Department of Biomaterials Science, Osaka University Graduate School of Dentistry, Osaka, Japan.
Department of Orthodontics and Dentofacial Orthopedics, Osaka University Graduate School of Dentistry, Osaka, Japan.
J Dent Res. 2023 Feb;102(2):207-216. doi: 10.1177/00220345221130682. Epub 2022 Oct 24.
Dental pulp stem cells (DPSCs) can differentiate into vascular endothelial cells and display sprouting ability. During this process, DPSC responses to the extracellular microenvironment and cell-extracellular matrix interactions are critical in regulating their ultimate cell fate. Heparan sulfate (HS) glycosaminoglycan, a major component of extracellular matrix, plays important roles in various biological cell activities by interacting with growth factors and relative receptors. However, the regulatory function of HS on vasculogenesis of mesenchymal stem cells remains unclear. The objective of this study was to investigate the role of HS in endothelial differentiation and vasculogenesis of DPSCs. Our results show that an HS antagonist suppressed the proliferation and sprouting ability of DPSCs undergoing endothelial differentiation. Furthermore, expression of proangiogenic markers significantly declined with increasing dosages of the HS antagonist; in contrast, expression of stemness marker increased. Silencing of exostosin 1 (EXT1), a crucial glycosyltransferase for HS biosynthesis, in DPSCs using a short hairpin RNA significantly altered their gene expression profile. In addition, -silenced DPSCs expressed lower levels of endothelial differentiation markers and displayed a reduced vascular formation capacity compared with control DPSCs transduced with scrambled sequences. The sprouting ability of -silenced DPSCs was rescued by the addition of exogenous HS in vitro. Next, we subcutaneously transplanted biodegradable scaffolds seeded with -silenced or control DPSCs into immunodeficient mice. Lumen-like structures positive for human CD31 and von Willebrand factor were formed by green fluorescent protein-transduced DPSCs. Numbers of blood-containing vessels were significantly lower in scaffolds loaded with -silenced DPSCs than specimens implanted with control DPSCs. Collectively, our findings unveil the crucial role of HS on endothelial differentiation and vasculogenesis of DPSCs, opening new perspectives for the application of HS to tissue engineering and dental pulp regeneration.
牙髓干细胞（DPSCs）可分化为血管内皮细胞，并表现出出芽能力。在此过程中，DPSC 对细胞外微环境的反应和细胞-细胞外基质的相互作用对于调节其最终的细胞命运至关重要。硫酸乙酰肝素（HS）糖胺聚糖是细胞外基质的主要成分，通过与生长因子和相对受体相互作用，在各种生物细胞活动中发挥重要作用。然而，HS 对间充质干细胞血管生成的调节作用尚不清楚。本研究旨在探讨 HS 在 DPSCs 内皮分化和血管生成中的作用。我们的结果表明，HS 拮抗剂抑制了正在进行内皮分化的 DPSCs 的增殖和出芽能力。此外，随着 HS 拮抗剂剂量的增加，促血管生成标记物的表达显著下降，而干性标记物的表达增加。使用短发夹 RNA 沉默 DPSCs 中 HS 生物合成的关键糖基转移酶外切聚糖 1（EXT1），显著改变了它们的基因表达谱。此外，与转导 scrambled 序列的对照 DPSCs 相比，沉默的 DPSCs 表达较低水平的内皮分化标记物，并且血管形成能力降低。体外添加外源性 HS 可挽救沉默的 DPSCs 的出芽能力。接下来，我们将用 -silenced 或对照 DPSCs 接种的可生物降解支架皮下移植到免疫缺陷小鼠中。绿色荧光蛋白转导的 DPSCs 形成了对人 CD31 和血管性血友病因子呈阳性的管腔样结构。在负载 -silenced DPSCs 的支架中，含血管的数量明显低于植入对照 DPSCs 的标本。总之，我们的研究结果揭示了 HS 对 DPSCs 内皮分化和血管生成的关键作用，为 HS 在组织工程和牙髓再生中的应用开辟了新的视角。
