Drug-free In Vitro Activation Combined With 3D-bioprinted Adipose-derived Stem Cells Restores Ovarian Function Of Rats With Premature Ovarian Insufficiency

Premature ovarian insufficiency（POI）is a common gynecological endocrine disease that signifiantly impacts the physical and psychological health of women in their child-bearing age.The final clinical manifestation is the accelerated depletion of ovarian follicles.Egg donation has been widely used for women with fertility demands,but it has a low success rate and does not allow them to have their own genetic offspring.In vitro activation（IVA）and drug-free IVA of residual dormant follicles are emerging as a novel effective approach for restoring fertility to women with POI,which enable them to conceive their own genetic offspring.However,the low pregnancy rates,loss of a substantial number of primordial follicles,and unimproved quality of age-associated oocytes have greatly restricted its application.The3D printing scaffold employing decellularized extracellular matrix（d ECM）-derived“bioink”containing adipose-derived stem cells（ADSCs）not only supports more cell retention and survival in the target tissue,but also exhibits a powerful pro-angiogenesis effect,which in turn reduces ischemic injury of the follicles and compensates for the disadvantages of IVA to some extent.This study aims to design a novel 3D-bioprined engineering ovary that com-bines the aforementioned two approaches,to investigate whether it could be applied to minimize ischemic injury of ovarian tissue by enhancing early angiogenesis and improving blood oxygen supply.By doing so,it aims to restore ovarian function and explore the underlying therapeutic mechanism responsible for angiogenesis,thereby improving the clinical effectiveness of IVA and bringing new hope for the recovery of ovarian function in POI patients.Part One Establishment of premature ovarian insufficiency rat model, and isolation and identification of adipose-derived stem cellsObjective:To establish the rat model of premature ovarian insufficiency（POI）and perform the isolation,culture,and identification of adipose-derived stem cells（ADSCs）from rat adipose tissue,in preparation for subsequent experimental investigations.Methods:1.Sprague-Dawley rats（8-10 weeks old,n=24）were randomly divided into two groups.The animal models of POI were induced by daily intraperi-toneal injection of CTX（50 mg/kg on the first day and 8 mg/kg/day for the following 14 consecutive days）.In the control group,the rats received equi-valent volumes of saline via intraperitoneal administration.Throughout the modeling process,the rats’body weight,hair condition,and estrous cycle were monitored.After the completion of modeling,twelve rats from both the POI group and the control group were sacrificed to harvest their ovaries and plasma.Ovarian morphology was examined using HE staining,while the apoptosis of ovarian cells was assessed using the TUNEL assay.The levels of serum hormones were measured using ELISA.2.ADSCs were isolated from the bilateral inguinal fat of rats through enzymatic digestion.Subsequently,the cells were cultured and passaged in vitro.The morphology of the cells was observed using inverted phase-contrast microscopy.Flow cytometry was employed to characterize the surface marker expression of ADSCs,including CD45,CD90,CD29,CD73,CD44,CD105and HLA-DR.Adipogenic and osteogenic differentiation of ADSCs was induced using specific differentiation kits,and the differentiation was comfirmed through Oil Red O and Alizarin Red staining.Results:1.Throughout the modeling process,noticeable changes were observed in the POI group,including a significant decline in body weight,hair loss with diminished luster,decreased activity,and irregular estrous cycles.HE staining revealed a remarkable reduction in the number of follicles at all developmental stages,accompanied by disordered arrangement of granulosa cells with nucl-ear pyknosis.ELISA results indicated a substantial decline in E₂hormone levels,a significantly increase in FSH levels,and a notable increase in TUNEL staining of apoptotic GCs in the POI group.Collectively,these findings confirmed that the successful creation of a rat model of chemotherapy-induced POI.2.ADSCs exhibited typical spindle-shaped fibroblast morphology and demonstrated adherent growth.Flow cytometry analysis revealed that the ADSCs were enriched with surface markers CD29（97.25%）,CD73（90.25%）,CD105（98.09%）,CD44（99.63%）,and CD90（99.66%）,which are indicative of mesenchymal stem cells（MSCs）.Conversely,only a small fraction of cells expressed CD45（0.24%）and HLA-DR（0.69%）.Moreover,these cells demonstrated multipotent characteristics of differentiating into adipocytes and osteoblasts when induced in vitro,confirming their capability for multilineage differentiation.Conclusions:1.This phase of the experiment successfully achieved the construction of the rat model for POI.2.In this stage of the experiment,ADSCs were successfully isolated and cultured using the enzyme digestion method.The cells exhibited appropriate morphology,and their surface markers were identified through flow cytomety analysis and multidirectional differentiation,meeting the identification criteri-a.3.In this part of the experiment,the construction of the POI rat models was successfully completed,and the culture and identification processes of ADSCs were established.Part Two Construction of 3D-Bioprinted Engineering Ovary in RatsObjective:The objective of this phase was to engineer a 3D-bioprinted scaffold utilizing ovarian decellularized extracellular matrix（d ECM）-based“bioinks”encapsulating ADSCs.The aim was to investigate whether the sca-ffold could enhance cell retention and survival in the target tissue and assess the biocompatibility between the ovarian tissue and the 3D scaffold.These investigations aimed to lay the foundation for the subsequent treatment of POI using the 3D-bioprinted engineering ovary.Methods:1.Porcine ovaries were subjected to physical,chemical,and enzymatic methods to remove cellular and nuclear components while preserving their biological components,mechanical integrity,and structural integrity.The ace-llular effectiveness of the porcine ovarian d ECM was evaluated through HE staining,DAPI staining,and Masson staining.2.A 3D-bioprinted scaffold was engineered using ovarian d ECM-based“bioinks”encapsulating ADSCs.The 3D-bioprinted engineering ovary was constructed by combining the 3D scaffold（8×8×3mm³）and POI ovarian frag-ments（1×1×1mm³）.The ovarian fragments were wrapped in the 3D scaffold,and the construct was placed on both sides of the back of the POI rats.Sam-ples were collected at 2 and 4 weeks after treatment for subsequent biocom-patibility examinations.3.Sixteen POI rats were randomly divided into two groups.CM-Dil lab-eled cells with ovarian fragments or 3D-bioprinted engineering ovary（CM-Dil labeled ADSCs）were placed on both sides of the back of the POI rats,respect-tively.The bilateral grafts were removed at 1 and 4 weeks after transplantatio-n,and frozen sections were prepared.The sections were incubated with DAPI and subsequently imaged under a fluorescence microscope.Results:1.HE staining and DAPI staining were performed to assess the preserva-tion of cellular components in native tissues and the absence of cellular com-ponents and nuclear material in decellularized tissues.Additionally,Masson staining verified the well-preserved collagen within the decellularized tissuee-s.2.In this study,we successfully designed a novel 3D-bioprinted engine-eering ovary for the first time.Visual examination revealed complete insertion of the ovarian fragments into the scaffold,and the 3D scaffold maintained its structural integrity.The tracing experiments using CM-Dil-labeled ADSCs demonstrated that the ADSCs were distributed within the interstitium of the ovaries rather than in the follicles.Furthermore,the retention ratio of ADSCs in the 3D-bioprinted engineering ovary group was significantly higher than that in the fragment-cell group,as observed at both 1 week and 4 weeks after transplantation.3.Following the surgical procefures,all rats survived without any com-plications.Complete removal of the grafts was achieved without any signs of infection,hematoma,or wound dehiscence.Ovarian fragments within the 3D scaffold exhibited normal growth without atrophy and tumor formation,and preovulatory follicles were observed within the grafts.HE staining revealed the presence of healthy-looking follicles at various developmental stages with-in the d ECM scaffold,with or without ADSCs,and no significant pathological response was observed.Conclusions:1.This phase of the experiment successfully completed the preparation and identification of porcine ovarian d ECM.2.In this stage of the experiment,a 3D-bioprinted scaffold was success-fully engineered using ovarian d ECM-based“bioinks”encapsulating ADSCs.Our findings confirmed that the 3D scaffold provided ADSCs with an approp-riate niche,leading to increased retention and survival of ADSCs within the target organ over an extended period.3.This part of the experiment successfully designed a novel 3D-biopri-nted engineering ovary and demonstrated its excellent biocompatibility.Part Three Drug-free in vitro activation combined with 3D-bioprinted adipose-derived stem cells restores ovarian function in rats with premature ovarian insufficiencyObjective:The objective of this study was to evaluate the effect of the subcutaneous transplantation of 3D-bioprinted engineering ovary on revascu-larization within the grafts and restoration of ovarian function in rats with POI.Additionally,we aimed to investigate the molecular mechanism underlying the proangiogenic effects of the system,within the ultimate goal of improving the clinical effectiveness of in vitro activation（IVA）techniques.Methods:1.Sixty rats with POI,exhibiting disrupted estrous cycles and weight reduction,were included in this study.They were randomly divided into five groups,each consisting of 12 rats（n=12）.The groups were as follows:1)untreated group:POI rats without any intervention（POI group）;2)Ovarian fragments alone group:POI rats receiving subcutaneous transplantation of ovarian fragments measuring 1×1×1mm³（fragment-alone group）;3)3D scaff-old combined with ovarian fragments group:POI rats receiving subcutaneous transplantation of a 3D scaffold measuring 8×8×3mm³along with ovarian fragments（scaffold-fragment group）;4)Ovarian fragments combined with ADSCs group:POI rats receiving subcutaneous transplantation of ovarian fragments combined with ADSCs（fragment-cell group）;5)3D-bioprinted engineeering ovary group:POI rats receiving subcutaneous transplantation of a 3D scaffold with ADSCs and ovarian fragments（3D-bioprinted engineer-ing ovary group）.Additionally,twelve normal rats without any treatment were included as the control group.Following the designated treatments,samples were collected from four rats in each group at 1,2,and 4 weeks after treatment for subsequent experiments.2.Ovarian samples were collected and subjected to histological examin-ation using HE staining to observe the morphological features of the ovaries and quantify the number of follicles.Immunofluorescence staining was per-formed to assess the expression of CD31（a marker of angiogenesis）and PCNA（a marker of cell proliferation）.Immunohistochemistry was employed to evaluate the protein expression levels of Ki67（a proliferation marker）and Caspase-3（an apoptotic marker）.TUNEL staining was utilized to determine the number of apoptotic cells in the ovarian tissues.Additionally,the serum hormone levels were analyzed using ELISA.Furthermore,changes in body weight and estrous cycle patterns were monitored throughout the entire treat-ment period.3.Ovarian samples were utilized to extract both total protein and m RNA.Western blot analysis was employed to determine the protein expression levels of molecules associated with the PI3K/AKT pathway,including VEGF,p-PI3K,PI3K,p-AKT and AKT.RT-PCR was carried out to assess the m RNA expression level of angiogenic factors,namely VEGF,FGF-2 and angiogenin.Results:1.This experimental segment demonstrated notable improvements in the weight of POI rats and the number of follicles,accompanied by a restoration of the estrous cycle and near-normal levels of serum hormones.Moreover,the transplantation of the 3D-bioprinted engineering ovary led to a significantly increased in cell proliferation within the ovary,while reducing the level of cell apoptosis.2.Macroscopic evaluation of the grafts revealed that the 3D-bioprinted engineering ovary group exhibited a higher presence of surrounding blood vessels compared to the other groups.Additionally,the degree of vasculari-zation in this group initiated earlier and persisted for a longer durationn.Immunofluorescence analysis further confirmed the presence of elevated CD31-positive signals and functioning blood vessels within the grafts of3D-bioprinted engineering ovary group,indicating enhanced vascularization.These preliminary findings suggested that the transplantation of the 3D-bioprinted engineering ovary promotes angiogenesis and restores ovarian functions,potentially mediated by the PI3K/AKT signaling pathway.Conclusions:1.This experimental segment provided evidence that the 3D-bioprinted scaffold loaded with ADSCs achieved a higher rate of vascularization and mitigated substantial follicle loss during the early grafting period.These findings offer a partial compensation for the lilmitations associated with IVA.2.The transplantation of the 3D-bioprinted engineering ovary effectively stimulated angiogenesis and restored ovarian functions through the activation of the PI3K/AKT signaling pathway.3.The application of 3D-bioprinted engineering ovary represents a promising and emerging approach for the restoration of impaired ovarian function in individuals with POI.