Characterization of cornea-specific bioink: high transparency, improved in vivo safety

Corneal transplantation is a typical surgical procedure for severe corneal diseases. However, the waiting time for a donor cornea has gradually increased due to a decrease in supply caused by an aging population and increased cases of laser-based surgeries. Artificial corneas were developed to meet the increase in demand; however, these approaches have suffered from material deterioration resulted by the limited tissue integration. Here, they introduce a cornea-derived decellularized extracellular matrix (Co-dECM) as a bioink for corneal regeneration. The developed Co-dECM bioink had similar quantitative measurement results for collagen and GAGs compared with that of the native cornea and also had the proper transparency for vision. The differentiation potential of human turbinate-derived mesenchymal stem cells (hTMSCs) to a keratocyte lineage was only observed in the Co-dECM group. Moreover, the developed bioink did not have any cytotoxic effect on encapsulated cells for three-dimensional (3D) culture and has great biocompatibility evident by the xeno-implantation of the Co-dECM gel into mice and rabbits for two and one month, respectively. An in vivo safety similar to clinical-grade collagen was seen with the Co-dECM, which helped to maintain the keratocyte-specific characteristics in vivo, compared with collagen. Taken together, the Co-dECM bioink has the potential to be used in various types of corneal diseases based on its corneal-specific ability and design flexibility through 3D cell printing technology.

To develop a hydrogel mimicking the native cornea-like environment, corneal ECMs were acquired through decellularization and dissolved in an acidic solution. The efficacy was verified through in vitro and in vivo examinations1.

Schematic of Co-dECM gel preparation and its validation.

Corneal ECMs, obtained by removing cells from the native corneas through chemical decellularization process, were validated by quantifying the amounts of remaining DNA, collagen, and GAG . The main purpose of the decellularization process is retaining only ECMs without cells, which can cause an immune response, the most problematic of xenotransplantation.27 To prevent immune rejection problems, decellularized tissue should have either less than 3% DNA relative to the native tissue, or no more than 50 ng/mg of double-stranded DNA content.28 The prepared Co-dECM powder was found satisfying the standards; the residual amount of DNA was 2.73% ± 0.009% of the original cornea. However, the chemicals used in the decellularization process not only remove cells and residues but can also cause some damages to the extracellular matrix. Thus, the efficacy of the decellularization process was quantified by measuring the amounts of collagen and GAG, typical components of corneal ECM, which were determined as 76.50% ± 0.043% and 62.08% ± 0.034%, respectively, relative to the original tissue. These results indicate that the prepared Co-dECM can provide complex cornea-specific biochemical cues similar to a native cornea while it reveals no serious immune response.

Quantification of remaining content in Co-dECM relative to the native cornea (**p < 0.01, ***p < 0.005).

To verify the functional suitability of Co-dECM gel for corneal regeneration studies, they examined the physical (transparency and microstructure) and chemical (internal biomolecular growth factors) properties, and investigated gene expression patterns when stem cells are cultured on the gel. In this experiment, they chose two control groups: collagen hydrogel (Col, widely used for corneal regeneration study) and native human cornea. The 500-µm thick (as the average thickness of native cornea) Co-dECM gel showed higher transparency than that of the Col in visible light wavelength range of 390–700 nm.

Optical properties of Co-dECM gel. (a) Gross images (scale bar: 2 mm). (b) Light transmittance variations of 2% Co-dECM gel, 2% Col, and human cornea at different wavelengths of visible light spectrum. (c) SEM micrographs of samples (scale bar: 10 µm). (d) Thicknesses of collagen fibers for Co-dECM gel (Co-dECM 1X), Co-dECM gel mixed with Col (Co-dECM 0.5X), and Col (Co-dECM 0X). *p < 0.05, ***p < 0.005.

Optical properties of Co-dECM gel. (a) Gross images (scale bar: 2 mm). (b) Light transmittance variations of 2% Co-dECM gel, 2% Col, and human cornea at different wavelengths of visible light spectrum. (c) SEM micrographs of samples (scale bar: 10 µm). (d) Thicknesses of collagen fibers for Co-dECM gel (Co-dECM 1X), Co-dECM gel mixed with Col (Co-dECM 0.5X), and Col (Co-dECM 0X). *p < 0.05, ***p < 0.005.

Inflammation test using mouse model. (a) Stained images on day 56 of implantation using May-Grunwald-Giemsa assay (scale bar: 200 µm). (b) Number of stained cells on various days.

H&E stained images using rabbit model. Optical micrographs, OCT images with H&E stained images on day 28, and the number of immune cells on days 14 and 28. Scale bar: 50 µm.

1. Kim H, Park M-N, Kim J, Jang J, Kim H-K, Cho D-W. Characterization of cornea-specific bioink: high transparency, improved in vivo safety. Journal of Tissue Engineering. January 2019. doi:10.1177/2041731418823382

cover photo: https://thomeye.com/corneal-disease-fargo-nd/