背景:牙髓再生是一种治疗未成熟恒牙牙髓坏死的新方法。这种技术包括干细胞的组合,脚手架,和增长因素。最近,干细胞衍生的细胞外囊泡(EV)已成为一种新的牙髓再生方法。新的证据已经证明,预处理是一种有效的方案,以修改电动汽车更好的治疗效力。同时,正确的脚手架对于保护电动汽车免受快速清除和破坏具有重要意义。这项研究旨在制造一种可注射的水凝胶,其中装有来自人脱落乳牙(SHED)的预分化干细胞的EV,并检查其对牙髓再生的影响。
结果:我们成功地使用了SHEDs的牙源性诱导培养基(OM)来产生功能性EV(OM-EV)。证明浓度为20µg/mL的OM-EV可促进牙髓干细胞(DPSC)的增殖和迁移。结果表明,在体外通过茜素红phalloidin,OM-EV比普通EV(CM-EV)具有更好的促进DPSCs牙源性分化的潜力,碱性磷酸酶染色,并评估牙源性相关标志物的表达。高通量测序表明,OM-EV的优异作用可能归因于AMPK/mTOR途径的激活。同时,我们制备了可光交联的甲基丙烯酰明胶(GelMA)来构建OM-EV封装的水凝胶。水凝胶显示出OM-EV的持续释放和DPSC的良好生物相容性。从水凝胶释放的OM-EV可以被DPSC内化,从而提高他们的生存和迁移。在裸鼠皮下移植的牙根切片中,发现OM-EV包封的水凝胶促进牙本质生成。8周后,有更多的矿化组织形成,以及较高水平的牙本质唾液酸糖蛋白(DSPP)和牙本质基质蛋白-1(DMP-1)。
结论:通过预处理SHED可以显著增强EV的作用。SHEDs联合GelMA的功能性EV能够通过上调DPSC的牙源性分化来有效促进牙本质生成。这为牙髓再生提供了一种有希望的治疗方法。
BACKGROUND: Pulp regeneration is a novel approach for the treatment of immature permanent teeth with pulp necrosis. This technique includes the combination of stem cells, scaffolds, and growth factors. Recently, stem cell-derived extracellular vesicles (EVs) have emerged as a new methodology for pulp regeneration. Emerging evidence has proven that preconditioning is an effective scheme to modify EVs for better therapeutic potency. Meanwhile, proper scaffolding is of great significance to protect EVs from rapid clearance and destruction. This investigation aims to fabricate an injectable hydrogel loaded with EVs from pre-differentiated stem cells from human exfoliated deciduous teeth (SHEDs) and examine their effects on pulp regeneration.
RESULTS: We successfully employed the odontogenic induction medium (OM) of SHEDs to generate functional EV (OM-EV). The OM-EV at a concentration of 20 µg/mL was demonstrated to promote the proliferation and migration of dental pulp stem cells (DPSCs). The results revealed that OM-EV has a better potential to promote odontogenic differentiation of DPSCs than common EVs (CM-EV) in vitro through Alizarin red phalloidin, alkaline phosphatase staining, and assessment of the expression of odontogenic-related markers. High-throughput sequencing suggests that the superior effects of OM-EV may be attributed to activation of the AMPK/mTOR pathway. Simultaneously, we prepared a photocrosslinkable gelatin methacryloyl (GelMA) to construct an OM-EV-encapsulated hydrogel. The hydrogel exhibited sustained release of OM-EV and good biocompatibility for DPSCs. The released OM-EV from the hydrogel could be internalized by DPSCs, thereby enhancing their survival and migration. In tooth root slices that were subcutaneously transplanted in nude mice, the OM-EV-encapsulated hydrogel was found to facilitate dentinogenesis. After 8 weeks, there was more formation of mineralized tissue, as well as higher levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1).
CONCLUSIONS: The effects of EV can be substantially enhanced by preconditioning of SHEDs. The functional EVs from SHEDs combined with GelMA are capable of effectively promoting dentinogenesis through upregulating the odontogenic differentiation of DPSCs, which provides a promising therapeutic approach for pulp regeneration.