Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Årstadveien 19, N-5009, Bergen, Norway.
Stem Cell Res Ther. 2018 Mar 21;9(1):69. doi: 10.1186/s13287-018-0815-3.
Angiogenesis is of utmost importance for tissue regeneration and repair. Human dental pulp stromal cells (hDPSCs) possess angiogenic potential, as they secrete paracrine factors that may alter the host microenvironment. However, more insight into how hDPSCs guide endothelial cells (ECs) in a paracrine fashion is yet to be obtained. Therefore, the current study aimed to investigate the effect(s) of conditioned medium derived from hDPSCs (hDPSC-CM) on EC behavior in vitro.
hDPSCs were harvested from third molars scheduled for surgical removal under informed consent. The angiogenic profile of hDPSC-CM was identified using human angiogenesis antibody array and enzyme-linked immunosorbent assay (ELISA). Using real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, the mRNA and protein expression level of specific angiogenic biomarkers was determined in human umbilical vein endothelial cells (HUVECs) exposed to hDPSC-CM. The effect of hDPSC-CM on HUVEC attachment, proliferation and migration was evaluated by crystal violet staining, MTT, transwell migration along with real-time cell monitoring assays (xCELLigence; ACEA Biosciences, Inc.). A Matrigel assay was included to examine the influence of hDPSC-CM on HUVEC network formation. Endothelial growth medium (EGM-2) and EGM-2 supplemented with hDPSC-CM served as experimental groups, whereas endothelial basal medium (EBM-2) was set as negative control.
A wide range of proangiogenic and antiangiogenic factors, including vascular endothelial growth factor, tissue inhibitor of metalloproteinase protein 1, plasminogen activator inhibitor (serpin E1), urokinase plasminogen activator and stromal cell-derived factor 1, was abundantly detected in hDPSC-CM by protein profiling array and ELISA. hDPSC-CM significantly accelerated the adhesion phases, from sedimentation to attachment and spreading, the proliferation rate and migration of HUVECs as shown in both endpoint assays and real-time cell analysis recordings. Furthermore, Matrigel assay demonstrated that hDPSC-CM stimulated tubulogenesis, affecting angiogenic parameters such as the number of nodes, meshes and total tube length.
The sustained proangiogenic and promaturation effects of hDPSC-CM shown in this in vitro study strongly suggest that the trophic factors released by hDPSCs are able to trigger pronounced angiogenic responses, even beyond EGM-2 considered as an optimal culture condition for ECs.
血管生成对于组织再生和修复至关重要。人牙髓基质细胞（hDPSCs）具有血管生成潜力，因为它们分泌旁分泌因子，可能改变宿主微环境。然而，hDPSCs 如何以旁分泌方式引导内皮细胞（ECs）的更多见解尚待获得。因此，本研究旨在探讨条件培养基（hDPSC-CM）对体外 EC 行为的影响。
从知情同意下计划手术切除的第三磨牙中提取 hDPSCs。使用人血管生成抗体阵列和酶联免疫吸附试验（ELISA）鉴定 hDPSC-CM 的血管生成谱。通过实时逆转录-聚合酶链反应（RT-PCR）和 ELISA 检测 hDPSC-CM 暴露后人脐静脉内皮细胞（HUVECs）中特定血管生成生物标志物的 mRNA 和蛋白表达水平。通过结晶紫染色、MTT、Transwell 迁移以及实时细胞监测分析（xCELLigence；Acea Biosciences，Inc.）评估 hDPSC-CM 对 HUVEC 附着、增殖和迁移的影响。Matrigel 测定用于检测 hDPSC-CM 对 HUVEC 网络形成的影响。内皮生长培养基（EGM-2）和添加 hDPSC-CM 的 EGM-2 作为实验组，而内皮基础培养基（EBM-2）作为阴性对照。
蛋白质谱阵列和 ELISA 检测到 hDPSC-CM 中存在广泛的促血管生成和抗血管生成因子，包括血管内皮生长因子、金属蛋白酶组织抑制剂 1 蛋白、纤溶酶原激活物抑制剂（serpin E1）、尿激酶型纤溶酶原激活物和基质细胞衍生因子 1。hDPSC-CM 显著加速了 HUVECs 的附着相，从沉降到附着和伸展，终点测定和实时细胞分析记录均显示增殖率和迁移率增加。此外，Matrigel 测定表明 hDPSC-CM 刺激了管腔形成，影响了节点、网格和总管长度等血管生成参数。
本体外研究显示 hDPSC-CM 持续的促血管生成和促成熟作用强烈表明，hDPSCs 释放的营养因子能够引发明显的血管生成反应，甚至超出 EGM-2，后者被认为是 ECs 的最佳培养条件。
