牙本质生物活性材料的相关性能及对牙髓干细胞周期及形态的影响

Properties of bioactive dentin matrix and its effect on cell cycle and morphology of dental pulp stem cells

目的 探讨经冷冻干燥处理的牙本质生物活性材料(FD)的生物、力学性能以及该材料对牙髓干细胞(DPSCs)细胞周期及形态特征的影响。方法 制备FD,检测FD、未冻干处理牙本质(D)、羟基磷灰石/磷酸三钙(HA)三组材料的挠曲强度及弹性模量;HE法观察材料的组织结构;ELISA法检测材料Ⅰ型胶原(COL-1)的释放量。将DPSCs分别培养于FD、D、HA及玻片四组材料表面,流式细胞术检测材料表面DPSCs周期,免疫荧光检测材料表面DPSCs的骨架特征。结果 挠曲强度D组为(194.1 ± 24.3)MPa,FD组为(166.1 ± 57.5)MPa,HA组为(6.2 ± 0.2)MPa;弹性模量D组为(5.8 ± 2.1)GPa,FD组为(4.7 ± 1.5)GPa,HA组为(0.05 ± 0.01)GPa。FD组和D组的挠曲强度及挠曲弹性模量差异无统计学意义( P >0.05),而HA组的平均值显著低于其他两组,且差异具有统计学意义( P <0.01)。组织学结果显示,冻干处理后牙本质小管形态较完整;FD组、D组二者COL-1释放差异无统计学意义( P >0.05);FD组与D组DPSCs细胞周期分布中各相细胞百分率无明显差异,HA表面DPSCs活性最低;荧光染色显示两个牙本质组表面DPSCs细胞骨架结构优于HA组及盖玻片/α-MEM组。结论 经冷冻干燥处理的牙本质生物活性材料具有与未冻干处理牙本质相似的力学性能和组织结构,对DPSCs周期分布及细胞骨架结构有积极作用。

Objective To explore the properties of freeze-dried dentin bioactive materials(FD) and their effects on cell cycle and morphology of dental pulp stem cells(DPSCs). Methods The flexural strength and elastic modulus of FD, untreated dentin(D) and hydroxyapatite/tricalcium phosphate(HA) were measured. The structure of the materials was observed by HE staining. The release of type Ⅰ collagen(COL1) was detected by ELISA. DPSCs were cultured with prepared FD, D, HA and slides, respectively. The cell periodic distribution of DPSCs cultured on the surface of materials mentioned above was detected by flow cytometry. The skeleton characteristics of DPSCs cultured on the surface of materials were detected in four groups by confocal microscopy. Results Flexural strength was (194.1 ± 24.3)MPa in D group,(166.1 ± 57.5)MPa in FD group and (6.2 ± 0.2)MPa in HA group. Flexural modulus was (5.8 ± 2.1)GPa in in D group,(4.7 ± 1.5)GPa in FD group, and (0.05 ± 0.01)GPa in HA group. There was no significant difference in flexural strength and flexural modulus between FD group and D group( P >0.05), but the flexural strength and flexural modulus were higher in FD group and D group than those in HA group( P <0.01). Histological results showed that samples in FD group and D group preserved the intact dentin tubule morphology. There was no significant difference in COL1 release rate between FD group and D group( P >0.05). The cell cycle results showed that there was no significant difference in the percentage of cell phases between FD group and D group, and the DPSCs activity in HA group was the lowest. Fluorescence staining showed that the structure of actin in DPSCs of the two dentin groups(FD group and D group) was better than that of HA group and coverslips/α-MEM group. Conclusion FD has similar mechanical properties and microstructure to untreated dentin, and has a positive effect on the periodic distribution and cytoskeletal structure of DPSCs.