Collagen-based conjunctival tissue equivalent: in vitro evaluation of biocompatibility and biomechanical properties

BACKGROUND: The development of a conjunctival equivalent capable of repairing tissue defects and preventing pathological scarring is a critical goal in biomaterial design for reconstructive procedures, in which collagen is frequently employed. Evaluating the biocompatibility and biomechanical properties of such materials supports the advancement of effective surgical approaches for conjunctival reconstruction. AIM: The work aimed to evaluate the biocompatibility of human conjunctival epithelial cells (hCECs) with a collagen membrane (CM) developed using an original technique in vitro; to confirm its noncytotoxicity toward hCECs; to characterize hCEC morphology and expression of specific proteins on CM; and to compare the biomechanical properties of CM with native conjunctiva. METHODS: A primary culture of hCECs was obtained using the explant method and verified through staining for the markers cytokeratin 7 and mucin 5AC. Two CM formulations (collagen concentration 10 mg/mL and 30 mg/mL) were tested. Cytotoxicity of the 30 mg/mL CM was evaluated by seeding hCECs at 50,000 cells/cm2 followed by MTS assay and viability testing with calcein-AM and Hoechst staining. hCECs morphology was analyzed using phase-contrast images of the cell culture after seeding on CM and immunocytochemical staining for cytokeratin 7 and mucin 5AC. Biomechanical properties of the 30 mg/mL CM and conjunctiva were assessed using static and dynamic tests on an indentation machine. RESULTS: Phase-contrast microscopy revealed that within 1 week after seeding, hCECs migrated into the inner layers of the 10 mg/mL CM, whereas on the 30 mg/mL CM, the cells formed a confluent monolayer on its surface. The metabolic activity assay revealed no significant difference between the control groups and 30 mg/mL CM. In the live/dead assay, 95% of cells in both groups stained positive with calcein. The immunocytochemical profile of hCECs remained unchanged: as in the control group, cells cultured on the CM expressed conjunctiva-specific cytokeratin 7 and mucin 5AC. The Young’s modulus values for the CM and bulbar conjunctiva were comparable: 0.0008739 ± 0.0004332 GPa and 0.0009472 ± 0.001323 GPa, respectively (p = 0.0549). The hardness of the CM was significantly higher than that of the conjunctiva (p 0.0001), while its viscosity was significantly lower compared to native tissue (p 0.0001). CONCLUSION: hCECs remained viable after seeding on the tested CM, confirming its noncytotoxicity. The migration of hCECs into the matrix indicates the material’s ability to create a favorable microenvironment for conjunctival epithelialization and to undergo resorption following cell colonization. Preservation of the molecular profile of hCECs indicates high biocompatibility of the CM with conjunctival epithelium. The 30 mg/mL CM exhibits elasticity comparable to that of conjunctiva, with higher hardness and lower viscosity. Optimization of CM biomechanical properties for specific clinical needs holds promise.