Expression of MMP-2, 9 and 13 in newly formed bone after sinus augmentation using inorganic bovine bone in human.

BACKGROUND AND OBJECTIVE: The aim of the present study was to analyse the expression of MMP-2, MMP-9 and MMP-13 in newly formed bone following maxillary sinus augmentation using inorganic bovine bone substitute, because these MMPs play a major role in bone remodeling and bone resorption. MATERIAL AND METHODS: Deproteinized bovine bone (Bio-Oss(®)) was used to fill cavities after elevating the sinus mucosa. Twenty patients with edentulous posterior maxilla were treated with 20 sinus-augmentation procedures using a two-stage technique. Forty-nine Straumann(®) endosseous implants were used to complete the implant-prosthetic rehabilitation. One cylinder-shaped bone biopsy from each patient was taken from the augmented maxillary region using trephine burs at the second stage of surgery, 8 months after grafting. A biopsy was also taken as a control from the upper molar region from six different patients who did not undergo the sinus procedure. All biopsies were subjected to biochemical analysis and staining for TRAP. RESULTS: No implant losses or failures occurred. The large number of TRAP-positive multinucleated osteoclasts in resorption lacunae indicated that the resorption was very active in all grafts, in contrast with the control group. Zymography and western blot analysis demonstrated a significantly increased expression of MMP-2, MMP-9 and MMP-13 in the newly formed bone compared with controls (p < 0.05). CONCLUSION: The quantity of osteoclastic cells and the increased expression of proteolytic enzymes suggest that 8 months after grafting, inorganic bovine bone is slowly resorbing and is the site of important remodeling of the newly formed bone by means of resorption and synthesis.

The defensive role of lysozyme in human gingiva in inflammatory periodontal disease.

BACKGROUND AND OBJECTIVE: The presence of lysozyme in human gingiva has not previously been demonstrated. In this study, we looked for evidence for the potential role of lysozyme as a protector of gingival elastic fibres. The objective of this study was also to determine the ex vivo susceptibility to hydrolysis of gingival elastic fibres from patients with or without periodontal disease by human leukocyte elastase and by human cathepsin G. MATERIALS AND METHODS: Using gingival tissue sections from eight control, 10 gingivitis and 10 periodontitis patients, we evaluated the area fraction occupied by gingival elastic fibres (after selective staining) by the use of automated image analysis. In the ex vivo experiments, serial tissue sections from four control, four gingivitis, four young periodontitis and four aged periodontitis patients were submitted to the action of human leukocyte elastase and cathepsin G, after which enzymatic activities were determined by image analysis. Indirect immunodetection of lysozyme was also done on tissue sections for all patients included in this study. RESULTS: Large variations of the area fraction occupied by elastic fibres were observed in human gingiva from young and aged patients with and without periodontal disease. In control and gingivitis patients, leukocyte elastase and cathepsin G had high comparable elastin solubilizing activities. With young and aged periodontitis patients, the two serine proteinases had weak elastin solubilizing activities. Lysozyme appeared to be present at the periphery of gingival elastic fibres in periodontitis patients. CONCLUSION: Lysozyme can be considered an important natural protector of elastic fibres in pathological gingiva.

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.

Potential effects of a low-molecular-weight fucoidan extracted from brown algae on bone biomaterial osteoconductive properties.

In this work, we first tested the influence of low-molecular-weight (LMW) fucoidan extracted from pheophicae cell wall on bidimensional cultured normal human osteoblasts' behaviors. Second, by impregnation procedure with LMW fucoidan of bone biomaterial (Lubboc), we explored in this bone extracellular matrix context its capabilities to support human osteoblastic behavior in 3D culture. In bidimensionnal cultures, we evidenced that LMW fucoidan promotes human osteoblast proliferation and collagen type I expression and favors precocious alkaline phosphatase activity. Furthermore, with LMW fucoidan, von Kossa's staining was positive at 30 days and positive only at 45 days in the absence of LMW fucoidan. In our three-dimensional culture models with the biomaterial pretreated with LMW fucoidan, osteoblasts promptly overgrew the pretreated biomaterial. We also evidenced that osteoblasts increased proliferation with pretreated biomaterial when compared with untreated biomaterial. Osteoblasts secreted osteocalcin and expressed BMP2 receptor on control material as well as with LMW fucoidan impregnated biomaterial. In conclusion, in our experimental conditions, LMW fucoidan stimulated expression of osteoblastic markers differentiation such as alkaline phosphatase activity, collagen type I expression, and mineral deposition; furthermore, cell proliferation was favored. These findings suggest that fucoidan could be clinically useful for bone regeneration and bone substitute design.

Elastin changes during chronological and photo-ageing: the important role of lysozyme.

Cutaneous ageing, as a result of combined chronological and photo-ageing in sun-exposed areas, is accompanied by major modifications of the elastic fibres. We aimed to investigate qualitative and quantitative changes of dermal elastin fibres during cutaneous chronological and photo-ageing and the involvement of lysozyme in these processes. Morphological, age-related changes and variations in the relative elastin content in sun-protected (buttock) and sun-exposed (forearm and face) skin of healthy volunteers were studied (145 samples). The deposition of lysozyme in elastin fibres was studied using light and immuno-electron microscopy and taking into consideration the relative efficacy of different UV wavebands (UVA or SSR (solar simulated radiation)). Our studies also included the proteolytic degradation of elastin by human leucocyte elastase (HLE) in situ. Our results indicate a reduction of elastin content with age in sun-protected and sun-exposed skin, associated for the latter with high elastin content, resulting in elastosis. Total UVA (320-400 nm), and in particular long wave UVA (UVA-1, 340-400 nm), induces lysozyme deposition in elastin fibres to a higher extent than solar simulated radiation (SSR, 280-400 nm). Immuno-electron microscopy revealed lysozyme association with the electron-dense granular amorphous elastin structures, corresponding to a basophilic degeneration induced by sun exposure. Lysozyme has no elastolytic activity in situ; however, its binding to elastin limits elastin degradation by human leucocyte elastase (HLE). In addition, a direct inhibitory effect of lysozyme on HLE was observed. Our data suggest that lysozyme prevents elastin degradation by HLE after binding to the damaged parts of the elastin network and by direct lysozyme-HLE interaction, which reduces HLE proteolytic activity. These observations contribute to a better understanding of the chronological and photo-induced changes of the dermal elastic network.

Vascular wall remodeling in patients with supravalvular aortic stenosis and Williams Beuren syndrome.

Supravalvular aortic stenosis (SVAS) and Williams Beuren syndrome (WBS) can be considered as inherited diseases affecting the whole arterial tree and causing narrowing of the vessels. It has been reported that abnormal deposition of elastin in arterial walls of patients with SVAS and WBS leads to increased proliferation of arterial smooth muscle cells (SMC), which result in the formation of hyperplastic intimal lesions. In this work, we conducted morphological and morphometrical analysis with stenotic aortas from patients suffering from SVAS and WBS and from healthy control subjects and demonstrated that the amount of elastic fibers and the loss of integrity of vascular elastic fibers in the aortas reflect similar changes in the skin of patients with SVAS or WBS, as reported in our previous work conducted on skin in these pathological states. On the other hand, we conducted investigations on metalloproteinases (MMP2, MMP9, MMP7) and their specific tissue inhibitors TIMP1 and TIMP2 to verify their possible involvement in the etiopathogeny of SVAS and WBS. We particularly evidenced an altered MMP9/TIMP1 balance in favor of matrix degradation which could facilitate SMC migration and neointimal hyperplasia. Our findings suggest that elastinolytic enzymes secreted by arterial SMC, possibly including matrilysin 1, are critical for the development of arterial lesions in SVAS and WBS and contribute to perpetuate arterial stenosis in either SVAS or WBS.

Protective effect of a vegetable extract from Lupinus albus (LU 105) on human gingival elastic fibers degradation by human leukocyte elastase.

The aim of this study was to determine if a vegetable extract from seeds of Lupinus albus (LU 105) has the capacity to inhibit human leukocyte elastase and/or protect gingival elastic fibers against proteolytic degradation. LU105 was extracted from seeds of L albus and is freely soluble in water. In this study the ex-vivo elastolytic activity of human leukocyte elastase and the potential inhibitory effect of LU 105 were determined using human gingival cryostat tissue sections and computerized morphometric analysis. The gingival tissue sections pre-treated or not with LU 105 were submitted to the action of human leukocyte elastase or submitted to the simultaneous action of human leukocyte elastase and LU 105, and then analyzed using automated image analysis. In such conditions, LU 105 at 0.1%, 0.01%, and 0.001% developed a dose dependent protection of gingival elastic fibers against enzymatic proteolysis due to human leukocyte elastase, and LU 105 at 0.1% or 0.01% was able to inhibit the elastolytic activity of leukocyte elastase itself. It is proposed that LU 105 is an option for the treatment of gingival inflammation in which leukocyte elastase is involved.

Antiproliferative polysaccharides modulate distribution and phenotypic expression of collagens by gingival fibroblasts.

Gingival fibroblasts are particularly involved in the physiologic maintenance and repair of periodontium. During these processes, cell proliferation and synthesis of a collagen-rich gingival matrix should be controlled. A dextran derivative, namely, carboxy methyl dextran benzylamide sulfonate (CMDBS), considered to be a functional analog of heparin, was previously described to regulate proliferation of different types of cells and independently to modulate the expression of collagen biosynthesis. In this report, we demonstrate that CMDBS and heparin inhibited gingival fibroblast proliferation. We then analyzed collagen biosynthesis by measuring the incorporation of the radiolabeled [3H]proline precursor into collagen by postconfluent gingival fibroblasts. Our results showed CMDBS did not alter total collagen synthesis; it induced the preferential accumulation of newly synthesized collagen into the pericellular matrix; and it decreased the expression of type III collagen, particularly in the cell layer. Taken together, our results suggest that by inhibiting cell proliferation, CMDBS could induce the synthesis of an extracellular collagenous matrix which forms a network between gingival fibroblasts.