| The repair of bone defect(BD)caused by trauma,congenital bone dysplasia,tumor,osteoporosis,etc.,is a major challenge for orthopaedic surgeons to solve urgently.Especially in recent years,as the coming of the aging society,bone defect caused by tumor,senile diseases such as osteoporosis fracture has been also increasing year by year.BD brings great pains and economic pressures for the patients’ families and the society.Using bone graft for the reconstruction is the effective way to the treatment of Bone defects at present.But it is quite difficult for bone graft repair materials to reach the level of bone microstructure.In the early stage,autologous bone was usually used as bone repair material.Although autologous bone has good biocompatibility and bone microstructure,and can better play the role of osteogenesis,bone induction and bone conduction,etc.,autologous bone has a limited amount,and problems such as pain and infection in the bone donor area that will bring great pain to patients.Allogeneic bone is a relatively good substitute for autologous bone.However,due to the problems of immune rejection,allogeneic bone will prevent the adhesion and growth of cells,and eventually lead to fracture nonunion and even infection.Metal has a good bone conduction effect,but its corrosion resistance and biocompatibility are poor,the ability of bone induction and bone formation is insufficient,and the elastic modulus does not match with natural bone.Therefore,when using metal material as bone graft,the situations of loose,fall off and rupture,or even collapse may be often found.Based on the above shortcomings,traditional bone repair materials gradually fade out of people’s attention.At present,tissue engineering has become a feasible choice in regenerative medicine.Artificial bone replacement material transplantation has become a medical focus to repair bone defects.However,the repair of bone defects and the regeneration of bone tissue are still a great challenge,because the bone tissue is hard and does not grow easily.Traditional artificial bone scaffolds promote bone healing mainly through the mechanism of osteogenesis,bone induction and bone conduction.In addition to the bone conduction,bone induction and bone regeneration,in the process of reconstruction,the establishment of new persistent functional blood vessels network in long bone grafts is the key to success which provides oxygen and nutrients for bone growth,differentiation and functional when bone defects,especially large bone defects.If vascularization is insufficient,local bone cells of transplant center will die from hypoxia and ischemia,leading to bone graft failure in the end.Therefore,it is urgent to solve the problem of how to rapidly form vascular network in bone graft materials.In tissue engineering,artificial bone scaffolds are used to provide necessary physiological and biological conditions for bone regeneration.These scaffold materials must be able to promote and support the infiltration of osteoblasts,vascular endothelial cells or other related biological factors,so as to accelerate the process of cell adhesion and proliferation,promote osteogenesis and vascularization,and ultimately promote the repair of bone defects.It have been confirmed that the surface morphology of materials can be changed to regulate cell behavior after protein modification,which can affect the microstructure and biological function of the new formed tissue.Polypeptides exert more precise control over their biological effects and are more likely to act on substrates than proteins.Specific peptides can promote the adhesion of endothelial progenitor cells on the surface of endothelial progenitor cells through the sequence of specific peptides of adhesion proteins or extracellular matrix molecules,which can reduce the rolling speed of endothelial cells in contact with them,support their dynamic adhesion,and trigger angiogenesis.SVVYGLR polypeptide,one of the important activity sequences of osteopontin,has the specific role of promoting vascularization,improving the adhesion and migration of endothelial cells,and inducing their differentiation.The activity of SVVYGLR polypeptide in promoting vascular regeneration can be comparable to or even stronger than vascular endothelial growth factor.Besides,SVVYGLR polypeptide has dual biological activities.In addition to promoting vascularization,it also inhibits the growth of osteoclasts,enhances the proliferation and differentiation of osteoblasts,and promotes the regeneration of bone tissue.Therefore,optimization of scaffold materials by peptide modification is a promising tissue engineering technology in the future,which may provide new methods and ideas for solving the problem of vascularization of bone transplantation.In this project,we combined OPN-derived SVVYGLR peptides(with dual role of promoting vascularization and promoting bone formation)with mesoporous calcium silicate powder material through covalent modification method,so as to activate the scaffold material.Using 3d printing technology,we built SVVYGLR-MCS compound artificial bone scaffold,and tested the physical and chemical performance of the composite bone scaffold.The biological effects on bone marrow mesenchymal stem cells and venous endothelial cells were examined,and the mechanism of promoting angiogenesis after modification of bone scaffolds by polypeptides was elucidated.Its toxicity and sensitization were tested in vivo,and its osteogenesis and vasculogenesis were verified.Through a lot of work above,a new type bone repair material of high biosafety can be developed to solve the problem of internal vascularization of bone graft material,and provide scientific basis for the further application of this kind of bone repair composite scaffold material in clinical practice.In this study,the following aspects were studied: 1.We prepared MCS,SVVYGLRMCS powders,artificial bone scaffolds,tested their physical and chemical properties to provide material basis for the next experiment.2.We observed the biological effects of scaffolds in the two groups on BMSCs and HUVECs,and studied the mechanism of SVVYGLR-MCS bone scaffolds promoting vascular formation,so as to provide cytological basis for further in vivo experiments.3.We observed the safety of SVVYGLR-MCS and MCS scaffolds in vivo and their respective ability to repair radial bone defects in rabbits,and compared the internal osteogenesis and blood vessel formation of the two groups.The results indicated that: 1.The MCS and SVVYGLR-MCS were successfully prepared,and they have highly ordered mesoporous channels.They are still highly ordered after amination,and the powder after grafting SVVYGLR polypeptide has not been greatly affected.The MCS has a 6 nm mesoporous channel,and the porosity of the MCS and SVVYGLR-MCS stents are(74.3±2.0)% and(65.6±2.0)%,respectively,and the compressive strengths of the MCS and SVVYGLR-MCS stents are(5.6±2.0)MPa and(5.3±1.0)MPa,respectively.The pH change of the simulated body fluid showed that the simulated body fluids of the MCS and SVVYGLR-MCS stents after 7 days were 7.65±0.01 and 7.61±0.01,respectively.After one month of soaking,the residual amounts of the MCS and SVVYGLR-MCS stents were respectively(94.36±0.62)% and(93.16±0.16)%.2.The effect of SVVYGLR-MCS on osteogenic differentiation of rat BMSCs was stronger than that of MCS.The difference of ALP staining,ARS staining,PCR gene expression detection and Western blotting protein expression was statistically significant(P<0.05).No significant difference was showed in HUVECs proliferation between the two groups(P>0.05).There were significant differences in the HUVECs adhesion and lumen formation between the two groups(P<0.05).The mechanism of SVVYGLR peptide promoting vascularization is to directly regulate the ERK pathway through PI3K/AKT as positive feedback signals.3.X-ray and general observation showed that the bone function of SVVYGLR-MCS was better than that of MCS.Bone density,trabecular thickness,bone volume fraction and trabecular separation degree of the SVVYGLR-MCS group were significantly better than those of the MCS group by micro-CT analysis of bone entry parameters,and the differences between the two groups were statistically significant(P<0.05).HE section staining and Masson staining showed infiltration of osteoblasts in the SVVYGLR-MCS group,and even showed a small amount of vascular structure.The neovascularization of the 12-week bone defect repair material was marked by CD34 and ZO-1 immunofluorescence staining,and the SVVYGLRMCS group was also more effective in promoting angiogenesis.Then,we can draw the conclusions:1.SVVYGLR-MCS artificial bone scaffold has good mechanical properties and is suitable for bone defect repair.2.SVVYGLR-MCS is beneficial to osteoblastic differentiation of BMSCs in rats,adhesion and lumen formation of HUVECs.The mechanism of SVVYGLR peptide promoting vascularization is to directly regulate the ERK pathway through PI3K/AKT as positive feedback signals.3.When SVVYGLR-MCS composite artificial bone scaffold is used to repair bone defects in vivo,its internal osteogenesis and angiogenesis are significantly superior to MCS. |
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