Expressions Of Hedgehog Signaling Molecules During Osteogenic And Chondrogenic Differentiation In Rat Bone Marrow Mesenchymal Cells

Recently, bone and cartilage damage due to mechanical, external force or its own factors has become one of the main symptoms of clinical treatment. Now, bone repair using of tissue engineering is on the hot of tissue defect and will become the development direction of treatment. Mesenchymal stem cells (bone marrow mesenchy stem cell, BMSCs)is one kind of non-hematopoietic multipotent in the bone marrow, which characteristics contains self-renewal and multiplex differentiation, and has become the first project organization seed cells because of source wide, drawn simple, immune rejection weak, genetic background stable. Many studies have shown that BMSCs can differentiate into cartilage cells and osteoblasts and other cells, so BMSCs are widely used for the treatment of tissue defect. However there was no induction standard of osteoblast differentiation and cartilage differentiation in BMSCs. Commonly, dexamethasone and TGF-β1 are used as osteoblast inducer and osteoblast cartilage inducer respectively.In vertebrate animals, there are three members in Hedgehog (Hh) protein family: Sonic Hedgehog (SHH), Indian Hedgehog (IHH) and Dersert Hedgehog (DHH). SHH establishes relevant with the differentiation of cells development in the body, the relationship and neural tube. IHH characteristically participates in chondrogenic differentiation. DHH plays a key role in the development of reproductive cells. Hedgehog signaling pathways not only play important readjustment function in bone growth process but also can promote osteogenesis related cell differentiation.So far, there was few molecular markers for the purification and enrichment of BMSCs. And in the advanced animals, roles of Hedgehog mechanism is more clear, but its mechanisms are unclear in osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro. Here to investigate the expressions of Hedgehog signaling molecules, Sonic Hedgehog (SHH) and Indian Hedgehog (IHH) , during osteogenic and chondrogenic differentiation in rat bone marrow mesenchymal stem cells (MSCs) induced by Dexamethasone and TGF-β1 respectively.Mainly results:1. Expressions of Hedgehog Signaling Molecules during Osteogenic and Chondrogenic Differentiation in Rat Bone Marrow Mesenchymal Cells To separate rat mesenchymal stem cells using direct stick wall separation, we found that the best way for cell growth state and cell proliferation is to culture cells in a plastics petri dishes with foreign womb bovine serum and DMEM-L liquor through comparing with four different culture enviroment. Most of cells were spindle and swirling growth after the third passage. The expression of CD44 and CD34 in the third passage cell were 90.16% and 2.27% respectively by flow cytometry detection.2. The positive expression of I type collagen were increased following osteoblastic differentiation in MSCs. After 14d Dexamethasone induction, the positive rate of I type collagen expression was higher than 90% and the staining intensity increased. Compared to non-induction group, the expression of SHH protein was significantly increased in induction group (P<0.05). With the induction time extension, the expression of SHH protein significantly increased (P<0.05). The expression of IHH protein was significantly lower in the induction group compared with non-induction group (P<0.05). There was no significantly changed of IHH among 7d, 14d and 21d induction groups(P>0.05). In the process of MSCs induction into bone, expression of Shh mRNA and Ihh mRNA had no significant change (P>0.05).3. The positive expression of II type collagen were increased following chondrogenic differentiation in MSCs. After 14d TGF-β1 induction, the positive rate of II type collagen expression was higher than 90% and the staining intensity increased. Compared to non-induction group, the expression of SHH protein was significantly decreased in induction group (P<0.05). With the induction time extension, the expression of SHH protein significantly increased (P<0.05). The expression of IHH protein was significantly higher in the induction group compared with non-induction group at 14 d and 21day(P<0.05). With the induction time extension, the expression of IHH protein significantly increased (P<0.05). The trend of Shh mRNA expression was similar to the expression of SHH. Expression of Ihh mRNA was significantly decreased in induction group compared to the non-induction group at 7d and 14d while significantly increased at 21d (P<0.05). Conclusion:1. After comparing four methods to get MSCs, the best way for cell growth state and cell proliferation is to culture cells in a plastics petri dishes with foreign womb bovine serum and DMEM-L liquor. These BMSCs can be applied directly to the following study.2. Expression ofⅠtype collagen in induction group showed that dexamethasone induced BMSCs to osteoblast. In induction group, SHH protein expression was significantly increased and IHH protein expression was significantly lower compare with non- induction group, which suggested that SHH and IHH may play different roles involving in the osteogenic differentiation of bone marrow stromal cells induced by Dexamethasone.3. Expression ofⅡtype collagen in induction group showed that TGF-β1 induced BMSCs to cartilage. In induction group, SHH protein expression was significantly decreased and IHH protein expression was significantly increased compared with non- induction group, which suggested that SHH and IHH may play different roles involving in the chondrogenic differentiation of bone marrow stromal cells induced by TGF-β1.In summary, this study first systematically investigated the expressions of hedgehog signaling molecules during osteogenic and chondrogenic differentiation in rat bone marrow mesenchymal cells. The results showed that expression of SHH protein and IHH protein were significantly increased during osteoblast differentiation and chondrogenic differentiation process respectively. These results suggests the expression pattern of SHH and IHH may be different in osteogenic and chondrogenic differentiation of rat bone marrow stromal cells.