Melatonin and endothelial cell-loaded alginate-fibrin hydrogel promoted angiogenesis in rat cryopreserved/thawed ovaries transplanted to the heterotopic sites

Abstract

Background: Ischemic niche can promote follicular atresia following the transplantation of cryopreserved/thawed ovaries to the heterotopic sites. Thus, the promotion of blood supply is an effective strategy to inhibit/reduce the ischemic damage to ovarian follicles. Here, the angiogenic potential of alginate (Alg) + fibrin (Fib) hydrogel enriched with melatonin (Mel) and CD144⁺ endothelial cells (ECs) was assessed on encapsulated cryopreserved/thawed ovaries following transplantation to heterotopic sites in rats.

Methods: Alg + Fib hydrogel was fabricated by combining 2% (w/v) sodium Alg, 1% (w/v) Fib, and 5 IU thrombin at a ratio of 4: 2: 1, respectively. The mixture was solidified using 1% CaCl₂. Using FTIR, SEM, swelling rate, and biodegradation assay, the physicochemical properties of Alg + Fib hydrogel were evaluated. The EC viability was examined using an MTT assay. Thirty-six adult female rats (aged between 6 and 8 weeks) with a normal estrus cycle were ovariectomized and enrolled in this study. Cryopreserved/thawed ovaries were encapsulated in Alg + Fib hydrogel containing 100 µM Mel + CD144⁺ ECs (2 × 10⁴ cells/ml) and transplanted into the subcutaneous region. Ovaries were removed after 14 days and the expression of Ang-1, and Ang-2 was monitored using real-time PCR assay. The number of vWF⁺ and α-SMA⁺ vessels was assessed using IHC staining. Using Masson's trichrome staining, fibrotic changes were evaluated.

Results: FTIR data indicated successful interaction of Alg with Fib in the presence of ionic cross-linker (1% CaCl₂). Data confirmed higher biodegradation and swelling rates in Alg + Fib hydrogel compared to the Alg group (p < 0.05). Increased viability was achieved in encapsulated CD144⁺ ECs compared to the control group (p < 0.05). IF analysis showed the biodistribution of Dil⁺ ECs within hydrogel two weeks after transplantation. The ratio of Ang-2/Ang-1 was statistically up-regulated in the rats that received Alg + Fib + Mel hydrogel compared to the control-matched groups (p < 0.05). Based on the data, the addition of Mel and CD144⁺ ECs to Alg + Fib hydrogel reduced fibrotic changes. Along with these changes, the number of vWF⁺ and α-SMA⁺ vessels was increased in the presence of Mel and CD144⁺ ECs.

Conclusions: Co-administration of Alg + Fib with Mel and CD144⁺ ECs induced angiogenesis toward encapsulated cryopreserved/thawed ovarian transplants, resulting in reduced fibrotic changes.

Keywords: Angiogenesis; Cryopreserved/thawed ovarian transplant; Fibrin-alginate hydrogel; Freezing; Ovarian tissue.