Collagen-Based Conjunctival Tissue Equivalent: In Vitro Evaluation of Biocompatibility and Biomechanical Properties



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Abstract

Background. Conjunctival damage leads to the formation of fibrosis and scarring of fornices, which reduces quality of life of patients. Collagen, as a natural component of the conjunctival stroma, has high biocompatibility and weak antigenicity, which makes it a promising material for clinical practice. Evaluation of the biocompatibility and biomechanical properties of collagen-containing materials contributes to the further development of surgical techniques.
Aim. To assess in vitro cytotoxicity of a collagen membrane (CM) for conjunctival epithelial cells (CECs), evaluate their morphology and protein expression after seeding onto CM, characterize the biomechanical properties of CM compared to conjunctival tissue.
Materials and methods. The study was conducted at the M.M. Krasnov Research Institute of Eye Diseases. Primary CEC's cultures were established using the explant method. Cell type was verified via CK7, MUC5 staining. CMs with collagen concentrations of 10 and 30 mg/ml were evaluated. Cytotoxicity was assessed by seeding CECs (50,000/cm²) onto 30 mg/ml CM, followed by: 1) metabolic activity analysis via MTS assay; 2) live/dead staining with Calcein-AM and Hoechst. Morphology was analyzed using phase-contrast imaging on 10 and 30 mg/ml CM and after immunocytochemical staining for CK7, MUC5. Biomechanical properties of CM and native conjunctiva were tested via static and dynamic indentation.

Results. CEC migration into the inner layers of 10 mg/ml CM within one week, while 30 mg/ml CM supported confluent monolayer growth on its surface. MTS assay showed no significant difference in metabolic activity between control and CM groups. Live/dead staining demonstrated 95% viability in both control and CM groups. Immunocytochemical profiles of CECs remained unchanged, with preserved CK7 and MUC5 expression, consistent with controls. Young’s modulus of CM (0.0008739 ± 0.0004332 GPa) and conjunctiva (0.0009472 ± 0.001323 GPa) were comparable (p=0.0549). CM exhibited higher hardness (p<0.0001) and lower viscosity (p<0.0001) than native tissue.

Conclusion. CECs maintained viability on CM. Migration into the matrix suggests CM provides a favorable microenvironment for epithelialization and post-implantation resorption. Preserved molecular markers indicate high biocompatibility. CM matches conjunctival elasticity, its greater hardness and lower viscosity highlight opportunities for biomechanical optimization to clinical needs.

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Author contribution. All authors confirm that their authorship meets the international ICMJE criteria (all authors have made a significant contribution to the development of the concept, research and preparation of the article, read and approved the final version before publication). The greatest contribution is distributed as follows: D. A. Krivolapova — performed experiments, collection and analysis of literary sources, collection and processing of material, preparation and writing of the text, editing of the article; A. Yu. Andreev — study’s concept and design, collection and processing of material, editing of the article; A. M. Subbot — study’s concept and design, collection and processing of material, editing of the article; S.E.Avetisov - study’s concept and design, editing of the article; E. O. Osidak — study concept and design, editing of the article; P. N. Ilyina and A. D. Panova — processing of material. All authors confirm that their authorship meets the international ICMJE criteria (all authors made a significant contribution to the development of the concept, conducting the study, and preparing the article, read and approved the final version before publication).

Acknowledgments. We thank the LLC “Imtek” company for supporting by collagen membranes.

Ethics approval. The study was approved by the local ethics committee of the M.M. Krasnov Research Institute of Eye Diseases (protocol №46/2 of November 8, 2022).

Funding source. This work was supported by the Research Foundation Flanders (№24-15-00387, https://rscf.ru/project/24-15-00387/).

Competing interests. The authors declare that they have no competing interests.

Statement of originality. During creating this article, the authors did not use previously published information (text, illustrations, data).

Data availability statement. The editorial policy regarding data sharing does not apply to this work; no new data was collected or created.

Generative AI. Generative AI technologies were not used for this article creation.

Figures

Fig.1. Graphical representation of primary (A) and secondary (B) hermetic packaging of collagen material. C – Image of the fabricated collagen membrane

Fig. 2. Primary cultures of conjunctival epithelial cells. A - monolayer of epithelial cells in the control group on the surface of culture plate on 4th day of cultivation, B - subconfluent monolayer formed on 7th day after cultivation with small cuboidal epithelial cells with a high nuclear-cytoplasmic ratio. Scale bar - 100 μm.

Fig.3. Representative visual fields of stained conjunctival epithelial cell culture of the 3rd passage on plastic at day 3 (A) and on CM at day 3 (B). А - CK7 - red staining, B – MUC5 (green staining). Scale bar – 100 µm.

Fig.4. Culture of conjunctival epithelial cells of the 3rd passage on collagen on the 13th day of cultivation (epifluorescence microscopy). Live cells are stained green: A - staining with calcein-AM in the control group, B - staining with calcein-AM after cultivation on collagen. Scale bar - 100 μm.

Fig.5. Primary cultures of conjunctival epithelial cells (phase-contrast microscopy). A, C – control group (plastic); B, D – cultured on CM at a concentration of 30 mg/mL (B) and 10 mg/mL (D) (phase-contrast microscopy). Scale bar – 100 µm.

Fig.6. Graphical representation of the results of colorimetric formazan test: control group — metabolic activity of cells (on plate); CM— metabolic activity of cells on collagen membrane.

Fig.7. Graphical representation of nanoindentation results in static mode: Con 0.75 – Young’s modulus of the bulbar conjunctiva under a load of 0.75 mN; CM 0.75 – Young’s modulus of CM under a load of 0.75 mN.

Fig.8. Graphical representation of nanoindentation results in dynamic mode: A – Con 0.75 – hardness of the bulbar conjunctiva under a load of 0.75 mN; CM 0.75 – hardness of CM under a load of 0.75 mN. B – Con 0.75 – viscous component of the bulbar conjunctiva under a load of 0.75 mN; CM 0.75 – viscous component of CM under a load of 0.75 mN.

 

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About the authors

Diana A. Krivolapova

M.M. Krasnov Research Institute of Eye Disease

Author for correspondence.
Email: dia.med94@gmail.com
ORCID iD: 0000-0001-6974-7872
SPIN-code: 4861-4771
Scopus Author ID: 58295322100
ResearcherId: IQV-7628-2023

Ophthalmologist, PhD student

Russian Federation, 11 A, Rossolimo St., 119021, Moscow, Russian Federation

Andrey Yu. Andreev

I.M. Sechenov First Moscow State Medical University (Sechenov University); M.M. Krasnov Research Institute of Eye Diseases; Imtek Ltd.

Email: docandreev@gmail.com
ORCID iD: 0000-0002-0267-9040
SPIN-code: 6410-7993
Scopus Author ID: 57211634906

MD, Cand. Sci. (Med)

Russian Federation, Moscow; Moscow; Moscow

Anastasia M. Subbot

M.M. Krasnov Research Institute of Eye Diseases

Email: kletkagb@gmail.com
ORCID iD: 0000-0002-8258-6011
SPIN-code: 3898-2570
Scopus Author ID: 55266556100

Candidate of Medical Sciences, Senior Researcher

Russian Federation, 11 A, Rossolimo St., 119021, Moscow, Russian Federation

Sergej Je. Avetisov

M.M. Krasnov Research Institute of Eye Diseases; I.M. Sechenov First Moscow State Medical University (Sechenov University)

Email: s.avetisov@niigb.ru
ORCID iD: 0000-0001-7115-4275
SPIN-code: 9784-0459
Scopus Author ID: 7004588108

MD, Dr. Sci. (Med.), рrofessor

Russian Federation, Moscow; Moscow

Egor O. Osidak

Imtek Ltd., Moscow, Russian Federation; Koltzov Institute of Developmental Biology of Russian Academy of Sciences; Dmitriy Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology Ministry of Healthcare of Russian Federation, Moscow

Email: eosidak@gmail.com
ORCID iD: 0000-0003-2549-4011
SPIN-code: 9995-3951
Scopus Author ID: 56062879600

Cand. Sci. (Biol.)

Russian Federation, Moscow; Moscow; Moscow

Polina N. Ilina

M.M. Krasnov Research Institute of Eye Disease

Email: polinailina1107@mail.ru
ORCID iD: 0009-0009-7926-7253
SPIN-code: 7893-4570

Clinical Research Assistant
Russian Federation, 11 A,B, Rossolimo St., 119021 Moscow, Russian Federation

Anna D. Panova

M.M. Krasnov Research Institute of Eye Disease

Email: ainushgnomello@gmail.com
ORCID iD: 0000-0002-9322-6273

Clinical Research Assistant
Russian Federation, 11 A,B, Rossolimo St., 119021 Moscow, Russian Federation

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