Experiment On Preparation Of Tissue Engineering Bone With Rabbit Bone Marrow Mesenchymal Stem Cell Cotransfected By BMP2 And BMP7 Adneoviral Vectors

Objective The purpose of this study is to establish an injectable tissue engineering bone with platelet-rich plasma (PRP) as scaffold, combined with rabbit bone marrow mesenchymal stem cells (BMSCs) coexpressed with BMP2 and BMP7 by adenoviral vectors.Methods Prepare the rabbit BMSCs with the method of direct way. And then make sure the identity of BMSCs by differentiation and FACs to determine the surface mark.Establish the BMP2 adenoviral vector and BMP7 adenoviral vector with pAd/CMV/V5-Dest system. Determine the proper MOI of each adenovial vectors. And then determine the BMP2 and BMP7 expression in the mRNA level and protein level by realtime PCR and immunofluorescence.Passege 3 BMSCs were divided into five groups: a. blank control; b. differentiation by osteo-induction culture medium; c. singlely transfected by BMP2 adenovirus; d. singlely trasfected by BMP7 adenovirus; e. cotranstected by both BMP2 and BMP7 adenovirus.Measure the mRNA level of Runx-2, Osx, AKP and Collagen I 7 and 14 days after transfection by realtime PCR. Measure the protein level of Osteocalcin and Collagen I 7 days after transfection by Western blot.Prepare PRP by Lendersberg way. Activate it after equably mixed with transfected BMSCs and then cultured for 1 week. And then scan electron microscope (SEM) was used to discover the three dimensional structure of the injectable tissue engineering bone. Then it was digested and cultured continuously.Results Preparation of BMSCs by direct way could help us obtain the BMSCs, which could differentiated to osteocyte, chondrocyte and adiocyte. It expressed CD29 and CD44 in its surface, but not expressed CD14, CD34, CD45 and CD90.Adenoviral vector of BMP2 and BMP7 we constructed could transfected rabbit BMSCs, and the proper MOI for BMP2 and BMP7 adenoviral vector were 200 and 50, respectively.7 days after transfection, BMP2 and BMP7 were all expressed in mRNA level and protein level.7 days after transfection, group e had higher level of Runx-2, Osx, AKP and Collagen I in mRNA level, than other four groups. Group c and d had higher level of Runx-2, Osx, AKP and Collagen I in mRNA level, than group a and b.14 days after transfection, group e had higher level of Runx-2, Osx, AKP and Collagen I in mRNA level, than other four groups. Group c and d had higher level of Runx-2, Osx, AKP and Collagen I in mRNA level, than group a and b. And mRNA level in day 14 of Runx-2, Osx, AKP and Collagen I were all higher than that in day 7. 14 days after transfection, group e had higher level of Osteocalcin and Collagen I in protein level, than other four groups.The concentration of platelet of PRP was 3.74 times of whole blood. 7 days after culture, SEM demonstrated that the BMSCs still alived in the PRP gels. After digestion, the BMSCs adhered to culture dish 1 day after culture. 7 days later, the cell prolifalated and kept the shape of spindle and polygon.Conclusion Preparation of BMSCs by direct way was proper.Adenoviral vector of BMP2 and BMP7 could transfect the BMSCs and express the BMP2 and BMP7.After cotransfected by both BMP2 and BMP7 adenoviral vector, BMSCs could express osteogenesis related gene higher in both mRNA level and protein level.Mixation with PRP, BMSCs could still kept alive and proliferation.