Optical Coherence Elastography (OCE).

Figure S10 shows a schematic of the custom-built OCE system. Wave excitation was generated using a 7 × 7 × 42 mm³ Piezo Stack Actuator (PiezoDrive, Newcastle, Australia) attached to a needle that was placed in direct contact with the hydrogel surface. Wave propagation was detected using a phase-sensitive Optical Coherence Tomography (PhS-OCT) system with ~9 μm axial resolution (in air), ~8 μm transversal resolution, and 0.28 nm of displacement stability. The A-line acquisition was set to 25 kHz during OCE acquisition. The piezo stack was driven by 5 pulses at 1 kHz, which was generated by a DG4162 Waveform Generator (RIGOL Technologies, Suzhou, China) and amplified by a PDu150 Three-Channel 150 V piezo drive (PiezoDrive, Newcastle, Australia). The burst of the signal was synchronized with the OCT frame trigger during the M-B mode scan to scan 500 points over 2.5 mm laterally with each M-mode scan consisting of 500 A-lines.

OCE is the elastography extension of optical coherence tomography (OCT), where the A-line constitutes the depth information (z-axis) of a sample at a given lateral position. The A-line is complex-valued, where the magnitude represents the depth-dependent intensity of the backscattered light, and its phase can be used to track the tissue displacement induced by the excitation in the tissue.⁵⁷ For two consecutive moments (t0 and t1), where t0<t1 at a particular lateral position (t0) and where a certain number of A-lines were acquired over time, the backscattered light’s axial temporal phase difference is Δφ(z)=φz,t1-φz,t2, and the particle velocity is calculated as vz(z,t)=Δφ(z,t)λ0/(4πΔt), where Δt is the resolution time and λ0 is the source central wavelength.⁵⁸ For all lateral positions, where in each of them, multiple A-lines were collected, the phase difference (Δφ) of the A-line complex values provides the particle velocity used to build spatiotemporal images.

Axial particle velocities vz were calculated based on the depth-dependent phase (φ) difference between two consecutive A-line complex values, vz(z,t)=Δφ(z,t)λ0/(4πΔt), using n=1.37 as the refractive index for phosphate-buffered saline gelation baths,⁵⁹ Δt≈40μs (temporal resolution), and λ0=840nm for the central wavelength of the OCT light source. Wave velocities were computed as the slope of the wave propagation on spatiotemporal images. Since K2 hydrogels were immersed in phosphate-buffered saline gelation baths, the scan and wave propagation areas were under liquid. Therefore, the Scholte wave model was used to describe the elastic properties of the hydrogels, where the shear wave velocity Cs is related to the Scholte wave speed Csch by Csch=0.846Cs.⁶⁰ The Scholte Young’s modulus (E) was calculated with the formula: E=3ρ/0.8462×Csch2, assuming a hydrogel density (ρ) of 1000 kg/m³.⁴¹ All calculations were performed in MATLAB 2020b (Mathworks Inc., Natick, MA).