1. Generation of Femtosecond Laser-Cut Decellularized Corneal Lenticule Using Hypotonic Trypsin-EDTA Solution for Corneal Tissue Engineering
- Author
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Hong Kyun Kim, Soo-Jin Yi, Jeongho Kim, Byeong-Ung Park, Kyoung-Pil Lee, and Man-Il Huh
- Subjects
0301 basic medicine ,Scaffold ,Decellularization ,Article Subject ,business.industry ,Trypsin ,Biocompatible material ,Staining ,Glycosaminoglycan ,03 medical and health sciences ,Ophthalmology ,030104 developmental biology ,0302 clinical medicine ,lcsh:Ophthalmology ,Corneal edema ,lcsh:RE1-994 ,030221 ophthalmology & optometry ,medicine ,Tonicity ,business ,Research Article ,medicine.drug ,Biomedical engineering - Abstract
Purpose. To establish an optimized and standardized protocol for the development of optimal scaffold for bioengineering corneal substitutes, we used femtosecond laser to process human corneal tissue into stromal lenticules and studied to find the most efficient decellularization method among various reagents with different tonicities. Methods. The decellularization efficacy of several agents (0.1%, 0.25%, and 0.5% of Triton X-100, SDS, and trypsin-EDTA (TE), resp.) with different tonicities was evaluated. Of all protocols, the decellularization methods, which efficiently removed nuclear materials examined as detected by immunofluorescent staining, were quantitatively tested for sample DNA and glycosaminoglycan (GAG) contents, recellularization efficacy, and biocompatibilities. Results. 0.5% SDS in hypertonic and isotonic buffer, 0.25% TE in hypotonic buffer, and 0.5% TE in all tonicities completely decellularized the corneal lenticules. Of the protocols, decellularization with hypotonic 0.25 and 0.5% TE showed the lowest DNA contents, while the GAG content was the highest. Furthermore, the recellularization efficacy of the hypotonic TE method was better than that of the SDS-based method. Hypotonic TE-treated decellularized corneal lenticules (DCLs) were sufficiently transparent and biocompatible. Conclusion. We generated an ideal protocol for DCLs using a novel method. Furthermore, it is possible to create a scaffold using a bioengineered corneal substitute.
- Published
- 2018
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