Experimental Study On Perfusion In Vivo In Inducing Bone Marrow Stromal Cells To Construct Tissue-Engineered Bone

Massive bone defect resulted from trauma, infection, tumor, burn, and etc is one of the troubling problems in clinic. The therapeutic efficacy treated by traditional methods, such as repair with autograft, alograft and biomaterial-filling and so on, is not optimal and hinders the widely clinical use. The rising of bone tissue engineering has provided a promising way to solve this problem, and has been one of the hotspots nowadays. However, the critical technology of vascularization and growth factorsËŠsustained release effect in large tissue engineered-bone still hasn't been solved idealy up to now.Obviously, the routine train of thoughts is faced up with large challenge in repaiering large bone defect. In order to find a clinically applicable tissue engineering way for the repair of large bone defects, the study group try hard to explore an innovative bone tissue engineering research way of which holism combines with reductionism and, furthermore, take the lead in setting up an approximate isologous model namely microenvironment system of osteogenisis induction by perfusion in vivo. Therefore, a series of experiment studies were performed as follows:Chapter 6: Culture of goat bone marrow stromal cells in VitroObjective:To explore the feasibility and application value of goat BMSC as seeding cells for bone tissue engineering,which were isolated from adult goat bone marrow and cultured in a defined condition to induce to osteoblast diferentiation, whose biological properties were observed. Method:Bone marrow was aspirated from the healthy adult goat and culture-expanded in vitro by the method of total marrow. Subsequently,passages of BMSC were induced to differentiate into osteoblast in the medium containing dexamethasone (10-8mol/L), beta-sodium glycerophosphate (10mmol/L) and ascorbic acid (50 mg/L), and were observed continually by inverted phase contrast microscope, stained by HE,growth curve of which was evaluated by MTT, surface marker proteins of which were analyzed by the flow cytometry.Alkaline phosphatase (ALP) and Calcifying nodule were strained by Gomori Calcium-Cobalt and Von Kossa methods respectively. Content of ALP was measured. Results: The goat bone marrow stromal cells cultured in primary and passage culture were proliferated actively. After BMSC's inducement for 2 to 3 weeks, these differentiated cells survived well, biochemical indexes were stable, biological properties and morphologic characteristics were similar to those of typical osteoblasts under light microscope. Flow sytometry analysis showed that the marker proteins of the surface such as CD29, CD44 and CD105 were positively expressed while CD14, CD34 and HLA-DR were negative. The result of stain reaction of ALP and calcifying nodule were positive respectively. The abilities of secreting ALP were enhanced (P<0.01). Conclusion:Goat BMSC can be aspirated from bone conveniently, and they can be induced to osteoblast diferentiation possessing typical morphologic characteristics and biological function in a defined condition, thus Goat BMSC can be used as an ideal resource of seeding cells for bone tissue engineering. Chapter 7: The effects of rhBMP-2 and rhbFGF on proliferation of bone marrow stromal cellsObjective:To evaluate the effects of rhBMP-2 and rhbFGF on proliferation of goat bone mesenchymal stem cells (BMSC) by using them alone and associatedly . Method: Cell culture technique and MTT colorimetric assay were used to evaluate the proliferation of BMSC on the 1st 3rd, 5th, 7th, and 10th day by adding rhBMP-2 and rhbFGF on diferent ways. Results:Statistical analysis showed that both rhBMP-2 and rhbFGF could enhance the proliferation of BMSC significantly in long term, and also in a dose-dependent manner. The efects of the two growth factors were significantly higher by using them associatedly than by using them alone . Conclusion : Both rhBMP-2 and rhbFGF could co-operatively enhance the proliferation of BMSC.Chapter 8: The effects of rhBMP-2 and rhbFGF on differentiation of bone marrow stromal cellsObjective: To evaluate the effects of rhBMP-2 and rhbFGF on diferentiation of rabbit BMSC by using them alone and associatedly. Method:Alkaline phosphatase (ALP) kit and Coomassie brilliant blue assay were used to evaluate ALP level and protein content of BMSC so as to reflect differentiation of the cells on the 2nd, 5th, 7th, and 10th day by adding rhBMP-2 and rhbFGF on diferent ways. Results:Statistical analysis showed that rhBMP-2 could enhance while rhbFGF could inhibit the differentiation of BMSC significantly in long term, and also in a dose-dependent manner. The efects of using rhBMP-2 alone was similar to that of using the two growth factors associatedly, and they were significantly higher than that of using rhbFGF alone . Conclusion:Using the rhBMP-2 and rhbFGF associatedly and using rhBMP-2 alone all have the powerful capability of enhancing the diferentiation of goat BMSC.Chapter 9: Bone marrow stromal cell transfected with enhanced green fluorescent protein gene plasmidObjective:To explore the feasibility of liposome-mediated pEGFP-N2 as a long term and steady tracer of BMSC. Method: Two Chinese green goats at 12 months of age were selected as donors to obtain bone marrow stromal cells( BMSC).Bacillus coli 109 strain ,which was transfected with pEGFP-N2, was prepared for plasmid extraction. The plasmid was identified by the restriction enzyme Avaâ…¡digesting. The plasmids were used to transfect the goat BMSC with lipofectamine 2000 . 24 hours and 6 months after transfection , the transfection efficiency was counted respectively . Results: The plasmid was digested into three bands which were at the length of 445bp,1938bp,2354bp respectively in gel electrophoresis. The plasmid was identified as pEGFP-N2. The green fluorescence expression efficiency was 35 percents after transfection 24 hours .After being screened by G418 ,the efficiency reached at 75 percents till the 6th month. Conclusion:The labeling technique of which BMSC were transfected with liposome-mediated pEGFP-N2 is an ideal tracer method.Chapter10: Enhanced green fluorescent protein gene plasmid labeling technology in monitoring tissue engineered bone formationObjective : To explore the feasibility of liposome-mediated enhanced green fluorescent protein(EGFP) plasmid as a tracer of seeding cells in monitoring tissue engineered bone formation. Method:Both labeled and unlabeled BMSC were induced to osteoblastic differentiation . The alkaline phosphatase (ALP) activity and calcium nodule dying were detected in both groups. The unlabeled BMSC were used as the control group. The labeled BMSC were seeded in coral scaffold to form BMSC-coral complexes. Under scanning electron microscope, these BMSC-coral complexes were observed at 4,7,14days respectively in vitro. The BMSC-coral complexes, which had been cultured for 7 days in vitro, were implanted into nude mice subcutaneously. They were harvested at 8 weeks, then were evaluated by HE staining and traced under fluorescence microscope. Results:Compared to the control group, the ALP expression activity was unaffected (P> 0. 05). Both the labeled and unlabeled BMSC have the capability of forming calcium nodules. The labeled-cells were able to grow into the coral scaffold and secret collagen fiber bundles .The mineralized nods were also observed in the scaffold. The tissue engineered bone had been formed over a 8-week period.There were newly formed trabeculae around the pores of coral. Fluorescence microscop detected that 1abeled cells existed in many newborn tissues,and coral was partly degraded.There were no green fluorescence expression in the control group. Conclusion: The labeled BMSC ,which were transfected with liposome-mediated pEGFP-N2,can be used to trace the formation of tissue engineered bone in nude mice. It may be an optimal labeling technique to monitor tissue engineered bone formation.Chapter11: Preparation of an experimental goat model of large tibia bone defectObjective:To provide an ideal animal model for tissue engineering.Method: The nine Chinese goats were divided into thre groups.The 25mm left tibia defect of every goat was made of and fixed with plate.Bone formation were evaluated by X-ray an d histology.Results:The Lane's scores of nine goats were all zero.There was no bone formed in histology.Conclusion:The goat tibia is adapt to preparation of segmental bone defect model and fixation with reconstructive plate. 25mm segment bone defect of tibia is ideal animal model for the bone tissue engineering study.Chapter12: The primary observation of the effect of approximate isologous model repairing goat tibia bone defectObjective: To explore the feasibility of approximate isologous model repairing goat tibia large bone defect.Method: The autologous bone marrow stromal cells were amplificated in vitro, then were seeded in coral scaffold to form BMSC-coral complexes and implanted into the site of large bone defect. By the use of combination of mobile micro pump and subcutaneously implanted drug delivery pump, the approximate isologous model were constructed . The approximate isologous model was the bone formation microenvironment system which the infusion of DMEM,rhbFGF and BMP-2 may be regulated by the bi-pump system.Results:X-ray:at 8 th weeks after implantation, bony callus was found in the bone defects and fused with the terminal of the defected bone in the experimental group. However, at 8 th weeks after implantation,the part of coral was partly degraded and absorbed in the control group. Histomorphometrical analysis showed new bony callus bone formed in experimental group at 8 th weeks after implantation. There was no bone formation but fibrous tissue was observed in the control group.Conclusion:The bone tissue engineering approximate isologous model has the feasibility of repairing diaphyseal large segmental bone defect of large animal(goat).