The Preliminary Experiment Research Of The Ossification And Vascularization In Vivo From Dog Bone Marrow Mesenchymal Stem Cells Seeded In Intensified β-TCP/PRP Gel Multiple-scaffold

Objective To investigate the ability of constructing tissue engineering bone and repairing bone defection as well as vascularizing with dog bone marrow mesenchymal stem cells in intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold implanted dog's tibia upper segment after directed differentiation of stem cells into osteoblasts in vitro.Methods(1) Preparing dog bone marrow mesenchymal stem cells in intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold including the isolation,purification and proliferation of BMSCs,the preparation of PRP and so on.(2) Dog BMSCs was seeded in and then cultured or osteo-induced with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold in vitro for 1 weeks.(3) The cells compound with alone beta-tricalcium phosphate(groupâ… ) or intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold(groupâ…¢) were randomly implanted in dog's left or right circular tibial upper segment and were compared with autogenetic Ilium(groupâ…¡).The changes of implants surface,the form of osteotylus,the reparation of bone defection and tissue reaction around the implant were observed.(4) All of implant were performed examination of HE stain and immunocytochemical stain of osteocalcin protein to observed the expression after operation 4,8,12 weeks,and to calculate microvessel density(MVD).(5) The expression of OCN,VEGF mRNA were detected by RT-PCR and the correlation of them was analyzed after operation 4,8,12 weeks.Results(1) The wound growth all right,inflammation and toxicity reaction in body or local part were not observed in all groups. The material in the group of the cells compound with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were degraded faster than groupâ… .(2)The numerical of microvessel density(MVD) in the group of the cells compound with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold nearly reached the level of the autogenetic ilium,and was higher than groupâ… .(3) The ossifications of new cartilages were observed in all groups by HE stain and immunocytochemical stain of osteocalcin protein.The expression of the cell/intensified beta-tricalcium phosphate/ platelet-rich plasma gel multiple-scaffold compounds were better positively than the cells/ intensified beta-tricalcium phosphate compounds.(4) The expression of osteocalcin,VEGF protein mRNA were showed positive at all groups aider operation for 4,8 and 12 weeks. The groupâ…¡,â…¢expressed all protein mRNA more positive than groupâ… at 8 and 12 weeks.And there was no different between the expression of groupâ…¡,â…¢,and there was positive correlation between the mRNA expression of OCN and VEGF.Conclusion(1)Good vascularization could be acquired after canine BMSCs seeded in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were transplanted in vivo.(2) There is close relationship between vascularization and ossification could after canine BMSCs seeded in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were transplanted in vivo.