Electrospun gelatin/PCL and collagen/PCL scaffolds for modulating responses of bone marrow endothelial progenitor cells
Electrospun gelatin/PCL and collagen/PCL scaffolds for modulating responses of bone marrow endothelial progenitor cells
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用于调节骨髓内皮祖细胞反应的电纺明胶/PCL和胶原/PCL支架
DOI：
10.3892/etm.2019.7387
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发表时间：
2019-05-01
影响因子：
2.7
通讯作者：
Fu, Yao
中科院分区：
文献类型：
--
作者：
Hu, Yang;Feng, Bei;Fu, Yao
确定角膜内皮移植的潜在可移植基质和替代细胞可以弥补角膜供体的不足。目前，关于构建生物可降解、生物相容性良好的组织工程化角膜基质及种子细胞的研究越来越多。在本研究中，静电纺丝明胶/聚己内酯（PCL）和胶原蛋白/PCL支架成功地建立。将骨髓内皮祖细胞（BEPC）在这些支架上培养，以确定支架是否可以促进BEPC的增殖以及保持干细胞特性。通过静电纺丝建立了两种混合支架，胶原/PCL（70%胶原和30%PCL）和明胶/PCL（70%明胶和30%PCL）。随后对两种支架的微观结构、亲水性和润湿性进行了研究。将BEPC分别在支架上培养，并接种在载玻片上作为对照组。此外，细胞形态;通过研究F-肌动蛋白表达水平确定的粘附;通过细胞计数试剂盒-8测定、Ki-67染色和溴脱氧尿苷（BrdU）染色确定的增殖;和通过分化簇（CD）-34和CD-133蛋白表达水平确定的干细胞标志物。此外，进行逆转录-定量聚合酶链反应（RT-qPCR）以确定基因表达。采用静电纺丝技术制备了两种具有预期亲水性、润湿性和生物相容性的生物支架。BEPC在胶原/PCL（70：30）和明胶/PCL（70：30）支架上铺展良好并牢固粘附。此外，Ki-67和BrdU染色结果显示，与对照组相比，两种杂交支架上的阳性点水平更高。与对照组相比，CD-34和CD-133蛋白染色显示支架上的荧光强度水平增加。此外，在两种支架上均检测到分化标记物的表达水平增加，如ATP结合盒亚家族G成员2、富含亮氨酸重复序列的G蛋白偶联受体5和CD 166。RT-qPCR结果表明，与细胞凋亡相关的caspase-3的表达在两种支架上与对照组相比有所减少。与对照组相比，明胶/PCL支架上炎性因子（包括白细胞介素（IL）-1）的表达显著降低;而胶原/PCL组和对照组所表现出的IL-1表达水平之间的差异无显著差异。电纺胶原/PCL和明胶/PCL支架具有增强BEPC粘附和增殖的潜力。在两种支架上培养的BEPC表现出增加的干细胞特性和分化潜力。静电纺丝明胶/PCL和胶原/PCL支架可能是组织工程角膜内皮的一种有前途的基质。
The determination of potential transplantable substrates and substitution cells for corneal endothelium transplantation may compensate for the shortage of cornea donors. Appropriate biodegradable and biocompatible tissue-engineered substratum with seed cells for endothelial keratoplasty has been increasingly studied. In the present study, electrospun gelatin/polycaprolactone (PCL) and collagen/PCL scaffolds were successfully established. Bone marrow endothelial progenitor cells (BEPCs) were cultured on these scaffolds to determine whether the scaffolds may promote the proliferation of BEPCs as well as maintain stem cell characteristics. Two variations of hybrid scaffolds, collagen/PCL (70% collagen and 30% PCL) and gelatin/PCL (70% gelatin and 30% PCL), were established via electrospinning. Microscopic structure, hydrophilicity and wettability of the two scaffolds were subsequently investigated. BEPCs were separately cultured on the scaffolds and were also seeded on glass slides to establish the control group. Furthermore, cell morphology; adherence, as determined by investigation of F-actin expression levels; proliferation, as determined via Cell Counting Kit-8 assays, Ki-67 staining and bromodeoxyuridine (BrdU) staining; and stem cell markers, as determined by cluster of differentiation (CD)-34 and CD-133 protein expression levels; were investigated. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine gene expression. The two nanofiber scaffolds were established using electrospun techniques with expected hydrophilicity, wettability and biocompatibility. BEPCs were revealed to spread well on and strongly adhere to the collagen/PCL (70:30) and gelatin/PCL (70:30) scaffolds. Furthermore, Ki-67 and BrdU staining results revealed greater levels of positive dots on the two hybrid scaffolds compared with the control group. CD-34 and CD-133 protein staining demonstrated increased levels of fluorescence intensity on scaffolds compared with the control group. Furthermore, increased expression levels of differentiation markers, such as ATP binding cassette subfamily G member 2, leucine rich repeat containing G protein-coupled receptor 5 and CD166, were detected on both scaffolds. RT-qPCR results demonstrated that the expression of caspase-3, which is associated with apoptosis, was decreased on the two scaffolds compared with in the control group. The expression of inflammatory factors, including interleukin (IL)-1, exhibited a significant decrease on the gelatin/PCL scaffold compared with in the control group; whereas the difference between the expression level of IL-1 exhibited by the collagen/PCL group and the control group were not markedly different. Electrospun collagen/PCL and gelatin/PCL scaffolds exhibited the potential to enhance the adherence and proliferation of BEPCs. BEPCs cultured on the two scaffolds demonstrated increased stem cell characteristics and differentiation potential. Electrospun gelatin/PCL and collagen/PCL scaffolds may represent a promising substratum in tissue-engineered corneal endothelium.