| Object:At present,HA/β-TCP biphasic calcium phosphate ceramic（BCP）has a wide range of application prospects in bone reconstruction surgery due to its advantages of high degradability,high bioabsorption rate and good biological properties.The sources of BCP materials are mostly artificial synthesis,which consumes a lot of chemical reagents and requires tedious processes.Existing studies have shown that BCP materials can be obtained from marine fish by simple calcination.In this study,BCP was extracted from butterfish bone by calcination,and its composition and microstructural characteristics were characterized,and the biocompatibility,osteoinductivity and osteoconduction of butterfish bone-derived BCP materials were explored through in vitro and in vivo experiments.The aim of this study was to explore the potential of butterfish bone-derived BCP materials as bone scaffolds.Methods:This study is mainly divided into two parts:Part one:Preparation and characterization of biphasic calcium phosphate ceramics derived from butterfish bone.In this experiment,the raw materials of butterfish bone were divided into five groups,placed in a quartz tube furnace under normal air conditions,and heated to 700°C,800°C,900°C,1000°C and 1100°C at a heating rate of 5°C min-1,respectively.The holding time was 1h,and finally cooled to room temperature naturally.Then,the composition of the calcined material was characterized by X-ray diffraction（XRD）,Fourier transform infrared spectroscopy（FTIR）,and inductively coupled plasma optical emission spectrometer（ICP-OES）.Scanning electron microscopy（SEM）and mercury intrusion porosimeter（MIP）were used to characterize the microstructure of the calcined materials.Part two:Osteogenic properties of biphasic calcium phosphate ceramics derived from butterfish bone.After preliminary characterization,we screened out four groups of calcined butterfish bones at 700°C,800°C,900°C,and 1000°C as the experimental group,and commercial Bio-oss bone meal as the control group,respectively loaded with human bone marrow mesenchymal stem cells（h BMSCs）.Subsequently,cell adhesion and survival were observed by scanning electron microscope（SEM）,and Western blot analysis was used to detect the expressions of osteogenic proteins ALP and RUNX2 in vitro.A rat femoral defect model was established and implanted with human bone marrow mesenchymal stem cells（h BMSCs）loaded with butterfish bone and Bio-oss bone powder calcined at 700°C and 900°C.Finally,micro-computed tomography（Micro-CT）and hematoxylin-eosin（HE）staining were used to detect bone regeneration and material degradation rate in a rat femoral defect model.Results:Part one:Preparation and characterization of biphasic calcium phosphate ceramics derived from butterfish bone.The main component produced by calcination of butterfish bone below 800℃is hydroxyapatite（HA）.When calcined above 800℃,BCP containing HA andβ-tricalcium phosphate（β-TCP）can be produced.In the range of 800℃to 900℃,the relative content ofβ-TCP increases with the increase of temperature.When the calcination temperature is in the range of 900℃to 1100℃,the relative content ofβ-TCP decreases with the increase of temperature.The relative content of TCP reached the highest at 900℃.The calcined butterfish bones contain a variety of trace metal elements.The BCP material produced by calcination of butterfish bone at above 800℃has concentrated distribution of macropores and micropores,high porosity,nanometer grain size,and the grain size and crystallinity increase with the increase of temperature.Part two:Osteogenic properties of biphasic calcium phosphate ceramics derived from butterfish bone.The human bone marrow mesenchymal stem cells（h BMSCs）loaded in each group of materials grew well and adhered closely to the materials.In vitro,the expression levels of bone formation-related markers Alp and Runx2 in butterfish bones after calcination at900℃were generally higher than those in other groups.In vivo,the osteogenic ability of butterfish bones calcined at 900°C was almost comparable to that of Bio-Oss,the most commonly used clinically,and significantly better than the other groups.Conclusions:Part one:Preparation and characterization of biphasic calcium phosphate ceramics derived from butterfish bone.1.Butterfish bone can produce BCP material with highβ-TCP content and good pore structure at lower calcination temperature.2.In all calcination systems,the optimal temperature is around 900°C,because the BCP organic matter produced at this calcination temperature is basically removed,partially carbonated hydroxyapatite（CHA）is retained,and the content ofβ-TCP is high,with natural uniformity.The pore structure and grains are mostly in the nanometer scale.Part two:Osteogenic properties of biphasic calcium phosphate ceramics derived from butterfish bone.1.The BCP bioceramic material extracted from butterfish bone has good biocompatibility and osteogenic ability in vitro and in vivo.2.The biological material has a wide range of sources and a simple production method.Compositionally,the material consists of two phases.Structurally,the material has nanostructure and high porosity.In addition,it has certain osteoconductive and osteoinductive properties.Therefore,it has a good development prospect as a new type of bone replacement graft. |
