In view of the current status of mesh application in pelvic floor reconstruction surgery, the ability to induce autologous tissue regeneration is the study and development direction of pelvic floor tissue engineering mesh. Considering the role of bFGF in the repair of pelvic floor tissue is obvious but uncontrollable, the study transduced the bFGF gene into ADSCs to produce sustained, stable, and long-term expression of bFGF, moreover avoiding some adverse reactions.When ADSCs is designed to overexpress growth factors such as bFGF, this paracrine effect may be amplified [16]. We observed that ADSCbFGF 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 [19–21] and induces pro-inflammatory responses in vitro[22].The oncogenic and proinflammatory 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 fibroblasts in tissue repair,we found that the chemotaxis ability of ADSCbFGF to fibroblasts did not increase, which may be related to the expression of chemokines didn't increase.
We found ADSCFGF has increased fibroblast 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 floor supporting tissues.Collagen I forms thick collagen fibers that provide continuous tension to the pelvic floor tissue. Collagen III mainly affects the flexibility 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 floor dysfunction disease in women [25], we observed that the ADSCFGF can be induced into fibroblasts and enhance the synthesize collagen capability,the microenvironment of the implantation site is improved༌which may mediates the reparation of pelvic floor tissue. Through the activation of fibroblasts, collagen synthesis is promoted, the collagen content of local tissues is improved,the ECM is reconstructed༌thereby༌The repair efficiency 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 floor 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 Ⅰ and Ⅲ collagen fibers by ADSCbFGF, and successfully induce themselves differentiate into fibroblasts [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 fibroblasts. Yin et al [32] colonized mesenchymal stem cells on interlaced scaffold materials. The cells could differentiate into ligament-like fibroblasts and express ligament-related collagens such as type Ⅰ, type Ⅲ collagen, and Tenascin-C gene. The study by Xiong et al [33] showed that bFGF can induce bone marrow mesenchymal stem cells differentiate into fibroblasts, and there is no significant statistical difference in cell morphology and collagen synthesis ability from cells derived from veins.We successfully induced the differentiation of ADSCs into fibroblasts through bFGF[13],and found that the ADSCbFGF gradually differentiated into fibroblasts and more efficient: the expression of the fibroblast 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 efficiency of collagen are higher. This provides a basis for the directed induction of differentiated stem cell in the direction of suitable pelvic floor tissue repair.
PI3-K/Akt is one of the important signaling pathways involved in the regulation of cell proliferation, self-renewal 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 finally 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 fibroblasts after bFGF transduction is unclear. We observed an increase in p-Akt levels of ADSCbFGF, indicating that ADSCbFGF can activate the PI3-K/Akt signaling pathway. In addition, the collagen expression of the cells after induction was significantly increased.Importantly, we found inhibition of PI3K with LY294002 reversed the effect of ADSCbFGF on the differentiation process,p-Akt levels decreased significantly, ADSCbFGF were negative for FSP1, after Ly294002 used to suppress the PI3-K/Akt signal pathway, the collagen expression was significantly 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 fibroblasts. 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 floor tissue engineering.
The inflammatory response is the first step after trauma, and plays an important role in the wound healing process, and the continuous inflammatory response will damage the wound healing process. Therefore, it is also crucial to suppress inflammation 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 defined 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 inflammatory or immune-related diseases [38]. A large body of medical literature indicates that MSCs repair damaged tissues because they respond to inflammation and migrate to injured sites and influence the microenvironment through the release of molecules involved in reparative processes and tissue regeneration [39]. Biomaterial-based delivery of MSCs may benefit organ and tissue repair through paracrine effects.These properties make MSCs an attractive source of cells for seeding on the engineered biomaterials to influence the foreign body reaction following implantation [40].We found that ADSCbFGF significantly inhibited the inflammatory response of damaged fibroblasts treated with lipopolysaccharide. The results show that ADSCbFGF can reduce inflammatory.