<figure><img src="image_1.png" /></figure>

# Contactless mechanical stimulation of the skin using shear waves

Na Qiao<sup>a,*</sup>, Virginie Dumas<sup>b</sup>, Alexandre Bergheau<sup>a</sup>, Lucas Ouillon<sup>a</sup>, Norbert Laroche<sup>c</sup>, Coralie Privet-Thieulin<sup>d</sup>, Jean-Luc Perrot<sup>e</sup>, Hassan Zahouani<sup>a</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Keywords:**

Shear wave stimulation

Mechanotherapy

Tissue-engineered skin

Skin mechanical behavior

Collagen

Elastin

Fibroblast

## ABSTRACT

The skin, the outermost organ of the human body, is vital for sensing and responding to stimuli through mechanotransduction. It is constantly exposed to mechanical stress. Consequently, various mechanical therapies, including compression, massage, and microneedling, have become routine practices for skin healing and regeneration. However, these traditional methods require direct skin contact, restricting their applicability. To address this constraint, we developed shear wave stimulation (SWS), a contactless mechanical stimulation technique. The effectiveness of SWS was compared with that of a commercial compression bioreactor used on reconstructed skin at various stages of maturity. Despite the distinct stimulus conditions applied by the two methods, SWS yielded remarkable outcomes, similar to the effects of the compression bioreactor. It significantly increased the shear modulus of tissue-engineered skin, heightened the density of collagen and elastin fibers, and resulted in an augmentation of fibroblasts in terms of their number and length. Notably, SWS exhibited diverse effects in the low- and high-frequency modes, highlighting the importance of fine-tuning the stimulus intensity. These results unequivocally demonstrated the capability of SWS to enhance the mechanical functions of the skin *in vitro*, making it a promising option for addressing wound healing and stretch mark recovery.

## 1. Introduction

The skin is an essential organ of the human body, serving as a protective barrier against various external factors and perceiving and adapting mechanical signals. (Wong et al., 2011) It is constantly subjected to various mechanical forces, such as stretching, compression, and shearing, during daily activities. (Biggs et al., 2020) One of its key functions is to respond to mechanical stimuli through mechanotransduction, a mechanism that converts mechanical signals to biochemical responses. (Martin, 1997; Iskratsch et al., 2014; Wang, 2017) Mechanotransduction is a fundamental biological mechanism in mechanotherapy, the practice of prescribing exercises to aid in healing and rehabilitation. (Khan and Scott, 2009; Dunn and Olmedo, 2016) Compression therapy, massage, and microneedling are typical forms of mechanotherapy for wounded skin. Compression therapy is beneficial for treating skin issues and is the benchmark of care for various lower-extremity conditions. (Park et al., 2020; Romanelli et al., 2023) Massage applies various types of force, such as compression and

stretching by hands or tools, to the human skin. (Moortgat et al., 2016; Okuda et al., 2022) By stimulating the production of collagen and elastin in the skin, microneedling improves the skin texture, reduces wrinkles and scars, and increases skin elasticity. (Alster and Graham, 2018) However, this requires a trained medical professional and can cause adverse reactions. (Khalid et al., 2019; Pajak et al., 2022) Generally, compression is a common force employed for mechanical stimulation of the skin. (Wu et al., 2007; Wang et al., 2013; Leyva-Mendivil et al., 2015) Nevertheless, these traditional techniques, which entail direct skin contact, have restricted applicability, being unsuitable for both *in vivo* and *in vitro* scenarios. Additionally, there is a potential risk of tissue damage, and their adaptability to various skin conditions is limited. Therefore, noncontact mechanical stimuli for skin therapy are advantageous.

Zahouani et al. (Boyer et al., 2012; Djaghloul et al., 2018; Bergheau et al., 2022) used air-generated waves to examine the mechanical properties of the skin. Ultrasound shear wave elastography is an emerging technology that employs shear waves formed in the tissue to

<footnote><sup>a</sup> Univ Lyon, Ecole Centrale de Lyon, CNRS, ENTPE, LTDS, UMR5513, 69130, Ecully, France</footnote>

<footnote><sup>b</sup> Univ Lyon, Ecole Centrale de Lyon, CNRS, ENTPE, LTDS, UMR5513, ENISE, 42023, Saint Etienne, France</footnote>

<footnote><sup>c</sup> INSERM U1059-SAINBIOSE, University of Lyon, Jean Monnet University, 42270 Saint Priest en Jarez, France</footnote>

<footnote><sup>d</sup> ECE OMNES Education Research Center, France</footnote>

<footnote><sup>e</sup> Département de Dermatologie, Centre Hospitalier Universitaire de Saint-Etienne, 42055, Saint-Etienne, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: naqiao1003@outlook.com, na.qiao@ec-lyon.fr (N. Qiao), hassan.zahouani@ec-lyon.fr (H. Zahouani).</footnote>
measure its elasticity. (Taljanovic et al., 2017; Davis et al., 2019) Therefore, we believe that shear waves can locally perturb the skin, and long-term perturbation may eventually alter the mechanical properties of mechanically responsive tissues, assisting tissue repair and remodeling.

Tissue-engineered skin has emerged as a valuable tool in skin research and offers advantages over traditional models. As a physiologically relevant human skin model, it allows researchers to study the skin biology and pathology in a controlled environment. Tissue-engineered skin can be tailored to mimic different skin layers, including epidermal, dermal, and full-thickness skin, providing a platform for investigating various skin-related topics, such as skin therapy and aging. (Shevchenko et al., 2010; Tamayol et al., 2013; Goodarzi et al., 2018) Alterations in the overall mechanical characteristics of the skin following mechanical stimulation can be examined at a macroscopic level using tissue-engineered skin, and changes in the internal composition of the skin can be explored at a microscopic level. In addition, skin samples can be obtained at various maturity levels, allowing investigation of the effects of identical mechanical stimuli on the skin in different states.

Therefore, this study demonstrated how shear wave stimulation (SWS), a noncontact mechanical stimulation technique, can affect the mechanical behavior of skin substitutes. The effects of SWS on shear modulus and constituents of skin were investigated using air-generated shear waves and a few compression waves (Djaghloul et al., 2020) to stimulate the reconstructed skin at two different developmental phases. The performance of SWS was compared with that of a commercial compression bioreactor to demonstrate its applicability in vitro.

## 2. Materials and method

### 2.1. Reconstructed skin

This study used T-Skin™ from EPISKIN (Lyon, France). (Bataillon et al., 2019) It is an *in vitro* model of reconstructed skin comprising a dermal equivalent containing human fibroblasts and collagen overlaid by a stratified and well-differentiated epidermis comprising normal human keratinocytes. The cells originated from adult donors. The T-Skin had internal and external diameters of 1.26 and 1.74 cm, respectively, and a thickness of approximately 0.5 mm (Fig. S1A).

The human full-thickness skin model was cultured on an inert

polycarbonate filter and had two stages of maturation: cultured for 11 days (SKINJ11) and 18 days (SKINJ18).

After receiving the samples, following the instructions provided by Episkin, the inserts containing the skin substitutes were placed in 6-well cell culture plates (Greiner standard, Falcon, U.S.) filled with 2 mL culture medium in each well (GDA3F + Culture medium for T-Skin, EPISKIN) and incubated at 37 °C, 5% CO₂ with 95% humidity. As depicted in Fig. S1B, the culture medium was level with the filter and was replaced daily. Following a 1 h incubation period, SKINJ11 and SKINJ18 underwent a 4-day experiment.

### 2.2. Shear wave stimulation

#### 2.2.1. Shear wave stimulation design

Using silicone allows for the intuitive presentation of the air-triggered stimulation mode. (Fig. 1A). Shear and compression waves are generated within the skin through air stimulation, which could activate mechanotransduction and alter the mechanical properties of the skin (Fig. 1B).

Three needles (VIEWEG GmbH Dosier-und Mischtechnik, Germany) with an internal diameter of 0.58 mm and bent at 90° were installed on a three-dimensional (3D)-printed support (Zortrax M200, Poland) to be arranged horizontally in a row at 5.5 mm intervals as air outlets. The diameter and spacing of the needles could be adjusted based on the specific samples being stimulated. The air outlets were connected to three solenoid valves (EMERSON-ASCO, France) that served as air switches and pressure regulators (FESTO, Germany) to control the pressure of the corresponding air source (Air Liquide, France). The opening and closing times were curbed using individual control relays (Meder Electronic, France). The valves were programmed to open three air outlets simultaneously using LabVIEW (National Instruments USB-6229, U.S.).

#### 2.2.2. Mechanical stimulation on reconstructed skin

Air outlets with 3D-printed supports were fixed to the robotic arm (UR3, Universal Robots, Denmark) and set approximately 15 mm above the skin sample (Fig. 1C). After performing preliminary experiments on the reconstructed skin, we determined the optimal air pressure to be 1 bar and set two different opening times: 10 and 20 ms. The closing times were set to 490 and 480 ms to ensure Y-movement during the closing time. To achieve this, the robotic arm was programmed to move 2 mm

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Illustration of the principle and Y-movement of the SWS: (A) Practical stimulus for SWS on silicone, (B) schematic diagram of the SWS principle on typical skin tissue, (C) schematic diagram of three air outlets fixed on a robotic arm, and (D) schematic of the movement of the outlets over the reconstructed skin along the Y-axis.</figcaption></figure>
along the Y-axis at each step, and the three air outlets began to return step-by-step after repeating the four steps, as illustrated in Fig. 1D. The movement was cycled for a 5 min total stimulation time.

## 2.3. Bioreactor

Due to the widespread use of compression therapy in skin healing, ElectroForce® 5270 BioDynamic® (BOSE) (Fig. S3) was used in order to contrast the effect of SWS in this field. This bioreactor can precisely apply controlled sinusoidal compression to skin tissue. It has been proved effective in imposing mechanical constraints on soft tissues, particularly on vascular tissues. (Trabelsi et al., 2020)

## 2.4. Experiments

The effects of SWS and the compression bioreactor on the two types of T-Skin were assessed using five experimental groups, as detailed in this section and summarized in Table 1.

### 2.4.1. Negative control group

Before any operation, a group of samples denoted as the negative control (NC) was subjected to all characterization methods. For the SKINJ11, a portion of the models in the NC group underwent line-field confocal optical coherence tomography (LC-OCT) imaging. Another portion was measured using UNDERSKIN; Section 2.5.1 discusses details of the UNDERSKIN methodology. Each remaining sample was divided into two parts: one part was fixed in 4% formalin solution for visualizing collagen and elastin, and the other was preserved in 80% ethanol at 4 °C for the paraffin section. The same procedure was implemented on the SKINJ18 samples in the NC group.

### 2.4.2. Four-day control group

In addition to stimulation, the SKINJ11 and SKINJ18 samples in the 4-day control group (D4-Control) were subjected to the same conditions as those of the constraint groups.

### 2.4.3. SWS groups

The SKINJ11 samples were split into two SWS groups: SWS10 and SWS20, with loading times of 10 and 20 ms, respectively. SWS10 and SWS20 loaded approximately 0.03 and 0.06 N of force, respectively. Both groups were stimulated twice daily, 6 h apart, for 4 consecutive days. During the interstimulus breaks, the samples were placed in an incubator at 37 °C, 5% CO₂, and 95% humidity.

One hour after all stimuli were completed, UNDERSKIN measurement was conducted on one part of the samples from each group, while the other part was prepared for histological and optical microscopic imaging. The third part of each group was examined using LC-OCT. The same protocol was used for the SKINJ18 samples in the SWS10 and SWS20 groups.

### 2.4.4. Compression bioreactor group

SKINJ11 and SKINJ18 samples underwent sinusoidal compression at a 1 Hz frequency, with a magnitude of 200 µm (0.2 N load) applied for 30 min each day for 4 consecutive days in this group.

After stimulation, samples at both maturity stages were characterized using the same procedure as those of the SWS groups.

<table><caption>**Table 1**
Summary of the experimental conditions for the two types of T-Skin.</caption><thead><tr><th colspan="7">SKINJ11/SKINJ18</th></tr><tr><th>Condition</th><th>NC</th><th>D4-Control</th><th>SWS10</th><th>SWS20</th><th>Bioreactor</th></tr></thead><tbody><tr><td>Daily stimulation</td><td>0</td><td>0</td><td>5 min, twice a day with 6 h intervals</td><td></td><td>30 min, once a day</td></tr><tr><td>Experiment duration (days)</td><td>0</td><td>4</td><td>4</td><td>4</td><td>4</td></tr></tbody></table>

## 2.5. Characterization methods

### 2.5.1. UNDERSKIN: mechanical behavior of the skin samples

Analyzing the mechanical properties of reconstructed human skin is crucial considering its many applications in plastic surgery, dermatology, and cosmetics. Accordingly, UNDERSKIN® 21, a modern, noninvasive, and contact-free methodology, was developed within the LTDS laboratory and patented (Patent: n°2,011,409 November 06, 2020; n°PCT/EP2021/080851 November 05, 2021). A precise, painless blast of air disrupts the reconstructed skin surface, creating ripples. UNDERSKIN® records and measures these ripples with an optical displacement sensor. The 7 mm long optical sensor contains 400 receivers spaced at $dx = 17.5 \ \mu m$ and is located at an offset distance $x_1$ of 0.3 mm from the source—the airflow. The air pressure, sampling rate $f_s$, and impact time were set to 3 bars, 8 kHz, and 7 ms, respectively. The airflow and sensors were attached to a multi-jointed robotic arm. Thus, UNDERSKIN records the displacement as a function of the time and space domain at each receiver location of the sensor line (Fig. 2A). (Bergheau et al., 2022)

Based on Fourier transform computations, UNDERSKIN signal processing enables analyzing surface wave dispersion by calculating the phase velocities as a function of frequency (Fig. 2B). Therefore, the dispersion curve output resulting from the recorded wavefield provides insights into how the particle movements of the medium propagate throughout the reconstructed skin depth. The extraction of dispersion curves from the dispersion image depends on the energy content within the recorded wavefield. Typically, the primary focus is on the dispersion characteristics of the fundamental mode because most inversion methods exclusively deal with the dispersion curve of this particular mode.

The phase velocity for each frequency is determined by identifying the maximum amplitude $A(V_r, f)$. Subsequently, each pair of $V_r, f$ values will be employed to deduce the corresponding wavelength $\lambda$ using the dispersion relation expressed in Equation (1).

$$ \lambda(f) = \frac{V_r(f)}{f} \qquad (1) $$

Currently, phase velocity has been the exclusive indicator of skin elasticity. (Bergheau et al., 2022) It can be converted into shear velocity to calculate the complex shear modulus. This complex shear modulus is significant for delineating the properties of soft tissues, such as skin. The real part of the complex modulus is referred to as the storage modulus.

This study derived the shear wave attenuation and established a model-independent complex modulus using surface wave velocity $V_{shear} = V_{phase} \sqrt{\frac{1}{(0.87+1.2\omega)^2}} \approx 1.02 V_{phase}$ and attenuation data $\beta_{shear}$. Consequently, the expressions for the shear storage $G'$ and loss modulus $G''$ are as follows:

$$ G' = \rho\omega^2 \frac{\left(\frac{\omega}{V_{shear}(\omega)}\right)^2 - \beta_{shear}(\omega)^2}{\left(\left(\frac{\omega}{V_{shear}(\omega)}\right)^2 + \beta_{shear}(\omega)^2\right)^2} \qquad (2) $$

$$ G'' = 2\rho\omega^2 \frac{\left(\frac{\omega}{V_{shear}(\omega)}\right)\beta_{shear}(\omega)}{\left(\left(\frac{\omega}{V_{shear}(\omega)}\right)^2 + \beta_{shear}(\omega)^2\right)^2} \qquad (3) $$
<figure><img src="image_4.png" /><figcaption>Fig. 2. Illustration of the signal processing of UNDERSKIN: (A) Reconstructed skin recorded wavefield, (B) reconstructed skin dispersion image, (C) shear modulus as a function of reconstructed skin depth, and (D) storage modulus map of reconstructed skin.</figcaption></figure>

These data provide supplementary insights into biomarkers for reconstructed skin analyses. Moreover, the parameter $\eta$, as derived from Equation (3), adds another dimension to this analysis, and it is directly related to the viscosity of the medium due to the loss modulus.

$$ \eta(\omega) = \frac{G'(\omega)}{\omega} \qquad (4) $$

Incorporating the inversion model and the viscoelastic model presented by Bergheau et al. (2022), the entire process yields key variations identifying the mechanical behavior across the reconstructed skin layers with a viscoelastic profile and/or a map; Fig. 2C and D shows genuine characterizations of these figures.

In addition, this methodology was validated using viscoelastic phantoms, as introduced by Bergheau et al. (2022) UNDERSKIN results presented identical estimations on the storage modulus and viscosity alike. Ten measurements with exact configurations were performed to ensure repeatability. The values of viscoelastic parameters for each experiment, their standard deviations (SD), and mean values were calculated. Silicon 1 and Silicon 2 have respectively a storage modulus of 4.99 kPa with 0.19 SD and 4.16 kPa with 0.16 SD, and a viscosity equal to 84.7 Pa s with 15.4 SD and 66.6 Pa s with 7.39 SD. The values given by the company who fabricated them were: Silicon 1 and Silicon 2 have respectively a storage modulus of 4.6 kPa and 3.9 kPa with a viscosity equal to 75 Pa s and 63 Pa s.

## 2.5.2. Histology, LC-OCT, and optical microscopic imaging

### 2.5.2.1. Hematoxylin and eosin staining and LC-OCT.
For histology, the samples were fixed in paraffin and cut into 5 µm thick sections. After deparaffinization in xylene and rehydration in graded ethanol, hematoxylin and eosin (H&E) staining was performed to identify and analyze the cellular structures and tissue morphology.

The deepLive™ (DAMAE MEDICAL, France) incorporates LC-OCT technology, which offers a distinctive 3D imaging capability. (Ogien et al., 2020a) This allows users to switch between a vertical mode resembling histology and a horizontal mode resembling confocal microscopy, enabling the recording of a 3D stack of tissue volumes in their native state. (Chauvel-Picard et al., 2022) Thus, 3D imaging was employed to obtain 3D images of T-Skin. Without any fixation, the

stratified layers of the skin samples were examined from the sample surface to a depth of approximately 500 µm with images sized 2 mm × 2 mm at an 800 nm wavelength.

### 2.5.2.2. Second-harmonic generation (SHG) and two-photon excitation fluorescence.
A two-photon confocal microscope (A1R MP Plus®, Nikon, Japan) was used to acquire 3D images of collagen and elastin separately using second-harmonic generation (SHG) and two-photon excitation fluorescence (TPF) signals. Two visualization channels were used at an 850 nm excitation wavelength. The blue channel had a 446 nm emission wavelength for imaging collagen in the skin reconstructed through SHG. The green channel had a 525 nm emission wavelength for elastin prepared using TPF signals. (Abraham and Hogg, 2010; Cui et al., 2014) The samples did not need to be fixed. Images were captured using a Z-stack with slice dimensions of 1024 × 1024 pixels and a 10 µm spacing between each slice. ImageJ software was used to quantify the density of each signal in the XY slice, and the cohesion indices of collagen and elastin were extracted from the XY slice to represent their tension.

### 2.5.2.3. HCS CellMask staining.
Cell staining was required to observe the impact of mechanical stimulation on the quantity, dimensions, and morphology of fibroblasts. In this regard, the HCS CellMaskTM stain (H32714, Thermo Fisher) was identified as suitable for marking the entire cell, thus facilitating a comprehensive analysis of its structure. The ZEISS LSM 800 Airyscan was utilized again, and the green channel had a laser wavelength of 488 nm to visualize cells in the sections. The acquisition area per image was 5 × 5 with 25 images mosaic. The objective had a magnification of 20 × . The number and length of fibroblasts in the images were collected by ImageJ.

## 2.6. Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). The UNDERSKIN data among multiple groups were analyzed using the multipaired sample Friedman test (N ≥ 150). Data from SHG and TPF imaging as well as cell staining in the four groups were compared using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) test (N = 6/10). Asterisks directly above the column graphs indicate the statistical significance of comparisons with the D4-
<figure><img src="image_5.png" /><figcaption>Fig. 3. Shear modulus of the T-Skin epidermis and dermis under the four experimental conditions: Representative LC-OCT images of T-Skin (A) offer information on the epidermis and dermis, according to which the corresponding area in the curve obtained by UNDERSKIN was selected for the calculation of the overall shear modulus within each layer and the results are present in (B) and (C). Significant differences in B, C: P < 0.01 are indicated with two asterisks (multipaired sample Friedman test, N ≥ 150).</figcaption></figure>

Control group, while brackets connected to each group and asterisks above them indicate comparisons between the two linked groups.

## 3. Results

### 3.1. Shear wave stimulations

The reconstructed skin was used for macroscopic skin mechanical behavior measurements and microscopic observations of collagen and elastin. Ensuring that the skin equivalents were uncontaminated during experiments was crucial. Therefore, the entire operation was performed in an aseptic room with sterile synthetic air and a biosafety cabinet. In addition, to prevent contamination, the samples were immediately placed in a sterilized incubator after each stimulus, and the culture medium was changed when necessary during the week-long experiment. These procedures ensured that the T-Skin was uncontaminated throughout the entire process, as shown in the histological images in Fig. S5. A comparison between the samples from the NC and D4-Control groups and a contaminated sample confirmed that the test samples remained uncontaminated throughout the 4-day experimental period. Consequently, the D4-Control group was selected for further analysis and comparison with the stimulation groups.

### 3.2. SKINJ11 and SKINJ18

Owing to the maturation duration, SKINJ11 had a thinner epidermis and dermis than SKINJ18, as shown in the histology and LC-OCT images in Fig. S6. Considering the variability of the skin *in vivo*, the data from SKINJ11 and SKINJ18 in each group are integrated in a later section. This enabled the investigation of the general applicability of SWS. Therefore, only the changes in the shear modulus, cohesion index and fibroblasts of the skin samples before and after mechanical stimulation were considered.

### 3.3. Effect of SWS on the T-Skin mechanical behavior

LC-OCT allows precise navigation over different layers of the reconstructed skin and offers a unique 3D imaging modality (Ogien et al., 2020b) to access the thickness of the epidermis and dermis (Fig. 3A). Moreover, UNDERSKIN can provide the shear modulus as a function of skin depth (Fig. 2C). By combining these two technologies, the T-Skin shear modulus can be analyzed layer-by-layer (Fig. 3B and C).

Based on the thicknesses of the epidermis and dermis acquired through LC-OCT, 150 data points of the shear modulus were selected throughout the depth of the epidermis to represent epidermal shear modulus, whereas 190 data points were selected to delineate the shear modulus of the dermis within the T-Skin model. The two frequencies of SWS significantly increased the shear modulus of the skin equivalent,

with the SWS10 exhibiting a 62.30% increase in the epidermis (P < 0.01) and 56.33% increase in the dermis (P < 0.01). The SWS20 exhibited a 67.95% increase in the epidermis (P < 0.01) and 108.55% increase in the dermis (P < 0.01). The SWS20 more significantly affected shear modulus, resulting in a more substantial change in dermal than in epidermal. Furthermore, a significant difference in the enhancement of skin shear modulus was observed between the compression bioreactor and SWS. Following stimulation with the bioreactor, the T-Skin exhibited a 41.13% increase in epidermal shear modulus. By contrast, a modest increase of 28.12% was observed in its dermal shear modulus.

### 3.4. Effect of SWS on the density of collagen and elastin

Collagen was visualized using SHG imaging (Fig. 4A), and elastin images were acquired using TPF signals (Fig. 4B).

The fluorescence of collagen and elastin was quantified using ImageJ (Fig. 4C and D). The results revealed that the SWS20 samples exhibited 62.22% (P = 0.05) more collagen fibers and 54.8% (P = 0.025) more elastin. By contrast, the collagen and elastin densities remained unchanged in the SWS10 samples. Elastin levels increased by 62.65% in the bioreactor (P = 0.011).

### 3.5. Effect of SWS on the fibroblasts in T-skin

IF staining for fibroblasts can label the entire cell for a more thorough portrayal of the anatomy of fibroblasts (Fig. 5A), which can serve for the analysis of changes in fibroblasts.

After 4 days of stimulation, SWS20 and the Bioreactor increased the number of fibroblasts by 24.34% (P = 0.037) and 23.25% (P = 0.046), respectively (Fig. 5B). However, the compression bioreactor hardly changed the shape of fibroblasts. Both SWS10 and SWS20 significantly led to around a 27% increase (P < 0.005) in fibroblast length (Fig. 5C).

## 4. Discussion

Compression therapy is a medical technique that involves applying pressure to a specific area of the body, often through the use of compression garments or bandages. One potential benefit of compression therapy is its ability to improve the mechanical behavior of skin, (Fournier and Pierard, 2000) particularly in cases where skin health is compromised, such as in specific medical conditions or after surgery. Moreover, compression bioreactors with regulated mechanical loads have been designed to enhance the metabolism of tissue-engineered grafts. (Powell et al., 2010) Dumas et al. (2009) demonstrated that osteoblasts respond well to low frequencies of compression. Tissue-engineered skin is more fragile and easily deteriorated than tissue-engineered bone. Therefore, the T-skin used in this study was compressed at a 1 Hz frequency and a 200 µm displacement. The
<figure><img src="image_6.png" /><figcaption>Fig. 4. Density of collagen and elastin in the T-skin under four experimental conditions. Representative images of collagen (A) and elastin (B) were acquired through biphoton confocal microscopy. Quantification of fluorescence provided information on collagen (C) and elastin (D) densities. Scale bars in (A) and (B): 100 µm. Significant differences in (C) and (D), *P* < 0.01, are denoted by two asterisks. A single asterisk denotes 0.01 < *P* < 0.05 (one-way ANOVA followed by LSD, N = 6).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. Number and length of fibroblast in the T-skin under four experimental conditions. Representative image of fibroblasts (A) was acquired through confocal microscopy. Fibroblasts counting (B) and measurement of the length of fibroblast (C) were carried out by ImageJ. Scale bars in (A): 200 µm. Significant differences in (B) and (C), *P* < 0.01, are denoted by two asterisks. A single asterisk denotes 0.01 < *P* < 0.05 (one-way ANOVA followed by LSD, N = 10).</figcaption></figure>

compression generated under these conditions was the minimum force that could be sensed by a bioreactor force sensor. In addition, by compression over several days, cells have time to react and remodel their extracellular matrix. Therefore, a compression stimulation duration of 30 min/day was set based on the phenomena observed in the pretests lasting 4 days. By contrast, to avoid damage to the reconstructed skin surface, air pressure of 1 bar and loading times of 10 and 20 ms were set for SWS. Therefore, the compression bioreactor applied approximately 0.2 N compression for 30 min/day, whereas SWS applied force ranging from 0.03 N to 0.06 N for 5 min, twice a day. These stimulation conditions differ significantly. However, SWS can achieve a better effect than that of the compression bioreactor in significantly increasing the T-skin shear modulus. It means that the skin becomes stiffer. In some cases, increasing the stiffness of the skin around a wound can provide structural support and stability, reducing mechanical stresses on the wound site. (Evans et al., 2013) This can be particularly beneficial during the early stages of healing to prevent further damage or disruption to the wound. The alteration in skin mechanical properties may also reflect changes in its organization, which are crucial for tissue regeneration and wound closure. (Chong et al., 2019)

At the microscopic level, skin's mechanical behaviors are primarily attributed to the reticular dermis, comprising a thick mesh of collagen bundles anchored by a tense network of elastic fibers. (Aziz et al., 2016) The presence of elastin fibers provides the skin with the ability to recoil,

(Mewes et al., 2007) crucial for the permanent tension of the skin. Scars and stretch marks are characterized by fewer elastin fibers. (Baumann et al., 2021) Collagen fibers offer skin tensile strength and stiffness (Cen et al., 2008) and are extensively used in various biomedical applications, including wound healing and skin regeneration. (Chattopadhyay and Raines, 2014) Therefore, this study also investigated these two elements. The results showed that SWS and the bioreactor increased elastin deposition. However, SWS also increased the collagen levels. The effect of SWS on skin substitutes provides a theoretical basis for its application in skin healing. Furthermore, fibroblasts serve as the principal cellular source for synthesizing the extracellular matrix (ECM) constituents in the skin, which encompass collagen, elastin, and diverse proteoglycans. (Tracy et al., 2016) The study demonstrated a rise in fibroblast count following SWS treatment, mirroring the observed effects of SWS on both collagen and elastin. Besides, some literature reported that mechanical stress can lead to an enlargement of fibroblasts and elongation of their shape, whereas aged skin tends to display a more rounded and collapsed morphology. (Fisher et al., 2016) Consequently, the length of fibroblasts was investigated in our study, and it turns out that the application of SWS results in the elongation of fibroblast length.

In addition, different stimulation effects were observed in the SWS10 and SWS20 groups, indicating the importance of appropriate adjustments to the stimulus intensity. Although a low frequency of SWS increased the shear modulus of the T-Skin, it did not amplify the density
of collagen and elastin. Therefore, this study found SWS20 as the best stimulation condition, and indicates that different stimulus intensities can be selected depending on the objectives.

SWS led to an increase in the shear modulus of both mature skin samples, similar to traditional stimulation methods. Simultaneously, SWS increased the density of two important components, collagen and elastic fibers, while also showing possibility to increase both the number and length of fibroblasts. Based on these two levels, we believe that SWS could be significant for skin healing and stretch mark repair. However, current data proving these applications are unavailable. Therefore, we subsequently conducted *in vivo* stimulation of the human skin in comparison with a well-established massage apparatus to investigate the effects of SWS *in vivo*. Further applications in injured skin models are necessary to prove its efficacy.

## 5. Conclusion

This study developed a novel mechanical stimulation system called SWS that applies shear waves triggered by air pressure to reconstructed skin. Similar to the existing stimulation method (compression device), SWS can elevate the ability of skin grafts to resist external forces, resulting in a microscopic increase in the density of collagen and elastin fibers, along with stimulating fibroblast production and instigating their geometric modifications. However, the SWS offers several key advantages. First, it is a contactless technique that avoids direct contact with the samples to provide a more versatile application. Second, the SWS device allows for the adjustment of stimulus frequency, with changes in air pressure, opening time, and distance from the sample, tailored to diverse needs. Finally, the SWS is mobile, and the robotic arm can be adapted to the size of the area of interest, allowing targeted stimulation of specific regions.

Therefore, SWS has significant potential for laboratory research and clinical therapy applications.

## Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

## CRediT authorship contribution statement

**Na Qiao:** Writing – review & editing, Writing – original draft, Visualization, Software, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Virginia Dumas:** Review, Validation, Supervision, Investigation. **Alexandre Bergheau:** Editing, Visualization, Software, Methodology. **Lucas Ouil-lon:** Software, Methodology, Investigation. **Norbert Laroche:** Resources. **Coralie Privet-Thieulin:** Supervision. **Jean-Luc Perrot:** Software, Resources. **Hassan Zahouani:** Supervision, Resources.

## 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.

## Data availability

Data will be made available on request.

## Acknowledgements

The authors would like to express their gratitude to Amaury Guil-lermin for providing guidance on Matlab, Ianis Ammam for assisting with characterisation, and Roberto Vargiolu for contributing to the design of the SWS. N.Q. would like to acknowledge the China

Scholarship Council for providing the scholarship.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jmbbm.2024.106597.

## Abbreviations

SWS shear wave stimulation

NC negative control

LC-OCT line-field confocal optical coherence tomography

SHG second-harmonic generation

TPF two-photon excitation fluorescence

ANOVA analysis of variance

LSD least significant difference

ECM extracellular matrix.

## References

Abraham, T., Hogg, J., 2010. Extracellular matrix remodeling of lung alveolar walls in three dimensional space identified using second harmonic generation and multiphoton excitation fluorescence. J. Struct. Biol. 171 (2), 189-196.

Alster, T.S., Graham, P.M., 2018. Microneedling: a review and practical guide. Dermatol. Surg. 44 (3), 397.

Aziz, J., Shezali, H., Radzi, Z., Yahya, N.A., Abu Kassim, N.H., Czernuszka, J., et al., 2016. Molecular mechanisms of stress-responsive changes in collagen and elastin networks in skin. Skin Pharmacol. Physiol. 29 (4), 190-203.

Bataillon, M., Lelievre, D., Chapuis, A., Thillou, F., Autourde, J.B., Durand, S., et al., 2019. Characterization of a new reconstructed full thickness skin model, T-Skin™, and its application for investigations of anti-aging compounds. Int. J. Mol. Sci. 20 (9), 2240.

Baumann, L., Bernstein, E.F., Weiss, A.S., Bates, D., Humphrey, S., Silberberg, M., et al., 2021. Clinical relevance of elastin in the structure and function of skin. Aesthetic Surgery Journal Open Forum 3 (3), ojab019.

Bergheau, A., Perrot, J.L., Vargiolu, R., Zahouani, H., 2022. Viscoelasticity assessment of tumoral skin with the use of a novel contact-free palpation methodology based upon surface waves. Sci. Rep. 12 (1), 18716.

Biggs, L.C., Kim, C.S., Miroshnikova, Y.A., Wickström, S.A., 2020. Mechanical forces in the skin: roles in tissue architecture, stability, and function. J. Invest. Dermatol. 140 (2), 284-290.

Boyer, G., Pailler Mattei, C., Molimard, J., Pericoi, M., Laquieze, S., Zahouani, H., 2012. Non contact method for in vivo assessment of skin mechanical properties for assessing effect of ageing. Med. Eng. Phys. 34 (2), 172-178.

Cen, L., Liu, W., Cui, L., Zhang, W., Cao, Y., 2008. Collagen tissue engineering: development of novel biomaterials and applications. Pediatr. Res. 63 (5), 492-496.

Chattopadhyay, S., Raines, R.T., 2014. Collagen-based biomaterials for wound healing. Biopolymers 101 (8), 821-833.

Chauvel-Picard, J., Bérot, V., Tognetti, L., Orte Cano, C., Fontaine, M., Lenoir, C., et al., 2022. Line-field confocal optical coherence tomography as a tool for three-dimensional in vivo quantification of healthy epidermis: a pilot study. J. Biophot. 15 (2), e202100236.

Chong, C., Wang, Y., Fathi, A., Parungao, R., Maitz, P.K., Li, Z., 2019. Skin wound repair: results of a pre-clinical study to evaluate electropsun collagen–elastin–PCL scaffolds as dermal substitutes. Burns 45 (7), 1639-1648.

Cui, J.Z., Tehrani, A.Y., Jett, K.A., Bernatchez, P., van Breemen, C., Esfandiari, M., 2014. Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy. J. Struct. Biol. 187 (3), 242-253.

Davis, L.C., Baumer, T.G., Bey, M.J., van Holmsbeek, M., 2019. Clinical utilization of shear wave elastography in the musculoskeletal system. Ultrasonography 38 (1), 2-12.

Djaghloul, M., Abdouni, A., Thieulin, C., Zahouani, H., 2018. Wave propagation as a marker of structural and topographic properties of human skin. Surf. Topogr. Metrol. Prop 6 (2), 024008.

Djaghloul, M., Abdouni, A., Thieulin, C., Pailler-Mattei, C., 2020. Surface wave investigation and phenomenological analysis: application on in vivo human cutaneous tissue. J. Mech. Behav. Biomed. Mater. 109, 103779.

Dumas, V., Perrier, A., Malaval, L., Laroche, N., Guignandon, A., Vico, L., et al., 2009. The effect of dual frequency cyclic compression on matrix deposition by osteoblast-like cells grown in 3D scaffolds and on modulation of VEGF variant expression. Biomaterials 30, 3279-3288.

Dunn, S.L., Olmedo, M.L., 2016. Mechanotransduction: relevance to physical therapist practice—understanding our ability to affect genetic expression through mechanical forces. Phys. Ther 96 (5), 712-721.

Evans, N.D., Oreffo, R.O.C., Healy, E., Thurner, P.J., Man, Y.H., 2013. Epithelial mechanobiology, skin wound healing, and the stem cell niche. J. Mech. Behav. Biomed. Mater. 28, 397-409.

Fisher, G.J., Shao, Y., He, T., Qin, Z., Perry, D., Voorhees, J.J., et al., 2016. Reduction of fibroblast size/mechanical force down-regulates TGF-β type II receptor: implications for human skin aging. Aging Cell 15 (1), 67-76.
Fournier, R., Pierard, G.E., 2000. Skin tensile strength modulation by compressive garments in burn patients. A pilot study. J. Med. Eng. Technol. 24 (6), 277-280.

Goodarzi, P., Falahzadeh, K., Nematizadeh, M., Farazandeh, P., Payab, M., Larijani, B., et al., 2018. Tissue engineered skin substitutes. Adv. Exp. Med. Biol. 1107, 143-188.

Iskratsch, T., Wolfenson, H., Sheetz, M.P., 2014. Appreciating force and shape — the rise of mechanotransduction in cell biology. Nat. Rev. Mol. Cell Biol. 15 (12), 825-833.

Khalid, F.A., Ahmad, S., Mehrose, M.Y., Saleem, M., Yousaf, M.A., Mujahid, A.M., et al., 2019. Efficacy of micro-needling on post acne scars. J. Ayub Med. Coll. Abbottabad 31 (3), 336-339.

Khan, K.M., Scott, A., 2009. Mechanotherapy: how physical therapists' prescription of exercise promotes tissue repair. Br. J. Sports Med. 43 (4), 247-252.

Leyva-Mendivil, M.F., Page, A., Bressloff, N.W., Limbert, G., 2015. A mechanistic insight into the mechanical role of the *stratum corneum* during stretching and compression of the skin. J. Mech. Behav. Biomed. Mater. 49, 197-219.

Martin, P., 1997. Wound healing-aiming for perfect skin regeneration. Science 276 (5309), 75-81.

Mewes, K.R., Raus, M., Bernd, A., Zöller, N.N., Sättler, A., Graf, R., 2007. Elastin expression in a newly developed full-thickness skin equivalent. SPP 20 (2), 85-95.

Moortgat, P., Anthonissen, M., Meirte, J., Van Daele, U., Maertens, K., 2016. The physical and physiological effects of vacuum massage on the different skin layers: a current status of the literature. Burns & Trauma 4, 1-12.

Ogien, J., Daures, A., Cazalas, M., Perrot, J.L., Dubois, A., 2020a. Line-field confocal optical coherence tomography for three-dimensional skin imaging. Front. Optoelectron. 13 (4), 381-392.

Ogien, J., Levecq, O., Azimani, H., Dubois, A., 2020b. Dual-mode line-field confocal optical coherence tomography for ultrahigh-resolution vertical and horizontal section imaging of human skin in vivo. Biomed Opt Express, BOE 11 (3), 1327-1335.

Okuda, I., Takeda, M., Taira, M., Kobayashi, T., Inomata, K., Yoshioka, N., 2022. Objective analysis of the effectiveness of facial massage using breakthrough computed tomographic technology: a preliminary pilot study. Skin Res. Technol. 28 (3), 472-479.

Pajak, J., Szepietowski, J.C., Nowicka, D., 2022. Prevention of ageing—the role of micro-needling in neck and cleavage rejuvenation: a narrative review. Int. J. Environ. Res. Publ. Health 19 (15), 9055.

Park, Y., Kwon, K., Kwak, S.S., Yang, D.S., Kwak, J.W., Luan, H., et al., 2020. Wireless, skin-interfaced sensors for compression therapy. Sci. Adv. 6 (49), eabe1655.

Powell, H.M., McFarland, K.L., Butler, D.L., Supp, D.M., Boyce, S.T., 2010. Uniaxial strain regulates morphogenesis, gene expression, and tissue strength in engineered skin. Tissue Eng. 16 (3), 1083-1092.

Romanelli, M., Manzo Margiotta, F., Michelucci, A., Granieri, G., Janowska, A., Dini, V., 2023. Compression therapy in dermatology. Curr Derm Rep 12 (2), 33-37.

Shevchenko, R.V., James, S.L., James, S.E., 2010. A review of tissue-engineered skin bioconstructs available for skin reconstruction. J R Soc Interface 7 (43), 229-258.

Taljanovic, M.S., Gimber, L.H., Becker, G.W., Latt, L.D., Klauser, A.S., Melville, D.M., et al., 2017. Shear-wave elastography: basic physics and musculoskeletal applications. Radiographics 37 (3), 855-870.

Tamayol, A., Akbari, M., Annabi, N., Paul, A., Khademhosseini, A., Juncker, D., 2013. Fiber-based tissue engineering: progress, challenges, and opportunities. Biotechnol. Adv. 31 (5), 669-687.

Trabelsi, O., Dumas, V., Breysse, E., Laroche, N., Avril, S., 2020. In vitro histomechanical effects of enzymatic degradation in carotid arteries during inflation tests with pulsatile loading. J. Mech. Behav. Biomed. Mater. 103, 103550.

Tracy, L.E., Minasian, R.A., Caterson, E.J., 2016. Extracellular matrix and dermal fibroblast function in the healing wound. Adv. Wound Care 5 (3), 119-136.

Wang, N., 2017. Review of cellular mechanotransduction. J. Phys. D Appl. Phys. 50 (23), 233002.

Wang, Y., Marshall, K.L., Baba, Y., Gerling, G.J., Lumpkin, E.A., 2013. Hyperelastic material properties of mouse skin under compression. PLoS One 8 (6), e67439.

Wong, V.W., Akaishi, S., Longaker, M.T., Gurtner, G.C., 2011. Pushing back: wound mechanotransduction in repair and regeneration. J. Invest. Dermatol. 131 (11), 2186-2196.

Wu, J.Z., Cutlip, R.G., Andrew, M.E., Dong, R.G., 2007. Simultaneous determination of the nonlinear-elastic properties of skin and subcutaneous tissue in unconfined compression tests. Skin Res. Technol. 13 (1), 34-42.