Collagen matrix for conjunctival reconstruction: an in vitro experimental study

Relevance Damage to the conjunctiva causes the development of fibrosis and scarring of the tissue, which makes the development of biomaterials for its reconstruction relevant [1]. Collagen, as a natural component of the stroma, has high biocompatibility and low antigenicity, which makes it promising for clinical use [2, 3], therefore, the assessment of its biocompatibility and mechanical properties contributes to the development of new surgical techniques [4, 5]. Objective To evaluate the cytotoxicity of the collagen membrane (CM) for conjunctival epithelial cells (EC) in vitro, their morphology and the pattern of characteristic proteins on the CM, to characterize the biomechanical properties of the CM in comparison with native conjunctival tissue. Material and methods The study was conducted at the Federal State Budgetary Scientific Institution M.M. Krasnov Research Institute of Clinical and Biophysical Research. The primary culture of conjunctival EC was obtained by the explant method, verified by CK7 and CK19. We studied CM with collagen 10 and 30 mg/ml. Cytotoxicity was assessed by the MTS test and cell viability when seeding 50 thousand/cm² on CM 30 mg/ml. Morphology was analyzed by phase-contrast microscopy and immunostaining (CK7, muc5). Biomechanical properties of CM and conjunctiva were studied by static and dynamic tests on an indenter. Results On CM 10 mg/ml, migration of EC into the carrier was observed within a week, on CM 30 mg/ml, the cells formed a confluent monolayer. Metabolic activity of cells on CM and in the control did not differ. In the live/dead test, 95% of the cells were viable in both groups (calcein staining). The immunocytochemical profile (CK7 and muc5 expression) was preserved on the CM, as in the control. Mechanical properties: Young's modulus of the CM (0.000706 ± 0.000513 GPa) and conjunctiva (0.00107 ± 0.000343 GPa) were comparable (p = 0.0566). The hardness of the CM exceeded that of the native tissue (p < 0.0001), and the viscosity was lower (p = 0.0002). Conclusion Conjunctival EC retained viability on the CM, confirming the absence of cytotoxicity. Cell migration deep into the matrix and preservation of the molecular profile (CK7, muc5) indicate the biocompatibility of the CM and its ability to support epithelialization. Biomechanically, CM is comparable to conjunctiva in elasticity, has greater rigidity and lower viscosity, which can be optimized for clinical use. Keywords: conjunctiva; conjunctival reconstruction; tissue engineering; conjunctival epithelium; collagen; biopolymers; indentation