DEPENDENCE OF THE ANGIOGENIC EFFECT OF THE AMNIOTIC MEMBRANE ON THE METHOD OF ITS PROCESSING

AIM: to investigate the effect of physical decellularization methods and glycerol pretreatment on the morphological structure and biological properties of lyophilized amniotic membrane.

MATERIALS AND METHODS. Two groups of human amniotic membrane fragments were studied. During pretreatment, fragments of the first group were immersed for 20 minutes in a 10% glycerol solution, while fragments of the second group received no glycerol treatment. Both groups were subsequently subjected to ultrasonic processing at a frequency of 35±10% kHz for 15 minutes, followed by lyophilization. Biological properties were assessed based on proteomic analysis of the preserved amniotic membrane and experimental studies in laboratory animals. Morphological examination was performed using light microscopy and a JEOL JSM-6390A Analysis Station scanning electron microscope (Japan).

RESULTS AND DISCUSSION. Destruction of epithelial cells and desquamation of the epithelial layer were identified, both more pronounced in amniotic membrane specimens not pretreated with glycerol; the stromal layers were flattened. Multidirectional fibers confer a three-dimensional architecture. In specimens of amniotic membrane lyophilized after glycerol impregnation, homogeneous, structureless substrates attached in clusters and forming a tree-like configuration were observed. Proteomic analysis revealed a greater number of angiogenesis inducers in amniotic membrane fragments not pretreated with glycerol. At the implantation site of biomaterial fragments not treated with glycerol, a greater number of mast cells and pronounced neoangiogenesis were noted.

CONCLUSION. Physical decellularization and preservation methods allow the anatomical structure and protein compounds that determine the biological activity of the amniotic membrane to be maintained. Glycerol treatment of decellularized lyophilized amniotic membrane promotes the retention of diverse protein compounds within the tissue structure, thereby sustaining its biological activity. Biomaterial not impregnated with glycerol contains a greater number of proteins, promotes greater cellular infiltration, and activates neoangiogenesis. By contrast, amniotic membrane pretreated with glycerol does not elicit a pronounced inflammatory response and does not lead to the formation of newly formed vessels.

1. Zhong Z, Balayan A, Tian J, Xiang Y, Hwang HH, Wu X, et al. Bioprinting of dual ECM scaffolds encapsulating limbal stem/progenitor cells in active and quiescent statuses. Biofabrication. 2021 Aug 13;13(4):10.1088/1758-5090/ac1992. https://doi.org/10.1088/1758-5090/ac1992. PMID: 34330126; PMCID: PMC8716326.

2. Farhadihosseinabadi B, Farahani M, Tayebi T, Jafari A, Biniazan F, Modaresifar K, et al. Amniotic membrane and its epithe-lial and mesenchymal stem cells as an appropriate source for skin tissue engineering and regenerative medicine. Artif Cells Nanomed Biotechnol. 2018;46(sup2):431-440. https://doi.org/10.1080/21691401.2018.1458730. Epub 2018 Apr 24. PMID: 29687742.

3. Riau AK, Beuerman RW, Lim LS, Mehta JS. Preservation, sterilization and de-epithelialization of human amniotic mem-brane for use in ocular surface reconstruction. Biomaterials. 2010;31(2):216-225. https://doi.org/doi.org/10.1016/j.biomaterials.2009.09.034

4. Murphy SV, Skardal A, Nelson RA Jr, Sunnon K, Reid T, Clouse C, et al. Amnion membrane hydrogel and amnion membrane powder accelerate wound healing in a full thickness porcine skin wound model. Stem Cells Transl Med. 2020 Jan;9(1):80-92. https://doi.org/10.1002/sctm.19-0101

5. Startseva O.I., Sinel′nikov M.E., Babayeva YU.V., Trushchenkova V.V. Detsellyulyarizatsiya organov i tkaney. Khirurgiya. Zhurnal im. N.I. Pirogova. 2019;(8):59-62. In Russian. https://doi.org/10.17116/hirurgia201908159

6. Tovpeko DV, Kondratenko AA, Astakhov AP. Detsellyulyarizatsiya organov i tkaney kak klyuchevoy etap sozdaniya bioso-vme-stimogo materiala. Vestnik Voyennogo innovatsionnogo tekhnopolisa «Era». 2023;4(4):342–346. In Russian. https://doi.org/10.56304/S2782375X23040150

7. Lin CH, Hsia K, Su CK, et al. Sonication-Assisted Method for Decellularization of Human Umbilical Artery for Small-Caliber Vascular Tissue Engineering. Polymers (Basel). 2021;13(11):1699. https://doi.org/10.3390/polym13111699

8. Koizumi N, Fullwood NJ, Bairaktaris G, Inatomi T, Kinoshita S, Quantock AJ. Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Invest Ophthalmol Vis Sci. 2000 Aug;41(9):2506-13. PMID: 10937561.

9. Koizumi NJ, Inatomi TJ, Sotozono CJ, Fullwood NJ, Quantock AJ, Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res. 2000 Mar;20(3):173-7. PMID: 10694891.

10. López-Valladares MJ, Touriño R, Vieites B, Gude F, Silva MT, Couceiro J. Effects of lyophilization on human amniotic mem-brane. Acta Ophthalmol. 2009 Jun;87(4):396-403. https://doi.org/10.1111/j.1755-3768.2008. 01261.x

11. Volova LT, Milyudin ES, Kuchuk KE. Patent RF na izobreteniye № 2835347. 24.02.2025 Byul. № 6. Sposob polucheniya al-logennoy amnioticheskoy membrany dlya rekonstruktivnoy meditsiny. Ssylka aktivna na 25.11.2025. In Russian. https://www.fips.ru/cdfi/fips.dll/ru?ty=29&docid=2835347

12. Milyudin E, Volova LT, Kuchuk KE, Timchenko EV, Timchenko PE. Amniotic Membrane Biopolymer for Regenerative Medi-cine. Polymers. 2023;15:1213. https://doi.org/10.3390/polym15051213

13. Ingraldi AL, Audet RG, Tabor AJ. The Preparation and Clinical Efficacy of Amnion-Derived Membranes: A Review. J Funct Biomater. 2023 Oct 20;14(10):531. https://doi.org/10.3390/jfb14100531. PMID: 37888195; PMCID: PMC10607219.

14. Bukhdruker S, Varaksa T, Orekhov P, Grabovec I, Marin E, Kapranov I, et al. Structural insights into the effects of glycerol on ligand binding to cytochrome P450. Acta Crystallogr D Struct Biol. 2023 Jan 1;79(Pt 1):66-77. https://doi.org/10.1107/S2059798322011019. Epub 2023 Jan 1. PMID:36601808

15. Niknejad H, Peirovi H, Jorjani M, Ahmadiani A, Ghanavi J, Seifalian AM. Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater. 2008 Apr 29;15:88-99. https://doi.org/10.22203/ecm.v015a07. PMID: 18446690.

16. Roy I, Gupta MN. Freeze-drying of proteins: some emerging concerns. Biotechnol Appl Biochem. 2004 Apr;39(Pt 2):165-77. https://doi.org/10.1042/BA20030133. PMID: 15032737.

17. The UniProt Consortium UniProt: the Universal Protein Knowledgebase in 2023 Nucleic Acids Res. 51:D523–D531 (2023).

18. The Gene Ontology Consortium. The Gene Ontology knowledgebase in 2023. Genetics. 2023 May 4;224 (1):iyad031. https://doi.org/10.1093/genetics/iyad031