Tissue-Engineered Bone Constructed By PLA Combined With The Co-Culture System Compose Of Marrow Stromal Cells And Endothelial Progenitor Cells: An Experimental Study In Vitro And Vivio

OBJECTIVE: Based on separation and culturing the Bone marrow stromal cells as well as the Endothelial progenitor cells in vitro, this dissertation researches the possibility of constructing tissue engineering bone through regarding the BMSCs and endothelial progenitor cells as the seed cells, PLA as the timbering material.METHORD:1) Mononuclear cells are isolated from bone marrow of Sprague Dawley rats (SD rats) and cultured in DMEM in vitro. The Marrow stromal cells are cultured in osteogenic medium from the second generation of cells. To test the characteristics of ossification,alkaline phosphatase( ALP) should be detected on the 1st,3rd,6th,9th,12th,14th,17th day and red calcium nodus staining should be done on the 14th day.2)Isolate EPCs from rat bone marrow by attachment-changing cultured method and seeded in EGM-2 medium. Observe the growth and shape of the cells by inverted microscope. EPCs are identified by flow cytometry and immunohistochemistry staining for the expressions of special surface marker,such as CD34,CD31 and vWF.3)Use technique of cell culture in vitro,the cells are seeded on PLA and divided into 3 group (group A:BMSCs+PLA; group B:BMSCs+EPCs+PLA; group C:EPCs+PLA).Bone marrow stromal cells and endothelial progenitor cells are seeded either alone or mixed in an 2:1 ratio into a polylactic acid (PLA) scaffold to a final concentration of 1×104cells/ml. Cell growth and reciprocity will be monitored by using MTT assay and SEM. RT-PCR is used to detect the expression of OPN mRNA.4) To investigate the ectopic osteoinduction and biocompatibility of cells-PLA composite, bone marrow stromal cells and endothelial progenitor cells are synthesized either alone or mixed in an 2:1 ratio into a polylactic acid (PLA) scaffold to a final concentration of 3×107/ml cells/ml. The cells-PLA construction should be implanted into the muscle pouches in the right and left pars dorsalis of SD rat respectively. In the 4th and 12th week after implanting,6 rats were sacrificed, the samples were harvested and the ectopic new bone formation was detected through histologic examination.Results:1) The value of ALP rapidly increased on the 3rd day,and reach to the top on the 14th day and the red calcium nodus were positive on the 14day after osteogenic induction.2) The P0 EPCs exhibited small spindle shape and were able to line up in the typical cord-like structure. It was revealed by flow cytometry, the positive CD34 cells was in 68% rate by the purified methods of attachment--changing culture from bone marrow .After 21days'culturing, these cells displayed the positive staining for CD31 and vWF vWF but negative for CD34.3) After seeded on PLA, the growth curve of cell on the PLA had an"S"shape in all group. The proliferation of every group increased gradually and reach tip on the 7th day, the highest was the co-culture cells group, it is significantly different comparing with the other groups(p<0.05) .Morphology investigation of adhered cells by SEM indicated that cells could adhere,proliferate on the surface of the composite presenting normal morphology, and the co-culture cells filling the surface of PLA .BMSCs/EPCs composite extract samples had a higher adhere rate(70.5±4.6%)comparing with the other samples (39.8±4.0% in BMSC cells group and41.3±3.1% in EPCs group)after 24 hours of incubation((p<0.05).We found that cells expressed OPN mRNA is more effective in BMSCs group and co-cultured cells group comparing with EPCs alone(p<0.05)4) Histologieally there was slight acute inflammation in the surrounding tissue of both types of implants at the early stage. New bone-like construction was evident in BMSCs and BMSCs /EPCs groups and the amount of new blood vessels were significantly higher in EPCs groups and BMSCs /EPCs groups.The Intergrated optical density(IOD)of steocalcin were significantly higher in BMSCs groups and BMSCs /EPCs groups comparing with the other groups。Conclusion:1) The endothelial progenitor cells can get from mononuclear cells of rat bone marrow and amplify after 21day in vitro, then differentiated into vascular endothelial cells.2) The BMSCs can differentiate into osteoblast-like cells by culturing in osteogenic medium.3) The co-culture cell can stimulate proliferation and ostified in vitro.4)Co-culture improve the adhesive and proliferation of cells on the PLA .After seeded 3days to 7days,the cells proliferation and the osteogenic potential increase rapidly, then the tissue-engineered bone constructed was fit for transplanted.5) Bone formation and the amount of new blood vessels in the co-culture cell group were the largest, vascular endothelial cells can improve not only the ectopic osteogenesis but also the hemoperfusion in vivo, which would be useful for repairing bone defects.