Experimental study of extracellular vesicle-small intestine submucosa novel biological composite for urethral stricture repair and reconstruction

Abstract

Background

Urethral stricture (US) is a common condition that considerably affects patients’ quality of life.

Objectives

This study aimed to explore the adoption value of the extracellular vesicle (EV)- small intestinal submucosa (SIS) complex in the repair of USs.

Methods

EVs were extracted from healthy male New Zealand white rabbits, and SIS was prepared using peracetic acid (PAA) oxidation and decellularization. The morphology and particle size of the prepared EV-SIS complex were evaluated using electron microscopy and qNano nanoparticle analyzer, and the labeled proteins of EVs were detected using Western blot method. EV-SIS the complex was implanted in a rabbit model of US, and urodynamic parameters were assessed.

Results

The EV-SIS complex displayed a full morphology, intact membrane structure, and uniform particle size. The protein concentration of EVs in the complex was approximately 0.351 µg/µL, with a yield of approximately 1.86 µg/10⁶ cells. The complex exhibited remarkable repair effects in the rabbit model of US, with bladder capacity, maximal urethral pressure, and minimal urethral pressure all markedly superior to those in the US group (P < 0.05).

Conclusion

The EV-SIS complex demonstrates potential clinical value in the repair of USs, improving urodynamic parameters, and offering a promising therapeutic option for patients with US.

Keywords

extracellular vesicle small intestine submucosa urethral stricture urodynamic urethral reconstruction

Introduction

Urethral stricture (US) is a common clinical problem characterized by narrowing or closure of the urethral lumen, leading to obstructed urine flow. It can be caused by various factors, including inflammation, infection, trauma, or surgery. The condition is often associated with symptoms such as reduced urine stream, difficulty in urination, urinary retention, and urinary frequency, considerably affecting the patients’ quality of life.^1,2 Despite the availability of several methodologies for US repair, such as urethral dilation, urethrotomy, and urethroplasty, these approaches still have limitations, as some traditional repair methodologies may result in postoperative stricture recurrence, functional impairment, or complications. Therefore, finding an effective, safe, and sustainable approach for US repair remains an imperative topic in the current clinical field.^3–5

Biological composite materials are a class of materials composed of natural biological tissues or synthetic biocompatible materials that exhibit excellent biocompatibility and bioactivity. These materials provide a scaffold structure that can support and direct cells during US repair.^6,7 They can mimic the three-dimensional structure of natural tissues, providing a suitable environment for cell adhesion, proliferation, and differentiation. Bioactive molecules within biological composite materials, such as cytokines, growth factors, and nucleic acids, can promote cell proliferation and differentiation, stimulating tissue regeneration and repair. These bioactive molecules can regulate cell function and behavior, facilitating the growth and reconstruction of new tissue. Biological composite materials have the potential to suppress inflammatory reactions and scar formation, which are common issues during US repair and may lead to stricture recurrence postsurgery. The bioactive molecules within the biological composite materials can mitigate the inflammatory response, promoting tissue remodeling and restoration during the repair process. Angiogenesis is a critical process in tissue repair and regeneration.^8,9 Biological composite materials can promote angiogenesis by providing a scaffold structure and releasing bioactive molecules, thereby ensuring sufficient blood supply and nutrients to the repair site. These materials possess favorable mechanical properties, offering essential mechanical support and stability to maintain the structural integrity of the US repair area. This aids in preventing stricture recurrence and promotes the proper formation and functional recovery of newly generated tissue. In short, biological composite materials play a multifaceted role in US repair. By optimizing the design and composition of these materials, the effectiveness of US repair can be further enhanced, providing improved clinical treatment options for patients.^10,11

Extracellular vesicles (EVs), known as exosomes, are a type of vesicular structure with a diameter of approximately 30–150 nanometers that are released by cells into the extracellular environment. Exosomes contain abundant proteins, nucleic acids, and bioactive molecules, which play essential biological functions in intercellular communication, signal transduction, and tissue repair.^12–14 In recent years, the combination of exosomes and small intestinal submucosa (SIS) to form a novel biocomposite material for US repair and reconstruction has garnered great interest. SIS is a biologically derived material sourced from animal tissue, subjected to decellularization, and retains the original collagen framework and bioactive substances, ensuring good biocompatibility and bioactivity.^15,16 The integration of exosomes and SIS provides an extracellular matrix scaffold that facilitates tissue regeneration and repair processes. Exosomes provide a rich source of bioactive molecules that can enhance cellular functions and repair capabilities. Simultaneously, SIS offers a stable scaffold structure, providing support and directional guidance to cells. By introducing the biocomposite material into the US injury site, the bioactive molecules from exosomes can promote cell proliferation and differentiation in the damaged area, stimulate tissue regeneration, and reduce inflammation and scar formation.^17,18 Additionally, the scaffold structure provided by SIS maintains tissue integrity and facilitates the growth and repair of new tissues. In this study, the effectiveness of the EV-SIS biocomposite material in US repair was evaluated, and its underlying mechanisms were investigated. It is hoped that this research can provide a novel and feasible repair approach for US treatment and offer substantial support and evidence for the adoption of biocomposite materials in US repair and other tissue engineering fields.

Materials and methodologies

Main reagents and equipment

The main reagents and equipment used in the experiment are presented in Table 1.

Table 1.

Main reagents and equipment.

Name	Model/article number	Manufacturer and place of origin
PAA (peracetic acid)	P1012	Sigma-Aldrich (Corp. of Hilmon, USA)
Triton X-100 (Polyethylene glycol octyl phenyl ether)	X100	Sigma-Aldrich (Corp. of Hilmon, USA)
Proteinase K	EO0491	Thermo Fisher Scientific (Thermo Fisher Scientific Shier Science & Technology Company, USA)
Phosphate buffer	1068181000	Millipore (American Philips Corporation)
Ultracentrifuge	5804R	Eppendorf (Ibendorf AG, Germany)
Magnetic stirrer	6795-220	Corning (Corning Corporation, USA)
Electron microscope	JEM-1400	JEOL (JEOL Corporation, Japan)
Low voltage desktop transmission electron microscope	LVEM5	Quantum Design (China)
Nano analyzer	qNano	IZON (IZON Technologies New Zealand)
Gel Doc EZ gel imager	1708270	BIO-RAD (USA)
CD63 antibody	ab217345	Abcam (Abcam company of America)
Alix antibody	2171S	Cell Signaling Technology (Cell Signal Technology Company, USA)
Goat anti rabbit antibody	111-005-003	Jackson ImmunoResearch Laboratories, Inc. (Jackson Immunological Research Laboratories, Inc)

Establishment of animal model

Healthy male New Zealand white rabbits, approximately 4 months old, with body weights ranging from 2.5 kg to 3.5 kg, were obtained from Beijing Beknts Experimental Rabbit Breeding Biotechnology Development Co., Ltd The animals were housed in a clean, hygienic, and well-ventilated animal laboratory, maintained at 25 °C between 16 °C and 25 °C, and maintained at suitable humidity levels between 40% and 70% to avoid excessive moisture or dryness. The light-dark cycle was simulated to mimic natural lighting conditions, with 12 h of light and 12 h of darkness. High-quality rabbit feed was provided to ensure that the rabbits received adequate nutrition and energy. Prior to the start of the experiment, the rabbits underwent body weight measurements and health checks to ensure that they were in good health. Ethical approval for this experiment was obtained, and relevant ethical and animal protection regulations were strictly followed. The use and handling of animals involved in the experiment complied with ethical standards. The experimental plan was submitted and approved by the ethics committee of the research institution (approval number: 20201034777).

3% pentobarbital sodium solution (2 mL/kg) was slowly injected (0.1 mL/s) into rabbits for anesthesia to ensure that rabbits were in a pain-free state. The rabbit was placed on the operating table, kept in the supine position, disinfected locally in the lower abdomen, some hair was shaved off, and the operating area was exposed. The bladder was rinsed with gentamicin saline solution (160,000 units/500 mL) 5 mL/wash until the rinse solution was clear. The ventral side of the rabbit was cut, making a 2 cm long longitudinal incision, and the subcutaneous tissue was cut with microscrissors. The skin was pulled to both sides to expose the urethra, and then the urethral cavernous body was pulled to expose the urethra. The operator pulled the urethral mucosa inward and peeled it off from the urethral cavernous body with elbow microscissors, with a length of 2 cm. After the injury was completed, the abdominal incision was sutured with 3-0 absorbent thread to ensure wound healing. The rabbit was put in the recovery room and observed until it woke up and resumed normal activities.

Extraction of exosomes

Rabbit primary cell lines (provided by Wuhan ShangEn Biotechnology Co., Ltd) were cultured, which had been tested for Mycoplasma, and there was no Mycoplasma infection. The cell line had passed STR verification, indicating that the identification of the cell line had been scientifically verified to ensure the accuracy of its source and characteristics. When the cell growth reached approximately 70% confluence, they were transferred to serum-free culture medium for exosomes (consisting of high-glucose Dulbecco's modified Eagle's medium (DMEM) medium supplemented with exosome-depleted serum and antibiotics) and cultured for 24 h. When the cells reached approximately 90% confluence, the supernatant was collected. The collected supernatant was transferred to centrifuge tubes. Low-speed centrifugation (300 xg, 10 min) was performed to remove cell debris and large particles. The supernatant was then transferred to new centrifuge tubes, avoiding inclusion of the precipitate. The supernatant was transferred to ultracentrifuge tubes, and ultracentrifugation was carried out using an ultracentrifuge at a maximum speed of 100,000 xg for 2 h. After ultracentrifugation, the supernatant was carefully discarded, leaving behind the exosome pellet. Phosphate-buffered saline (PBS) was applied to the exosome pellet to resuspend it. The exosome pellet was then subjected to low-speed centrifugation at 10,000 xg for 30 min to pellet the exosomes, and the supernatant was removed. This washing step was repeated 2–3 times to remove impurities and contaminants as much as possible. After removal of the wash buffer, the exosome pellet was retained. Finally, the exosome pellet was resuspended in PBS buffer, transferred to sterile EP tubes, and stored at −80 °C in a refrigerator.

Detection of exosome marker proteins by western blot

The exosome sample (1 μg) was subjected to protein lysis using RIPA buffer and denatured at 95 °C in a dry heat block for 10 min. Protein samples were prepared and loaded into the wells of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, with 5 μL of Marker applied to the first well, and the remaining wells were loaded with the samples. Protein loading buffer was applied to serve as a molecular weight marker for determining the migration positions of the proteins. Subsequently, SDS-PAGE was performed at 100 V for approximately 15 min until the marker bands appeared, followed by an increase in voltage to 150 V for a total running time of approximately 40–60 min. A transfer apparatus was prepared, and the gel was transferred onto a polyvinylidene fluoride (PVDF) membrane using a semidry transfer apparatus at 15 V for 35 min, ensuring successful transfer when the bands on the gel appeared on the PVDF membrane.

The transferred membrane was washed three times with washing buffer and then placed in Tris buffered saline Tween (TBST) buffer containing BSA to block nonspecific binding, followed by incubation on a horizontal shaker for 10 min. The membrane was then incubated with primary antibodies (CD63, Alix) overnight at 4 °C at a dilution of 1:1000. After multiple washes with TBST buffer to remove unbound antibodies and nonspecific binding, each wash lasted approximately 3 min, and the membrane was incubated with the corresponding secondary antibodies (all goat anti-rabbit) at a dilution of 1:1000 at 25 °C in the dark for 1 h to form antibody-antigen complexes. Finally, the gel imager was used for exposure. After the image was saved, the quantitative analysis was carried out with Image J.

Detection of exosome particle size and concentration

Exosome size analysis was performed as follows. The qNano nanoparticle analyzer and nanopore filter membrane were prepared. Ultracentrifugation was implemented to pellet the exosomes from the sample. The exosome sample was suspended in a suitable buffer to maintain their stability and activity. An appropriate nanopore size was selected for the filter membrane and inserted into the qNano nanoparticle analyzer. The exosome sample was loaded into the qNano nanoparticle analyzer following the instrument's operation manual. Finally, the qNano nanoparticle analyzer was used to perform size analysis and record the results.

Preparation of ev-SIS

The terminal ileum (near the ileocecal junction) of healthy domestic pigs (≥200 kg) was obtained (Tianjing Village Pig Breeding and Breeding Base in Jiucaizhuang Township, Hohhot City, Inner Mongolia Autonomous Region) and thoroughly cleaned within 4 h, and the small intestine was dissected along the mesentery to obtain approximately 10 cm long segments. The serosal and muscular layers of the small intestine were gently scraped using a surgical knife to ensure complete removal of debris, followed by thorough rinsing with distilled water to remove any residual contaminants and cellular debris.

Following the isolation of the submucosa from the small intestine, the tissue was placed on a magnetic stirrer and stirred at 800 rpm for 48 h at 4 °C. The sample was then rinsed with distilled water to remove any residual chemical agents. Subsequently, the sample was immersed in solutions of 0%, 1%, 3%, and 5% PAA for 4 h to promote decellularization. After this treatment, the sample was placed in a 1% Triton X-100 solution and stirred at 800 rpm for an additional 48 h at 4 °C. Following this, the SIS scaffold was continuously rinsed with distilled water on a magnetic stirrer at 800 rpm for 48 h to ensure the thorough removal of any remaining chemicals. For sterilization, the SIS scaffold was immersed in a solution containing 0.1% PAA and 20% ethanol for 2 h, followed by multiple rinses with distilled water to eliminate any residual disinfectant. Finally, the processed SIS samples were stored in sterile distilled water at 4 °C for subsequent experimental use.

Hematoxylin-eosin (He) staining of SIS

A suitable amount of tissue samples was taken from the SIS specimens stored in a 4 °C refrigerator and fixed in 10% buffered formalin for 48 h. The fixed SIS tissue samples were transferred to a 70% ethanol solution for dehydration. The ethanol concentration was gradually increased, including 80% ethanol, 95% ethanol, and absolute ethanol. The tissue samples were then transferred to xylene as a clearing agent to allow tissue penetration and infiltration with paraffin. Subsequently, the tissue samples were embedded in paraffin for impregnation. The paraffin-embedded tissue samples were then cut into 4–6 μm thick sections using a rotary microtome, baked for 5 h at approximately 60 °C, placed in deionized water to remove residual paraffin, and transferred to a pretreatment box.

The sections were immersed in hematoxylin solution for 5–10 min to stain the cell nuclei. Subsequently, the sections were transferred to an alcohol wash jar to remove excess hematoxylin and then rinsed under running tap water. The sections were then placed in eosin solution for 5–10 min to stain the cytoplasm and collagen fibers. After that, the sections were washed again in an alcohol wash jar to remove excess staining and rinsed under running tap water for 30 min. The sections were gradually dehydrated in ascending alcohol concentrations (70%, 80%, 95%). They were then infiltrated with xylene and embedded in paraffin using a tissue embedding machine. Finally, the paraffin-embedded sections were placed on glass slides, fixed to the slides using melted wax, and formed into glass slides with tissue sections. The stained sections were visualized under a microscope, and various magnifications were adopted for detailed observation and image capture. The cell nuclei staining and tissue structure in the sections were analyzed to evaluate the morphology and tissue structure of SIS.

Detection of residual DNA content of SIS treated with PAA at various concentrations

The obtained SIS samples were treated with different concentrations of PAA, including 0%, 3%, 5%, and 10% treatment groups. Each treatment group's SIS sample (25 mg) was placed in a centrifuge tube. To the sample tissue block, 20 μL of proteinase K was applied and thoroughly mixed. Then, 250 μL of Buffer TG-A was applied, and the mixture was vigorously shaken approximately 20 times to ensure homogenization. The sample was placed in a 56 °C water bath, and after periodic observations, it was removed once the tissue block was completely digested. Next, 250 μL of Buffer TG-B was applied, and the mixture was vigorously shaken approximately 20 times to ensure homogenization. The sample was placed in a 70 °C water bath and kept for 10 min. The sample was centrifuged at 25 °C and 12,000 × g for 2 min, and the supernatant was transferred to another 1.5 mL centrifuge tube. Then, 250 μL of anhydrous ethanol was applied to the supernatant and mixed thoroughly, and the mixture was transferred to a DNA binding column (C30) and placed in a collection tube. The DNA binding column (C30) was centrifuged at 25 °C and 7000 × g for 1 min and then placed into another new collection tube.

A total of 500 μL of WAG buffer was applied, and the mixture was centrifuged at 7000 × g for 1 min at 25 °C. The collection tube was then discarded, and the DNA binding column (C30) was transferred into a new collection tube. Subsequently, another 500 μL of Buffer WB1 was applied, and the DNA binding column (C30) was centrifuged at 7000 × g for 1 min at 25 °C. The waste liquid was discarded, and the DNA binding column (C30) was placed back into the collection tube. This process was repeated once more. Then, the DNA binding column (C30) was centrifuged at 12,000 × g for 2 min at 25 °C and subsequently placed into a 1.5 mL centrifuge tube provided with the DNA kit. Next, 100 μL of TE was applied to the center of the silica gel mold, and after 2 min of standing, it was centrifuged at 12,000 × g for 1 min in a high-speed centrifuge. Finally, the obtained sample was placed into a DNA concentration meter, and the DNA concentration was determined following the instrument's guidelines. The DNA concentration of each sample was recorded based on the measurement results.

Compound and morphological structure detection of ev-SIS

According to the experimental requirements, the EV and SIS samples were adjusted to the same concentration using 200 μL of PBS buffer, ensuring comparable sample concentrations for better mixing. The adjusted EV and SIS samples were placed in separate centrifuge tubes on ice to maintain a low temperature of 4 °C. The EVs were gradually applied to the SIS sample, drop by drop, with gentle agitation or shaking to ensure thorough mixing. The EVs and SIS samples were allowed to incubate at 25 °C for 24 h to promote complex formation. After incubation, the complex samples were centrifuged at 10,000 xg for 2 min using an ultracentrifuge to separate the complex from any unbound EVs or residual SIS. The complex samples in the supernatant were collected and transferred to a new centrifuge tube. The EV-SIS complexes were then squeezed into a mold with a diameter of 1 cm and a depth of 0.5 cm and transferred to a 24-well plate, each well containing 200 μL of PBS for immersion. Daily, the PBS in each well was replaced with an equal volume. To detect the morphology and structure of EV-SIS, the EV-SIS sample was diluted tenfold and a 10 μL suspension was applied to a copper mesh-supported membrane to allow absorption. After 10 min, the excess liquid was removed using filter paper and the EV-SIS sample was placed on a transmission electron microscope (TEM). Then, a 1% uranyl acetate staining solution was applied to each copper grid and stained for 10 min, and the excess liquid was removed using filter paper. The sample was air-dried and TEM was performed to adjust the electron acceleration voltage and parameters to observe and capture the EV-SIS morphology.

Autologous replantation of the exosome-SIS complex

During the autologous reimplantation of the ex vivo-SIS composite, rabbits were initially anesthetized with a 3% pentobarbital sodium solution (2 mL/kg) to ensure they were pain-free and unconscious. Subsequently, the surgical area was prepared through surface disinfection. Under sterile conditions, the pre-prepared EV-SIS composite samples were recovered to ensure their sterility. A longitudinal incision was made in the skin at the urethral region, exposing and excising the narrowed portion of the urethra to create an approximately 1.5 cm tubular defect. The EV-SIS composite was then carefully implanted longitudinally into the urethra, ensuring its secure placement within the narrowed area. The composite was fixed in place using 5-0 absorbable sutures, followed by interrupted suturing of the subcutaneous tissue and skin. Postoperatively, the rabbits were transferred to a recovery area for observation and monitoring, receiving supportive care, including antibiotics, to facilitate their recovery. In this context, considering the advantages of using synthetic materials, traditional blood flow protection procedures may no longer be necessary.

The methodology for EV implantation and SIS implantation was as described above.

Urodynamic detection

Each group of five rabbits was selected, ensuring that the rabbits had appropriate body weights and health conditions for the experiments. Prior to the experiment, the rabbits were fasted to ensure an empty bladder during the experiment. The rabbits were anesthetized with a 3% sodium pentobarbital solution (2 mL/kg) to ensure that they were in a painless and unconscious state. The rabbits were immobilized on the surgical table for ease of measurement. The urodynamic measurement instrument was prepared, and its calibration and proper functioning were ensured. The bladder was completely emptied by catheterization, and then physiological saline was slowly infused into the bladder using a syringe while recording the volume of saline infused. The infusion was continued until the rabbits showed a cessation of micturition reflex, and the volume of infused physiological saline at that point represented the bladder capacity. With the bladder filled, the F-7 catheter with a pressure transducer was placed into the urethra to ensure good contact between the urethra and the pressure transducer. The pulling speed of the pressure catheter was controlled at 1 mm/s while recording the readings of the maximum and minimum pressures in the urethra. Each rabbit underwent repeated measurements (at least 3 times) to obtain accurate and reliable data.

Statistical analysis

The experimental results were denoted as the mean ± standard deviation (x̅±s). Data analysis was performed using SPSS 20.0. One-way analysis of variance (ANOVA) was used to compare multiple groups, and P < 0.05 indicated statistically significant differences.

Results

Identification results of exosomes

In Figure 1 below, Western blot analysis showed that the exosome marker proteins CD63 and Alix were positive.

Figure 1.

Western blot detection of exosomes.

Western blot analysis revealed positive expression of the EV-specific proteins CD63 and Alix. The qNano nanosizer analysis (Figure 2) demonstrated that the size of the EV particles was 90 ± 14.5 nm, with a maximum size of 194 nm, and the particle concentration was measured to be 9.96 × 10¹⁰ particles/mL.

Figure 2.

Particle size and concentration of exosomes.

Scanning observation by Ev-SIS electron microscopy and urethroscopy

After the preparation of the EV-SIS composite, the morphological and structural characteristics of the composite were observed using TEM. In Figure 3(A), the EVs within the composite exhibited a robust morphology with a certain degree of fullness. The vesicles displayed intact membrane structures, encapsulating internal biomolecules. The size distribution was relatively uniform, and the particle features appeared mature, displaying typical characteristics of EVs with a structure similar to those observed in pure EV samples.

Four weeks after implantation of the EV-SIS composite, the morphology of the rabbit urethra observed by urethroscopy is shown in Figure 3(B) below. The urethral wall surface appeared relatively smooth and uniform, with the composite exhibiting stability within the urethral tissue. The cytoplasm of the EV-SIS composite appeared full, and the surface membrane structure remained intact, with minimal surrounding tissue reaction. The size of the composite remained similar to that at the time of implantation, with no apparent signs of degradation. At twelve weeks postimplantation, the presence of the composite was still evident within the urethral tissue, maintaining a stable morphology. The surrounding tissue reaction continued to be relatively mild, without evident signs of inflammation or immune responses. Overall, through electron microscopy observations, successful implantation of the EV-SIS composite in the rabbit US model was confirmed.

SIS staining results and residual DNA detection

In Figure 4(A) below, when observed under a microscope, the stained SIS sample exhibited a bright and clear cellular structure. The cytoplasm of the cells presented a light pink color, and the tissue cells were tightly arranged, presenting a regular and orderly structure. In Figure 4(B), the DNA content in the SIS samples treated with 0%, 3%, 5%, and 10% PAA oxidation demonstrated a clear negative correlation with the PAA concentration. With increasing PAA concentration, the DNA content in the SIS considerably decreased, and this decrease exhibited a considerable difference (P < 0.05). Particularly, in the SIS samples treated with 5% and 10% PAA oxidation, the DNA content was remarkably low, indicating that at these two concentrations, the oxidative action of PAA effectively removed cellular components from the SIS, achieving a more thorough decellularization of the SIS.

Figure 3.

EV-SIS electron microscopy and urethroscopy. Note: A shows the electron microscopy examination of EV-SIS (200 nm), while B shows the observation of EV-SIS implantation under urethroscopy.

Figure 4.

SIS staining results and residual DNA detection. Note: A shows HE staining (200 nm), and B shows DNA quantity. *P < 0.05 vs. Normal SIS.

Urodynamic detection

Twelve weeks after implantation, five rabbits were selected from each group for urodynamic testing under anesthesia. In Figure 5, the bladder capacity, urethral maximum pressure, and urethral minimum pressure in the EV group (Group A), SIS group (Group B), and EV-SIS composite group (Group C) were markedly superior to those in the US group (P < 0.05). Additionally, the urodynamic parameters in the EV-SIS composite group were markedly superior to those in the EV group and SIS group (P < 0.05).

Figure 5.

Urodynamic detection. Note: *P < 0.05 vs. US group; ^#P < 0.05 vs. between EV group and SIS group.

Urethral manometry

Figure 6 shows the urethral pressure measurements of each group. Pura is the pressure measurement of the urethra, reflecting the pressure inside the urethra. 0 represents the reference value, which is the basic value before inserting the urethral probe, used to compare and evaluate the pressure changes inside the urethra. Numbers such as 56 ^ and 45 ^ indicate the peak pressure measurement in the urethra, which is the highest pressure point that occurs during urethral pressure measurement. cm H₂O is the unit of pressure, representing centimeters of water column. It is used in urethral manometry to represent the pressure inside the urethra. The above data was automatically generated during testing. The curve in the red circle in the figure represents the urethral pressure measurement in each group. The urethral pressure in the US group was higher than that in the other groups, but the difference between the EV group and the SIS group was not significant.

Figure 6.

Urethral manometry. Note: A shows the US group, B shows the EV group, C shows the SIS group, and D shows the EV-SIS group. The red circle in the figure represents the pressure measurement situation.

Discussion

US may arise from various factors, including infection, urethral trauma, and urethral stones. Severe US can lead to urine retention and impaired urine flow and subsequently affect kidney function, considerably impacting the patient's quality of life and health.¹⁹ To seek more effective methodologies for US repair, this study aimed to explore the role of EV-SIS composites in urethral repair. EVs are vesicular structures produced by cells and contain a rich array of bioactive substances, such as proteins, nucleic acids, and growth factors, playing a crucial role in intercellular communication and the regulation of cellular functions. SIS is a decellularized matrix derived from the submucosal layer of healthy porcine small intestines, known for its excellent biocompatibility and tissue scaffolding properties, and has been widely applied in tissue repair and regeneration fields. The objective of this experiment was to evaluate the reparative efficacy and safety of the EV-SIS composite in a rabbit model of US and explore its potential clinical adoption in US repair. By comparing the urodynamic parameters and urethral pressures between the EV group, SIS group, and EV-SIS composite group, the repair effects of the composite and its impact on urethral function can be assessed.

According to the Western blot analysis, the expression of the characteristic EV proteins CD63 and Alix was positive, demonstrating the successful detection of these two marker proteins in the EV samples. This indicates that the extraction and purification process of EVs was relatively effective, and the protein extraction was successful.²⁰ The results obtained from the qNano nanosizer revealed that the EVs had a particle size of 90 ± 14.5 nm, with a maximum size of 194 nm and a particle concentration of 9.96 × 10¹⁰ particles/mL. These findings indicate a certain degree of uniformity in the size of EVs, with particle sizes concentrated at approximately 90 nm and a relatively high particle concentration. The qNano nanosizer is an instrument used for measuring nanoparticles, and this information is crucial for understanding the particle size and concentration of EVs.

Electron microscopy observation revealed that the prepared EV-SIS composite exhibited a full morphology with a certain degree of integrity. The EVs displayed intact membrane structures that encapsulate internal bioactive molecules. The size distribution of the particles was relatively uniform, and the particle characteristics appeared mature, displaying typical EV morphology and structure. These findings indicate the successful combination of EVs with SIS during the preparation process, and the composite maintained the typical morphological and structural features of EVs. Additionally, the intact membrane structure of the vesicles within the composite suggests high stability, which may contribute to the protection of internal bioactive molecules.²¹ Within four weeks after implantation, no notable degradation of the composite was observed in the rabbit urethra, indicating good binding of the composite with the urethral tissues. The successful implantation may be attributed to the appropriate choice of the model and the adaptability of the EV-SIS composite, providing preliminary evidence for the feasibility of the composite in clinical adoptions. At twelve weeks postimplantation, the stability of the composite within the urethra is likely supported by the SIS scaffold and protected by EVs, enabling its long-term presence without inducing evident rejection or immune responses. These results provide robust support for the potential adoption of the composite in the repair of USs.

PAA, an abbreviation for peracetic acid, is a commonly used oxidizing agent extensively applied in the decellularization process of biomaterials. The action of PAA in decellularization mainly involves oxidizing biomolecules such as DNA, RNA, lipids, and proteins in cells, leading to the loss of cellular activity and ultimately achieving complete removal of cells. In this experiment, by treating SIS samples with various concentrations of PAA (0%, 3%, 5%, 10%), the extent of cellular decellularization was controlled, thereby affecting the DNA content in SIS. The experimental results demonstrated that SIS samples subjected to 5% and 10% PAA oxidation exhibited considerably low DNA content, indicating that at these concentrations, the oxidative action of PAA can more thoroughly remove cellular components from SIS. Consequently, SIS treated with 5% and 10% PAA oxidation achieved more thorough decellularization. This is of paramount importance for the preparation of SIS composite materials and their adoption in US repair, as completely decellularized SIS can reduce the risk of immune reactions and rejection, thereby enhancing the biocompatibility and safety of the composite material in the body. For SIS samples treated with 0% and 3% PAA oxidation, relatively higher DNA content was observed, possibly due to the lower extent of cellular decellularization at these concentrations, retaining a certain amount of cellular components. However, caution should be exercised in selecting PAA concentrations, as excessively high concentrations may lead to tissue structure damage and injury. Therefore, in further adoptions, a comprehensive consideration of the decellularization extent and biocompatibility of the composite material is necessary when choosing the appropriate PAA concentration for treatment.

In the experiment conducted after twelve weeks of implantation, the urodynamic parameters, including bladder capacity, maximum urethral pressure, and minimum urethral pressure, were considerably higher in the EV group, SIS group, and EV-SIS composite group than in the US group. This indicates that by implanting exosomes, SIS, and EV-SIS composite, the urodynamic parameters of the rabbit US model were improved. Furthermore, the urodynamic parameters in the EV-SIS composite group were markedly superior to those in the EV group and SIS group, suggesting that the EV-SIS composite had a more pronounced effect compared to the individual adoption of exosomes or SIS, leading to a more effective improvement in urodynamic parameters. Urethral pressure measurement results showed that the impact of exosomes and SIS on urethral pressure might be similar, while the EV-SIS composite group exhibited lower urethral pressure, which aligns with the urodynamic parameter results. This indicates that the EV-SIS composite may have a positive effect on US repair, resulting in a more effective improvement in urodynamic function in the US model. Wang et al.²² suggested that the regeneration of urethral defects has been challenging in clinical practice. They prepared a collagen/poly (lactide-co-caprolactone) nanofiber scaffold for the delivery of adipose-derived stem cell-derived exosomes (ADSC-exos). The measurement results revealed that the 50% concentration of the ADSC-exo nanofiber scaffold considerably improved the cell viability of fibroblasts, as evidenced by human foreskin fibroblasts (HFFs) and human urethral scar fibroblasts showing good biocompatibility with the ADSC-exo nanofiber scaffold. The reduced production of the inflammatory factors IL-6 and Col 1A1 indicated that ADSC-Exos had minimal cellular inflammatory effects. At four weeks postsurgery, the use of ADSC-exo nanofiber scaffolds for urethral repair showed no signs of US or scar formation. There was minimal collagen deposition, and multilayered epithelial cells were formed. Therapeutic stimulation with ADSC-exos facilitated epithelialization and vascular formation, promoting the transition from an inflammatory state to a regenerative state. The EV-SIS composite may combine the advantages of both exosomes and SIS. Exosomes contain various bioactive substances that aid in cell proliferation and repair, while SIS exhibits good biocompatibility and tissue scaffold function. The composite might activate the regenerative and repair processes in urethral tissue, improving urethral wall stability and elasticity and thereby enhancing urodynamic parameters. Moreover, implantation of the composite may help reduce immune and rejection reactions, thereby promoting the repair process. In addition, this study still has certain limitations. Firstly, although the experimental results showed that the composite material had good effects in urethral repair, its long-term efficacy and safety require further long-term follow-up research. Secondly, the experimental design did not consider other possible confounding factors that may affect the results, such as environmental changes and individual differences in the rabbit body. The future research focus should be on optimizing the preparation process of EV-SIS composite materials to improve their biological activity and tissue compatibility. In addition, larger scale animal experiments and clinical trials should be conducted to verify the repair effectiveness and safety of composite materials. Furthermore, it is possible to consider combining it with other biomaterials or growth factors to further improve the repair effect of composite materials.

Conclusion

In this experiment, EV-SIS composite materials were successfully prepared and applied to the rabbit US model. Through a series of experiments and evaluations, the performance of this composite material and its role in urethral repair were thoroughly studied. The results indicated that the EV-SIS composite material can effectively improve urodynamic parameters, including bladder capacity, maximum urethral pressure, and minimum urethral pressure. Compared to using foreign materials or SIS alone, composite materials exhibited more significant effects, demonstrating good biocompatibility and repair functions. The successful implantation of EV-SIS composite material in the rabbit US model demonstrates its potential clinical application value. Overall, the research and adoption of EV-SIS composite materials have brought new opportunities and challenges to the field of US repair, providing better clinical treatment options for US patients.

Footnotes

Author contributions

Dan Wang: Conceptualization, Project administration. Xiaojun Zhu and Buhe Siqin: Writing – original draft, Formal analysis. Chao Ren: Investigation, Resources. Ming Chang and Ligang Bai: Data curation, Writing – review & editing. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Funding

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The research was supported by the National Natural Science Foundation of China (No. 81960131) and the Affiliated Hospital of Inner Mongolia Medical Doctoral Initiation Fund program (No. NYFY BS 202103).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

Gelman

Furr

. Urethral stricture disease: evaluation of the male urethra. J Endourol 2020; 34: S2–S6.

Wessells

Morey

Souter

, et al. Urethral stricture disease guideline amendment. J Urol 2023; 210: 64–71.

Verla

Oosterlinck

Spinoit

, et al. A comprehensive review emphasizing anatomy, etiology, diagnosis, and treatment of male urethral stricture disease. BioMed Res Int 2019; 2019: 9046430.

Campbell

Vanni

Kowalik

. An update on female urethral stricture disease. Curr Urol Rep 2022; 23: 303–308.

Shi

Xue

, et al. Comparison of the diagnostic value of capsule endoscopy in two positions for esophageal lesions in the elderly. Acta Med Mediterr 2019; 35: 3389–3394.

Wang

, et al. Preparation and characterization of nano-laponite/PLGA composite scaffolds for urethra tissue engineering. Mol Biotechnol 2020; 62: 192–199.

Yang

Zhang

Fang

, et al. Urine-microenvironment-initiated composite hydrogel patch reconfiguration propels scarless memory repair and reinvigoration of the urethra. Adv Mater 2022; 34: e2109522.

Sterling

Schardein

Joshi

, et al. Long-term outcomes from a multi-institutional experience with prefabricated composite gracilis-buccal mucosal flap for reconstruction of devastated urethras. Int Urol Nephr 2022; 54: 1047–1052.

Zhou

, et al. Nano-carbon-based application of parecoxib sodium combined with hydromorphone in preventing anesthesia hyperalgesia caused by remifentanil after thyroidectomy. Cell Mol Biol 2022; 3: 68.

10.

Cunnane

Davis

Cunnane

, et al. Mechanical, compositional and morphological characterisation of the human male urethra for the development of a biomimetic tissue engineered urethral scaffold. Biomaterials 2021; 269: 120651.

11.

Rakhmatulina

Boldyreva

Lipova

, et al. Evaluation of the composition of the microbiota of the urethra in men with sexually transmitted infections. Urologiia 2019; 6: 31–37.

12.

Wang

Fei

, et al. microRNA-23B inhibits non-small cell lung cancer proliferation, invasion and migration via downregulation of RUNX2 and inhibition of Wnt/Β-catenin signaling pathway. J Biol Reg Homeos Agents 2020; 34: 825–835.

13.

Kim

Choi

Kim

. Mesenchymal stem cell-derived exosomes for effective cartilage tissue repair and treatment of osteoarthritis. Biotechnol J 2020; 15: e2000082.

14.

Fan

Liu

Chen

, et al. Exosomes-loaded electroconductive hydrogel synergistically promotes tissue repair after spinal cord injury via immunoregulation and enhancement of myelinated axon growth. Adv Sci (Weinh) 2022; 9: e2105586.

15.

Fitzgerald

Laurent

Funaro

, et al. Defining the role of T lymphocytes in the immunopathogenesis of neuromyelitis optica spectrum disorder. Discov Med 2020; 29: 91–102.

16.

Chen

Ning

Yin

, et al. Balloon-assisted endoscopic submucosal dissection for treating small intestinal lipomas: report of two cases. World J Clin Cases 2021; 9: 1631–1638.

17.

Burke

Lewis

Schreiter

, et al.

Is porcine small intestinal submucosal extracellular matrix (ECM) a suitable material for right ventricular outflow tract reconstruction in association with pulmonary valve replacement?

World J Pediat Congenit Heart Surg 2022; 13: 518–521.

18.

Totten

Manzoor

Yancey

, et al. Comparison of small intestinal submucosal graft and autologous tissue in prevention of CSF leak after posterior fossa craniotomy. J Neurol Surg Part B, Skull Base 2021; 82: 695–699.

19.

Mangir

Chapple

. Recent advances in treatment of urethral stricture disease in men. F1000Research 2020; 9: F1000 Faculty Rev-330(1–7).

20.

Mathieu

Névo

Jouve

, et al. Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. Nat Commun 2021; 12: 4389.

21.

Chen

Yang

, et al. The application of pulp tissue derived-exosomes in pulp regeneration: a novel cell-homing approach. Int J Nanomed 2022; 17: 465–476.

22.

Wang

Cheng

Zhu

, et al. Electrospun nanoyarn and exosomes of adipose-derived stem cells for urethral regeneration: evaluations in vitro and in vivo. Colloid Surface B-Biointerfaces 2022; 209: 112218.