| BackgroundsInfected bone defect is one of the knotty problems. With the increases of open fracture incidence, the occurrence of infected bone nonunion will also increase. It is reported that about 5%~10% of bone fractures will develop into bone nonunion. But, in infected bone nonunion, infection and bone defect go hand in hand, is very hard to cure thoroughly which has brought serious physical and mental health injury and economic loss to the patients and the society. Staphylococcus aureus(S.aureus) are the main pathogenic bacteria for infected bone nonunion and can be detected from over 70% cases of osteomyelitis. Currently, the modalities for treating infected bone nonunion are mainly anty-infectious, debridement and the subsequent repair and reconstruction for bone defect, all these means are contronted long treatement term, high expenditure and long term pain etc. Thus, it will be of great importance of investigating the relevant mechanism and new treatements of infected bone nonunion.During the bone repair process, the bone fracture places need enogh mesenchymal stem cells(MSCs) which may affect the bone formation and bone repair ability for its osteogenic differentiation. Under infectious condition, a great lumber of bacteria dissociate due to the fuctions of body immune system or antibiotis. The body component of bacteria produce biological effects when they enter into the fracture regions. However, it is generally believed that only the bacteria can hardly give rise to osteomylitis, and when the obstacle of microcirculation occure. Hence, there are any factors for infected bone nonunion, for example the osteogenic ability of h MSCs, a procures of osteoblasts, declines. S.aureus protein A, an component of S.aureus wall, is an important virulence of S.aureus.SPA may inhibit the bone formation by inhibiting the expression of alkaline phosphatase, collagen I, osteocalcin and osteopontin after adhering to the osteoblasts. In osteomyelitis, SPA may hinder the proliferation of ostoblasts and induce apotosis and the bone formation. From the standpoints of development and heredity, the precurse of osteoblasts, hMSCs determine the number and quality of osteoblasts to some extent depending on its osteogenic differentiation ability. Whether may SPA affects the osteogenic potential of hMSCs and the bone repair? Until now, none similar studies have published.Wnt,a rich source of L-cysteine, plays an important modulat role in the differentiation of MSCs. But Wnt11 takes part in the relseasing Ca2+ and modulating development, tumor and the differentiation of stem cells mainly by phospholipase C(PLC) and protein kinase C(PKC) pathway. WNT11 overexpressing can enhance the chondrogenic differentiation of h MSCs in collaboration with TGF-β, this demonstrats that WNT11 play an important role in the differentiation of stem cells.Research objective:(1) To investigate the effects of SPA on the osteogenic differentiation of h MSCs in vivo and vitro, and further explicit whether SPA may significantly down regulate the osteogenic ability of hMSCs.(2) To investigate the role and mechanism of WNT11/Ca2+ signal pathway in the osteogenic differentiation of h MSCs in present with SPA stimulis and to illustrate whether SPA down regulate the osteogenic ability of hMSCs by mediating WNT11/Ca2+ signal pathway.(3) To investigate the mechanism of osteogenic differentiaion of hMSCs from the gene trasduce modulate standpoint of WNT11; To invesitigate whether inhibiting or overexprssion WNT11 or its upstream proteins mdodulate the effects of osteogenic differentiaion of SPA on h MSCs.(4) To construct the WNT11 RNAi or expressing h MSCs and further investigate the roles in the bone formaiont and bone repair of WNT11 gene in vitro or in vivo and in the infected bone defect animal model.We wish illustrate the role or possible modulate mechanism of WNT11 and non canonical β-catenin signal pathwy of hMSCs in the infectios condition, this may provide experimental basis and new idea for the treatment of osteomyelitis.Method1. Large lumber of h MSCs are perchursed form Caye company and culture and proliferate in vitro, which will used for lentivirus transduction. The factors for the steogenic ability of hMSCs in present with SPA was checked by cell culture, stem cell differentiation, cell staining, ALP activity test, quantitative RT-PCR and Western Blotting technologies.2. The construction of WNT11 RNAi or expressing h MSCs and evaluation the efficacy. The used technologies include cell culture, stem cell differentiation, cell staining, bioinformatics analysis, ALP activity test, quantitative RT-PCR and Western Blotting technologies.3. The evaluation of tissue engineere bone for treating infected bone defect model. The tiss tissue engineere bones were constructed by different hMSCs with PHA/FN/ALG composite scaffolds and then were implanted into the rabbit infected bone defect model. One month after implantation, those animals were tested by X-ray and mircro CT. The bone formation was observe from the slice and quantitative RT-PCR. The final results were made by comprehensive analysis.The data were processed for statistical analysis with SPSS 13.0 version software, One-way analysis of variance(ANOVA) was applied among different groups at one specific time point, and repeated-measures ANOVA was conducted to analyze the differences among these groups in general. The paired-samples t test was used to compare within group at different time points. P values less than 0.05 were considered to be significant.Results1. SPA can significant dow-regulates the osteogenic ability of hMSCs in vitro and down-regulates the non canonical β-catenin signal pathway by down-regulated WNT11.2. WNT11 positively regulates the osteogenic differentiation of h MSCs in vitro in present with SPA virulus, may be regarded as a potential target of signal pathway and enhance the osteogenic potential of hMSCs.3. WNT11 plays an positive regulation in the osteogenic differentiation of h MSCs in present with 100ng/mLSPA stimulus.4. The tissue engineered bone constructed with WNT11 overexpressing h MSCs showed the more potent bone repair ability in the infected bone defect model.Conclusion1. SPA may down-regulate the WNT11 mediated non-canonical WNT signal path way, and also significant down-regulate the osteogenic differentiation ability of h MSCs in vitro.2. WNT11 plays an important positive regulation role in the osteogenic differentiation process of h MSCs under inflammantory condition in vitro.3. During osteogenesis process, Wnt11 may enhance the bone formation of hMSCs under the inflammatory condition in vitro or in vivo and may be regarded as an potential therauputic target4. During the osteogenic differentiation under SPA stmimulis, the non-canonical WNT signal pathway is correlated with canonical WNT signal pathway, wich demonstrates an cooperative effect.5. During the osteogenic differentiation under SPA stmimulis, non-canonical WNT signal pathway may inhibit NF-κB. |
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