Study On The BMP9-induced Bone Formation Process Of Mesenchymal Stem Cells Mediated By PPCN-G And TAZ

Vertebrates have an endoskeleton made of mineralized extracellular matrix. Bone is a highly specialized connective tissue which confers protection to the internal organs, hosts the bone marrow, acts as a reservoir of calcium and phosphate, and is an important regulator of energy metabolism. Osteochondroprogenitors, osteoblasts, and osteocytes are three cell types essential to skeletogenesis. The differentiation of osteoblasts plays an important role in the bone formation and the osteoblasts derive from the mesenchymal stem cells(MSCs).The bone tissue engineering emerged with the deeper understanding of bone development and bone disease and the progress of science and technology. In the bone tissue engineering, high cell density osteoblasts, mesenchymal stem cells and chondrocyte from the body were amplified and cultured in a biocompatible and biodegradable scaffold with some cell factors. In the scaffold, cells can develop in 3D space and exchange nutrient and air with outside. Then transfer the mixture of cells, scaffold and cell factors into the bone defective site. As the biomaterial scaffold being degraded, the osteogenic differentiation and bone formation are in the progress to repair the bone defect.Successful bone tissue engineering requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of the mesenchymal stem cells. We also successfully immortalized a kind of mesenchymal stem cells iMEFs, thus we want to find a biocompatible scaffolding material to 3D culture the BMP9-induced iMEFs on it and it will promote the bone formation.Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-Nisopropylacrylamide)(PPCN) doped with gelatin(PPCN-G) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The material PPCN will become solid at 37℃ to be fixed in vivo and become liquid at 21℃ to be mixed with cells and cell factors in vitro.The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line iMEFs, we found that PPCN-G facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs+PPCN-G group was significantly more pronounced than that of the direct cell injection group. Accordingly, VEGF expression was shown to be significantly higher in the bony masses recovered from the iMEFs+PPCN-G group. Taken together, our results suggest that PPCN-G may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.Recently research revealed that the BMP9 inducing bone formation had the different molecular mechanism with other BMPs. Some signaling pathway involved in the development played important roles in the BMP9-induced bone formation. Despite many factors which mediate the bone formation have been functional reported, for instance, TAZ promotes the osteogenesis and inhibits the adipogenesis definitely, but the role of TAZ in the BMP9-induced bone formation is still unclear.Here we constructed the adenovirus of siRNA knockdown TAZ and got the adenovirus of overexpression TAZ. Then we infected BMP9-induced iMEFs with knockdown or overexpression adenovirus. We did the ALP activity assay to test whether TAZ can promote the BMP9-induced bone formation in vitro. We also mixed the infected iMEFs with PPCN-G and injected subcutaneously in nude to test whether TAZ can promote the BMP9-induced bone formation in vivo.The in vitro and in vivo data revealed that both overexpression and knockdown TAZ can promote the BMP9-induced bone formation. And knockdown TAZ can promote the bone formation much more significantly, so we test the proliferation of infected iMEFs in vitro and in vivo. The proliferation of knockdown TAZ group was facilitated than other groups. We also collected the RNA of the infected iMEFs and iMEF-OKmTAZ and did the reverse-transcription and qPCR to detect the lineage markers and TEAD of iMEFs and iMEF-OKmTAZ. The qPCR results showed that knockdown TAZ promoted the bone formation through increasing the expression of Runx2. On the other hand, TEAD was inhibited by knockdown TAZ.Totally, we demonstrated that we can promote the BMP9-induced bone formation both by using the thermosensitive material PPCN-G and by knockdown TAZ.