Osteogenesis Of Marrow Stroma Cells With VEGF Gene Transfection And Porous Calcium Phosphate

Tissue engineering is an interdisciplinary science of co-development and combination of modern cell biology, biomaterial science and engineering. The aim of tissue engineering is to investigate and restore tissue and organ substitutes. In this study, bone formation was conducted using the method of tissue engineering.1. The experiment of bone Marrow stroma cells of cultured in vitro.Method: SD rat MSCs were cultured in vitro, cell proliferation and ALP activity were observed. Bone formation potentiality of MSCs in mineralized condition was investigated by ALP staining. Results: Cells occupied the whole bottom of cell culture bottle in about 5-6 days. When cultured up to 30 days, von Kossa staining showed there was calcium deposition in the nodules.2. Construction and identification of eukaryotic expression plasmid of humanVEGF,65.Methods: In this experiment, the human VEGFies gene in pSP73 plamsid was restricted by BamH I and Xho I, then the VEGFies gene was cloned into eukaryotic expression pcDNA3.l. The eukaryotic expression plamsid pcDNA3.1 was transformed into E.colidh5a which was cultured overnight, then plamsid pcDNA3.l-VEGFies was substracted as well as purificated largely. Results: The positive clone by identification of recombinant and sequencing showed that the human VEGF165 was cloned correctly into the eukaryotic expression plamsid.3. Construction and expression of eukaryotic expression vector containinggene in rat bone marrow stroma cells.Methods: The recombinant eukaryotic expression vector pcDNA3.1-VEGFi65 was transfected into the rMASCs by and positive clones were screened with G418. The expression of VEGFies gene in the transfected cells was detected by immunocytochemical staining. Results: The expression of hVEGF165 gene in the transfected cells had been demonstrated by immunocytochemical staining. Therefore, it is possible to use the rMSCs expressing hVEGF^s gene as seeded cells in the bone tissue engineering. 4. The experiment of porous calcium phosphate ceramic combined with SD rat bone marrow stroma cells in vitro.Methods: Bone marrow stroma cells were cultured with DMEM containing 10%FBS. After inoculating the cells onto the surface of porous calcium phosphate ceramic, we surveyed the characteristic of proliferation by cell counting. Using scanning electronic microscope, we observed cellular morphology. Results: Rat bone marrow stroma cell could be attached to and extended on the surface of porous calcium phosphate ceramic, and normally grown, proliferated. Porous calcium phosphate ceramic could promote cell proliferation. Conclusion: The results show that porous calcium phosphate ceramic has good biocompatibility.With rat bone marrow stroma cells, they can be used as biomaterials in bone tissue engeering.5. SD rat marrow stroma cells with VEGF)65 gene transfection loading on porous calcium phosphate ceramic scaffolds.Methods: Autogenous marrow stromal cells were obtained from femurs and tibias of 12 male adult SD rats under general anesthesia and sterile condition and cultured in DMEM supplemented with 10%FBS. VEGFi65 gene was transfectedinto stroma cells by means of LipofectAMINE?000 reagent transfection. The stably gene expressive cells were screened with G-418 for fourteen days. The cell mixture were seeded in porous calcium phosphate bioceramic and cultured in vitro for another 7 days. The cell-ceramic compound was implanted subcutaneously and intramuscularly in the corresponding rat.