Fabrication And Performance Evaluation Of Large Salmon Fish Bone Demineralized Bone Matrix

Objective:The optimal preparation conditions of large salmon fish bone demineralized bone matrix(L-DBM)was explored,and the performance of L-DBM bone particles and bone powder were studied separately.The treatment effect of L-DBM bone powder for tibial defect in SD rats was preliminarily verified.Methods:Firstly,using large salmon bones as raw materials,L-DBM bone particles were prepared by sodium hydroxide(Na OH)degreasing,hydrochloric acid(HCl)decalcification and other processes;The effects of different variables on the decalcification rate of large salmon bones were explored through single factor experiments,and then the decalcification process parameters were optimized through orthogonal experiments,and the optimal process conditions for the preparation of L-DBM bone particles were finally obtained.At the same time,the properties of L-DBM bone particles were preliminarily tested,including microstructure,mechanical properties,porosity and in vivo degradation tests.Then,on the basis of L-DBM bone particles,L-DBM bone powder was continuously prepared,that is,the product obtained by crushing L-DBM bone particles,and its physical and chemical properties and biological safety were verified.The physical and chemical properties include microstructure,hydroxyproline content determination,infrared spectrum detection,thermal stability analysis;biological safety including cytotoxicity,mouse systemic acute toxicity test,cell viability assay,skin sensitization test,hemolysis test,pyrogen test and intradermal reaction test.Finally,SD rat tibial defect model was prepared by electric drilling method,and the repair effect of L-DBM bone powder on SD rat tibial defect was preliminarily evaluated by H?E staining and Masson staining,and its in vivo biosafety was also evaluated.Results:The L-DBM bone particles were obtained by the processes of alkali degreasing and acid decalcification.The results of single factor experiment showed that the decalcification rate of L-DBM bone particles reached the maximum value in each of the conditions of solid-liquid ratio of 1:30(w/v),hydrochloric acid concentration of 0.1 mol/L,decalcification time of 48 h,and decalcification temperature of 25?.The results of orthogonal experiments showed that when the concentration of hydrochloric acid is 0.05mol/L,the decalcification time is 48 h,the ratio of solid to liquid is 1:30(w/v),and the decalcification temperature is 25?,the decalcification rate of L-DBM bone particles reached the maximum value.L-DBM bone particles have a porous structure(large pores of about 40?70?m can be observed),good mechanical properties(compressive strength is3.8±0.6 MPa),high porosity(70.22±3.76%),and in vivo good biocompatibility and biodegradability.The main component of L-DBM bone powder is type I collagen;the particle size is mainly distributed in 20?60?m and the size is uneven;the hydroxyproline content is high(8.6±0.4%),and it has relatively stable thermal stability(T_i is 50.1?,T_d is55.4?),no cytotoxicity,no acute toxicity,no pyrogen reaction,no sensitization,no hemolysis(0.43%)and no intradermal irritation.The H?E and Masson staining results of SD rat tibial defect experiment showed that L-DBM bone powder had a certain effect on promoting the healing of bone defects.Meanwhile,L-DBM bone powder has good in vivo biosafety.Conclusion:L-DBM bone powder has good physical and chemical properties,biocompatibility and osteogenic activity,which can achieve the goal of bone tissue engineering materials.