| In clinical situation, bone defects commonly occurred following tumor resection, trauma and inflammation, causing severe loss of structure and function, and becoming one of the serious clinical problems. Autologous bone graft has been considered as the golden standard in treatment of bone defects. However, due to the limited sources of autologous bone, limitations of shape and size, and various complications of donor sites, the application of autologous bone graft has been confined. Therefore, tissue engineering bone went through rapid development as a substitute of autologous bone graft. There are three elements in bone tissue engineering:scaffold, seed cells and bio-factors.Models of bone defects are indispensible in researches of bone tissue engineering. During the past decades, a great variety of bone defect models has been introduced, including murine calvarial defect, murine femoral segmental defect and mandibular defect and so on. This study investigated the availability of bone defect with certain size during a certain obervation period,using a novel murine femoral segmental defect model based on customized fixation system.Connective tissue growth factor, also known as CCN2, the second member of the CCN superfamily, is a secretory matrix protein, widely participates in bone development and regeneration. CTGF was highly expressed in development of bone and cartilage, and plays important roles in regulating the whole process of bone development and cartilage development. Moreover, CTGF also took part in the healing process of bone and cartilage. Lots of studies, both in vitro and in vivo, demonstrated that CTGF was able to induce and promote osteogenic differentiation in various cell cultures and in animal models. Based on the abovementioned evidences, CTGF was considered as a promising bio-factor for bone tissue engineering.MC3T3-E1 cell line, a mouse calvarial preosteoblast cell line, was often used as a model cell source for osteoblasts related studies in vitro and in vivo. In most of cases, MC3T3-E1 cells acted as preosteoblasts, and studies concernd the cell subpopulation of MC3T3-E1 cells showed that subclone 4 and subclone 14 of this cell line expressed the highest osteogenesis potential. The osteogenesis potential made MC3T3-E1 cells possible for acting as a source of seed cell in bone tissue engineering. Here in my study, MC3T3-E1 cells was transinfected with lentivirus carried the recombinant mouse CTGF gene to overexpress mouse CTGF, and then alterations of osteogenic phenotype were studied in vitro; subsequently these CTGF overexpressing MC3T3-E1 cells were seeded into a chitosan/β-tricalcium phosphate scaffold and were used to restore mouse femoral segmental defect, investigating the practicability of CTGF overexpressing cells in restoration of bone defects.Study I:The overexpression of CTGF on the proliferation, differentiation and migration of MC3T3-E1 cellsObjective:To investigate the effect of lentivirus mediated CTGF overexpression on osteogenic differentiation of MC3T3-E1 cells, and to explore the alterations of proliferation and migration of CTGF overexpressing MC3T3-E1 cells.Material and Methods:Recombinated mouse CTGF gene was carried in a GFP tagged lentivirus, and transinfected into MC3T3-E1 cells to obtain CTGF overexpressing cells. Then total RNA and total protein were extracted after different culture period of osteogenic medium. Real time PCR and western blot were performed to determine the expression level of several osteogenic markers. The ALP activity was qualitatively analyzed by the diazotization-coupling method and quantitatively analyzed by the PPNP method. Mineralization was assessed by alizarin red staining. Additionally, the proliferation was measured by cell counting kit 8 assay to assessed the effect of CTGF overexpression on the proliferation of MC3T3-E1 cells, and cell migration capability was assessed by transwell assay.Results:The lentivirus mediated transfection successfully overexpressed mRNA and protein of CTGF in MC3T3-E1 cells. Real time PCR analysis showed that in CTGF overexpressing cells the osteogenic gene markers, including osteopontin, Runx2, and osterix were significantly upregulated. Western blot analysis showed that the osteogenic proteic markers of CTGF overexpressing cells were also significantly increased. The qualitative and quantitative analysis of ALP acitivity showed that ALP activity of the CTGF overexpressing cells was significantly promoted. Alizarin red assay showed that the CTGF overexpressing cell formed moremineralized nodules than the control group. Cell counting kit 8 assay showed that, comparing to the control group, CTGF overexpressing cells were not significantly altered in proliferation and transwell assay showed that the migration capability was enhanced in CTGF overexpressing cells.Conclusion:Overexpression of CTGF was demonstrated to enhance osteogenic differentiation of MC3T3-E1 cells, while promote the migration and has no significant effect on proliferation. All these results, taken together, proved that CTGF overexpressing MC3T3-E1 cells were available to act as a source of seed cells for restoration of bone defects.Study Ⅱ:Establishment of mouse femoral segmental bone defect model and fabrication of cell-scaffold complexObjective:To investigate the capability of self-repairing in mouse femoral segmental bone defect, and to determine the suitable size of defect with a observation period of 5 weeks.To fabricate and shape the chitosan/β-tricalcium phosphate scaffold, and to inoculate CTGF overexpressing or control MC3T3-E1 cells into the scaffold so as to form a cell-scaffold complex and then to confirm the inoculating efficacy.Material and methods:Mouse femoral segmental bone defect model was established using customized rigid internal fixation system. The healing condition of 1.2 mm sized bone defect till 5 weeks was assessed by MicroCT analysis and histological analysis. The chitosan/β-tricalcium phosphate scaffolds were fabricated using the lyophilization method. Cell-scaffold complex was produced following the soaking and centrifugation protocol. The distribution of seed cells in the scaffold was observed by frozen section and fluorescent staining.Results:MicroCT results histological analysis showed that the 1.2 mm size defect was not apparently healed, and little new bone was formed by 5 weeks.The fluorescent staining showed that the inoculated seed cells adhered to the walls of pores, and well distributed in scaffold.Conclusion:The 1.2 mm sized segmental defect showed no self-healing till 5 weeks, and so it was suitable for bone defect reparing studies. The inoculation method was demonstrated to be effective and available to use in further studies. Study Ⅲ:Restoration of mouse femoral segmental defect using lentivirus mediated CTGF overexpressing MC3T3-E1 cells as seed cells. Objective:To determine the restoration effeciency of CTGF overexpressing MC3T3-E1 cells in the abovementioned mouse femoral segmental defect model. Material and methods:. Mouse femoral segmental bone defect model of 1.2 mm size was established using customized rigid internal fixation system. The chitosan/β-tricalcium phosphate scaffolds were fabricated using the lyophilization method. Cell-scaffold complex was produced following the soaking and centrifugation protocol, and subsequently transplanted into the mouse femoral segmental defect to restore it. After 2 weeks and 5 weeks postoperatively, femurs of experimental animal were dissected as samples. The healing condition was determined by Micro-CT. The histological healing status was determined by H-E staining and Van Gieson staining.Results:Micro-CT analysis and histological staining were performed to evaluate the healing progresses of defects at 2 and 5 weeks, respectively. The 3D reconstruction images of μ-CT showed that the healing of S-C group was dramatically better than those of the other two groups at 5 weeks postoperatively. However, no great difference was observed at 2 weeks. Setting the defect area as area of interest, quantitative data of μ-CT showed that group S-C was significantly higher than the other two groups in bonemineral density (BMD), bone volume/tissue volume (BV/TV), trabecula number (Tb.N), and connectivity density (Conn.D), but lower in trabecula separation (Tb.Sp) at 5 weeks. Higher BMD, BV/TV, Tb.N and Conn.D, as well as lower Tb.Sp, represented better bone healing. Meanwhile, both S-N and S-C group showed the same tendency as S-C group versus S-N group in BMD, BV/TV, Tb.N, Conn.D, and Tb.Sp compared to S group at 5 weeks, thereby indicating that cell-loaded scaffold had better restoration effects than non-cell scaffold. All the quantitative data approximated null, and showed no significant difference at 2 weeks. However, trabecula thickness (Tb.Th) showed no significant difference among all groups.Masson’s trichrome staining images showed that at 2 weeks, osteoid and bone remodeling occurred in the defect of S-C group, whereas in the S-N group cells accumulated in scaffold In the S group, only scaffold structure was present. At 5 weeks all groups formed osteoid in defect area, in group S-C one side of the defect was sufficiently close to be bridged by newly formed bone, whereas in group S-N small amount of osteoid formed and in group S the defect remained unhealed.Conclusion:. In conclusion, we demonstrated that the overexpression of CTGF enhanced osteogenesis in vivo. CTGF overexpression cells serve as efficient seeding cell source for bone regeneration.In summary:.MC3T3-E1 cells are capable of overexpressing CTGF protein with lentivirus mediated transinfection. Compared to the control group, the CTGF overexpressing cells expressed higher osteogenic markers both on mRNA level and on protein level, demonstrating that CTGF overexpression enhanced the osteogenic differentiation of MC3T3-E1 preosteoblast cell line. Moreover, the migration capability of MC3T3-E1 cells was also promoted by CTGF overexpression, while the proliferation was not significantly altered. The CTGF overexpressing cells were inoculated into the chitosan/β-TCP scaffold as seed cells, and facilitated the restoration of mouse femoral segmental defect model. So the CTGF overexpressing cells were suitable for bone tissue engineering. |
