Background Bombyx mori silk fibroin is a biomacromolecule that allows the assembly of scaffolds for tissue engineering and regeneration purposes due to its cellular adhesiveness, high biocompatibility and low immunogenicity. Earlier work showed that two types of 3D silk fibroin nonwovens (3D-SFnws) implanted into mouse subcutaneous tissue were promptly vascularized via undefined molecular mechanisms. The present study used nontumorigenic adult human dermal fibroblasts (HDFs) adhering to a third type of 3D-SFnws to assess whether HDFs release exosomes whose contents promote neoangiogenesis. Methods Electron microscopy imaging and physical tests defined the features of the novel carded/hydroentangled 3D-SFnws. HDFs were cultured on 3D-SFnws and polystyrene plates in an exosome-depleted medium. DNA amounts and D-glucose consumption revealed the growth and metabolic activities of HDFs on 3D-SFnws. CD9-expressing total exosome fractions were from conditioned media of 3D-SFnws and 2D polystyrene plates HDF cultures. Angiogenic growth factors (AGFs) in equal amounts of the two groups of exosomal proteins were analysed via double-antibody arrays. A tube formation assay using human dermal microvascular endothelial cells (HDMVECs) was used to evaluate the exosomes’ angiogenic power. Results The novel features of the 3D-SFnws met the biomechanical requirements typical of human soft tissues. By experimental day 15, 3D-SFnws-adhering HDFs had increased 4.5-fold in numbers and metabolized 5.4-fold more D-glucose than at day 3 in vitro. Compared to polystyrene-stuck HDFs, exosomes from 3D-SFnws-adhering HDFs carried significantly higher amounts of AGFs, such as interleukin (IL)-1α, IL-4 and IL-8; angiopoietin-1 and angiopoietin-2; angiopoietin-1 receptor (or Tie-2); growth-regulated oncogene (GRO)-α, GRO-β and GRO-γ; matrix metalloproteinase-1; tissue inhibitor metalloproteinase-1; and urokinase-type plasminogen activator surface receptor, but lesser amounts of anti-angiogenic tissue inhibitor metalloproteinase-2 and pro-inflammatory monocyte chemoattractant protein-1. At concentrations from 0.62 to 10 μg/ml, the exosomes from 3D-SFnws-cultured HDFs proved their angiogenic power by inducing HDMVECs to form significant amounts of tubes in vitro. Conclusions The structural and mechanical properties of carded/hydroentangled 3D-SFnws proved their suitability for tissue engineering and regeneration applications. Consistent with our hypothesis, 3D-SFnws-adhering HDFs released exosomes carrying several AGFs that induced HDMVECs to promptly assemble vascular tubes in vitro. Hence, we posit that once implanted in vivo, the 3D-SFnws/HDFs interactions could promote the vascularization and repair of extended skin wounds due to burns or other noxious agents in human and veterinary clinical settings.

中文翻译：

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在 3D 丝素蛋白无纺布上培养的成人成纤维细胞外泌体强烈刺激新血管生成

背景 家蚕丝素蛋白是一种生物大分子，由于其细胞粘附性、高生物相容性和低免疫原性，可以组装用于组织工程和再生目的的支架。早期的工作表明，植入小鼠皮下组织的两种 3D 丝素蛋白非织造布 (3D-SFnws) 通过未定义的分子机制迅速血管化。本研究使用粘附于第三种 3D-SFnws 的非致瘤成人真皮成纤维细胞 (HDF) 来评估 HDF 是否释放其内容物促进新血管生成的外泌体。方法 电子显微镜成像和物理测试定义了新型梳理/水刺 3D-SFnws 的特征。HDF 在 3D-SFnws 和聚苯乙烯板上在外泌体耗尽的培养基中培养。DNA 量和 D-葡萄糖消耗揭示了 HDF 在 3D-SFnws 上的生长和代谢活动。表达 CD9 的总外泌体部分来自 3D-SFnws 和 2D 聚苯乙烯板 HDF 培养物的条件培养基。通过双抗体阵列分析等量的两组外泌体蛋白中的血管生成生长因子 (AGF)。使用人真皮微血管内皮细胞 (HDMVECs) 的管形成试验用于评估外泌体的血管生成能力。结果 3D-SFnws 的新特性满足了人体软组织的典型生物力学要求。到实验第 15 天，与体外实验第 3 天相比，粘附 3D-SFnws 的 HDF 数量增加了 4.5 倍，代谢的 D-葡萄糖增加了 5.4 倍。与聚苯乙烯粘合的 HDF 相比，来自 3D-SFnws 粘附 HDF 的外泌体携带显着更高量的 AGF，例如白细胞介素 (IL)-1α、IL-4 和 IL-8；血管生成素-1 和血管生成素-2；血管生成素-1 受体（或 Tie-2）；生长调节癌基因 (GRO)-α、GRO-β 和 GRO-γ；基质金属蛋白酶-1；组织抑制剂金属蛋白酶-1；和尿激酶型纤溶酶原激活物表面受体，但较少量的抗血管生成组织抑制剂金属蛋白酶-2和促炎单核细胞趋化蛋白-1。在 0.62 到 10 μg/ml 的浓度下，来自 3D-SFnws 培养的 HDF 的外泌体通过诱导 HDMVEC 在体外形成大量管子证明了它们的血管生成能力。结论梳理/水刺 3D-SFnws 的结构和机械性能证明了它们适用于组织工程和再生应用。与我们的假设一致，3D-SFnws-adhering HDFs 释放携带几种 AGFs 的外泌体，这些 AGFs 诱导 HDMVECs 在体外迅速组装血管。因此，我们假设一旦植入体内，3D-SFnws/HDFs 相互作用可以促进血管化和修复人类和兽医临床环境中由于烧伤或其他有害物质引起的扩展皮肤伤口。