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

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% CaCl. 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 × 10 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% CaCl). 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.