Acceleration of Pelvic Tissue Construction by Overexpression of Basic Fibroblast Growth Factor in the Stem Cells

Background Pelvic organ prolapse (POP) is a common debilitating condition affecting about 30–40% of women. The application of stem cells therapy and growth factor has greatly promoted the development of pelvic tissue engineering, which remains a promising approach, but there is no consensus on the therapeutic mechanism of stem cells and the application of growth factors. Stem cells were mainly used as seed cells to differentiate into target tissue cells, fuse with target tissue after transplantation and paracrine effect to play a therapeutic role in pelvic tissue engineering. However, whether stem cells can be differentiated into target tissue cells is still to be a question,in this regard, the contemporary trend is to investigated the effect of adipose-derived stem cells (ADSCs) as the seed cells of pelvic tissue engineering on the repair of POP and the underlying mechanisms. Methods In the present study,we evaluated the therapeutic potential of gene-modied ADSC that overexpress basic broblast growth factor (cid:0) bFGF (cid:0) and evaluated its effects on paracrine function and directional differentiation ability. Results The results showed that following ADSCs are designed to continuously release controllable levels of growth factors during the control period of repair, taking advantage of the paracrine function of stem cells to accelerate cell growth and extracellular matrix (ECM) reconstruction during the early stage of stem cell implantation, and then stem cells are differentiated into target tissues-broblasts to accelerate the reconstruction of pelvic oor tissues. Conclusions We suggest that the observed effects are determined by pleiotropic effects of bFGF, along with the multifactorial paracrine action of ADSC which remain viable and functionally active within the engineered cell construct.Thus, we demonstrated the high therapeutic potential of the utilized approach for pelvic tissue engineering.


Introduction
POP is the herniation of pelvic organs into the vagina with symptoms of bladder, bowel, and sexual dysfunction [1].For women who suffering from POP, surgery is the primary treatment option and recurrence is common with ungrafted methods [2].Several complications such as foreign body reactions, excessive in ammatory responses, and vaginal erosion affect the use of synthetic mesh or biological grafts in clinical practice [3].Therefore, tissue engineering methods using stem cells for POP are rapidly emerging as potential strategies to treat patients the application of stem cells has greatly promoted the development of tissue engineering,which has been successfully applied to the regeneration of bone, cartilage, skin, muscle and other organs [4,5].A variety of studies have reported differences between various types of stem cells, including bone marrow mesenchymal stem cells (BMSCs), endometrial mesenchymal stem cells (EMSCs), and ADSCs, which showed promising safety and e cacy for POP in vitro and in vivo [6].Adipose tissue is easily obtained by liposuction which has been shown to have more stem cells/progenitor cells than bone marrow tissue, and ADSC has a faster expansion capacity than stem cells derived from other tissues,A recent study compared the differences of ADSCs and BMSCs, they found ADSCs as a more stable and controllable stem cell source, was more adaptable and exhibited superiority in regulating in ammation [7]. ADSCs are more active than BMSCs, secrete more autocrine cytokines and immunoregulatory factors, and exhibit lower immunogenicity, which is ideal for cell therapy [8][9][10].The main therapy mechanism of stem cell is directional differentiation and self-renewal ability, which can be used as seed cells to differentiate into target tissue cells or fuse with target cells after transplantation. With proper conditions, they can display strong tissue and organ repair capabilities.
However, if stem cell are not properly controlled, they may not achieve the goals, and may even cause safety hazards such as tumors. Therefore, how to induce stem cells in a direction which suitable for pelvic oor supporting tissues-broblast,is also the current study bottleneck.
Research has demonstrated that ADSCs can be induced into broblasts by growth factors in vitro [11][12].Growth factor is an important factor that constitutes the cell micro-environment. FGF is one of the most widely studied among many cell growth factors. It is one of the important wound healing factors in the body, has a wide range of biological activities and can affect early in ammation, granulation and the entire wound repair process such as tissue regeneration, epithelialization, and tissue remodeling,thus, has an important application value [13].Our data indicate successfully induced the ADSCs into broblasts by bFGF in vitro, and con rmed that bFGF can signi cantly increase the proliferation of differentiated broblasts, moreover the expression of collagen.However, the growth factor half-life is short and the biological permeability is poor, so it is very easy to be inactivated, and repeated dosing is required to maintain the local concentration [13]. Additionally, studies have shown that bFGF injection is associated with severe anemia, which limits the systemic application of bFGF [14][15]. Therefore, it is necessary to achieve continuous release and an effective concentration of bFGF, in order to provide a suitable microenvironment for the directed differentiation of stem cells.
Therefore, we hypothesized that the use of ADSC to overexpress the bFGF gene and provide a reasonable microenvironment of bFGF which can accelerate the repair and reconstruction of pelvic oor tissue. When ADSCs are designed to continuously release controllable levels of growth factors during the control period of injury repair, take advantage of the paracrine function of stem cells to accelerate cell growth and extracellular matrix reconstruction during the early stage after stem cell implantation, and later stem cells are differentiated into target tissues-broblasts under the continuous stimulation of bFGF to accelerate the reconstruction of pelvic oor tissues. The combination of gene and cell therapy will accelerate the reconstruction of pelvic oor tissue through a combination of paracrine and directed differentiation mechanisms, and may be more effective than systematically giving individual growth factors.The results of this basic study may indicate a new strategy to speedily ameliorate POP.

Construction lentivirus and transfection
Lentiviral constructs expressing rat bFGF were prepared by Hanbio(shanghai)using HBLV-r-Fgf2-Null-ZsGreen-PURO.ADSCs at passage 2 were co-transduced or single-transduced with lentiviral constructs at a multiplicity of infection (MOI) of 100 and were screened with 4 µg/mL puromycin. After 72 h of screening, green uorescence were observed using an immuno uorescence microscope to con rm successful transfection of ADSCs. In addition, the expression of bFGF was detected using Real time RT-PCR and western blot. Real time RT-PCR was carried out by isolation of the total RNA cultured on the all groups using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). For synthesize of the cDNA, random hexamer primer and M-MuLV Reverse Transcriptase kit (Fermentas, Helsingborg, Sweden) was applied based on the manufactured protocol. Finally, real time RT-PCR was carried out using SYBR premix ExTaq (Takara Bio, Shiga, Japan) and an ABI StepOne system. All primers were used in this study was bFGF:Forward5'-ACGCCTGGAGTCCAATAACTACAAC-3'Reverse5'-AGGCCCCGTTTTGGATCCGAGTTTA − 3';β-Actin:Forward5'-TGGGTATGGAATCCTGTGGCA − 3',Reverse5'-TGTTGGCATAGAGGTCTTTACGG − 3') 2.3 Preparation of cell culture media(CM) ADSC, ADSC bFGF were cultured in 6-well plates (105 cells/well). When the cells reached 90% con uence, the medium was replaced with 1 mL of serum-free medium, and the cells were incubated for 24 hours. The supernatant was collected after centrifugation at 12 000 g for 10 minutes and stored at − 80 °C.

CCK8 assay.
When the con uency of the ADSCs and ADSC bFGF reached ~ 80%, the cells were washed with PBS twice and treated with 0.25% trypsin, followed by preparation of a single cell suspension. After counting, the cells were seeded in a 96-well plate(Costar) with a cell density of 2 × 10 3 cells per well (0.2 ml, with 6 replicate wells), which were placed in a 5% CO2 cell culture medium and kept at 37 °C and saturated humidity; 10 ul CCK-8 was added to each well followed by a further incubation at 37 °C for 2 h. The medium was then removed, and the dye intensity was then read on a micro-plate reader (Bio-Rad) at 450 nm.

Co-culture assay
To con rm the paracrine effect of ADSCs and ADSC bFGF for broblasts, we used the co-culture system ( Fig. 1).The co-culture groups were established between ADSCs (5 × 103cells/well) in Cell culture inserts (Cell Culture inserts, 0.4 µm pores, BD Falcon™) and broblasts (5 × 103cells/well) in twelve-well plates (Cell Culture Insert Companion Plates, BD Falcon™) in DMEM.The control group was established between no cells in Cell culture inserts and broblasts only in twelve-well plates (5 × 103cells/well) in DMEM.

Wound healing assay
For the chemotaxis of ADSCs and ADSC bFGF ,migration assay of broblasts, the broblasts were prepared by Yubio(shanghai)in ADSCs-CM treated and ADSC bFGF -CM for 24 h, perspectively.The broblast monolayers were carefully scratched using a 10-µl pipette tip. After 24 h, the wounded area was photographed. The empty area which indicates the wound region was calculated.

Transwell assy
Cell transwell was assayed using transwell chambers (Costar, Cambridge, MA) with 8-mm-pore polycarbonate lters. Fibroblasts(10,000 cells/chamber) were seeded in the top of the cylindrical cell culture inserted in the DMEM without FBS. ADSCs-CM and ADSC bFGF -CM was placed in the wells to allow the cells to migrate through the lter for 36 h at 37 °C in 5% CO2,perspectively. Cells that did not migrate were removed from the upper surfaces of the chambers by scrubbing with a cotton swab. Cells that migrated to the lower membrane were xed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Invitrogen) for 30 min. Invasion cells were counted under a microscope.

Human umbilical vein endothelial cell tube formation assay
The human umbilical vein cell line, EA. hy926 (HUVEC), was were prepared by Yubio(shanghai).Tube formation was evaluated by culturing HUVEC on BD Matrigel (BD Biosciences). After incubating the wells with 80 µL of Matrigel for 1 hour, the HUVEC were resuspended in theADSCs-CM and ADSC bFGF -CM and DMEM medium alone as negative control (NC) into 96-well plates (5000 cells per well), then the number of tube-like structures were analysed 4 hours later. Quantitative analysis based on the number of lumens in each high-power eld.

Western blot analysis
Total protein was extracted from cells with a radio-immunoprecipitation assay buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing PMSF, incubated on ice for 30 min and centrifuged at 4˚C for 10 min (12,000 x g). The supernatant was obtained. The protein concentration of the supernatant of each sample was determined using a bicinchoninic acid kit (cat. no. 23225; Thermo Fisher Scienti c, Inc.), and deionized water was then used to adjust the amount of protein. Next, a 10% SDS-PAGE gel (cat. no. P0012A; Beyotime Institute of Biotechnology, Shanghai, China) was prepared and 50 µg of the protein sample was added to each well. Electrophoresis was conducted at a constant voltage of 80 V for 2 h. The proteins were transferred onto polyvinylidene uoride (PVDF) membrane (cat. no. ISEQ00010; EMD Millipore, Billerica, MA, USA) with a voltage of 110 V for 2 h. The PVDF membrane was blocked with TBST buffer containing 5% skimmed milk powder for 2 h. Then, the membrane was washed with TBST and incubated with rabbit polyclonal anti-body ColIA1 (1:100; cst), rabbit polyclonal antibody Col III A1(1:100; NOVUS), and GAPDH antibody (1:100;Abcam) The gray intensity of protein bands was quantitated with Image J and normalized to GAPDH. The p-Akt (Thr308), Akt, p-PI3K (Tyr458),PI3K, antibodies were purchased from Cell Signaling. bFGF growth factor antibodies were purchased from peprotech, at 4℃overnight, followed by three washed with TBST, 10 min each time for 3 times. The membrane was then washed with 0.1% PBS/Tween-20 (PBST) 3 times at room temperature, 10 min each time, immersed in enhanced chemiluminescence reaction solution (cat. no. WBKLS0100; EMD Millipore), and developed GAPDH was used as the internal reference, and the ratio of the gray values of the target protein band to the internal reference band was used as the relative protein expression.

The ADSCs differentiate into broblasts
ADSCs and ADSC bFGF from the third generation were selected and divided into three groups, with the cell density adjusted to 1 × 10 5 /ml: Control group (cells inoculated and cultured in DMEM-F12 containing 5% FBS),ADSCs group (cells inoculated and cultured in DMEM-F12 containing 5% FBS and 20 ng/ml bFGF) and ADSC bFGF group (cells inoculated and cultured in DMEM-F12 containing 5% FBS). Three parallel samples were used for each group. The medium was replaced every day. The survival condition of cells was observed after 1-4 weeks under an inverted microscope, followed by subsequent studies.The cells were then identi ed by immunohistochemistry of broblastspeci c protein1 (FSP1). SPSS 21.0 statistical software (IBM Corp. Armonk, NY, USA) was used to analyze the data, which were processed with normal distribution and homogeneity of variance tests. Measurement data are expressed as the mean ± standard deviation. Comparisons between two groups were conducted by t-test. Comparisons among multiple groups were assessed by one-way analysis of variance. The pairwise comparisons among groups were assessed by Tukey post hoc test. Enumeration data are presented as % and were assessed by the chi-square test. P < 0.05 was considered to indicate a statistically signi cant difference.

Statistical analysis
3. Results

ADSC bFGF exhibited a higher bFGF level and cell viability
Primary ADSCs were isolated from adipose tissue of 2-week-old Sprague-Dawley rats cultured in vitro. Spindle morphology and broblast-like morphology were observed at passage 1, and these characteristics were more pronounced at passage 3 (Fig. 1a). Fluorescence microscopy showed that most cells expressed green uorescent protein, which indicated successful transduction. Furthermore, qPCR and western blot analysis showed that the expression of bFGF was signi cantly higher in ADSCs transfected with bFGF than that in vehicle-treated ADSCs (Fig. 1F,G,H). Western blot and qPCR results con rmed that bFGF were expressed at higher levels in ADSC bFGF than in vehicle (Fig. 1L,M,N).CCK8 assay demonstrate that after transfected with bFGF,the proliferation of ADSC bFGF correspondingly increased.
2. ADSC paracrine ability and function was further increased after bFGF transfected To con rm the paracrine effect of ADSCs on broblasts, their cell viability was evaluated using a coculture system (Fig. 2a).The cell viability of broblasts co-cultured with ADSC bFGF was higher than that of broblasts cultured with ADSCs and alone (Fig. 2b). ADSCs made tubule formation of HUVECs and chemotaxis of broblasts( Fig. 2c-g).Then we examined the effect of ADSC on the broblast collagen synthesis ability and found both collagen I and III expression levels were increased ,collagen synthesis ability were further increased after ADSC transfected with bFGF Fig. 2 h-i).

ADSC bFGF differentiate into broblasts with a higher e ciency
In order to further verify the differentiation e ciency of ADSCs differentiate into broblasts after transfection, The three group differentiation following treatment with or without bFGF for 4 weeks. The control group and ADSC bFGF group vehicle-treated ,bFGF group treated with 20 ng/ml stimulation.The results showed that that the ADSC bFGF cell morphology gradually becomes long spindle after 2 weeks of culture,present a radial growth with compact cytoplasm that was different from the loose cytoplasm of ADSCs under a light microscope,the ADSC group have a large and loose cytoplasm, short protrusions and without morphological change until 4 weeks culture.Most cells have the typical broblast cell morphology on the 4 weeks, indicating that the cells gradually differentiate into broblasts cells (Fig. 3a).After 4 weeks, immunocytochemical staining was performed on the three groups of cells ADSC bFGF were positive for FSP1 and FGF group FSP-1 positive about 50%,the control group FSP-1 were negative (Fig. 3b). The collagen expression ability was tested on the 2th and 4th week immuno uorescence and WB showed that the collagen expression ability of ADSC bFGF increased from the 2nd week and reached the highest in the 4th week, which was signi cantly higher than the bFGF stimulation group (Fig. 3c-f).Together these results suggest that ADSCs overexpress bFGF may induce ADSC differentiation in vitro more effectively than with bFGF or alone. 4. ADSC bFGF activated PI3K/Akt signaling pathway to promote ADSC differentiate into broblast PI3K phosphorylase is a class of enzymes that phosphorylate phosphatidylinositol and can mediate intracellular signal transduction. The study found that growth factors can mediate stem cell differentiation by activating PI3K /Akt signaling and regulating Akt phosphorylation levels.Thus, we examined if the PI3K/Akt signaling pathway was involved in the process of ADSC bFGF mediate differentiation. We detected the changes in this pathway via Western blotting. Consistent with our hypothesis,we found in ADSC bFGF further increased the phosphorylation of PI3K, and Akt proteins compared with the ADSCs group (Fig. 5a,b,c). Then to elucidate if the PI3K/Akt signaling pathway is necessary for the differentiation process, we treated the two groups with or without PI3K inhibitor, LY294002.We found that LY294002 decreased the collagen expression which was consistent with our data (Fig. 4d,e), LY294002 reversed the increased expression of collagen.These data demonstrated that ADSC bFGF accelerated the differentiation process via increasing the activity of PI3K/Akt signaling pathway in a time-dependent manner.

The ADSC bFGF decreased the in ammation response in the injured broblasts
ADSC has been widely used for the anti-in ammatory therapy and wound healing and bFGF is an important damage repair factor. And it has been demonstrated that immune response plays important roles in the process of wound healing. Thus, we wanted to know if the immune response was also regulated by ADSC bFGF during the promotion of wound healing. We treated the injured broblasts isolated for 0,3,and 5 days post injury and treated them with LPS, which could induce the immune response ,together co-culture with or without ADSC bFGF .After 1 day, we examined the expression of in ammation factors, TNF-αandIL-6, by CBA.Indeed, we found that LPS signi cantly induced the immune response ,with the up-regulation of IL-1,TNF-αand IL-6,while ADSC bFGF dramatically repressd the increase in IL-1, IL-6 and TNF-α (Fig. 5). These data demonstrated that ADSC bFGF could suppress the in ammation in vitro.The present study demonstrated that ADSC bFGF provided strong pleiotropic effects (Fig. 6), contributing to more effective morphological and functional recovery. This therapeutic strategy that could be applied as seed cells which holds great promise for the pelvic oor tissue engineering.

Discussion
In view of the current status of mesh application in pelvic oor reconstruction surgery, the ability to induce autologous tissue regeneration is the study and development direction of pelvic oor tissue engineering mesh. Considering the role of bFGF in the repair of pelvic oor tissue is obvious but uncontrollable, the study transduced the bFGF gene into ADSCs to produce sustained, stable, and longterm expression of bFGF, moreover avoiding some adverse reactions.When ADSCs is designed to overexpress growth factors such as bFGF, this paracrine effect may be ampli ed [16]. We observed that ADSC bFGF increased its own proliferation and the ability to induce angiogenesis also increased, which is consistent with the results of our observation of increased levels of bFGF after transfection. BFGF stimulates blood vessel growth and has a synergistic effect with VEGF and platelet-derived growth factor (PDGF) during angiogenesis, which is important for wound healing [17][18].Moreover, prolonged exposure to protein mitogens, such as FGFs, is associated with increased risk for cancer [19][20][21] and induces pro-in ammatory responses in vitro [22].The oncogenic and proin ammatory effects of bFGF were observed with continuous exposure to bFGF at the dose of 10 ng/mL or larger, which were approximately at least 1,000-fold higher than the level of bFGF being released from MSCs [23] .ADSCs play an important role in the activation and migration of broblasts in tissue repair,we found that the chemotaxis ability of ADSC bFGF to broblasts did not increase, which may be related to the expression of chemokines didn't increase.
We found ADSC FGF has increased broblast proliferation and collagen expression. Fibroblasts can synthesize many ECM proteins, such as collagen, remodeling enzymes and their inhibitors. Fibrous collagen I and III are the main components of the vaginal and pelvic oor supporting tissues.Collagen I forms thick collagen bers that provide continuous tension to the pelvic oor tissue. Collagen III mainly affects the exibility and expansibility of tissues to overcome cyclical stress. Therefore, Col-I and III play an important role in repairing POP [24]. Studies have shown that abnormal metabolism of collagen may be one of the reasons that cause pelvic oor dysfunction disease in women [25], we observed that the ADSC FGF can be induced into broblasts and enhance the synthesize collagen capability,the microenvironment of the implantation site is improved which may mediates the reparation of pelvic oor tissue. Through the activation of broblasts, collagen synthesis is promoted, the collagen content of local tissues is improved,the ECM is reconstructed thereby The repair e ciency of tissue engineering has been improved [26].Increasingly evidence shows that during wound healing, collagen mediates the ECM to promote tissue regeneration [27]. Fibroblasts play a key role in wound healing by precisely regulating their function [28].
A key mechanism for ADSCs to promote tissue repair is the ability to differentiation. With appropriate conditions, they can display strong tissue and organ repair capabilities. However, if they are not properly controlled, we can not reach the goal, and may even cause safety hazards such as tumors. Therefore, how to induce stem cells to a directional differentiation which suitable for the repair of pelvic oor supporting tissues is a key for pelvic tissue engineering mesh, and is also the current research bottleneck. In this study have shown that indirect co-culture can promote the synthesis of type and collagen bers by ADSC bFGF , and successfully induce themselves differentiate into broblasts [29][30]. Lee et al [31] added connective tissue growth factor to the culture medium to differentiate bone marrow umbilical cord mesenchymal stem cells into broblasts. Yin et al [32] colonized mesenchymal stem cells on interlaced scaffold materials. The cells could differentiate into ligament-like broblasts and express ligamentrelated collagens such as type , type collagen, and Tenascin-C gene. The study by Xiong et al [33] showed that bFGF can induce bone marrow mesenchymal stem cells differentiate into broblasts, and there is no signi cant statistical difference in cell morphology and collagen synthesis ability from cells derived from veins.We successfully induced the differentiation of ADSCs into broblasts through bFGF [13],and found that the ADSC bFGF gradually differentiated into broblasts and more e cient: the expression of the broblast surface marker fsp-1 has been observed in about two weeks, most of the cells expressed fsp-1in fourth week.Then we found the production ratio and e ciency of collagen are higher. This provides a basis for the directed induction of differentiated stem cell in the direction of suitable pelvic oor tissue repair.
PI3-K/Akt is one of the important signaling pathways involved in the regulation of cell proliferation, selfrenewal and multi-directional differentiation potential [34]. PI3-K mainly are activated by platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), insulin, FGF, guinea pig hepatocyte growth factor (HGF), recombinant human epidermal growth factor (EGF), bone morphogenetic protein 2 (BMP-2) and other receptors by binding to their receptors. Activated PI3-K phosphorylates the 3rd hydroxyl group of the inositol ring to generate three 3-phospholipid inositol phospholipids. The product of PI3-K nally phosphorylates Ser473 and Thr308 on the Akt protein sequence to make it fully activated, which in turn activates the Akt downstream signaling pathway, mediating cell proliferation, migration, differentiation and survival [35]. Recently, the role of PI3-K/Akt signaling pathway in stem cell differentiation has received attention. Whether the PI3-K/Akt signaling pathway plays a regulatory role in the induction of ADSC differentiation into broblasts after bFGF transduction is unclear. We observed an increase in p-Akt levels of ADSC bFGF , indicating that ADSC bFGF can activate the PI3-K/Akt signaling pathway. In addition, the collagen expression of the cells after induction was signi cantly increased.Importantly, we found inhibition of PI3K with LY294002 reversed the effect of ADSC bFGF on the differentiation process,p-Akt levels decreased signi cantly, ADSC bFGF were negative for FSP1, after Ly294002 used to suppress the PI3-K/Akt signal pathway, the collagen expression was signi cantly reduced. The results of this experiment indicate that after ADSC overexpresses bFGF gene, it activates PI3-K /Akt signaling pathway and promotes the differentiation of ADSCs into broblasts. Therefore, we believe that by regulating the expression of bFGF and the activation of the PI3-K/Akt pathway, it promotes the induction and differentiation of ADSCs, thereby speeding up the process of tissue repair and provides a new treatment strategy for pelvic oor tissue engineering.
The in ammatory response is the rst step after trauma, and plays an important role in the wound healing process, and the continuous in ammatory response will damage the wound healing process. Therefore, it is also crucial to suppress in ammation during wound healing.In recent years, adipose tissue has become an attractive source of MSCs for cell-based therapies and regenerative medicine. ADSCs can be harvested from an ever increasing number of liposuction procedures.ADSCs have similar properties to BMSCs but do not decline with the age of the donor and are an alternative source of MSCs in regenerative medicine [36].Regardless of their origin, MSCs are usually de ned by their trophic, paracrine and immunomodulatory functions [37]. These non-stem cell properties appear to have the greatest therapeutic impact, evidenced by the large number of MSC-based clinical trials conducted for several life-threatening in ammatory or immune-related diseases [38]. A large body of medical literature indicates that MSCs repair damaged tissues because they respond to in ammation and migrate to injured sites and in uence the microenvironment through the release of molecules involved in reparative processes and tissue regeneration [39]. Biomaterial-based delivery of MSCs may bene t organ and tissue repair through paracrine effects.These properties make MSCs an attractive source of cells for seeding on the engineered biomaterials to in uence the foreign body reaction following implantation [40].We found that ADSC bFGF signi cantly inhibited the in ammatory response of damaged broblasts treated with lipopolysaccharide.
The results show that ADSC bFGF can reduce in ammatory.

Conclusion
There were multiple bene cial effects for use of ADSC bGF as seed cells in pelvic tissue engineering.In rst,as a direct ADSCs supplement;secondly,ADSC bGF stimulated trophic factors such as bFGF that stimulate angiogenesis, broblast differentiation; and thirdly,the early stage after ADSC bGF implantation, the paracrine function was used to accelerate cell growth and extracellular matrix reconstructon,in the late stage, ADSCs were differentiated into broblasts to accelerate the reconstruction of pelvic oor tissue. The combination of gene and cell therapy will accelerate the repair of pelvic oor tissue through a combination of paracrine and directed differentiation mechanisms, and may be more effective than systematically giving individual growth factors.Taken together, ADSC bGF may serve as a potential cellbased treatment for POP repair as it can provide an environment rich in stem cells,growth factors, and matrix proteins over a short time period, which can promote pelvic oor regeneration.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding authors upon reasonable request.
Ethics approval and consent to participate Not applicable Consent for publication Not applicable Figure 1 After transfected with bFGF,ADSC exhibited a higher bFGF level and cell viability a:Morphology of ADSCs in passage 3. b Immuno uorescence analysis of the expression of GFP in transfected ADSCs. C,d,e:Levels of mRNA and protein of bFGF in ADSCs were examined48h,72h after transduction respectively. The value is shown in each graph as mean ± SD. *p < 0.05, **p < 0.01 ***p < 0.001 and ****p < 0.0001.f:the proliferation after ADSCs transfection. The ADSCbFGF paracrine after bFGF transfected ADSCs transfected with bFGF enhanced the tubule formation of HUVEC both upregulated the broblast proliferation and collagen expression.a:The coculture system is comprised of ADSCs in transwell inserts and broblasts in six-well plates. ADSCs itself cannot pass cell culture chamber, but can secreted factors and affect broblasts. b:The broblast proliferation ratio with ADSC,ADSCbFGF or alone. c:Tubule formation of HUVECs cultured with different supernatants.d: Quantitative analysis of tubular formation of HUVECs. e:The HUVECs proliferation ratio in response to different ADSC cell culture supernatants. f,g: Quanti cation of migrated cells. h,i:collagen I and III expression levels .Each bar depicts the mean ± SD (***P < 0.001, **P < 0.01 and *P < 0.05, n = 3); one-way ANOVA followed by the S-N-K test.   ADSCbFGF reversed the in ammation of the injured broblasts a: ADSCbFGF decreased the IL-1 protein level after the injured broblasts were treated with LPS b:ADSCbFGF decreased the IL-1 protein level after the injured broblasts were treated with LPS c:ADSCbFGF decreased the IL-1 protein level after the injured broblasts were treated with LPS Error bars represent the mean±S.D.; *P<0.05 and **P<0.01. Figure 6