UNASSIGNED: Gelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia.
UNASSIGNED: Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The
islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively.
Islets were cultured under hypoxia (1%O 2) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability.
Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O 2) and hypoxia (1%O 2) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions, respectively.
UNASSIGNED: GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat
islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group ( P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C ( P<0.05).
UNASSIGNED: GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.
UNASSIGNED: 制备甲基丙烯酰化明胶(gelatin methacryloyl,GelMA)/甲基丙烯酰化透明质酸(hyaluronic acid methacryloyl,HAMA)/壳寡糖(chitosan oligosaccharide,COS)水凝胶,用于构建胰岛仿生微环境,并探讨GelMA/HAMA/COS水凝胶对低氧下胰岛活性和功能的改善作用。.
UNASSIGNED: 以组织培养板培养为对照组,COS浓度分别为0、1、5、10、20 mg/mL的GelMA/HAMA/COS水凝胶为实验组,扫描电镜观察微观形态,流变仪评价成胶性能,接触角检测亲水性,水凝胶浸提液培养L929细胞评估生物相容性 [采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法]。从8周龄SD大鼠胰腺中提取胰岛,双硫腙染色和葡萄糖刺激胰岛素释放(glucose-stimulated insulin secretion,GSIS)实验分别鉴定胰岛纯度和功能。胰岛在低氧(1%O 2)下分别培养24、48、72 h,使用钙黄绿素-乙酰甲氧基甲酯/碘化丙啶(Calcein-acetyl methyl/propidium iodide,Calcein-AM/PI)染色评价低氧对胰岛活性的影响。将胰岛在不同COS浓度的GelMA/HAMA/COS水凝胶中培养48 h,活性氧试剂盒评价COS在常氧(20%O 2)和低氧(1%O 2)条件下拮抗胰岛活性氧产生情况,Calcein-AM/PI染色评价COS在低氧(1%O 2)条件下对胰岛活性的影响。将胰岛分别在组织培养板(A组)、GelMA/HAMA水凝胶(B组)和GelMA/HAMA/COS水凝胶(C组)中培养48 h,免疫荧光和GSIS实验分别评价COS在低氧(1%O 2)条件下对胰岛活性的影响。.
UNASSIGNED: GelMA/HAMA/COS水凝胶呈多孔结构,流变仪检测提示其具有较好的成胶性能,接触角表现出良好亲水性,CCK-8法检测示各组水凝胶均具有良好的生物相容性。分离的大鼠胰岛呈类圆形,具有较高的胰岛纯度和胰岛素分泌能力。胰岛在低氧条件下处理24、48、72 h,Calcein-AM/PI染色示死细胞比例随时间延长逐渐增多,均显著高于未低氧处理组( P<0.001)。活性氧染色示不同COS浓度的GelMA/HAMA/COS水凝胶在常氧和低氧条件下均能拮抗活性氧的产生,且该能力与COS浓度成正相关。Calcein-AM/PI染色示,不同COS浓度的GelMA/HAMA/COS水凝胶在低氧条件下均能改善胰岛活性,细胞活性与COS浓度成正相关。免疫荧光染色示GelMA/HAMA/COS水凝胶在低氧条件下能促进胰岛功能相关基因的表达;GSIS实验结果显示C组胰岛在低氧条件胰岛素分泌量显著高于A、B组( P<0.05)。.
UNASSIGNED: GelMA/HAMA/COS水凝胶具有良好的生物相容性,通过抑制活性氧提高胰岛的存活和功能,是构建胰岛仿生微环境用于胰岛培养及移植的理想载体。.