Introduction: The cornea is the most exterior, transparent surface of the eye which is roughly 500 µm thick in the center and the diameter is about 12 mm. It is one of the avascular tissues in the body, a property essential for its transparency[1]. Stroma is the thickest part of the cornea which plays a major role in the protection, transmission and refraction by the cornea. Corneal keratocytes are mesenchyme-derived cells distributed throughout the collagen-proteoglycan matrix (3-10%, v/v) of the stroma. They play a major role in the maintenance of transparency of the cornea both by expression of crystalline proteins and also by preserving the organization of the stroma by producing proteoglycans continuously[2]. In this study, isolated human corneal keratocytes are mixed with methacrylated gelatin (GelMA) in order to form a cell embedded transparent hydrogel to be used in repairing corneal stroma damages.
Experimental Methods: Surface markers (CD34, CD45, CD73 and CD90) of isolated human keratocytes (HK) were analyzed with flow cytometry (BD Accuri C6). GelMA was synthesized from type A porcine skin gelatin and to study purity and degree of methacrylation 1H NMR spectra were obtained at room temperature on a Bruker DPX 400 spectrometer after dissolving Gelatin and GelMA in D2O at 40 oC. 10% GelMA was prepared in HK growth medium and mixed with HK at a density of 40x103 cells/hydrogel. Solution was crosslinked under UV for 1 min in the presence of photoinitiator (2-(hydroxyl)-4-(2-hydroxyethoxy)-2-methylpropiophenone). Cell numbers of the hydrogels were determined with Alamar Blue assay for 21 days. Live dead assay and DAPI Phalloidin-FITC stainings were applied to the gels at day 21 and samples were analyzed under fluorescent and confocal laser scanning microscopes, respectively.
Results and Discussion: Flow cytometry results showed that more than 95% of the cells express CD73 and CD90 and their fluorescence intensities were significantly higher than unstained and isotype controls. However, cells were negative for both CD34 and CD45 surface markers where less than 5% expression was observed. These results were in correlation with the literature where CD73 and CD90 were reported as positive markers and CD34 and CD45 as negative markers for HK[3] and results showed that isolated HK maintained their keratocyte function. For this reason isolated HK was used in the rest of the study.
1H NMR spectra of the Gelatin and GelMA samples showed additional peaks due to methacrylic anhydride double bond hydrogens around 5 ppm and a reduction in the area of the peak around 7 ppm which represents peaks due to aromatic residues of the gelatin. The ratio of the double bond hydrogens to unreacted aromatic residues gives the degree of methacrylation and in this study a 60% methacrylation was achieved.
The number of the cells in the GelMA hydrogels increased proportionally in 21 days incubation period and live dead assay at day 21 showed that cells were all alive. The morphology of the cells was similar with the ones incubated in TCPS and cells formed a mesh in all over the hydrogel as observed with DAPI- Phalloidin FITC staining.
Results were promising in the way of creating stromal layer of the cornea.
The authors would like to acknowledge to BIOMATEN and the financial support from METU BAP-01-08-2013-003 and BAP08-11-DPT-2011-K120350 projects and TUBİTAK for the BİDEB 2211 Scholarship.
References:
[1] Nishida, T. Cornea: Fundamentals, Diagnosis, and Management (2nd edition) (pp. 3-11), 2005
[2] Ruberti J. W. et al., Principles of tissue engineering (3rd edition) (pp. 1025-1047), 2007.
[3] Choong P. F. Et al., Cytotherapy (pp. 252-258), 2007.