal disease is characterized by marked inflammation, affecting both the epithelial and connective tissues, causing degeneration of the dermal papilla, increase in the number of inflammatory cells, destruction of reticulin fibers, and accumulation of dense collagen fibers (fibrosis). These changes were worsened by diabetes, apparently by hampering the inflammatory response and affecting tissue repair of the affected tissues.     

Pertinent cell population to characterize periodontal disease

The purpose of this in situ study is to quantify the inflammatory cell subsets and the area fraction (AA%) occupied by collagen fibers in human healthy and diseased (four different stages) gingival connective tissue in order to establish a possible correlation between periodontal disease resulting in collagen breakdown and specific inflammatory cell subsets.Paraffin gingival tissue sections from eight healthy controls (group 0), 10 patients with gingivitis (group 1), 10 patients with moderate periodontitis (group 2) and 10 patients with severe periodontitis (group 3) were immunohistochemically investigated using antibodies against CD-45+, CD-3+, CD-8+, CD-20+, CD-68+, and EMA+ (plasma cells).The AA% occupied by gingival collagen fibers significantly decreased from 54.12% in group (0) to 38.58% in group (1), to 31.87% in group (2), and to 25.46% in group (3). In progressive lesions of periodontal disease, CD-3+ and CD-8+ cell numbers were increased in early stages within the connective tissue, while CD-20+ cell numbers were increased only in late stages. On the other hand, EMA+, CD-68+ and CD-45+ cell numbers were progressively increased from group (0) to group (3). We demonstrated that CD-68+ monocyte/macrophages, CD-45+ leukocyte common antigen and notably EMA+ plasma cells are pertinently correlated with the severity of periodontal disease and related collagen breakdown.     

Immunohistochemical study of types I, III and IV collagen in diseased human gingiva of patients with rapidly progressive periodontitis: a light and electron microscopic study

The distribution of type I, III and IV collagens and their ultrastructural organization have been studied in diseased gingival connective tissue of patients with rapidly progressive periodontitis. This disease is characterized by acute destruction of the gingival collagenous components. The use of an immunofluorescent procedure has shown that the diseased connective tissue was made up of both type I and III collagens but that type III collagen was less resistant to acute inflammation. Ultrastructural immunolabelling, using the peroxidase procedure has shown that the large, dense bundles of type I collagen of PI, the main pattern of organization of the gingival connective tissue offered a better resistance to acute destruction than PII, a loose pattern of organization mainly composed of type III collagen. Type IV collagen was exclusively located in degraded lamina densa of basement membrane.     

Transglutaminase 2 expression is significantly increased in cyclosporine-induced gingival overgrowth

Cyclosporine A is a potent immunosuppressant used to prevent organ transplant rejection and treat various autoimmune diseases. However, cyclosporine A can also induce gingival overgrowth, which is characterized by increased extracellular matrix due to an altered balance between collagen synthesis and degradation. This study proposed to verify whether trans-glutaminase 2, an enzyme thought to be responsible for the assembly and remodelling of extracellular matrix, plays any role in the pathogenesis of cyclosporine A-induced gingival overgrowth. Cyclosporine A-induced gingival overgrowths were collected from 21 liver transplant patients and case-controlled with 20 non-hyperplastic gingival biopsies from healthy patients who had previous periodontal treatment. In both groups, the presence and tissue distribution of transglutaminase 2 were determined by immunohistochemistry and analyzed in comparison with the tissue morphology and expression of lymphocyte-related antigens (CD3 and CD20) and a vessel-related marker (CD34). Transglutaminase 2 expression showed a significant increase (2.6-fold) in the stromal component of cyclosporine A-treated patients compared with controls (p<0.001), which suggested that transglutaminase 2 had a role in the pathogenesis of the disease. Further studies should investigate the therapeutic effect of anti-transglutaminase 2 drugs (putrescine or 1,4-diamino-butane) in these patients.     

A comparative analysis of structure and spatial distribution of decorin in human leiomyoma and normal myometrium

Leiomyoma is a benign smooth muscle tumor of the uterus that affects many women in active reproductive life. It is composed by bundles of smooth muscle cells surrounded by extracellular matrix. We have recently shown that the glycosylation of extracellular matrix proteoglycans is modified in leiomyoma: increased amounts of galactosaminoglycans with structural modifications are present. The data here presented show that decorin is present in both normal myometrium and leiomyoma but tumoral decorin is glycosylated with longer galactosaminoglycan side chains. Furthermore, these chains contain a higher ratio D-glucuronate/L-iduronate, as compared to normal tissue. To determine if these changes in proteoglycan glycosylation correlates with modifications in the extracellular matrix organization, we compared the general structural architecture of leiomyoma to normal myometrium. By histochemical and immunofluorescence methods, we found a reorganization of muscle fibers and extracellular matrix, with changes in the distribution of glycoproteins, proteoglycans, and collagen. Thin reticular fibers, possibly composed by types I and III collagen, were replaced by thick fibers, possibly richer in type I collagen. Type I collagen colocalized with decorin both in leiomyoma and normal myometrium, in contrast to type IV collagen that did not. The relative amount of decorin was increased and the distribution of decorin and collagen was totally modified in the tumor, as compared to the normal myometrium. These findings reveal that not only decorin structure is modified in leiomyoma but also the tissue architecture changed, especially concerning extracellular matrix.     

Alterations in sarcomere structure, collagen organization, mitochondrial activity, and protein metabolism in the avian low score normal muscle weakness

Skeletal muscle fibers are surrounded by an extracellular matrix. The extracellular matrix is composed of glycoproteins, collagen, and proteoglycans. Proteoglycans have been suggested to play an important functional role in tissue differentiation. However, an understanding of how the extracellular matrix affects skeletal muscle development and function is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), a late embryonic increase in the expression of decorin is followed by a subsequent increase in collagen crosslinking. The sarcomere organization, collagen fibril diameter and organization were investigated using transmission electron microscopy. Measurements were made at 20 days of embryonic development and 6 weeks posthatch. These studies showed changes in sarcomere organization and deterioration of muscle fibril structure in the LSN pectoral muscle. In vitro satellite cell cultures were developed and assayed for mitochondrial activity, and protein synthesis and degradation. In these analyses, mitochondrial activity from LSN satellite cells was significantly higher than those from normal pectoral muscle satellite cells. Protein synthesis rates between the normal and LSN satellite cell-derived myotubes were similar, but protein degradation rates were higher in the LSN cultures. Based on the reported functions of decorin as a regulator of cell proliferation and collagen fibril organization, it is possible that the late embryonic increase in decorin may be influencing the alterations in LSN sarcomere and collagen organization.     

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen or fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by η-amino-caproic acid (ηACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva. © 1996 Wiley-Liss, Inc.     

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-alpha and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior.     

Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts.

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Ultrastructural and immunocytochemical study of bone-derived cells cultured in three-dimensional matrices: Influence of chondroitin-4 sulfate on mineralization

Abstract. Bone-derived cells were cultured in three-dimensional reconstituted matrices made of type I collagen or type I collagen chondroitin-4-sulfate. As observed by microscope, their characteristics were as follows: The cells deposited a faint extracellular matrix mainly composed of type I collagen. In the collagen-chondroitin-sulfate sponge fibers, a calcification process, which involved the deposition of hydroxyapatite crystals, was demonstrated. Mineralization occurred only in collagen chondroitin sulfate sponge fibers when seeded with bone-derived cells and was not seen with nonosteogenic cells, such as gingival fibroblasts. Gla protein was intracellularly visualized in both types of sponges seeded with bone-derived cells while an extracellular secretion was seen only in the collagen chondroitin sulfate sponge fibers where calcification occurred. These results suggest that collagen chondroitin sulfate promotes in vitro mineralization of three-dimensional collagen matrices when seeded with bone-derived cells.     

人牙龈成纤维细胞与牙周膜细胞的生物活性

采用组织块法分离培养牙周膜细胞和牙龈成纤维细胞,测定二者的增殖特性和ALP活性,利用免疫组化和FCM方法比较Ⅰ、Ⅲ型胶原、BMP的表达情况,以观察对比两种细胞的生物学特性的异同。找出牙龈成纤维细胞和牙周膜细胞在胶原基质合成方面存在差异,发现Ⅰ、Ⅲ型胶原可作为鉴别两种细胞的标志物,ALP与BMP可作为鉴别两种细胞的标志,牙周膜细胞比牙龈成纤维细胞具有较强的成骨能力。从而为今后改良两种细胞成为牙周组织工程的种子细胞奠定基础。     

Inflammatory cells do not decrease the ultimate tensile strength of intact tendons in vivo and in vitro: protective role of mechanical loading.

Although inflammatory cells and their products are involved in various pathological processes, a possible role in tendon dysfunction has never been convincingly confirmed and extensively investigated. The goal of this study was to determine whether or not an acute inflammatory process deprived of mechanical trauma can induce nonspecific damages to intact collagen fibers. To induce leukocyte accumulation, carrageenan was injected into rat Achilles tendons. We first tested the effect of leukocyte recruitment on the concentrations or activities of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. Second, we analyzed at the biochemical, histological, and biomechanical levels the impact of leukocyte invasion on tendons. Finally, collagen bundles isolated from rat-tail tendons were exposed in vitro to mechanical stress and/or inflammatory cells to determine if mechanical loading could protect tendons from the leukocyte proteolytic activity. Carrageenan-induced leukocyte accumulation was associated with an increased matrix metalloproteinase activity and a decreased content of tissue inhibitors of matrix metalloproteinases. However, hydroxyproline content and load to failure did not change significantly in these tendons. Interestingly, mechanical stress, when applied in vitro, protected collagen bundles from inflammatory cell-induced deterioration. Together, our results suggest that acute inflammation does not induce damages to intact and mechanically stressed collagen fibers. This protective effect would not rely on increased tissue inhibitors of matrix metalloproteinases content but would rather be conferred to the intrinsic resistance of mechanically loaded collagen fibers to proteolytic degradation.     

Ultrastructural aspects of corneal fibrous tissue in the scheie syndrome

An ultrastructural study of the corneal fibrous tissue was performed in a case of Scheie's syndrome. Mucopolysaccharidosis deposits in keratocytes were observed as electron-clear and electron-dense inclusions. Modifications of the extracellular space included modifications of lamellar collagen organization and local hypertrophy of collagen bundles; presence of microfibrillar dense material isolating large irregular collagen fibers; and presence of fibrous long spacing type collagen fibers. The significance of these changes is discussed. This special form of collagen organization is supposed to appear in a modified microenvironment, that is the presence of an abnormal concentration of proteoglycans.     

Distribution of newly synthesized aggrecan in explant cultures of bovine cartilage treated with retinoic acid.

This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when explant cultures of articular cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In cartilage explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.     

Contributions of Glycosaminoglycans to Collagen Fiber Recruitment in Constitutive Modeling of Arterial Mechanics

The contribution of glycosaminoglycans (GAGs) to the biological and mechanical functions of biological tissue has emerged as an important area of research. GAGs provide structural basis for the organization and assembly of extracellular matrix (ECM). The mechanics of tissue with low GAG content can be indirectly affected by the interaction of GAGs with collagen fibers, which have long been known to be one of the primary contributors to soft tissue mechanics. Our earlier study showed that enzymatic GAG depletion results in straighter collagen fibers that are recruited at lower levels of stretch, and a corresponding shift in earlier arterial stiffening (Mattson et al., 2016). In this study, the effect of GAGs on collagen fiber recruitment was studied through a structure-based constitutive model. The model incorporates structural information, such as fiber orientation distribution, content, and recruitment of medial elastin, medial collagen, and adventitial collagen fibers. The model was first used to study planar biaxial tensile stress-stretch behavior of porcine descending thoracic aorta. Changes in elastin and collagen fiber orientation distribution, and collagen fiber recruitment were then incorporated into the model in order to predict the stress-stretch behavior of GAG depleted tissue. Our study shows that incorporating early collagen fiber recruitment into the model predicts the stress-stretch response of GAG depleted tissue reasonably well (rms = 0.141); considering further changes of fiber orientation distribution does not improve the predicting capability (rms = 0.149). Our study suggests an important role of GAGs in arterial mechanics that should be considered in developing constitutive models.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.     

YKL-40 secreted from adipose tissue inhibits degradation of type I collagen

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.