Search

Objective Our poor understanding of how inflammatory mediators can affect osteoblast behavior led us to investigate the tumor necrosis factor ( TNF) α-induced focal adhesion kinase ( FAK) and Src phosphorylation. Material and Methods MC3T3-E1 pre-osteoblast cells were harvested at specific time points after either TNF α treatment or RAW267 stimulated conditioned medium, and thereafter cell extracts were prepared for Immunoblotting assay. ELISA detected TNF α content at conditioned medium. Tumor necrosis factor- α-neutralizing antibodies also were used. Results It was possible to show that TNF α provokes attenuation at Y-phosphorylation of both FAK (at Y397) and Src (at Y416) proteins ( P < 0.05), suggesting a decrease in their activities. The very similar profile was observed when osteoblasts were incubated with conditioned medium from lipopolysaccharide ( LPS)-stimulated macrophages, it being significantly different than control ( FAK and Src, P < 0.05). Nevertheless, in order to validate these findings, we decided to pre-incubate osteoblasts with anti- TNF α neutralizing antibody (2 μg ml−1) prior exposing to conditioned medium. Importantly, our results revealed that there was a diminution on those conditioned medium effects when the same biological parameters were evaluated ( P < 0.05). Moreover, we also showed that TNF α impairs osteoblast adhesion, suggesting an interesting role on osteoblast performance. Conclusions Altogether, these results suggest that LPS-stimulated macrophage mediators attenuate both FAK and Src activations in osteoblast, suggesting a novel role for TNF α on osteoblast performance. [ABSTRACT FROM AUTHOR]

Bone tissue is capable of self-healing in the event of injuries. However, situations such as the patient's age or size of the damage can compromise natural healing, bringing to the fore the need for interventions to treat bone injuries. Tissue engineering presents treatment alternatives involving the use of cells and associated biomaterials. In this context, studies using biomaterials claim that carbonated hydroxyapatite (CHA) is more resorbable than hydroxyapatite (HA) and has biological similarity to bone hydroxyapatite. Parameters of indirect contact (cytotoxicity, proliferation, and migration) of the extract obtained from spheres of carbonated hydroxyapatite associated with murine bone marrow stem cells (BMSCs) in vitro were evaluated. Then the osteogenic potential of CHA extract in murine BMSCs in vitro was assessed by quantification of calcium matrix, alkaline phosphatase levels and gene expression Col1A1 and Bglap. Non-critical size bone defects were performed in rat femur. After 14 days of healing, bone blocks were obtained for analysis from treated animals and compared to non-treated animals by histological and histomorphometric evaluation. The CHA extract showed osteoinductive properties in vitro without the need to add osteogenic inducing medium. The ability to induce osteogenic differentiation in undifferentiated cells was confirmed by increased activity of alkaline phosphatase, formation of mineralized nodules and increased expression of the Col1A1 and Bglap genes after 7 and 14 days of differentiation. All groups presented bone neoformation at the injury site 14 days after the operation. Histomorphometric analysis showed no statistical difference between groups in the parameters of bone formation, biomaterial and connective tissue. The CHA spheres have in vitro osteoinductive properties and, when loaded with murine BMSCs, have the potential for bone repair and regeneration in vivo, presenting biocompatibility and osteoconductive, making it a promising alternative for bone tissue engineering applications. [ABSTRACT FROM AUTHOR]