Introduction

Peyronie’s disease (PD) is an acquired fibro-connective tissue disorder that affects between 0.4 and 11% of the men [1]. Men with PD suffer from penile curvature, which can lead to significant pain, sexual dysfunction, and emotional burden [2]. While the specific pathophysiology is not completely elucidated, extravascular protein deposition and transformation of tunical collagen leads to plaque formation [3]. PD plaques are composed primarily of collagen, fibrin, and, in some, deposits of calcium [4]. Currently, Collagenase Clostridium Histolyticum (CCh) is the only FDA-approved medication for PD and contains a mixture of two collagenase proteins, AUX-1 and AUX-II [5, 6]. CCh is approved for patients who have palpable PD plaques with curvature greater than 30 degrees [6]. Early clinical trials excluded patients with calcified plaques, and suggested that CCh may be less effective in reducing curvature in men with calcified plaques, however more recent studies refute this [7,8,9].

To date, no in vitro study has explored whether PD plaque calcification decreases the enzymatic ability of CCh. Understanding how plaque calcification impacts the efficacy of CCh will help physicians better treat these patients. Our objective was to analyze the collagen content in calcified plaques before and after digestion with CCh in vitro to determine if calcification affects the action of CCh. We hypothesized that CCh would lead to plaque breakdown in calcified plaques. Findings from our study will help elucidate the role of CCh in the treatment of calcified plaques.

Materials and methods

IRB approval from the University of Miami was obtained (#20150740) for collection of noncalcified penile tissue. Human materials transfer agreement (#MTA00000300) was approved to obtain calcified PD plaque tissue from Mayo Clinic. All patients consented to participation based on institutional protocol. We identified ten men undergoing penile surgery that consented for collection of penile tissue for laboratory evaluation. Five men with erectile dysfunction undergoing penile prosthesis surgery and no history of PD served as control tissue of corpus cavernosum. Calcified plaque tissue was collected from five males undergoing plaque excision and grafting procedures for PD. Calcification was detected preoperatively using ultrasound. Tissue samples were stored at −80 °C until thawed for analysis. Samples were divided into equal weights and one half of each sample was injected with 0.25 ml PBS (Gibco 10010023, Thermo Fischer Scientific, Waltham, MA, USA) or 0.25 ml CCh (0.58 mg CCh reconstituted in 0.39 ml sterile saline, Endo Pharmaceuticals, Malvern, PA) through a 27-gauge needle to mimic the intralesional administration of CCh for PD treatment. After 24-h, the tissue was removed from the solution, dried and weighed, and solutions (PBS and CCh) were collected and stored at 4 °C. The tissue was made into paraffin slides. The slides were subjected to Picrosirius Red (Ab150681, Abcam, Boston, MA, USA) staining to allow for qualitative and quantitative analysis of collagen as described by Junquiera and validated by Lattouf [10, 11]. Slides were viewed using polarized light microscopy and collagen amount and type of collagen (type I and III) were measured. Type I collagen was identified by a strong yellow-red birefringence whereas type III was identified with a weak, green birefringence (Fig. 1). Images were taken and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) to provide relative quantities of type I and type III collagen. Color specific filters in ImageJ were utilized to isolate and quantify pixels corresponding to the color of type I and type III collagen as described by Courtoy et al. [12]. Next, soluble collagen was measured from the retained incubation solutions (PBS and CCH) using the Sircol Soluble Collagen Assay Kit (S5000, BioColor, Carrickfergus, UK) according to the manufacturer’s instructions. In brief, Sircol Dye Reagent was added to each sample to allow formation of collage-dye complexes that precipitate during incubation and were isolated by centrifugation, eluted, and measured. For statistical analysis, t-tests were performed in Excel.

Fig. 1: Polarized light photos of collagen staining in different experimental conditions.
figure 1

Type I collagen fibers are bright, red–yellow, and type III fibers appear green.

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Results

The average weight specimens was 0.01 g in both groups. When looking at digestion of calcified plaques between incubation solutions (CCh vs. PBS), the mean soluble collagen level was significantly higher in CCh compared to PBS (CCh: 301.07 ug ± 21.28 vs. PBS: 32.82 ug ± 3.68, p = 0.02) (Table 1a). Similarly, there was significantly less collagen (type I and III) staining in calcified plaque tissues incubated in CCh compared to PBS (CCh: 0.12 ± 0.08 vs. PBS: 0.44 ± 0.17, p = 0.002) (Table 1b).

Table 1 Comparison of soluble collagen and collagen staining between control and experimental groups.
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When comparing different tissue types (control penile tissue vs. calcified plaque) incubated in the CCh solution, though calcified plaques demonstrated less soluble collagen after incubation, there were no significant difference in amount of soluble collagen (control: 316.12 ug ± 34.91 vs. calcified: 286.01 ug ± 10.85, p = 0.06). In the PBS incubation, there were similar findings with no significant difference in amount of soluble collagen between the control and calcified groups (control: 30.22 ug ± 16.60 vs. calcified: 35.42 ug ± 47.69, p = 0.4) (Table 2).

Table 2 Measurement of soluble collagen in calcified vs. noncalcified, control penile tissue.
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Additionally, though higher mean collagen staining was seen in the calcified group, similar to the amount of soluble collagen, there was no significant difference found in the collagen staining of the CCh group between control and calcified tissue (control: 0.08 ± 0.02 vs. calcified: 0.17 ± 0.09, p = 0.08). Similarly, mean collagen staining in the PBS incubation was higher in the control tissue compared to calcified, but this did not reach significance (control: 0.50 ± 0.23 vs. calcified: 0.39 ± 0.39, p = 0.23) (Table 3).

Table 3 Collagen staining in noncalcified, control penile tissue vs. calcified, Peyronie’s disease plaques.
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Discussion

We aimed to determine if calcification impacts enzymatic breakdown of collagen in calcified PD plaques treated with CCh. To study this, we performed in vitro digestion of calcified and normal penile tissue with CCh and quantified change in collagen level and staining. We found the amount of collagen released into solution was similar between control and calcified tissues. As was the amount of Type I and Type III collagen remaining after incubation in CCh. Though we saw differences in amount and staining of soluble collagen between calcified and noncalcified plaques exposed to CCh, these did not reach significance. This suggests that plaque calcification does not change the enzymatic activity of CCh.

In the first large, randomized clinical trials assessing CCh for PD (IMPRESS I and II), severe plaque calcification was an exclusion criteria [13]. Post hoc analysis of the IMPRESS trials showed that men with noncalcified plaques experience significant improvement with CCh compared to placebo and calcified plaques did not, despite similar overall change in penile curvature in treatment groups [14]. Interestingly, in this post hoc analysis, men with calcified plaques receiving placebo had greater curvature improvement compared to noncalcified receiving placebo suggesting that the action of passing the needle through the plaque may have been beneficial. Further retrospective, single institution studies showed that calcification, may be associated with less improvement in curvature as well as subjective symptom scoring [7, 15]. Despite these concerns about the clinical effect of CCh on calcified plaques, there is minimal research into the biochemical basis of these findings and the pathogenesis of plaque calcification.

The first in vitro study assessing CCh in the treatment of PD plaques came from a 1980 study by Gelbard et al. who showed that PD plaques were successfully digested by CCh [16]. Shortly thereafter, a 1982 pilot study published by the same group showed that 3 PD plaque samples decreased in weight when injected with a CCh but preserved other penile structures [17]. Del Carlo et al. corroborated these results with an in vitro study presented at the 2009 American Urologic Association national meeting [18]. Finally, an immunohistochemical analysis of PD plaques showed degradation of collagen type I, III, and IV (with type I and III as the most abundant collagen types in both calcified and noncalcified PD plaques) after exposure to CCh [19]. Their study helped elucidate the mechanism by which CCh leads to improvement in PD [19]. To our knowledge, our study is the first in vitro study to quantitatively analyze the impact of CCh on calcified PD plaques.

Our study has limitations. The small sample size of five patients per tissue type group limits the strength of our conclusions and may be underpowered to draw conclusions about the definitive role of CC in the management of calcified PD plaques. Small sample size may allow for individual participant characteristics (i.e. severity or duration of disease) to significantly impact the results of the study and create confounders. Though we tried to mitigate these potential sources of bias and minimize the heterogeneity of the participants by using the same patient’s tissue in all experimental and control conditions. Additionally, our control group was normal penile tissue (corpus cavernosum without PD plaque) rather than noncalcified PD plaque due to unavailability of PD plaque at our institution. We hope that further translational research can clarify the utility of CCh on calcified PD plaques compared to noncalcified plaques that may be obtained during plaque excision and grafting which we are unable to offer. Additionally, we hope that further research into the role of CCh on nonclassical PD, as well as alternative treatments for men with calcified PD plaques, can be developed.

It is not fully understood if CCh is clinically efficacious in the treatment of calcified plaques. The presence of calcium in PD plaques has been hypothesized to prevent CCh from collagen digestion. However, our studies results suggest that calcification does not significantly impact the enzymatic digestion of Type I and Type III collagen by CCh.