Malheiro Afonso, Morgan Francis, Baker Matthew, Moroni Lorenzo, Wieringa Paul
Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, 6200 MD, Maastricht, the Netherlands.
Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, 6200 MD, Maastricht, the Netherlands.
Biomaterials. 2020 Oct;257:120230. doi: 10.1016/j.biomaterials.2020.120230. Epub 2020 Jul 11.
In vitro peripheral nerve models provide valuable tools to study neurobiology questions and assess drug performance, in a regenerative or pathology context. To this end, we have developed a representative model of the peripheral nerve that displays three-dimensional (3D) neural anisotropy and myelination, which we showcase here as a simple and low-cost platform for drug screening. The model is composed of three main parts, including rat primary Schwann cells (SCs) seeded onto an electrospun scaffold to create bands of Büngner (BoB), primed PC12 cells as neuronal cell population, and a fibrin hydrogel to provide three-dimensionality. We also validated the use of primed PC12 as a neuron population by comparing it to rat dorsal root ganglions (DRGs) neurons. In both models we could obtain well aligned neurites and mature myelin segments. In short term cultures (7 days), we found that the addition of exogenous SCs enhanced neurite length and neurite growth area, compared to scaffolds with a laminin coating only. Addition of fibrin also lead to increased outgrowth of DRG and primed PC12 neurites, compared to 2D cultures. Moreover, neurite outgrowth in fibrin cultures was simultaneously multiplanar and anisotropic, suggesting that the SC-seeded scaffold can direct not only the growth of adjacent neurites, but also those growing above it. These results highlight the feasibility of the combination of a SC pre-seeded scaffold with a fibrin hydrogel, to direct and improve neurite growth in 3D. To demonstrate the model potential, we tested our platform at an immature (7 days in vitro) and mature state (28 days in vitro) of development. At the immature stage we could inhibit neurite growth through protein blocking (via antibody binding) and show suramin (200 μM) neurotoxicity on cells. At the mature stage, when myelin is compact, we exposed cells to hyperglycemic conditions (45 mM glucose) to mimic diabetic conditions and showed that myelin deforms consequently. Moreover, we demonstrated that by supplementing cultures with epalrestat (1 μM), myelin deformation can be partly prevented. In sum, we developed a biomimetic nerve platform using an affordable and accessible cell line as neuronal population, which displays similar results to primary neurons, but does not require recurrent animal sacrifice. This platform holds great promise as it can be used to conveniently and inexpensively perform drug screenings on peripheral nerve-like tissue, in a normal or pathological state.
体外周围神经模型为研究神经生物学问题和评估药物性能提供了有价值的工具，可用于再生或病理研究。为此，我们开发了一种具有代表性的周围神经模型，该模型展现出三维（3D）神经各向异性和髓鞘形成，在此我们将其作为一种简单且低成本的药物筛选平台进行展示。该模型由三个主要部分组成，包括接种到电纺支架上以形成许旺氏带（BoB）的大鼠原代雪旺细胞（SCs）、作为神经元细胞群体的预处理PC12细胞，以及用于提供三维结构的纤维蛋白水凝胶。我们还通过将预处理的PC12细胞与大鼠背根神经节（DRG）神经元进行比较，验证了其作为神经元群体的用途。在这两种模型中，我们都能获得排列良好的神经突和成熟的髓鞘节段。在短期培养（7天）中，我们发现与仅涂有层粘连蛋白的支架相比，添加外源性雪旺细胞可增加神经突长度和神经突生长面积。与二维培养相比，添加纤维蛋白也能促进DRG和预处理PC12神经突的生长。此外，纤维蛋白培养中的神经突生长同时具有多平面性和各向异性，这表明接种了雪旺细胞的支架不仅可以引导相邻神经突的生长，还能引导其上方神经突的生长。这些结果突出了预先接种雪旺细胞的支架与纤维蛋白水凝胶相结合在三维空间中引导和改善神经突生长的可行性。为了证明该模型的潜力，我们在发育的不成熟阶段（体外7天）和成熟阶段（体外28天）对我们的平台进行了测试。在不成熟阶段，我们可以通过蛋白质阻断（通过抗体结合）抑制神经突生长，并显示苏拉明（200μM）对细胞的神经毒性。在成熟阶段，当髓鞘致密时，我们将细胞暴露于高血糖条件（45mM葡萄糖）以模拟糖尿病状态，并表明髓鞘会因此变形。此外，我们证明通过向培养物中添加依帕司他（1μM），可以部分防止髓鞘变形。总之，我们使用一种经济实惠且易于获取的细胞系作为神经元群体开发了一种仿生神经平台，其结果与原代神经元相似，但无需反复牺牲动物。该平台具有很大的潜力，因为它可用于在正常或病理状态下方便且廉价地对外周神经样组织进行药物筛选。
