## 2 Materials and Methods
### Fabrication of Composite Collagen-Fibronectin Hydrogels  
Collagen hydrogels were polymerized in the presence of ultrasound, as described previously [28] with the following modifications. Neutralized collagen solutions were prepared by combining equal volumes of type I rat tail collagen (Corning, Lowell, MA, USA) with 2X [PERSON]'s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA). Collagen concentration was adjusted to 2 mg/mL by addition of fibroblast culture media (1:1 mixture of AimV (Invitrogen) and SF Medium (Corning) containing 25 mM HEPES). The pH of the solution was adjusted to 7.4 by addition of 0.1 N NaOH. Collagen solutions were maintained on ice until gel fabrication. Fibronectin was purified from outdated human plasma (American Red Cross, Rochester, NY, USA) using gelatin-Sepharose (GE Healthcare, New York, NY, USA) affinity chromatography, as described [18]. Fibronectin (25 mg/mL final concentration) was added to soluble, neutralized collagen immediately prior to ultrasound exposure.  
Collagen-fibronectin hydrogels were fabricated within one well of a modified elastomer-bottomed tissue culture plate. Plates were placed within the acoustic exposure tank using a three-axis positioner (Velmex, Leatherhead, UK, Series B4000 Unislide) such that the center of the well was in contact with the water bath and the acoustic beam was centered within the well. The temperature of the water in the exposure tank was set to 18, 25, or 37 \({}^{\circ}\)C. Hydrogel polymerization was initiated in response to the temperature rise in the sample upon transferring collagen-fibronectin solutions to the exposure well [29]. Samples were exposed to continuous wave ultrasound for 15 min, which was sufficient  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Water Tank** & **Acoustic Intensity** & **Peak Positive** & **Peak Negative** & **Final Temperature** \\
**Temperature** & **(I\({}_{\text{SPPA}}\))** & **Pressure** & **Pressure** & **Pressure** & **Final Temperature** \\ \hline
18 \({}^{\circ}\)C & 8.2 \(\pm\) 0.1 W/cm\({}^{2}\) & 0.76 \(\pm\) 0.02 MPa & 0.44 \(\pm\) 0.02 MPa & 24.2 \(\pm\) 0.3 \({}^{\circ}\)C \\
25 \({}^{\circ}\)C & Sham (0 W/cm\({}^{2}\)) & - & - & 24.6 \(\pm\) 0.1 \({}^{\circ}\)C \\
25 \({}^{\circ}\)C & 3.8 \(\pm\) 0.1 W/cm\({}^{2}\) & 0.48 \(\pm\) 0.02 MPa & 0.28 \(\pm\) 0.02 MPa & 27.7 \(\pm\) 0.1 \({}^{\circ}\)C \\
25 \({}^{\circ}\)C & 8.2 \(\pm\) 0.1 W/cm\({}^{2}\) & 0.76 \(\pm\) 0.02 MPa & 0.44 \(\pm\) 0.02 MPa & 32.3 \(\pm\) 0.2 \({}^{\circ}\)C \\
37 \({}^{\circ}\)C & Sham (0 W/cm\({}^{2}\)) & - & - & 34.5 \(\pm\) 0.1 \({}^{\circ}\)C \\ \hline \hline \end{tabular}
\end{table}
Table 1: Summary of acoustic exposure conditions. Ultrasound fields were generated using an unfocused piezoceramic transducer with diameter of 1 cm and a fundamental frequency of 8.8 MHz. Fields were calibrated using a capsule hydrophone at a position in the free field 10.5 cm from the surface of the transducer. The temperature within a polymerizing collagen sample (2 mg/mL) was measured under each set of exposure conditions using a wire thermocouple embedded at the center of the gel. Data are presented as mean \(\pm\) SEM for \(n\geq\) 9 (intensity and pressure) or \(n\geq\) 3 (temperature) measurements per condition on independent experimental days.  
time for the sample to transition from a fluid solution to a solid hydrogel. Sham-exposed hydrogels were fabricated using the same exposure system and identical protocols, but the ultrasound was not activated. Collagen-fibronectin hydrogels were placed in a humidified incubator (37 \({}^{\circ}\)C, 8% CO2) overnight to ensure complete collagen polymerization before subsequent experiments.