Dual optical elastography detects TGF - β -induced alterations in the biomechanical properties of skin scaffolds

Abstract

Significance: The skin's mechanical properties are tightly regulated. Various pathologies can affect skin stiffness, and understanding these changes is a focus in tissue engineering. Ex vivo skin scaffolds are a robust platform for evaluating the effects of various genetic and molecular interactions on the skin. Transforming growth factor-beta ( $\mathrm{TGF}\text{-}\beta$ ) is a critical signaling molecule in the skin that can regulate the amount of collagen and elastin in the skin and, consequently, its mechanical properties.

Aim: This study investigates the biomechanical properties of bio-engineered skin scaffolds, focusing on the influence of $\mathrm{TGF}\text{-}\beta$ , a signaling molecule with diverse cellular functions.

Approach: The $\mathrm{TGF}\text{-}\beta$ receptor I inhibitor, galunisertib, was employed to assess the mechanical changes resulting from dysregulation of $\mathrm{TGF}\text{-}\beta$ . Skin scaffold samples, grouped into three categories (control, $\mathrm{TGF}\text{-}\beta$ -treated, and $\mathrm{TGF}\text{-}\beta$ + galunisertib-treated), were prepared in two distinct culture media-one with aprotinin (AP) and another without. Two optical elastography techniques, namely wave-based optical coherence elastography (OCE) and Brillouin microscopy, were utilized to quantify the biomechanical properties of the tissues.

Results: Results showed significantly higher wave speed (with AP, $p<0.001$ ; without AP, $p<0.001$ ) and Brillouin frequency shift (with AP, $p<0.001$ ; without AP, $p=0.01$ ) in $\mathrm{TGF}\text{-}\beta$ -treated group compared with the control group. The difference in wave speed between the control and $\mathrm{TGF}\text{-}\beta$ + galunisertib with ( $p=0.10$ ) and without AP ( $p=0.36$ ) was not significant. Moreover, the $\mathrm{TGF}\text{-}\beta$ + galunisertib-treated group exhibited lower wave speed without and with AP and reduced Brillouin frequency shift than the $\mathrm{TGF}\text{-}\beta$ -treated group without AP, further strengthening the potential role of $\mathrm{TGF}\text{-}\beta$ in regulating the mechanical properties of the samples.

Conclusions: These findings offer valuable insights into $\mathrm{TGF}\text{-}\beta$ -induced biomechanical alterations in bio-engineered skin scaffolds, highlighting the potential of OCE and Brillouin microscopy in the development of targeted therapies in conditions involving abnormal tissue remodeling and fibrosis.

Keywords: Brillouin microscopy; bioengineered skin; elasticity; optical coherence elastography; tissue scaffold.

Fig. 1

(a) Schematic of the ACUS-OCE system consisting of a hemispherical ACUS transducer to induce elastic waves in the skin scaffold and a PhS-OCT system to track the mechanical wave. The ACUS transducer had a $\sim 10\text{-}\mathrm{mm}$ apical opening, which allows confocal OCT imaging. During imaging, the transwell was inverted with the basal side facing the imaging and excitation beams, and hence, the dermis layer was directly imaged. (b) Schematic of the Brillouin/OCT system. OCT was used to align the sample for Brillouin imaging. ARF, acoustic radiation force; AT, acoustic transducer; C, collimator; DAC, digital to analog converter; D, dichroic mirror; EMCCD, electron-multiplying CCD; FC, fiber coupler; FG, function generator; GS, 2D galvo scanner; L, lens; OL, objective lens; PBS, polarizing beam splitter; PC, polarization controller; RF, power amplifier; RM, reference mirror; S, SPEC, spectrometer; SLD, superluminescent diode; T, Transwell insert; VA, variable attenuator; VIPA, virtually imaged phase array; $\lambda /4$, quarter wave plate. MS Word 2010 Home ribbon. The red arrow indicates where to access the Styles window.

Fig. 2

(a) 3D rendering of a typical skin scaffold sample obtained via 3D OCT scanning from the inverted (basolateral side) of the transwell. (b) Representative cross-sectional image of the skin scaffold showing various in-depth regions, including the dermis, epidermis, and membrane layers. Elastic wave speed analysis was performed in the upper portion of the dermis layer, identified as OCE ROI in the cross-sectional image.

Fig. 3

Typical B-mode images (top row), wave propagation snapshots (middle row), and speed maps (bottom row) for (a) CTL, (b) $\mathrm{TGF}\text{-}\beta$, and (c) $\mathrm{TGF}\text{-}\beta$ + galunisertib with aprotinin (AP). ACUS excitation points are indicated by the yellow arrows. In the middle panel, the wave propagation snapshots ($t=7.48\mathrm{ms}$ for CTL, $t=6.08\mathrm{ms}$ for $\mathrm{TGF}\text{-}\beta$, and $t=7.16\mathrm{ms}$ for $\mathrm{TGF}\text{-}\beta$ + galunisertib after the start of excitation) show differences in the wavelengths of the waves. The bottom row depicts elastic wave speed maps overlaid on the B-mode images. The scale bar is $100\mu \mathrm{m}$.

Fig. 4

Brillouin frequency shift map of (a) CTL, (b) $\mathrm{TGF}\text{-}\beta$, (c) $\mathrm{TGF}\text{-}\beta$ + galunisertib with AP, (d) CTL, (e) $\mathrm{TGF}\text{-}\beta$, and (f) $\mathrm{TGF}\text{-}\beta$ + galunisertib without AP bio-engineered skin samples. Image size: $1.3\mathrm{mm}\times 0.3\mathrm{mm}$ (width × height).

Fig. 5

The biomechanical properties of the bio-engineered skin scaffolds, represented by (a) elastic wave speed and (b) average Brillouin frequency shift measured using OCE and Brillouin microscopy, respectively. The samples were categorized into two groups: with aprotinin (AP) and without (no AP). Outliers are indicated by *. The number of samples, $N$, vary between 12 and 19 among groups, as certain samples were discarded because of damage.