可注射性藻酸锶凝胶应用于骨组织工程的体外研究

IN VITRO STUDY ON INJECTABLE ALGINATE-STRONTIUM HYDROGEL FOR BONE TISSUE ENGINEERING

目的体外研究藻酸锶凝胶作为骨组织工程支架材料的应用潜力，为可注射骨组织工程支架材料的制备提供依据。方法制备2．0wt％藻酸钠溶液，分别与0．2mol／LSrCl2及CaCl2胶凝液离子交联形成藻酸锶和藻酸钙凝胶，扫描电镜观察微观结构并测其压缩模量、溶胀率及降解率。采用全骨髓培养法分离培养兔BMSCs并鉴定，将第5代BMSCs分别接种于藻酸锶凝胶（实验组）和藻酸钙凝胶（对照组），观察其生长、黏附及增殖情况；两组凝胶来源的细胞成骨诱导后采用细胞化学法检测ALP活性，荧光定量RT-PCR检测成骨相关基因Osterix（OSX）、I型胶原、Runx2mRNA表达，茜素红染色、VonKossa染色检测钙盐沉积情况，并设同法制备的藻酸钙凝胶来源的BMSCs作为空白对照组。结果藻酸锶与藻酸钙凝胶微观结构相似，均有均匀的孔隙结构；藻酸锶及藻酸钙凝胶压缩模量分别为（186．53-4-8．37）、（152．14-4-7．45）kPa，比较差异有统计学意义（t=6．853，P=0．002）；溶胀率分别为14．32％±1．53％和15．25％±1．64％，比较差异无统计学意义（t=-0．737，P=0．502）；两种凝胶的体外降解性均较好，第20、25、30天藻酸锶凝胶的降解率明显低于藻酸钙凝胶（P〈0．05）。BMSCs于两组凝胶中1～4d时呈悬浮生长，之后逐渐黏附生长并形成大量细胞集落。三维培养第21天，实验组细胞的DNA含量为（4．38±0．24）g，显著高于对照组的（3．25±0．21）g（t=-8．108，P=0．001）。成骨诱导培养第12天，实验组ALP活性为（15．28±1．26）U／L，显著高于对照组的（12．07±1．12）U／L（P〈0．05）。实验组OSX、I型胶原、Runx2mRNA相对表达量均高于对照组，差异有统计学意义（P〈0．05）。成骨诱导培养第21天，两组茜素红染色及VonKossa染色均可见钙盐沉积，实验组的钙结节数量及磷酸盐沉积面积均显著高于对照组（P〈0．05）。结论藻酸锶凝胶具有优良的理化特性，对BMSCs增殖及成骨分化有显著促进作用，可作为可注射骨组织工程支架材料。

Objective To investigate the application potential of alginate-strontium （St） hydrogel as an injectable scaffold material in bone tissue engineering. Methods The alginate-Sr/-calcium （Ca） hydrogel beads were fabricated by adding 2.0wt% alginate sodium to 0.2 mol/L SrCh/CaCh solution dropwise. Microstructure, modulus of compression, swelling rate, and degradability of alginate-Sr/-Ca hydrogels were tested. Bone marrow mesenchymal stem cells （BMSCs） were isolated from femoral bones of rabbits by flushing of marrow cavity. BMSCs at passage 5 were seeded onto the alginate-Sr hydrogel （experimental group） and alginate-Ca hydrogel （control group）, and the viability and proliferation of BMSCs in 2 alginate hydrogels were assessed. The osteogenic differentiation of cells embeded in 2 alginate hydrogels was evaluated by alkaline phosphate （ALP） activity, osteoblast specific gene [Osterix （OSX）, collagen type I, and Runx2] expression level and calcium deposition by fluorescent quantitative RT-PCR and alizarin red staining, Von Kossa staining. The BMSCs which were embeded in alginate-Ca hydrogel and cultured with common growth medium were harvested as blank control group. Results The micromorphology of alginate-Sr hydrogel was similar to that of the alginate-Ca hydrogel, with homogeneous pore structure; the modulus of compression of alginate-Sr hydrogel and alginate-Ca hydrogel was （186.53±8.37） and （152.14± 7.45） kPa respectively, showing significant difference （t=6.853, P=0.002）; there was no significant difference （t=0.737, P=0.502） in swelling rate between alginate-Sr hydrogel （14.32% ±1.53%） and alginate-Ca hydrogel （15.25% ±1.64%）. The degradabilities of 2 alginate hydrogels were good; the degradation rate of alginate-Sr hydrogel was significantly lower than that of alginate-Ca hydrogel on the 20th, 25th, and 30th days （P 〈 0.05）. At 1-4 days, the morphology of cells on 2 alginate hydrogels was spherical and then the shape was spindle or stellate. When three-dimensional cultured for 21 days, the DNA content of BMSCs in experimental group [（4.38±0.24） g] was significantly higher than that in control group [（3.25 ± 0.21） g ] （t=8.108, P=0.001）.On the 12th day after osteogenic differentiation, the ALP activity in experimental group was （15.28 ＋ 1.26） U/L, which was significantly higher than that in control group [（12.07 ＋ 1.12） U/L] （P 〈 0.05）. Likewise, the mRNA expressions of OSX, collagen type I, and Runx2 in experimental group were significantly higher than those in control group （P 〈 0.05）. On the 2 lth day after osteogenic differentiation, alizarin red staining and Von Kossa staining showed calcium deposition in 2 groups; the calcium nodules and phosphate deposition in experimental group were significantly higher than those in control group （P 〈 0.05）. Conclusion Alginate-Sr hydrogel has good physicochemical properties and can promote the proliferation and osteogenic differentiation of BMSCs, so it is an excellent injectable scaffold material for bone tissue engineering.