| Bone defect is a common and refractory disease to treat in orthopedics. The causes of this disease include trauma, tumour and some clinical surgical complication. Autografts, depending on the size of the bone deficiency, is currently the gold standard for treating such defects in clinics. However, Autografts is not suitable for the treatment of a large defect because of the limitation of bone amount. The major risks and disadvantages in using allografts are viral disease transmission and bacterial infection. The transplantation of bone graft substitute is an important treatment for bone defect. Titanium alloy is a vital major bone graft substitute. However, traditional titanium alloy has some disadvantages, such as poorer osteoconduction and mechanics conductivity, etc. In recent years, with the development of 3D copying technology, porous titanium alloy(pTi) provides a new idea for bone defect repair in clinics. As compared with traditional titanium alloy, pTi has similiar structure characteristics with natural trabecular bone. It can provide effective pores for bone tissue fluid in the transmission channel. However, because of the inert metal titanium, pTi can only combine with the bone tissue in the form of mechanical riveting, rather than a more stable chemical bonds combination way. Therefore, pTi can not yet achieve early and rapid osseointegration. The efficiency and quality of bone defect repair are still unsatisfactory.Low-level whole-body vibration(WBV) is a kind of safe, economic and non-intrusive physical factor. Different parameters of WBV stimulus to animal and human skeleton have shown significant regulatory effects. A large amount of research at home and abroad have shown that WBV can effectively restrain osteoporosis of disuse patients and postmenopausal women, inhibit bone loss, enhance bone strength, and prevent the occurance of fractures. Some clinical investigations have also indicated that WBV can significantly improve the whole-body bone mass not only in healthy people but also in patients with osteoporosis, and thus regulate bone turnover. At the same time, many preclinical studies confirmed that WBV can significantly promote the fracture healing. However, there is no literature reported at home and abroad whether WBV can improve osseointegration and ingrowth of p Ti and thus improve the efficiency and quality of bone defect repair. In our present study, we firstly explored the cytoskeletal structures, cellular behaviour, biological activity and expression of osteogenesis-related genes and proteins for in vitro osteoblasts seeded in pTi followed by stimulatation with WBV. Secondly, we explored the effect and potential mechanism of WBV synergy with pTi in the repair of bone defects in the long bones of rabbits via in vivo experiment. The aim of the present study is to systematically reveal the effect and mechanism of WBV on osseointegration and osteogenesis with p Ti, and thus providing novel ideas and strategy for bone defect repair in clinics. Moreover, our study is expected to reduce the cost of repairing treatment spend in bone defect, and aslo improve survival quality of patients with bone defect. Our study not only has important reference value in scientific research, but also poses important clinical value. The whole project can be divided into the following two parts:Experiment ?:Effects of behavior, function and mechanism of osteoblast in three-dimensional pTi stimulized by WBVMethod: PTi(12mm×2.5mm, 70% porosity, 1mm pore diameter) was designed by CAD and manufactured via electron beam melting technology, after ultrasonic cleaning, high temperature and high pressure sterilization, was used in this experiment. Primary osteoblast from New Zealand rabbit were seeded into 3-D pTi discs. After WBV stimulation, proliferation was detected via the MTT assay, adhesion was detected via DAPI staining and SEM examination, cytoskeletal structures was detected via FITC staining, and osteogenesis-related signaling pathway gene and protein expression was detected by qRT- PCR and Western-blot.Result: The results of MTT assay detection displayed that WBV stimulation can significantly promote the proliferation of the cells in pTi surface(P< 0.05). DAPI staining and SEM results showed that the number of osteoblasts and its pseudopodiums in pTi+WBV group were significantly more than the Control group. FITC cytoskeleton fluorescence staining results showed that the cell attachment number of pTi+WBV group was significantly higher than the Control group(P< 0.05), and cells in p Ti+WBV group can stretch out more microfilament as compared with the Control group(P< 0.05). The result of qRT-PCR and Western-blot showed that WBV upregulated the expression of osteogenesis-related genes and proteins and canonical Wnt pathway genes and proteins.Conclusion: The in vitro experimental results showed that WBV stimulation could accelerate the speed of proliferation and increase the number osteoblast adhesion, enhance the osteogenesis-related gene and protein expression for osteoblasts seeded into the pTi implants.Experiment ?: Effect of combined application of WBV and pTi on rabbits long bone defect repair and the potential mechanism researchMethod: 3D models of pTi were designed by CAD(6mm×8mm, 70% porosity, 1 mm pore diameter) in this experiment. The pTi implants were fabricated by using the electron beam melting system. After ultrasonic cleaning and 60 cobalt sterilization, pTi can be used as the implants of in vivo experiments. Female New Zealand white rabbits, weighting 3.0±0.4kg, were anesthetized by 3% sodium pentobarbital solution(1ml/kg) and xylazine hydrochloride injection(0.1ml/kg) via intramuscular injections. The pTi were then implanted into left hind legs and the animals were randomly divided into the pTi group and pTi +WBV group according to the body weight. The rabbits in the pTi+WBV group were subjected to 1 h/day WBV stimulation with 30 Hz frequency and 0.5 g acceleration. After WBV stimulation for 6 weeks and 12 weeks, rabbits in each group were euthanatized. All animals received intramuscular injections of 8% calcein solution(1ml/kg) before sacrifice. Samples were examined by X-ray, Micro-CT, fluorescent double standard analysis, Masson staining and qRT-PCR detection, toluidine blue staining.Result: X-ray examination showed that the location of implant was correct. Micro-CT examination showed that after 6 weeks and 12 weeks WBV stimulation, the pTi implant osseointegration in pTi +WBV group is superior to the Control group. The values of BV/TV, Tb.N were higher than those in the Control group(P< 0.05). The levels of BS/BV and Tb.Sp were lower than those in the Control group(P< 0.05). The Masson staining results showed that the quantity of bone in pTi+WBV was more than that in the Control group. The result of fluorescence double labelling results showed that the values of MAR, MS/BS and BFR/BS in the pTi+WBV group were significantly higher than those in the Control group. After 6-week and 12-week WBV stimulation, the expression levels of osteogenesis related genes, including ALP, OCN, Runx2, BMP2, OPG were significantly higher than those in the Control group(P< 0.05). The Sost and RANKL gene expression levels were significantly lower than the Control group(P< 0.05). The canonical Wnt signaling pathway genes, including Wnt3 a, Lrp6 and ?-catenin were significantly higher than those in the Control group(P< 0.05).Conclusion: Through the observation of rabbit bone defect repair model, we can draw a conclusion that, after WBV stimulation, implant osseointegration in pTi+WBV was more obvious than the Control group. There are a large number of trabecular bones adhered around and growed into the pores of pTi.WBV improved osseointegration on the microscopic levels, improved the effect of repair and stability of the implant to reduce the loose and inflammation. |
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