Xiangguo Lv

VEGF-activated miR-144 regulates autophagic survival of prostate cancer cells against Cisplatin

Cisplatin is a commonly used chemotherapy drug for prostate cancer (PC). However, some PCs are resistant to cisplatin treatment, while the molecular mechanisms underlying the resistance of PCs to cisplatin are not completely understood. In this study, we found that cisplatin dose-dependently activated Beclin-1 in two PC cell lines, PC3 and LNCap. Autophagy suppression significantly increased the cisplatin-induced cell death of these PC cells in a CCK-8 assay. Moreover, microRNA (miR)-144 levels were significantly downregulated in cisplatin-treated PC cells, in a VEGF-dependent manner. Bioinformatics analysis showed that miR-144 targeted the 3′-UTR of Beclin-1 mRNA to inhibit its translation, which was confirmed by luciferase reporter assay. In PC patients after cisplatin treatment, low miR-144 levels appeared to predict poor outcome of patients’ survival. Together, these data suggest that cisplatin may induce VEGF to suppress miR-144 levels in PC cells, which subsequently upregulates Beclin-1 to increase autophagic cell survival against cisplatin-induced cell death. Upregulation of miR-144 or suppression of cell autophagy may improve the outcome of cisplatin therapy in PC.

Improved cell infiltration and vascularization of three-dimensional bacterial cellulose nanofibrous scaffolds by template biosynthesis

A significant problem limiting the application of bacterial cellulose (BC) nanofibrous scaffolds for tissue regeneration is the nanoscale pores that inhibit cell infiltration and vascularization in their three-dimensional (3D) structure. In this paper, a facile method was used to fabricate 3D microporous nanofibrous gelatin/BC composite scaffolds (Gel/BC) by stationary cultivation Gluconacetobacter xylinus using microporous gelatin scaffold as a template. The Gel/BC scaffolds with highly interconnected micropore (171 ± 71 μm) and surface decorated on the micropore walls by BC nanofibers (25.2 ± 7.0 nm) were fabricated, which are remarkably similar in structure to the native extracellular matrix (ECM). Cell distribution, viability and morphology were evaluated by seeding adipose-derived stem cells (ADSCs) on the scaffolds, using the 3D laser scanning confocal microscopy (3D-LSCM), LIVE/DEAD® viability/cytotoxicity assay and field emission scanning electron microscopy (FE-SEM). In vivo biocompatibility was evaluated by subcutaneous implantation using a dog model for 2 weeks. These results indicate that the 3D microporous nanofibrous scaffolds exhibit good biocompatibility, promoting cellular attachment, proliferation and maintain cellular phenotype, improving cellular infiltration and vascularization. It is anticipated that this 3D microporous nanofibrous scaffold can be applied in the fields such as medical implants, cell supports, and materials, which can be used as instructive 3D environments for tissue regeneration.

Urethral reconstruction with a 3D porous bacterial cellulose scaffold seeded with lingual keratinocytes in a rabbit model.

The goal of this study was to evaluate the effects of urethral reconstruction with a three-dimensional (3D) porous bacterial cellulose (BC) scaffold seeded with lingual keratinocytes in a rabbit model. A novel 3D porous BC scaffold was prepared by gelatin sponge interfering in the BC fermentation process. Rabbit lingual keratinocytes were isolated, expanded, and seeded onto 3D porous BC. BC alone (group 1, N  =  10), 3D porous BC alone (group 2, N  =  10), and 3D porous BC seeded with lingual keratinocytes (group 3, N  =  10) were used to repair rabbit ventral urethral defects (2.0   ×   0.8 cm). Scanning electron microscopy revealed that BC consisted of a compact laminate while 3D porous BC was composed of a porous sheet buttressed by a dense outer layer. The average pore diameter and porosity of the 3D porous BC were 4.23   ±   1.14 μm and 67.00   ±   6.80%, respectively. At 3 months postoperatively, macroscopic examinations and retrograde urethrograms of urethras revealed that all urethras maintained wide calibers in group 3. Strictures were found in all rabbits in groups 1 and 2. Histologically, at 1 month postoperatively, intact epithelium occurred in group 3, and discontinued epithelium was found in groups 1 and 2. However, groups 2 and 3 exhibited similar epithelial regeneration, which was superior to that of group 1 at 3 months (p  <  0.05). Comparisons of smooth muscle content and endothelia density among the three groups revealed a significant increase at each time point (p  <  0.05). Our results demonstrated that 3D porous BC seeded with lingual keratinocytes enhanced urethral tissue regeneration. 3D porous BC could potentially be used as an optimized scaffold for urethral reconstruction.

Urethral Reconstruction with Small Intestinal Submucosa Seeded with Oral Keratinocytes and TIMP-1 siRNA Transfected Fibroblasts in a Rabbit Model.

Background: To evaluate the effect of tissue inhibitor of metalloproteinase-1 small interfering RNA (TIMP-1 siRNA) transfected fibroblasts (FB) for urethral reconstruction. Materials and Methods: A ventral urethral mucosal defect was created. Substitution urethroplasty was performed with small intestinal submucosa (SIS) alone (8 rabbits, group 1), autogenic oral keratinocytes (OK)-seeded SIS (8 rabbits, group 2) or autogenic OK and TIMP-1 siRNA transfected FB-seeded SIS (8 rabbits, group 3). At 1 and 6 months after surgery (4 rabbits at each time point), retrograde urethrogram and histologic analysis were performed to evaluate the outcomes of urethroplasty. Results: TIMP-1 siRNA transfected FB decreased the secretion of type I collagen. Under retrograde urethrography, 5 rabbits in group 1, 6 in group 2 and 7 in group 3 maintained a wide urethral caliber. Histologically, inflammation and fibrosis were observed at 6 months in group 1. The speed of urothelium, smooth muscle and vessel regeneration in group 3 was faster than that in group 2. Comparison of smooth muscle-to-collagen ratio, epithelial layers, smooth muscle content and microvessel density among three groups revealed a significant increase (p < 0.05). Conclusions: TIMP-1 siRNA transfected FB could be used as a source of seed cell for urethral tissue engineering and could prevent the proliferation of urethral scar tissue.

Use of heparinized bacterial cellulose based scaffold for improving angiogenesis in tissue regeneration

Vascularization is a prerequisite to achieve tissue regeneration especially for long-term survival of a scaffold. During the regeneration process, the delivery of angiogenic factors is very important for developing a vascular network. In this paper, vascular endothelial growth factor (VEGF)-loaded 3D porous bacterial cellulose/gelatin (B/G) scaffolds modified with heparin were firstly prepared. The pro-angiogenic effects of scaffolds towards proliferation and migration of endothelial cells (PIECs) were evaluated as well as in vivo implantation. Results showed that the B/G scaffold modified with heparin could provide a prolonged release of VEGF for two weeks. In vitro cellular assays showed that proliferation and migration were promoted in the presence of VEGF. Subcutaneous implantation demonstrated that angiogenesis was significantly improved for the heparinized scaffolds loaded with VEGF (V-B/G/H), compared to B/G scaffold. The resulting scaffold with sustained delivery of VEGF could be potential and effective tissue engineered candidates in tissue regeneration for future clinical applications.

Adipose-derived stem cells-seeded bladder acellular matrix graft-silk fibroin enhances bladder reconstruction in a rat model

The unfavourable clinical outcomes of host cell-seeded scaffolds for bladder augmentation warrant improved bioactive biomaterials. This study aimed to examine the feasibility of adipose-derived stem cells (ASCs)-seeded bilayer bladder acellular matrix graft (BAMG)-silk fibroin (SF) scaffold in enhancing bladder reconstruction. Sprague Dawley rats were randomly divided into three groups: the BAMG-SF-ASCs group, the acellular BAMG-SF group and the cystotomy group. The BAMG-SF-ASCs group was sampled at 2, 4 and 12 weeks, and compared with the other groups at 12 weeks. In the BAMG-SF-ASCs group, the normal bladder contour was reformed similar to that in the cystotomy group, with abundant urothelium and smooth muscle regeneration, as well as a suitable scaffold degradation speed, and trivial fibrosis and inflammation. The ASCs seeded in BAMG-SF were maintained in the regenerated region during the 12-week experimental period and significantly enhanced the vessel density, nerve regeneration and bladder function compared with acellular BAMG-SF. In addition, the BAMG-SF-ASCs group presented elevated levels of SDF-1α, VEGF and their receptors, with an obvious increase in ERK 1/2 phosphorylation. BAMG-SF is a promising biomaterial for ASCs seeding to facilitate bladder augmentation and demonstrated an enhanced angiogenic potential possibly related to the SDF-1α/CXCR4 pathway via ERK 1/2 activation.

Comparative study of different seeding methods based on a multilayer SIS scaffold: Which is the optimal procedure for urethral tissue engineering?

Seeding cells efficiently and uniformly onto three-dimensional scaffolds is key for engineering urological tissue with an ideal histological structure in vitro. Using an optimized seeding technology allows cells to cooperate positively with biomaterials, resulting in successful reconstructive surgery. In this study, we used four different types of seeding methods in a scaffold of small intestinal submucosa (SIS). The efficiency of the sandwich co-culture, layered co-culture, static-agitation seeding, and centrifugation seeding methods were compared. It was demonstrated that dynamic seeding methods, such as static-agitation and centrifugation seeding, had superior cell-matrix infiltration and mechanical properties. The seeding time could be reduced by 5-10 min using the centrifugation method. Furthermore, functional assessment of the barriers revealed that this function was better in the centrifugation seeding method than in any other method. Our study suggests that both the static-agitation and centrifugation methods are suitable for cell seeding on SIS. There is no significant change in surface area of SIS with different seeding methods. These methods reinforce the physiological and mechanical properties of biomaterials and allow for the future in vivo study of tissue-engineered urethral reconstruction.

A smart bilayered scaffold supporting keratinocytes and muscle cells in micro/nano-scale for urethral reconstruction

Rationale: In urethral tissue engineering, the currently available reconstructive procedures are insufficient due to a lack of appropriate scaffolds that would support the needs of various cell types. To address this problem, we developed a bilayer scaffold comprising a microporous network of silk fibroin (SF) and a nanoporous bacterial cellulose (BC) scaffold and evaluated its feasibility and potential for long-segment urethral regeneration in a dog model. Methods: The freeze-drying and self-assembling method was used to fabricate the bilayer scaffold by stationary cultivation G. xylinus using SF scaffold as a template. The surface morphology, porosity and mechanical properties of all prepared SF-BC scaffolds were characterized using Scanning electron microscopy (SEM), microcomputed tomography and universal testing machine. To further investigate the suitability of the bilayer scaffolds for tissue engineering applications, biocompatibility was assessed using an MTT assay. The cell distribution, viability and morphology were evaluated by seeding epithelial cells and muscle cells on the scaffolds, using the 3D laser scanning confocal microscopy, and SEM. The effects of urethral reconstruction with SF-BC bilayer scaffold was evaluated in dog urethral defect models. Results: Scanning electron microscopy revealed that SF-BC scaffold had a clear bilayer structure. The SF-BC bilayer scaffold is highly porous with a porosity of 85%. The average pore diameter of the porous layer in the bilayer SF-BC composites was 210.2±117.8 μm. Cultures established with lingual keratinocytes and lingual muscle cells confirmed the suitability of the SF-BC structures to support cell adhesion and proliferation. In addition, SEM demonstrated the ability of cells to attach to scaffold surfaces and the biocompatibility of the matrices with cells. At 3 months after implantation, urethra reconstructed with the SF-BC scaffold seeded with keratinocytes and muscle cells displayed superior structure compared to those with only SF-BC scaffold. Principal Conclusion: These results demonstrate that the bilayer SF-BC scaffold may be a promising biomaterial with good biocompatibility for urethral regeneration and could be used for numerous other types of hollow-organ tissue engineering grafts, including vascular, bladder, ureteral, bowel, and intestinal.

Three-Dimensional Computerized Model Based on the Sonourethrogram: A Novel Technique to Evaluate Anterior Urethral Stricture

Purpose: The sonourethrogram is a useful alternative to the traditional retrograde urethrogram to evaluate anterior urethral strictures. With the development of 3‐dimensional reconstructive techniques 3‐dimensional urethral imaging can provide more accurate and useful information to enable the surgeon to make the best surgical decisions. We evaluated the accuracy and efficacy of a 3‐dimensional reconstructed digital model of the urethra based on the sonourethrogram to assess anterior urethral disease. Materials and Methods: A total of 50 patients with an anterior urethral stricture and 10 healthy volunteers were enrolled in this study from April 2014 to January 2017. All patients and volunteers underwent sonourethrogram and retrograde urethrogram. Three‐dimensional urethral models were reconstructed based on the sonourethrogram. Stricture length and location on retrograde urethrogram or sonourethrogram based images were compared with those found at operation. Results: The 3‐dimensional digital model revealed the entire anterior urethra, including the navicular fossa, and the penile and bulbar parts. The semitransparent model clearly demonstrated the structure of the corpus spongiosum and inside the urethral lumen. Further information on spongiofibrosis could also be seen in the 3‐dimensional digital model. There was no significant difference in stricture length or location in the 3‐dimensional model compared with retrograde urethrogram imaging and actual surgical findings. However, the latest technique could only reconstruct the short segment of the anterior urethra due to the probe width limitation. Conclusions: The 3‐dimensional computerized model based on the sonourethrogram is a novel and effective technique of evaluating anterior urethral strictures.

Posterior Urethroplasty Complexity and Prognosis Can be Described by a Novel Method: Posterior Urethral Stenosis Score

OBJECTIVE To develop a standardized PU-score (posterior urethral stenosis score), with the goal of using this scoring system as a preliminary predictor of surgical complexity and prognosis of posterior urethral stenosis. PATIENTS AND METHODS We retrospectively reviewed records of all patients who underwent posterior urethral surgery at our institution from 2013 to 2015. The PU-score is based on 5 components, namely etiology (1 or 2 points), location (1-3 points), length (1-3 points), urethral fistula (1 or 2 points), and posterior urethral false passage (1 point). We calculated the score of all patients and analyzed its association with surgical complexity, stenosis recurrence, intraoperative blood loss, erectile dysfunction, and urinary incontinence. RESULTS There were 144 patients who underwent low complexity urethral surgery (direct vision internal urethrotomy, anastomosis with or without crural separation) with a mean score of 5.1 points, whereas 143 underwent high complexity urethroplasty (anastomosis with inferior pubectomy or urethrorectal fistula repair, perineal or scrotum skin flap urethroplasty, bladder flap urethroplasty) with a mean score of 6.9 points. The increase of PU-score was predictive of higher surgical complexity (P = .000), higher recurrence (P = .002), more intraoperative blood loss (P = .000), and decrease of preoperative (P = .037) or postoperative erectile function (P = .047). However, no association was observed between PU-score and urinary incontinence (P = .213). CONCLUSION The PU-score is a novel and meaningful scoring system that describes the essential factors in determining the complexity and prognosis for posterior urethral stenosis.