Harnessing the regenerative effects of human amniotic stem cells (hAFSCs) on restoring erectile function in a bilateral cavernous nerve crush (BCNC) injury rat model

Abstract

Background

Intracavernous (IC) injections of stem cells has been shown to ameliorate cavernous nerve (CN)-induced erectile dysfunction (ED). However, the regenerative effects underlying the recovery of erectile function (EF) in human amniotic fluid-derived stem cells (hAFSCs) remain unclear. In the bilateral cavernous nerve crushing (BCNC) injury rat paradigm, we sought to ascertain the effects of hAFSC treatment on EF recovery during the incipient phase.

Methods

Three groups of 45 male rats were used in this study: sham (Group 1), saline IC injection after BCNC (Group 2), and hAFSC intracavernous injection (ICI) after BCNC (Group 3). hAFSCs from the fourth passage showed potential to differentiate into trilineage cells. All animals were subjected to EF analysis on the 28th day post-injection and tissues were retrieved for histopathological and immunohistochemical analyses.

Results

IC injections of hAFSC significantly improved EF parameters in BCNC-ED rats at 28 days post-injury. AFSC treatment enhanced the smooth muscle condition and increased the smooth muscle/collagen ratio, as evidenced by histological analysis. Immunohistology revealed increased expression of 𝛼-SMA andvWf in the corpus cavernosum and enhanced expression of nNOS in the dorsal penile nerve in BCNC-ED rats (p < 0.05). Western blotting showed that hAFSC treatment significantly increased α-SMA expression in the hAFSC group compared with that in the BCNC group. Electron microscopy revealed significantly elevated myelination in the CN (p < 0.05), maintenance of smooth muscle structures, and restoration of EF in BCNC-ED rats treated with hAFSC.

Discussion and conclusions

hAFSC treatment increased EF in BCNC-ED rats at a single dose. As BCNC-ED resembles ED caused by radical prostatectomy (RP), this therapy has high potential for ED patients after RP surgery.

Graphical Abstract

Introduction

Globally, prostate cancer (PC) is the second most common cancer type in men and sixth most common cancer type in Taiwan [1]. The preferred treatment for locally advanced PC in sexually active men is nerve-sparing radical prostatectomy (RP), and erectile dysfunction (ED), which is caused by cavernous nerve (CN) damage despite technical and anatomical advancements, continues to be a significant side effect of this procedure. ED is an intricate disorder characterized by a persistent inability to achieve and sustain erection strong enough to satisfy the sexual desire of a partner [2]. Following bilateral nerve-sparing radical prostatectomy, phosphodiesterase type 5 (PDE5) inhibitor medication is frequently used as first-line treatment for ED in ad hoc and erectile rehabilitation programs. However, these medications are generally ineffective because of restrictions on nerve regeneration [3].

An array of surgical and non-surgical treatments are available, ranging from oral PDE5 inhibitors and vacuum pumps to intraurethral medications, intracavernosal injections (ICI), penile prostheses, immunotherapy, and PRP therapy [4,5,6,7,8]. Despite their availability, none of these therapies can eradicate the underlying pathology, often resulting in adverse effects, complications, or a decline in sexual spontaneity, which are common reasons for increased rates of discontinuation and dissatisfaction.

In recent years, stem cell-based therapy has attracted attention as a potentially effective treatment for ED following CN injury because these stem cells can regenerate damaged penile neurovascular and endothelial tissues [9]. Several types of stem cell therapies have been investigated for their potential to improve erectile function, including mesenchymal stem cells (MSCs), adipose-derived stem cells (ADSCs), and hematopoietic stem cells (HSCs) [10,11,12]. In particular, human amniotic fluid-derived stem cells (hAFSCs) are a new and appealing source of stem cells and represent an attractive alternative to embryonic and adult stem cells because of their degree of plasticity, safety, ease of accessibility, and minor ethical concerns [13]. hAFSCs offer the potential for effective tissue repair and regeneration without ethical concerns associated with other stem cell sources.

These hAFSCs can be obtained at 14–18 weeks of gestation from amniotic fluid [14] and are generally multipotent because they can develop into non-mesodermal lineages in addition to mesoderm-derived lineages (bone, fat, cartilage, muscle, and hematopoietic) (endothelial, hepatic, and neuronal). The ability to produce progenitors from a variety of lineages and the lack of tumorigenicity in accessible primitive stem cell types make hAFSCs appealing candidates for regenerative medicine-based therapies for both congenital and acquired diseases. The exclusive mesenchymal nature of hAFSCs has clear implications for their prospective application in regenerative medicine. Remarkably, specific cells within the amniotic fluid exhibit the ability to activate Oct-4 and Rex-1 promoters, which are recognized markers of stem cells derived from the amniotic fluid. This finding provides additional support for the presence of stem cell populations within the amniotic fluid [15]. ICI of stem cells has been demonstrated to ameliorate CN injury-induced ED [16]. In the present study, we demonstrated the effects of hAFSC on CN regeneration and erectile function (EF) recovery in a bilateral cavernous nerve crush (BCNC) injury rat model by enhancing its potential translation into therapeutic opportunities.

Materials and methods

Culture, characterization, and hAFCS isolation from mesenchymal stem cells (MSCs)

In this study, hAFSCs were procured from U-Neuron Biomedical, Inc. (Hsinchu, Taiwan) and cultured in a commercial serum-free, chemically defined mesenchymal stem cell (MSC) medium at 37 °C in a humidified 5% CO₂ incubator. Upon reaching 80–90% confluency, the cells were detached using recombinant trypsin solution and subsequently replanted in the same culture medium. The biological characteristics of the AFSCs used in this study were examined by flow cytometry at passage 4. Phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated mouse anti-human monoclonal antibodies targeting CD44, CD73, CD90, CD105, HLA-ABC, Nestin, and Sox2, along with appropriate isotype controls, were used for this analysis. The cells were incubated with varying concentrations of primary or isotype control antibodies at 4 °C for 30–60 min, followed by analysis using a BD FACS flow cytometer (all antibodies were procured from BD Pharmingen). Flow cytometry analysis was conducted using a FACSCalibur instrument and the data were processed using CellQuest software (Becton Dickinson, NJ, USA).

Experimental animals

A total of 45 male 8-week-old Sprague‒Dawley rats were used in this study. The Fu Jen Catholic University Animal Care and Use Committee authorized the study (IACUC approval no: A10966; Dated: 2021/04/16), and the rats were procured from BioLasco Taiwan Co., Ltd. (Taipei, Taiwan). The study methodology and procedures adhered to the regulations outlined in the Declaration of Helsinki. All experimental protocols and reports were conducted in accordance with ARRIVE 2.0, ensuring transparency and reproducibility in the reporting of in vivo animal experiments.

Study design

All animals were equally separated into three groups (n = 15/group) and kept at the Fu Jen Catholic University animal house in standard cages at a temperature of 25 °C with a cycle of 12 h light and 12 h darkness in an aseptic environment and with an unlimited supply of food and water. All animals, including those in the sham group, underwent a laparotomy. In the BCNC group, the CNs were clamped for two minutes to eliminate the EF. No specific surgical manipulations were performed in the sham-operated group. Intracavernous pressure (ICP) was used to evaluate the EF. Immunohistochemistry and transmission electron microscopy (TEM) were performed on erectile tissues and CN nerves of each rat. The erectile tissues and CNs of the rats in each group were compared with respect to the EF.

Animal surgery procedure and measurement of erectile function

An intraperitoneal dose of sodium pentobarbital (40 mg/kg) was administered to anesthetize animals. Subsequently, the surgical site was completely shaved and sterilized with iodine-based solution, and a lower abdominal incision was made. During surgery, the prostate gland was initially identified before the CN and the major pelvic ganglia were located. Following CN separation using hemostatic forceps, the CN was crushed for 2 min on either side to induce BCNC injury. Furthermore, 2 × 10⁶/0.2 mL of hAFSC or vehicle (normal saline) was injected into the corpus cavernosum (CC), and a suture was made in the abdomen of each rat. For the measurement of EF, all animals were subjected to EF analysis to assess EF recovery four weeks before penile tissue collection. The EF measurements were similar to those reported previously [17, 18].

Briefly, penile nodules were located after opening the abdomen and the CNs were identified and isolated. A 24G needle containing 100 U/mL heparin solution was inserted into one side of the crus before being connected to polyester-50 tubing for ICP measurement using an MP 36 pressure transducer (Biopac System Inc., Goleta, CA, USA) and BSL software, version 3.7.3. A hook electrode made of bipolar stainless steel was used to stimulate the CNs distally from the crush injury site (each pole was 0.2 mm in diameter and was separated by 1 mm). A computer-controlled DS3 constant-current isolated stimulator was used to produce a monophasic rectangular pulse (AutoMate Scientific Inc., CA, USA). The stimulus amplitude was 7.5 mA, frequency was 20 Hz, pulse width was 0.2 ms, and duration was 60 s. The trial was performed in duplicate to ensure accuracy.

Histopathological analysis

For histological and immunohistological analyses, eight rats were randomly selected from each group and the analysis was duplicated for accuracy. An overdose of pentobarbital sodium solution via IP injection was used to euthanize the animals. Subsequently, tissues from the middle of the penis were removed, and fixed in 10% formaldehyde (v/v). Tissue samples were immersed in formaldehyde solution for 24 h. The materials were then carefully embedded in paraffin blocks for the microtomy procedure, dried, post-fixed, and cut into 5 mm-thick slices. The slices were then deparaffinized in preparation for staining and hydrated in ethanol solutions of varying concentrations (100%, 95%, 80%, and 70%) and double-distilled water. As mentioned in our earlier investigation, tissue samples were stained with hematoxylin and eosin.

Immunohistological analysis

After fixation with 10% formaldehyde for 24 h, the tissue from the middle part of the penis was dehydrated, post-fixed, and embedded. For three treatments, the embedded penile cross-sectional tissue was deparaffinized in xylene twice and then hydrated in a graded alcohol series. The slides were incubated for one hour at room temperature in blocking buffer (Sigma-Aldrich). Furthermore, the slides were treated for 1 h at room temperature with the following antibodies: mouse anti-neuron-specific 𝛽-III tubulin, rabbit anti-neuronal nitric oxide synthase (nNOS), and anti- 𝛼-smooth muscle actin (𝛼-SMA) (Abcam). The tissues were exposed to secondary antibodies conjugated with Alexa Fluor 488 or Texas Red (Invitrogen, Carlsbad, CA, USA) for 1 h following primary antibody incubation, and the results were analyzed using fluorescence microscopy. The ratio of the area of nNOS-positive cells to the 𝛽-III tubulin area of the neuron and nerve fibers and the 𝛼-smooth muscle actin area of the DPN group were computed at 400 and 100× magnification, respectively. We also investigated the smooth muscle cell (SMC) content. ImageJ software was used for all histomorphometric evaluations of the nerves (National Institutes of Health, Bethesda, MD, USA) The experiment was duplicated to attain accuracy.

Western blot analysis

Penile tissues were homogenized in cell lysis buffer containing phosphatase and protease inhibitors. Protein concentrations were determined using the Bradford assay. For each sample, 20 µg of the protein lysate was loaded onto a sodium dodecyl sulfate-polyacrylamide gel, separated, and transferred to a polyvinylidene difluoride membrane. After transfer, membranes were blocked and incubated with primary antibodies: anti-nNOS (1:1000, sc-648, Santa Cruz Biotechnology, Santa Cruz, California, USA), anti-eNOS (1:1000, sc-654, Santa Cruz Biotechnology, Santa Cruz, California, USA), anti-α-SMA (1:1000, 14395-I-AP, Proteintech, Rosemont, Illinois, USA), and β-actin (1:1000, 60008-1-Ig, Proteintech, Rosemont, Illinois, USA). Secondary antibodies conjugated to horseradish peroxidase were used for detection, visualized using the UVP Chemstudio Plus (Analytik Jena, Thuringia, Germany), and quantified with Image Lab 6.0 (Bio-Rad, Hercules, California, USA). The signal intensities were normalized to those of β-actin. Detailed methods are described in our previous study [19].

TEM analysis

Six animals were used for TEM studies, and the sample preparation for TEM was carried out according to our previous studies [20]. Penile tissues were sliced into small pieces and maintained in 2.5% phosphate-buffered glutaraldehyde overnight (0.1 M, pH 7.2). They were subsequently postfixed in 1% phosphate-buffered osmium tetroxide (0.1 M, pH 7.2). The samples were carefully embedded in Epon-812 after dehydration in various concentrations of ethanol. Toluidine blue was first used to stain 1 μm semithin slices. Lead citrate and uranyl acetate were used to stain extremely thin slices from the selected blocks. A JOEL JEM-1400 transmission electron microscope was used to observe each segment (JOEL, Tokyo, Japan). The TEM analysis was performed in duplicate for accuracy.

Statistical analysis

SPSS (version 14.0) statistical software was used for statistical analyses. One-way ANOVA was used to compare the means of different treatment groups, and Scheffe’s post hoc analysis was used to determine the results. Data are presented as mean ± standard error of the mean (SEM). All comparisons were deemed statistically significant if the p-value was less than 0.05 (*p < 0.05).

Results

Characterization of hAFSCs via Flow Cytometry

hAFSCs were characterized by flow cytometry after passage four. The cells expressed the pluripotent marker SOX2+ (91.9%), as well as the mesenchymal markers CD73+ (99.8%), CD44+ (99.5%), CD90+ (100%), CD105+ (99.3%), HLA-ABC+ (99.4%), and Nestin+ (100%). Notably, CD31, CD34, CD45, and HLA-DR were not expressed by these cells (data not shown). Moreover, these hAFSCs demonstrated trilineage differentiation potential and successfully differentiated into osteocytes, chondrocytes, and adipocytes under suitable culture conditions (Fig. 1A).

Fig. 1

Immunophenotyping and trilineage differentiation capability of hAFSCs. A) Flow cytometry results depicting the pluripotent and mesenchymal markers of hAFSCs. (B)hAFSC profiles exhibiting typical MSC characteristics of various origins, including osteocytes (Alizarin Red staining), adipocytes (Oil Red staining), and chondrocytes (arrows indicate acidic polysaccharides). *Passage 4 cells were used for the analysis

The hAFSCs demonstrated trilineage potential by differentiating into osteogenic, adipogenic, and chondrogenic lineages, as confirmed by positive staining for Alizarin Red, Oil Red, and Alcian blue after three weeks of cultivation in the appropriate induction media. Alizarin red staining revealed characteristic calcium deposits indicative of osteogenic differentiation, whereas control cells showed no such staining. Adipogenic differentiation was assessed by culturing cells at approximately 90% confluence for three weeks in adipogenic differentiation media, followed by the visualization of neutral lipid inclusions with Oil Red O staining. Successful differentiation into osteogenic and adipogenic lineages was confirmed by Alizarin Red and Oil Red O staining. Chondrocyte induction resulted in the accumulation of acidic polysaccharides (Fig. 1B).

hAFSC treatment restores the EF in BCNC-ED rats

In a recent study, we successfully demonstrated that ED consistently develops 7–28 days after BCNC damage in all animals [17]. The same procedure was used in this study. Electrical stimulation of the CNs and measurement of ICP were used as indicators of EF recovery. The effect of IC injection of hAFSCs on EF was shown in Fig. 2A & B. Generally, electrically stimulated CNs respond to the EF of the penis, as demonstrated by the ICP. The ICP in the BCNC group was lower than that in the sham control group. In contrast, hAFSC IC injection-treated rats showed increased ICP compared to the vehicle-treated BCNC group. The maximum ICP (64.55 ± 4.37 cmH₂O), delta ICP (max ICP – min ICP) (42.20 ± 4.81 cmH₂O), and area under the curve (AUC) (1947.71 ± 253.67 cmH₂O*sec) were significantly lower in the BCNC group than in the sham group. In contrast, these parameters (ICP – 93.51 ± 4.82 cmH₂O; ∆ICP − 61.41 ± 4.29 cmH₂O; AUC − 3942.37 ± 560.66 cmH₂O*sec) were significantly greater in the hAFSC group than in the BCNC group (Fig. 2A). The MAP of the BCNC vehicle-treated group (139.24 ± 4.87 cmH₂O) and BCNC treated with hAFSC (149.60 ± 3.73 cmH₂O) did not significantly differ from that of the sham group of rats (156.96 ± 2.83 cmH₂O). In addition, the maximum ICP/MAP of the BCNC vehicle-treated group (0.47 ± 0.03) was significantly lower than that of the sham group (0.78 ± 0.02). In contrast, the hAFSC-treated group (0.63 ± 0.03) showed a remarkably significant increase in value compared to the BCNC vehicle-treated group. Similar results were observed for the ∆ICP/MAP of the sham rats (0.58 ± 0.02), vehicle-treated BCNC rats (0.31 ± 0.04), and hAFSC-treated BCNC rats (0.41 ± 0.03) (Fig. 2B).

Fig. 2

2A and B: Comparison of EF among the sham, BCNC, and BCNC + hAFSC-treated groups. (A) Intracavernous pressure (ICP) (red) and blood pressure (BP) (blue) in sham, BCNC, and BCNC rats + hAFSC. Green bars indicate electrical stimulation for 60 s. (B) Quantitative results of EF parameters (MAP, ICP, and AUC) calculated for each group (sham rats, BCNC rats, and BCNC rats + hAFSC). (n = 15). * p < 0.05, compared to sham rats; # p < 0.05, compared to BCNC rats. (AUC, area under the curve; ICP, intracavernous pressure; MAP, mean arterial pressure)

Protective effects of hAFSCs on preserving the smooth muscle content and collagen ratio in the CC

H&E staining revealed that the corporal smooth muscle had the worst condition with loosely packed smooth muscle content, as its intact structure was lacking in BCNC rats. In addition, BCNC rats treated with hAFSCs exhibited tightly arranged tissue content, similar to that of the sham group (Fig. 3A). Masson’s trichrome staining revealed a smooth muscle-to-collagen ratio in the corpus cavernosal tissue; the penile smooth muscle/collagen structure was disrupted in the BCNC group, and a large amount of extracellular matrix was deposited. In the present study, the BCNC rats treated with vehicle exhibited a significant decrease in the smooth muscle cells (area)/collagen (area) (%) ratio (0.18 ± 0.01) compared with the sham group of rats (0.28 ± 0.01 smooth muscle cells (area)/collagen (area) (%)). In contrast, the hAFSC-treated group exhibited an increase in the smooth muscle and collagen ratio (0.21 ± 0.02 smooth muscle cells (area)/collagen (area) (%)) compared with the BCNC rats treated with vehicle only (Fig. 3B).

Fig. 3

(A) Micrograph showing hematoxylin and eosin and Masson’s trichrome staining of the corpus cavernosum (40× magnification). H&E staining revealed varying degrees of damage and poor tissue arrangements exclusively in the BCNC group (blue demarcated area), whereas no similar damage was observed in BCNC rats treated with hAFSC. The smooth muscle/collagen ratio was determined using Masson’s trichrome-stained corpus cavernosum (green demarcated area). (B) Quantification of the smooth muscle/collagen ratio in the sham, BCNC, and BCNC rats treated with hAFSC. BCNC refers to bilateral cavernous nerve crush injury and hAFSC denotes human amniotic fluid stem cells. * Indicates a significant difference from the sham rats (p < 0.05) and ^# indicates a significant difference from the BCNC rats (p < 0.05). For each group, 8 animals were used in this study (n = 8)

IC hAFSC treatment recovers the CN status in BCNC rats

According to the TEM results, a significant decrease in mature and well-structured myelinated nerve axons was observed in the BCNC vehicle-treated group compared to that in the sham group. In addition, the BCNC exhibited a significant increase in myelinated axons after hAFSC treatment (Fig. 4A). Semi-quantification analysis also revealed significant differences between the hAFSC-treated (52.6%) and vehicle-treated (6.2%) BCNC groups (Fig. 4B). This indicates that hAFSC treatment substantially increased the content of myelinated axons after BCNC.

Fig. 4

(A) Transmission electron microscopy of the cavernous nerve (CN) in sham rats, BCNC rats, and BCNC rats treated with hAFSC magnified 2500×. The image illustrates an increased number of myelinated sheaths (indicated by red arrows) and demyelinated nerve axons (indicated by green arrows) observed in BCNC rats treated with hAFSC compared to the vehicle-treated BCNC group. The scale shown in Fig. 3A is 5 μm. (B) Graph showing statistical results of the number of myelin sheaths in each group of rats. BCNC refers to bilateral cavernous nerve crush injury and hAFSC denotes human amniotic fluid stem cells. * Indicates a significant difference from the sham rats (p < 0.05) and ^# indicates a significant difference from the BCNC rats (p < 0.05). Six rats from each group were used for this study (n = 6)

The neuroprotective effects of hAFSCs preserve nNOS-positive nerve fibers in BCNC rats

The dorsal penile nerve (DPN) has been demonstrated to interact and communicate with the cavernous nerve. The DPN was immunostained for β-III-tubulin to identify nerve fibers positive for neuronal nitric oxide synthase (nNOS) and quantify their nNOS content in the sample (Fig. 5). In the post-injury episode, the expression of nNOS in DPN was lost; however, no significant changes were observed in the expression of β-III-tubulin after BCNC (Fig. 5A). Semi-quantitative analysis revealed that the percentage of BCNC-treated rats was significantly lower than that of sham- and hAFSC-treated rats (p < 0.05). In addition, the ratio of nNOS/β-III-tubulin was also significantly lower in the BCNC group of rats (0.32 ± 0.02 nNOS (area)/𝛽-III-tubulin (area) (%) than in the sham group of rats (0.50 ± 0.01) nNOS (area)/𝛽-III-tubulin (area) (%) (p < 0.05). However, there was a significant increase in the ratio of nNOS/β-III-tubulin in the hAFSC-treated group (0.44 ± 0.001) nNOS (area)/𝛽-III -tubulin (area) (%) (p < 0.05) compared with that in the BCNC group (Fig. 5B).

Fig. 5

(A) The illustration depicts Immunofluorescence staining of control, nNOS in the DPN of sham rats, BCNC rats, and BCNC rats treated with hAFSC 28 days post-BCNC. Representative images of the control and experimental groups are shown (nNOS in green, β-III tubulin in red) with an original magnification of 400x. nNOS-positive nerve fibers in DPN were quantified as the area of nNOS-positive nerve fibers/β-III tubulin. (B) Quantitative analysis revealed a decrease in the number of nNOS-positive nerve fibers in the BCNC vehicle-treated group compared to the sham and BCNC groups treated with hAFSC. * Indicates a significant difference from the sham rats (p < 0.05), and ^# indicates a significant difference from the BCNC rats (p < 0.05). For each group, eight rats were used in this study (n = 8)

Potential rescue effect of hAFSC on EF is caused by preservation of corporal smooth muscle in BCNC rats

The condition of the CC was investigated by 𝛼-SMA staining to measure the SMC structure (Fig. 6A). The underlying assessment after the BCNC injury revealed smooth muscle atrophy and potential CC fibrosis. α-SMA expression was diminished in vehicle-treated BCNC rats, and semiquantitative results confirmed this finding (0.12 ± 0.05). However, the BCNC group of rats treated with hAFSC showed a significantly greater increase in 𝛼-SMA expression (p < 0.05) than the BCNC vehicle-treated group of rats (0.15 ± 0.02) (Fig. 6B). Additionally, as shown in Fig. 6A, BCNC vehicle-treated rats had significantly fewer cells (0.06 ± 0.01) expressing the endothelial marker Von Willebrand factor (vWF) than did rodents treated with hAFSCs (0.09 ± 0.01) (p < 0.05) (Fig. 6B).

Fig. 6

(A) illustrates the expression of 𝛼-smooth muscle actin (𝛼-SMA) and Von Willebrand Factor (vWF) in assessing the endothelial contents in the corpus cavernosum by immuno- histological analysis in normal control, BCNC and the BCNC + hAFSC group of rats. The immuno-histopathological images indicate the expressions of 𝛼-SMA in red colour and the expression of vWF in green fluorescent colour. (B) Semi-quantification analysis of the expression of 𝛼-SMA and vWF in the corpus cavernosum of normal control, BCNC, and BCNC + hAFSC group of rats. *p < 0.05 – compared with normal sham rats; ^#p < 0.05 – compared with BCNC rats. In each group, eight rats were used in the study (n = 8)

Fig. 7

(A) Western blot analysis of (the middle section of the corpus cavernosum tissue) was used to evaluate the protein expression of nNOS, eNOS, and α-SMA across different groups, with β-actin serving as the loading control. (B) Quantification of nNOS expression by Western blot analysis showed an increase in the BCNC + hAFSC group and a low level in the BCNC group. Compared with the sham group, both the BCNC + hAFSC and BCNC groups were lower. (C) A similar pattern was observed for eNOS expression, an increase in the BCNC + hAFSC group, a low level in the BCNC group, and low levels in both the BCNC + hAFSC and BCNC groups compared to the sham group. (D) A significantly increased level of α-SMA was observed in the BCNC + hAFSC group compared to the BCNC and sham groups. level of significance: *p < 0.05 - compared to sham rats; ^#p < 0.05 – compared to BCNC rats. n = 4 animals per group for Western blot analysis

Histological evaluation and subsequent western blot analysis for nNOS and eNOS were conducted on the middle section of the corpus cavernosum tissue from four rats per group (n = 4). The analysis revealed significantly higher nNOS and eNOS expression in the hAFSC group compared to the BCNC group (0.48 ± 0.15 vs. 0.27 ± 0.11; 0.94 ± 0.38 vs. 0.30 ± 0.06). However, there was no significant difference in nNOS and eNOS expression across all the groups (Fig. 7A and C). Western blot analysis of α-SMA (Fig. 7A and D) was performed on 3–5 samples per group. The BCNC group exhibited a significant decrease in α-SMA expression relative to the sham group (0.82 ± 0.28 vs. 1.91 ± 0.16; p < 0.05). The hAFSC-treated group showed a significant increase in α-SMA expression compared with in that the BCNC group. Additionally, α-SMA expression in the hAFSC-treated group was significantly higher than in the sham group (2.88 ± 0.32 vs. 1.91 ± 0.16; p < 0.05). As a result of the hAFSC IC injection, there was a notable increase in the sinusoids of the CC in BCNC rats.

IC hAFSC treatment improves the smooth muscle structure of the CC in post-BCNC injury rats

Ultrastructural analysis of the CC was performed using TEM (Fig. 8), and the results revealed that the sham group showed no discernible alterations in the adherent junctions of the SMC and exhibited tightly packed muscle content that was intact with collagen. The endothelial layer was visible without any damage. In the BCNC vehicle-treated rats, the muscle contents were loosely packed with a large gap between the collagen layer and the CC. Furthermore, adherent junctions were disordered and not distinctly visible. The endothelial layer is severely disrupted. However, in BCNC rats treated with hAFSCs, the smooth muscle contents were tightly packed and had proper adherent junctions with a distinct endothelial layer similar to those in the sham group. In addition, a few hAFSCs fused with SMCs were observed. These observations suggest that hAFSC treatment potentially improved the smooth muscle structure of the CC in BCNC-ED rats.

Fig. 8

Transmission electron microscopy image of the corporal smooth muscle in sham rats, BCNC rats, and BCNC rats treated with hAFSC at a magnification of 2500×. Sham control rats showed normal structures, characterized by intact adherent junctions and preserved smooth muscle cells. BCNC rats exhibited abnormal endothelial layer structures (indicated by red arrows) with disrupted adherent junctions and chasm between the smooth muscle layers (indicated by red double-sided arrows). In contrast, BCNC rats treated with hAFSC displayed tightly packed normal smooth muscle structures with well-defined adherent junctions. The yellow demarcated area represents the integration of hAFSC into the smooth muscle layer. Six rats were used for each group (n = 6)

Discussions

This study highlights the beneficial effects of hAFSC IC injection therapy for restoring EF after BCNC injury. The effective mechanisms of hAFSC include enhancing nerve repair, preventing apoptosis in the corpora cavernosa, and promoting hAFSC differentiation. The neuroprotective benefits were accompanied by restored EF, increased number of nNOS-positive nerve fibers, normal morphological appearance in the DPN, and regeneration of myelinated axons 28 days post-injury. Furthermore, hAFSC injection demonstrated tissue-protective effects by increasing smooth muscle cell and endothelium composition in the corpora cavernosa sinusoids. The significant increase in smooth muscle cell numbers in the corpora cavernosa after hAFSC treatment indicates the potential of hAFSCs for in vivo transformation and differentiation.

The effectiveness of stem cells in animal ED investigations has been demonstrated in numerous studies [21, 22]. This study aimed to demonstrate the effectiveness of hAFSCs in a BCNC damage model of ED and to clarify the processes through which this cell-based regenerative therapy works. The present study shows in detail how hAFSCs exert their effects in a BCNC-ED model. The occurrence of ED after BCNC differs significantly because of its heterogeneity, making it highly challenging to evaluate the mechanism of ED and determine therapeutic measures and optimal treatment timing when using this model [23]. ADSCs, bone marrow-derived stem cells (BMDS), and muscle-derived stem cells are three different stem cell types that have been studied in ED. Stem cell therapy can restore EF by enhancing intracavernosal pressure/MAP ratio. Furthermore, bone marrow mononuclear cells and the adipose-derived stromal vascular fraction, which are uncultured stem cells, appear to be less effective than cultured stem cells in enhancing EF [24].

Several mesenchymal markers have been used to confirm pluripotency of hAFSCs. Moreover, a significant proportion of hAFSCs, ranging from 90 to 99% for CD90 and 85% for CD44, express mesenchymal stem cell surface markers, indicating a high level of multipotency. Flow cytometry analysis further revealed the absence of the hematopoietic markers CD31 and CD45 in these cells. hAFSCs have demonstrated potential as precursors to various differentiated cell lineages [25], as demonstrated by their successful differentiation into osteoblasts, adipocytes, and chondrocytes in vitro. This acquisition of lineage-specific functionality suggests its versatility in cellular differentiation. The observed differences in pluripotency among hAFSCs, leading to trilineage differentiation, highlight the influence of dynamic microenvironments or stem cell niches on cellular behavior. This underscores the importance of considering anatomical origins and molecular responses to regeneration capacity when evaluating tissue-forming potential [26].

The electrical stimulation-induced ICP response is currently the most commonly used indicator of EF, and it represents hemodynamic alterations related to the erectile response. The following factors are the fundamental ICP parameters and their implementation in EF measurement, and the amount of time the CN needs to take after electrical stimulation to reach its maximal ICP has been used to quantify the CN response quality. The maximal ICP has been used to assess the capacity of the cavernous body in penile venococclusions. The AUC was used to evaluate the capacity to maintain erection after continuous electrical stimulation. Blood pressure fluctuations in individual rats and the impact of blood pressure suppression after anesthesia can be decreased using the carotid arterial pressure of each rat to correct ICP [1]. Overall, all selected ICP studies reported similar functional outcomes in terms of EF enhancement following intervention. This progress was typically measured using the penile ICP/MAP ratio in animal studies, with increases ranging from 2- to 3-fold compared with the control group. In this study, the ICP and ICP/MAP ratios of vehicle-treated BCNC rats were generally lower than those of sham control rats. Additionally, rats in the BCNC group treated with hAFSC exhibited a significant increase in both ICP and ICP/MAP. This result clearly demonstrates that hAFSC can improve the EF. Generally, the control of erection is influenced by nerve control, sinusoid relaxation, arterial dilation, and venous compression, which are the results of various events [27]. hAFSC can increase the intracavernosal pressure of the corpus cavernosum, suggesting that the therapeutic properties of hAFSCs might repair CC tissue and nerve damage, and restore proper blood flow in BCNC-induced ED rats [13].

The corporal smooth muscle plays a critical role in the erectile process by increasing arterial inflow and restricting venous outflow from the corpora when relaxed [24]. Healthy cavernosal tissue with normal erectile function relies on flawless sinusoids with a standard microvascular framework. The CC consists of SMCs, fibrous connective tissue matrix, blood vessels, vascular lacunae, and numerous non-myelinated and pre-terminal autonomic neurons. In addition, intracavernous injections of both autologous and allogeneic mesenchymal stem cells have been shown to restore EF in rats with cavernous nerve injury by improving the mean maximum intracavernosal pressure in animal models [28]. In general, the smooth muscle, the most abundant component, attaches to the fibrous skeleton of the corpora cavernosa and is arranged in bundles in all directions. The fibrous skeleton, mainly composed of collagenous fibers, includes the tunica albuginea, its columns, and sheaths of perineural/periarterial fibrous and intracavernosal fibrous frameworks [29]. Smooth muscles are essential for erection by controlling blood flow in and out of the sinusoids. In this study, the smooth muscle/collagen ratio was decreased in the BCNC vehicle-treated group, resulting in severe ED. Injury to the penile cavernous nerve and vascular system decreases the perfusion of the cavernous artery and causes tissue hypoxia. Chronic hypoxia damages the cavernous endothelium and smooth muscle cells, and induces fibroblasts to produce extracellular matrix, leading to tissue fibrosis and worsening hypoxia. A decreased smooth muscle/collagen ratio indicates significant extracellular matrix deposition due to BCNC-induced damage to cellular structural integrity, potentially causing endoplasmic reticulum enlargement, basement membrane rupture, or mitochondrial swelling [30]. However, the BCNC rats treated with hAFSC exhibited an increased smooth muscle/collagen ratio. Recent research has demonstrated that hAFSCs enhance collagen content at injury sites, aiding healing [31]. Additionally, embryonic lineage stem cells, which can become multipotent mesenchymal stem cells, have been ubiquitously identified in living systems. These stem cells support local tissue growth and regeneration and exert positive effects in a paracrine-dependent manner [28]. In this study, hAFSCs were shown to enhance the smooth muscle cell-to-collagen ratio in the corpus cavernosum, which is crucial for maintaining erectile function. This effect is achieved by differentiating hAFSCs into smooth muscle cells and secreting factors that promote smooth muscle cell proliferation [32]. Our results demonstrate that hAFSCs promote cavernosal smooth muscle cell proliferation and limit collagen deposition through the dual mechanisms of paracrine effects and cell differentiation.

The MPG, a primary reservoir of NOS-positive nerve fibers in penile erectile tissue, is characterized by a high density of NOS-positive neurons. NOS, an enzyme crucial for NO production and erectile function, has been found in rat penile neurons that innervate the CC and in the neuronal plexuses of the penile arterial adventitia [17]. DPN interacts with CN, highlighting the importance of analyzing nNOS in the penile tissues of patients with diabetic peripheral neuropathy to understand the mechanisms through which CN damage causes ED. In this study, preservation of nNOS-positive neurons in the MPG, which is vital for neuronal development and survival, was used to illustrate the neuroregenerative effects of hAFSCs following IC injection. As expected, the number of nNOS fibers in the DPN decreased in the BCNC group owing to cavernous nerve damage and reduced relaxation ability of the CC smooth muscle. Prolonged ischemia and hypoxia in the penile cavernous body may also damage the CC smooth muscle and endothelial cells [33]. Our findings indicate that the number of NOS-positive neurons increased in the DPN of the BCNC group receiving hAFSC IC injections compared to that in the vehicle-treated group. This increase in nNOS-positive nerve fibers in the hAFSC-treated group strongly suggests the neuroprotective benefits of hAFSC treatment [34]. This study confirmed that hAFSCs facilitate penile erection by preserving neuronal nitric oxide synthase and enhancing NO synthesis in the corpus cavernosum.

The CC extracellular matrix is necessary for the normal erection process, and the corporal smooth muscle is a major target of NO action in the corpus cavernosum and has been associated with many forms of ED. Apoptosis of the smooth muscle in the corpus cavenosum leads to veno-occlusive dysfunction [35]. As shown in Fig. 6A, the number of 𝛼-SMA-positive cells in the BCNC group was significantly lower than that in the sham control group. In contrast, the BCNC group of rats treated with hAFSCs exhibited a significantly increased number of 𝛼-SMA-positive cells, similar to the sham group of rats. These findings suggest that hAFSCs may be essential for preventing CC smooth muscle atrophy. These findings were confirmed by determining the collagen/muscle ratio in the CC by Masson’s trichrome staining. Additionally, Western blotting revealed that hAFSC treatment significantly enhanced smooth muscle actin expression compared to that in the sham group. This finding aligns with the results of studies on adipose-derived stem cell differentiation [36]. A likely explanation is that hAFSCs target the cavernous tissue and differentiate into corporal smooth muscle cells. These differentiated cells may play a critical role in the regeneration of corporal smooth muscle and improvement of erectile function.

Under physiological circumstances, the vascular endothelium produces a variety of substances that are directly linked to hemostasis, fibrinolysis, the production of growth factors, and the control of vessel tone and permeability. vWF is produced and stored in the endothelial cells. These vWf levels have been suggested as potential biomarkers of endothelial dysfunction [37]. After surgery, immunofluorescence examination revealed that the endothelial content in the CC of BCNC rats was significantly reduced. After hAFSC administration, the BCNC group exhibited significantly increased vWf expression in CC. The elevated vWf expression in the BCNC hAFSC rats suggests that IC injections of hAFSC might have promoted regional vascularization by initiating vascular endothelial regeneration and restoring EF through the endogenous nitric oxide pathway. A similar study by Songet al. [38]. revealed that a combination of autologous stromal vascular fraction and adipose-derived stem cells promotes endothelial regeneration and restores EF. In this study, we demonstrated the endothelial protective effects of intracavernosal injection of hAFSCs following BCNC injury.

According to the TEM analysis, BCNC rats had significantly more demyelination of the CN following clamping than sham rats. Demyelination is a core indication of irreversible transformation in erectile tissue, which may result in irreversible ED [39]. In this study, BCNC rats that underwent hAFSC treatment exhibited significantly less demyelination than those treated with vehicle alone. This demonstrates the neuroprotective properties of the hAFSCs. To support this finding, Shanet al. [40]. reported that high concentrations of growth factors, including BDNF and NGF, play a vital role in stimulating nerve regeneration and that the recovery of EF is contingent upon successful nerve regeneration and suppression of cell apoptosis in the cavernous tissue. In addition, the ability of stem cells to differentiate into neuronal or glial cells, promote axonal growth, and regenerate myelin is the main factor in selecting a particular type of stem cell. A recent study revealed that hAFSCs greatly improve bladder function by promoting myelin regeneration during neural development [41]. In the present study, the ultrastructural findings of the CN and CC collectively revealed the potential of hAFSC treatment to increase the content of myelinated axons in the CN and to maintain the intact smooth muscle structure of the CC after BCNC injury.

Adherent junctions are a set of interrelated polypeptides found in the ‒ junctions of diverse cell types, including epithelial, endothelial, and muscle cells. They are crucial for intercellular tissue connection, mechanotransduction, and smooth muscle contraction. The multipotent nature of hAFSCs has been shown to allow the formation of adherent junctions [18, 42]. TEM further revealed that IC injection of hAFSCs preserved the adherens junction structure of the smooth muscles, which is essential for smooth muscle contraction. Smooth muscle contraction is a key phenomenon in the EF process [43]. Hence, by restoring smooth muscle contraction in the CC, hAFSC re-established EF in the BCNC rats. Notably, a small number of hAFSCs integrated into the smooth muscle layer enriched intracellular tissue connections and reestablished the smooth muscle structure. This observation reinforces our study as hAFSC are known to differentiate into diverse cell and tissue types.

Limitations and prospects

The current study has certain limitations. First, the rats were observed for only four weeks during the research period. A longer investigation might produce additional findings in new dimensions, particularly considering the possibility of observational studies on hAFSC transdifferentiation. Such a study would enable the examination of both functional and histomorphometric changes over a longer period and address a wide range of issues. Furthermore, owing to the short study duration, it was not possible to determine the effects of hAFSCs on comorbid conditions of ED, such as hypertension and hyperlipidemia. Finally, the rats received a single dose of the injection; however, it may be necessary to augment further doses to determine the additional effects of successive treatments. It would be more advantageous to assess the viability and repeated usage conditions of frozen hAFSCs and grow them by culturing if repeated injections were demonstrated to have further effects. Although pharmacological alterations of stem cells have not yet been attempted for the treatment of ED, they may influence the capacity for self-renewal and extend the therapeutic effect of these cells in the future. The optimal procedure for the cellular therapy of ED following CN injury is still being determined in additional studies, such as western blotting. Nevertheless, our study is the first to demonstrate EF using hAFSC therapies in a rat model of BCNC-induced ED. These results implied that intracavernous injection of hAFSCs is a promising therapeutic strategy for the treatment of BCNC-induced ED.

Conclusions

BCNC injury in rats results in severe ED in response to significant damage to CN. hAFSC administration effectively restored EF by increasing blood flow and angiogenesis in the CC. In addition, hAFSC therapy significantly decreased smooth muscle atrophy, elevated the smooth muscle/collagen ratio, and increased the expression of 𝛼-smooth muscle actin (α-SMA) and endothelial factor (vWF) in the CC. The neuroprotective benefits of hAFSCs were demonstrated by an increase in the number of nNOS-positive fibers in the DPN group and by an increase in the number of myeline-rich axons in the CN group. Finally, hAFSC treatment significantly maintained SMC content while preserving the integrity of adherens junctions in the CC and faultlessly restored EF in BCNC-induced ED rats. As indicated in this study, this hAFSC therapeutic approach successfully treats BCNC-induced ED, which is comparable to RP-induced ED. Thus, this therapeutic approach has strong potential for patients with RP-induced ED with appropriate improvements.