An Experimental Study On Percutaneous Autologous Cultured Bone Marrow Mesenchymal Stem Cell Grafting For Bone Defect

Objectives: To establish a reproducible system for the culture and osteogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSC s ) in vitro,and also to improve the aspiration and cultivation of BMSCs. To determine in vivo osteogenic potential in size-critical bone defect after percutaneous autologous grafting of culture-expanded rabbit autologous BMSC s combined with those induced by osteogenic supplement (OS).Methods: 1 . BMSCs were cultured and then induced with OS medium meanwhile they were observed daily and cell number were analyzed. ALP enzyme activity was also measured so as to find out the exact phase when MSC would have the maxium osteogenic potential. The cell number and ALP enzyme activity were compared before and after osteogenic induction.2. Rabbits were divided into 4 groups at random, myeloid puncture needles were applied in Group A, B, C during bone marrow aspiration,and common 20ml injection syringe with circular cone-shaped pinhead were applied in group D.In group A,B,C,D, culture medium was DMEM, DMEF-12 a-MEM, DMEM respectively. The cell number after cultivatgion for 10 days and cell growth curves of P2 were investigated.3. the 15mm bone defect experimental model at both radius of rabbit were divided into 5 groups at random. BMSCs with and without OS induction were collected and percutaneously autologous injected respectively into radius bone defects. The graft in Group A was 1 X 10~6 BMSCs+1 X 10~6 osteo-inducedBMSCs ,in group B was 2 X 10~6 osteo-induced BMSCs,and 2 X 10~6 BMSCs in group C .5mg BMP combined with fibrin sealant was implanted in group D and 0.9%NaCl solution in E. Osteogenesis at the defect area was observed by regular radiography, histology and biomechanics.Results: 1. there was no differnence on cell number,shape,growth curve of BMSCs between myeloid puncture needles and common 20ml injection syringe with circular cone-shaped pinhead2. BMSCs show much higher growth rate and greater osteogenic potential when cultured in a - MEM medium ,in comparison with DMEM DMEF-12.3. A large quantity of callus was found in the 4 groups while the blank control group show little callus 4 weeks after cell trasplantation. At the 12th week GroupA, which was transplanted with BMSCs+osteo-induced BMSCs achived complete bone healing with medullary cavity united. Group A had the largest quantity of new bone measured by X-ray analysis, and also their maximal load were better than those in other groups.Conclusion: the common 20ml injection syringe with circular cone-shaped pinhead could completely substitute myeloid puncture needles in bone marrow aspiraton at iliac crest of rabbit, in the cultivation and osteogenic induction of BMSCs, a -MEM medium was superior to DMEM and DMEF-12 . The osteo-induced BMSCs and BMP have excellent osteogenic potential in vivo while the bone-forming ability of rabbit osteo-induced BMSCs combined with BMSCs in bone defect is superior to that of BMP or BMSCs grafting.