Experimental Studies On One New Tissue-engineering Bone Using Bone Marrow Mesenchymal Stem Cells, Bovine Bone Morphorgenetic Protein And Silk Fibroin

With the appearance and development of bone tissue engineering, it maybe the new and ideal therapy in spinal fusion. So we practice three parts of experimental studies on bone tissue engineering: constructing tissue-engineering bone with bone mesenchymal stem cells, bovine bone morphorgenetic protein and silk fibroin in vitro, enhancing spinal fusion in vivo. To elementally explore the feasibility about constructing tissue-engineering bone with three elements of bone tissue engineering. PART I: Experimental study on the biological features of rabbit's bone marrow mesenchymal stem cells in vitro Objective To explore the best experimental conditions for isolation and cultivation of bone marrow mesenchymal cells (BMSCs) and observe its'biological features in vitro. Methods BMSCs were separated by gradient centrifugation on percoll from bone marrow of New Zealand white rabbits, then were cultivated and sub-cultivated. Cell morphon and vigor were estimated through invert microscope and cell growth curves. After cultivated continuously in mineralization medium for 4 weeks, the cells of 3rd passage were stained by calcifying nodule/Von-kossa and alkaline phosphates/Calcium-cobalt methods. Results BMSCs were able to grow with normal morpha and vigor. The results of stain reaction were positive in inducing group, and negative in control group. Conclusion The method of isolation and cultivation of bone marrow mesenchymal cells is relatively simple and feasible. Bone marrow mesenchymal cells can be introduced to be osteoblasts directionally, and maybe one potential seed cells of bone tissue engineering. PART Ⅱ: Biologic feature of bone marrow mesenchymal cells in carriers combined with bovine bone morphorgenetic protein and silk fibroin Objective To observe experimentally the biocompatibility of bone marrow mesenchymal stem cells (BMSCs) with the delivery system of bovine bone morphorgenetic protein (BMP) in conjunction with porous silk fibroin (SF) in vitro, and elementarily explore its practical significance. Method We produced the delivery system with bBMP and SF in laboratory. Then 5ml bone marrow was drawed by puncture from New Zealand white rabbit 4 weeks old, the BMSCs were isolated and cultivated as before until affluent cells were collected. We seeded these cells in the delivery system with the density of 5×105/ml. In order to promote attachment, cells were allowed to adhere to the system for 4 hours prior to the addition of media. Then the cells were continuously cultivated for 8 days at 5% CO2 and 370C. Meanwhile control group were made with only SF as above. A series examinations including scanning electron microscope (SEM), laser scan confocal microscope (LSCM) were carried out, and at eighth day cells were stained by calcifying nodule Vonkossa and alkaline phosphatase (ALP)/ Calcium-Cobalt methods. Result BMSCs could grow with normal morphon and vigor in both groups in vitro. Cells attached to the material with creeping morphon after 24 hours, and at 8th day cells surrounded with reticulation and granules expanded, inosculated, and sheeted on materials. Cells stained by calcifying nodule Vonkossa and alkaline phosphatase (ALP)/ Calcium-Cobalt methods expressed positive reaction in experimental group, and negative in control group. Conclusion Porous silk fibroin have perfect biocompatibility, can serve as not only extracellular matrix but also carrier system of growth factor. PART Ⅲ: Experimental study on spinal fusion using the new tissue-engineering bone Objective To observe the enhancement on spinal fusion using the tissue-engineering bone constructed with bone marrow mesenchymal stem cells, bovine bone morphorgenetic protein and silk fibroin in vivo, and explore the feasibility and significance about the newtissue-engineering bone. Method We established the model of intercentrum process fusion of 60 New Zealand white rabbits posteriorly. Rabbits were separated into five groups (12 rabbits each group) at random: tissue-engineering bones constructed with bone marrow mesenchymal stem cells, bovine bone morphorgenetic protein and silk fibroin were transplant in vivo in group A, composites of bone morrow mesenchymal stem cells and silk fibroin were transplanted in group B, carrier system constructed with bovine bone morphorgenetic protein and silk fibroin were transplanted in group C, and silk fibroin were transplanted in group D, untreated in group E. A series of examinations, including radiograph, histomorphology were carried out at the time of 2, 4, 8 and 12 weeks. At the time of 12 weeks all rabbits were killed to perform manual palpation, biomechanical testing. Result At the time of 12 weeks 100% spinal fusion rate was achieved in group A, 25% fusion rate in group B, 83.3% fusion rate in group C, 16.7% fusion rate in group D, 0 fusion rate in group E. Group A exhibited better ossification and higher spinal fusion rate than other groups. Conclusion Tissue-engineering bone constructed with bone marrow mesenchymal stem cells, bovine bone morphorgenetic protein and silk fibroin exhibited better ossification and spinal fusion rate in vivo, could be prospective in further clinical usage.