背景:生物材料必须允许血运重建才能成功进行组织再生。由细胞外基质(ECM)配制的生物材料由于其优越的生物相容性而在组织工程中获得了普及,由于它们的流变特性,ECM-水凝胶可以很容易地应用于受损区域,允许细胞定植并整合到宿主组织中。猪膀胱ECM(pUBM)保留功能信号和结构蛋白,是再生医学的绝佳选择。甚至一些小分子,例如抗微生物的cathelicidin衍生的LL-37肽已被证明具有血管生成性质。
目的:本研究的目的是评估用LL-37肽(pUBMh/LL37)生物功能化的源自猪膀胱(pUBMh)的ECM-水凝胶的生物相容性和血管生成潜力。
方法:巨噬细胞,成纤维细胞,和脂肪组织间充质干细胞(AD-MSC)暴露于pUBMh/LL37,并通过MTT法评价其对细胞增殖的影响,通过定量乳酸脱氢酶释放和活/死细胞成像测定的细胞毒性。此外,巨噬细胞产生IL-6、IL-10、IL-12p70、MCP-1、INF-γ、使用基于珠子的细胞计数阵列定量TNF-α细胞因子。pUBMh/LL37通过背侧皮下注射直接植入Wistar大鼠24小时以评估生物相容性,和pUBMh/LL37负载的血管反应器植入21天以评估血管生成。
结果:我们发现pUBMh/LL37不影响细胞增殖,并且与所有测试的细胞系细胞相容,但在巨噬细胞中诱导TNF-α和MCP-1的产生。在体内,这种ECM-水凝胶诱导材料内的成纤维细胞样细胞募集,在48小时没有组织损伤或炎症。有趣的是,在21天观察到血管反应器内部的血管系统的组织重塑。
结论:我们的结果表明pUBMh/LL37在细胞学上是相容的,并在体内诱导血管生成,显示组织再生疗法的潜力。
BACKGROUND: Biomaterials must allow revascularization for a successful tissue regeneration process. Biomaterials formulated from the extracellular matrix (ECM) have gained popularity in tissue engineering because of their superior biocompatibility, and due to their rheological properties, ECM-hydrogels can be easily applied in damaged areas, allowing cell colonization and integration into the host tissue. Porcine urinary bladder ECM (pUBM) retains functional signaling and structural proteins, being an excellent option in regenerative medicine. Even some small molecules, such as the antimicrobial cathelicidin-derived LL-37 peptide have proven angiogenic properties.
OBJECTIVE: The objective of this study was to evaluate the biocompatibility and angiogenic potential of an ECM-hydrogel derived from the porcine urinary bladder (pUBMh) biofunctionalized with the LL-37 peptide (pUBMh/LL37).
METHODS: Macrophages, fibroblasts, and adipose tissue-derived mesenchymal stem cells (AD-MSC) were exposed pUBMh/LL37, and the effect on cell proliferation was evaluated by MTT assay, cytotoxicity by quantification of lactate dehydrogenase release and the Live/Dead Cell Imaging assays. Moreover, macrophage production of IL-6, IL-10, IL-12p70, MCP-1, INF-γ, and TNF-α cytokines was quantified using a bead-based cytometric array. pUBMh/LL37 was implanted directly by dorsal subcutaneous injection in Wistar rats for 24 h to evaluate biocompatibility, and pUBMh/LL37-loaded angioreactors were implanted for 21 days for evaluation of angiogenesis.
RESULTS: We found that pUBMh/LL37 did not affect cell proliferation and is cytocompatible to all tested cell lines but induces the production of TNF-α and MCP-1 in macrophages. In vivo, this ECM-hydrogel induces fibroblast-like cell recruitment within the material, without tissue damage or inflammation at 48 h. Interestingly, tissue remodeling with vasculature inside angioreactors was seen at 21 days.
CONCLUSIONS: Our results showed that pUBMh/LL37 is cytologically compatible, and induces angiogenesis in vivo, showing potential for tissue regeneration therapies.