Unusual glycosaminoglycans from a deep sea hydrothermal bacterium improve fibrillar collagen structuring and fibroblast activities in engineered connective tissues.

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair.

Clinical, histological, and histomorphometrical analysis of maxillary sinus augmentation using inorganic bovine in humans: preliminary results.

The aim of the present study was to evaluate bone formation after maxillary sinus augmentation using bovine bone substitute material Bio-Oss alone by means of clinical, histological, and histomorphometrical examination of human biopsies. Deproteinized bovine bone (DPBB, Bio-Oss) was used to fill cavities after elevation of the sinus mucosa following major sinus pneumatization. Twenty patients with edentulous posterior maxillae were treated with 20 sinus augmentation procedures using a 2-stage technique. Residual lateral maxillary bone height was less than 3 mm. Forty-nine Straumann endosseous implants were used to complete the implant-prosthetic rehabilitation. Forty cylinder-shaped bone biopsies were taken from the augmented maxillary region 8 months after grafting during the second-stage surgery before implant placement. All implants were loaded 3 months after insertion, and no failures were recorded. Histomorphometrical analysis showed an average percentage of newly formed bone of 17.6% (± 2.8%) and a proportion of residual bone substitute material of 29.9% (± 4.9%) of the total biopsy area. Intimate contact between newly formed bone and Bio-Oss was detected along 28.2% (± 6.8%) of the particle surfaces. The results also showed that in all cases, the DPBB granules had been interconnected by bridges of vital newly formed bone. Inorganic bovine bone appears to be biocompatible and osteoconductive, and it can be used with success as a bone substitute in maxillary sinus augmentation procedures.

Inherited connective tissue diseases highlight macromolecular network interdependences in skin extracellular matrix: a histomorphometric study.

Mutation of just a single extracellular matrix protein, a receptor or enzyme involved in connective tissue metabolism is sufficient to cause systemic pathologies and failure of tissues that are subjected to strong mechanical stresses. Skin histological and computerized image analyses can provide a good qualitative and quantitative indication of these inherited connective tissue diseases. In this study, skin biopsies from young (10 to 25 years) and middle-aged patients (26 to 50 years) suffering from Ehlers-Danlos syndromes (EDS), Marfan syndrome (MS) or pseudoxanthoma elasticum (PXE) were studied after specific staining of both the collagen and elastic networks. Findings from the histomorphometric analyses conducted on skin sections of the patients with EDS, MS and PXE were compared to skin sections of healthy subjects from the same age groups. Our results show that both the collagen and the elastic networks were affected in all the studied pathological cases, but that the adverse changes to the elastic network in older patients were distinct from the physiological changes observed during aging process for healthy subjects. This degenerative process may be explained by an added phenomenon involving a general connective tissue proteolysis.

Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering.

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.

Human bone marrow-derived cells: an attractive source to populate dermal substitutes.

We have previously shown the importance of dermal fibroblasts within skin substitutes for promoting the emergence of a functional neodermis after grafting in humans. However, the use of fibroblasts from sources other than the dermis needs to be evaluated for patients with extensive skin loss. Here we examined the capacity of human bone marrow-derived cells (BMDCs), selected for their ability to adhere to plastic culture dishes, to behave like human dermal fibroblasts when incorporated within a 3D in vitro reconstructed tissue that promotes dermal fibroblast differentiation. Like dermal fibroblasts, BMDCs contracted a collagen matrix and were growth regulated by the matrix environment. They had the same shape and their nuclei had the same form factor as dermal fibroblasts. In addition, both cell types expressed desmin and vimentin but not alpha-smooth muscle actin. BMDCs deposited collagen types I and III, and fibrillin-1 with similar efficiency to dermal fibroblasts. In addition, BMDCs have the potential to regulate this deposition, as they produced metalloproteinases (MMP1, MMP2, and MMP9) and metalloproteinase inhibitors (TIMP1) very similarly to dermal fibroblasts. BMDCs can thus be induced to express functions resembling those of dermal fibroblasts, including those involved in the wound healing process.

Comparison of extracellular matrix in skin and saphenous veins from patients with varicose veins: does the skin reflect venous matrix changes?

Varicose vein disease is a frequently occurring pathology with multifactorial causes and a genetic component. An intense remodelling of the varicose vein wall has been described and could be at the origin of its weakness and altered elasticity. We have described previously a dysregulation of collagen synthesis in cultured smooth muscle cells from saphenous veins and in dermal fibroblasts from the skin of patients with varicose veins, suggesting a systemic defect in their connective tissue. The present study describes comparative morphological and immunohistochemical data in both the skin and saphenous veins of eight control subjects (undergoing coronary bypass surgery) and eight patients with varicose veins. Histological staining of glycoproteins, the elastic fibre network and collagen bundles showed that the remodelling and fragmentation of elastic fibres observed in varicose veins were also present in the skin of the patients. When compared with control subjects, we observed in both the veins and skin of patients with varicose veins (i) an increase in the elastic network, as quantified by image analysis; (ii) an accumulation of collagen type I, fibrillin-1 and laminin; and (iii) an overproduction of MMP (matrix metalloproteinase)-1, MMP-2 and MMP-3, analysed by immunohistochemistry, but normal levels of other MMPs (MMP-7 and MMP-9) and their inhibitors (TIMP-1, TIMP-2 and TIMP-3). An imbalance of extracellular matrix production/degradation was thus observed in veins as well as in the skin of the patients with varicose veins and, taken together, these findings show that remodelling is present in different organs, confirming systemic alterations of connective tissues.

Do Langerhans cells behave similarly in elderly and younger patients with chronic periodontitis?

OBJECTIVE: The purpose of this study was to compare the number, the distribution and the expression of markers of maturation of Langerhans cells (LC) in elderly and younger patients with chronic periodontitis in order to evidence the effect of aging on LC in inflammatory gingival tissue. METHODS: Gingival tissue specimens presenting chronic periodontitis from 8 elderly patients aged >75 (group E) and from 8 younger patients aged 50-60 (considered as controls, group C) were used for immunohistochemistry with monoclonal antibodies against CD45RB (leucocytes), CD1a (LC), markers of LC maturation (DC-LAMP, CD83) and number of immunolabelled cell subsets was evaluated using image analysis. RESULTS: The difference in the number of CD45RB+ leucocytes in the upper connective tissue between groups was not significant. In group E, the number of CD1a+ LC was significantly decreased (P<0.002) in the epithelium and significantly increased (P<0.0004) in the upper connective tissue. Furthermore, in group E, intraepithelial CD1a+ LC are more often observed in the upper epithelium and their dendritic processes were shorter and less numerous. Concerning the expression of markers of maturation, the numbers of intraepithelial DC-LAMP+ cells and CD83+ cells were significantly increased (P<0.0007 and P<0.02, respectively) in group E. CONCLUSION: During chronic periodontitis in elderly patients, the decrease in the number of intraepithelial LC and the alteration of dendritic processes could be balanced by a cellular distribution often observed in the upper epithelium associated with changes in cell maturation in response to bacterial elements.

Dense fibrillar collagen matrices: a model to study myofibroblast behaviour during wound healing.

Fibroblastic cells play an important part in wound healing. Human dermal fibroblasts seeded onto three-dimensional fibrillar collagen matrices migrate into the collagen network and differentiate into myofibroblasts. In order to evaluate the use of collagen matrices as model systems for studying myofibroblast phenotype during wound healing, myofibroblast behaviour migrating into dense or loose matrices was compared. The effect of collagen concentration on cell morphology, remodelling, proliferation and apoptosis of human myofibroblasts was evaluated. Myofibroblasts within dense collagen matrices (40 mg/ml) were spindle shaped, similar to cells observed during tissue repair. In contrast, cells within loose matrices (5mg/ml) were more rounded. Matrix hydrolysis activities (MT1-MMP and MMP2) did not differ between the two collagen concentrations. The myofibroblast proliferation rate was measured after 24h bromodeoxyuridine incorporation (BrdU). Cells in dense collagen matrices proliferated at a higher rate than cells in loose matrices at each culture time point tested. For example, 40% of cells in dense matrices were replicating compared to 10% of cells in loose matrices after 28 days in culture. Apoptotic cells were only detected in dense matrices from day 21 onwards when cells had already migrated into the collagen network. Taken together, these results show that a high collagen concentration has a stimulatory effect on myofibroblast proliferation and apoptosis, two important events in wound healing. Thus, dense matrices can be used to create controlled conditions to study myofibroblast phenotype.

Fibroblast populated dense collagen matrices: cell migration, cell density and metalloproteinases expression.

Dense collagen matrices obtained by using the property of type I collagen to form liquid crystals at high concentrations, were shown to be colonized by human dermal fibroblasts (Biomaterials 23 (2002) 27). In order to evaluate them as possible tissue substitutes, we investigated in this study the mechanism of cell colonization. Fibroblasts were seeded at the surface of collagen matrices at concentrations of 5 and 40 b mg/ml. Cell density and migration were estimated from histological sections over 28 days within 500 microm thick matrices. At day 14, migration started in the 40 mg/ml matrices, attaining 320 microm in distance and 5500 cell/mm(3) in density at day 28. As zymography and western blot techniques demonstrated production of collagenase 1 (MMP1) and gelatinase A (MMP2) in culture medium, collagen hydrolysis was required for cells to penetrate the collagen network. Furthermore, the presence of MMP1 and MMP2 and their tissue inhibitors TIMP1 and TIMP2 was revealed by immunohistochemistry. We presently show that 40 mg/ml collagen matrices are colonized by human dermal fibroblasts and reach, at day 28, a density close to that measured in human dermis.

Lupinus albus, a novel vegetable extract with metalloproteinase inhibitory properties: a potential periodontal therapy.

BACKGROUND: In this study we examine the properties of a vegetable extract from seeds of Lupinus albus (LU 105). In previous works we demonstrated that LU 105 reduced the expression, by gingival fibroblasts, of both matrix metalloproteinase (MMP)-2 and MMP-9. We decided to study the impact of LU 105 on cell proliferation and morphology. Using organ culture media we also studied the MMP and tissue inhibitors of metalloproteinases (timp) expression AND THE cytokines secretion. METHODS: Healthy and inflamed gingival biopsies were placed in appendage culture with or without LU 105. The organ culture media were analyzed using Western blottings (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, TIMP-1, and TIMP-2) and gelatine zymography. A reverse transcription polymerase chain reaction (RT-PCR) was also performed on healthy and inflamed gingival biopsies, which were maintained in culture with or without LU 105 0.1%. Then, we decided to determine the amount of cytokines present in the organ culture media such as interleukin (IL)-1 beta, IL-4, IL-6, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha. RESULTS: When gingival biopsies derived from inflamed tissues were cultured with LU 105 0.1% in the culture media, the MMP and TIMP expression and activity decreased significantly when compared to cultures without LU 105. Moreover, we did not note any statistical difference in the cell proliferation compared with human gingival fibroblast cultures without LU 105. Furthermore, IL-1 beta, IL-6, TGF-beta, and TNF-alpha amounts in the culture media decreased significantly, whereas IL-4 increased significantly when LU 105 0.1% was added to the culture media. CONCLUSION: LU 105, a novel metalloproteinase inhibitor with few consequences on cell proliferation and morphology, is a vegetable extract with potential clinical capacity.

Skin expression of metalloproteinases and tissue inhibitor of metalloproteinases in sibling patients with recessive dystrophic epidermolysis and intrafamilial phenotypic variation.

A number of COL7A1 mutations have now been reported in recessive dystrophic epidermolysis bullosa patients, and the analysis of phenotype-genotype correlations showed evidence for interfamilial and intrafamilial phenotypic variability, occurring for the same mutation. Collagenase and stromelysin activities have been found to be overexpressed in skin cultures of some recessive dystrophic epidermolysis bullosa patients, and tissue destruction in the disease process might result from an imbalance of metalloproteinases (MMP) over tissueinhibitor of metalloproteinases (TIMP). So we suspected that the phenotypic variability for the same mutation could be linked to other genetic or environmental factors, as a particular balance between MMP and TIMP. Organ cultures were performed using explants from the skin of three patients from the same family with recessive dystrophic epidermolysis bullosa to reveal and quantify the expression of MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MMP-3 (stromelysin 1), TIMP-1, and TIMP-2, and to compare the results with those obtained with two human control skins, with the same experimental conditions. Increased amounts of all metalloproteinases investigated were observed in the skin of the three recessive dystrophic epidermolysis bullosa affected sibling brothers, both in lesioned and in apparently nonlesioned skin, compared with controls. The amounts of MMP-1, MMP-2, MMP-3, and MMP-9 increased particularly in the skin of the more clinically affected patient. Furthermore for this patient we evidenced higher amounts of MMP-1 and also a lower TIMP-1 amount in his unlesioned and lesioned skin compared with the other two affected patients and with healthy control donors. So we can suspect that recessive dystrophic epidermolysis bullosa phenotypic variability could be related to patients' collagenase activity heterogeneity, linked to imbalance between MMP-1 and TIMP-1.

Effect of adenovirus-mediated overexpression of decorin on metalloproteinases, tissue inhibitors of metalloproteinases and cytokines secretion by human gingival fibroblasts.

Decorin is a small leucine-rich proteoglycan that plays a role in control of cell proliferation, cell migration, collagen fibrillogenesis and modulation of the activity of TGF-beta. In the present study, we investigated the effects of decorin on the production of metalloproteinases (MMP-1, -2, -3, -9 and -13), tissue inhibitors of metalloproteinases (TIMP-1, -2) and cytokines (TGF-beta, IL-1beta, IL-4 and TNF-alpha). Decorin was overexpressed in cultured human gingival fibroblasts using adenovirus-mediated gene transfer. Decorin infection resulted in decreased protein levels of MMP-1 and MMP-3 whereas MMP-2 and TIMP-2 secretion was increased. MMP-9, MMP-13 and TIMP-1 were not affected by decorin infection. Cytokine measurements by ELISA showed that decorin overexpression reduced TGF-beta and IL-1beta. In contrast, IL-4 and TNF-alpha levels were markedly increased in decorin-infected cells. These results suggest that decorin could modulate the expression of certain metalloproteinases and their inhibitors, as well as the production of cytokines. Altogether, our data suggest that decorin might play a pivotal role in tissue remodeling by acting on the balance between extracellular matrix synthesis and degradation.

Activation of latent transforming growth factor beta 1 and inhibition of matrix metalloprotease activity by a thrombospondin-like tripeptide linked to elaidic acid.

Impaired wound healing and skin aging are characterized by neutral protease-mediated destruction of matrix macromolecules associated with disturbance in tissue repair. We synthesized a fatty acyl-peptide derivative at aims to simultaneously activate latent TGF-beta through its peptide domain, KFK, and inhibit MMPs through its lipophilic moiety, elaidic acid. Elaidyl-KFK as well as KFK were shown to activate LAP-TGF-beta both in vitro, using a solid phase assay with immobilized LAP-TGF-beta, and ex vivo using human dermal fibroblasts cultures. In both assays, as much as up to 10% of LAP-TGF-beta added could be recovered as active form. KQK, KQFK as well as their lipopeptide counterparts were inactive. Elaidyl-KFK-mediated LAP-TGF-beta activation led to up-regulation of collagen and TIMP-1 production and down regulation of PMA-induced MMP-1 expression in fibroblasts cultures. Those effects could be suppressed by supplementing cell culture medium with blocking TGF-beta antibody. Elaidyl-KFK inhibited MMP-2, MMP-9, MMP-3, MMP-1, in vitro with IC(50) equal to 1.2, 1.0, 0.24 and 8.9 microM, respectively. Its ex vivo inhibitory capacity, as assessed using skin tissue sections, towards the elastin-degrading capacity of MMP-9 was even more pronounced. At a 1 microM concentration, the lipopeptide decreased by up to 80% enzyme activity. Thus, "Lipospondin," i.e. elaidyl-KFK might be considered as a promising model compound to prevent age-associated dermal alterations.

Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy and diseased human gingiva.

BACKGROUND: The purpose of this study was to quantify the amount of matrix metalloproteinases such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 expressed by human gingival explants in culture media and the area fraction (AA%) of gingival collagen fibers according to the degree of inflammation, to investigate a possible correlation between these enzymes and collagen loss. METHODS: Gingival tissue specimens from 6 healthy controls (group 1), 17 patients with mild gingival inflammation (group 2), 10 patients with moderate gingival inflammation (group 3), and 9 patients with severe gingival inflammation (group 4) were placed in organ culture for 3 days. The MMPs and TIMPs in the culture media were quantified using zymography, dot blotting, and Western blotting. Paraffin gingival sections were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis. RESULTS: The AA% occupied by collagen fibers significantly decreased from group 1 (53%) to group 4 (35%). The decrease in collagen fibers was inversely correlated with the significant increase in MMP-1, MMP-9, and MMP-13 (dot blotting analysis), with the increase of the active form of MMP-2, and with the active form and proform of MMP-9 (zymography analysis). CONCLUSION: The present study showed that metalloproteinases, particularly MMP-2, MMP-9, MMP-1, and MMP-13, are involved in the gingival extracellular matrix degradation during periodontitis.

Are cytokines linked to collagen breakdown during periodontal disease progression?

BACKGROUND: Evidence of the role of cytokines produced by resident and inflammatory cells during inflammation is well established. The aim of this study was to quantify in healthy and diseased human gingiva the area fraction (AA%) occupied by collagen fibers and the amount of cytokines such as interleukin (IL)-1beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and epidermal growth factor (EGF) to investigate a possible correlation between such cytokines, collagen degradation, and the gingival index. METHODS: Gingival tissue specimens from 6 healthy controls (group 1), 6 patients with mild gingival inflammation (group 2), 6 patients with moderate gingival inflammation (group 3), and 6 patients with severe gingival inflammation (group 4) were cultured for 72 hours, and the cytokines present in the culture media were quantified using an enzyme-linked immunosorbent assay (ELISA). Paraffin gingival sections from the 24 subjects were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis. RESULTS: The present study revealed significant differences (P < 0.05) between means of AA% in group 1 (53%), group 2 (41%), group 3 (39.5%), and group 4 (35%) for collagen fibers. Compared to controls, there were significant increases of IL-1beta (groups 3 and 4), IL-6, and TNF-alpha (group 3); a significant decrease of IL-4 (groups 2, 3, and 4) and TGF-beta (groups-2 and, 3); and no change of EGF. The collagen AA% was significantly correlated with the amounts of IL-4 and TGF-beta, and significantly inversely correlated with the amounts of IL-1beta for all 3 inflamed groups and IL-6 and TNF-alpha for groups 2 and 3. CONCLUSION: The present study showed that EGF was not changed in inflamed gingival tissue and that IL-1beta and IL-4 were particularly and intensively correlated with collagen loss. These 2 cytokines could be markers of clinical severity during active periodontitis.

Langerin+ versus CD1a+ Langerhans cells in human gingival tissue: a comparative and quantitative immunohistochemical study.

Langerhans cells (LC) are dendritic cells of the immune system able to capture intraepithelial pathogens and migrate to regional lymph nodes to present them to naive T cells. Up to now immunohistological studies on human gingival LC have been carried out using antibodies against HLA-DR or CD1a molecules. A new marker of LC called Langerin (CD207) and described, among other subcellular localisations, in the Birbeck granules is now available in immunohistochemistry. The purpose of this in situ study was to quantify and to compare Langerin+ versus CD1a+ LC number in order to show differences in the expression of these molecules, if any, and to determine which marker is the most specific. The present study was conducted using nine frozen healthy gingival samples. Double immunofluorescence procedures were performed with an anti-Langerin antibody revealed by FITC and with an anti-CD1a-PE antibody. Mounted slides were analysed by fluorescence microscopy and quantifications were performed on projected slides associated with a grid of 0.015 mm(2). Our results have shown that 1/ the number of CD1a+ LC was significantly increased (P=0.01) when compared with Langerin+ LC 2/ 92% of Langerin+ LC co-expressed CD1a 3/ only 82% of CD1a+ cells co-expressed Langerin 4/ a positive correlation was noted between CD1a+ and Langerin+ LC numbers. The present study has revealed the heterogeneity in the phenotype of gingival LC population and shown that Langerin seems the most specific marker for the study of LC.

Magnesium and connective tissue.

Magnesium (Mg2+) is the fourth most abundant cation and the second most abundant intracellular cation in vertebrates. Magnesium plays a critical role in cellular functions such as: cell adhesion via integrins on various macromolecular substrates, cell migration, DNA transcription and protein synthesis.

Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues.

Specific staining of the extracellular matrix components is especially helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quantitative morphometric analysis. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histological example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly depends on the orientation of the collagen bundles.

Epigallocatechin gallate's protective effect against MMP7 in recessive dystrophic epidermolysis bullosa patients.

The analysis of phenotype-genotype correlations of patients suffering from recessive dystrophic epidermolysis bullosa (RDEB) evidenced intrafamilial and interfamilial phenotype variability occurring for the same mutation of COL7A1; this underscores the role of other genetics environmental factors in the expressivity of the disease. In this work, we checked whether matrilysin 1 (matrix metalloproteinase (MMP)7) could take part in the epidermal detachment in RDEB. Furthermore, we investigated epigallocatechin 3 gallate (EGCG) to determine whether it could inhibit matrilysin activities on collagen type VII and fibrillin 1 known to be associated with the dermo-epidermal junction. In this work, matrilysin 1 was detected in affected and unaffected skins of the three RDEB patients; furthermore, MMP7 was shown to degrade ex vivo on healthy normal skin collagen VII and fibrillin 1. Thus, we suspect that MMP7 could take an active part in the epidermal detachment occurring during RDEB. We evidenced that EGCG in in vitro as well as in ex vivo experiments was a good inhibitor of MMP7 and developed a good protection of collagen type VII and fibrillin 1 susceptible of being degraded by MMP7. We therefore propose that EGCG could be used beneficially in patients suffering from RDEB.

Dextran derivatives modulate collagen matrix organization in dermal equivalent.

Dextran derivatives can protect heparin binding growth factor implied in wound healing, such as transforming growth factor-beta1 (TGF-beta1) and fibroblast growth factor-2 (FGF-2). The first aim of this study was to investigate the effect of these compounds on human dermal fibroblasts in culture with or without TGF-beta1. Several dextran derivatives obtained by substitution of methylcarboxylate (MC), benzylamide (B) and sulphate (Su) groups were used to determine the effects of each compound on fibroblast growth in vitro. The data indicate that sulphate groups are essential to act on the fibroblast proliferation. The dextran derivative LS21 DMCBSu has been chosen to investigate its effect on dermal wound healing process. Fibroblasts cultured in collagenous matrices named dermal equivalent were treated with the bioactive polymer alone or associated to TGF-beta1 or FGF-2. Cross-sections of dermal equivalent observed by histology or immunohistochemistry, demonstrated that the bioactive polymer accelerates the collagen matrices organization and stimulates the human type-III collagen expression. This bioactive polymer induces apoptosis of myofibroblast, property which may be beneficial in treatment of hypertrophic scar. Culture media analyzed by zymography and Western blot showed that this polymer significantly increases the secretion of zymogen and active form of matrix metalloproteinase-2 (MMP-2), involved in granulation tissue formation. These data suggest that this bioactive polymer has properties which may be beneficial in the treatment of wound healing.