Osteoblast Proliferation And Apoptosis In Osteoarthritis Subchondral Bone Remodeling

Background:Osteoarthritis (OA) is the most common joint disease and affects a large proportion of the population. OA is a multifactorial disease characterized by breakdown of articular cartilage and subchondral plate thickening. Several tissues of the joint, including the cartilage, the synovial membrane and the subchondral bone, play significant roles in the development/progression of OA pathology.It provides substantial evidence that changes in the metabolism and the process of regeneration of the subchondral bone are an integral part of the OA disease process and that these alterations are not merely secondary manifestations, but are part of a more active component of OA. Subchondral bone alterations including sclerosis, osteopenia of the underlying trabecular bone. Osteoblast activity responsible for the the bone formation through the synthesis of bone matrix, osteoclast activity accountable for degrading the bone microenvironment. It is now well established that OA osteoblasts show an altered phenotype and secrete more ALP, OC, interleukin (IL)-6, transforming growth factor (TGF)-b1, insulin-like growth factor (IGF)-1 and prostaglandin (PG)E2 than normal osteoblasts. Osteoblasts that produce and mineralize matrix during the process of regeneration, eventually become the lining cells that cover quiescent surfaces and some become osteocytes. It is well documented that the number of osteoblasts, is determined by the balance between their birth from mesenchymal progenitors and their death by apoptosis. The balance of osteoblast proliferation, differentiation, and apoptosis determines the fate of the osteoblast at any given time. During bone remodeling of lamellar bone, the sequence of cell events is known but control of osteoblast number through the dual effects of proliferation and apoptosis often has not been considered. There are numerous molecules that are involved in proliferation and apoptosis, and the fate of the osteoblast depends on how these molecules interact with each other. An improved understanding of the osteoblast apoptotic pathways and their functional outcomes in subchondral bone alterations and OA may facilitate development of more targeted therapeutics to control bone sclerosis in patients with OA.An abundance of literature strongly indicated that chondrocyte apoptosis is related to the progression of OA. However, it should be noted that the relative contribution of apoptotic cell death in the pathogenesis of OA is still difficult to assess because of the chronic nature of the disease process. With the refinement of technologies for detecting apoptosis, however, the best option to clarify the role of apoptosis as a pathogenetic mechanism of OA is not clear. Recent studies showed that the pathways and patterns of apoptotic cell death are much more diverse than originally observed. Small changes in the prevalence of osteoblast apoptosis in a bone section indicate extensive changes in the absolute number of these cells in the live tissue.Over the last few years, the emergence of protein arrays has allowed the simultaneous detection of multiple protein expression cells or tissues. Many different protein array technologies have been developed, each with particular advantages, disadvantages, and optimal applications. This technology combines multiple advantages, including specificity and ease of use. Antibody-based protein arrays applicating include the identification of new proteins, the identification of new disease targets by differential analysis between healthy and pathological samples, the discovery of biomarkers. The analysis of multiple protein secretion by osteoblast using proteomic approaches are scarce.Prostaglandins (PGs) are multifunctional regulators of bone metabolism that stimulate both bone resorption and formation. These complex actions on bone metabolism were depended on interactions with different types and subtypes of receptors. Osteoblasts show abnormal phenotypic characteristics possibly responsible for bone sclerosis and that two subgroups of OA patients can be identified by low or high endogenous production of PGE2 by OA osteoblasts. These data suggest that elevated endogenous PGE2 levels in OA Ob could be important for bone sclerosis in OA. Understanding the mechanisms and regulation of OA osteoblasts apoptosis will enhance our knowledge of osteoblast function and help us to develop better therapeutics for OA.Objective:The present study takes advantage of the development of a new antibody-based protein array, human apoptosis antibody array to evaluate and assess the potential functions of the osteoblast apoptosis on OA We also investigate the effect of prostaglandin E2 (PGE2) on the induction of p21 and heat shock protein 27 (HSP27) in osteoblast-like MC3T3-E1 cells, assess the effects of PGE2 on the proliferation and apoptosis of MC3T3-E1 cells in a well-characterized model system that have been used for studies of osteoblast.Method:1. The expression of 43 human apoptosis cytokines was analyzed by a commercial sandwich-type assay that uses membrane-bound cytokine-specific antibodies to capture cytokines from biological fluids. Cartilage and subchondral bone are obtained from one healthy and three knee osteoarthritic joints. Protein get from articular cartilage and subchondral bone rubbing with liquid nitrogen. Array membranes were incubated for 2h in 2 ml blocking buffer, further incubated for 12h in 4°with 1 ml of the protein fluids, and washed. Then, a cocktail of Biotin-Conjugated Antibody Mix was added for 120 min at 1 ml per array membrane. Membranes were washed and sandwiched antigens were detected by incubation for 90 min with 2 ml of a HRP-conjugated Streptavidin solution diluted 1:1000. Proteins were detected by enhanced chemiluminescence and signals were captured on X-ray films. Array images were acquired at a resolution of 300 ppi on an scanner.2. The murine calvaria-derived osteoblastic cell line MC3T3-E1 were challenged with 10μmol/LPGE2 for 30 min,2 h and 4 h respectively. Detection of p21 and HSP27 mRNA by Real-time RT-PCR. Cell were flushed with 1 ml of TRIzol reagent, and RNA was isolated according to the manufacturer's instructions, Real-time PCR was performed. Primer sequences and annealing conditions are available on request. For investigating the effect of PGE2 on p21 and HSP27 protein expression in MC3T3-E1 cells, western blot was performed.Result:1. Many differences were observed when microarrays of OA joints were compared to the normal joint array. However, since the possibility to obtain cartilage from healthy joints is very scarce, we emphasize that the single experiment on normal, although informative, should be interpreted with caution. Cartilage specimens from different donors were used to study multiple protein expression by human OA chondrocytes just apoptosis.38 apoptosis related proteins are high in OA cartilage compared with normal (P<0.05), only BID was tone down, other did not changed. But in OA subchondral bone many apoptosis related protein have not different with normal. There were 9 proteins high in OA.2. The expression levels of p21 and HSP27 of MC3T3-E1 cells by real-time RT-PCR analysis of RNA isolated directly from cell. This showed a strong amplification product for p21 and HSP27 in PGE2 treated cells. p21 and HSP27 mRNA higher than normal with time for longer. Western blot analysis indicated that the expression of p21and HSP27 protein in the stimulated cells were apparently higher (P<0.01) than that of the controls after 10μmol/L PGE2 treated 2h.Conclusion:In summary, this study shows more apoptosis in the OA cartilage and subchondral bone. We are aware that further investigations are needed to identify the factor(s) involved in the osteoblast effects. Antibody array will boost research about OA. The effects of PGE2 on the proliferation of MC3T3-E1 cells can may be via increase expression levels of p21 and HSP27. Findings with regard to the physiological and pathological mechanisms of OA will make it possible to better target therapeutic approaches that could lead to the development of treatments to reduce or stop the progression of the disease.