The Research On Preparation And Properties Of Fishbone-derived BCP Porous Ceramic Bone Scaffolds

ObjectIn this study,a variety of fish bones were used as raw materials,and high-temperature calcinations were used to screen out fish that could produce biphasic calcium phosphate?BCP?.The effects of different calcining temperatures on the composition and structure of BCP derived from fish bones were studied,and the reason for the formation of BCP after calcination was in-depth discussed as well.Then a series of convenient and feasible methods were used to prepare a series of BCP porous ceramic bone scaffolds derived from fish bones,and their composition,structure,cytotoxicity,the biocompatibility as well as osteoinductive properties in animals were studied.Through the research of this topic,it lays a foundation for the future development and clinical use of new bone defect repair materials.MethodThe first part of the experiment was to screen and prepare fishbone-derived BCP by high temperature calcination.The fish bones of six kinds of fishes including Salmo salar,Anoplopoma fimbria,Sardine,Channa argus,Carassius auratus and Pangasisus haniltoa were used as raw materials.High-temperature calcining combined with XRD analysis was used to screen out fish that could produce biphasic calcium phosphate after calcination.By changing the calcining temperature,X-ray diffraction?XRD?,Fourier transform infrared spectroscopy?FTIR?and scanning electron microscopy?SEM?were used to find out the influence of composition,crystallite size,and microstructure of BCP derived from fish bones at different calcining temperatures,thus to determine the optimal calcination temperature for further preparation of fishbone-derived BCP porous ceramic scaffold material.Thermogravimetric analyzer?TGA?was used to study the thermal stability and compositional changes during the calcination of fish bones.The inductively coupled plasma emission spectrometer?ICP-OES?and X-ray fluorescence spectrometer?XRF?were used to determine the Ca/P ratio and trace elements of fishbone-derived BCP.The second part of the experiment is to prepare and study the properties of the BCP porous ceramic scaffold materials.Fishbone-derived BCP porous ceramic bone scaffolds were prepared through a series of processes such as calcination,screening,cleaning,drying,and disinfection.The three-dimensional pore structure,microscopic surface structure and specific surface areas of the scaffold materials were determined by SEM,mercury porosimeter,and BET method.Human bone marrow mesenchymal stem cells?hBMSCs?were extracted and co-cultured with the material extracts for 1,3,and 5 days according to the national standard GBT₁6886.5-2003.CCK-8 Assay and Live/dead staining was used to quantitatively and qualitatively evaluate the influence of materials on cell proliferation ability.The intramuscular ectopic osteogenesis of fishbone-derived BCP porous ceramic scaffolds was also studied.An intramuscular ectopic osteogenic model of mice was constructed by surgery on the hip muscle of the mice and the different materials were implanted into the muscle.The animals were sacrificed at 8 weeks after surgery.The materials were taken out together with the surrounding tissue to make paraffin sections.The biocompatibility and osteoinductive properties of the scaffold materials were further clarified by HE and Masson staining.ResultsIn the first part of the experiment,six kinds of fish bones were sintered at 1000°C,including Salmo salar,Anoplopoma fimbria,Sardine,Channa argus,Carassius auratus and Pangasisus haniltoa.The inorganic phase of the calcined fish bones was analyzed by XRD.It was found that fish bones of Salmo salar,Anoplopoma fimbria,Sardine could generate biphasic calcium phosphate after calcination while the remaining three kinds of fish bones could only generate HA.The effects of temperature on the BCP were further investigated.It was found that at a temperature of 700°C or above,the three kinds of fish bones of Salmo salar,Anoplopoma fimbria,Sardine calcined could transformed into biphasic phosphates.The?-TCP content in BCP varied from 12.1%to 49.0%depending on the fish and calcination temperature,and the Ca/P ratio was between 1.5 and 1.67.Carbonated hydroxyapatite and various trace elements also existed.As the calcination temperature increased,most of the organic components were removed,and the HA/?-TCP content ratio in the BCP followed the temperature change.When the calcination temperature exceeded900°C,the carbonated hydroxyapatite disappeared.The increase of the calcination temperature also continuously caused the change of grain size and inter-particle mesoporous structure of BCP.Therefore,800-900°C is the optimum temperature for preparation of fishbone-derived BCP ceramic scaffold materials.At this temperature,most of the organics in the fish bone are removed,and the BCP product contains more?-TCP components.The carbonated hydroxyapatite is retained,the formed BCP crystals are uniform in size,the crystallinity is high,and there is an even distribution of natural nanoscale mesoporous structure.In the second part of the experiment,through a series of simple and feasible preparation processes such as high-temperature calcination,sieving,washing and drying,and sterilization,we converted the fish bones of salmo salar,anoplopoma fimbria,and sardine into biphasic calcium phosphate porous ceramic bone scaffolds?Salmo salar calcined at 900°C and named as Sa 900,Anoplopoma fimbria calcined at 800°C and named as An 800,Sardine calcined at 900°C and named as Sd 900?.Through the comparative study of the composition and structure of the three scaffold materials,it was found that the three materials are different,among which Sa 900 has the most?-TCP component,reaching 49.0%,An 800 has more open macropores and the highest total porosity,reaches 74.8%and the Sd 900 has the highest specific surface area,reaching23.527m²g^-1.The CCK-8 assay and Live/dead cell staining confirmed that the three materials had not only no obvious cytotoxicity to cells,among which Sa 900 also had some pro-proliferative effects on human bone marrow mesenchymal stem cells.In the HE and Masson staining of mice intramuscular ectopic osteogenesis model,no signs of inflammatory response directly related to the implants was observed which again demonstrated the excellent biocompatibility of materials.At the periphery of particles of materials,some compacted cuboidal osteoblast-like cells could be seen neatly arranged along the interface between the material and fibrous tissue,where thin bands of osteoid could be found as well.Although no new bone tissue was formed,the results of this experiment can also indicate that the fishbone-derived BCP porous ceramic bone scaffolds have some osteoinductivity.ConclusionThrough high-temperature calcination and XRD analysis,it was found that most of the organic components in fish bones were removed during the calcination at 800-900°C.Fish bones of Salmo salar,Anoplopoma fimbria,and Sardine were transformed into biphasic calcium phosphate which contained relatively more?-TCP components,carbonated hydroxyapatite and various trace elements.The grain size of BCP were uniform with high crystallinity and there was a uniform distribution of natural nanoscale mesoporous structure.Using these three kinds of fish bones as raw materials,biphasic calcium phosphate porous bone scaffolds were prepared through a series of convenience and feasible processes such as high-temperature calcination,screening,washing and drying,and sterilization.The porous ceramic scaffolds retains the natural three-dimensional pore structure of the fish bone,and has a high porosity and specific surface area.The cytotoxicity experiment confirmed that these materials have good biocompatibility.Intramuscular ectopic osteogenesis experiments in mice found that these materials have some osteoinductivity,and have the possibility of becoming a new type of clinical medical bone scaffolds.