Initial angiogenesis within the first 3 days is critical for healing ischemic diseases such as myocardial infarction. Recently, decellularized extracellular matrix (dECM) has been reported to provide tissue-derived ECM components and can be used as a scaffold for cell delivery for angiogenesis in tissue engineering. Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, decreased presence of various ECM proteins and cytokines, and loss of mechanical properties. To overcome these limitations, in this study, we developed a supercritical carbon dioxide and ethanol co-solvent (scCO₂-EtOH) decellularization method, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM, and tested on rat heart tissues. The heart tissue was placed into the scCO₂ reactor and decellularized at 37°C and 350 bar. After scCO₂-EtOH treatment, the effects were evaluated by DNA, collagen, and glycosaminoglycan (GAG) quantification and hematoxylin and eosin and immunofluorescence staining to determine the absence of nucleic acids and preservation of heart ECM components. Similar to the native group, the scCO₂-EtOH group contained more ECM components such as collagen, GAGs, collagen I, laminin, and fibronectin and angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and others in comparison to the detergent group. In addition, to estimate angiogenesis of the dECM hydrogels, the neutralized dECM solution was injected in a rat subcutaneous layer (n=6 in each group: collagen, scCO₂-EOH, and detergent group), after which the solution naturally formed gelation in the subcutaneous layer. After 3 days, the gels were harvested and estimated by immunofluorescence staining and the ImageJ program for angiogenesis analysis. Consequently, blood vessel formation and density of vWF and α-SMA in the scCO₂-EtOH group were significantly greater than that in the collagen group. Here we suggest that heart-derived decellularized extracellular matrix (dECM) with scCO₂-EtOH treatment is a highly promising angiogenic material for healing in ischemic disease.

Statement of significance

Supercritical carbon dioxide (scCO₂) in a supercritical phase has low viscosity and high diffusivity between gas and liquid properties and is known to be affordable, non-toxic, and eco-friendly. Therefore, scCO₂ extraction technology has been extensively used in commercial and industrial fields. Recently, decellularized extracellular matrix (dECM) was applied to tissue engineering and regenerative medicine as a scaffold, therapeutic material, and bio-ink for 3D printing. Moreover, the general decellularization method using detergents has limitations including eliminating tissue-derived ECM components and disrupting their structures after decellularization. To overcome these limitations, heart tissues were treated with scCO₂-EtOH for decellularization, resulting in preserving of tissue due to the various ECM and angiogenic factors derived. In addition, initiation of angiogenesis was highly induced even after 3 days of injection.

中文翻译：

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超临界二氧化碳脱细胞心脏ECM水凝胶改善血管生成

前3天内的初始血管生成对于治愈缺血性疾病（例如心肌梗塞）至关重要。最近，据报道脱细胞的细胞外基质（dECM）提供了组织衍生的ECM成分，可以用作组织工程中血管生成的细胞递送支架。通过各种去污剂（例如十二烷基硫酸钠（SDS）和Triton X-100）进行脱细胞可以去除组织器官中的细胞核。然而，这导致ECM结构变性，各种ECM蛋白和细胞因子的存在减少以及机械性能的损失。为了克服这些限制，在这项研究中，我们开发了一种超临界二氧化碳和乙醇助溶剂（scCO ₂-EtOH）脱细胞法，这是一种无洗涤剂的系统，可防止ECM结构破坏并在心脏dECM中保留各种血管生成蛋白，并已在大鼠心脏组织上进行了测试。将心脏组织放入scCO ₂反应器中，并在37°C和350 bar下脱细胞。scCO ₂ -EtOH处理后，通过DNA，胶原蛋白和糖胺聚糖（GAG）定量，苏木精和曙红以及免疫荧光染色评估效果，以确定是否存在核酸并保留了心脏ECM成分。与本地组类似，scCO ₂与洗涤剂组相比，-EtOH组含有更多的ECM成分，例如胶原蛋白，GAG，胶原蛋白I，层粘连蛋白和纤连蛋白，以及血管生成因子，包括血管内皮生长因子，成纤维细胞生长因子和血小板衍生的生长因子等。另外，为了估计dECM水凝胶的血管生成，将中和的dECM溶液注入大鼠皮下层（每组n = 6：胶原，scCO ₂ -EOH和去污剂组），然后溶液自然形成凝胶。皮下层。3天后，收集凝胶并通过免疫荧光染色和用于血管生成分析的ImageJ程序进行估计。因此，scCO ₂中的血管形成和vWF和α-SMA的密度-EtOH组显着大于胶原蛋白组。在这里，我们建议用scCO ₂ -EtOH治疗心脏源性脱细胞细胞外基质（dECM）是治疗缺血性疾病的高度有希望的血管生成材料。

重要声明

超临界相中的超临界二氧化碳（scCO ₂）具有低粘度和气液特性之间的高扩散性，并且已知价格低廉，无毒且对环境友好。因此，scCO ₂提取技术已广泛用于商业和工业领域。最近，脱细胞的细胞外基质（dECM）作为支架，治疗材料和生物墨水用于3D打印，已应用于组织工程和再生医学。此外，使用去污剂的常规脱细胞方法具有局限性，包括消除组织来源的ECM成分并在脱细胞后破坏其结构。为了克服这些限制，用scCO ₂处理心脏组织-EtOH用于脱细胞，由于各种ECM和衍生的血管生成因子而导致组织保存。另外，甚至在注射3天后也高度诱导了血管生成的开始。