In this study, we compared four decellularization protocols and finally developed an optimized one through which a porcine bladder acellular matrix (BAM) with well-preserved extracellular bioactive factors had been prepared. In this protocol, the intact bladder was treated with trypsin/ethylenediaminetetraacetic acid to remove the urothelium, then with hypotonic buffer and Triton X-100 in hypertonic buffer to remove the membranous and cytoplasmic materials, and finally with nuclease to degrade the cellular nuclear components. Bladder distention and mechanical agitation were simultaneously used to facilitate cell removal. Meanwhile, several preservative techniques, including limitation of wash time, supplement with inhibitors of proteinase, control of the pH value and temperature of the wash buffer, ethylene oxide sterilization, and lyophilization of the scaffold for storage, were used to protect the extracellular bioactive factors. This decellularization protocol had completely removed the cellular materials and well preserved the extracellular collagen, sulfated glycosaminoglycan (GAG), and bioactive factors. The preserved bioactive factors had a great potential of promoting the proliferation and migration of both human bladder smooth muscle cell and human umbilical vein endothelial cell. It was also found that the amount of two representative bioactive factors, platelet-derived growth factor BB and vascular endothelial growth factor, was positively correlated with the sulfated GAG content in the porcine BAM, implying that the amount of sulfated GAG might be a determinant for preservation of bioactive factors in the decellularized tissues. In conclusion, the porcine BAM with well-preserved extracellular bioactive factors might be a favorable scaffold for tissue engineering applications.

It is widely accepted that inflammation is an important risk for the development of prostate cancer (PCa). The objective of this study was designed to investigate the potential molecular mechanism of NLR family, pyrin domain-containing protein 3 (NLRP3) inflammasome in the malignant progression of PCa. The expression level of NLRP3 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization. The effects of NLRP3 in the development of PCa by applying gain- and loss-of-function assays in LNCaP and PC3 cell lines were detected by CCK-8, TUNEL, and Transwell migration assays. The underlying mechanism of NLRP3 and caspase-1 in PCa was examined by the rescue experiments, western blotting, and qRT-PCR assays. In addition, the promoting effect of NLRP3 inflammasome was performed with an animal subcutaneous tumorigenesis experiment in vivo. The upregulation of NLRP3 was confirmed in PCa tissues and cell lines. Functionally, using CCK-8, TUNEL, and Transwell migration assays, these results showed that activation of NLRP3/caspase-1 inflammasome by LPS + ATP could enhance the ability of proliferation and migration; and decrease the apoptosis of LNCaP and PC3 cell lines. Western blotting assay showed that the activation of caspase-1 would increase after the stimulation of NLRP3 inflammasome by LPS + ATP. Moreover, the overexpression of NLRP3 promoted, while the knockdown of NLRP3 inhibited the malignant progression in PCa cell lines by positively regulating caspase-1. In addition, the rescue experiments revealed the association among NLRP3 and caspase-1, which showed that the overexpression vectors/inhibitors of caspase-1 could reverse the effect of knockdown/overexpression of NLRP3 in PCa cell lines in vitro. Finally, In in vivo experiment, the suppression of NLRP3 knockdown impaired tumor growth of PCa. Collectively, these results indicated that NLRP3 inflammasome played a vital role in promoting the malignant progression of PCa via the activation of caspase-1. Together, our findings provided insight into the mechanisms of NLRP3/caspase-1 inflammasome and revealed an alternative and potential target for the clinical diagnosis and treatment of PCa.

Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.

Tissue-engineering techniques have brought a great hope for bladder repair and reconstruction. The crucial requirements of a tissue-engineered bladder are bladder smooth muscle regeneration and vascularization. In this study, partial rabbit bladder (4×5 cm) was removed and replaced with a porcine bladder acellular matrix (BAM) that was equal in size. BAM was incorporated with platelet-derived growth factor-BB (PDGF-BB) and vascular endothelial growth factor (VEGF) in the experimental group while with no bioactive factors in the control group. The bladder tissue strip contractility in the experimental rabbits was better than that in the control ones postoperation. Histological evaluation revealed that smooth muscle regeneration and vascularization in the experimental group were significantly improved compared with those in the control group (p<0.05), while multilayered urothelium was formed in both groups. Muscle strip contractility of neobladder in the experimental group exhibited significantly better than that in the control (p<0.05) assessed with electrical field stimulation and carbachol interference. The activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the native bladder tissue around tissue-engineered neobladder in the experimental group was significantly higher than that in the control (p<0.05). This work suggests that smooth muscle regeneration and vascularization in tissue-engineered neobladder and recovery of bladder function could be enhanced by PDGF-BB and VEGF incorporated within BAM, which promoted the upregulation of the activity of MMP-2 and MMP-9 of native bladder tissue around the tissue-engineered neobladder.