The quest for finding a suitable scaffold system that supports cell survival and function and, ultimately, the regeneration of the pulp–dentin complex remains challenging. Herein, we hypothesized that dental pulp stem cells (DPSCs) encapsulated in a collagen-based hydrogel with varying stiffness would regenerate functional dental pulp and dentin when concentrically injected into the tooth slices. Collagen hydrogels with concentrations of 3 mg/mL (Col3) and 10 mg/mL (Col10) were prepared, and their stiffness and microstructure were assessed using a rheometer and scanning electron microscopy, respectively. DPSCs were then encapsulated in the hydrogels, and their viability and differentiation capacity toward endothelial and odontogenic lineages were evaluated using live/dead assay and quantitative real-time polymerase chain reaction. For in vivo experiments, DPSC-encapsulated collagen hydrogels with different stiffness, with or without growth factors, were injected into pulp chambers of dentin tooth slices and implanted subcutaneously in severe combined immunodeficient (SCID) mice. Specifically, vascular endothelial growth factor (VEGF [50 ng/mL]) was loaded into Col3 and bone morphogenetic protein (BMP2 [50 ng/mL]) into Col10. Pulp–dentin regeneration was evaluated by histological and immunofluorescence staining. Data were analyzed using 1-way or 2-way analysis of variance accordingly (α = 0.05). Rheology and microscopy data revealed that Col10 had a stiffness of 8,142 Pa with a more condensed and less porous structure, whereas Col3 had a stiffness of 735 Pa with a loose microstructure. Furthermore, both Col3 and Col10 supported DPSCs’ survival. Quantitative polymerase chain reaction showed Col3 promoted significantly higher von Willebrand factor (VWF) and CD31 expression after 7 and 14 d under endothelial differentiation conditions ( P < 0.05), whereas Col10 enhanced the expression of dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and collagen 1 (Col1) after 7, 14, and 21 d of odontogenic differentiation ( P < 0.05). Hematoxylin and eosin and immunofluorescence (CD31 and vWF) staining revealed Col10+Col3+DPSCs+GFs enhanced pulp–dentin tissue regeneration. In conclusion, the collagen-based concentric construct modified by growth factors guided the specific lineage differentiation of DPSCs and promoted pulp–dentin tissue regeneration in vivo.

中文翻译：

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用于牙髓-牙本质再生的可注射组织特异性水凝胶系统


寻找合适的支架系统来支持细胞存活和功能，并最终支持牙髓-牙本质复合物的再生仍然具有挑战性。在此，我们假设封装在具有不同硬度的胶原基水凝胶中的牙髓干细胞（DPSC）在同心注射到牙齿切片中时会再生功能性牙髓和牙本质。制备浓度为 3 mg/mL (Col3) 和 10 mg/mL (Col10) 的胶原水凝胶，并分别使用流变仪和扫描电子显微镜评估其硬度和微观结构。然后将 DPSC 封装在水凝胶中，并使用活/死测定和定量实时聚合酶链反应评估它们对内皮细胞和牙源性谱系的活力和分化能力。在体内实验中，将具有或不具有生长因子的不同硬度的 DPSC 封装的胶原水凝胶注射到牙本质牙片的牙髓腔中，并植入严重联合免疫缺陷（SCID）小鼠的皮下。具体来说，将血管内皮生长因子 (VEGF [50 ng/mL]) 加载到 Col3 中，将骨形态发生蛋白 (BMP2 [50 ng/mL]) 加载到 Col10 中。通过组织学和免疫荧光染色评估牙髓-牙本质再生。相应地使用单向或双向方差分析（α = 0.05）对数据进行分析。流变学和显微镜数据显示，Col10 的刚度为 8,142 Pa，具有更凝聚和更少的孔隙结构，而 Col3 的刚度为 735 Pa，具有松散的微观结构。此外，Col3 和 Col10 都支持 DPSC 的存活。 定量聚合酶链反应显示，在内皮分化条件下7和14天后，Col3显着促进血管性血友病因子（VWF）和CD31表达升高（P<<0.05），而Col10则增强牙本质唾液酸磷蛋白（DSPP）、碱性磷酸酶（ALP）的表达)、runt 相关转录因子 2 (Runx2) 和胶原蛋白 1 (Col1) 在牙源分化 7、14 和 21 d 后 (P < 0.05)。苏木精和伊红以及免疫荧光（CD31 和 vWF）染色显示 Col10+Col3+DPSCs+GF 增强了牙髓-牙本质组织再生。总之，经生长因子修饰的基于胶原蛋白的同心结构引导 DPSC 的特定谱系分化，并促进体内牙髓-牙本质组织再生。