## 3 Results
### Acoustic Modification Reduces Initial Adhesion to Collagen-Fibronectin Hydrogels
Quantification of cell-occupied area demonstrated a significant reduction in cell-occupied area on reduced-temperature, ultrasound-exposed gels compared to the corresponding, temperature-matched sham conditions (Figure 5B, 24-25 \({}^{\circ}\)C). In contrast, no difference in cell-occupied area was observed on the temperature-matched ultrasound and sham-exposed gels polymerized under conditions that exceeded 30 \({}^{\circ}\)C, wherein both gels supported microtissue formation (Figure 5B, 32-35 \({}^{\circ}\)C). Thus,  
Figure 4: Ultrasound exposure decreases initial cell attachment strength. Neutralized solutions of collagen (2 mg/mL) and fibronectin (25 \(\upmu\)g/mL) were co-polymerized during exposure to 8.8-MHz ultrasound at an intensity of 8.2 W/cm\({}^{2}\) in a 25 \({}^{\circ}\)C water bath, or polymerized under sham conditions in a heated water bath to achieve polymerization temperatures corresponding to the temperature achieved at the center or edge of the ultrasound-exposed gel. The peak temperature achieved within the gel sample under each set of polymerization conditions is indicated in parentheses. FN-null MEFs (2 \(\times\) 10\({}^{4}\) cells/cm\({}^{2}\)) were seeded onto polymerized gels and allowed to adhere for 10 min before the number of adherent cells was determined. Data are presented as mean \(\pm\) SEM for \(n\geq\) 7 gels fabricated on 4 independent experimental days. Significantly different means, \({}^{*}p<\) 0.05 vs. 25 \({}^{\circ}\)C sham by one-way ANOVA with [PERSON]’s post-hoc test. at fabrication temperatures below \(\sim\)30 \({}^{\circ}\)C, ultrasound-mediated effects on the functional properties of collagen-fibronectin hydrogels are mediated by non-thermal mechanisms. To investigate non-thermal effects of ultrasound on fibronectin structure within 3D hydrogels, low-temperature, ultrasound-exposed (8.8 MHz, 8.2 W/cm\({}^{2}\), 24 \({}^{\circ}\)C peak temperature) composite hydrogels were fabricated, and the structures of both fibronectin and collagen were visualized with multiphoton microscopy. Under these fabrication conditions, high-intensity fibrillar fibronectin staining was observed (Figure 6, 8.2 W/cm\({}^{2}\), arrows). These fibronectin fibrils localized primarily  
Figure 5: Mechanical and thermal effects of ultrasound can be uncoupled by reducing polymerization temperature. Neutralized solutions of type I collagen (2 mg/mL) and fibronectin (25 mg/mL) were exposed either to 8.8-MHz ultrasound at an intensity of 8.2 W/cm\({}^{2}\), or sham-exposure conditions. The peak temperature achieved within the sample was adjusted by manipulating the water tank temperature and is reported for each condition in parentheses. FN-null MEFs (\(4\times 10^{4}\) cells/cm\({}^{2}\)) were seeded on the surfaces of fully polymerized hydrogels. (**A**) Phase-contrast images were collected 24 h post-seeding at a location corresponding to the center of the acoustic exposure. Scale bar = 100 \(\upmu\)m. (**B**) The area occupied by cells within a ROI corresponding to the center of the gel was quantified by tracing the outlines of cell clusters and measuring the percentage of the cell-occupied surface area for ultrasound-exposed (black) and sham (white) gels for each polymerization condition. Data are presented as mean \(\pm\) SEM for \(n\geq 3\) gels fabricated on independent days. Significantly different means, \(p<0.05\) by two-tailed t-test. n.s., not significant. to the periphery of pores outlined by dense collagen fibers, and bridged gaps between adjacent collagen fiber bundles (Figure 6, 8.2 W/cm\({}^{2}\), Merge, arrows). In contrast, gels polymerized under temperature-matched, sham-exposure conditions exhibited diffuse, homogeneous fibronectin staining that largely co-localized with the underlying short collagen fibers, with no evidence of fibronectin fibril formation (Figure 6, Sham, Merge).