| Background:According to statistics,millions of patients with bone defects due to various reasons need bone transplantation surgery every year in the world,and the frequency of application ranks second only to blood transfusion among all the transplantation operations.In clinical practice,the requirements of bone defect grafts should not only achieve the bone fusion effect but also have reliable safety,be easy to use adaptable to various clinical needs.From the initial use of autologous bone grafts to the current use of allogeneic bone grafts,there seem to be unavoidable problems of their own.In synthetic bone grafts,no matter ceramic,metal,bone cement,or polymer materials,the single function is not enough to meet the comprehensive needs of defect repair.The original intention of our research is to combine the functions of synthetic materials.Bone tissue engineering can accomplish the goal of bone defect repair simply and effectively.From the perspective of the three factors of tissue engineering,growth factors are costly and may cause unpredictable complications and reject reactions in the process of use;seed cells are complicated to extract and laborintensive to store and transport.Meanwhile,the possibility of disease transmission can not be ruled out;scaffold materials are the basis for the use of the former two.Hence,the use performance and biological performance of the materials should be the focus of our research.Methods:In this study,we attempted to select a more suitable CaPcs /PLGA composite for bone defect repair by regulating the phase composition of calcium phosphate ceramics with chemical synthesis,and the solvent-mixing method was applied to prepare CaPcs/PLGA composites with different phase compositions of calcium phosphate by phase separation in NMP solvent.Firstly,the phase composition of single and double phase CaPcs was analyzed by XRD,and the microscopic morphology characteristics of the particles were observed in SEM.Then the microstructure and pore structure of the composite scaffolds were observed by Micro-CT,and the porosity of the scaffolds was calculated.We also compared the hydrophilicity of different composites;secondly,we also studied the degradation performance of the composites.The changes of macrostructure,microstructure,p H of the solution,and weight loss rate were compared at different time points;thirdly,we compared the biomineralization properties of different composite scaffolds,including the mass change,hydrophilic change,microscopic morphology,and mineral deposition effect in each group;fourthly,we investigated the differences in cell adhesion,proliferation,and differentiation by cultured mouse embryonic osteogenic precursors on different composite surfaces;finally,CaPcs /PLGA composite scaffolds with different phase compositions were implanted into the tibia defect of rats.Morphological and histological changes of bone repair were observed and analyzed at different time points.In this study,a complete experimental evaluation of CaPcs/PLGA composites was conducted,which proved that the biological properties of the composites could be affected by the different phase composition of calcium phosphate.Results:1.Preparation of calcium phosphate ceramics(CaPcs)with different phase compositions and construction of CaPcs /PLGA composite scaffolds.On the premise of calculating Ca/P,the calcium phosphate ceramic precursors were prepared by adjusting the p H of the reaction.The calcinating temperature was adjusted several times at different temperatures.Finally,the calcium phosphate ceramic particles with accurate phase composition and uniform morphology were successfully prepared at 850℃.The CaPcs/PLGA composite scaffolds with different phase compositions were prepared by the phase separation method.The internal pore structure of the scaffold presented a typically finger-like connected pore channel with an inner diameter of more than 100μm.At high magnification,there were also abundant micropores in the scaffolds.From micro-CT images,we could find that CaPcs particles were evenly dispersed inside the scaffold without an obvious agglomeration phenomenon.The pore porosity of all scaffolds was more than 50%.By measuring the contact angle of the materials,we found that the composite material effectively improved the hydrophilicity of the polymer,which was conducive to cell adhesion and proliferation on the material surface.2.Degradation characteristics of CaPcs/PLGA composite scaffolds with different phase compositions of calcium phosphate.The size of scaffolds decreased with the progress of degradation,and the morphology of the scaffold gradually became irregular and finally disintegrated.Correspondingly,the mass of scaffolds decreased due to degradation,and the mass loss rate of composite scaffolds with different compositions was different.In general,the degradation rate of composite scaffolds was slower than that of polymer scaffolds.It could be seen from the p H change of solution that the composite scaffold had a small range of p H change in the process of degradation.The higher the proportion of the β-TCP phase,the smaller the overall change of p H value in the solution.This performance indicated that the synchronous degradation of calcium phosphate ceramics could play a role of “self-buffering” in the degradation of materials.In terms of the scaffold microstructure,the pore size inside the scaffold gradually increased,which was conducive to the repair of surrounding cells and tissues.The maintenance of pore-wall structure in composite materials also provided a frame structure for tissue growth,which enabled osteogenesis to take place on the inner surface of the materials and played a role in bone conduction.3.Study on biomineralization properties of CaPcs/PLGA composite scaffolds with different phase compositions of calcium phosphate.The overall mass of the materials did not increase continuously due to mineralization throughout the process.However,the total mass of the materials increased and decreased with the degradation process,and the mass change was synergistic by the degradation and mineralization process.From the contact angle test,it could be seen that the hydrophilicity of the materials after mineralization was significantly improved compared with the initial state(p < 0.05).Even pure PLGA materials also improved the original hydrophobic characteristics with the help of calcium phosphate deposition.The hydrophilicity of the composite materials was significantly better than that of PLGA(p < 0.05),indicating that calcium phosphate ceramics could help the composites to form a bone-like apatite layer in SBF.We observed the morphology of mineral deposition on the surface of materials by SEM.Furthermore,the analysis results of the elemental composition of the sediments by EDS confirmed the existence of calcium and phosphate,and the calculation results of Ca/P in the sediments suggested that the sediments of composite materials should be bone-like apatite and its derivatives.4.Cell behaviour evaluation of CaPcs/PLGA composites with different phase compositions of calcium phosphate in vitro.The result of the cell adhesion experiment showed that the composite containing calcium phosphate ceramics could promote the adhesion of cells,which indicated the good biocompatibility of the composites.In terms of the number of cell proliferation,the effect of composite material was significantly better than that of PLGA alone.Moreover,the cell proliferation rate of the composites containing β-TCP phase was significantly better than that of HA/PLGA,indicating that the ions released by calcium phosphate contributed to the proliferation of osteoblasts.The intracellular calcium ion concentration of the cells on the surface of BCP2/PLGA,BCP3/PLGA,and β-TCP/PLGA materials was significantly higher than that of the control group.It was suggested that the degradation of the β-TCP phase released extracellular calcium ions,which could increase intracellular calcium ion deposition through calcium influx.In the experiments of ALP activity and calcium deposition,the cells on the surface of BCP3/PLGA had the highest ALP activity and the amount of calcium deposition.The ability to induce osteoblast differentiation in all composite materials was significantly better than that of the control group(p < 0.05).We also found that the composite containing calcium phosphate ceramics could significantly increase the expression of osteogenic genes through real-time quantitative PCR analysis.By further analyzing the expression differences of the marker genes at each time point,we speculated that the composite material rich in the β-TCP phase could play a better role in promoting the differentiation process of osteoblasts.5.Repair of bone defects in vivo by CaPcs/PLGA composite scaffolds with different phase compositions of calcium phosphate.The morphological changes of tibia defects were characterized and analyzed by micro-CT.The composite materials were better than the control group in BV/TV,BS/TV,and the number of trabeculae,and BCP3/PLGA material had the best effect on bone repair.Histological staining showed that the new bone tissue in the control group only existed on the surface of the material,with only a small amount of fibrous tissue inside.In the experimental groups,there were different degrees of new bone growth inside the materials,as well as granulation tissue and fibrous callus.In the BCP3/PLGA group,not only the compact bone around the scaffolds could be seen,but also the woven bone could be found inside the material.The results of in vitro experiments could be verified by immunofluorescence.Among all the materials,the defect of BCP3/PLGA material had the highest fluorescence intensity and the widest coverage area,which suggested that it could promote the secretion of OCN and accelerate the formation of new bone.In summary,the repair effect of the BCP3/PLGA group was the best in all groups.Conclusion:In this study,CaPcs with different phase compositions were prepared with chemical synthesis,and CaPcs/PLGA composites were synthesized by phase separation.We evaluated the degradation performance,biomineralization properties,cell function and bone defect repair of composite materials with different phase compositions.The results proved that the ability of composite materials to repair bone defects was determined by the combined effect of material degradation,biomineralization,ion release,and osseointegration,instead of a single property.In summary,the performance of BCP3/PLGA composite can better meet the needs of bone tissue engineering and promote the repair of bone defects. |
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