Fabrication Of Free-scaffold Tissue Engineering Vascularized Bone Based On Cell Sheet Technology

The purpose of this study is to explore a new approach of developing free-scaffold tissue engineering vascularized bone based on rat BMSCs, and assess its feasibility to repair rat skull critical defect. Method (1):Isolation and culture of rat bone marrow stem cells(BMSCs):rat BMSCs from femoral marrow were isolated by using the method of whole bone marrow adherence, microscope was used to observe the morphology of BMSCs.(2):Rat BMSCs were differentiated into Endothelial cells(ECs):the BMSCs were cultured in ECs medium for 14 days, the morphology of BMSCs were observed by microscope, flow cytometry was used to evaluate the induction efficiency.(3):construction of BMSCs cell sheet:BMSCs were seeded on six well plates at a density of 1 × 105/ cm2 and cultured for 2 weeks to form undifferentiated cell sheet and osteogenic cell sheet. Then the induced ECs were seeded on the undifferentiated cell sheet at α density of 5×104/ cm2 for 3 days. Immunochemistry staining for CD-31 were used to evaluate the vascularized cell sheet. ALP staining and Alizarin red staining were performed to evaluate the osteogenic properties of BMSCs cell sheet(4) repairing rat skull critical defect using compound cell sheet:The pre-vascularized cell sheet were harvested and covered the osteogenic cell sheet surface, then the double layersoft cell sheet were folded to fabricated three-dimisioncompound constructs(OM/ECs/UM group). At the same time, the composite construct of the undifferentiated cell sheet and the osteogenic cell sheet was defined as OM/UM group. The rat model of 8mm diameter defect skull was established.Then OM/ECs/UM and OM/UM construct was transplanted into the defects respectively. At the time of point of 2,4,8 weeks after implantation, gross observation, X-ray, HE staining,CD-31 immunohistochemistry and Van Gieson staining were used to detect the ability of vascularization and oteogenesis of the implants. Results:(1):It’s an effective method to harvest BMSCs by using whole bone marrow adherence, and the BMSCs showed long spindle under the microscope.(2):BMSCs could be induced into ECs, and the rate of expression for CD-31 positive reached to 38% by Flow cytometry detection.(3):when ECs were seeded onto undifferentiated cell sheet,the ECs migrated and rearranged on the BMSCs cell sheet, after 3days, CD31 staining showed cavity structure on surface.(4):After 2 weeks implantation, OM/ECs/UM group had more blood vessels formation than OM/UM and blank group.After 4 weeks and 8 weeks implantation,the OM/ECs/UM group showed more osteogenic potential. Conclusion:It is feasible to fabricate the three-dimension free-scaffold tissue engineering vascularized bone and repair the skull critical defects by using cell sheet engineering technology.