磷酸钙骨水泥复合物/脐带间充质干细胞凝胶构建组织工程骨

An injectable calcium phosphate-alginate hydrogel with umbilical cord mesenchymal stem cell composite for bone tissue engineering

目的构建新型可注射强化型磷酸钙骨水泥复合物/脐带间充质干细胞凝胶组织工程骨,探讨其力学性能,细胞活性和成骨作用。方法选用第四代hUCMSCs,1.2%海藻酸钠水凝胶构建hUCMSCs水凝胶微球。高温煅烧钙/磷比约为1.9的磷酸氢钙和碳酸钙混合物,按摩尔质量比以1:1混合制备CPC粉末。15%Chitosan,8mm长度可吸收纤维用于提高CPC复合物力学强度。实验组分为四组:(1)单纯hUCMSCs微球;(2)CPC+hUCMSCs微球;(3)CPC+chitosan+hUCMSCs微球;(4)CPC+chitosan+可吸收纤维+hUCMSCs微球,分别检测力学性能,细胞活性和成骨作用。结果新型组织工程骨力学性能显著增强,抗弯曲强度提高到(11.7±2.1)MPa,弹性模量提高到(2.0±0.4)GPa,断裂功提高到(1.65±0.66)kj/m2(P<0.05)。hUCMSCs的ALP活性第7天时明显增高,第14天时达峰,第21天时有所减弱,各组间比较无明显统计学差异(P>0.1)。第7天时,所有实验组中的hUCMSCs均见少量矿物合成。第14天和第21天时,矿物合成数量明显增多。hUCMSCs中ALP基因表达培养第1天最低,第4天达峰,第8天时稍减弱。OC基因表达第8天达峰。结论构建完成新型可注射强化型磷酸钙骨水泥/脐带间充质干细胞凝胶构建组织工程骨。水凝胶微球中hUCMSCs在CPC中具有良好的成骨作用。CPC-chitosan-可吸收纤维组织工程骨力学性能满足松质骨力学要求,支持水凝胶微球中hUCMSCs的细胞活性和成骨作用。为组织工程骨研究和临床应用提供新思路和新方法。

Objective To develop a novel injectable composite of calcium phosphate-alginate hydrogel and umbilical cord mesenchymal stem cells for bone tissue engineering,and evaluate its mechanical properties and osteogenesis.Methods The fourth generation hUCMSCs and 1.2% sodium alginate hydrogel were used to build hUCMSCs beads.High-temperature calcinationdicalcium phosphate with a Ca/P ratio of approximately 1.9 was mixed with calcium carbonate in the molar ratio of 1：1 for calcium phosphate cement（CPC） powder.15% of chitosan and absorbable fibersin of 8 mm in length were used to enhance the mechanical strength of CPC complex.The experimental group was divided into four subgroups：（1）hUCMSCs beads;（2）CPC＋hUCMSCs beads;（3）CPC＋chitosan＋hUCMSCs beads;（4） CPC＋chitosan＋fiber＋hUCMSCs beads.Results The flexural strength of the hUCMSCs encapsulating calcium phosphate cement（CPC） with 20% of polyglactin fibers increased to（11.7±2.1） MPa.The elastic modulus increased to（2.0±0.4） GPa.The work of fracture increased to（1.65±0.66）kj/m2（P0.05）.ALP of hUCMSCs was dramatically increased in the osteogenic media.ALP at seventh day was much higher in osteogenic media,peaked at 14th day,and slightly decreased at 21st day（P0.1）.The encapsulated hUCMSCs inside four different constructs successfully synthesized bone minerals.The percentage of mineral area synthesized by the encapsulated hUCMSCs increased from about seventh day to 21st day.The mineral staining amount increased at 14th day and 21st day.hUCMSCs elevated alkaline phosphatase（ALP） and osteocalcin（OC） gene expressions.ALP peaked at 4th day,and slightly decreased at eighth day.OC peaked at eighth day.Conclusions This study demonstrated that hUCMSCs encapsulated in the bioengineered scaffolds osteo-differentiated and synthesized bone minerals.The self-setting CPC-chitosan-fiber scaffold supported the viability and osteogenic differentiation of the encapsulated hUCMSCs,and had mechanical strength matching that of cancellous bone.