Novel Three-Dimensional Hydrogel Model from Ovine Aortic Valve ECM to Study ECM Remodeling
Novel Three-Dimensional Hydrogel Model from Ovine Aortic Valve ECM to Study ECM Remodeling
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绵羊主动脉瓣 ECM 的新型三维水凝胶模型用于研究 ECM 重塑
DOI：
10.1055/s-0037-1598740
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Akhyari P
中科院分区：
文献类型：
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作者：
Raschke S;Nehrenheim L;Barth M;Barbian A;Lichtenberg A;Akhyari P
目的：主动脉瓣（AV）狭窄是西方世界最常见的瓣膜疾病，与病理生理刺激诱导的瓣膜间质细胞（VIC）转分化有关。然而，大多数分析病理机制的生化研究都是使用经典的二维细胞培养技术进行的，缺少细胞外基质（ECM）蛋白的复杂网络和信号传导。本研究的目的是建立一种新的，创新的水凝胶AV ECM的三维（3D）培养的VICs具有高度相似性，在体内条件下，以揭示新的方面CAVD.Methods：羊主动脉瓣脱细胞十二烷基硫酸钠，脱氧胆酸和叠氮化钠，然后用DNase消化。然后，将AV-ECM冻干，机械均质化（6，800 rpm，30秒，三次重复）以产生均匀的粉末并使用胃蛋白酶/HCL溶液消化。调节pH后，ECM水凝胶在37 ℃下聚合1小时并进一步分析。与I型胶原和基质胶相比，在AV-ECM水凝胶内培养绵羊VIC。细胞活力进行了评估，使用BrdU，LDH，和生死assay.Results：扫描电子显微镜显示，在1，4和6 mg/mL的AV-ECM水凝胶的典型的纤维状外观。比较AV-ECM的水凝胶，I型胶原和Matrigel浊度随着浓度的增加而增加。对于I型胶原蛋白，在4 mg/mL的浓度下测量最高浊度，其次是AV-ECM水凝胶。I型胶原蛋白（8.2±0.4 ng/mL）和GAG（0.89±0.18 µg/mL）保存在AV-ECM中，而在Matrigel中未检测到I型胶原蛋白。与细胞培养塑料（4.24倍，n= 9，p< 0.01）或基质胶（3.07倍，n = 9，p< 0.001）相比，在AV-ECM水凝胶上2D培养的VIC的增殖显著增强，而LDH释放没有增加。有趣的是，与AV-ECM和胶原相比，VIC在基质胶上显示出发散的恒星生长模式。VIC在3D水凝胶中培养时表现出较高的活力，并通过生死染色进行分析。结论：我们的数据表明，AV-ECM代表了一种新的独特的工具，以研究维克信号在3D的方式。因此，AV-ECM是一种有前途的临床前模型，用于研究介导导致主动脉瓣狭窄的不良反应的病理生理机制和潜在信号通路。
Objective: Aortic valve (AV) stenosis is the most common valve disease in the Western world and associated with trans-differentiation of valvular interstitial cells (VICs) induced by pathophysiological stimuli. Nevertheless, most biochemical studies analyzing the pathomechanisms are conducted using classical two-dimensional cell culture techniques missing the complex network and signaling of extracellular matrix (ECM) proteins. The aim of this study was to establish a novel, innovative hydrogel of AV ECM for three-dimensional (3D) cultivation of VICs with high similarity to in vivo conditions to reveal new aspects about CAVD.Methods: Ovine aortic valves were decellularized using sodium dodecyl sulfate, deoxycholate acid and sodium azide followed by DNase digestion. Afterwards, AV-ECM was lyophilized, mechanically homogenized (6,800 rpm, 30 seconds, three repeats) to create homogenous powder and digested using pepsin/HCL solution. After adjusting for pH, ECM hydrogel was polymerized at 37 C for 1h and further analyzed. Ovine VICs were cultured within the AV-ECM hydrogel compared with collagen type I and Matrigel. Cell vitality was assessed using BrdU, LDH, and life–dead assay.Results: Scanning electron microscopy showed a typical fibrillar appearance of the AV-ECM hydrogel at 1, 4 and 6 mg/mL. Comparing the hydrogels of AV-ECM, collagen type I and Matrigel turbidity raised with increasing concentration. Highest turbidity was measured at a concentration of 4 mg/mL for collagen type I, followed by AV-ECM hydrogel. Collagen type I (8.2±0.4 ng/mL) and GAG (0.89±0.18 µg/mL) were preserved in AV-ECM, while collagen I was not detected in Matrigel. Proliferation of VICs cultured 2D on AV-ECM hydrogel was significantly enhanced compared with cell culture plastic (4.24-fold, n= 9, p< 0.01) or Matrigel (3.07-fold, n= 9, p< 0.001), while LDH release was not increased. Interestingly, VICs showed divergent, stellar growth pattern on Matrigel compared with AV-ECM and collagen. VICs showed high vitality when cultured in 3D hydrogel and analyzed by life-dead-staining.Conclusion: Our data show that AV-ECM represents a novel unique tool to study VIC signaling in a 3D manner. Thus, AV-ECM is a promising pre-clinical model to study pathophysiological mechanisms and underlying signaling pathways that mediate adverse effects leading to aortic valve stenosis.