Preparation Of Fish Decalcified Bone Matrix And Evaluation Of Its Properties

Object:Bone tissue repair has always been a hot point in biomedical research.The search for artificial bone materials with good biocompatibility,compliant mechanical strength and strong osteoinductive ability has been a popular topic in this field,which is of great significance to promote the health development of our country and even the whole world.Compared with terrestrial animals,marine animals are rich in sources,easy to extract and have a higher safety level.At the same time,China is rich in marine resources and there are many fish processing enterprises of various types.If we can make full use of the characteristics of fish bones and process them into decalcified bone matrix with a certain spatial structure,we can not only obtain an ideal raw material for medical bone repair materials,but also significantly improve the added value of fish bones and realise the conversion of old and new dynamic energy of fish processing enterprises.Methods:In this study,a Fish Decalcified Bone Matrix（FDBM）was prepared using HCl decalcification and fresh flounder fish bone as the base material,through the treatment of impurity removal,decellularization,defatting and decalcification,which can be used for the repair of various non-load-bearing bone defects and the repair of load-bearing bone defects.The performance of the product was optimized by using the decalci-fication rate as a variable.The decalcification rate was controlled by real-time detection of Ca²⁺in the decal-cification solution by atomic absorption spectrophotometer,and the cell adhesion growth,mechanical strength,porosity,water absorption and in vitro degradation rate of FDBM were evaluated at different decal-cification rates.The optimal decalcification rate was selected and the FDBM was tested for biocompatibility and safety with reference to national testing standards for implantable medical devices.A rat model with a critical size femoral defect was established and a commercially available Bovine Decalcified Bone Matrix（BDBM）was used as a control to observe the changes in the implant material and the repair of the defect area from the imaging and histological aspects,and to study its bone repair ability and degradation properties.Results:In this study,a method for the preparation of FDBM was designed and developed.By comparing all the data,the FDBM has optimal performance when the decalcification rate is 80%.The cell adhesion rate was 85.00%and the porosity and water absorption rates were 75.73%and 330.16%respectively.The in vitro degradation time is approximately 8-10 weeks.The results of the cytological and ex vivo safety tests showed a FDBM cytotoxicity grade of Class I,representing no cytotoxicity,as well as no haemolysis（haemolysis rate=1.55%）and no pyrogenic reaction.No immune rejection or allergic reactions such as redness,swelling or septicemia were observed in rats implanted in vivo.In the bone defect repair test,combined with CT imaging and histological staining,the FDBM group showed that the surface of the defect area was largely intact at 12 weeks postoperatively,with no bone crust structures present,and mature new bone tissue had formed at the edges and inside the defect area.Semi-quantitative analysis showed that the FDBM group had a score of 2.35,which was higher than the BDBM group（2.07）and much higher than the self-healing group（0.91）.Conclusion:At the optimum decalcification rate,FDBM has an ideal structure,good mechanical strength,high porosity and water absorption,and a degradation rate that matches the human osteogenesis rate,meeting the basic requirements for an artificial bone material.The safety of FDBM was then further investigated.FDBM did not cause any adverse reactions in cellular and in vivo tests,and met the relevant standards for implantable medical devices,with good biocompatibility.In conclusion,FDBM is an artificial bone material with good physicochemical properties,biocompatibility and bone repair ability.