Effect of Adipose-derived Mesenchymal Stem Cells Combined With Urinary Bladder Matrix Scaffold on the Structure and Function of Autografted Rat Ovaries


 Background: Ovarian transplantation has unique advantages in the preservation of female fertility, especially in young women with cancer who need chemotherapy. However, a large number of follicles are lost because of ischemia during ovarian tissue transplantation. While it has been reported that adipose-derived mesenchymal stem cells (ADSCs) accelerate angiogenesis, the transplanted ADSCs usually diffuse quickly from the target tissue. Urinary bladder matrix (UBM) is an extracellular matrix biomaterial that has a complete basement membrane and provides a foundation for transplanted cells to anchor, migrate, and function. In this study, ADSCs on UBM scaffolds (UBM/ADSCs) were transplanted during ovary autotransplantation in rats to test whether collagen/ADSCs have a better therapeutic effect than transplantation of ADSCs alone. Method: A total of 30 rats were divided into 5 groups of 6 rats in each . untreated-control, oophorectomy , autograft , autograft + ADSCs (ADSC) and autograft + UBM/ADSCs(UBM/ADSC). 28 days after ovary autografting, follicle number, serum concentrations of follicular stimulating hormone and anti-Mullerian hormone and apoptosis rate were also estimated. At 7and 28 days post ovary autografting, angiogenesis was detected. The estrous cycle recovery was measured.The results were analyzed using one-way analysis of variance (ANOVA) and Tukey test, and the means were significantly different at P< 0.05.Results: The number of both growing follicles and primordial follicles in rats in the ADSC/UBM and ADSC groups was significantly higher than that in rats in the autograft group (P <0.05). Follicle stimulating hormone levels in rats in the ADSC/UBM group were significantly decreased and anti-Müllerian hormone levels increased compared to control rats (P <0.05). Apoptosis rate in the UBM/ ADSC group was lower than the autograft group (P<0.05). The angiogenesis was accelerated following ADSC/UBM transplantation.Rats in the ADSC/UBM and ADSC groups showed better estrous cycle recovery than rats in the autograft group (P<0.05).Conclusions: UBM increases the retention of ADSCs in ovaries and contributes to long-term restoration of ovarian function. UBM/ADSC transplantation may be a promising candidate for ovarian transplantation.

tissues of the heart, kidneys [8], skin [9] and liver [10]. Malek et al. [11] demonstrated that ADSC transplantation improves the function of rat grafted ovaries through angiogenesis acceleration; however, ADSC therapy is restricted by insu cient settlement of transplanted cells in the target tissue [2]. The cells usually diffuse quickly to the surrounding organs or tissues, and the interplay of biological factors, including in ammation, apoptosis, and ischemia of the injected areas, make cell retention more di cult in the target tissue [12]. The application of scaffolds for the delivery and support of grafted cells represents a promising method for retaining stem cells in grafted organs [13]. Urinary bladder matrix (UBM), a decellularized extracellular matrix (ECM), is used clinically in a variety of applications. Clinical indications of commercialized UBM include the reinforcement of abdominal wall repair [14], management of diabetic ulcers [15], and management of deep wounds [16]. The immune microenvironment created by the UBM alters the presence of various cytokines and growth factors that can contribute to stem cell differentiation and tissue regeneration [14]. Therefore, in the present study, ovary autografting under the rat kidney capsule was performed and ADSCs in UBM were transplanted to the graft site, and the endocrine function and structure of the autografted ovaries were investigated.

ADSCisolation and culture
Rat ADSCs were isolated and cultured as previously described [34]. Brie y, adipose tissues were collected from the inguinal area of Sprague-Dawley male rats at the age of 8-10 weeks. The tissues were digested with 0.25% (v/v) collagenase type I (Gibco, USA) at 37°C for 30 min. The suspension was centrifuged at 250 × g for 20 min to separate oating adipocytes from the SVF. The SVF was suspended with highglucose Dulbecco's modi ed Eagle's medium (HG-DMEM; Gibco), containing 10% fetal bovine serum (v/v) (Hyclone, USA), 50 U/ml penicillin (Gibco) and 50 mg/ml streptomycin (Gibco) in a humidi ed environment with 5% CO 2 (v/v) at 37℃. The culture medium was changed every 48-72 h until the cells reached 90% con uence. Isolated cells were broblast-like under light microscopy and attached to the bottom of the plate (Fig. 2C ).

Differentiation of ADSCsand ow cytometry analysis
The ability of isolated cells to differentiate into osteocytes and adipocytes was evaluated as described previously [35]. In brief, to attain osteogenic differentiation, cells were plated in 24-well plates in an osteogenic differentiation medium containing HG-DMEM, and the medium was changed every 3 days [36]. After 2 weeks, cells were xed and stained with Alizarin Red Suzhou Saiye Biotechnology [37]. Adipogenic differentiation of ADSCs was performed as previously described using Oil Red O staining Suzhou Saiye Biotechnology [37]. To determine cellular characteristics, cultured ADSCs were labeled with appropriate uorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC)conjugated primary antibodies for 30 min at 48°C and analyzed by ow cytometry (Becton Dickinson, USA). The antibodies detected were CD90, CD73, CD45, CD44, CD34, and CD11b (BD Pharmingen, USA).

Preparation of the urinary bladder matrix
The UBM was derived from porcine urinary bladder provided by ZhuoRuan Medical Technology Co., Ltd.
(Suzhou, China). Brie y, excess adipose and collagen connective tissue was removed from the urinary bladder, and a rectangular-shaped sheet was formed by opening the bladder from the neck to the dome region. The tunica serosa, tunica muscularis externa, tunica submucosa, and muscularis mucosa were removed by mechanical delamination, leaving only the basement membrane and tunica propria intact. The remaining tissue was then soaked in deionized (DI) water and the layers of the urinary bladder that remained constituted the UBM. The UBM appeared white and consisted of soft akes ( Fig. 2A). Under a scanning electron microscope, the UBM appears loose and porous, and has a high porosity and a wide range of pore diameters, which are conducive to the colonization and proliferation of cells (Fig. 2B).

Suspended ADSCs on the UBM and observation
ADSCs (1×10 6 ) were suspended on the UBM approximately 3×3 mm 2 , for 48 h, whereafter the ADSC/UBM fragments were studied by scanning electron microscopy (SEM). For SEM analysis, the ADSC/UBM fragments were dehydrated in a graded series of ethanol and dried with hexamethyldisilane 21 as described previously [38]. In this study, under a scanning electron microscope, attened cells were well developed with extended cytoplasmic processes, and lopodia on UBM were observed (Fig. 2D).

Ovarian autografting
For the autograft, rats in the ADSC and ADSC/UBM groups were anesthetized with intraperitoneal injections of 3% pentobarbital sodium at a dose of 0.1 ml/100g. Under aseptic conditions, a bilateral ovariectomy was performed and the ovary was resected. The ovarian tissue was divided into two parts: a small incision was made in the ipsilateral renal capsule, and the two separated ovarian tissues were pushed under the renal capsule. Aliquot suspensions (40 ml) were injected into the core of the ovaries using 0.33 mm (29G) needles on BD Ultra-FineTM 1.0-ml disposable insulin syringes (Becton Dickinson and Company, Franklin Lakes, NJ, USA) [13]. For the autograft group, 40 ml of PBS was injected per ovary tissue. For the ADSC group, 40 ml PBS with 1 × 10 6 ADSCs was injected per ovary tissue. The ADSC/UBM group received 1 × 10 6 ADSCs in 40 ml PBS suspended on 3×3 mm 2 UBM. The muscles and skin were sutured in an interrupted fashion.

Estimation of the number of follicles
Twenty-eight days post-transplantation, rats were anesthetized and the ovaries were removed from the renal capsule, xed in Bouin's xative for 24 h, and dehydrated using ascending concentrations of ethanol. Ovaries were embedded in para n and sectioned at 5 μm, and morphological grading and follicle counts were performed following hematoxylin and eosin staining (Merck, Darmstadt, Germany). Every fth section obtained from each ovarian para n block was evaluated to avoid a consecutive assessment of the same follicle; follicles were counted in ovaries from each rat in each group. Follicle assessment and follicle counting were performed according to the accepted published standards [39].
Primordial follicles were de ned as oocytes surrounded by a single layer of at granulosa cells. All other follicles at more advanced stages of maturation were grouped and de ned as growing follicles. All counted primordial, primary, secondary, and tertiary follicles were classi ed as normal or atretic according to the following criteria: (i) primordial, single layer of attened pre-granulosa cells; (ii) primary, single or few layers of cuboidal granulosa cells where the antrum is absent; (iii) secondary, multiple layers of cuboidal granulosa cells where a multifocal antrum is present; (iv) tertiary, multiple layers of cuboidal granulosa cells where a single antrum is present.
Immuno uorescence At 7 and 28 days post-ovary autograft, CD31 was detected using immunohistochemical techniques. For this purpose, rats were anesthetized as mentioned above, and the ovaries were removed, xed in Bouin's xative for 24 h, and dehydrated using ascending concentrations of ethanol (70-100%). Slides were depara nized in xylene and rehydrated with decreasing concentrations of ethanol. The slides were steamed in citrate buffer (10 mM, pH 6.0) for 30 min for antigen retrieval and exposed to 3% hydrogen peroxide for 30 min. Slides were blocked in 5% BSA for 1 h and incubated with primary antibodies against CD31 (Bioworld, USA) overnight. For immuno uorescence analysis, slides were labeled with CD31 and stained with Cy3-labeled anti-rabbit IgG secondary antibodies (1:800)  Hormone assay Twenty-eight days after transplantation, blood samples were collected and centrifuged at 3000 × g for 5 min, and the levels of serum anti-Mullerian hormone (AMH) and follicle-stimulating hormone (FSH) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Xi Tang, China) according to the manufacturer's instructions.

Vaginal smear examination
Seven days post-autograft, sterile pipettes and sterile normal saline were used to gently wash the vaginal wall of each rat. Then, the cells were smeared onto a clean glass slide and observed under a light microscope (BX51; Olympus) at 100× magni cation.

Data analysis
Data were statistically analyzed using SPSS software (version 19) and one-way analysis of variance (ANOVA) and Tukey's test, and the means were considered signi cantly different at P < 0.05.

Characteristics of ADSCs
Flow cytometry revealed that approximately 90% of the cells were negative for CD34, CD11b, and CD45, and positive for CD105, CD73, and CD90, indicating a high purity of ADSCs (Fig. 1A-J). Alizarin Red and Oil Red O staining showed that the isolated cells could differentiate into osteoblasts and adipocytes ( Fig.  1K-L).

Appearance of transplanted ovarian tissue
On the 28th day post-operation, the kidney tissue was exposed along the original back incision to assess the transplanted ovaries. All ovarian tissues in the three autograft groups survived, and new blood vessels grew from the kidney to the graft on the surface. The autograft ovarian tissues were fresh and alive. The blood supply in rats in the ADSC and ADSC/UBM groups was more abundant than that of rats in the autograft group. There was more neovascularization in rats in the ADSC/UBM group (Fig. 3).

The number of follicles
The mean number of primordial and growing follicles was evaluated and counted in all groups 28 days post-autograft. The primordial follicle numbers in the ADSC and ADSC/UBM groups improved compared to those in the autograft group (P<0.05), and the number of primordial follicles in ADSC/UBM rats increased signi cantly compared to that in ADSC rats. The outcome of growing follicles in rats in each group was similar. However ,the follicle numbers in rats in each transplantation group was signi cantly lower than that in rats in the control group (P<0.05; Fig. 4).

Immunohistochemistry with CD31
On day 7 post-autograft, CD31 expression in rats in the ADSC group increased signi cantly compared to that in rats in the autograft group. When rats were transplanted with ADSC/UBM, CD31 expression increased signi cantly compared to that in rats that received ADSCs only (P <0.05; Fig. 5). On day 28 post-autograft, CD31 positive cells were further increased in rats in each group, and the expression in ADSC/UBM rats was increased signi cantly compared to that in rats in the other two groups (P<0.05).

Apoptosis rate
The apoptotic rate was calculated using Image Pro Plus 6 software (Media Cybernetics, Silver Spring, USA). When transplanted with ADSCs, the apoptotic rate decreased compared to that in the autograft group (P>0.05). When transplanted with ADSC/UBM, apoptosis was decreased signi cantly compared to the other two groups (P<0.05; Fig. 6 ).

Hormone assay and vaginal cytology
A signi cant increase in the serum concentration of FSH was detected in rats in the autograft group compared with to rats in the control group, although it signi cantly decreased in rats in the ADSC/UBM group when compared to rats in the autograft group (P < 0.05). The serum concentration of AMH signi cantly decreased in rats in all the surgery groups compared to control rats (P < 0.05), and concentrations in rats in the ADSC and ADSC/UBM groups were signi cantly higher than those in rats in the autograft group (P < 0.05; Table 1). The estrous cycle recovered in all rats that underwent surgery, but the starting day of the estrous cycle was signi cantly more rapid in rats in the ADSC/UBM group than in rats in the autograft group (P < 0.05; Table 1).

Discussion
Angiogenesis plays an important role in tissue development, repair, and regeneration. In many diseases, such as myocardial and peripheral ischemia, diabetic ulcers, retinal diseases, and chronic wounds, the pathophysiological problem lies in the decreased blood supply [17], which induces the death of tissue cells. The rapid formation of new capillaries is essential for tissue engineering and regenerative medicine. Capillaries deliver essential nutrients and oxygen to the cells and remove waste. Similarly, adequate blood perfusion is essential for the survival and functional life of the transplanted ovaries. How to quickly restore the blood supply of the grafts, promote the formation of new blood vessels, and reduce the loss of ovarian follicles is the key to the success of ovarian transplantation. In recent years, stem cell therapy has become a promising and advanced scienti c research area [18], and has already shown potential application in several diseases, such as diabetes mellitus [19], heart failure [20], and disorders of the nervous system [21]. ADSCs, isolated within the stromal vascular fraction (SVF) [22] in a less invasive and reproducible manner, were demonstrated to differentiate into the adipogenic lineage, and their multipotency is suitable for ectodermic and endodermic tissue repair [23]. Evidence suggests that ADSCs induce neoangiogenesis during tissue repair and wound healing. They secrete factors such as vascular endothelial growth factor (VEGF), Hepatocyte growth factor(HGF) , and Basic broblast growth factor (bFGF) [24,25], which can stimulate the differentiation of ADSCs into endothelial cells. Recently, ADSCs were con rmed to be able to induce angiogenesis and restore the number of ovarian follicles in ovary transplantation in mice [11]. However, the seed cells quickly diffused away from the target organ, while local in ammation and ischemia further decreased the viability of the remaining stem cells, which resulted in a low survival rate of the transplanted cells [26]. Many researchers have reported that ADSCs undergo massive cell loss after injection, and a large number of engrafted cells die within 1 month after transplantation [27]. Using biomaterials, ADSCs can in ltrate the constructed scaffold and differentiate into specialized newborn young cells, and the combination of ADSCs and elastin-like recombinamers (ELR)-based hydrogels have been reported to induce the formation of new blood vessels within the biomaterials, thus providing nutrients and oxygen support [28]. UBM, a decellularized ECM, is used clinically in a variety of applications, and current evaluations of UBM composition have shown that it is composed primarily of collagens, interspersed with proteoglycan ECM, growth factors, and cytokines [29]. UBM can support multiple tissues and cell types [30] and provide a suitable niche for transplanted cells to anchor, migrate, and function [31]. In this study, we demonstrated that ADSCs grow well on the UBM, and the effect of ADSCs combined with the UBM on ovarian tissue grafted into a rat model was analyzed after ovary autotransplantation. The results of this study suggest that ADSCs improve angiogenesis in ovarian grafts 7 and 28 days post-transplantation. Additionally, the effects of ADSC/UBM were found to increase graft survival by triggering angiogenesis and reducing apoptosis. The present study con rmed that ADSCs trigger the angiogenic process, which can initiate the formation of new blood vessels either by sprouting, intussusception, or elongation via the incorporation of circulating endothelial cells. In the context of the physiological angiogenic process of folliculogenesis, most of these processes are effective. Ovarian tissue grafts are exposed to ischemic damage during the post-transplantation period until the vasculature develops. Vascular connections between the host and an ovarian strip grafted into the murine ovary were observed 5 days after transplantation [32]. To determine whether the addition of ADSCs and the UBM had a bene cial effect on vascular recruitment and limited the period of tissue hypoxia, transplanted fragments were analyzed 7 and 28 days after grafting. The results showed that graft vascularization was effectively improved as early as three days post-transplantation. Using cell-speci c CD31 immunohistochemistry, the data con rmed the proangiogenic effect of ADSC/UBM, which modulated the follicular number and altered the brosis and apoptosis index. In this study, follicular morphology was evaluated in several sections using light microscopy, as previously described [33]. The results showed that, in rats in the ADSC/UBM group, the density of CD31-positive cells was highest after 7 and 28 days compared to rats in the autograft and ADSC-only groups. There is probably a relationship between the presence of VEGF, FGF, and IGF in the antral follicles and corpus lutea with angiogenesis in the cortex of mature ovaries [42]. The production of these factors by the transplanted ADSCs [43] may lead to increased angiogenesis in the cortex of grafted ovaries, which is essential for folliculogenesis and the production of steroids [44,45]. UBMs can promote the attachment of exogenous stem cells, recruit stem cells from the host, and provide an ideal microenvironment for their proliferation and differentiation.

Conclusions
This study demonstrated, for the rst time, that the UBM increases the retention of ADSCs in ovaries and contributes to long-term restoration of ovarian function, including estrus cycles, hormone levels, and follicle numbers. ADSC transplantation on UBMs improved the outcome of rat ovarian transplantation.
UBM/ADSC transplantation is a promising candidate for ovarian transplantation.