## 3 Results
### Effects of Ultrasound on Microtissue Assembly are Temperature-Dependent and Spatially Localized  
Previous studies demonstrated that ultrasound can influence collagen structure and function through both thermal [27] and non-thermal mechanisms [28]. Thus, studies were conducted to determine whether the ability of ultrasound-fabricated collagen/fibronectin hydrogels to accelerate microtissue formation were mediated via thermal or non-thermal mechanisms. To do so, we first measured the temperature rise within collagen gels that occurred in response to the acoustic exposure conditions used in this study. Collagen gels polymerized in the presence of 8.8-MHz ultrasound at an intensity of 8.2 W/cm\({}^{2}\) heated to a final temperature of 32.3 \({}^{\circ}\)C, representing a 7.7 \({}^{\circ}\)C increase above corresponding sham-exposures (Table 1). Sham-exposed collagen gels polymerized in a 37 \({}^{\circ}\)C water tank heated to a temperature of 34.5 \({}^{\circ}\)C by the end of the 15 min exposure period (Table 1). Therefore, in subsequent experiments, sham-exposed gels polymerized in a 37 \({}^{\circ}\)C water were included as temperature-matched control conditions to recapitulate the maximal temperature rise induced by ultrasound at an intensity of 8.2 W/cm\({}^{2}\).  
Ultrasound-induced heating within collagen samples is highest at the center of the beam and lower at locations outside of the half-maximal beam width [27]. Thus, we next asked whether microtissue formation on ultrasound-fabricated gels was spatially localized to the acoustic beam area. Cells that had adhered to the center of collagen-fibronectin gels fabricated in the presence of 8.8-MHz ultrasound (8.2 W/cm\({}^{2}\)) formed microtissues within 24 h of culture (Figure 3A, 8.2 W/cm\({}^{2}\), center). In contrast, cells that had adhered to regions of the gel outside of the area affected by the acoustic beam formed interconnected adherent networks rather than 3D microtissues (Figure 3A, 8.2 W/cm\({}^{2}\), edge). To quantify differences in microtissue formation as a function of acoustic beam width, cell-occupied area was determined for sham- and ultrasound-exposed gels within non-overlapping ROIs beginning at the center of the gel and moving off-center in 1.6-mm steps. On ultrasound-exposed gels, a gradient of microtissue formation occurred along the radius of the gel, with less than 10% cell-occupied area at the acoustic exposure location increasing to \(\sim\)18% at the gel edge (Figure 3, 8.2 W/cm\({}^{2}\), gray bars). Sham-exposed gels, polymerized at a temperature similar to that achieved at the center of the ultrasound beam, supported microtissue formation uniformly across the entire gel surface (Figure 3A, 35 \({}^{\circ}\)C Sham) and exhibited a similar cell-occupied area as the center ROI on ultrasound-exposed gels (Figure 3B). In contrast, cells seeded on sham-exposed gels, polymerized at a temperature comparable to that observed outside of the acoustic beam, did not form microtissues and (Figure 3A, 25 \({}^{\circ}\)C Sham) and occupied greater than 20% of the surface area at all locations (Figure 3B). Together, these data suggest that changes in collagen-fibronectin gel structure that support accelerated microtissue formation are mediated through a thermal acoustic mechanism, and furthermore, are spatially localized to regions within and immediately adjacent to the half-maximal acoustic beam width.