海藻酸盐与异种骨构建组织工程载体的适宜浓度

背景:研究认为,载体的结构在很大程度上影响着载体的功能。目的:探讨不同浓度的海藻酸盐凝胶与去抗原异种骨构建组织工程载体的生物学性能。设计:观察性实验。单位:解放军第四军医大学西京医院全军创伤骨科研究所。材料:海藻酸盐,氯化钙,骨髓基质细胞,异种骨。方法:取小牛松质骨,制成骨粒,脱脂、脱蛋白,真空冻干;选择孔隙直径在300～500μm左右的骨粒,环氧乙烷消毒。将提纯后的海藻酸钠以DMEM培养液为溶剂,配制成0.5%,2%,8%和16%的海藻酸钠DMEM溶液;将诱导后的骨髓基质细胞以1×1012L-1浓度等体积与海藻酸钠DMEM溶液充分混合,在0.5MPa的负压状态下复合,使细胞悬液充分占据松质骨的空隙,以50g/L的葡萄酸钙浸渍30s,以使海藻酸钠获得交联。置于CO2培养箱中,培养4d。观察异种骨孔隙中凝胶复合情况及其中细胞生长情况。观察不同藻酸密度复合松质骨粒中细胞生长情况。主要观察指标:观察海藻酸盐凝胶与去抗原异种骨复合情况,复合后的载体中细胞生长状态和基质分泌情况。结果:海藻酸盐的浓度为0.25%,1%时,凝胶在异种骨中均匀复合,浓度为4%和8%时藻酸仅复合在松质骨表面。体外培养4d后,0.25%海藻酸盐复合异种骨的表面大部分凝胶脱落,1%的海藻酸盐均匀复合在异种骨的骨孔中,细胞形态饱满,基质分泌;4%的海藻酸盐中,细胞生长受到限制;8%的凝胶的质地不均匀,无法见到细胞结构。结论:1%的海藻酸盐适宜与异种骨复合构建骨组织工程载体。

Suitable concentration of alginate with xenograft for constructing bone tissue engineering carrier

BACKGROUND:The structure of tissue engineering carrier affects the bio-action of cells greatly.OBJECTIVE: To investigate the biological characteristics of bone marrow stem cells (MSCs) in different concentrations of alginate combined with de-antigen bone xenograft (DBX).DESIGN: Observational trial.SETTING: PLA Institute of Trauma and Orthopedics, the Fourth Military Medical University of Chinese PLA.MATERIALS: Alginate, calcium chloride, MSCs, bone xenograft.METHODS: Bovine cancellous bone was out into cubes, which were degreased, deproteinized and then lyophilized.Cubes in pore size within 300-500 μm were selected for use after ethylene oxide sterilization. The purified sodium alginate was dissolved in DMEM cell culture medium of concentrations as different as 0.5%, 2%, 8% and 16%; 1×1012 L-1 induced MSCs were blended with isopyknic alginate-DMEM and compounded with DBX at a status of 0.5 Mpa negative pressure for 5 minutes in order to make a cell suspension fully fill into the pores of the cancellous bone. Then alginate was crosslinked with 50 g/L calcium gluconic acid for 30 seconds. The complex was put into a CO2 incubator and cultured for 4 days. The gel compound and cell growth in the pores of the complex were grossly observed with an inverted microscope. Status of cell growth in the complex with different concentrations of alginate was observed with scanning electron microscopeMAIN OUTCOME MEASURES: Compound status of alginate and bone xenograft, cell growth status and matrix secretion in compound carries.RESULTS: When the concentration of alginate was 0.25% or 1%, alginate was equally combined in DBX, while that of 4% and 8% only combined on the surface of cancellous bone. After in vitro cultured for 4 days, alginate of 0.25% were broken off from DBX surface. But alginate of 1% was equally combined with DBX pores with cells secreting well in alginate. Development of cells in alginate of 4% was restricted and no cells were seen in alginate of 8%.CONCLUSION: Alginate of 1% is suitable for constructing the carrier of bone tissue engineering with bone xenograft.