In situ polymerizable hydrogel incorporated with specific pathogen-free porcine platelet-rich plasma for the reconstruction of the corneal endothelium

- A hydrogel composed of hyaluronic acid and Pluronic F-127 is proposed.
- Platelet-rich plasma (PRP) is activated using collagen, thrombin and CaCl2.
- Cytotoxicity and functions of hydrogel with activated PRP are investigated.
- Activated PRP can replace fetal bovine serum for BCECs culture.
- Combination of activated platelet-rich plasma and hydrogel is benefit for corneal endothelium reconstruction.

Corneal inflammation and injury result in scarring and the loss of transparency of the cornea, accounting for a million cases of blindness worldwide each year. Hence, tissue engineering for the reconstruction of the corneal endothelium is a potential therapy. In this study, hydrogels composed of various ratios of hyaluronic acid (H) and Pluronic F-127 (F) were synthesized. These hydrogels can transit between liquid below 15°C and hydrogel at 37°C and were injectable for treating corneal wounds via in situ polymerization. The hydrogel preparations were assessed by Fourier transform infrared spectroscopy and scanning electron microscopy, and their physical-chemical properties, including transparency, viscoelastic properties, biodegradation and swelling properties, were also evaluated. The results demonstrated that the two ratios of hydrogels, H:F = 1:15 (H1F15) and 1:20 (H1F20), exhibited thermal responsive rheological properties, biodegradation and swelling properties. Specially, the H1F15 hydrogel incubated with 10,000 U of a lysozyme solution still retained over 70% of its remaining weight for 7 days. Moreover, this study further compared the characteristics of bovine corneal endothelial cells (BCECs) cultured on the hydrogels with and without porcine platelet rich plasma (P-PRP) using cytotoxicity, migration, apoptosis/necrosis and cell cycle assays. The results showed that the H1F15 hydrogel with P-PRP displayed higher cell viability and cell migration. In addition, the incorporation of P-PRP positively affected the hydrogel to reduce apoptosis and to enhance the cell cycle of BCECs. Taken together, these results confirmed that the H1F15 hydrogel lacks cytotoxicity and is biodegradable. BCECs can survive, grow and retain normal characteristics in the H1F15 hydrogel with P-PRP. These results provide an opportunity for corneal endothelium reconstruction using a tissue engineering approach with the H1F15 hydrogel with P-PRP.

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