Exploration Of The Optimal Ratio Of CollagenⅠ/Large-particle True Bone Ceramic Constructing Cartilage Tissue Engineering Scaffolds And Preliminary Study On Physicochemical Properties

Background: The number of patients with cartilage defects caused by traffic accidents,construction accidents,industrial injuries,tumors and various diseases is increasing,and how to repair the lost cartilage has been a popular topic of research by domestic and international research teams.Currently,there are two main types of repair methods: autologous/allogeneic transplantation and tissue-engineered cartilage.However,cartilage autologous/allogeneic transplantation suffers from limited donor,low number of transplantable cartilage,and immune rejection,resulting in very limited results in the treatment of large cartilage defects.The discovery and research of cartilage tissue engineering has provided a new therapeutic direction for the treatment of cartilage defects.One of the three important components of tissue engineering is the choice of scaffold materials,which are mainly divided into artificial and natural materials.The advantages of synthetic materials are low price,controllable mechanical properties and degradation rate,but there are also many disadvantages: poor biocompatibility,residual toxic reagents,acidic degradation products leading to inflammatory reactions,etc.Natural materials have irreplaceable advantages over synthetic materials due to their low immunogenicity,absorbable degradation products and good biocompatibility.Bovine calcined bone powder is formed by calcination of natural bone,similar to the composition of human bone,the main composition is hydroxyapatite,a large number of pore channels is its own structural characteristics,cut with good biocompatibility and induce cell differentiation osteogenesis,its many advantages make it an ideal scaffold material,is one of the early biological materials used in bone and cartilage repair;large particles of calcined bone powder compared to ordinary calcined bone powder,the advantages of calcined bone powder.Compared with ordinary calcined bone powder,large particle calcined bone powder has a larger pore size structure,which is more suitable for cell adhesion and growth,while retaining its original advantages.Collagen is prevalent in human body and is the main organic component of cartilage,easily degradable,low immunogenicity and easy to bind with cells,and can induce chondrocytes to differentiate into tissues.Therefore,in this study,we proposed to construct a tissue-engineered scaffold using natural materials: type I collagen and large-particle calcined bone powder,and to explore the optimal ratio of collagen/large-particle calcined bone powder in the scaffold,aiming to provide options for the optimal scaffold for tissue-engineered cartilage.Objective: Different ratios of large particle TBC/Co L Ⅰ composite scaffolds were constructed to evaluate their physicochemical properties and biological performance,and to obtain the optimal TBC/Col Ⅰ ratio to provide a theoretical basis for the replacement material of cartilage defects in clinical practice.Methods:(1)Type I collagen is dissolved in acetic acid,and after modified cross-linking and modified lyophilization process,the disorganized and dense spatial structure is transformed into a more orderly,more porous and looser structure suitable for cell attachment and growth.(2)By observation and comparison during the preparation process,the scaffolds with relatively better morphology of different ratios of collagen/bone powder were initially selected for the next detailed testing and comparison.(3)Determination of physicochemical properties and biocompatibility of the selected scaffolds,including: general observation of scaffold morphology,SEM microstructure observation,determination of elastic modulus and water absorption,porosity,density,degradation rate,DIO fluorescence staining observation,and in vivo degradation experiments in animals.Results:(1)Type I collagen dissolved in acetic acid,after modified cross-linking and modified lyophilization process,the disordered and dense spatial structure was transformed into a more orderly,more porous and looser structure suitable for cell attachment and growth.(2)The scaffolds formed by different collagen-bone powder ratios were compared experimentally,and the collagen concentration of 6 mg/ml and collagen-bone powder ratios of 1:7,1:9 and 1:11 were selected for the next comparative tests.(3)The results of porosity,water absorption,elastic modulus,fluorescence staining observation and animal experiments showed that the scaffold performance was best with the collagen/bone powder ratio of 1:9,and the biocompatibility and physicochemical properties were more compounded with the requirements of tissue engineering scaffolds.Conclusions:(1)Different ratios of type I collagen/large particles of calcined bone powder scaffolds were successfully constructed.(2)It was demonstrated that a 1:9 ratio with a collagen/acetic acid solution concentration of 6 mg/ml is the best ratio of collagen/bone powder.Through the improved mixing and stirring process and cross-linking and lyophilization methods,scaffolds with higher porosity,better biocompatibility,lower degradation rate and stronger elastic modulus can be obtained,so they are more suitable as an ideal tissue engineering scaffold.