Advances in cancer treatment have improved survival rates for patients of childbearing age, but chemotherapy and radiation can cause irreversible damage to the ovaries, leading to infertility. Ovarian tissue cryopreservation (OTC) and transplantation (OTT) are fertility preservation options, but ischemia-reperfusion injury after transplantation reduces primordial follicle survival. While complete ovarian transplantation with arteriovenous anastomosis can reduce ischemic injury, clinical application is limited by factors such as the lack of appropriate cryoprotectants and the complexity of vascular anastomosis. Co-transplantation with exogenous endothelial cells (ExECs) accelerates tissue perfusion, but a biomaterial-based co-transplantation system is lacking. Graphene oxide (GO) has a large surface area and abundant oxygen functional groups that promote cell-tissue interaction, intracellular signaling, and angiogenesis. Combining GO with biomaterials like poly-L-lactic acid (PLLA), a biocompatible and degradable polymer, could create a composite material to aid organ transplantation. However, PLLA's low initial strength and rapid strength decay need to be addressed. GO's unique electrical and mechanical properties can enhance the mechanical properties of polymers. This study aimed to create GO/PLLA nanofiber scaffolds to enhance ovarian tissue transplantation by promoting angiogenesis and improving tissue fusion.

The literature review section of the paper cites previous studies on fertility preservation in cancer patients, ovarian tissue cryopreservation and transplantation techniques, the challenges of ischemia-reperfusion injury, and the angiogenic properties of graphene oxide. It also highlights the use of poly-L-lactic acid (PLLA) in tissue engineering and the potential of combining GO with PLLA to enhance the properties of the biomaterial.

The study involved several key steps: 1. **Preparation and Characterization of GO/PLLA Nanofiber Scaffolds:** Graphene oxide (GO) was synthesized from natural graphite using a modified Hummer's method. GO/PLLA nanofiber scaffolds with varying GO concentrations (0.0, 0.5, 1.0, and 4.0 wt%) were prepared using electrospinning. The scaffolds' morphology, chemical structure, mechanical properties (Young's modulus and strength), hydrophilicity (contact angle), and porosity were characterized using various techniques such as atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and contact angle goniometry. 2. **In Vitro Degradation Assay:** The degradation behavior of the scaffolds was evaluated by immersing them in different media (water, PBS, DMEM, and DMEM with FBS) and observing changes in morphology and microstructure over time. 3. **In Vitro Cytotoxicity Study:** The cytotoxicity of GO nanosheets and the GO/PLLA nanofiber scaffolds on granulosa cells (GCs) was assessed using CCK-8 and Live/Dead staining assays to determine cell viability. 4. **POI Model Construction:** A primary ovarian insufficiency (POI) mouse model was created by administering cisplatin intraperitoneally for 10 days. Ovarian atrophy, reduced follicle numbers, and altered hormone levels confirmed the success of the model. 5. **Ovarian Tissue Transplantation:** Normal ovarian tissue was transplanted into the ovarian cysts of POI mice, either alone or co-transplanted with GO/PLLA nanofiber scaffolds (0.0 wt% and 1.0 wt%). 6. **Oocyte Collection and Maturation:** Oocytes were collected from transplanted ovaries and subjected to in vitro maturation (IVM) to evaluate their developmental potential. 7. **Immunohistochemistry:** Immunohistochemistry was performed to assess the expression of CD31 and CD34 (markers of angiogenesis) in transplanted ovarian tissue and on the scaffolds themselves. 8. **Hormone Level Assay:** Serum hormone levels (E2, AMH, FSH, and LH) were measured by ELISA to evaluate ovarian function recovery. 9. **Follicle Counting:** Follicle numbers in transplanted and contralateral ovarian tissues were counted to assess follicle development. 10. **TUNEL Assay:** A TUNEL assay was used to measure apoptosis in ovarian tissue. 11. **Western Blotting:** Western blotting was performed to detect the expression levels of eNOS and p-eNOS in transplanted ovarian tissue. 12. **NO Detection:** Nitric oxide (NO) levels were measured in the supernatant of granulosa cells co-cultured with GO/PLLA nanofiber scaffolds of varying concentrations. 13. **Statistical Analysis:** Data were analyzed using appropriate statistical methods such as one-way or two-way ANOVA, Student's t-test, and chi-square tests.

The key findings of the study are as follows: 1. **Scaffold Characterization:** The GO/PLLA nanofiber scaffolds exhibited a continuous, uniform, and defect-free structure with a hydrophilic surface and high porosity, suitable for cell infiltration. The incorporation of GO enhanced the mechanical properties of the PLLA, particularly at a 1.0 wt% concentration. The scaffolds showed minimal degradation in water and PBS but displayed controlled degradation in cell culture media. The 1.0 wt% GO/PLLA concentration demonstrated optimal properties. 2. **In Vitro Cytotoxicity:** GO nanosheets exhibited minimal cytotoxicity to granulosa cells. The 1.0 wt% GO/PLLA scaffold showed the best support for cell growth and proliferation. 3. **In Vivo Ovarian Tissue Survival and Function:** Co-transplantation of ovarian tissue with the 1.0 wt% GO/PLLA scaffold significantly improved the survival rate of transplanted ovarian tissue compared to transplantation alone or co-transplantation with the 0.0 wt% scaffold. The 1.0 wt% GO/PLLA group showed improved tissue fusion, follicle development, and significantly higher E2 and AMH levels while showing lower FSH levels compared to the control group. Oocytes derived from the 1.0 wt% GO/PLLA group exhibited higher maturation rates than those from other groups. 4. **Angiogenesis:** Co-transplantation with the 1.0 wt% GO/PLLA scaffold significantly promoted angiogenesis in the transplanted ovarian tissue, as evidenced by increased expression of CD31 and CD34. Angiogenesis was also observed on the scaffold itself. 5. **eNOS/NO Pathway:** The improved survival and function were linked to increased expression of phosphorylated endothelial nitric oxide synthase (p-eNOS) and nitric oxide (NO) production, suggesting the involvement of the eNOS/NO signaling pathway in the angiogenic effects of the GO/PLLA scaffolds.

This study demonstrated that GO/PLLA nanofiber scaffolds, particularly at a 1.0 wt% GO concentration, significantly enhance the success of ovarian tissue transplantation. The improved survival and function of the transplanted tissue are attributed to enhanced angiogenesis, likely mediated by the eNOS/NO pathway. The controlled degradation of the scaffold materials and their biocompatibility further contribute to the positive outcome. The results suggest a novel approach to improve ovarian tissue transplantation, addressing the limitations of current methods by reducing ischemia-reperfusion injury and promoting vascularization. This approach may have broad implications for fertility preservation and potentially other organ transplantation procedures.

This study presents a novel GO/PLLA nanofiber scaffold for enhancing ovarian tissue transplantation. The 1.0 wt% GO/PLLA scaffold significantly improved transplanted ovarian tissue survival, function, and angiogenesis, likely through the eNOS/NO pathway. This provides a new strategy for fertility preservation and a potential platform for other organ transplantation applications. Future research could focus on optimizing scaffold design and exploring other applications of this biomaterial.

The study was conducted in a mouse model, and the results may not directly translate to humans. Further research is needed to confirm the findings in larger animal models and eventually in human clinical trials. The specific mechanisms underlying the interaction between the scaffold and ovarian tissue require more investigation.