Abstract：
Objective.To develop a clinically relevant injectable hydrogel derived from decellularized porcine peripheral nerves and with mechanical properties comparable to native central nervous system (CNS) tissue to be used as a delivery vehicle for Schwann cell transplantation to treat spinal cord injury (SCI).Approach.Porcine peripheral nerves (sciatic and peroneal) were decellularized by chemical decellularization using a sodium deoxycholate and DNase (SDD) method previously developed by our group. The decellularized nerves were delipidated using dichloromethane and ethanol solvent and then digested using pepsin enzyme to form injectable hydrogel formulations. Genipin was used as a crosslinker to enhance mechanical properties. The injectability, mechanical properties, and gelation kinetics of the hydrogels were further analyzed using rheology. Schwann cells encapsulated within the injectable hydrogel formulations were passed through a 25-gauge needle and cell viability was assessed using live/dead staining. The ability of the hydrogel to maintain Schwann cell viability against an inflammatory milieu was assessedin vitrousing inflamed astrocytes co-cultured with Schwann cells.Mainresults. The SDD method effectively removes cells and retains extracellular matrix in decellularized tissues. Using rheological studies, we found that delipidation of decellularized porcine peripheral nerves using dichloromethane and ethanol solvent improves gelation kinetics and mechanical strength of hydrogels. The delipidated and decellularized hydrogels crosslinked using genipin mimicked the mechanical strength of CNS tissue. The hydrogels were found to have shear thinning properties desirable for injectable formulations and they also maintained higher Schwann cell viability during injection compared to saline controls. Usingin vitroco-culture experiments, we found that the genipin-crosslinked hydrogels also protected Schwann cells from astrocyte-mediated inflammation.Significance. Injectable hydrogels developed using delipidated and decellularized porcine peripheral nerves are a potential clinically relevant solution to deliver Schwann cells, and possibly other therapeutic cells, at the SCI site by maintaining higher cellular viability and increasing therapeutic efficacy for SCI treatment.
摘要：
目的：开发一种临床相关的可注射水凝胶，其来自脱细胞猪周围神经，具有与天然中枢神经系统（CNS）组织相当的机械性能，可用作施万细胞移植治疗脊髓损伤（SCI）的递送载体。
方法：使用我们小组先前开发的脱氧胆酸钠和DNase（SDD）方法将猪周围神经（坐骨神经和腓骨）脱细胞。使用二氯甲烷和乙醇溶剂将去细胞化的神经脱脂,然后使用胃蛋白酶消化以形成可注射的水凝胶制剂。京尼平用作交联剂以增强机械性能。可注射性，机械性能，使用流变学进一步分析了水凝胶的凝胶化动力学。将包封在可注射水凝胶制剂内的施万细胞通过25号针,并使用活/死染色评估细胞活力。使用与施万细胞共培养的发炎星形胶质细胞在体外评估水凝胶维持施万细胞针对炎症环境的活力的能力。
结果：SDD方法有效地去除细胞并保留去细胞化组织中的细胞外基质。使用流变学研究，我们发现，用二氯甲烷和乙醇溶剂脱去细胞猪外周神经可以改善水凝胶的凝胶化动力学和机械强度。使用京尼平交联的脱脂和脱细胞水凝胶模拟了CNS组织的机械强度。发现水凝胶具有可注射制剂所需的剪切稀化性质,并且与盐水对照相比,它们在注射期间还保持较高的施万细胞活力。采用体外共培养实验，我们发现,京尼平交联的水凝胶还可以保护雪旺氏细胞免受星形胶质细胞介导的炎症.
结论：使用去脂化和脱细胞的猪周围神经开发的可注射水凝胶是递送雪旺氏细胞的潜在临床相关解决方案，可能还有其他治疗细胞，通过维持较高的细胞活力和增加SCI治疗的治疗功效，在SCI部位。
方法：使用我们小组先前开发的脱氧胆酸钠和DNase（SDD）方法将猪周围神经（坐骨神经和腓骨）脱细胞。使用二氯甲烷和乙醇溶剂将去细胞化的神经脱脂,然后使用胃蛋白酶消化以形成可注射的水凝胶制剂。京尼平用作交联剂以增强机械性能。可注射性，机械性能，使用流变学进一步分析了水凝胶的凝胶化动力学。将包封在可注射水凝胶制剂内的施万细胞通过25号针,并使用活/死染色评估细胞活力。使用与施万细胞共培养的发炎星形胶质细胞在体外评估水凝胶维持施万细胞针对炎症环境的活力的能力。
结果：SDD方法有效地去除细胞并保留去细胞化组织中的细胞外基质。使用流变学研究，我们发现，用二氯甲烷和乙醇溶剂脱去细胞猪外周神经可以改善水凝胶的凝胶化动力学和机械强度。使用京尼平交联的脱脂和脱细胞水凝胶模拟了CNS组织的机械强度。发现水凝胶具有可注射制剂所需的剪切稀化性质,并且与盐水对照相比,它们在注射期间还保持较高的施万细胞活力。采用体外共培养实验，我们发现,京尼平交联的水凝胶还可以保护雪旺氏细胞免受星形胶质细胞介导的炎症.
结论：使用去脂化和脱细胞的猪周围神经开发的可注射水凝胶是递送雪旺氏细胞的潜在临床相关解决方案，可能还有其他治疗细胞，通过维持较高的细胞活力和增加SCI治疗的治疗功效，在SCI部位。
