聚羟基乙酸支架的表面改性及在胰岛培养和移植中的应用
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摘要
聚羟基乙酸（Polyglycolic acid, PGA）作为医用的生物可吸收高分子材料是目前生物降解高分子材料中最活跃的研究领域，已经获得美国食品与药品管理局（FDA）的许可。本研究为了提高PGA细胞支架的粘附性，对PGA支架材料进行了表面改性的研究，为胰岛细胞在PGA支架表面能粘附生长提供了良好的粘附条件，从而使PGA支架能更好的应用于胰岛细胞的体外培养与移植的研究中。
通过XPS分析、拉伸性能测试和浸润性测试研究了不同功率、不同时间的等离子体处理对于PGA支架性能的影响。结果表明：处理功率240W，处理时间4min时，PGA支架的拉伸性能以及与生理盐水和血清培养液之间的浸润性较好。采用拉伸性能测试和浸润性测试分别研究了不同浓度的蔗糖、L-谷氨酸和多聚赖氨酸涂层处理对于PGA支架性能的影响，结果表明：涂覆有2mg/ml的多聚赖氨酸涂层的PGA支架的拉伸性能以及与生理盐水和血清之间的浸润性相对于其它涂层要好。最后采用处理功率240W的等离子体处理结合浓度为2mg/ml的多聚赖氨酸涂层处理PGA支架，通过对其拉伸性能以及浸润性能分析，结果表明：等离子体结合多聚赖氨酸涂层处理的PGA支架的拉伸性能和浸润性能比采用单一方法处理的PGA支架的各方面性能都要好。因此，最终采用等离子体结合多聚赖氨酸涂层对PGA支架进行处理，改进其与细胞培养液之间的浸润性，从而提高PGA细胞支架与细胞之间的粘附性。
将大鼠胰岛细胞种植在经表面改性的PGA支架上进行体外培养（静止培养和微重力培养），实验结果显示，与无支架静止培养组胰岛相比，倒置显微镜下可见PGA支架上的胰岛细胞形态完整，生长状态良好，胰岛能与支架紧密粘附，胰岛细胞死亡少。双硫腙（DTZ）染色后PGA支架上的胰岛细胞纯度较好。吖啶橙（AO）和碘化丙啶（PI）荧光双染色法和放射免疫法检测结果显示，PGA支架上的胰岛细胞的生存率及胰岛素含量较高。噻唑蓝比色法（MTT）结果显示，PGA支架材料与胰岛细胞的生物相容性较好。扫描电镜下可见胰岛细胞在PGA支架上能粘附生长，胰岛之间的间隙较大，胰岛可充分地获取营养，PGA支架上胰岛细胞形态良好。激光共聚焦显微镜下可清楚的看到PGA支架上绿色的胰岛β细胞和红色的胰岛α细胞。
PGA-胰岛复合物经过体外共培养5天后，移植到糖尿病大鼠的腿部肌肉内，对PGA-胰岛移植物移植治疗的效果进行比较观察。从血糖和血清胰岛素含量检测结果显示，支架微重力培养移植组的PGA-胰岛细胞移植物，移植后可使糖尿病大鼠体内的血糖值和血清胰岛素含量恢复到正常。扫描电镜、共聚焦显微镜及病理检测结果显示，PGA支架上胰岛细胞的形态良好，支架材料与胰岛细胞的生物相容性较好，胰岛细胞间可见到细胞外基质的形成，呈网状细纤维，PGA-胰岛细胞周围空间较大，胰岛细胞未受到肌肉组织的挤压，在PGA支架之间有新生的毛细血管，胰岛细胞周围可见到红细胞，说明肌肉内的血液供应较丰富，支架上的胰岛细胞能得到充分的营养供应。移植后30天左右，PGA支架开始逐渐断裂，PGA的降解及降解产物对机体没有造成损害。
将PGA支架与猪胰岛细胞共培养，观察猪胰岛细胞体外生存的质量、生物学特征及猪胰岛细胞与PGA支架材料的生物相容性。与静止培养组胰岛相比，AO-PI荧光双染法结果显示，PGA支架可改善猪胰岛细胞的活性，可使猪胰岛生存率增高。放射免疫方法胰岛功能检测结果显示，PGA支架上猪胰岛细胞的胰岛素释放指数较高。MTT检测结果显示，PGA支架与猪胰岛细胞具有较好的生物相容性。倒置显微镜及免疫荧光显微镜观察结果显示，PGA支架上猪胰岛细胞形态良好，猪胰岛细胞在PGA支架上能粘附生长。
实验结果表明，PGA细胞支架可作为胰岛细胞外基质，为胰岛细胞体外生存提供良好的生长环境。尤其是PGA支架微重力培养条件更适合于胰岛细胞的体外生存与体内移植。
Polyglycolic acid (PGA), as a medical biologically-adaptive polymer approvedby FDA, is one of the most studied bio-degradation materials. This research aimed toimprove the adhesion of PGA scaffold via re-modifying the surface materials of PGAscaffolds and to provide better adhesion condition for islet cells on PGA scaffoldswith implication for PGA scaffolds to be applied in the fields of islet culture in vitroand transplantation.
The effect of treating power and time on the PGA holder performance was wasstudied with XPS analysis, mechanical analysis and wetting ability analysis. Theresults indicated that the mechanical performance and the wetting ability of PGAholder were better when the treating power was240W and treating time was4min.The effect of different concentration coatings of sucrose, L-glutaminic acid andpoly-lysine acid on the PGA holder performances were carried on throughmechanical analysis and wetting ability analysis, respectively. It is found that theperformance and wetting ability of PGA treated by poly-lysine acid (2mg/ml) werebetter than those of PGA holder treated by other coatings. In the end, the plasma andthe poly-lysine acid coating were combined to treat PGA. The mechanicalperformances and wetting ability of PGA holder was better than that of PGA holdertreated by either single. Thus, the method that plasma and the poly-lysine acidcoating method was employed in order to improve the wetting ability between PGAholder and culture fluid and further increase the adhesion of PGA scaffolds and cells..
Islet cells on the modified PGA scaffolds were cultured in vitro (stasis andmicrogravity culture, respectively) and observed under inverted microscope. Theresult showed that the islet cells which were in good shape and well growth adheredtightly to the scaffold with less dead cells. Dithizon (DTZ) staining results indicatedthat the purity of islet cells on PGA scaffolds was quite high. Acridine Orange (AO)and Propidium Iodide (PI) fluorescence double-staining method and radioimmunitydetection results demonstrated that survival rates and insulin contents of islet cells onPGA scaffolds were relatively high. MTT detection results showed thatbio-compatibility between PGA scaffold materials and islet cells was excellent.Observations under scanning electron microscopy (SEM) exhibited that islet cellscould grow adhesively to PGA scaffolds and large interspaces between islet cellshelped islet cells acquire nutrition sufficiently and enabled islet cells to exhibit goodmorphology. Under laser confocal microscopy, green islet β and red α cells were clearly seen on PGA scaffolds.
The islet cells were co-cultured with PGA scaffolds for five days in vitro andthen PGA-islet grafts were transplanted into the muscle of diabetic wistar rats toobserve the treatment effect of PGA-islet graft. The blood glucose and serum insulincontent results indicated that PGA-islet graft in PGA-microgravity culture group canrecover blood glucose and serum insulin content to the normal condition. Observationunder SEM and confocal microscopy, pathological detection results as well showedthat islet cells on PGA scaffolds were in good shape, the bio-compatibility betweenislet cells and PGA scaffolds was good, extracellular matrix like reticular fibers wasformed and visible between islet cells. In addition, interspaces around PGA-islet werelarge and no islet cells were crushed in muscular tissues. Nascent capillary were seenbetween PGA scaffolds and red cells were visible around islet cells, which indicatedsufficient blood supply and plenitudinous nutrition supply to islet cells in the muscles.After30days’ transplantation, PGA fiber scaffolds began to collapse, PGA pieces andits degradable products had no harm to body.
After porcine islet cells and PGA scaffolds were co-cultured, the quality,biological characteristics of porcine islet cells survival in vitro and bio-compatibilitywere studied. Compare with the islet cells in the static condition, the AO-PIfluorescence double staining results showed that PGA scaffold can improve theviability of porcine islet cells and increase survival rates of porcine islet cells.Radioimmunity detection result demonstrated high insulin releasing index of porcineislet cells on PGA scaffolds and MTT detection result showed good bio-compatibilityPGA scaffold and porcine islet cells had. Observations under the inverted microscopyand immunofluorescence microscopy indicated that porcine islet cells on PGAscaffold exhibited good morphology and adhesive growth.
The experimental results demonstrated that PGA scaffold was acted asextracellular matrix, providing good culture condition for islet cells in vitro.Especially, the PGA-microgravity culture condition was a better choice for islet cellsin vitro survival and transplantation in vivo.
通过XPS分析、拉伸性能测试和浸润性测试研究了不同功率、不同时间的等离子体处理对于PGA支架性能的影响。结果表明：处理功率240W，处理时间4min时，PGA支架的拉伸性能以及与生理盐水和血清培养液之间的浸润性较好。采用拉伸性能测试和浸润性测试分别研究了不同浓度的蔗糖、L-谷氨酸和多聚赖氨酸涂层处理对于PGA支架性能的影响，结果表明：涂覆有2mg/ml的多聚赖氨酸涂层的PGA支架的拉伸性能以及与生理盐水和血清之间的浸润性相对于其它涂层要好。最后采用处理功率240W的等离子体处理结合浓度为2mg/ml的多聚赖氨酸涂层处理PGA支架，通过对其拉伸性能以及浸润性能分析，结果表明：等离子体结合多聚赖氨酸涂层处理的PGA支架的拉伸性能和浸润性能比采用单一方法处理的PGA支架的各方面性能都要好。因此，最终采用等离子体结合多聚赖氨酸涂层对PGA支架进行处理，改进其与细胞培养液之间的浸润性，从而提高PGA细胞支架与细胞之间的粘附性。
将大鼠胰岛细胞种植在经表面改性的PGA支架上进行体外培养（静止培养和微重力培养），实验结果显示，与无支架静止培养组胰岛相比，倒置显微镜下可见PGA支架上的胰岛细胞形态完整，生长状态良好，胰岛能与支架紧密粘附，胰岛细胞死亡少。双硫腙（DTZ）染色后PGA支架上的胰岛细胞纯度较好。吖啶橙（AO）和碘化丙啶（PI）荧光双染色法和放射免疫法检测结果显示，PGA支架上的胰岛细胞的生存率及胰岛素含量较高。噻唑蓝比色法（MTT）结果显示，PGA支架材料与胰岛细胞的生物相容性较好。扫描电镜下可见胰岛细胞在PGA支架上能粘附生长，胰岛之间的间隙较大，胰岛可充分地获取营养，PGA支架上胰岛细胞形态良好。激光共聚焦显微镜下可清楚的看到PGA支架上绿色的胰岛β细胞和红色的胰岛α细胞。
PGA-胰岛复合物经过体外共培养5天后，移植到糖尿病大鼠的腿部肌肉内，对PGA-胰岛移植物移植治疗的效果进行比较观察。从血糖和血清胰岛素含量检测结果显示，支架微重力培养移植组的PGA-胰岛细胞移植物，移植后可使糖尿病大鼠体内的血糖值和血清胰岛素含量恢复到正常。扫描电镜、共聚焦显微镜及病理检测结果显示，PGA支架上胰岛细胞的形态良好，支架材料与胰岛细胞的生物相容性较好，胰岛细胞间可见到细胞外基质的形成，呈网状细纤维，PGA-胰岛细胞周围空间较大，胰岛细胞未受到肌肉组织的挤压，在PGA支架之间有新生的毛细血管，胰岛细胞周围可见到红细胞，说明肌肉内的血液供应较丰富，支架上的胰岛细胞能得到充分的营养供应。移植后30天左右，PGA支架开始逐渐断裂，PGA的降解及降解产物对机体没有造成损害。
将PGA支架与猪胰岛细胞共培养，观察猪胰岛细胞体外生存的质量、生物学特征及猪胰岛细胞与PGA支架材料的生物相容性。与静止培养组胰岛相比，AO-PI荧光双染法结果显示，PGA支架可改善猪胰岛细胞的活性，可使猪胰岛生存率增高。放射免疫方法胰岛功能检测结果显示，PGA支架上猪胰岛细胞的胰岛素释放指数较高。MTT检测结果显示，PGA支架与猪胰岛细胞具有较好的生物相容性。倒置显微镜及免疫荧光显微镜观察结果显示，PGA支架上猪胰岛细胞形态良好，猪胰岛细胞在PGA支架上能粘附生长。
实验结果表明，PGA细胞支架可作为胰岛细胞外基质，为胰岛细胞体外生存提供良好的生长环境。尤其是PGA支架微重力培养条件更适合于胰岛细胞的体外生存与体内移植。
Polyglycolic acid (PGA), as a medical biologically-adaptive polymer approvedby FDA, is one of the most studied bio-degradation materials. This research aimed toimprove the adhesion of PGA scaffold via re-modifying the surface materials of PGAscaffolds and to provide better adhesion condition for islet cells on PGA scaffoldswith implication for PGA scaffolds to be applied in the fields of islet culture in vitroand transplantation.
The effect of treating power and time on the PGA holder performance was wasstudied with XPS analysis, mechanical analysis and wetting ability analysis. Theresults indicated that the mechanical performance and the wetting ability of PGAholder were better when the treating power was240W and treating time was4min.The effect of different concentration coatings of sucrose, L-glutaminic acid andpoly-lysine acid on the PGA holder performances were carried on throughmechanical analysis and wetting ability analysis, respectively. It is found that theperformance and wetting ability of PGA treated by poly-lysine acid (2mg/ml) werebetter than those of PGA holder treated by other coatings. In the end, the plasma andthe poly-lysine acid coating were combined to treat PGA. The mechanicalperformances and wetting ability of PGA holder was better than that of PGA holdertreated by either single. Thus, the method that plasma and the poly-lysine acidcoating method was employed in order to improve the wetting ability between PGAholder and culture fluid and further increase the adhesion of PGA scaffolds and cells..
Islet cells on the modified PGA scaffolds were cultured in vitro (stasis andmicrogravity culture, respectively) and observed under inverted microscope. Theresult showed that the islet cells which were in good shape and well growth adheredtightly to the scaffold with less dead cells. Dithizon (DTZ) staining results indicatedthat the purity of islet cells on PGA scaffolds was quite high. Acridine Orange (AO)and Propidium Iodide (PI) fluorescence double-staining method and radioimmunitydetection results demonstrated that survival rates and insulin contents of islet cells onPGA scaffolds were relatively high. MTT detection results showed thatbio-compatibility between PGA scaffold materials and islet cells was excellent.Observations under scanning electron microscopy (SEM) exhibited that islet cellscould grow adhesively to PGA scaffolds and large interspaces between islet cellshelped islet cells acquire nutrition sufficiently and enabled islet cells to exhibit goodmorphology. Under laser confocal microscopy, green islet β and red α cells were clearly seen on PGA scaffolds.
The islet cells were co-cultured with PGA scaffolds for five days in vitro andthen PGA-islet grafts were transplanted into the muscle of diabetic wistar rats toobserve the treatment effect of PGA-islet graft. The blood glucose and serum insulincontent results indicated that PGA-islet graft in PGA-microgravity culture group canrecover blood glucose and serum insulin content to the normal condition. Observationunder SEM and confocal microscopy, pathological detection results as well showedthat islet cells on PGA scaffolds were in good shape, the bio-compatibility betweenislet cells and PGA scaffolds was good, extracellular matrix like reticular fibers wasformed and visible between islet cells. In addition, interspaces around PGA-islet werelarge and no islet cells were crushed in muscular tissues. Nascent capillary were seenbetween PGA scaffolds and red cells were visible around islet cells, which indicatedsufficient blood supply and plenitudinous nutrition supply to islet cells in the muscles.After30days’ transplantation, PGA fiber scaffolds began to collapse, PGA pieces andits degradable products had no harm to body.
After porcine islet cells and PGA scaffolds were co-cultured, the quality,biological characteristics of porcine islet cells survival in vitro and bio-compatibilitywere studied. Compare with the islet cells in the static condition, the AO-PIfluorescence double staining results showed that PGA scaffold can improve theviability of porcine islet cells and increase survival rates of porcine islet cells.Radioimmunity detection result demonstrated high insulin releasing index of porcineislet cells on PGA scaffolds and MTT detection result showed good bio-compatibilityPGA scaffold and porcine islet cells had. Observations under the inverted microscopyand immunofluorescence microscopy indicated that porcine islet cells on PGAscaffold exhibited good morphology and adhesive growth.
The experimental results demonstrated that PGA scaffold was acted asextracellular matrix, providing good culture condition for islet cells in vitro.Especially, the PGA-microgravity culture condition was a better choice for islet cellsin vitro survival and transplantation in vivo.
引文
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