Hong-Bin Li

Urethral Reconstruction With Tissue Engineering and RNA Interference Techniques in Rabbits

OBJECTIVE To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes and transforming growth factor β (TGF-β)1 siRNA transfected fibroblasts seeded on bladder acellular matrix graft (BAMG) to reconstruct urethra. METHODS Autologous oral keratinocytes and TGF-β1 siRNA transfected fibroblasts were seeded onto BAMGs to obtain a tissue-engineered mucosa. The tissue-engineered mucosa was assessed using morphology and scanning electron microscopy. In 27 male rabbits, a ventral urethral mucosal defect was created. Urethroplasty was performed with autogenic oral keratinocyte and TGF-β1 siRNA transfected fibroblast-seeded BAMGs (9 rabbits, group 1), with autogenic oral keratinocyte-seeded BAMGs (9 rabbits, group 2) or with BAMGs with no cell seeding (9 rabbits, group 3). Retrograde urethrography and histological analyses were performed to evaluate the results of urethroplasty. RESULTS In vitro, oral keratinocytes and TGF-β1 siRNA transfected fibroblasts had good biocompatibility with BAMGs. In vivo, the urethra kept a wide caliber in groups 1 and 2. Strictures were observed in group 3. Histologically, the retrieved urethra in group 3 showed fibrosis and inflammation during 6 months. Stratified epithelial layer regenerated in group 2, whereas there was no evidence of formation of capillary in the epithelial lower layer during the study period. Stratified epithelial layer and formation of capillary in the epithelial lower layer were evident after 6 months in group 1. CONCLUSION Our study suggested that oral keratinocytes and TGF-β1 siRNA transfected fibroblasts could be used as a source of seed cells for urethral tissue engineering.

Preparation of PCL/silk fibroin/collagen electrospun fiber for urethral reconstruction

PurposeTo prepare polycaprolactone (PCL)/silk fibroin/collagen electrospun nanofiber scaffold and test its effects on growth and proliferation of oral mucosal epithelial cells.MethodsRegenerated silk fibroin film, water-soluble collagen powder, and PCL, at mass ratios of 1:1:4, 1:1:8, and 1:1:10, were dissolved in hexafluoroisopropanol, and electrostatic spinning method was adopted to prepare PCL/silk fibroin/collagen electrospun nanofiber scaffold. In vitro cultured oral mucosal epithelial cells were inoculated on the material surface, MTT assay and scanning electron microscopy were adopted to study the growth and proliferation of oral mucosal epithelial cells on the material surface, and cell compatibility of PCL/silk fibroin/collagen electrospun nanofiber was evaluated.ResultsThe result of MTT assay showed that oral mucosal epithelial cells were growing well on the PCL/silk fibroin/collagen electrospun nanofiber scaffold. Scanning electron microscopy showed that the prepared electrospun fiber was uniform in diameter and presented an interconnected porous net structure, and oral mucosal epithelial cells had a good growth form on the surface of the modified material.ConclusionsPCL/silk fibroin/collagen electrospun nanofiber scaffold has appropriate pore size and porosity, is suitable for the growth of oral mucosal epithelial cells, has good cell compatibility, and is a good scaffold for tissue engineering urethral reconstruction.

TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation.

Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT)-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1), Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

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.

Reconstruction of Penile Urethra With the 3-Dimensional Porous Bladder Acellular Matrix in a Rabbit Model

OBJECTIVE To evaluate the effect of reconstruction of penile urethra with the 3-dimensional (3-D) porous bladder acellular matrix (BAM) in a rabbit model. MATERIALS AND METHODS In 30 male rabbits, a ventral urethral mucosal defect (1.5 × 0.8 cm) was created. Substitution urethroplasty was performed with 5% peracetic acid (PAA)-treated BAM (3-D porous BAM; 15 rabbits, PAA-treated BAM group) and non-PAA-treated BAM (15 rabbits; non-PAA-treated BAM group) in an onlay fashion. At 1, 2, and 3 months after surgery (5 rabbits at each time point) in the 2 groups, retrograde urethrogram and histologic analysis were performed to evaluate the outcomes of urethroplasty. RESULTS In the PAA-treated BAM group, 13 rabbits maintained a wide urethral caliber without a fistula or stricture. In contrast, 10 rabbits kept a wide caliber in the non-PAA-treated BAM group. Histologically, at 1, 2, and 3 months after the surgery, the speed of urothelium regeneration in the PAA-treated BAM group was faster than that in the non-PAA-treated BAM group. The smooth muscle-to-collagen ratio and the content of smooth muscle in the PAA-treated BAM group were significantly higher than that in the non-PAA-treated BAM group at each time point (P <.05). The endothelium density between the non-PAA-treated BAM and the PAA-treated BAM groups revealed a significant increase at all 3 time points (P <.05). CONCLUSION Our results confirmed that PAA-treated BAM urethroplasty enhanced urothelium, smooth muscle regeneration and neovascularization compared with those of the non-PAA-treated BAM. The 3-D porous BAM as an optimized biological scaffold may be used for cell-based tubular and long-segmental urethral reconstruction in the future.

Electrocautery-induced cavernous nerve injury in rats that mimics radical prostatectomy in humans

To investigate the early and delayed effects of cavernous nerve electrocautery injury (CNEI) in a rat model, with the expectation that this model could be used to test rehabilitation therapies for erectile dysfunction (ED) after radical prostatectomy (RP).

Preliminary experimental study of urethral reconstruction with tissue engineering and RNA interference techniques

This study investigated the feasibility of replacing urinary epithelial cells with oral keratinocytes and transforming growth factor-β1 (TGF-β1) small interfering RNA (siRNA)-transfected fibroblasts seeded on bladder acellular matrix graft (BAMG) in order to reconstruct tissue-engineered urethra. Constructed siRNAs, which expressed plasmids targeting TGF-β1, were transfected into rabbit fibroblasts. The effective siRNA was screened out by RT-PCR and was transfected into rabbit fibroblasts again. Synthesis of type I collagen in culture medium was measured by enzyme-linked immuno sorbent assay (ELISA). Autologous oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto BAMGs to obtain a tissue-engineered mucosa. The tissue-engineered mucosa was assessed morphologically and with the help of scanning electron microscopy. The TGF-β1 siRNA decreased the expression of fibroblasts synthesis type I collagen. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto sterilized BAMG to obtain a tissue-engineered mucosa for urethral reconstruction. The compound graft was assessed using scanning electron microscope. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had a good compatibility with BAMG. The downregulation of fibroblasts synthesis type I collagen expression by constructed siRNA interfering TGF-β1 provided a potential basis for genetic therapy of urethral scar. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had good compatibility with BAMG and the compound graft could be a new choice for urethral reconstruction.

Intermediate-Term Outcomes and Complications of Long Segment Urethroplasty with Lingual Mucosa Grafts

Purpose: We evaluated outcomes and donor site complications in male patients with complex urethral strictures who underwent urethroplasty using with long strip oral mucosal grafts. We also analyzed whether a lingual mucosa graft is a good substitute for repairing long segment urethral strictures. Materials and Methods: This retrospective study was done in 81 male patients with complex urethral strictures who underwent oral mucosal graft urethroplasty. Patients with long segment (8 cm or greater) anterior urethral strictures who were considered candidates for long strip lingual mucosa graft urethroplasty were included in study. Results: Oral mucosal graft urethroplasty was performed in 81 patients with complex urethral strictures between August 2006 and December 2014. Mean urethral stricture length was 12.1 cm (range 8 to 20). A single 9 to 12 cm long strip lingual mucosa graft was used in 52 patients, a lingual mucosa graft greater than 12 cm was placed in 17 and a lingual mucosa graft combined with a buccal mucosal graft was used in 12. Mean followup was 41 months (range 15 to 86) postoperatively. The overall urethroplasty success rate was 82.7%. Urethral complications developed in 14 patients (17.3%), including urethral strictures in 10 and urethrocutaneous fistulas in 4. At 12 months 5 patients (6.2%) reported minimal difficulty with fine motor movement of the tongue. Conclusions: Lingual mucosa harvested from the ventrolateral surface of the tongue can provide a wide and long graft that is an excellent urethral substitute. Donor site complications are primarily limited to postoperative year 1. Our study confirms that the lingual mucosa graft is a good substitute for urethral reconstruction and lingual mucosa graft urethroplasty is a valuable procedure to treat long anterior urethral strictures.

One-stage dorsal lingual mucosal graft urethroplasty for the treatment of failed hypospadias repair.

The aim of this study was to retrospectively investigate the outcomes of patients who underwent one-stage onlay or inlay urethroplasty using a lingual mucosal graft (LMG) after failed hypospadias repairs. Inclusion criteria included a history of failed hypospadias repair, insufficiency of the local skin that made a reoperation with skin flaps difficult, and necessity of an oral mucosal graft urethroplasty. Patients were excluded if they had undergone a failed hypospadias repair using the foreskin or a multistage repair urethroplasty. Between January 2008 and December 2012, 110 patients with failed hypospadias repairs were treated in our center. Of these patients, 56 underwent a one-stage onlay or inlay urethroplasty using LMG. The median age was 21.8 years (range: 4-45 years). Of the 56 patients, one-stage onlay LMG urethroplasty was performed in 42 patients (group 1), and a modified Snodgrass technique using one-stage inlay LMG urethroplasty was performed in 14 (group 2). The median LMG urethroplasty length was 5.6 ± 1.6 cm (range: 4-13 cm). The mean follow-up was 34.7 months (range: 10-58 months), and complications developed in 12 of 56 patients (21.4%), including urethrocutaneous fistulas in 7 (6 in group 1, 1 in group 2) and neourethral strictures in 5 (4 in group 1, 1 in group 2). The total success rate was 78.6%. Our survey suggests that one-stage onlay or inlay urethroplasty with LMG may be an effective option to treat the patients with less available skin after failed hypospadias repairs; LMG harvesting is easy and safe, irrespective of the patient′s age.