In present study, we first demonstrated that ad-SVF spheroids significantly improved bladder functions and attenuated bladder injury in UAB rats, and enhanced engraftment of ad-SVF. Meanwhile, we also found that the ad-SVF spheroids promoted angiogenesis in bladder tissues, which was involved in the paracrine of bFGF, HGF, and VEGF-A, and part endothelial progenitor cells in ad-SVF. Furthermore, the results also showed that the ad-SVF spheroids promoted cell proliferation and anti-apoptosis via the activation of AKT/GSK-3β pathway. Following clinical studies on the proof of the safety and effectiveness of ad-SVF transplantation for the functional recovery of damage organs[25–27], our study provides important clinical significance for the improvement of underactive bladder induced by partial bladder outlet obstruction.
Underactive bladder is common but under researched bladder disorder, which is known to the change of bladder morphology and function[10, 28]. Recently, studies are devoted to exploring the effective therapies to treat UAB, due to its complex and multifactorial etiology. Study has reported that MSC could improve the structure and function of UAB. In our study, bladder functions were evaluated by cystometry and the results showed that administration of ad-SVF spheroids was also able to attenuate the histological changes and improve bladder function, indicating the potential effect of ad-SVF spheroids on UAB. However, as contractility of bladder smooth muscle strips is also an important factor to evaluate functional recovery of UAB, it is necessary to detect the contractility of bladder smooth muscle strips in the future.
Ad-SVF provide a rich of stem cells, which consists of heterogeneous cell populations[12, 14, 31]. Many studies demonstrated that ad-SVF could promote tissue regeneration and organ function recovery[19, 25, 32]. Previous research demonstrated that adipose-derived regenerative cells transplantation improved bladder dysfunction and attenuated the changes of bladder histology. Furthermore, in clinical trial, ad-SVF were confirmed as a safe and effective therapy[27, 33]. Therefore, in current study, ad-SVF spheroids were applied to attenuate histological damage, improve bladder function and stimulate bladder tissue regeneration. However, we only explored the early therapeutic effects of ad-SVF spheroids treatment, and the long-term protective effects of ad-SVF spheroids in UAB induced by PBOO also need to be verified.
Currently, various of stem cells are studied in bladder tissue recovery and regeneration, but the underly mechanisms of cellular recovery and regeneration remain poorly understood. One possible mechanism is the secreted growth factors of implanted ad-SVF into bladder wall, which may play a crucial role in cell migration and proliferation. Research has investigated that ad-SVF could secrete various of growth factors including bFGF, HGF, VEGF-A, and so on. In this study, ad-SVF spheroids could secrete more growth factors than ad-SVF and promote endothelial cell migration, which indicate a better recovery effect in UAB induced by PBOO. bFGF, as a member of FGFs, is demonstrated to suppress cell apoptosis and promote proliferation. HGF was reported to be a potential anti-fibrotic factor in damaged tissues and pro-angiogenic factor, which has similar effects with VEGF-A[36, 37]. In our experiments, UAB induced by PBOO exhibited serious bladder muscle bundles injury and bladder fibrosis. Ad-SVF spheroids administration attenuated the damage of bladder and intermuscular collagen accumulation. Meanwhile, the percent of TUNEL-positive cells reduced and the percent of PCNA-positive cells increased after ad-SVF spheroids transplantation. Additionally, we found that ad-SVF spheroids facilitated the formation of microvessels through Matrigel plug angiogenesis assay and immunohistological analysis. Furthermore, AKT/GSK-3β pathway may act as a crucial role in cell apoptosis and proliferation. bFGF has been reported to be related to cell survival by regulating downstream pathways, which involves in the increased expression of phosphorylating AKT and GSK-3β[30, 38]. Our results also showed that the phosphorylation of AKT and GSK-3β expression level both increased in the ad-SVF group and ad-SVFsp group. Other possible mechanism may relate to the direct differentiation of implanted stem cells. Several studies reported that stem cells could differentiate into smooth muscle and endothelial cells to promote the regeneration of bladder tissue[39, 40]. According to the immunofluorescence staining, we observed that part CM-DiI labelled ad-SVF differentiate into endothelial cells to promote blood supply recovery in bladder.
To the best of our knowledge, several strategies to improve the survival rate and transplantation rate of stem cells have been explored, including stem cell genetic engineering modification, tissue engineering scaffolds[13, 41], and stem cell combined with growth factor transplantation. Transplanting ad-SVF as 3D spheroid may enhance cell survival and improve therapeutic effect in bladder. Compared with dissociated stem cells, spheroid could maintain more extracellular matrix (ECM) without enzymatic treatments. Our previous research demonstrated that ECM enhance the therapeutic effects of stem cells in ischemic tissue through its bioactive ingredients. In addition, cells inside the spheroid are under mild hypoxia, which naturally preconditioned to transplant environment. Therefore, in this study, we demonstrated that ad-SVF spheroids enhanced cell survival rate and retention rate in bladder after cell transplantation. However, the underlying mechanism of 3D spheroid needs more experimental proofs.
Although ad-SVF spheroids show great potential therapeutic effects in UAB, there are some limitations of this study. Firstly, the diversity of animal models indicates the complexity of exploring the etiology of UAB, like aging models, bladder outlet obstruction models, diabetic bladder dysfunction models, and neurogenic models. Further researches need to prove whether ad-SVF spheroids are effective for various of UAB. In addition, only the short-term cell tracking used in this study due to the transient labelling of CM-DiI and DiI-C18(5)-DS. The long-acting labelling technique to track ad-SVF cell should be considered in the subsequent studies. At last, our data confirmed that ad-SVF spheroids could attenuate bladder injury and improve bladder functions attributing to the paracrine effect of ad-SVF spheroids. Further investigation on inhibiting the release growth factors from ad-SVF spheroids is needed to identify.