Yara İyileşmesine Yönelik Ekzozom Entegre Biyoaktif Jelatin Süngerlerin Geliştirilmesi

Abstract

Exosomes are intercellular signal transduction vesicles, that play a role in many biological processes such as angiogenesis, antigen transport, apoptosis, coagulation, cellular hemostasis, inflammation, and intercellular signal transduction through various biomolecules they carry in their cargo. Because of these effects, exosomes gained great interest in the diagnosis and treatment of neurodegenerative, cancer, cardiovascular diseases, bone regeneration, drug and vaccine research. In wound healing, it is known that exosomes are effective in hemostasis, inflammation, angiogenesis, collagen deposition, granule tissue formation, and epithelialization phases. However, preserving their stability and biological functions is critical for exosomes to have a therapeutic effect. For this purpose, the preservation of stability and activity of exosomes by loading them into carrier systems attracts attention in the field of regenerative medicine. The thesis aim is to load rat bone marrow mesenchymal stem cell (rt-MSC) exosomes into gelatin sponges for wound healing, to preserve their biological functions, and provide ease of application in the wound area. For this purpose, exosomes were isolated by ultracentrifugation method from rt-MSC. Particle sizes of exosomes were in the range of 160-180 nm. Also flow cytometry analysis showed that exosomes express CD81 and CD63 proteins. The cell source and culture condition affects on exosome yield were investigated. Results showed that rat age is effective in exosome yield, and exosome yield isolated from 8-week-old rat MSC is two times higher than that of 5- and 3-week-old rats. In addition, the use of exosome-free fetal bovine serum (Exodep FBS) increased exosome yield by two times compared to the medium without FBS (p<0.05). When exosomes were applied to keratinocyte cells (HaCaT) at different concentrations (0-10 µg/mL), cell proliferation increased by 10% at a concentration of 0.25 µg/mL and significantly decreased at higher concentrations (>1 µg/mL). The effect of rt-MSC isolated exosomes on cell migration was investigated at different concentrations (0.25, 1, and 10 µg/mL). At the end of 24 hours, when 10 µg/mL exosome was applied to human fibroblast cells (hFB), the wound area was reduced by 51%, while 36% was in the control group. In HaCaT cells, however, no effect of exosomes on migration was observed. The pro-angiogenic effects of exosomes were tested on the aortic ring and chorioallantoic membrane (CAM) analysis. Exosomes increased endothelial sprouting and new vessel formation in a concentration-dependent manner. Gelatin sponges with an average pore diameter of 200–250 µm and high water absorption capacity were prepared by freeze-drying. The biological effects of exosomes were investigated by loading them into gelatin sponges. CAM analyzes showed that 5 µg/mL exosome-integrated gelatin sponges increased vessel density by approximately 50% (p<0.05) compared to the control. In the aortic ring experiment, however, exosome-integrated gelatin sponges showed a higher angiogenic response than free exosomes. As a result, gelatin sponges would be an effective strategy in chronic/complex wound treatments with ease of application by preserving exosome stability and biological properties.