Anterior substitutional urethroplasty using a biomimetic poly‐l‐lactide nanofiber membrane: Preclinical and clinical outcomes

Abstract The aim of this study is to investigate the feasibility and efficacy of a novel biomimetic poly‐l‐lactide (PLLA) nanofiber membrane in repairing anterior urethral strictures from both preclinic and clinic. Biomimetic PLLA membrane was fabricated layer by layer according to the structure of human extracellular matrix. Microstructure, tensile strength, and suture retention strength were fully assessed. Before the clinical application, the safety and toxicology test of the biomimetic PLLA membrane was performed in vitro and in experimental animals. The patients underwent urethroplasty used dorsal onlay or lateral onlay technique. Then, they were followed up for 1 month, 3 months, 6 months, and then annually after the surgery. The mechanical experiments showed well property for application. Biomimetic PLLA membrane was safe according to the in vitro and animal studies. Then, a total of 25 patients (mean age 48.96 years) were included in the study from September 2016 to December 2018. After a mean follow‐up of 33.56 months, 20 patients successfully treated with biomimetic PLLA membrane. Five patients (2 bulbar and 3 penile) suffered postoperational urethral stricture recurrence. None of infection or urinary fistula or any other adverse events related to the use of biomimetic PLLA membrane were observed during the follow‐up period for all patients. The preliminary result confirmed the feasibility and efficacy of the biomimetic PLLA membrane as a novel material for anterior urethral repair. The long‐term effects with more patients should be investigated in further studies.


| INTRODUCTION
Anterior urethral strictures can occur due to various reasons. In China, trauma accounts for the majority of urethral strictures. The incidence of urethral strictures caused by iatrogenic injury has increased. 1,2 The management of a complex and long-segment urethral stricture is one of the most challenging issues at present for reconstructive urologists.
The most frequently used method for this purpose is substitution urethroplasty using autologous tissues. Various autologous tissues, such as penis skin flaps, bladder mucosa, and oral mucosa, have been proposed for substitution urethroplasty. 3,4 However, the harvest of these autologous grafts inevitably has led to lesions at the donor site and has limited availability. The use of acellular matrix has provided new ways for urethral repair, such as bladder acellular matrix graft, 5,6 small intestine submucosa, 7 and urethral extracellular matrix (ECM). 8 After implantation, the acellular matrix acts as a scaffold for cell growth and tissue regeneration and provides a suitable microenvironment for cell growth. Finally, it can gradually degrade and be eventually replaced by new tissue. However, the source of human-or animal-derived acellular matrix material is relatively limited. Moreover, the potential risks of ethics, transmission of disease, and immunological problems have aroused concerns.
Poly-L-lactide (PLLA) is a kind of synthetic polymer, which approved by the European Union and FDA for medical use. The good biocompatibility, controllable mechanical properties, degradation rate, and topological microstructure make PLLA an extraordinary promising synthetic biomaterial in tissue regeneration. Various manufacture technologies had been applied on PLLA scaffolds preparation. There is a difference between the microstructure of human tissue and traditionally processed PLLA membrane, such as weaving, casting, and hot pressing membranes, which have relatively poorer elasticity, flexibility, cell adhesion and infiltration. The PLLA membrane used in the present study was prepared via additive manufacturing technology. And it is composed of biomimetic PLLA fibers that resemble the ECM in humans. The novel ECM structure has shown good regeneration capacity in tissue repair. The biomimetic PLLA membrane has been preliminarily applied in dura mater repair and complex wound healing. [9][10][11] However, only few bioactive materials finally applied in clinic, especially in urethral reconstruction field. In the present study, we aimed to investigate the preclinical and clinical outcomes of the novel biomimetic PLLA fiber graft in anterior urethral substitutional urethroplasty.

| Materials and characterization
The biomimetic PLLA nanofiber membrane used in this study was provided by Medprin Regenerative Medical Technologies Co. Ltd.
(Guangzhou, China), which were manufactured through additive manufacturing technology as described in previous report. 10 Briefly, bioresorbable PLLA fibers were fabricated and deposited layer by layer to form the fiber-structured membrane, which resembled the extracellular matrix of human soft tissue.
The microstructure of the nanofiber membrane was observed using the scanning electron microscope (SEM, JEOL JSM-5600LV, Japan). Membrane was cut into small pieces and attached on conductive carbon tape, and gold-spraying was processed prior to SEM observation. The thickness of the membrane was tested using a commercial hand-held thickness gauge. Pore size and pore size distribution was tested using automatic mercury porosimeter (MA-3000, NIPPON, Japan). The tensile strength and suture retention strength were evaluated using a universal material testing machine (Instron 5567, Instron, USA) to represent the mechanical properties of membrane. Tensile strength was examined according to ISO 527-3 standard. Membranes were soaked in distilled water for at least 1 min to fully hydrate and then cut into stripes with length of 6 cm and wide of

| Cell experiments
Human urothelial cells (HUCs, Sciencell, Cat 4320) were used to evaluate the effect of the nanofiber membrane on cell viability, attachment, and morphology. The HUCs were cultured in cell culture dishes in an incubator (37 C, 5% CO 2 ) with a humidified atmosphere.
Urothelial cell medium (HUM, Sciencell, Cat 4321) was used for cell culture and refreshed every 2 days. Cells at passage 4 were used for the cytotoxicity and attachment experiments.
The sterile membranes were cut into round pieces with diameter of 1 cm and introduced into 48-well plates. HUCs in 50 μl HUM were added onto the samples with a cell density of 2 Â 10 4 cells per well.
Four hours later, 500 μl HUM was replenished into each wells.
Cytotoxicity was evaluated using a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) after HUCs seeded on samples and cultivated for 24 h, following the manufacturer's instructions. CCK-8 working solutions were added into each well to replace the HUM, and incubated at 37 C for 1 h. An enzyme-linked immune sorbent assay (ELISA) plate reader (Thermo Scientific, Thermo3001, USA) was used to read the absorbance of CCK-8 reaction solution. Six replicates were prepared for the assay and HUCs seeded directly onto the surface of plate were treated as control group.
Cell attachment morphology was observed using SEM after HUCs cultured on membranes for 4, 8, 24, and 48 h. At each time point, cells on membranes were washed with PBS twice and fixed with 4% paraformaldehyde for at least 4 h. After dehydrated with gradient ethanol and dried, cell morphology was viewed via SEM.

| Animal experiments
A total of 12 male New Zealand white rabbits (weighing 2.0-3.0 kg) were divided into two groups. Six rabbits were in experiment group and treated with biomimetic PLLA membrane. Other six rabbits were in control group. All surgeries were performed by the same surgeons using procedures described previously. 12

| Follow-up
The patients were followed up after 1 month, 3 months, 6 months, and then annually. Clinical evaluations included uroflowmetry or voiding function report. Urethrography or urethroscopy was performed if the maximum urine flow rate was continuously lower than 15 ml/s or selectively performed before the removal of the suprapubic catheter in some patients. Success was defined as a maximum flow rate of >15 ml/s and without the need for further surgical interventions, such as dilatation or optical urethrotomy.
Stricture recurrence was defined as recurrent symptomatic stricture requiring further operative intervention following initial intervention. F I G U R E 8 Representative hematoxylin and eosin staining (HE), Masson trichrome staining, AE1/AE3 (expression in the urothelium) immunohistochemical staining, and CD31 (expression in the vascular endothelium) immunohistochemical staining at 3 months after implantation in the two groups. The red arrows indicate surgical sites. The red triangles indicate blood vessels retentions of the scaffolds were 11.8% and 1.66 N in the dry state and 54.5% and 2.26 N in the wet state (Figure 4c). The suture retention strength of the biomimetic PLLA membrane was more than or close to 2.0 N in the wet state, which was generally accepted where suturing was required with tissue in implantation cases. 13

| Cell experiments
OD values of cytotoxicity experiment analyzed using CCK-8 kit were shown in Figure 5. There is no significant difference between the nanofiber membrane sample and the control group. And the relatively OD value of sample to the control group was 92.2%, which meant the cytotoxicity level of nanofiber membrane to human urothelial cells was zero.
The adhesion and distribution of HUCs after seeded on nanofiber membranes for 4, 8, 24, and 48 h were shown in Figure 6. Cell conjugation formed after 48 h of culture. HUCs had a relatively high crawling and proliferation rate on samples.

| Clinical application
Twenty-five patients were included in the study. The mean age of patients was 48.96 years (range from 19 to 83). Table 1     There are several studies reported clinical application of tissueengineered material in substitution urethroplasty. In 2011, a tissueengineered autologous urethra was reported used in patients with urethral defects. 16 The muscle and epithelial cells were harvest from the patients, expanded, and seeded onto tubularized PGA scaffolds.
After a mean 71 months follow-up, none of the patients suffer the recurrence. This is a great approach in urethral reconstruction. In 2018, a tissue-engineered oral mucosa graft named MukoCell ® was applied in anterior urethroplasty. 17 It also had an overall high success rate. Unlike these studies, we fabricate the biomimetic PLLA nanofiber membrane via additive manufacturing technology, mimicking the ECM structure of humans. Without harvesting harm, long-time tissue culture, and bio-safety problems, synthetic material provides a valid alternative to tissue-engineered ones owing to their lower costs (up to 10 times less), wider availability, and fewer ethical concerns.
Besides, the pore size, construction, and absorbability could be adjusted according to demands. It is a good graft choice for the urethral substitution. Meanwhile, several studies also showed that the ultrastructure and 3D architecture of collagen fibers of the acellular matrix were important in modulating the cells' ability to migrate into the scaffold or influence tissue-specific cell phenotype. 18,19 Thus, the exploration and development of a new urethral substitution material that can mimic the structures of an acellular matrix are urgently needed in clinic.
Using additive nanomanufacturing technology to prepare biomimetic scaffolds is realistic with the rapid development of technology in recent years. A biomimetic PLLA nanofiber membrane is sequentially fabricated in layers to form a 3D structure. The cell experiment showed a high biocompatibility (Figures 5 and 6), which is coincident with previous study. 9,10 Compared with the acellular matrix, the biomimetic 3D structure with a highly adjustable porous network can facilitate the passage and exchange of nutrients and gases, which are important for cellular growth and tissue regeneration. 20  Sufficient blood supply is beneficial to the surrounding cell infiltration and material degradation. In animal experiment, the biomimetic PLLA membrane was used in bladder substitution. Bladder had a better blood supply, lots of red blood cells, and a few of white blood cells filled in the space of the biomimetic PLLA membrane ( Figure S3). This phenomenon might also account for the quick degradation of biomimetic PLLA membrane in animal model. While, in human, the cavernous in bulbar urethra is stronger and thicker than penile urethra, and supplies with more blood. The residual urethral plate appearance of bulbar urethra is normally appearing much better than that of penile urethra after opening the narrowed urethral lumen. The anatomical difference led to the insufficient blood supply of the penile urethra, which might be one reason for the more failed urethroplasties in penile urethral strictures. Besides, the spongiofibrotic degree could be another influence factor for the blood supply. As our center is one of the biggest tertiary referral urethral reconstruction centers in China, before the patients came to our center, some of them had already received several interventions. The prior interventions could contribute a lot to the spongiofibrotic degree. Tissue regeneration was based mainly on the nutrition supply and inosculation of the urethral plate. 22 Thus, the spongiofibrotic segment of the urethra was not good enough to support the regeneration of urethral epithelial cells inside the lumen, which might be an important reason for the failed urethroplasties in the biomimetic PLLA membrane.
As for polymer materials, PLLA was confirmed to be degraded in vivo. Due to the ethical concern and for human rights protection, biopsy and extra examinations were not performed to analyze the degradation state. However, in the animal experiment, we found the biomimetic PLLA membrane was vanished in the rabbit bladder in 2 weeks, the fixed area showed no significant different in gross observation ( Figure S2). Besides, the stress-strain curves of postoperative bladder were also similar to the normal bladder ( Figure S5) In this study, the biomimetic PLLA membrane was used for substitution urethroplasty, which also seemed to be promising in urethra reconstruction. However, this study had some limitations. The sample size was not large enough to identify statistically significant differences between the succeed and failed patients. For safety concern, we only used our biomimetic PLLA membrane in patients with nonobliterated urethral stricture, which possess a well blood supply urethral bed. Thus, the indication of substitution urethroplasty using biomimetic PLLA membrane should be future investigated. Moreover, this finding requires confirmation in an adequately powered prospective randomized controlled trial with a long-term follow-up.

| CONCLUSIONS
This study showed that the biomimetic PLLA membrane was a feasible and effective novel material for the anterior urethral repair.
Urethral reconstruction using the biomimetic PLLA membrane should only be carefully considered with proper indications, including the stricture location, thickness of scar, and diameter of the remaining urethra lumen. Moreover, the long-term effects with more patients should be investigated in further studies.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.