Osteogenic/odontogenic differentiation ability of human dental pulp stem cells under photocrosslinked composite hydrogel scaffold

doi:10.12307/2025.071

Abstract

Abstract: BACKGROUND: The composite hydrogel scaffold formed by crosslinking of gelatin-methacryloyl (Gel-MA) and treated dentin matrix (TDM) under a certain proportion of ultraviolet light has good porosity, mechanical properties, swelling properties, and biodegradation rate, which provides a new idea and method for clinical pulp regeneration of young permanent teeth.
OBJECTIVE: To explore the effect of Gel-MA/TDM composite hydrogel scaffold with 1:2 mass ratio on the proliferation ability and osteogenic/odontoblast differentiation ability of human dental pulp stem cells.
METHODS: The passage 3 dental pulp stem cells were inoculated into the Gel-MA/TDM composite hydrogel scaffold with a mass ratio of 1:2. The proliferation ability of human dental pulp stem cells in the composite hydrogel scaffold was detected by CCK-8 assay. Dental pulp stem cells at passage 3 were cultured in Gel-MA/TDM composite hydrogel scaffold with a mass ratio of 1:2 for osteogenic induction. The formation of mineralized nodules was observed by alkaline phosphatase and alizarin red staining. The gene expression levels of odontogenic factors (dentin matrix protein 1, dentin sialophosphoprotein), and osteogenic factors (osteocalcin, Runt-related transcription factor 2) were detected by RT-PCR.
RESULTS AND CONCLUSION: (1) The results of CCK-8 assay showed that the proliferation ability of dental pulp stem cells increased significantly in the first 7 days, and slowed down on day 10. (2) The results of alkaline phosphatase staining and alizarin red staining showed that the alkaline phosphatase activity and the formation of mineralized nodules of dental pulp stem cells in the Gel-MA/TDM composite hydrogel group were stronger than those in Gel-MA hydrogel group (P < 0.05). (3) RT-PCR results showed that the gene expression levels of dentin matrix protein 1, dentin sialophosphoprotein, osteocalcin, and Runt-related transcription factor 2 in dental pulp stem cells in Gel-MA/TDM composite hydrogel group were significantly higher than those in Gel-MA hydrogel group (P < 0.05). The gene expression level at 14 days was significantly higher than that at 7 days (P < 0.05). The results conclude that the dental pulp stem cells cultured on Gel-MA/TDM composite hydrogel scaffolds with a mass ratio of 1:2 exhibit a good proliferation ability, which can strengthen the osteogenic and odontogenic differentiation abilities of dental pulp stem cells.

Key words: dental pulp regeneration, gelatin-methacryloyl, dentinal matrix, composite scaffold, dental pulp stem cell, osteogenesis, odontogenic

CLC Number:

Yang Dujuan, Cheng Mengke, Liu Jia. Osteogenic/odontogenic differentiation ability of human dental pulp stem cells under photocrosslinked composite hydrogel scaffold[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(19): 4022-4028.

Figures/Tables 2

2.1 人牙髓干细胞的培养及鉴定结果培养第 7 天可见有部分牙髓干细胞贴壁，见图 1A。细胞传代后 24 h 镜下可见大部分细胞贴壁完成，细胞呈长梭形，形态似成纤维细胞。传代至第 3 代可见细胞生长良好，形态均匀，见图 1B。流式细胞技术鉴定结果显示，细胞表面标记物 CD29 和 CD105 的阳性率为 99.84% 和 82.81%，CD14 和 CD45 的表达为阴性，见图 2。 2.2 Gel-MA/TDM 复合水凝胶的溶胀性能不同质量比的 Gel-MA/TDM 复合水凝胶大体观如图 3 所示，1 ∶ 2 Gel-MA/ TDM 复合水凝胶呈微黄色圆柱形的凝胶状，Gel-MA 浓度越高，复合水凝胶的颜色越透明。单纯 Gel-MA 水凝胶为透明的圆柱形凝胶状。不同质量比的复合水凝胶及单纯 Gel-MA 水凝胶的溶胀曲线如图 4 所示，1 ∶ 2 质量比的 Gel-MA/TDM 复合水凝胶溶胀比最高，在 4 h 左右达到溶胀平衡，4 h 溶胀比为 (629.36±83.27)%。在初始 4 h 内可见水凝胶迅速吸收溶胀 (0.5，2，4 h 溶胀比明显增高， P < 0.05)，然后溶胀速度开始减慢，直至溶胀平衡 (12， 24 h 溶胀比未见明显变化，P > 0.05)。 2.3 Gel-MA/TDM 复合水凝胶的孔隙率及生物降解率孔隙率检测结果如图 5 所示，4 组水凝胶的孔隙率差异有显著性意义 (P < 0.05)。其中 1 ∶ 2 Gel-MA/TDM 复合水凝胶支架的孔隙率最高，为 (44.43±1.42)%，其次是 1 ∶ 1 Gel-MA/TDM 复合水凝胶支架和 2 ∶ 1 Gel-MA/TDM 复合水凝胶支架，分别为 (34.41±1.07)% 和 (23.04±1.27)%，单纯 Gel-MA 组的孔隙率最低，为 (13.77±1.07)%。生物降解率如图 6 所示，4 组支架的生物降解率差异有显著性意义 (P < 0.05)。在第 14 天时，1 ∶ 2 Gel-MA/TDM 复合水凝胶支架的生物降解率最高，达到了 25% 以上，为 (27.71±0.51)%。 2.4 细胞活力 CCK-8 细胞增殖实验显示：牙髓干细胞在 1 ∶ 2 质量比 Gel-MA/TDM 复合水凝胶支架中，第 3，5， 7 天可见明显增殖 (P < 0.05)，第 10 天可见增殖速度减缓，第 7 天与第 10 天增殖无明显差异 (P > 0.05)，见图 7。与对照组相比，在第 1 天未见明显差异 (P > 0.05)，第 3，5， 7，10 天时可见在复合水凝胶支架中的牙髓干细胞增殖能力明显增强。 2.5 碱性磷酸酶染色和茜素红染色结果牙髓干细胞在成骨诱导液中连续培养 7，14 d 进行碱性磷酸酶染色，结果显示 14 d 染色强度明显高于 7 d(P < 0.05)，且在 1 ∶ 2 质量比 Gel-MA/TDM 复合水凝胶支架中碱性磷酸酶活性更高 (P < 0.05)，见图 8。连续成骨诱导 14，21 d 后进行茜素红染色，结果显示 21 d 染色强度明显强于 14 d (P < 0.05)，与对照组相比，1 ∶ 2 质量比 Gel-MA/TDM 复合水凝胶支架内可见更明显的矿化结节生成(P < 0.05)，见图9。 2.6 RT-PCR 检测成骨诱导 14 d 的成骨 / 成牙本质相关基因表达量明显高于 7 d(P < 0.05)。1 ∶ 2 质量比 Gel-MA/ TDM 复合水凝胶支架中的成骨 / 成牙本质相关基因表达高于对照组及单纯 Gel-MA 组，单纯 Gel-MA 组中成骨 / 成牙本质相关基因表达高于对照组 ( 均 P < 0.05)，见图 10。"

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