## 3 Results
### Acoustic Modification Reduces Initial Adhesion to Collagen-Fibronectin Hydrogels  
Cellular self-assembly in response to fibronectin is influenced, in part, by the adhesive properties of the substrate, wherein reduced adhesion strength correlates with enhanced microtissue assembly [35; 36]. To determine whether ultrasound exposure affects adhesive properties of acoustically modified collagen-fibronectin hydrogels, cell adhesion assays were conducted. As shown in Figure 4, the number of adherent cells was significantly reduced on collagen-fibronectin gels fabricated using 8.8-MHz ultrasound at an intensity of 8.2 W/cm\({}^{2}\) compared to sham-exposed gels polymerized at 25 \({}^{\circ}\)C (Figure 4, 25 \({}^{\circ}\)C sham vs. 8.2 W/cm\({}^{2}\)). A similar reduction in cell adhesion was observed on gels polymerized under sham conditions that mimicked the temperature rise induced by ultrasound exposure (Figure 4, 25 vs. 35 \({}^{\circ}\)C sham). These results are consistent with previous demonstrations that decreases in the initial cell-substrate binding strength are permissive for spheroidal microtissue formation [35],  
Figure 3: Accelerated microtissue formation on acoustically modified gels is localized to the acoustic beam width and dependent on polymerization temperature. Neutralized solutions of type I collagen (2 mg/mL) and fibronectin (25 \(\upmu\)g/mL) were co-exposed to 8.8-MHz ultrasound at an intensity of 8.2 W/cm\({}^{2}\) in a 25 \({}^{\circ}\)C water tank. Sham-exposed samples were polymerized with the water temperature set to either 25 or 37 \({}^{\circ}\)C to achieve polymerization temperatures corresponding to the temperature achieved at the center (32 \({}^{\circ}\)C) or edge (25 \({}^{\circ}\)C) of the ultrasound-exposed gel. FN-null MEFs (4 \(\times\) 10\({}^{4}\) cells/cm\({}^{2}\)) were seeded on the surfaces of polymerized hydrogels and cultured for 24 h. (**A**) Phase-contrast images were collected beginning at a position corresponding to the acoustic exposure location (position 0), and moving off-center radially in 1.6 mm steps to the edge of the gel (positions 1–4). Scale bar = 200 \(\upmu\)m. (**B**) The area occupied by cells within each ROI was quantified by tracing the outlines of cell clusters and measuring the percentage of the surface area occupied by cells. Data are presented as mean \(\pm\) SEM for \(n=4\) gels fabricated on independent days. Significantly different means, \({}^{*}p<0.05\) by repeated measures ANOVA with [PERSON]’s post-hoc test. and provides further evidence that ultrasound exposure can accelerate microtissue formation via thermal effects on the collagen-fibronectin substrate. Mechanical Effects of Ultrasound on Fibronectin Structure are Observed at Reduced Polymerization Temperature  
The conformations of both collagen and fibronectin are sensitive to temperature, as is the affinity of the interaction between the two proteins [22; 23; 37]. As temperatures are reduced from 33 to 30 \({}^{\circ}\)C, intact fibronectin undergoes a conformational change that increases its affinity for collagen by \(\sim\)10-fold [22]. Therefore, we next performed experiments to investigate the bioactivity of acoustically modified collagen-fibronectin hydrogels fabricated under temperature conditions that were not allowed to exceed 30 \({}^{\circ}\)C. To do so, collagen-fibronectin solutions were exposed to 8.8-MHz ultrasound (8.2 W/cm\({}^{2}\)) in a water tank cooled to 18 \({}^{\circ}\)C. Under these conditions, the peak temperature achieved within the gel sample was 24 \({}^{\circ}\)C, similar to the temperature achieved within a 25 \({}^{\circ}\)C water bath under sham-exposure conditions (Table 1). Cells cultured for 24 h on the surface of these reduced-temperature, ultrasound-exposed collagen-fibronectin gels assembled into elongated, tightly adherent microtissues (Figure 5A, 8.2 W/cm\({}^{2}\), 24 \({}^{\circ}\)C). In contrast, cells cultured for 24 h on temperature-matched sham-exposed gels formed 2D interconnected networks (Figure 5A, 25 \({}^{\circ}\)C Sham).