用于牙髓-牙本质再生的可注射组织特异性水凝胶系统

Journal of dental research Pub Date : 2024-04-01 Epub Date: 2024-02-27 DOI:10.1177/00220345241226649
Y Han, J Xu, H Chopra, Z Zhang, N Dubey, W L Dissanayaka, J E Nör, M C Bottino
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引用次数: 0

摘要

寻找一种合适的支架系统,以支持细胞的存活和功能,并最终支持牙髓-牙本质复合体的再生,仍然具有挑战性。在此,我们假设将牙髓干细胞(DPSCs)包裹在不同硬度的胶原蛋白水凝胶中,集中注射到牙片中,可再生出功能性牙髓和牙本质。制备了浓度为 3 毫克/毫升(Col3)和 10 毫克/毫升(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了它们的硬度和微观结构。然后将 DPSCs 包囊在水凝胶中,使用活/死检测法和定量实时聚合酶链反应评估它们的存活率以及向内皮细胞和牙本质细胞系分化的能力。在体内实验中,将含有或不含生长因子的不同硬度胶原水凝胶包裹的 DPSC 注入牙本质牙齿切片的牙髓腔,并植入严重联合免疫缺陷(SCID)小鼠的皮下。具体来说,在 Col3 中注入血管内皮生长因子(VEGF [50 ng/mL]),在 Col10 中注入骨形态发生蛋白(BMP2 [50 ng/mL])。通过组织学和免疫荧光染色对牙髓-牙本质再生进行评估。数据采用单因子或双因子方差分析(α = 0.05)进行分析。流变学和显微镜数据显示,Col10 的硬度为 8,142 Pa,结构更加凝结,孔隙较少;而 Col3 的硬度为 735 Pa,微观结构疏松。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,Col3能在7天和14天后显著提高von Willebrand因子(VWF)和CD31的表达量(P < 0.05),而Col10则能在牙本质分化7天、14天和21天后提高牙本质磷蛋白(DSPP)、碱性磷酸酶(ALP)、runt相关转录因子2(Runx2)和胶原蛋白1(Col1)的表达量(P < 0.05)。血红素和伊红以及免疫荧光(CD31 和 vWF)染色显示 Col10+Col3+DPSCs+GFs 增强了牙髓-牙本质组织再生。总之,生长因子修饰的胶原同心构建物引导了 DPSCs 的特异性系分化,促进了体内牙髓-牙本质组织的再生。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Injectable Tissue-Specific Hydrogel System for Pulp-Dentin Regeneration.

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.

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