目的:制备羟乙基壳聚糖(glycol-chitosan, GC)基单/双网络水凝胶,比较人牙髓细胞(human dental pulp cells,hDPCs)在水凝胶内三维培养增殖和分化情况,探究GC基单/双网络水凝胶及其理化性能对hDPCs生物学行为的影响。方法:制备不同组成配比的GC基单网络水凝胶(GC31)和GC基双网络水凝胶(DN3131、DN6262)。用双联注射器在恒力下推注GC基单/双网络水凝胶,测定其可注射性能。用失重法测定水凝胶的体外耐降解性能,并用万能力学试验机测定材料断裂应力。在水凝胶内包封hDPCs进行三维培养,用CCK-8法和Calcein-AM/PI活死细胞染色法测定hDPCs的增殖情况。成牙本质向诱导培养14 d后,用实时荧光定量PCR(real-time quantitative reverse transcription PCR, RT-PCR)测定各组成牙本质向分化相关基因牙本质涎磷蛋白(dentin sialophosphoprotein, DSPP)、牙本质基质蛋白(dentin matrix protein-1, DMP-1)和矿化相关基因碱性磷酸酶(alkaline phosphatase, ALP)的表达变化,用Von Kossa染色法观察矿化结节的形成。结果:3组水凝胶均具有良好的可注射性能,GC31推注时间最短,DN6262推注时间较DN3131长(P<0.05)。GC31水凝胶在体外降解速率高于双网络水凝胶组(P<0.05), DN3131和DN6262的降解速率差异无统计学意义(P>0.05)。GC31断裂应力为1.10 kPa,DN3131和DN6262断裂应力分别为7.33 kPa和43.30 kPa,双网络水凝胶的抗压缩性能较单网络水凝胶有明显增强。hDPCs在3种水凝胶内均处于良好的增殖状态,GC31组的增殖能力高于DN3131和DN6262组(P<0.05),DN3131组的增殖能力高于DN6262组(P<0.05)。在成牙本质向诱导培养14 d后,DN3131和DN6262组的DSPP、DMP-1、ALP表达水平较GC31组升高(P<0.05), DN3131和DN6262组的DMP-1和ALP表达水平差异无统计学意义(P>0.05)。DN3131和DN6262组体外培养2周后均可见明显生成棕黑色的团块状矿化结节,而GC31组仅观察到浅棕色钙沉积染色,为零星颗粒状散在分布。结论:不同组成配比的GC基单/双网络水凝胶均满足可注射需求,GC单网络水凝胶力学性能较低,hDPCs在其中表现出更好的增殖能力;而双网络水凝胶具备更好的耐降解性能和更高的力学性能,hDPCs在其中表现出更好的成牙本质向分化潜力和矿化潜力。

Objective: To prepare glycol-chitosan (GC)-based single /dual-network hydrogels with different composition ratios (GC31, DN3131 and DN6262) and to investigate the effects of hydrogel scaffolds on biological behavior of human dental pulp cell (hDPC) encapsulated. Methods: GC-based single-network hydrogels (GC31) and GC-based dual-network hydrogels (DN3131, DN6262) with different composition ratios were prepared. The injectability was defined as the average time needed to expel a certain volume of hydrogel under a constant force. The degradation of the hydrogel was determined by the weight loss with time. The fracture stress was measured using a universal testing machine. The proliferation of hDPCs in hydrogels was detected using the cell counting kit-8 (CCK-8) method and Calcein-AM/PI Live/Dead assay. After 14 days of odontoblastic induction, the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) was detected by real-time quantitative reverse transcription PCR (real-time RT-PCR) and the mineralized nodules was observed by Von Kossa staining. Results: The injectability of all three groups of hydrogels was acceptable. The time of injection of GC31 was the shortest, and that of DN6262 was longer than DN3131 (P<0.05). The degradation rate of GC31 hydrogel in vitro was significantly faster than that of the dual-network hydrogel groups (P<0.05). There was no significant difference between DN3131 and DN6262 (P>0.05). The compressive resistance failure point of GC31 group was 1.10 kPa, while it was 7.33 kPa and 43.30 kPa for DN3131 and DN6262. The compressive strength of dual-network hydrogel was significantly enhanced compared with single-network hydrogel. hDPCs were in continuous proliferation in all the three groups,and the GC31 group showed a higher proliferation rate (P<0.05). The expression levels of DSPP, DMP-1 and ALP in the dual-network hydrogel groups (DN3131, DN6262) were significantly higher than that of GC31 after culturing for 14 days (P<0.05), there was no difference in the expression levels of DMP-1 and ALP between DN3131 and DN6262 (P>0.05); Von Kossa staining showed that more mineralization deposition and mass-shaped mineralized nodules formed in DN3131 and DN6262, while only light brown calcium deposition staining was observed in GC31 group, which was scattered in granular forms. Conclusion: GC-based single/dual network hydrogels with different composition ratios met the injectable requirements. GC31 group had a lower mechanical properties, in which hDPCs exhibited a higher proliferation rate. Dual-network hydrogels had slower degradation rate and higher mechanical properties, in which hDPCs exhibited better odontoblastic differentiation potential and mineralization potential.