The Preparation And Characterization Of CO₃^2--Containing BCP Bioceramics From Salmo Salar Bone

Object:At present,biphasic calcium phosphate?BCP??HA/?-TCP?ceramic is an ideal material for repairing bone defects.Most BCPs are artificially synthesized,the preparation method is complicated,the technology is fine,and the production cost is high.In recent years,due to the availability of raw materials and simple processes,research has focused on natural porous BCP bioceramics derived from Salmo salar bone.In this experiment,Salmo salar bone was used as a raw material,processed in vaious gas by a high-temperature calcination method.The objective is to prepare CO₃^2--containing BCP microporous ceramic and screen a convience and feasible method.The mechanism of CO₃^2--containing BCP porous bioceramics produced by calcining Salmo salar bone in vaious gas was in-depth discussed.BCP bioceramics have been widely used in clinical repair of bone defects,and they have good osteoconductive properties,but osteoinductive properties are still controversial in the literature.In order to explore the effect of microstructure of BCP bioceramics on osteoinductive properties,we compared the differences in the chemical composition and physical structure of two BCP bioceramics with and without CO₃^2-from Salmo salar bone by a series of characterizations.In vitro cell experiments were performed to compare the osteoinductive properties of the two groups of materials.The effects of microscopic changes in chemical composition and physical structure on osteoinductive performance were also analyzed.The purpose of this study is to modify BCP porous bioceramics from Salmo salar bone to make close to the physical and chemical properties of human bone tissue,to have better biocompatibility,and better osteoinductive properties.The clinical application of BCP porous bioceramics from Salmo salar bone provides the basis for previous experiments.Method:In this experiment,the raw materials are calcined in groups,and the physical structure and chemical composition of the calcined samples are characterized.First,Salmon Salar bone was divided into five groups A,B,C,D,and E,and calcined using a quartz tube furnace.Groups A,B,and C are calcined in CO₂ gas?1L/min?for 1 hour at temperatures of 900°C,1000°C,and 1100°C.Group D is calcined in air at 600°C for 1 hour,then,it was calcined in CO₂ gas at 900°C for 1 hour;group E was not calcined as a control group.Then,the above five groups of calcined samples are characterized by X-ray diffraction?XRD?and Fourier infrared spectroscopy?FTIR?.XRD is used to detect the crystallinity and phase composition of materials,and FTIR analyzes the main characteristic functional groups of materials.This experiment is intended to calcinate Salmo salar bones in groups according to the method determined by previous experiments,and to test the physical structure,chemical composition and biological properties of the obtained samples.First,the Salmo salar bones were divided into Groups A and B.Group A was first calcined in air at A high temperature of 600°C for 1 hour,and then in CO₂ gas at A high temperature of 900°C for 1 hour.Group B was first calcined in air at a high temperature of 600°C for 1 hour,and then in air at a high temperature of 900°C for 1 hour.Then,the composition and physical structure are characterized.XRD is used to detect the powder crystallinity and phase composition of the two groups of samples,and the cell parameters of the two groups of materials are obtained through Rietveld refinement.The calculated density is to reflect the compactness of the atomic arrangement and the regularity of the structure of the cells;The main feature functional groups of the samples were identified by FTIR analysis,the CO₃^2-content can be estimated by comparing the extinction coefficient?E?of CO₃^2-and PO₄^3-on the FTIR and by using the carbon analyzer?CHN?;the microstructure and crystal size of the two groups of materials are observed by Electron scanning electron microscopy?SEM?.In addition,the icp-oes is used to determine the Ca/P ratio and other trace elements in the two groups of materials.Finally,the biological properties were tested by in vitro cell experiments.Extracts of the two materials were first prepared according to the international standards for the evaluation of medical devices?ISO standard,10993-12?and then co-cultured with human bone marrow mesenchymal stem cells?h BMSCs?.The cytotoxicity of the samples and their effect on cell proliferation are detected by cck-8method.The early osteogenic transcription factor Runx2 and the specific protein Smad4 in the osteogenic differentiation pathway are detected by Western blot.In addition,alizarin red staining is used to evaluate the effect of two groups of samples on osteogenic differentiation activity of h BMSCs.Result:All four groups of fish bones were changed after high temperature treatment.Visual observation showed that the black color of Groups A and B and the darker color of Group A,indicated that the organic matter in Groups A and B had not been completely removed,so thet were not tested.The Groups C and D were chalky.The characterization results of the Groups C,D,and E?uncalcined?were found as follows:The XRD patterns showed that the Groups C and D contained characteristic peaks of HA and?-TCP,indicated that BCP had been generated,but only the characteristic peaks of HA appeared in Group E.Compared with the standard card,the HA peak on the Group D spectrum is shifted,and is shorter and wider.In addition to the HA and?-TCP diffraction peaks,there are other diffraction peaks in the Group C spectrum,indicated Impurities were generated.In the FTIR spectrum,the absorption peaks of CO₃^2-could be observed in Groups C,D,and E,but there are absorption peaks of organic impurities in Groups C and E,which was consistent with the XRD spectrum results.The results showed that the chemical composition,physical structure and biological properties of CO₃^2--containing BCP bioceramics changed.In the FTIR spectrum,the absorption peak of CO₃^2-in Group A was obvious.According to the FTIR spectrum,the content of CO₃^2-in Group A was estimated to be3.74wt%,while there was no absorption peak of CO₃^2-in Group B.The content of CO₃^2-measured by CHN analyzer was 3.2 wt%in Group A and 0.068wt%in Group B,which was close to the ideal range of human bone tissue content.In the XRD pattern,compared with Group B,the diffraction peaks of Group A HA are short,wide and offset,indicated the presence of dopants.Phase quantitative analysis showed that the relative content of?-TCP in group A was 45.3wt%,while that in Group B was 38.2wt%.Rietveld finishing results showed that the a-axis and b-axis cell parameter in Group B was 9.4845nm,while that in Group A was 9.2942nm,indicated that the cell structure of Group A was incomplete.The calculated density of Group B is 3.114 g/cm³,while that of Group A is 2.882 g/cm³,indicated that the atoms in Group B are more closely arranged and more regular structure.At high power SEM,the grain size of Group A was significantly smaller than that of Group B.The total porosity of natural micropores measured by mercury injection instrument was 64.06%in group A and 49.6%in Group B.The specific surface area was17.161m²/g in Group A and 5.829 m²/g in Group B.In vitro cell experiments showed that the two groups of materials had no cytotoxicity and promoted proliferation of h BMSCs.WB results indicated that compared with Group B,the expression of Runx2 was increased and the expression of Smad4 was also enhanced in the cells cultured in Group A.Alizarin red staining showed that Group A contained more clusters of red calcification nodules.Conclusion:1.By improving the preparation method,Salmo Salar bone could be used to CO₃^2--containing BCP bioceramics and remove the organic matter.2.The method is to calcine Salmon Salar bone in 600?air and then 900?CO₂ gas for one hour.3.In CO₃^2--containing BCP bioceramics,the relative content of?-TCP increases,the grain size decreases,the crystallinity decreases,the total porosity and the specific surface area increase;4.CO₃^2--containing BCP bioceramics had better biocompatibility and better bone osteoinductive performance.