Study On The Mineralization Method Of Chitosan Scaffolds Regulated By Alkaline Phosphatase And The Enhanced Osteo-regenerability Of Composite Scaffolds

The compatibility of materials and host tissue as well as its mineralization performance in vivo are the key problems of bone tissue repair and replacement.In this study,according to the requirements of mineralization performance of scaffold materials for bone tissue repair and regeneration,a nano-hydroxyapatite/chitosan?n-HAP/CS?composite scaffolds material with structure and composition close to the natural bone was prepared by using the method of‘dialysis mineralization regulated by alkaline Phosphatase?ALP?'and its biology performance was evaluated preliminarily.In this paper,a three-dimensional CS with graded porosity was used as the matrix material,the three-dimensional n-HAP/CS composite scaffolds material was prepared with the method of‘dialysis mineralization regulated by ALP'.The main innovations are:ALP was used as the enzyme of regulation,while calcium glycerophosphate?Ca-GP?and Ca Cl₂ as the calcium and phosphorus sources in the mineralization process.With the slow,mild and deep process of mineralization under the regulation of ALP,a new type of bone tissue repair material with structure and composition close to natural bone was prepared.The results showed that the surface of scaffolds was deposited by a layer of n-HAP particles uniformly after mineralization for 48 hours in the mineralized solution with the concentration of 0.4mg/m L?ALP?,95 mmol/L?Ca-GP?and 64 mmol/L?Ca Cl₂?,respectively.The compression modulus of n-HAP/CS scaffolds were 4.38±0.31 MPa which was significantly higher than that of the pure CS scaffolds?2.65±0.39 MPa?.To explore the effect of n-HAP/CS on cell behavior and the feasibility of bone tissue repair in vivo,this research has evaluated the biological properties of n-HAP/CS both in vitro and vivo.The results of in-vitro studies indicated that the pre-osteoblasts?MC3T3-E1?showed good adhesion and proliferation on n-HAP/CS.After MC3T3-E1 were cultured for 12 hours,the adhesion ratio of CS and n-HAP/CS?mineralized for 24,48 and 72 hours?were 80.10±0.96%?81.50±1.03%?86.13±0.64%and 87.25±0.59%,respectively.The results of osteogenic gene also showed that the n-HAP/CS scaffolds could promote the MC3T3-E1 differentiate into osteoblasts significantly,compared with CS scaffolds.The in vivo results showed that the new bone volume in n-HAP/CS composite scaffolds model group were up to24.35±2.65%?8 weeks?and 54.32±3.41%?12 weeks?,while that in pure CS scaffolds were only 13.41±3.12%?8 weeks?and 26.68±4.22%?12 weeks?.The results indicated that the n-HAP/CS could contributed better effect to strengthen the regeneration of bone tissue comparing with the simple CS scaffolds materials in vivo.In order to study the effects of the n-HAP/CS on stem cell behavior and the compatibility of the composite system in tissue repair and regeneration,this study carried out a method of co-culture of human Umbilical Cord Mesenchymal Stem Cells?h UCMSCs?and n-HAP/CS to explore the effect of n-HAP/CS on h UCMSCs behavior and the effect of composite system in the model of lumbar defect.The results indicated that h UCMSCs had better proliferation and adhesion status on the n-HAP/CS composite scaffolds.After cltured for 7 days,the ALP activity value of CS group was 13.52±1.41 U/g,while the n-HAP/CS group was 20.72±1.12 U/g.After cultured for 14 days,the ALP activity value were 14.65±1.02 U/g for CS and 23.34±0.57 U/g for n-HAP/CS.The n-HAP/CS scaffolds showed significant ability to promote the differentiation of stem cells into osteoblasts.The results in vivo showed that,n-HAP/CS composite scaffolds can promote bone tissue regeneration to achieve the effect of lumbar fusion,while n-HAP/CS/h UCMSCs bionic composite system can contribute not only to bone tissue regeneration promotion,but also to the repair of intervertebral annular fibrosus ring in a certain extent.Most living tissues and organs have a certain degree of liquid crystal properties.However,there are few reports of mineralization behavior of liquid crystal.In order to further simulate the mineralized microenvironment in vivo,this paper innovatively studied the effect of liquid crystal state on mineralization behaviors.Thermogenic cholesteric liquid crystal(LC₃₀)and LC₃₀/CS composite scaffolds,which were in the liquid crystal state at the physiological temperature,were prepared by the binary blending of cholesterol oleyl carbonate?COC?and cholesterol nonanoic acid ester?CN?.Subsequently,the n-HAP/LC₃₀/CS?HLC?composite scaffolds were obtained by the method of‘dialysis mineralization regulated by ALP'.The results showed that the cholesteric liquid crystal phase temperature range of CN-COC was from 29.50 to49.40?while the CN content was 30%.The cholesteric liquid crystal phase temperature range of LC₃₀/CS scaffolds material was from 27.81 to 44.60?while the content of LC₃₀ was 40%.Meanwhile a continuous liquid crystal domains was formed on the surface of the material,the scaffolds material present liquid crystal property as a whole.The HLC composite scaffolds were obtained through the method of‘dialysis mineralization regulated by ALP',the n-HAP particles covering on the scaffolds surface of HLC were different with HC in structure and morphology.