Histopathological, histoenzymological, immunohistochemical and immunofluorescence analysis of tissue response to sealing materials after furcation perforation.

AIM: To evaluate in vivo tissue responses after sealing furcation perforations in dog's teeth with either Biodentine™, mineral trioxide aggregate (MTA) or gutta-percha, by means of histopathological, histoenzymological, immunohistochemical and immunofluorescence analysis. METHODOLOGY: After root canal treatment, perforations were created in the central region of the pulp chamber floor using a round diamond bur and filled with one or other of the materials. The animals were euthanized after 120 days, and the teeth (n = 30) were processed for histopathological analysis of new mineralized tissue formation and collagen fibre reinsertion, immunohistochemical analysis of osteopontin (OPN) and alkaline phosphatase (ALP) and immunofluorescence analysis for bone morphogenetic protein (BMP-2), cementum attachment protein (CAP), bone sialoprotein (BSP), osteocalcin (OCN) and cementum protein1 (CEMP1). Histoenzymology was performed for TRAP activity and osteoclast count. Data were analysed statistically (α = 0.05) using chi-square and Kruskal-Wallis tests. RESULTS: Gutta-percha did not induce mineralized tissue formation. MTA and BiodentineTM formed mineralized tissue in 88% and 92% of specimens, respectively, with no significant difference (P > 0.05). Gutta-percha was associated with scattered collagen fibres parallel to the perforations. Groups treated with MTA or BiodentineTM had partial fibre reinsertion perpendicular to the newly formed mineralized tissue. All materials induced OPN and ALP expression, weakest for gutta-percha and strongest for MTA (P < 0.05). Only MTA induced BMP-2, BSP, OCN, CAP and CEMP1 expression. Osteoclast counts were similar in all groups (P = 0.97). CONCLUSIONS: Mineral trioxide aggregate and BiodentineTM were biocompatible, with formation of mineralized tissue and partial reinsertion of collagen fibres. In addition, the participation of several molecules by which calcium silicate-based materials induce the formation of mineralized tissue were noted, with expression of ALP and OPN mineralization markers, without interference in the number of osteoclasts. Only MTA stimulated the expression of proteins associated with the formation of a cementum-like mineralized tissue.

Cementum protein 1 transfection does not lead to ultrastructural changes in nucleolar organization of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Transfection of cementum protein 1 (CEMP1) into human gingival fibroblasts (HGFs) notably increases cell metabolism and results in overexpression of molecules related to biomineralization at transcriptional and protein levels. Therefore, HGF-CEMP1 cells are considered as putative cementoblasts. This represents a significant advance in periodontal research because cementum neoformation is a key event in periodontal regeneration. In addition, it is well known that important changes in cell metabolism and protein expression are related to nucleolar structure and the function of this organelle, which is implicated in ribosome biogenesis. The aim of this study was to determine the effect of transfecting CEMP1 gene in human HGF on the ultrastructure of the nucleolus. MATERIAL AND METHODS: Cells were processed using the conventional technique for transmission electron microscopy, fixed with glutaraldehyde, postfixed with osmium tetraoxide, and embedded in epoxy resin. Semi-thin sections were stained with Toluidine blue and observed by light microscopy. Thin sections were stained with uranyl acetate and lead citrate. For ribonucleoprotein detection, the staining method based on the regressive effect of EDTA was used. In addition, the osmium ammine technique was used for specific staining of DNA. RESULTS: The results obtained in this study suggest that transfection of CEMP1 into HGFs does not produce changes in the general nucleolar ultrastructure because the different components of the organelle are present as fibrillary centers, and dense fibrillar and granular components compared with the control. CONCLUSION: The transfection of CEMP1 into HGFs allows these cells to perform cementoblast-like functions without alteration of the ultrastructure of the nucleolus, evaluated by the presence of the different compartments of this organelle involved in ribosomal biogenesis.

High glucose concentrations alter the biomineralization process in human osteoblastic cells.

Diabetes mellitus (DM) may alter bone remodeling, as osteopenia and osteoporosis are among the complications. Moreover, DM increases the risk and severity of chronic inflammatory periodontal disease, in which bone resorption occurs. Broad evidence suggests that chronic inflammation can contribute to the development of DM and its complications. Hyperglycemia is a hallmark of DM that may contribute to sustained inflammation by increasing proinflammatory cytokines, which are known to cause insulin resistance, via toll-like receptor (TLR)-4-mediated mechanisms. However, the mechanisms by which bone-related complications develop in DM are still unknown. Studies done on the effect of high glucose concentrations on osteoblast functions are contradictory because some suggest increases (although others suggest reductions) in the biomineralization process. Therefore, we evaluated the effect of high glucose levels on biomineralization and inflammation markers in a human osteoblastic cell line. Cells were treated with either physiological 5.5 mM or increasing concentrations of glucose up to 24 mM, and we determined the following: i) the quantity and quality of calcium-deposit crystals in culture and ii) the expression of the following: a) proteins associated with the process of biomineralization, b) the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG), c) cytokines IL1, IL6, IL8, IL10, MCP-1 and TNF alpha, and d) TLR-2, -3, -4 and -9. Our results show that high glucose concentrations (12 mM and particularly 24 mM) alter the biomineralization process in osteoblastic cells and provoke the following: i) a rise in mineralization, ii) an increase in the mRNA expression of RANKL and a decrease of OPG, iii) an increase in the mRNA expression of osteocalcin, bone sialoprotein and the transcription factor Runx2, iv) a diminished quality of the mineral, and v) an increase in the expression of IL1beta, IL6, IL8, MCP-1 and IL10 mRNAs. In addition we found that both high glucose levels and hyperosmotic conditions provoked TLR-2, -3, -4 and -9 overexpression in osteoblastic cells, suggesting that they are susceptible to osmotic stress.

Immunolocalization of a human cementoblastoma-conditioned medium-derived protein.

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

Preliminary characterization of epithelial root sheath cells in vitro.

Murine root sheath cells from CD-1 mice were isolated and propagated in culture in both monolayer and tridimensional system using basement membrane components as substrata. Cells were grown for a period of seven days. The epithelial cells cultured in monolayer exhibited the typical cobblestone-like feature and also were cytokeratin positive when they were immunostained with the specific antibody. The histological analysis of the cells cultured in basement membrane components revealed differentiated behavior of the cells, and they organized in a round structure with a center of material probably representing keratin. Also the cells differentiated and organized into a squamous stratified epithelia, with a basal, intermedium and superficial layers. The preliminary data obtained with this model could be useful as a new approach to study root formation in the murine periodontum.