The tendon/ligament-to-bone transition (enthesis) is a highly specialized interface tissue with structural gradients of extracellular matrix composition, collagen molecule alignment and mineralization. These structural features are essential for enthesis function, but are often not regenerated after injury. Tissue engineering is a promising strategy for enthesis repair. Engineering of complex tissue interfaces such as the enthesis is likely to require a combination of biophysical, biological and chemical cues to achieve functional tissue regeneration. In this study, we cultured human primary adipose-derived mesenchymal stem cells (AdMCs) on biphasic silk fibroin scaffolds with integrated anisotropic (tendon/ligament-like) and isotropic (bone/cartilage like) pore alignment. We functionalized those scaffolds with heparin and explored their ability to deliver transforming growth factor β2 (TGF-β2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-β2 and GDF5 remaining attached to the scaffold matrix and resulted in biological effects at low growth factor doses. We analyzed the combined impact of pore alignment and growth factors on AdMSCs. TGF-β2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-β2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity. Altogether, the data obtained in this study improves current understanding on the combined effects of biological and structural cues on stem cell fate and presents a promising strategy for tendon/ligament-to-bone regeneration.

Statement of significance

Regeneration of the tendon/ligament-to-bone interface (enthesis) is of significance in the repair of ruptured tendons/ligaments to bone to improve implant integration and clinical outcome. This study proposes a novel approach for enthesis regeneration based on a biomimetic and integrated tendon/ligament-to-bone construct, stem cells and heparin-based delivery of growth factors. We show that heparin can keep growth factors local and biologically active at low doses, which is critical to avoid supraphysiological doses and associated side effects. In addition, we identify synergistic effects of biological (growth factors) and structural (pore alignment) cues on stem cells. These results improve current understanding on the combined impact of biological and structural cues on the multi-lineage differentiation capacity of stem cells for regenerating complex tissue interfaces.

中文翻译：

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肝素功能化可增加TGF-β2和GDF5在双相丝素蛋白支架上的保留，用于肌腱/韧带到骨的组织工程

肌腱/韧带到骨的过渡（增强）是一种高度专业化的界面组织，具有细胞外基质组成，胶原分子排列和矿化的结构梯度。这些结构特征对于综合功能必不可少，但通常在受伤后不会再生。组织工程学是修复前体的有前途的策略。复杂的组织界面（例如生物体）的工程化可能需要结合生物物理，生物学和化学线索，以实现功能性组织再生。在这项研究中，我们在具有整合的各向异性（肌腱/韧带样）和各向同性（骨骼/软骨样）孔隙排列的双相丝素蛋白支架上培养了人类原发于脂肪的间充质干细胞（AdMC）。我们用肝素功能化这些支架，并探讨了它们传递转化生长因子β2（TGF-β2）和生长/分化因子5（GDF5）的能力。肝素功能化增加了附着在支架基质上的TGF-β2和GDF5的​​数量，并在低生长因子剂量下产生了生物学效应。我们分析了孔排列和生长因子对AdMSCs的综合影响。TGF-β2和毛孔各向异性协同增加了肌腱/韧带标志物的表达和胶原I蛋白的含量。另外，TGF-β2和GDF5的​​联合递送增强了各向同性孔隙度基质上的软骨标志物的表达和II型胶原蛋白的含量，而在各向异性/各向同性孔隙度混合区域中的合成标志物得到了增强。共，

重要声明

肌腱/韧带-骨界面的再生（修复）在修复断裂的肌腱/韧带至骨骼以改善植入物整合和临床效果方面具有重要意义。这项研究提出了一种基于仿生和整合的肌腱/韧带至骨骼构建物，干细胞和肝素为基础的生长因子递送的新的骨架再生方法。我们表明，肝素可以在低剂量时保持生长因子局部和生物活性，这对于避免超生理剂量和相关的副作用至关重要。此外，我们确定了生物学（生长因子）和结构（孔排列）线索对干细胞的协同作用。