González-Andrades Miguel, Carriel Victor, Rivera-Izquierdo Mario, Garzón Ingrid, González-Andrades Elena, Medialdea Santiago, Alaminos Miguel, Campos Antonio
Tissue Engineering Group, Department of Histology, University of Granada, Spain and Instituto de Investigación Biosanitaria ibs.GRANADA, Spain ; Division of Ophthalmology, University Hospital San Cecilio, Granada, Spain.
Tissue Engineering Group, Department of Histology, University of Granada, Spain and Instituto de Investigación Biosanitaria ibs.GRANADA, Spain.
Transl Vis Sci Technol. 2015 Apr 10;4(2):13. doi: 10.1167/tvst.4.2.13. eCollection 2015 Apr.
In this work, we decellularized whole porcine corneas including the sclerocorneal limbus (SCL) and we evaluated regional differences in order to identify an efficient method to decellularize whole corneas for future clinical use.
We analyzed the efficiency of four decellularization protocols based on benzalkonium chloride (BAK), Igepal, sodium dodecyl sulfate (SDS), and Triton X-100 detergents on whole porcine corneas.
Results showed that the decellularization efficiency of most protocols was low, with the specific protocol resulting in more efficient levels of decellularization being 0.1% SDS for 48 hours, especially in the medium and posterior cornea regions. A significant correlation was found between the decellularization efficiency and the concentration of agent used ( = 0.0174; = 0.1540), but not for the incubation time ( > 0.05). The analysis of cornea components preservation demonstrated that all protocols were able to preserve the integrity of the Bowman's layer and Descemet's membrane. Although the collagen structure was partially altered, the global decellularization groups showing highest preservation of the ECM collagen contents and orientation were Igepal and SDS, with differences among the three regions of the cornea. All global groups showed high levels of proteoglycan and glycoprotein preservation after decellularization, with the best results were found in the SDS group followed by the Igepal group.
These results suggest that very powerful protocols are necessary for whole-cornea decellularization. For the generation of lamelar corneas for clinical use, decellularization regional differences should be taken into account.
Decellularized whole corneas may be potential therapeutic agents for lamelar keratoplasty.
在本研究中，我们对包括巩膜角膜缘（SCL）在内的整个猪角膜进行了去细胞处理，并评估了区域差异，以确定一种有效的去细胞方法，用于未来的临床应用，对整个角膜进行去细胞处理。
我们分析了基于苯扎氯铵（BAK）、Igepal、十二烷基硫酸钠（SDS）和 Triton X - 100 去污剂的四种去细胞方案对整个猪角膜的效率。
结果表明，大多数方案的去细胞效率较低，其中导致更高去细胞效率的特定方案是 0.1% SDS 处理 48 小时，特别是在角膜中后区域。去细胞效率与所用试剂浓度之间存在显著相关性（ = 0.0174； = 0.1540），但与孵育时间无关（ > 0.05）。角膜成分保存分析表明，所有方案都能够保留 Bowman 层和 Descemet 膜的完整性。尽管胶原蛋白结构部分改变，但在 ECM 胶原蛋白含量和取向方面保存最好的整体去细胞组是 Igepal 和 SDS，角膜三个区域之间存在差异。所有整体组在去细胞后都显示出高水平的蛋白聚糖和糖蛋白保存，其中 SDS 组效果最佳，其次是 Igepal 组。
这些结果表明，对于整个角膜的去细胞处理，需要非常有效的方案。对于用于临床的板层角膜的生成，应考虑去细胞区域差异。
去细胞化的全角膜可能是板层角膜移植的潜在治疗剂。
