Regenerative Biology Group, Oral and Biomedical Sciences, School of Dentistry, Cardiff Institute of Tissue Engineering and Repair (CITER), College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK.
Melbourne Dental School, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Australia.
Biomed Res Int. 2020 Sep 10;2020:3034727. doi: 10.1155/2020/3034727. eCollection 2020.
Dental pulp stem cells (DPSCs) are increasingly being advocated for regenerative medicine-based therapies. However, significant heterogeneity in the genotypic/phenotypic properties of DPSC subpopulations exist, influencing their therapeutic potentials. As most studies have established DPSC heterogeneity using 2D culture approaches, we investigated whether heterogeneous DPSC proliferative and contraction/remodelling capabilities were further evident within 3D type I collagen gels . DPSC subpopulations were isolated from human third molars and identified as high/low proliferative and multipotent/unipotent, following culture expansion and population doubling (PD) analysis. High proliferative/multipotent DPSCs, such as A3 (30 PDs and 80 PDs), and low proliferative/unipotent DPSCs, such as A1 (17 PDs), were cultured in collagen gels for 12 days, either attached or detached from the surrounding culture plastic. Collagen architecture and high proliferative/multipotent DPSC morphologies were visualised by Scanning Electron Microscopy and FITC-phalloidin/Fluorescence Microscopy. DPSC proliferation (cell counts), contraction (% diameter reductions), and remodelling (MMP-2/MMP-9 gelatin zymography) of collagen gels were also evaluated. Unexpectedly, no proliferation differences existed between DPSCs, A3 (30 PDs) and A1 (17 PDs), although A3 (80 PDs) responses were significantly reduced. Despite rapid detached collagen gel contraction with A3 (30 PDs), similar contraction rates were determined with A1 (17 PDs), although A3 (80 PDs) contraction was significantly impaired. Gel contraction correlated to distinct gelatinase profiles. A3 (30 PDs) possessed superior MMP-9 and comparable MMP-2 activities to A1 (17 PDs), whereas A3 (80 PDs) had significantly reduced MMP-2/MMP-9. High proliferative/multipotent DPSCs, A3 (30 PDs), further exhibited fibroblast-like morphologies becoming polygonal within attached gels, whilst losing cytoskeletal organization and fibroblastic morphologies in detached gels. This study demonstrates that heterogeneity exists in the gel contraction and MMP expression/activity capabilities of DPSCs, potentially reflecting differences in their abilities to degrade biomaterial scaffolds and regulate cellular functions in 3D environments and their regenerative properties overall. Thus, such findings enhance our understanding of the molecular and phenotypic characteristics associated with high proliferative/multipotent DPSCs.
牙髓干细胞 (DPSCs) 越来越多地被提倡用于基于再生医学的治疗方法。然而，DPSC 亚群的基因型/表型特性存在显著的异质性，影响其治疗潜力。由于大多数研究都是使用二维培养方法来建立 DPSC 异质性，因此我们研究了在 3D 型 I 胶原凝胶中是否存在不同的 DPSC 增殖和收缩/重塑能力。从人第三磨牙中分离出 DPSCs 亚群，并在经过培养扩增和群体倍增 (PD) 分析后，将其鉴定为高/低增殖和多能/单能。高增殖/多能 DPSCs，如 A3（30 PD 和 80 PD）和低增殖/单能 DPSCs，如 A1（17 PD），在胶原凝胶中培养 12 天，要么附着在周围培养塑料上，要么与之分离。通过扫描电子显微镜和 FITC-鬼笔环肽/荧光显微镜观察胶原的结构和高增殖/多能 DPSCs 的形态。还评估了 DPSCs、A3（30 PD）和 A1（17 PD）的增殖（细胞计数）、收缩（直径减少百分比）和重塑（MMP-2/MMP-9 明胶酶谱）。出乎意料的是，尽管 A3（80 PD）的反应显著降低，但 DPSCs、A3（30 PD）和 A1（17 PD）之间没有增殖差异。尽管 A3（30 PD）的胶原凝胶迅速分离收缩，但 A1（17 PD）的收缩率确定相似，尽管 A3（80 PD）的收缩明显受损。凝胶收缩与独特的明胶酶谱相关。A3（30 PD）具有优于 A1（17 PD）的 MMP-9 和相当的 MMP-2 活性，而 A3（80 PD）的 MMP-2/MMP-9 显著降低。高增殖/多能 DPSCs、A3（30 PD），在附着的凝胶中进一步表现出成纤维细胞样形态，变成多边形，而在分离的凝胶中失去细胞骨架组织和成纤维细胞形态。这项研究表明，DPSCs 在凝胶收缩和 MMP 表达/活性能力方面存在异质性，这可能反映了它们降解生物材料支架和调节 3D 环境中细胞功能以及整体再生能力的差异。因此，这些发现增强了我们对与高增殖/多能 DPSCs 相关的分子和表型特征的理解。
