The impact of absorbable hemostatic agents on wound healing in an experimental penile fracture rat model

Abstract

Objective

This study aimed to evaluate the effects of two absorbable hemostatic agents, oxidized regenerated cellulose (ORC) and gelatin sponge, on wound healing in a rat model of penile fracture.

Materials and methods

A total of 32 Wistar albino rats were divided into four groups: Control (C), Primary Suturing (PS), ORC, and Gelatin Sponge (GS). A penile fracture model was created in all rats, and wound healing was assessed histopathologically after two weeks. Key parameters assessed included primary healing, fibrosis, inflammation, and cavernous tissue healing.

Results

The ORC group showed significantly higher primary healing rates (100%) compared to the other groups (p < 0.01). Fibrosis was more common in the GS group (87.5%), whereas no fibrosis was observed in the ORC group (p < 0.01). In terms of cavernous tissue healing, the group treated with the ORC absorbable hemostatic agent demonstrated significantly higher healing rates compared to the control group (p = 0.000). No significant differences were observed between groups regarding inflammation.

Conclusion

ORC absorbable hemostatic agents significantly promote primary wound healing and reduce fibrosis in an experimental penile fracture model, whereas the gelatin sponge was associated with increased fibrosis and did not improve healing. These findings suggest that ORC may have potential clinical applications in penile fracture repair. Further clinical studies are necessary to validate these results.

Introduction

Penile fracture is a urological emergency characterized by rupture of the cavernosal tunica albuginea, usually resulting from trauma to an erect penis, most often during sexual intercourse. Although it is reported in the literature that sexual intercourse accounts for the majority of cases (77%), trauma during erection or sudden reverse movements during masturbation can also lead to penile fracture [1]. While penile fracture can occur in any sexual position, the basic mechanism involves the penis failing to re-enter the vagina properly after exiting, leading to sudden bending and high-pressure trauma [2]. Patients typically hear a popping sound, followed by rapid loss of erection, swelling, and subcutaneous ecchymosis [3]. Ecchymosis may extend to the anterior abdominal wall and scrotum, depending on the severity and location of the rupture. Approximately one-fifth of patients with penile fractures also experience urethral injury [4]. The recommended treatment approach is immediate surgical exploration and repair within the first 24 h; however, surgery should not be ruled out even for patients presenting later [5]. While complication rates are between 4 and 12% for early surgical repair, they are significantly higher for patients treated conservatively or with delayed surgery [2]. Some studies report complication rates as high as 80% in non-surgically managed penile fracture cases [6]. These complications include penile curvature, abscess formation, non-healing hematomas, erectile dysfunction, urethral stricture, and fistula formation due to missed urethral injury [2]. The high complication rate with non-surgical management and delayed surgery underscores the importance of early intervention [7, 8]. Postoperative fibrosis can lead to penile curvature, erectile dysfunction, and pain during intercourse [6]. Therefore, strategies that reduce fibrosis and promote primary healing are critical to the successful surgical treatment of penile fractures.

Absorbable hemostatic agents are specialized biomaterials designed to control bleeding when traditional methods such as cauterization, ligation, or suturing are insufficient. These agents are absorbed by the body over time and offer several advantages during surgery, including reducing blood loss, minimizing the time spent on hemostasis, controlling bleeding in difficult-to-repair parenchymal organs, and reducing the need for blood transfusions [9]. Although many different types of absorbable hemostatic agents have been described in the literature, the most well-known are oxidized regenerative cellulose (ORC)-based, collagen-based, gelatin-based, and fibrin-based sealants [10]. ORC-based absorbable hemostats, such as Surgicel™, are derived from wood pulp cellulose [9]. They promote hemostasis by using cellulosic acid, which has a strong affinity for hemoglobin, to form an artificial clot [11]. Spongostan™ is an absorbable, gelatin-based hemostatic agent that stops bleeding by rapidly absorbing blood and initiating the coagulation process by trapping platelets [10]. It helps stop bleeding by providing a mechanical buffer effect in the area where it is applied. It creates a mechanical barrier in the applied area and is fully absorbed by the body within 4–6 weeks [10]. This agent promotes wound healing and reduces infection risk with minimal tissue reaction [10].

This study aims to evaluate the effects of two widely used absorbable hemostatic agents, ORC absorbable hemostat (Surgicel™) and gelatin-based absorbable hemostatic sponge (Spongostan™), on wound healing in an experimental penile fracture rat model.

Materials and methods

Ethical approval

After the initial planning phase, an application was submitted for ethics committee approval. The study was initiated following the necessary permissions from the Local Ethics Committee (Ethical approval number: 16.04.2021/0065/660).

Experimental procedure

Animal groups

This study included 32 Wistar Albino rats, weighing between 220 and 250 g, obtained from the Ankara Training and Research Hospital Experimental Animals Laboratory. The same surgical team performed all surgical procedures consecutively under sterile conditions, using a surgical loop with 2.5X magnification. The rats were randomly divided into four groups of 8 each: Group C (Control), Group PS (Primary Suturing), Group ORC, and Group GS (Gelatin Sponge).

Preoperative preparation

Ceftriaxone (Desefin^® 1gr, Deva Holding Company, Türkiye) was administered as a prophylactic antibiotic. Following a 12-hour fasting period, a single dose of 20 mg/kg was given intramuscularly to the rats 1 h before surgery. General anesthesia was induced by intramuscular injection of 50 mg/kg ketamine (KETALAR^® 500 mg/10 ml Solution for Injection, Pfizer PFE Drugs Company, Türkiye) 15 min before the surgical procedure.

Creating the penile fracture model

Once adequate anesthesia was achieved, the rats were placed in the supine position on the surgical field. The genital area was carefully shaved to avoid skin injury, and the penis was cleansed with 4% chlorhexidine digluconate solution (Dermanios Scrub Chlorhexidine^®, Laboratoires Anios, France). A penile fracture model was created by making a single incision on the proximal dorsal part of the penis using a size 15 scalpel (Fig. 1a).

Fig. 1

Surgical procedures in the experimental penile fracture rat model. a Creation of the penile fracture with a single incision on the proximal dorsal penis using a size 15 scalpel. b Group PS (Primary Suturing). The tunica albuginea was sutured with 5 − 0 polyglactin (Vicryl^®). c Group GS (Gelatin Sponge). After suturing, a gelatin sponge (SPONGOSTAN™) was placed on the fracture site. d Group ORC (Oxidized Regenerated Cellulose). After suturing, an ORC absorbable hemostat (SURGICEL™) was placed on the fracture site

Surgical procedures

Group C (n = 8)

The tunical incision was left unrepaired, allowing for secondary healing.

Group PS (n = 8)

The tunical incision was closed with primary suturing using 5 − 0 polyglactin suture (Vicryl^® Polyglactin 910 Suture, Ethicon, USA) (Fig. 1b).

Group GS (n = 8)

After primary suturing with 5 − 0 polyglactin, an absorbable gelatin-based hemostatic sponge (SPONGOSTAN™ Absorbable Gelatin Sponge, Ethicon, USA) was applied to the fracture site (Fig. 1c).

Group ORC (n = 8)

After primary suturing with 5 − 0 polyglactin, an ORC absorbable hemostat (SURGICEL™ Original Absorbable Hemostat, Ethicon, USA) was applied to the fracture site (Fig. 1d).

Postoperative care

Once the rats awoke from general anesthesia, they were housed individually in a controlled environment maintained at 22–24 °C with 50–55% humidity and a 12/12-hour light/dark cycle. All groups were provided ad libitum access to food and water.

Sacrification and sample collection

After a two-week postoperative follow-up period, the rats were sacrificed using an overdose of pentobarbital sodium (100 mg/kg intraperitoneally). Penectomy materials were excised from the proximal portion of the penis to include the entire fracture site. These specimens were fixed in 10% formalin and individually labeled.

Histopathological evaluation of Penile tissues

The penile tissues were fixed in 10% formalin, sectioned into 4 mm thick slices, and embedded in paraffin. Sections 4 microns thick were cut using a microtome. The tissues were stained with hematoxylin-eosin (HE) and Masson trichrome, and slides were prepared for microscopic examination. The samples were numbered to ensure blinding and examined by an experienced pathologist using white light microscopy (Olympus^® Microscope, Olympus^® DP72 Camera, Tokyo, Japan). Parameters assessed included primary tissue healing (yes/no), degree of cavernous tissue healing (Grade 0–4), fibrosis development (yes/no), and inflammation (yes/no).

Statistical analysis

Data analysis was performed using SPSS^® (version 22 for Windows^®, USA). Categorical variables were expressed as frequencies and percentages. Fisher’s exact test was used for group comparisons, with p < 0.05 considered statistically significant.

Results

No mortality occurred during the experiment period among the 32 animals across the 4 groups of 8 rats each. No major complications were observed that would affect the results. The efficacy of the methods used in the study was evaluated through histopathological examination of penile tissue samples, which were assessed by an experienced pathologist. Examples of microscopic images from the tissue examinations are provided (Fig. 2).

Fig. 2

Histopathological images of penile tissue samples from each experimental group, stained with Hematoxylin and Eosin (HE) at different magnifications. a Group C (Control): HE stain, 40x magnification, showing fibrosis in the tissue. b Group PS (Primary Suturing): HE stain, 200x magnification, showing fibrosis in the tissue after primary suturing of the tunica albuginea. c Group GS (Gelatin Sponge): HE stain, 200x magnification, showing significant fibrosis after the application of absorbable gelatin sponge (Spongostan™). d Group ORC (Oxidized Regenerated Cellulose): HE stain, 100x magnification, showing evidence of primary wound healing after the application of ORC absorbable hemostat (Surgicel™)

Primary healing was observed in two rats in Group C (25%), one rat in Group PS (12.5%), and one rat in Group GS (12.5%). However, primary healing was observed in all eight rats in Group ORC (100%). Pairwise comparisons between groups revealed that Group ORC had a statistically significantly higher rate of primary healing compared to Groups C, PS, and GS (p = 0.007, p = 0.001, p = 0.001, respectively). No statistically significant differences were detected between the other groups (p > 0.05).

When the results were evaluated in terms of fibrosis, fibrosis was detected in three rats in Group C (37.5%), two rats in Group PS (25%), and seven rats in Group GS (87.5%). No fibrosis was observed in the tissues of rats in Group ORC. Pairwise comparisons revealed that the absence of fibrosis in Group ORC was statistically significant compared to Groups PS and GS (p = 0.007, p = 0.001, respectively). No statistically significant differences were found between the other groups (p > 0.05).

Inflammation was observed in three rats in Group C (37.5%), five rats in Group PS (62.5%), three rats in Group GS (37.5%), and one rat in Group ORC (12.5%). No statistically significant differences were detected in pairwise comparisons between the groups (p > 0.05).

Regarding cavernous tissue healing, Grade 0 healing was observed in seven rats in Group C, while Grade 3–4 healing was observed in seven rats in Group ORC. Pairwise comparisons showed that Group ORC had significantly better healing scores compared to Group C (p = 0.000), while no significant differences were detected in the other comparisons (p > 0.05). Data from the histopathological assessments for each group are presented in the (Table 1).

Table 1 Distribution of the data obtained from the study according to rat groups and comparisons between groups

Discussion

In our study investigating the effects of ORC absorbable hemostatic agents and gelatin-based absorbable hemostatic sponges on wound healing in a rat model of experimental penile fracture, we observed that ORC absorbable hemostats significantly enhanced primary wound healing and reduced fibrosis formation. In contrast, the application of gelatin-based hemostatic sponges did not contribute to wound healing and was associated with increased fibrosis during the healing process. These findings hold significant clinical implications, as the development of fibrosis and scarring in penile tissue may result in complications such as curvature and erectile dysfunction, which can adversely affect the quality of life [12].

Regarding primary healing, the group treated with the ORC absorbable hemostatic agent had significantly better outcomes than the other groups. Additionally, when evaluating cavernous tissue healing, the ORC-treated group demonstrated significantly higher healing rates compared to the control group. ORC absorbable hemostatic agents support hemostasis by providing a physical matrix that facilitates clot formation at the application site [10]. These agents are biodegradable and generally absorbed quickly by the body, minimizing foreign body reactions and promoting favorable healing conditions. These agents have also been employed in other tissue types, such as vascular, neurosurgical and abdominal surgeries, with favorable outcomes [13]. Nonetheless, some studies have suggested that residual non-absorbable ORC fragments may induce granuloma formation, potentially leading to chronic inflammation and impairing the wound-healing process [9]. However, this was not observed as a significant finding in our study.

The gelatin-based hemostatic agents, while known to induce rapid hemostasis, may exacerbate inflammatory responses when interacting with surrounding tissues, potentially increasing the risk of fibrosis [10]. These findings align with the results of our study. No significant differences were observed between groups in terms of inflammation. Achneck et al. have noted that absorbable hemostatic agents like ORC may induce short-term inflammatory responses, but over the long term, these agents are absorbed by the body without significant impact on inflammation [10]. Our study’s relatively small sample size may have also limited the ability to detect small differences between groups regarding inflammation.

In a study conducted by Wolfe et al., the use of ORC in penile surgeries was shown to be effective in improving hemostasis and reducing postoperative complications [13]. According to the findings of this study, the application of ORC pledgets during corporotomy closure in inflatable penile prosthesis implantation significantly reduced postoperative scrotal drain output, thereby decreasing the risk of hematoma formation. Additionally, the study highlighted that the low pH of ORC provides bactericidal properties, which can lower the risk of prosthetic infections, including those caused by antibiotic-resistant microorganisms. These findings suggest that ORC’s pliable structure enables easy application in surgical defects, ensuring efficient hemostasis without increasing the risk of infection or other complications.

From a clinical perspective, our findings suggest that ORC absorbable hemostatic agents may be a preferred option for penile fracture repair to minimize fibrosis and promote primary healing. Given the challenges associated with penile curvature, scar tissue, and fibrotic plaques in conservatively managed patients, strategies that enhance healing and limit fibrosis are vital [14].

Future research should focus on addressing the limitations of this study. Increasing sample sizes and extending follow-up durations would provide a more comprehensive understanding of long-term outcomes, including chronic inflammation, fibrosis, and functional recovery. Furthermore, evaluating ORC and gelatin-based agents in diverse tissue environments and in human clinical settings would enhance the generalizability and applicability of these findings.

Limitations

One limitation of this study is the relatively small sample size in the experimental groups. Although a larger sample was desired to achieve more statistically robust results, the number of animals was restricted by the local ethics committee’s guidelines, particularly under the 3R principles (Replacement, Reduction, and Refinement) in animal research. Consequently, the sample size was capped at the maximum permissible level. Future studies with larger sample sizes would help validate these findings and provide greater statistical power.

Another limitation is the short follow-up period of two weeks, which restricts the ability to evaluate long-term outcomes, including chronic inflammation, persistent fibrosis, and functional recovery. This follow-up duration was dictated by the experimental design, physical conditions, and resource availability. Extending the follow-up period in future studies would enable a more comprehensive assessment of the healing process and potential complications over time.

Moreover, although this study focused on penile fracture repair, ORC and gelatin-based hemostatic agents are widely used in various surgical fields. Evaluating their effects in different tissue environments would enhance the generalizability of these findings and provide broader clinical insights.

Lastly, while the methodology incorporated a robust experimental design and advanced analytical techniques, histological evaluations could benefit from additional markers to more thoroughly assess fibrosis and inflammation. Including such markers in future studies could provide a deeper understanding of the underlying mechanisms and therapeutic implications of these agents.

Conclusion

This study, utilizing an experimental penile fracture model in rats, demonstrated that ORC absorbable hemostatic agents significantly contributed to primary wound healing and played a critical role in preventing fibrosis formation. In contrast, the application of absorbable gelatin sponges did not promote wound healing and was associated with increased fibrosis. Further clinical studies are needed to confirm the potential for routine use of ORC hemostatic agents in the treatment of penile fractures, given their promising results in the rat model.