Experimental Study Of Revascularization And Osteogenic Ability Of Rabbit Bone Mesenchymal Stem Cells Seeding Onto Xenogenic Bone In Vivo

Objective:In recent years,the characteristics and advantages of tissue engineering which could renew tissues or organs have been widely recognized.As an important area of tissue engineering,bone tissue engineering which has achieved many exciting research results and preliminary clinical application,is considered to be one of the most promising and feasibility areas.The subject is according to the basic principles of bone tissue engineering.BMSCs and calf bone composites implanted into rabbit ilium,and then observe the osteogenesis and revascularization during the process of bone healing.The ideal is to provide a theoretical basis for building the initial tissue-engineered bone products.Methods:1.The isolation,cultivation and identification about the rabbit marrow mesenchymal stem cellCells were isolated from the New Zealand white rabbits' bone marrow via density gradient centrifugation.Light and electron microscope was used to observed the cell morphology and Flow cytometry was used to examine the expression of cell surface antigen.The cell culture growth curve was obtained based on observation of the proliferation status of 3^rd,6^th cell generation by MTT method.2.Study of osteoblastic differentiation potentiality of the rabbit bone marrow mesenchymal stem cell in vitroThe well 3^rd generation cells were utilized for osteoblasts differentiation in osteogenic inductive conditioned medium,which was changed every three days.Light and electron microscope was used to observed the cell morphology.Cells were collected for determination of intracellular alkaline phosphatase respectively in the first 2,4,6,8,10,12, 14 days culture.TypeⅠcollagen and osteocalcin(OCN) assayed with immunocytochemical stain and calcium node detected by staining after several days' culture BMSCs under osteogenic inductive condition.Using RT-PCR detection of BMP-2,VEGF mRNA content and using Western-blot detection of BMP-2,VEGF protein content after 0 days,7 days,14 days,21 days osteogenic induction.3.Study of histocomapatibility of cortical strut with partial cancellous bone and its potentiality used as the carrier for BMSCsThe well 3^rd generation cells were seeded in calf cortical strut with partial cancellous bone,and then the adhesion ratio was detected in the first 4,8,12,24 hours.BMSCs were cultured in calf bone leach liquor with different concentration.Then the cells were observed by contrast phase microscope,RGR(relative growth rate) were measured by MTT and cytotoxicity was graded after 2 days,4 days,7 days culture.The growth, proliferation and matrix secretion of BMSCs in calf cortical strut were observed by scanning electron microscope after 2 weeks culture.4.An Experimental study of rabbit BMSCs combined with calf bone implants into rabbitsBMSCs induced osteogenic composite calf bone(groupⅠ),Simple calf bone group(groupⅡ) or autogenous iliac implant(groupⅢ) were randomly implanted in rabbit ilium.The changes of implants surface and tissue reaction around the implant were observed.All of implants were performed X-ray examination and observed the changes after 4,8,12 and 24 weeks.All of implants were performed examinations of HE stain and immunohistochemistry staining of VEGF and BMP-2 to observe the expression after operation 4,8,12,24 weeks..The expression of VEGF and BMP-2 mRNA were detected by RT-PCR after operation 4,8,12,24 weeks.Results:1.BMSCs were very purified and all in the whirlpool shape.Flow cytometry showed that more than 90%cells expressed CD44 and CD29, while only 5%of the cells expressed CD34.Cell culture growth curve showed that cells of the 3^rd and 6^th generation have high proliferation while the 10^th generation have lower proliferation.2.During rabbit BMSCs osteogenic induction,cell morphology gradually changed from a long fusiform to short fusiform,polygonal,and finally stacked into colony growth.In the induction process,ALP content was gradually increased,compared with the control group,there is a significant difference(P＜0.05.Immunocytochemical staining showed TypeⅠcollagen and OCN positive.Calcium node detected by staining was also positive.RT-PCR and Western-Blot showed that BMP-2 and VEGF were expressed in induced group and control group.The strongest expression of BMP-2 and VEGF was in the 7^th day after BMSCs osteogenic induction.There is a significant difference(P＜0.05) in the in the 7^th day, 14^th day,21^th day after BMSCs osteogenic induction compared to the control group.3.The adhesion ratio were 47%±4%,65%±2%,77%±3%,86%±2%, respectively;after seeding 4,8,12,24 hours.The rabbit BMSCs showed normal morphology and proliferation increased with culture time.The toxicity gradation was zero to one.The BMSCs could be adhered the surface of calf cortical strut and extended in the cancellous bone,and extracellular matrices were found in it.4.The wound was well,inflammation and toxicity reaction in body or local part were not observed in all groups.The X-ray showed that groupⅠhas reached bone healing after implant 12 weeks,groupⅡalmost got host bone junction healing after implant 24weeks,while groupⅢwere not satisfying healing.The process of ossifications from cartilages were observed in all groups by HE stain and BMP-2 and VEGF were positive by immunocytochemical stain.BMP2,VEGF mRNA were expressed in all groups.Compared between groupⅠ,Ⅱ,Ⅲ,there is statistical significance of BMP2 expression in each group(P＜0.05).Compared groupⅡwith groupⅠandⅢ,there is statistical significance of BMP2 expression(P＜0.05).Compared groupⅠwith groupⅢ,there is statistical significance of BMP2 expression(P＞0.05). Conclision:1.The method of isolation and culture of BMSCs is simple and feasible.BMSCs could be induced into obteoblasts with stably expression of osteoblastic phenotype in vitro.BMSCs may be accorded as a kind of seed cells for tissue engineering.During BMSCs osteogenic induction, BMP-2 and VEGF were first expressed increased and then decreased.The expression of VEGF may play a role in promoting angiogenesis.2.The calf cortical strut with partial cancellous bone could be used for cells carrier in bone tissue engineering.It was an innocuity,better histocompatibility material and could be doubled as osteo-conductibility and osteo-inductivity for satisfying the demand of bone tissue engineering.The cortical strut with partial cancellous bone was an ideally material for bone tissue engineering which was satisfied in histocompatibiity,the speed of degradation,the efficiency of biomechanics and ossification.Rabbit BMSCs seeded in the cortical strut with partial cancellous bone could construct tissue engineering bone by osteoinductation in vivo.