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Corresponding author. Department of Urology, Shanghai Jiaotong University Affiliated Sixth People's Hospital, No 600, Yishan Road, Shanghai, People's Republic of China 200233. Tel.: +86 18930177466; fax: +86 021 64083783.
Corresponding author. State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai, People's Republic of China. Tel.: +86 21 67792954; fax: +86 21 67792855.
To investigate the feasibility of urethral reconstruction using tissue-engineered buccal mucosa (TEBM) with silk fibroin (SF) matrices in a canine model.
Materials and methods
Autologous oral keratinocytes and autologous fibroblasts were isolated, expanded, and seeded onto SF matrices to obtain TEBM. The TEBM was assessed using hematoxylin and eosin staining and scanning electron microscopy. A 5-cm urethral mucosal defect was created in 10 female canines. Urethroplasty was performed using TEBM in five canines in the experimental group and with SF matrices without cells in the five canines in the comparison group. Retrograde urethrography was performed after 6 mo of grafting. The urethral grafts were analyzed grossly and histologically.
The oral keratinocytes and fibroblasts exhibited good biocompatibility with the SF matrices. TEBM could be constructed using SF matrices. The canines implanted with the tissue-engineered mucosa voided without difficulty. The retrograde urethrography revealed no sign of stricture. The histologic staining showed that epithelial cells developed gradually and exhibited stratified epithelial layers at 6 mo. In the comparison group, the canines had difficulty voiding, and the retrograde urethrography showed urethra stricture. The histologic staining showed that one to two layers of epithelial cells developed.
The TEBM using SF matrices could be a potential material for urethra reconstruction.
]. Harvesting BM is associated with donor site morbidity and prolongs the length of surgery. The amount of BM tissue that can be safely obtained is limited. Tissue engineering technology is progressing rapidly to provide a possible solution to avoid limitations on the use of BM tissue. Li et al. combined bladder acellular matrix grafts with keratinocytes to construct tissue-engineered buccal mucosa (TEBM) in a rabbit model for urethral reconstruction [
] repaired a urethral stricture secondary to lichen sclerosus in human patients with DED in vitro cultured keratinocytes and fibroblasts; fibrosis and contraction occurred in two of the five patients. One of the reasons could be that DED is not the ideal scaffold for reconstructing tissue-engineered tissue. Acellular tissue matrices such as DED, bladder acellular matrix graft, and small-intestinal submucosa inevitably have heterologous genetic substances that cause inflammatory reactions, and these materials present the potential danger of disease transmission. The degradation products of the most synthetic polymeric materials, such as polylactide-co-glycolide, are acidic, which is not conducive to the growth of the surrounding cells [
Previous studies have shown that silk fibroin (SF) has excellent biocompatibility and low inflammatory potential; it is a protein that comprised up to 90% of the amino acids glycine, alanine, and serine [
]. In this study, we introduced electrospun SF matrices as scaffolds for TEBM that could comprise a potential scaffold for urethral reconstruction.
We developed TEBM based on electrospun SF matrices with autologous keratinocytes and fibroblasts and evaluated the application of TEBM as a potential graft for urethra-tissue engineering in a canine model.
2. Methods and materials
This study was conducted with the approval of the Institutional Animal Care and Use Committee of our institute.
2.1 Preparation of the electrospun silk protein matrices
The electrospinning procedure was described in previous studies [
]. Briefly, natural cocoons of Bombyx mori (Tongxiang, China) were degummed twice with a 0.5 wt % Na2CO3 aqueous solution at 100°C for 30 min and rinsed with deionized water to remove the sericin. The degummed silk was dissolved in a 9.0 M LiBr aqueous solution. After dilution, the resultant regenerated SF solution was dialyzed against deionized water at 10°C with a cellulose semipermeable membrane (the molecular weight cutoff was 14,000). The SF aqueous solution was concentrated to a 33% wt. The prepared SF aqueous solution was transferred to a 2.5 mL syringe capped with a 6-G needle (Inner diameter = 0.6 mm) as a spinneret. The electrospinning was performed using a voltage of 25 kV, a flow rate of 0.3 mL/h, and grounded aluminum foil placed at a distance of 9 cm to collect the random fibers.
To improve the mechanical properties, the electrospun matrices were mechanically stretched in a 90% ethanol aqueous solution at a stretched rate of 0.1 mm/s and a stretched ratio of 1.4×. The matrices were immersed in an identical solution for 30 min with a fixed length. The morphology of the electrospun SF fibers was observed using a JSM-5600LV (JEOL Co, Tokyo, Japan) scanning electron microscope (SEM).
2.2 Oral keratinocyte culture and characterization
The canines were anesthetized under general anesthesia with an intravenous injection of pentobarbital, and 0.5 × 0.5 cm2 specimens of canine BM were taken. The specimens were washed with phosphate-buffered saline (100 IU/mL of penicillin and 100 μg/mL of streptomycin). The BM was digested by dispase II enzyme (Roche) at 4°C overnight. The oral keratinocytes were isolated from the epidermis after trypsinization. The dermis samples were retained to isolate the fibroblasts, and 5 × 105/cm2 i3T3 cells were preseeded for 24 h. The keratinocytes were seeded at a density of 2 × 106/cm2 in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, New York) and defined-KSFM (Gibco) medium in a 1:1 ratio, supplemented with 10% fetal bovine serum (Gibco). The oral keratinocytes were refed every 2 d until they were 80%–90% confluent. Before the cell passaging, the residual fibroblasts were removed with a dispase II enzyme (Roche, Roche Mannheim Germany), and the keratinocytes were identified by the AE1/AE3 antibody (Abcam, Cambridge). The generation 2–3 cells were used for the experiments.
2.3 Fibroblasts culture and characterization
The epidermis was removed from the BM for the keratinocyte culture, and the dermis was retained for culturing the fibroblasts. The dermis was cut into pieces (of approximately 1 mm3) and placed in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) in a humidified 5% CO2 incubator. After 3–4 d, spindle cells appeared around the dermic tissue. The cells could be passaged until they were 50%–60% of the confluent. The fibroblasts were identified with vimentin. For the expansion, the fibroblasts were cultured and refed every 3 d, and the generation 3–5 cells were used for the experiments.
2.4 Oral keratinocytes and fibroblasts were seeded onto the SF matrices
Before use, the matrices were sterilized with 75% ethanol for 2 h, washed three times in sterilized phosphate-buffered saline, and filled in 5 mL DMEM (Gibco) overnight at 37°C.
The oral fibroblasts were seeded on one side of the scaffolds at 1 × 105 cells/cm2. At 3 d, the scaffolds were reversed, and the keratinocytes were seeded on to the other surface of the scaffold at 3 × 106 cells/cm2, and the coculture was cultured for an additional 7 d. The compound graft was cultured at an air-fluid level for the final 3 d. The culture medium was refreshed every 2 d, and the compound grafts were assessed using hematoxylin and eosin (H&E) staining and SEM.
2.5 Canine urethral reconstruction
Ten female beagle dogs (at an average age of 1.3 y, 11.5 kg in weight) were divided randomly into a comparison group (five animals) that received SF matrices as the comparison substitution and an experimental group (five animals) that received constructed the TEBM. The urethras were retrieved at 6 mo after implantation.
The surgeries were performed by the same surgeons, and the surgical procedures were described before in our previous study [
]. Briefly, the canines were under general anesthesia by the intravenous injection of pentobarbital. The urethras were incised longitudinally between the bladder and the pubic symphysis, and a 5 × 1.5 cm2 section of the urethra mucosa was excised through an abdominal incision. The 5.0 × 1.5 cm2 TEBM was positioned into the urethra defect and its margins were anastomosed with the mucosal edge of the normal urethra using 6/0 Vicryl sutures (Ethicon, New Jersey) (Fig. 1A). An 8F catheter was inserted into the urethra, and the corpus spongiosum was closed. The wound was closed in layers with a routine method. The in-dwelling catheter was left to provide bladder drainage for 7 d after the surgery, and ampicillin G (2 g/d) was given intravenously for 3 d after the surgery.
2.6 Follow-up of the animal experiments
The retrograde urethrography of all the animals was observed at 6 mo after surgery. After the retrograde urethrography, the animals were humanely killed. When urethral stricture or fistulae developed and caused severe voiding difficulty, the animals were humanely killed. After the sacrifice, the urethras were removed and fixed in 4% (vol/vol) formalin. The formalin-fixed samples were embedded in paraffin, and serial sections of 8 μm were generated and stained with H&E and Masson trichrome (MT) staining. The H&E staining was performed to study the presence of epithelial and inflammatory cells, whereas the MT staining allowed highlighting of the extracellular matrix distribution as well as the detection of epithelial cells. Immunohistochemical staining with the CD68 antibody was used to identify the inflammatory cells in the paraffin sections.
The H&E staining slides were observed using an Axioplan 2 microscope (Zeiss, Oberkochen, Germany). The epithelial areas of the H&E staining were measured by the KS400 Image Analysis System in 10 different locations at each time point. The epithelial areas of H&E staining are presented as the means ± standard deviations. A single factor analysis of variance test with 95% confidence interval was used to determine the significant differences between the two groups, and P values of <0.05 were considered statistically significant.
3.1 Characteristics of the electrospun SF matrices
The SEM figure (Fig. 1B) of the SF showed that the SF fibers formed a highly interconnected and porous structure. The general size of the pores observed was 41.74 ± 5.32 μm; the fiber was smooth, and the uniform fiber diameter was 800–1200 nm.
3.2 Characteristics of the keratinocytes
The keratinocytes showed a typical cobblestone shape (Fig. 1C). The immunofluorescence staining showed that the cells were positive to the AE1/AE3 antibody (Fig. 1D).
3.3 Characteristics of fibroblasts
Fibroblasts began to appear around the dermic tissue after 3–4 d, and the cells were typically spindle shaped (Fig. 1E). After approximately 5–6 d, the cells began to achieve fusion and presented a typical fibroblast colony. After 7–9 d, the fibroblasts could pass, and the cells were positive to the vimentin antibody (Fig. 1F).
3.4 Evaluation of the tissue-engineered construct
The H&E staining of TEBM (Fig. 2A) showed that the epidermal cellular layers and the fibroblast layers were easily distinguished in the sections. The keratinocyte layers were compact, and the fibroblast layers were loose. The oral keratinocytes grew well and formed a multilayer epithelium on the surface of the SF matrices. The SEM showed that the oral keratinocytes were ellipsoid or polygonal in shape and adhered tightly on the surface of the materials. The cells stretched out peripherally with pseudopodia and were connected with each other (Fig. 2B).
3.5 Surgical outcomes
The dogs in the experimental group implanted with the TEBM survived until sacrifice, and they voided without difficulty or signs of bladder distension after the catheter removal. The 6-mo retrograde urethrography revealed the maintenance of a wide urethral caliber without any sign of strictures in the experimental group (Fig. 3A). The macroscopic examination of the urethra confirmed the absence of ulceration, stricture, and fistula. At 6 mo, the urethra demonstrated normal-appearing tissue without any evidence of fibrosis or scarring. In the histologic examination at 6 mo, well-developed stratified urothelial cells that had the appearance of normal urethral cells were present (Fig. 4A). In contrast, five dogs in the comparison group developed dysuria, and we found bladder distension after the catheter removal. The urethrography demonstrated urethra strictures in varying degrees (Fig. 3B), and fibrosis and shrinkage were observed. The five animals in the tissue-engineered group healed well, whereas the five animals in the scaffold-only group developed strictures; a two-tailed Fisher exact statistical test identified the outcomes as different with P = 0.0072. Histologically, the cellular layer did not complete regeneration (one to two cell layers) during the study period (Fig. 4A). The inflammatory response, evaluated by H&E staining, against the cellular grafts were not observed; however, inflammatory cells could be detected at the graft sites in the five canines implanted with the acellular grafts (Fig. 4B). The connective tissue regeneration was investigated using MT staining, and the connective tissue in the experimental group appeared to be a lighter blue-green (Fig. 4C). Extensive fibrosis could be observed in the comparison group. The MT staining showed that the epidermis developed gradually and showed typical transitional epithelial layers at 6 mo postoperatively (Fig. 4C). In the comparison group, the cellular layer did not completely regenerate at 6 mo postoperatively (Fig. 4D), and CD68-positive cells were present in the experimental group (Fig. 5).
The image analysis of the epithelial areas in the H&E staining was shown in (Fig. 4A and B). At 6 mo, the epithelial areas regenerated in the experimental group were significantly increased over that in the comparison group (48,209.50 ± 9897.267 μm2versus 165762.20 ± 16,547.637 μm2, P = 0.042 < 0.05).
Various autologous tissues have been used for urethral reconstruction. The complications and the limited amount of available autologous donor tissue restrict urethral reconstruction, especially for those patients with a long segment of strictures (>3 cm). For these shortages, many attempts have been made to identify alternative tissues that would be adequate urethral substitutes. Tissue engineering techniques might be a suitable option. BM is one of the most widely used grafts for the repair of urethral strictures [
] because in clinical series, BM has been easy to harvest, has a thick epithelium adapted to a wet environment, has relatively less morbidity at the donor site, has a thin lamina propria for the prompt establishment of vascular supply to the graft, and has favorable medium-term outcomes [
]. Keratinocytes and fibroblasts were selected as the seeded cells to construct TEBM. The disparity between the experimental group and the comparison group was that SF has seeded cells. In the experimental group, the keratinocytes that were positive to the AE1/AE3 antibody grew well and formed a multilayer epithelium on the surface of the SF matrices. The new epithelium acted as a barrier to protect the underlying tissues from the caustic properties of urine. In the comparison group, no epithelial cells regenerated at the beginning. Urine thus permeated the urethral defect and caused inflammation, urethral fibrosis, and shrinkage, which resulted in urethral stricture in the comparison group. Fibroblasts could strengthen the mechanical properties of the grafts with efficiency in keratinocyte expansion, although this mechanism has not been extensively investigated. The mechanism is possibly related to a series of growth factors and the cytokines and types of collagen that are secreted by the fibroblasts. The fibroblasts are positive to the vimentin antibody, which indicated that the cells we obtained were fibroblasts and not smooth muscle cells.
SF is a well-known natural biomaterial that has excellent biocompatibility and low potential for inflammation; it is obtained from B mori cocoons and might have potential for use in urethra reconstruction. SF is a protein that comprised up to 90% of the glycine, alanine, and serine amino acids that can be fully degraded by naturally occurring proteolytic enzymes [
]. This degradation time is suitable for urethra reconstruction, and the degradation products could be conducive to cell growth. Plasticity in terms of silk processing methods allows for the construction of a variety of configurations including films, foams, hydrogels, gel-spun matrices, and woven or nonwoven meshes. Electrospinning might be an appropriate technique for urethra reconstruction because it can create three dimensions and a highly porous scaffold in a conformation that mimics the properties of the native extracellular cell matrix structure in vivo [
]. The three-dimensional porous structure promotes a microenvironment in which cells live and allows for the movement of air, nutrients, waste products, and cell-signaling molecules. The poor mechanical properties of electrospun SF matrices limit their specific applications in tissue engineering.
In this study, we used electrospun SF matrices that were stretched in ethanol after SF electrospinning. The post-processing of this material substantially increases its mechanical strength. The posttreated matrices showed a breaking strength of 8.6 MPa, which is approximately 5-fold the breaking strength of the naturally spun materials [
]. The posttreated matrices could be sutured to the tissue, and the suture retention strength should be >0.8 N to implant. The stretched electrospun SF with 1.4× were 1.3 N, which could be used for implantation [
In this study, TEBM was constructed successfully and seeded with autologous keratinocytes and fibroblasts. The keratinocytes formed a multilayered epithelium on one side of the scaffold, and the fibroblasts formed a corresponding layer on the other surface. The keratinocytes stretched out pseudopodia peripherally and connected with each other tightly in a matrix that could act as permeability barrier. A urine barrier protected the underlying tissues from the caustic effects of urine, which could prevent urethral hyper-fibrosis and urethral stricture recurrence.
In an animal experiment, we implanted the TEBM into the canine model. The animals in the experiment group did not show signs of urine stricture. The histology test showed that the epithelial cells covered the defect and formed stratified layers (three to four cell layers) at 6 mo. In the comparison group, there were two to three cell layers formed at 6 mo. In the experimental group, the keratinocytes could act as a barrier to protect the underlying tissues from the caustic effects of urine. In the comparison group, urine permeated the urethral defect that caused severe inflammation and extensive fibrosis. These factors affected the growth of the epithelium.
In this study, we built the animal urethra stricture model by removing 5 cm2 of urethra mucosa. There is a long urethra mucosa defect in the canine model. It is suggested that this TEBM presents a potential application in the treatment of long urethral strictures.
We successfully constructed tissue-engineered mucosa combined with autologous keratinocytes and fibroblasts. Our study confirmed that TEBM could serve as a promising alternative graft for urethra reconstruction and as an application for tissue engineering.
The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant No. 81170641) and the Doctor Innovation fund of Shanghai Jiaotong University School of Medicine (Grant No. BXJ201239).