| Although hydroxyapatite(HA)has excellent biocompatibility and osteoconductivity,its degradation and osteogenesis are not sufficient.To overcome these insufficiencies,this study aims to prepare calcium phosphate materials that are closer to the structure,composition and property of biological apatite from the perspective of bionics,which is also based on the "material genome" project.In this paper,a series of calcium phosphates were obtained under different preparation methods and conditions,such as poorly crystalline HA with gradient change of calcium phosphorus ratio(Ca/P ratios)and corresponding highly crystalline calcium phosphate with different phase ratios after calcinating,HA with different crystallinity and aspect ratios were obtained.The effects of these factors on the physical and chemical properties and biological properties of calcium phosphate materials were studied.After screening the experiment groups with better performance,the material structures and properties are correlated to further optimize the preparation schemes to enrich the material genetic bank.Firstly,poorly crystalline HA with a gradient of Ca/P ratio(1.5,1.55,1.6,1.67)was prepared by chemical precipitation method,the highly crystalline calcium phosphates with various phase ratio of HA/β-TCP(0/10,3/7,6/4 and 10/0)were obtained by calcining the former,and whose The phase composition and morphology were characterized by XRD,FTIR,SEM and so on.Then the physicochemical and cytological properties were studied and the results showed as follows.Reducing Ca/P ratio could effectively improve the protein adsorption performance of poorly crystalline HA,and the poorly crystalline calcium deficient HA with the Ca/P ratio of 1.5 demonstrated the best protein adsorption performance;the protein adsorption properties of highly crystalline calcium phosphate after calcinating decreased with the β-TCP content increased and that of pure β-TCP was the worst;the protein adsorption property of poorly crystalline HA was obviously superior to that of the highly crystalline calcium phosphate.The P release levels of poorly crystalline HA and highly crystalline calcium phosphate gradually increased with the decrease of Ca/P ratio,while the release of Ca varied greatly.Calcium deficiency promoted the adsorption of Ca ions by poorly crystalline HA,leading to a decrease of Ca ion concentration in the corresponding solution;the Ca ion released from highly crystalline calcium phosphate increased as the content of β-TCP increasing.Poorly crystalline HA had almost no mineralization,while compared with other groups,high crystallinity biphasic calcium phosphate(3/7BCP)with 3HA/7β-TCP had obvious mineralization,which may be related to its special micro-nano structure.Poorly crystalline CDHA with Ca/P ratio of 1.55 had the best cell proliferation activity,and its alkaline phosphatase(ALP)activity and the expression of osteogenic-related genes were also the highest;the cytological properties of the corresponding 3/7BCP group was also the best in all highly crystalline calcium phosphate after calcinating,and its proliferation activity and the expression of osteogenic-related genes are the highest;comparing the cytological properties of poorly crystalline HA and calcined highly crystalline calcium phosphate,it was found that there was little difference in cell proliferation activity between them,but poorly crystalline HA could better improve the ALP activity of mesenchymal stem cells(BMSCs)and promote the expression of osteogenic-related genes.Then,the in vitro aging experiment of poorly crystalline CDHA with Ca/P ratio of 1.5 was carried out,it was found that with the extension of aging time,the crystallinity and Ca/P ratio of poorly crystalline CDHA increased,the grain became larger,and transformed from spherical to long rod.With the aging time prolonging,poorly crystalline CDHA enhanced the expression of osteogenesis-related genes greatly.Among them,poorly crystalline CDHA aging for 14 days had the most significant upregulation effect on osteogenic genes of BMSCsTwo groups of poorly crystalline HA(1.55 HA and 1.67HA)and two groups of highly crystalline calcium phosphate(β-TCP and 3/7BCP)after calcination were selected according to the previous in vitro osteogenesis experiment and Polylactic acid(PLA)was used as the binder.Porous scaffolds were prepared by 3D printing and implanted into the femoral defect of rabbits to investigate the in vivo osteogenic properties of these scaffolds.The experimental results show that the poorly crystalline HA composite scaffold had better osteogenic effect than the high crystalline calcium phosphate composite scaffold.A large amount of new bone formed at 6 weeks,and the materials degraded significantly at 12 weeks,while the high crystalline calcium phosphate composite scaffold had a little or no osteogenesis at 6 weeks;the osteogenic properties of poorly crystalline HA composite scaffold with Ca/P of 1.55 and1.67 were very excellent,but there was not significant difference;The osteogenesis properties of 3/7BCP composite scaffold was better than β-TCP at 12 weeks,and the latter was wrapped by a large amount of fibrous tissue,resulting in the decrease of osteogenesis.The high throughput method and hydrothermal method were combined to prepare 20 groups of HA in a batch at one time by changing the hydrothermal temperature and the initial Ca/P of raw materials,then the obtained products were characterized.The result showed that HA with high purity could be synthesized by hydrothermal method,with the increase of hydrothermal temperature,the crystallinity of product HA increased,but the Ca/P ratio would also decrease,and the aspect ratio of rod-like HA increased and transformed into hexagonal plate at 170 ℃;with the increase of the initial Ca/P ratio in the synthesis process,the crystallinity of HA increased slightly,and the morphology began to change from long rod-like to short rod-like and spherical-like.After phase detection and morphology observation,three groups of HA with different aspect ratios and five groups of HA with different crystallinity at different hydrothermal temperatures were selected for further study.With the increase of the aspect ratio of HA grains,the protein adsorption property was improved,but the released Ca ion concentration was low,which affects the osteogenic differentiation of cells.Therefore,the spherical-like HA grains significantly up regulate the expression of osteogenic-related genes of m BMSCs;with the increase of crystallinity,the protein adsorption and ion release properties of HA decreased,therefore,the HA hydrothermally prepared at 90 ℃ had better osteogenic activity.Poorly crystalline HA with a gradient of Ca/P ratio(1.5,1.55,1.6 and 1.67)was prepared by chemical precipitation method,hydrothermal method and heat treatment after preparation by chemical precipitation method,respectively.The effects of preparation method,process and Ca/P ratio on the physicochemical and cytological properties of HA were compared.The results show that the crystallinity of HA prepared by hydrothermal method was higher than that prepared by chemical precipitation method,and the morphology was mainly long rod,which gradually shortened with the increase of Ca/P ratio;the crystallinity of HA after heat treatment prepared by chemical precipitation method increased,and which was more obvious with the increase of Ca/P ratio,and the grain size of HA also increases,but the increase range decreased with the increase of Ca/P ratio.Compared with HA prepared by chemical precipitation,the protein adsorption performance of HA prepared by hydrothermal method increased,and the performance can be further improved by reducing the Ca/P ratio of HA;the protein adsorption performance of HA after heat treatment prepared by chemical precipitation and decreased,but this performance could be improved with the increase of Ca/P ratio of HA.Compared with HA prepared by chemical precipitation,the concentration of Ca ion released by HA prepared by hydrothermal method decreased,but with the increase of Ca/P ratio,the concentration of Ca ion released increased,while the concentration of P released decreased;The ion release concentration of HA after heat treatment prepared by chemical precipitation increased significantly,and decreased with the increase of Ca/P ratio.The cells proliferation activity of HA with Ca/P ratio of 1.5 prepared by chemical precipitation method and hydrothermal method was lower than that of other groups;There was no significant difference in the cell proliferation activity of HA before and after heat treatment prepared by chemical precipitation,but when the Ca/P ratio was 1.5,its cell proliferation activity was optimum;poorly crystalline CDHA with the Ca/P ratio of 1.55 prepared by chemical precipitation,poorly crystalline CDHA with the Ca/P ratio of 1.6 prepared by hydrothermal method and poorly crystalline CDHA after heat treatment prepared by chemical precipitation with Ca/P ratio of 1.55 had optimum osteogenic activity.Poorly crystalline CDHA with the Ca/P ratio of 1.55 was sintered at low temperature after isostatic pressing to obtain poorly crystalline CDHA ceramics.Compared with high crystallinity submicron HA ceramics sintered at conventional temperature,the results showed that: the mechanical strength of poorly crystalline nano CDHA ceramics was obviously lower than that of high crystallinity HA ceramics,which were 38.8 MPa and 69.5 MPa,respectively.However,the former had better protein adsorption capacity,degradation rate,and could stimulate and promote the expression of multiple osteogenesis-related genes of m BMSCs. |
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