Table_1_Silencing VEGFR-2 Hampers Odontoblastic Differentiation of Dental Pulp Stem Cells.docx

Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.牙髓干细胞（Dental pulp stem cells，简称DPSCs）是成体干细胞的重要来源，对牙本质与牙髓组织的维持与再生至关重要。既往体内移植研究表明，DPSCs可分化为成牙本质样细胞，形成牙本质/牙髓样结构，并诱导血管生成。值得注意的是，牙本质形成与血管生成密切相关。我们既往研究证实，DPSCs诱导的血管生成依赖于血管内皮生长因子受体2（VEGFR-2）。VEGFR-2可能在DPSCs的成牙本质细胞分化、牙齿形成与再生中发挥重要作用。然而，VEGFR-2信号通路在DPSCs成牙本质细胞分化中的作用仍未完全阐明。 因此，本研究旨在通过敲低DPSCs中VEGFR-2的表达，并在体外与体内探究其成牙本质细胞分化能力，以明确VEGFR-2在DPSCs成牙本质细胞分化中的作用。 小鼠DPSCs的分离与鉴定方法参照既往研究方案。 采用VEGFR-2短发夹RNA（shRNA）病毒载体转染DPSCs（感染复数MOI=10:1）以沉默VEGFR-2的表达。 CopGFP标记的DPSCs中GFP阳性表达被用作替代指标，以评估转染效率，并验证病毒载体不会影响VEGFR-2的表达。 与正常DPSCs相比，VEGFR-2敲低的DPSCs中，通过定量实时聚合酶链反应（Q-RT-PCR）与免疫荧光检测到的VEGFR-2表达水平显著降低，证实了病毒转染的有效性。 与CopGFP标记的DPSCs相比，VEGFR-2 shRNA转染的DPSCs不仅VEGFR-2表达水平极低，其配体血管内皮生长因子A（VEGF-A）的转录与翻译水平也显著降低。 将DPSCs在添加了100 ng/ml骨形态发生蛋白2（BMP-2）的成骨-成牙本质诱导培养基中体外诱导分化21天，结果显示，CopGFP标记的DPSCs可呈碱性磷酸酶（Alkaline Phosphatase，ALP）染色阳性，并形成茜素红S（Alizarin Red S）染色阳性的矿化结节，而VEGFR-2 shRNA转染的DPSCs则无此表现。 与VEGFR-2 shRNA转染的DPSCs相比，诱导分化后的CopGFP标记的DPSCs中，牙本质基质蛋白1（Dentin Matrix Protein 1，Dmp1）、牙本质涎蛋白（Dentin Sialoprotein，Dspp）与骨涎蛋白（Bone Sialoprotein，Bsp）的表达水平均显著上调，提示VEGFR-2 shRNA转染的DPSCs的成牙本质细胞分化功能受损。 将DPSCs与羟基磷灰石/磷酸三钙（Hydroxyapatite/Tricalcium Phosphate，HAp/TCP）复合后进行体内皮下移植，持续5周后结果显示，CopGFP标记的DPSCs可分化为长梭形、极化的成牙本质样细胞，形成富含血管的、类似牙髓组织的疏松结缔组织，该结果通过苏木精-伊红（Hematoxylin-Eosin，H&E）染色、茜素红S（Alizarin Red S）染色与牙本质基质染色得以证实。 与之相反，VEGFR-2 shRNA转染的DPSCs移植组中未观察到成牙本质样细胞，仅见胶原纤维替代了牙本质/牙髓样结构。 上述结果表明，VEGFR-2在牙本质再生中发挥重要作用，并凸显了通过调控VEGFR-2以增强牙本质再生与组织工程的潜力，为临床应用提供了新思路。