The Study Of The Effect Of Hypoxia On Osteogenesis Of Bone Arrow Mesenchymal Stem Cells In Rats

Chronic periodontitis is a bacteria-associated chronic inflammationwithin periodontal support tissues, resulting teeth mobility and looseness.Nowadays, traditional treatment pronciples of chronic periodontitis mainlyincludes scaling and root planing, periodontal flap surgery and GTR.Howerver the outcome is not successful. Although we can control theinflammation of periodontal tissues and stop the progression of disease, wecould not get the periodontal regeneration. With the rapid development oftissue engineering, especially bone tissue engineering, we have a newmethod to treat the chronic periodontitis, that's periodontal tissueengineering.Mesenchymal stem sells (MSCs) have received extensive attention in thefield of bone tissue engineering due to their distinct biological capability todifferentiate into osteogenic lineage cells. Physiologicalmicroenvironments, such as oxygen concentration and hypoxia, play acrucial role in the process of MSC-related bone regeneration anddevelopment. Hypoxia-inducible factor1alpha (HIF-1α), the hallmark ofhypoxia response factor, plays an important role in the differentiation ofMSCs into osteogenic lineages. The expression of HIF-1α was increasedwith the time of hypoxia, whereas the expression of core binding factor alpha1(Cbfα1), another key transcription regulator involved in thechondrocyte differentiation and ossification, was increased gradually.However, other report showed that temporary exposure to hypoxia wouldinhibit the expression of Cbfα-1/Runx2in MSCs. Differentiation of MSCsinto osteogenic lineages and the osteogenesis-related change of MSCs arethe complicated processes, and are regulated by many growth factors,hormones, protein kinases and inhibitors. The regulation of growth factorsand hormones, such as transform growth factor beta (TGF-β), fibroblastgrowth factor (FGF), insulin-like growth factor1(IGF-1), bonemorphogenetic protein (BMP) and parathormone (PTH), on MSCsproliferation and differentiation have been studied thoroughly.It has been reported that MSCs cultured in vitro would differentiate intoosteoblasts spontaneously. But whether HIF-1α and Cbfα1are involved inthe proliferation and differentiation of MSCs as well as theosteogenesis-like changes under hypoxia has not been revealed, and alsolittle is known about the regulation of Cbfα1during the differentiation ofMSCs into osteogenic lineage cells.In the present study, MSCs were grown under low-pO2conditions (2%,4%,6%and8%oxygen tension) or air (21%oxygen tension). The effectsof hypoxia on proliferation and osteogenesis of MSCs and the expressionlevels of HIF-1α and Cbfα1in MSCs were examined by using MTT,enzyme-linked immunoreaction, immunochemistry, RNA interference,real-time PCR, Western blotting, et al.The results indicated:1. Hypoxia can promote significantly the proliferation of MSCs inoxygen tension with2%,4%,6%and8%, especially in4%oxygen tension；2. Hypoxia decreased the expression of ALP in MSCs significantly inoxygen tension with2%,4%,6%and8%；3. Hypoxia decreased the expression of Col I in MSCs significantly inoxygen tension with2%,4%,6%and8%；4. Hypoxia increased the expression of HIF-1α but decreased theexpression of Cbfα1in MSCs significantly；5. Chemically synthesized siRNA targeting HIF-1α can suppress theexpression of HIF-1α mRNA in rat MSCs. Transfected MSCs can promotethe expression of Cbfα1in4%oxygen tension. Down-regulation of HIF-1αinduces Cbfα1expression in hypoxia with4%oxygen tension.In conclusion, we have shown that hypoxia markedly increases theproliferation of MSCs; hypoxia markedly decreases ALP activity andproduction of CoI; down-regulation of HIF-1α expression with siRNAobviously induced Cbfα1expression under hypoxia. We therefore suggestthat hypoxia may promote MSCs to differentiate into osteogenic lineagecells and that HIF-1α may negatively regulate the Cbfα1expression.