# Mechanical and microstructural characterization of the human tunica albuginea

Pierre-Hugo Minster, Clément Parat, Paul Neuville, Damien Carnicelli, Nicolas Morel-Journel, Karine Bruyère-Garnier

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## To cite this version:

Pierre-Hugo Minster, Clément Parat, Paul Neuville, Damien Carnicelli, Nicolas Morel-Journel, et al.. Mechanical and microstructural characterization of the human tunica albuginea. Journal of the mechanical behavior of biomedical materials, 2026, 175, pp.107280. 10.1016/j.jmbbm.2025.107280. hal-05408493

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Submitted on 19 Dec 2025

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# Mechanical and microstructural characterization of the human tunica albuginea

Pierre-Hugo Minster<sup>a</sup>, Clément Parat<sup>b, c</sup>, Paul Neuville<sup>b</sup>, Damien Carnicelli<sup>b</sup>, Nicolas Morel-Journel<sup>b</sup>, Karine Bruyère-Garnier<sup>a, * d</sup>

## ARTICLE INFO

### Keywords:

Human tunica albuginea

Microstructure

Tensile test

Corpora cavernosa

## ABSTRACT

The tunica albuginea (TA) is a fibrous connective membrane surrounding the corpora cavernosa (CC), which plays a crucial role in the erection. In case of erectile dysfunction, inflatable penile prosthesis (IPP) may be a treatment of choice and mechanical interactions occur between prostheses and these penile tissues. There is still much to be learned about their mechanical behavior to help to improve IPP and penile surgical techniques. This paper presents the characterization of the TA mechanical behavior combined with the observation of its microstructural organization, as well as the mechanical behavior of the cavernous tissue. Uniaxial tensile tests were performed on 40 TA samples and 17 CC samples collected from 5 post mortem human subjects. TA samples were cut along both longitudinal and circumferential directions, and in both proximal and distal regions. Histological slices were produced from biopsies contiguous to the samples to observe the collagen fiber organization in the TA. We observed that this fiber organization usually schematized by 2 layers of perpendicular fibers is more complex, with some dispersion in the fiber orientations and interlacing of the 2 layers. The mechanical characterization of the TA samples revealed no clear anisotropy but different properties for the proximal and distal locations, whereas the CC showed a very low elastic modulus. These data complement those already published and further analysis of the microstructure of the TA will be needed to explain the variability of the mechanical behavior of the TA in view of selecting and identifying nonlinear behavior models.

## 1. Introduction

The tunica albuginea (TA) is a fibrous connective membrane surrounding the corpora cavernosa (CC) of the penis, playing a crucial role in the mechanical rigidity required for erection (Brock et al., 1997). Various conditions, including cancer, physical trauma, and Peyronie's disease can impair its structural integrity and function, leading to erectile dysfunction, which corresponds to the inability to achieve or sustain an erection adequate for sexual activity (Chung et al., 2020; Salonia et al., 2021). When oral and injectable treatments for erectile dysfunction are ineffective, one treatment of choice is a penile prosthesis, such as inflatable penile prostheses (IPPs) (Mobley et al., 2017). However, complications like tissue fibrosis, infection, erosion, and mechanical malfunctions of IPPs may damage the TA (Bose et al., 2023). Mechanical interactions also occur between prostheses and the surrounding tissues, both CC and TA. Additionally, TA reconstruction may

prove beneficial for certain procedures, such as incision-patch surgery for Peyronie's disease (Chierigo et al., 2022).

Prior to the 2020's the mechanical behavior of the human TA remained insufficiently studied and quantified. Early studies, such as Bitsch et al. (1990), reported values for elastic modulus and ultimate stress using inflation tests (Bitsch et al., 1990). Similarly, Hsu et al. (1994) measured ultimate tensile stress using puncture tests (Hsu et al., 1994). More recently, values for elastic modulus and ultimate tensile stress have been reported for tissues from healthy persons (Berardo et al., 2024; Kandabarow et al., 2022; Khorshidi et al., 2024; Luet et al., 2023) or those with Peyronie's disease (Brady et al., 2022; Colombo et al., 2024) using uniaxial tensile tests. These studies highlighted the nonlinear behavior of the TA but results are still difficult to combine and to generalize due to the use of various experimental protocols.

The microstructure of the human TA has been described through optical and electron microscopy analyses, revealing a bilayer structure

<footnote><sup>a</sup> Univ Lyon, Univ Gustave Eiffel, Université Claude Bernard Lyon 1, LBMC UMR_T9406, F-69622 Lyon, France</footnote>

<footnote><sup>b</sup> Service d'Urologie, Hôpital Lyon Sud, Hospices Civils de Lyon, 165 Chemin du Grand Revoyet, 69310, Pierre-Bénite, France</footnote>

<footnote>* Corresponding author. LBMC UMR_T9406, Université Gustave Eiffel, 25, Av. François Mitterrand 69675 Bron, France.</footnote>

<footnote>E-mail addresses: pierre-hugo.minster@univ-lyon1.fr (P.-H. Minster), clement.parat@chu-lyon.fr (C. Parat), paul.neuville@chu-lyon.fr (P. Neuville), damien.carnicelli@chu-lyon.fr (D. Carnicelli), nicolas.morel-journel@chu-lyon.fr (N. Morel-Journel), karine.bruyere-garnier@univ-eiffel.fr (K. Bruyère-Garnier).</footnote>
consisting of an internal layer of circumferential collagen fibers and an external layer of longitudinal one, both composed of collagen bundles interspersed with elastic fibers, with global thickness varying along the penis (Bitsch et al., 1990; Hsu et al., 1994; Loreto et al., 2012).

Similar to other fibrous connective membranes, such as skin or deep fascia, it is admitted that the microstructural organization of the TA largely explains its mechanical behavior. The variability of this microstructural organization along the penis and among subjects could contribute to the variability of the mechanical behavior of the TA. Animal studies have demonstrated mechanical anisotropy consistent with the TA's suggested microstructure of the various species (Bielajew et al., 2023; Bose et al., 2024; Kandabarow et al., 2021; Kelly, 1999). Recent studies on human TA concluded to the anisotropy of its mechanical behavior with the circumferential direction as the dominant one (Berardo et al., 2024; Khorshidi et al., 2024) whereas no specific anisotropy was observed by others (Hou, 2019; Luet et al., 2023). Beyond the hypothesis of a schematic organization of collagen fibers in human TA, no in-depth observation of collagen fiber organization of the tested TA samples was proposed to explain the resulting mechanical behavior.

Other penile tissues, including Buck's fascia, urethra, bladder, and prostate, have been tested in tension (Berardo et al., 2024; Cunnane et al., 2021; Masri et al., 2018; Natali et al., 2016). By contrast, the cavernous tissue has been exclusively tested in compression without reaching failure (Khorshidi et al., 2024). Furthermore, Khorshidi et al. (2024) reported recently radial compression values for penis sections at various orientations (Khorshidi et al., 2024).

Currently, understanding the mechanical behavior of the TA requires additional data to achieve a consensus in observations (Bose et al., 2023; Schmidt et al., 2023). This lack of consensus may be explained by substantial heterogeneity in experimental methods and testing conditions. Protocols vary in the inclusion of pre-loading, pre-cycling or relaxation phases; information on the initial sample strain state is also often limited; and differences in samples' cutting and preparation may occur. For instance, CC residues adhering to the TA samples may distort the assessment of the TA mechanical properties (Luet et al., 2023). This last observation justifies testing some CC samples under the same conditions as TA samples. Moreover, the CC is compressed and forms a dense, thin layer between the wall of the implant cylinder and the TA in the presence of an inflatable implant. In this configuration, the rigidity of the CC may influence the mechanical response of the entire implanted penis, therefore, a better understanding of its mechanical behavior seems also valuable.

In this context, the aim of this study is to quantify the mechanical behavior of the tunica albuginea and corpora cavernosa under uniaxial tensile loading, associated to some observations of the microstructural organization of collagen fibers within the TA. Specific emphasis is placed on specific methodological aspects for the assessment of the mechanical behavior of the TA including the assessment of the sample geometry and the definition of the zero-strain state of the samples.

## 2. Materials and methods

### 2.1. Sample preparation

Five penises from post-mortem human subjects (PMHS) prepared with a formaldehyde-free preservation fluid (Safebalm®), were obtained from donors aged 81–98 years (median age 90 years) from the Department of Anatomy Rockefeller of the University of Lyon (Lyon, France) with a post-mortem interval of 4–10 days. Ethical approval was not required for this study since it was not required by French law at that time. The cadaver subjects were obtained after the deceased gave their informed consent to donate them to science during their lifetime (law article R2213-13). The authors ensure that these experiments were performed with respect for these remains, and in correct health and safety conditions. Initial preparations involved assessing for penile

anomalies (e.g., penile curvature during erection), removing the skin and superficial fascia, separating the urethra and glans, and transecting the penis at its base. The specimens were then frozen and stored at -18 °C until further dissection.

Prior to dissection, each specimen was thawed at 4 °C for 12 h. First, the specimen was sectioned longitudinally along the urethra path (see Fig. 1a). Then, corpora cavernosa were excised, opened longitudinally to remove the central artery, and cut into between two and four rectangular samples of CC tissue.

Secondly, the tunica albuginea was isolated removing Buck's fascia and as much CC tissue as possible. From each penis, at least 8 I-shaped TA samples (12 for one specimen, 8 for others) were die-cut following a specific pattern designed to obtain at least 2 longitudinal and 2 circumferential direction samples for both proximal and distal areas (Fig. 1b). Each sample was then stored in saline solution at 4 °C until testing. The offcuts between the I-shaped samples were retained as biopsies for microstructural analysis (Fig. 1b).

Before testing, each sample was brought to room temperature during 2 h in saline solution. Immediately prior to testing, several dots of black ink were applied along the gauge length of each sample to enable strain measurements by an optical method. Overall, 45 samples of TA and 18 samples of CC were tested.

Tensile tests were performed using an INSTRON 8802 machine (High Wycombe, England). Loads were measured with a 5N uniaxial load cell (STM1 5N, DOERLER Mesures, France) for CC samples and a 250N uniaxial load cell (CDP-04 25/500 daN, TME, France) for TA samples (Fig. 2a).

For strain measurements, the experiments were recorded using a SA3 PHOTRON black and white camera (Tokyo, Japan) equipped with a Sigma 105 mm lens (Kawasaki, Japan). The camera resolution was 1024 by 256 pixels, allowing for approximately 10 pixels per millimeter in the region of interest. The black ink dots on the samples were tracked with TrackImage software (version 3.6, ORME, France) to measure local sample stretch.

Two zero-strain states of each sample were considered (Fig. 2b). First, a “suspended” zero-strain state was measured from an image of the sample suspended in the upper grip of the machine, named $L_0$ “suspended”. In this configuration the sample was only submitted to the gravity and the load cell was set to zero. By post-processing of the records, a “preload” zero-strain state was measured from an image of the sample submitted to a 0.1 MPa tensile stress, named $L_0$ “preload”.

Experiments were conducted in air at ambient temperature. To prevent slippage inside grips, emery cloth was used. A lighter, lower grip compatible with the load cell capacity of 5N was used for tests on CC samples. After positioning the samples in both grips, they were sprayed with saline solution. No preconditioning cycles were applied. A uniaxial stretch was applied at a strain rate of $0.01 \text{ s}^{-1}$ until failure. This strain rate is similar to those reported in previous studies (Bielajew et al., 2023; Khorshidi et al., 2024).

### 2.2. Geometry measurements

Initial cross-sectional areas of each sample suspended in the upper grip were measured by a 3D scanning (Go!SCAN 3D, Creaform-AMETEK, Lévis, QC, Canada). From the 3D reconstruction of each sample, the average cross-sectional area was calculated over the gauge length of the sample, from 10 measurements, to obtain the scanned initial cross-section, named $S_0$ (Fig. 2c).

### 2.3. Microstructure observations

Each of the 40 biopsies was divided into two pieces and then placed in histological cassettes (Fig. 1b). The cassettes were immersed in formalin (formaldehyde 4 % m/v, pH 7) for 40 h, then rinsed in phosphate-buffered saline (PBS) at 4 °C for a maximum of 24 h and sent for embedding. One piece of each biopsy was embedded for a 5 µm slice
<figure><img src="image_4.png" /><figcaption>Fig. 1. Preparation of samples. a) Human penis before dissection along the urethra path. b) Pattern for tunica albuginea (TA) sampling after corpora cavernosa (CC) removal, showing tensile test I-shape samples and biopsy sites. c) Schemes of expected transversal and tangential histological slices of a distal circumferentially oriented biopsy. LONG = longitudinal, CIRC = circumferential, DIST = distal, PROX = proximal.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 2. Set up and measurement of sample's dimensions, a) picture of the set up. b) reference dimensions used for initial length L₀ "suspended" and L₀ "preload". c) reference dimensions averaged to compute the initial scanned cross-section (S₀) in the "suspended" configuration.</figcaption></figure>

cut in the transversal plane of the biopsy and the other was embedded for a 5 µm slice cut in its tangential plane (Fig. 1c). The slices were stained with Picrosirius red and Azan-Mallory to facilitate the identification of collagen fibers. The slices were imaged at 2.5x magnification under both white and circular polarized light.

Furthermore, as feasibility test, one unique biopsy underwent a detailed three-dimensional reconstruction. Following a clearing process (CUBIC process), it was imaged using light sheet microscopy.

## 2.4. Mechanical behavior assessment

To standardize the curves for post-processing, each force-displacement curve was resampled to 200 points from the start of the test to the maximum force. Nominal stress-stretch curves were computed from force and marker displacement recordings.

The stretch was computed using either the L₀ "suspended" as zero-strain state or the L₀ "preload". Moreover, the first histological slices revealed the presence of residual corpora cavernosa in the thickness of the TA samples as observed by Luet et al. (2023), suggesting that the real section of the TA contributing to the mechanical response of the sample,
differed from the scanned initial cross-section $S_0$. To compute a more realistic cross-section of the TA in each sample, we assumed that the two samples adjacent to a given biopsy share the same microstructure, and we used the ratio of CC tissue/total thickness estimated on the histological slices to estimate an effective fibrous section of each TA sample, named $S_0$ without CC.

Nominal stress-stretch curves were plotted, and the following properties were determined: the elastic modulus (E in MPa) as the slope of the steepest linear part of the curve, the ultimate tensile stress (UTS in MPa) defined as the maximal tensile stress prior to failure, the stretch at UTS ($\lambda_{UTS}$), and the strain energy (U in MJ/m³) represented by the area under the curve (Fig. 3). For curves plotted using the $L_0$ "suspended", the low stress stretch (LSS) was defined as the stretch applied between the suspended configuration and the preloaded configuration (nominal stress = 0.1 MPa).

## 2.5. Statistical analysis

All statistical analyses were performed using the Python library scipy.stats (Virtanen et al., 2020). Data are expressed in terms of mean and standard deviation. The Shapiro-Wilk test was used to assess data normality.

To compare the results obtained from each TA sample using different post-processing methods, paired tests were performed: Student t-test or Wilcoxon depending on the normality of the data.

To compare the TA properties obtained for the two loading directions (longitudinal or circumferential) and the two locations (proximal or distal), independent mean comparisons were assessed using a Student t-test or a nonparametric Mann Whitney test depending on the normality of the data.

The differences were considered statistically significant for p-value $\le 0.05$.

# 3. Results

## 3.1. Microstructural organization of the TA

Histological transversal slices from two TA biopsies obtained from the same subject in the proximal and the distal region are illustrated in Fig. 4a and b. In the proximal biopsy, three distinct layers are clearly visible: some residual CC tissue, an internal layer of circumferential collagen fibers and an external layer of longitudinal collagen fibers. When collagen fibers are cut across their section, localized folding

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Typical nominal stress-stretch curve of a TA sample with the zero-strain state for the suspended configuration. The measured properties include elastic modulus (E), low stress stretch (LSS) calculated between stretch = 1 and stretch at preload (nominal stress = 0.1 MPa), ultimate tensile stress (UTS), stretch at UTS ($\lambda_{UTS}$), and strain energy (U).</figcaption></figure>

occurs in the histological slice, creating artifacts that help to identify these three different layers. Under polarized light, Sirius Red staining enhances the alignment of the collagenic fibers bundles, with the most oriented bundles in dark orange. In the distal biopsy, the circumferential fiber bundles of the internal layer appear thinner compared to the longitudinal fiber bundles of the external layer. Moreover, circumferential fibers appear within the longitudinal bundles, suggesting an interlaced organization of the two layers. Among all the biopsies (n = 20), the ratio of the thickness of the residual CC tissue to the total thickness was estimated to be 0, 1/2 (case of the distal biopsy on Fig. 4b) or 1/3 (case of the proximal biopsy on Fig. 4a).

Tangential slices obtained from biopsies contiguous to those cut in the transverse plane are illustrated on Fig. 4a and d. In the proximal biopsy, the slice shown is not strictly tangential but forms an angle with the biopsy external and internal sides. This angle allows to identify different regions on a unique slice, corresponding to different location in the thickness of the biopsy (i.e. different layers): an external layer containing long and oriented bundles of longitudinal fibers; an intermediate layer containing various directions of shorter fiber bundles; an internal layer containing an alignment of bundles in the circumferential direction, and residual CC tissue. In contrast, the distal biopsy slices seem more tangential to the internal and external surfaces, showing more homogeneous slices. The slice closer to the external side of the biopsy shows wavy bundles variously oriented, without clearly identified oriented bundles of longitudinal fibers. The slice closer to the internal side of the biopsy, shows bundles of circumferential fibers typically wavy arranged and relatively thick and oriented.

The 3D image of the cleared TA biopsy obtained with 3D light sheet microscopy revealed an intermediate layer of significant thickness showing a clear fiber bundle crossing in the tangential plane, between thin layers of fibers bundles totally oriented in the longitudinal and circumferential directions (Fig. 5).

## 3.2. Mechanical behavior

Among the 63 tested samples, three samples (two TA and one CC) slipped in the clamps, which led to the test being stopped. Consequently, 43 TA samples and 17 CC samples were retained for analysis.

### 3.2.1. Mechanical behavior of the corpora cavernosa (CC)

The nominal stress-stretch curves obtained for the CC samples are illustrated in Fig. 6a. The behavior of the CC samples shows a large variability with some more or less nonlinear behavior. As the samples were rectangular, their failure occurred mainly inside the grips, thus we chose not to report failure properties. Due to the variability of the curve shapes, the low stress stretch was not assessed. Both intra and inter-subject variabilities were observed, with an average elastic modulus of $1.08 \pm 0.93$ MPa (n = 17) (Fig. 6b).

### 3.2.2. Mechanical behavior of the tunica albuginea (TA)

The nominal stress-stretch curves obtained for TA samples and the associated mechanical properties are illustrated on Fig. 7. These results were obtained using the zero-strain state $L_0$ "suspended" and using the initial sections $S_0$ without CC. Three outlier curves were not considered in the rest of the study. Both intra and inter-subject variabilities were observed.

### 3.2.3. Effect of the post-processing method on TA properties

As mentioned in the "Material and methods" section, a preload configuration was introduced, defining a $L_0$ "preload", which was significantly superior to the $L_0$ "suspended" (p < 0.001). The choice of the zero-strain state impacts the values of the elastic modulus of the TA, with values of E = $86.4 \pm 41.8$ MPa with the $L_0$ "suspended" vs. $94.1 \pm 45.1$ MPa with the $L_0$ "preload" (p < 0.001), but no specific effect of the $L_0$ choice on the variability of the elastic modulus has been observed, with a coefficient of variation of 0.5 for both. The choice of the zero-
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Histological slices of TA biopsies oriented along the circumferential direction from one subject. a) Transversal slices of the biopsy from the proximal region, b) Transversal slices of biopsy from the distal region. Asterisks (*) on images of Sirius Red-stained slices (observed with polarized light) indicate cutting artifacts, which are also visible in the images of Azan-Mallory trichrome stained slices. c) Tangential slices of biopsy from the proximal region. This slice is not perfectly tangential to the biopsy, revealing different fiber organizations along the thickness of the biopsy, d) Tangential slices of biopsy from the distal region. These three slices spaced by 90 µm and 180 µm are more tangential to the biopsy and appear more homogeneous, various fiber organizations can be seen on these different slices. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

strain state also impacts the stretch at UTS, with values of 1.22 ± 0.07 with the L₀ “suspended” vs. 1.12 ± 0.03 with the L₀ “preload” (p < 0.001), and the variability in stretch at UTS with the L₀ “suspended” was reduced when considering the L₀ “preload”, with coefficient of variation of respectively 0.06 and 0.03. Using the preload configuration as zero-strain state conforms to previous studies (Bielajew et al., 2023; Brady et al., 2022; Khorshidi et al., 2024), but using the suspended configuration allows to record the TA mechanical behavior at low stress and to assess a low stress stretch (LSS).

Furthermore, as the cross section of the TA samples increases when

considering the total scanned section (S₀) rather than the effective section of fibers (S₀ without CC), it impacts the nominal stress values. Thus, it decreases significantly (p < 0.001 for all parameters), the elastic modulus from E = 86.4 ± 41.8 MPa (n = 40) to E = 65.8 ± 38.9 MPa (n = 40), the UTS, from UTS = 7.7 ± 3.5 MPa (n = 40) to UTS = 5.8 ± 3.0 MPa (n = 40) and the strain energy, from U = 0.43 ± 0.27 MJ/m³ to U = 0.32 ± 0.21 MJ/m³ (n = 40). Moreover, the variability of these properties did not appear to be impacted by the use of scanned sections (S₀) or effective fibrous sections (S₀ without CC) of the samples. However, as the CC tissue showed a 100 times lower elastic modulus than the TA, it
<figure><img src="image_8.png" /><figcaption>Fig. 5. 3D reconstruction of the cleared TA biospy oriented along the circumferential direction and obtained from light sheet microscopy. The five tangential sections shown were taken at different thickness levels of the 3D reconstruction: from the corpora cavernosa (CC) tissue (z = 510 µm), through the area of circumferential fibers (z = 897 µm), to the area of longitudinal fibers (z = 2097 µm). The white arrows indicate the orientation of some fiber bundles.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 6. Corpora cavernosa mechanical behavior using L₀ “suspended” and the scanned initial cross-section S₀, a) Nominal stress-stretch curves and b) elastic modulus distribution.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 7. TA mechanical behavior using L₀ “suspended” and S₀ without CC. Nominal stress-stretch curves and distributions of mechanical properties: elastic modulus (E), low stress stretch (LSS), ultimate tensile stress (UTS), stretch at UTS (λUTS), and strain energy (U). The outlier curves (dashed lines) and their properties (squares) are given for information and not considered in the statistical analysis.</figcaption></figure>

seemed relevant to consider the effective fibrous section (S₀ without CC) of each TA sample, instead of the scanned section (S₀), for the computation of the TA nominal stress.

Fig. 8 illustrates the stress-stretch curves obtained using two post-processing methods, one using an effective fiber section (S₀ without CC) and a L₀ “suspended”, and one using the scanned section S₀ and a L₀ “preload” which conforms to previous studies.

### 3.2.4. Mechanical anisotropy and location effect

The dataset was divided according to the sample loading directions (longitudinal and circumferential) and location (distal or proximal). These groups are identified by different colors on Fig. 8. Visually, no clear tendency of loading direction or localization discrimination was observed on these nominal stress-stretch curves.

For the quantitative analysis of the anisotropy and the location effect on TA properties, we considered the nominal stress-stretch curves computed using an effective fiber section (S₀ without CC) and a L₀ “suspended” (Fig. 8b).

Regarding the effect of loading direction (Fig. 9 - orange and green bars), the average elastic modulus was higher in the circumferential direction (E = 93.6 ± 42.7 MPa) than in the longitudinal direction (E = 77.6 ± 38.9 MPa), with similar coefficients of variability around 0.48. However, it is not statistically significant. For all the properties of the TA, no significant differences were observed according to the loading direction.

Regarding the effect of the location (Fig. 9 - red and purple bars), there were significant differences (p < 0.05) in the main properties of the TA. Elastic modulus and ultimate tensile stress were higher in the proximal area than in the distal area: respectively E = 101.7 ± 47.2 MPa

and E = 69.5 ± 29.6 MPa; and respectively UTS = 8.8 ± 4.2 MPa and UTS = 6.4 ± 1.8 MPa. The mean values of the low stress stretch and the stretch at UTS were lower in the proximal area than in the distal one: respectively LSS = 0.07 ± 0.05 and LSS = 0.10 ± 0.04; and resp. λUTS = 1.20 ± 0.08 and λUTS = 1.24 ± 0.06.

## 4. Discussion

The primary objective of this study was to characterize the mechanical behavior of the tunica albuginea (TA) and the corpora cavernosa (CC), while also investigating the microstructure of the TA samples.

For the observation of the TA microstructural organization from histological slices, a specific attention was paid to the orientation of the transversal biopsy sections according to the anatomical directions. These slices allowed to estimate an effective fibrous section of the TA samples by accounting for the residual cavernous tissue present on the contiguous biopsy.

In an original way on human TA, tangential histological slices allowed to observe the orientations of the fiber bundles in the different layers of the TA. Despite being limited to two-dimensional observations, these tangential slices suggest that the TA's fibrous organization, previously described as two layers of orthogonal fibers (Bitsch et al., 1990), is more complex, making it difficult to quantify the thicknesses of the 2 layers of the TA and the main orientations of their respective fiber bundles. This observation was supported by the 3D image obtained on one cleared biopsy with 3D light sheet microscopy that showed an intermediate layer of significant thickness with fiber bundle's crossing in the tangential plane. This observation also brings to mind Kelly's work
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Nominal stress-stretch curves of tunica albuginea samples computed using a) $L_0$ in the suspended configuration and So without CC, b) $L_0$ in the preload configuration and the scanned $S_0$ to conform to the literature. Color codes indicate the loading direction and the sample location. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

on the nine-banded armadillo penis (Kelly, 1997). The author measured the thicknesses of its TA's outer layer, inner layer, but also of a thin transition layer that contains randomly arranged fibers. Further 3D observations of the human TA are required to confirm our preliminary findings and automated or semi-automated procedures need to be developed to identify the different layers of fibers, the distribution of their orientation with respect to the tensile direction and their waviness, either from histological slices or from 3D reconstructions.

For the mechanical tests, we emphasized the measurement of the external geometry of each specimen and the use of local strain measurements using non-contact methods. Measuring the external geometry of soft biological tissues with a caliper gauge can be challenging and may lack reproducibility, while local strain measurements avoid potential bias associated with sample slippage in the grips.

The cavernous tissue showed a more or less nonlinear mechanical behavior in tension, with a low average elastic modulus in the steepest linear parts of the nominal stress-stretch curves (1.20 ± 0.98 MPa, n = 17). Initially, it seems reasonable to assume that this tissue is homogeneous and isotropic due to its spongy architecture (Goldstein et al., 1985; Goldstein and Padma, 1990). To our knowledge, these are the first reported tensile data for CC samples, complementing earlier work by Khorshidi et al. (2024) on radial compression (Khorshidi et al., 2024). Although the loading modes of the corpora cavernosa during erection

are more complex than uniaxial tension, our data may help for developing numerical models of implanted penis, where the CC is submitted to non-physiological loadings that may imply both radial compression and longitudinal tension during implant inflation.

The tunica albuginea exhibited a characteristic J-shaped nonlinear mechanical behavior, typical of fibrous connective membranes, often described in four phases: a toe region, a heel region, a linear phase and a failure phase (Khorshidi et al., 2024). This is similar to the previous observations on TA (Berardo et al., 2024; Bitsch et al., 1990; Brady et al., 2022; Colombo et al., 2024; Hou, 2019; Kandabarow et al., 2022; Khorshidi et al., 2024; Luet et al., 2023). In terms of average elastic moduli in the linear phase, we obtained.

$E = 93.6 \pm 42.7$ MPa (n = 22) and $E = 77.6 \pm 38.9$ MPa (n = 18), respectively in the circumferential and in the longitudinal directions, regardless of the location. These values are of the same order of magnitude as those reported by Bitsch et al. (1990) from inflation tests on TA, which was considered as isotropic (i.e. 100 MPa). But our values are higher than those obtained by Khorshidi et al. (2024), and Berardo et al. (2024) obtained from uniaxial tensile tests. Our choice to use an effective TA fibrous section, excluding residual cavernous tissue, for the computation of the nominal stress largely contributes to the obtention of higher average elastic moduli and ultimate stresses than those reported in previous studies. This approach was driven by the observation that
<figure><img src="image_12.png" /><figcaption>Fig. 9. Mean values and standard deviations of the TA properties as a function of location and loading direction: elastic modulus (E), low stress stretch (LSS), ultimate tensile stress (UTS), stretch at UTS ($\lambda_{UTS}$), and strain energy (U). \*p < 0.05, \*\*p < 0.01; n. s.: not significant.</figcaption></figure>

the average elastic modulus of the cavernous tissue is 100 times lower than the average elastic moduli of the TA, and that CC remnants were not negligible in terms of thickness. Nevertheless, adjusting the sample cross-sections in this manner did not reduce the overall variability in the measured properties.

The different experimental conditions may contribute to these discrepancies: our tests were performed in air at room temperature without cycling, whereas these authors applied several cycles of loading before measuring properties. Moreover, Khorshidi et al. performed their tests in a bath at 37 °C and computed the sample stretch from the clamp displacement (Khorshidi et al., 2024).

Preconditioning is commonly applied to soft tissues to improve the reproducibility and comparability of mechanical test results, as it is generally assumed that preconditioning cycles homogenize the stiffness of samples at larger deformations. However, in the present study, we chose not to apply preconditioning cycles for several reasons.

First, it was important to quantify the TA behavior under small deformations during the first loading, particularly to quantify the length of the toe region in the stress-stretch curve. Indeed, we expected this parameter to show some degree of anisotropy, as observed by Kelly on the penis of the nine-banded armadillo (Kelly, 1999). In addition, the length of this low stress region, that we characterized by the Low Stress Stretch parameter, proved to be highly variable. Because the tests were performed under displacement control, it was difficult to apply identical pre-cycling loading conditions to all TA samples, and thus, to guarantee that the first cycle would reach a sufficiently high stretch to enter the steepest linear region of the stress-stretch curve. Moreover, on several connective membranes, as skin (Caro-Bretelle et al., 2015) or esophagus wall (Durcan et al., 2022), stress softening is observed leading to changes in the mechanical behavior after cycling. We can hypothesis that this stress softening is related to irreversible re-arrangement of the

microstructure, or even to micro-damage, that we preferred to avoid. Lastly, unlike other fibrous connective membranes such as the arterial wall, the TA is not subjected to significant cyclic loading in vivo.

All mechanical tests were conducted in air, for practical reasons. At the time of experimentation, equipment for testing in a temperature-controlled fluid bath was not available in our laboratory. Nevertheless, special care was taken to preserve sample hydration throughout testing, and the duration of each test was short, as no preconditioning cycles were applied. Testing in a 37 °C bath could yield slightly different mechanical behavior of the TA. Regarding the CC, the presence of interstitial fluid within the sample may further influences its mechanical behavior in a fluid environment. In this study, the CC and the TA were tested under identical conditions to assess the effect of the remaining CC on the TA sample, and the interstitial fluid movement was free. In addition, in the configuration where the CC is compressed and forms a dense thin layer between the wall of an inflatable implant and the TA, the spongyous structure of the CC is altered, thereby limiting the influence of fluid effects on its mechanical behavior.

By using the configuration where the sample is suspended as the zero-strain state, we introduced a low stress-stretch (LSS) parameter that quantifies the length of the toe region of the nominal stress-stretch curves. In practice, defining the zero-strain state without any applied load may be a challenge (Gao and Desai, 2010), and gravity cannot be eliminated in air. Therefore, considering that the specimens have the same shape geometry and assuming that the weight of the samples is similar, the suspended sample configuration seemed easy to apply and to reproduce. In previous studies on the TA, a preload configuration of the sample may be used as the zero-strain state (Khorshidi et al., 2024). Apart the differences obtained on the computed properties depending on the chosen zero-strain state, using a preload configuration shortens the recording of the mechanical behavior of the sample under low stresses.
On the other hand, recording the mechanical behavior of a sample from its suspended configuration makes it possible to update the stress-stretch curve with any chosen preload and to compare results among studies.

As already mentioned, the distribution of the LSS values that we obtained shows a great variability. Contrary to what was observed on the nine-banded Armadillo by Kelly (1999), the LSS did not depend significantly on the direction of loading but it was dependent on the sample location. Further analysis will be needed to explain this variability of toe region length in the nominal stress-stretch curves. One hypothesis could be that the dispersion of the collagen fiber orientations can lead to more or less rapid re-orientation along the tensile direction or the effect of the waviness of some fibers; but only an analysis of the 3D microstructure of each sample could help to verify this hypothesis.

Although the circumferential samples exhibited higher elastic moduli, and a lower failure stretch than the longitudinal samples, our results did not show a statistically significant anisotropy in the mechanical behavior of the TA. The low number of samples tested in each direction (around 20) associated to the variability of the measured properties could explain that we did not observe a statistically significant difference. However, in the literature, based on studies involving no more than samples, the mechanical anisotropy of the human TA remains inconclusive. Some authors reported significant differences (Berardo et al., 2024; Kandabarow et al., 2022; Khorshidi et al., 2024), while others did not (Hou, 2019; Luet et al., 2023). In contrast, animal studies indicate a more pronounced mechanical anisotropy of the TA (Bielajew et al., 2023; Bose et al., 2024; Kandabarow et al., 2021). This low mechanical anisotropy of the human TA may be explained by a microstructural variability, which appears more complex than two simply distinct layers of perpendicular fiber bundles, as described in the literature (Brock et al., 1997; Hsu et al., 1994).

To our knowledge, no previous study has compared the mechanical behavior of distal and proximal samples. We obtained significant differences between the 2 locations with samples in the proximal region showing higher mean values of elastic moduli and ultimate stresses, and lower low stress stretch and stretch at ultimate tensile stress. The literature indicates that the thickness ratio of the two fibers layers depends on the location (Brock et al., 1997; Hsu et al., 1994), this may explain these differences. A more detailed analysis of this thickness ratio at the different locations would have been valuable; however, the interlacing of the two layers prevented an easy quantification of this ratio from histological slices in the present work.

Despite the precautions taken in measuring the geometry of the specimens and in measuring local deformations on the specimens, variability in the behavior of the albuginea specimens is observed. The anisotropy of mechanical behavior between the circumferential and longitudinal directions was not very pronounced but significant differences were observed between TA samples from the proximal and the distal regions. The number of donors involved in our study combined with a large inter-subject variability, results in low statistical power that may limits the robustness of our conclusions.

Aggregating data from various experimental studies on the TA could enhance the understanding of its mechanical behavior. However, methodological variations among studies limit the reliability of a simple data compilation and compromises relevant comparisons.

First, preservation methods may differ from a study to another and many studies were published to assess the impact of sample preservation before mechanical testing. The effect of the preservation method depends on the tissue type, the freezing/thawing procedure (freezing temperature, freezing speed, number of cycles, storage duration, thawing protocol). Freezing at -20 °C is used by many teams in the literature and it was the case in the recent studies on penile tissues (Berardo et al., 2024; Khorshidi et al., 2024), to which we compared our results. The donors involved in our study are also prepared with a SafeBalm® solution to preserve the bodies and to facilitate their use. SafeBalm® is a range of formaldehyde-free preservation fluids and previous analysis on samples of ilio-tibial bands shown that the differences in the mechanical

behavior of the samples taken before and after the embalmment of the body with SafeBalm® were minor, and not statistically significant (Gras et al., 2018). A thorough analysis of the impact of TA preservation methods on the mechanical properties of the tunica albuginea would be valuable, as performing a large number of experiments on fresh tissue remains challenging.

Moreover, testing methods on soft biological tissues differ among teams even for a uniaxial tensile test. In a recent study, Famaey et al. (2026) investigated the methodological variations of several teams to conduct uniaxial tensile test of pig aorta samples and assessed the variability of the resulted mechanical properties. In a first round of tests, various methods were applied for the environment of the samples during tests (for instance in bath at 37 °C or humified by a spray), for the sample shape (dog-bone, rectangular or hourglass), for the loading (with pre-conditioning or not), for the load cell taring (before or after mounting), for the strain measurement (on sample or from actuator displacement) and for the choice of the zero-strain state (before or after mounting, after preload). As expected, the measured mechanical properties shown variances but any correlation between the protocol variations and the results were identified. To overcome these limitations, we plan to carry out further experiments to assess the present findings on TA against data obtained from tests performed in bath at 37 °C, with preconditioning cycles.

This study is also based on samples from bodies donated to science at a very advanced age, ranging from 81 to 98 years. In comparison, other recent studies on the TA have involved slightly younger donors. Khorshidi et al. examined samples from donors aged between 75 and 89 years (Khorshidi et al., 2024), while the only donor involved in the study by Berardo et al. (2024) is 77 years old. There are limited opportunities to test samples from younger donors. To our knowledge, the only study providing mechanical properties for subjects under 70 years old is that of Bitsch et al. (1990) who tested TA from subjects aged between 33 and 83 years. Moreover, the age range of patients undergoing prosthesis implantation appears to be at least as wide. A recent meta-analysis on the sexual satisfaction of patients with penile prosthesis, involving 12 132 patients, reports ages between 20 and 77 years. with a mean of 59 years (Corona et al., 2025). It is well established that ageing alters the microstructure of the soft fibrous connective tissues leading to various pathologies. These changes are associated with the degradation of the elastin fibers (Heinz, 2021) and the stiffening of the collagen structures at the molecular and tissue scales (Bailey, 2001; Gautieri et al., 2017; Sloseris and Forde, 2025). More specifically regarding the tunica albuginea, a decrease in elastin concentration with age has been demonstrated (Akkus et al., 1997). Taken together, these observations suggest that mechanical testing alone is insufficient and that a quantitative analysis of the collagen and elastin content and arrangement of the tested samples would be highly valuable. It would allow to personalize a structural-based model of the TA mechanical behavior, that could then be extrapolated to younger patients.

Beyond the characterization of the mechanical behavior of the TA under uniaxial tension, numerous analyses of its mechanical behavior under different loading modes are still needed in order to correctly represent it in models for the evaluation of penises with inflatable penile implants: behavior in plane tension and biaxial tension, or relaxation behavior. Moreover, for a better knowledge of the initial strain state of the TA in an implanted penis, and to better know its loading path during inflation, an in-situ analysis of its strain mechanisms and levels would be of great interest for an efficient selection and identification of nonlinear behavior models.

## 5. Conclusions

Our results complement recent findings by other authors on the mechanical behavior of cavernous tissue and human TA. A non-linearity in the mechanical behavior of TA was observed, with a high degree of variability. Despite different mean values of the mechanical properties
for the longitudinal and circumferential tensile directions, the anisotropy of the mechanical behavior of the TA did not appear to be statistically significant. On the contrary, TA properties differed significantly depending on the proximal or distal location of the samples. The 2D observation of the microstructure of the TA, made as close as possible to the tested samples, showed that the organization of the collagen fibers in the external and internal layers of the TA is not so schematic. Despite the limitations of this study, the data obtained can be used to identify non-linear structural-based models of the TA behavior. Although initially validated using a cadaveric tissue model, such a mechanical behavior model could be further personalized to the TA microstructure of a broader population. Such constitutive material model and its associated properties are fundamental elements for the future development of comprehensive finite element models of the implanted penis. These models would enable to investigate how various factors (penile and implant geometry, mechanical properties of the TA and the implant cylinder wall, internal cylinder pressure ...) impact the resulting penile deformations and axial rigidity. Understanding the mechanical behavior of TA can also help to validate TA substitute materials (e.g., for Peyronie's disease treatment) or to optimize the wall of inflatable implants. The ultimate properties of TA are also essential values in view of automating the inflation of inflatable implants. To overcome the actual limitations, and enhance the understanding of the mechanical behavior of the TA, further work is needed. To get new insights into its microstructure, 3D imaging of cleared TA samples using fluorescence microscopy will allow to quantify more accurately the organization of the collagen fibers. On mechanical aspects, future work will aim to assess some methodological variations, to analysis samples from younger donors, and to quantify the mechanical behavior of TA under more realistic loads, such as plane tension and in situ inflation.

### CRediT authorship contribution statement

**Pierre-Hugo Minster:** Writing - review & editing, Writing - original draft, Visualization, Software, Resources, Methodology, Investigation, Data curation.
**Clément Parat:** Writing - review & editing, Writing - original draft, Resources, Methodology, Investigation, Formal analysis, Conceptualization.
**Paul Neuville:** Writing - review & editing, Writing - original draft, Resources, Investigation, Formal analysis, Conceptualization.
**Damien Carnicelli:** Writing - review & editing, Writing - original draft, Resources, Investigation, Formal analysis, Conceptualization.
**Nicolas Morel-Journel:** Writing - review & editing, Writing - original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.
**Karine Bruyère-Garnier:** Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This work was supported by a grant from the French National Research Agency as part of the "Investissements d'Avenir ExcellencES" program from France 2030 (SHAPE-Med@Lyon; ANR-22-EXES-0012).

We acknowledge the contribution of SFR Santé Lyon-Est (UAR3453 CNRS, US7 INSERM, UCBL) facility: CIQLE, as LyMIC member and member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-24-INBS-0005 FBI BIO-GEN), for the analysis of the microstructure of the tunica albuginea;

especially, Annabelle Bouchardon and Batoule Smatti for producing the histological sections and digitizing them; and Bruno Chapuis and Camilla Luccardini for clearing the sample and acquiring the light-sheet microscopy images. Zeiss Axio Scan.Z7 and LightSheet Z7 used for this study were partially funded by the Fonds européen de développement régional (FEDER).

The authors also sincerely thank those who donated their bodies to science so that research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their families deserve our highest gratitude (Iwanaga et al., 2021).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jmbbm.2025.107280.

### Data availability

I have shared the link to my data in the manuscript.

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