Chen Yantian, Zhang Fengli, Fu Qiang, Liu Yong, Wang Zejian, Qi Nianmin
Cell Culture and Bioprocess Engineering Lab, School of Pharmacy, Shanghai Jiao Tong University, China.
Cell Culture and Bioprocess Engineering Lab, School of Pharmacy, Shanghai Jiao Tong University, China
J Biomater Appl. 2016 Sep;31(3):317-27. doi: 10.1177/0885328216661566. Epub 2016 Aug 5.
Injectable thermo-sensitive hydrogels have a potential application in bone tissue engineering for their sensitivities and minimal invasive properties. Human dental pulp stem cells have been considered a promising tool for tissue reconstruction. The objective of this study was to investigate the proliferation and osteogenic differentiation of dental pulp stem cells in injectable thermo-sensitive chitosan/β-glycerophosphate/hydroxyapatite hydrogel in vitro. The chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel were prepared using the sol-gel method. The injectability of chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel was measured using a commercial disposable syringe. Scanning electron microscopy was used to observe the inner structure of hydrogels. Then dental pulp stem cells were seeded in chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel, respectively. The growth of dental pulp stem cells was periodically observed under an inverted microscope. The proliferation of dental pulp stem cells was detected by using an Alamar Blue kit, while cell apoptosis was determined by using a Live/Dead Viability/Cytotoxicity kit. The osteogenic differentiations of dental pulp stem cells in chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel were evaluated by alkaline phosphatase activity assay and mRNA expression of osteogenesis gene for 21 days in osteogenic medium. The results indicated that there was no significant difference between chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel in injectability. Cells within the chitosan/β-glycerophosphate/hydroxyapatite hydrogel displayed a typical adherent cell morphology and rapid proliferation with high cellular viability after 14 days of culture. Dental pulp stem cells seeded in chitosan/β-glycerophosphate/hydroxyapatite hydrogels had a higher alkaline phosphatase activity and better up-regulation of gene expression levels of Runx-2, Collagen I, alkaline phosphatase and osteocalcin than in chitosan /β-glycerophosphate hydrogels after osteogenic differentiation. These results demonstrated that the chitosan/β-glycerophosphate/hydroxyapatite hydrogel had excellent cellular compatibility and the superiority in promoting dental pulp stem cells osteogenic differentiation in vitro, showing that the combination of dental pulp stem cells and chitosan/β-glycerophosphate/hydroxyapatite hydrogel has the potential to be used for bone tissue engineering.
可注射热敏水凝胶因其敏感性和微创特性在骨组织工程中具有潜在应用价值。人牙髓干细胞被认为是组织重建的一种有前景的工具。本研究的目的是体外研究牙髓干细胞在可注射热敏壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶中的增殖和成骨分化情况。采用溶胶-凝胶法制备壳聚糖/β-甘油磷酸酯水凝胶和壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶。使用商用一次性注射器测量壳聚糖/β-甘油磷酸酯水凝胶和壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶的可注射性。用扫描电子显微镜观察水凝胶的内部结构。然后将牙髓干细胞分别接种于壳聚糖/β-甘油磷酸酯水凝胶和壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶中。在倒置显微镜下定期观察牙髓干细胞的生长情况。用阿拉玛蓝试剂盒检测牙髓干细胞的增殖情况，用活/死细胞活力/细胞毒性试剂盒测定细胞凋亡情况。在成骨培养基中培养21天，通过碱性磷酸酶活性测定和骨生成基因的mRNA表达评估壳聚糖/β-甘油磷酸酯水凝胶和壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶中牙髓干细胞的成骨分化情况。结果表明，壳聚糖/β-甘油磷酸酯水凝胶和壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶在可注射性方面无显著差异。壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶中的细胞呈现典型的贴壁细胞形态，培养14天后增殖迅速且细胞活力高。成骨分化后，接种于壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶中的牙髓干细胞比接种于壳聚糖/β-甘油磷酸酯水凝胶中的牙髓干细胞具有更高的碱性磷酸酶活性和更好的Runx-2、I型胶原、碱性磷酸酶和骨钙素基因表达水平上调。这些结果表明，壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶具有优异的细胞相容性，在体外促进牙髓干细胞成骨分化方面具有优势，表明牙髓干细胞与壳聚糖/β-甘油磷酸酯/羟基磷灰石水凝胶的组合有潜力用于骨组织工程。
