Lujie Song

Urethral Reconstruction Using Oral Keratinocyte Seeded Bladder Acellular Matrix Grafts

PURPOSE We investigated the feasibility of urethral reconstruction using oral keratinocyte seeded bladder acellular matrix grafts. MATERIALS AND METHODS Autologous oral keratinocytes were isolated, expanded and seeded onto bladder acellular matrix grafts to obtain a tissue engineered mucosa. The tissue engineered mucosa was assessed using morphology and scanning electron microscopy. In 24 male rabbits a ventral urethral mucosal defect was created. Urethroplasty was performed with autogenic oral keratinocyte seeded bladder acellular matrix grafts in 12 rabbits in the experimental group or with bladder acellular matrix grafts with no cell seeding in 12 in the control group. Retrograde urethrography was performed 1, 2 and 6 months after grafting. The urethral grafts were analyzed grossly and histologically. RESULTS Oral keratinocytes had good biocompatibility with bladder acellular matrix grafts. Rabbits implanted with oral keratinocyte seeded bladder acellular matrix grafts voided without difficulty. Retrograde urethrography revealed no sign of strictures at 1, 2 and 6 months. In the control group the urethra with repaired defects was accompanied by strictures. Histological examination showed that grafts seeded with oral keratinocytes formed a 1-layer structure by 1 month, and at 2 and 6 months the keratinocytes had formed multiple layers. There was an evident margin between graft oral keratinocytes and host epithelium. The oral keratinocytes at basilar layers of the grafts expressed P63, as shown by immunocytochemistry. In the control group histopathological evaluation revealed that no 1-layer or stratified epithelium cells had developed at the repaired defect sites, whereas an inflammatory reaction was found in 2 rabbits. CONCLUSIONS Oral keratinocytes had good biocompatibility with bladder acellular matrix grafts. Urethral reconstruction with these grafts was better than with bladder acellular matrix grafts alone.

3D bioprinting of urethra with PCL/PLCL blend and dual autologous cells in fibrin hydrogel: An in vitro evaluation of biomimetic mechanical property and cell growth environment

OBJECTIVE Urethral stricture is a common condition seen after urethral injury. The currently available treatments are inadequate and there is a scarcity of substitute materials used for treatment of urethral stricture. The traditional tissue engineering of urethra involves scaffold design, fabrication and processing of multiple cell types. METHODS In this study, we have used 3D bioprinting technology to fabricate cell-laden urethra in vitro with different polymer types and structural characteristics. We hypothesized that use of PCL and PLCL polymers with a spiral scaffold design could mimic the structure and mechanical properties of natural urethra of rabbits, and cell-laden fibrin hydrogel could give a better microenvironment for cell growth. With using an integrated bioprinting system, tubular scaffold was formed with the biomaterials; meanwhile, urothelial cells (UCs) and smooth muscle cells (SMCs) were delivered evenly into inner and outer layers of the scaffold separately within the cell-laden hydrogel. RESULTS The PCL/PLCL (50:50) spiral scaffold demonstrated mechanical properties equivalent to the native urethra in rabbit. Evaluation of the cell bioactivity in the bioprinted urethra revealed that UCs and SMCs maintained more than 80% viability even at 7days after printing. Both cell types also showed active proliferation and maintained the specific biomarkers in the cell-laden hydrogel. CONCLUSION These results provided a foundation for further studies in 3D bioprinting of urethral constructs that mimic the natural urethral tissue in mechanical properties and cell bioactivity, as well a possibility of using the bioprinted construct for in vivo study of urethral implantation in animal model. SIGNIFICANCE OF STATEMENTS The 3D bioprinting is a new technique to replace traditional tissue engineering. The present study is the first demonstration that it is feasible to create a urethral construct. Two kinds of biomaterials were used and achieved mechanical properties equivalent to that of native rabbit urethra. Bladder epithelial cells and smooth muscle cells were loaded in hydrogel and maintained sufficient viability and proliferation in the hydrogel. The highly porous scaffold could mimic a natural urethral base-membrane, and facilitate contacts between the printed epithelial cells and smooth muscle cells on both sides of the scaffold. These results provided a strong foundation for future studies on 3D bioprinted urethra.

Oral complications after lingual mucosal graft harvesting for urethroplasty in 110 cases.

Study Type – Therapy (case series) 
Level of Evidence 4

Preliminary Experimental Study of Tissue-Engineered Urethral Reconstruction Using Oral Keratinocytes Seeded on BAMG

Introduction: This study investigates the feasibility of replacing urinary epithelial cells with oral keratinocytes seeded on a bladder acellular matrix graft (BAMG) to reconstruct a tissue-engineered urethra. Materials and Methods: After enzymatic treatment of a small segment of rabbit buccal mucosa (1.0 × 0.4 cm), the epidermis and dermis were separated mechanically. Oral keratinocytes were isolated from the epidermis and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin (i3T3) or on a culture dish without i3T3, respectively. These cells were expanded and applied to sterilized BAMG to obtain a tissue-engineered mucosa. Oral keratinocytes were assessed by growth curve, morphology and immunofluorescence staining. The tissue-engineered mucosa was assessed using morphology, immunohistochemistry staining and scanning electron microscopy. Results: Oral keratinocytes seeded onto a feeder layer of i3T3 had finer morphous and better amplification capability, and could be passaged for 8 generations. Oral keratinocytes that were cultured without i3T3 could only be subcultured 2 generations before ageing. Passage 2 oral keratinocytes cultured with i3T3 were expanded and seeded onto sterilized BAMG to obtain a tissue-engineered mucosa for urethral reconstruction. The compound graft was assessed using HE staining and scanning electron microscopy. Oral keratinocytes had good compatibility with BAMG. Conclusions: Rabbit oral keratinocytes of can be cultured in vitro and attain magnitude in quantity. Oral keratinocytes also had good compatibility with BAMG and the compound graft achieved could become a new choice in urethral reconstruction.

Evaluation of stretched electrospun silk fibroin matrices seeded with urothelial cells for urethra reconstruction

BACKGROUND We investigated the feasibility of urethral reconstruction using stretched electrospun silk fibroin matrices. MATERIALS AND METHODS A novel electrospun silk fibroin matrix was prepared. The structure of the material was assessed by scanning electron microscopy and a porosity test. Canine urothelial cells were isolated, expanded, and seeded onto the material for 1 wk to obtain a tissue-engineered graft. The tissue-engineered graft was assessed using hematoxylin and eosin staining and scanning electron microscopy. A dorsal urethral mucosal defect was created in nine female beagle dogs. In the experimental group, tissue-engineered mucosa was used to repair urethra mucosa defects in six dogs. No substitute was used in the three dogs of the control group. Retrograde urethrography was performed at 1, 2, and 6 mo after grafting. The urethral grafts were analyzed grossly and histologically. RESULTS Scanning electron microscope and a porosity test revealed that the material had a three-dimensional porous structure. Urothelial cells grew on the material and showed good biocompatibility with the stretched silk fibroin matrices. Canines implanted with tissue-engineered mucosa voided without difficulty. Retrograde urethrography revealed no signs of stricture. Histologic staining showed gradual epithelial cell development and stratified epithelial layers at 1, 2, and 6 mo. The canines in the control group showed difficulty in voiding. Retrograde urethrography showed urethra stricture. Histologic staining showed that no or only one layer of epithelial cells developed. A severe inflammatory reaction was also observed in the control group. CONCLUSIONS Stretched electrospun silk fibroin matrices have good biocompatibility with urothelial cells, which could prove to be a potential material for use in urethra reconstruction.

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest‐site morbidity. The goal of this study was to assess whether human urine‐derived stem cells (USCs), obtained through non‐invasive procedures, can differentiate into skeletal muscle linage cells (Sk‐MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25–55. Expression profiles of cell‐surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA‐4 and STRO‐1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from ‘rice‐grain’‐like cells to spindle‐shaped cells. The USCs expressed specific Sk‐MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk‐MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk‐MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright

Construction of cavernosum smooth muscle using umbilical artery smooth muscle cells seeded on acellular corporal collagen matrices

The objective of this study was to investigate the feasibility of tissue engineering of corpus cavernosal smooth muscle. Acellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting immunological reaction. Human umbilical artery smooth muscle cells (HUASMCs) were isolated from human umbilical arteries through explant techniques and expanded in vitro. Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30 x 10(6) cells/mL. Then, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyse the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues. The decellularization process successfully extracted all cellular components while preserving the original collagen fibers. The immunological reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro. Histological analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64 +/- 0.18 g/100 mg and 2.50 +/- 0.21 g/100 mg, respectively. Our study demonstrates that HUASMCs can be seeded on three-dimensional ACCM scaffolds and will develop tissues similar to that of the native corpus cavernosum smooth muscle.

Dual-factor loaded functional silk fibroin scaffolds for peripheral nerve regeneration with the aid of neovascularization

To improve the therapeutic effect of a damaged peripheral nerve, aligned scaffolds based on regenerated silk fibroin (RSF) were fabricated by electrospinning, and were simultaneously loaded with dual factors of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). An ELISA assay measurement in vitro demonstrated that the release of dual factors from the scaffolds was well maintained up to 2 weeks. Schwann cell (SC) morphologies on the RSF scaffolds were obtained using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) after cell seeding. Cell viability and proliferation on the scaffolds were investigated using an MTT assay. Furthermore, the scaffolds were implanted in a mouse model to support nerve regeneration and angiogenesis in vivo, which was evaluated by histological and immunohistochemical (IHC) analyses at 4 and 8 weeks after implantation. In all retrieved scaffolds, de novo innervation and vascularization, indicated by a positive endothelial marker (von Willebrand factor, vWF) and an innervation marker (S-100 protein), were evident without chronic inflammatory responses. Compared to the control group, the dual-factor loaded scaffolds significantly promoted nerve regeneration and possessed great potential in peripheral nerve repair and regeneration, and for the repair of other tissues.

Changing trends in the causes and management of male urethral stricture disease in China: an observational descriptive study from 13 centres.

To determine whether there have been any changes in the causes and management of urethral strictures in China.

Combined Buccal and Lingual Mucosa Grafts for Urethroplasty: An Experimental Study in Dogs

BACKGROUND To evaluate combined buccal mucosa and lingual mucosa grafts for urethroplasty in a dog model. MATERIALS AND METHODS Seven female mongrel dogs were randomly selected. After a segment of proximal urethra mucosa (4 cm × 1cm) was excised and onlayed urethroplasty was performed using the combined free buccal mucosa (2 cm × 1cm) graft harvested from the inferior cheek and free lingual mucosa graft (2 cm × 1cm) harvested from the inferior lateral surface of the tongue. A 12F urethral catheter was kept inside the urethra for a mean of 7 d. Retrograde urethrography was done and urethra diameter was calibrated with a 10Fr catheter before animals were sacrificed at wk 12. After sacrifice of all dogs, the grafted areas excised and evaluated by gross and histopathological examination. RESULTS All dogs survived the procedure and there were no tongue and oral complications. One of the seven dogs developed a severe urethral stricture near the proximal anastomosis. The remaining six dogs voided spontaneously with no difficulty. Retrograde urethrography showed that no strictures or fistulas had formed. The combined buccal mucosa graft and lingual mucosa graft shortened by 10.2% after surgery, from a mean (SD) of 4 (0.15) to 3.59 (0.23) cm (statistical significance, P < 0.05). No stricture was found in the connection of the buccal mucosa and lingual mucosa grafts. Histologic examination showed that the combined buccal mucosa and lingual mucosa grafts were well-incorporated into the urethral walls and covered by a keratinized squamous epithelium. CONCLUSIONS We successfully developed a dog model for combined buccal mucosa graft and lingual mucosa graft and showed the feasibility of this approach for urethral substitution.