Influence of protein-peptide bioregulator isolated from bovine sclera and incorporated into an albumin-based cryogel on the sclera in a model cultivation of a posterior eye segment

Abstract

Delivering bioactive substances to certain spots in the human and animal body is a crucial task. To address this problem, we have developed a delayed-release bioactive substance carrier – an albumin-based cryogel obtained by cryostructuring. It was tested on an organotypic culture model of the posterior eye segment of a newt.Objective: to study the effectiveness of porous albumin-based cryogel obtained by cryostructuring and loaded with a bioregulator isolated from bovine sclera in different quantities in maintaining eye tissue integrity and preserving Iberian ribbed newt fibroblasts on an organotypic culture model.Materials and methods. Albumin sponges were obtained after being denatured at temperatures –15 °C, –17.5 °C, and –20 °C, with albumin levels 40 mg/mL, 50 mg/mL, and 60 mg/mL in a thermostatic cooler. Their modulus of elasticity was measured. Eye tissues were isolated from adult sexually mature Iberian ribbed newts of both sexes. The posterior segment of each eye was placed on a sponge sample of albumin cryogel in penicillin vials, sealed and placed in a thermostat. At the end of cultivation, the samples were fixed, washed, dehydrated, and embedded in paraffin. Paraffin sections were made, followed by staining. A Leica microscope (Germany) with an Olympus DP70 camera (Japan) was used to view histological sections. Fibroblast count in the histological sections was estimated using the ImageJ program.Results. Cryogel with initial albumin solution levels of 50 mg/mL obtained at –20 °C with 4.50 kPa elastic modulus, was chosen for the organ culture experiment. Histological studies showed that eye tissue integrity was maintained in the experiment when albumin-based scaffold was loaded with the bioregulator at doses of 2.46 × 10–5, 2.46 × 10–7, 2.46 × 10–9, 2.46 × 10–13, 2.46 × 10–15 μg. Moreover, the statistically significant difference for fibroblast count per unit area in the sclera partially correlates with the qualitative state of the posterior eye tissue itself. Groups where bioregulator isolated from the sclera had a dose of 2.46 × 10–7, 2.46 × 10–9 and 2.46 × 10–15 μg, showed the best result as compared with the control group.Conclusion. Albumin-based scaffold as a carrier with a bioregulator adsorbed on it (doses of 2.46 × 10–5, 2.46 × 10–7, 2.46 × 10–9, 2.46 × 10–13, 2.46 × 10–15 μg) is effective in maintaining eye tissue integrity and preserving Iberian ribbed newt fibroblasts. Albumin cryogen is an effective carrier for delayed release of bioactive substances.