Transplant Division, Department of Surgery, Indiana University School of Medicine, Indianapolis, Indianapolis, United States of America.
Department of Ophthalmology, Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indianapolis, United States of America.
PLoS One. 2021 Mar 5;16(3):e0243682. doi: 10.1371/journal.pone.0243682. eCollection 2021.
The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization.
Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium.
All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity.
Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.
本研究旨在比较三种已应用的传统猪角膜脱细胞方法，并展示通过脱细胞化保留角膜缘的重要性。
使用三种不同的方法对新鲜的、野生型（有或没有）带角膜缘的猪角膜进行脱细胞处理，包括(i)十二烷基硫酸钠（SDS）、(ii)高渗盐水（HS）和(iii) N2 气体（NG）。通过组织学、残留核材料和超微结构分析进行治疗后评估。甘油用于帮助减少脱细胞化的不良影响。角膜在角膜储存液中保存两周。
三种脱细胞方法在不同程度上减少了基质组织中角膜细胞的数量。然而，除 SDS 外，所有方法均导致大量细胞和细胞碎片的保留。SDS 方法（0.1% SDS，48h）导致无角膜缘的角膜几乎 100%脱细胞。NG 方法（<50%）的低脱细胞能力使其不利。尽管 HS 方法具有更平衡的损伤-脱细胞比，但它的脱细胞能力低于 SDS 方法。保留角巩膜缘可部分防止结构损伤和水肿，但会降低脱细胞能力。
我们的结果表明 SDS 是一种非常强大的脱细胞方法，但会对角膜造成不可逆转的损伤。保留角巩膜缘会降低脱细胞效率，但也会降低损伤程度。
