Abstract：
In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF β2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF β2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF β2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHAs retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with increasing process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF β2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONH astrocyte-encapsulated hydrogel to investigate astrocyte behavior in response to injury.
摘要：
在青光眼中，视神经乳头（ONH）内的星形胶质细胞重排其肌动蛋白细胞骨架，同时变得反应性和上调中间丝胶质纤维酸性蛋白（GFAP）。转化生长因子β2（TGFβ2）水平升高与青光眼ONH功能障碍有关。使用常规2D培养研究ONH星形胶质细胞行为的关键限制是不能忠实地复制体内ONH微环境。这里,我们设计了3DONH星形胶质细胞水凝胶，以更好地模拟体内小鼠ONH星形胶质细胞（MONHA）形态，并使用TGFβ2测试MONHA反应性的诱导。从C57BL/6J小鼠中分离原代MONHA并确认细胞纯度。为了设计3D充满细胞的水凝胶，MONHAs与光活性细胞外基质成分（I型胶原蛋白，透明质酸）并使用光引发剂（0.025％核黄素）和紫外线（405-500nm，10.3mW/cm2)。MOHA包裹的水凝胶培养3周，然后用TGFβ2(2.5、5.0或10ng/ml)处理7天以评估反应性。封装后，通过活/死染色和MTS测定确定,MONHAs在水凝胶中保持高细胞活力并持续增殖超过4周。Sholl分析表明,水凝胶内的MONHA随着时间的推移随着过程长度的增加而发展出增加的过程复杂性。细胞进程与相邻细胞相连，与星形细胞过程中的Connexin43表达一致。如通过改变的F-肌动蛋白细胞骨架形态确定的，用TGFβ2处理在MOHA包裹的水凝胶中诱导的反应性，GFAP表达增加，纤维连接蛋白和胶原IV沉积升高。我们的数据为将来使用这种3D仿生ONH星形胶质细胞包裹的水凝胶研究星形胶质细胞对损伤的反应奠定了基础。
