Department of Chemical and Materials Engineering, Chang Gung University, Taoyuan 33302, Taiwan, ROC.
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan 33302, Taiwan, ROC; Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan, ROC; Department of Materials Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan, ROC.
Acta Biomater. 2018 Jan;65:123-136. doi: 10.1016/j.actbio.2017.11.018. Epub 2017 Nov 8.
To offer an ideal hospitable environment for corneal keratocyte growth, the carrier materials can be functionalized with incorporation of signaling molecules to regulate cell biological events. This study reports, for the first time, the development of gelatin/ascorbic acid (AA) cryogels for keratocyte carriers in vitro and in vivo. The cryogel samples were fabricated by blending of gelatin with varying amounts of AA (0-300 mg) and carbodiimide cross-linking via cryogelation technique. Hydrophilic AA content in the carriers was found to significantly affect cross-linking degree and pore dimension of cryogels, thereby dictating their mechanical and biological stability and AA release profile. The cryogel carriers with low-to-moderate AA loadings were well tolerated by rabbit keratocyte cultures and anterior segment eye tissues, demonstrating good ocular biocompatibility. Although higher incorporated AA level contributed to enhanced metabolic activity and biosynthetic capacity of keratocytes grown on cryogel matrices, the presence of excessive amounts of AA molecules could lead to toxic effect and limit cell proliferation and matrix production. The cytoprotective activity against oxidative stress was shown to be strongly dependent on AA release, which further determined cell culture performance and tissue reconstruction efficiency. With the optimum AA content in carrier materials, intrastromally implanted cell/cryogel constructs exhibited better capability to enhance tissue matrix regeneration and transparency maintenance as well as to mitigate corneal damage in an alkali burn-induced animal model. It is concluded that understanding of antioxidant molecule-mediated structure-property-function interrelationships in gelatin/AA cryogels is critical to designing carrier materials for potential use in corneal stromal tissue engineering.
Multifunctional cryogel material can offer an ideal hospitable environment for cell-mediated tissue reconstruction. To our knowledge, this is the first report describing the use of gelatin/ascorbic acid (AA) cryogels as keratocyte carriers for corneal stromal tissue engineering. The AA loading during cryogel fabrication is found to have a significant effect on cross-linking degree and pore dimension, mechanical and biological stability, ocular biocompatibility, cell culture performance, and cytoprotective activity, giving comprehensive insight into fine-tuning the structure-property-function interrelationships of keratocyte carrier material. Using an alkali burn-induced animal model, we present evidence that with the optimum AA loading into cryogel materials, intrastromally implanted cell/carrier constructs exhibited better capability to enhance tissue matrix regeneration and transparency maintenance as well as to mitigate corneal damage.
为了为角膜基质细胞的生长提供理想的亲和环境，可以将载体材料进行功能化，以掺入信号分子来调节细胞的生物事件。本研究首次报道了明胶/抗坏血酸（AA）冷冻凝胶在体外和体内作为角膜细胞载体的发展。通过将明胶与不同量的 AA（0-300mg）混合并通过冷冻凝胶技术进行碳二亚胺交联来制备冷冻凝胶样品。载体中亲水性 AA 的含量显著影响冷冻凝胶的交联程度和孔径，从而决定了它们的机械和生物稳定性以及 AA 的释放特性。低至中等 AA 负载的冷冻凝胶载体可被兔角膜细胞培养物和眼前节眼组织耐受，表现出良好的眼生物相容性。尽管较高的掺入 AA 水平有助于增强在冷冻凝胶基质上生长的角膜细胞的代谢活性和生物合成能力，但过多的 AA 分子会导致毒性作用并限制细胞增殖和基质产生。抗氧化应激的细胞保护活性强烈依赖于 AA 的释放，这进一步决定了细胞培养性能和组织重建效率。在载体材料中具有最佳 AA 含量的情况下，间质内植入的细胞/冷冻凝胶构建体表现出更好的增强组织基质再生和透明度维持以及减轻碱烧伤诱导的动物模型中角膜损伤的能力。结论是，了解明胶/AA 冷冻凝胶中抗氧化分子介导的结构-性质-功能关系对于设计用于角膜基质组织工程的载体材料至关重要。
