## 4 Discussion  
Previous work demonstrated that ultrasound can influence the microstructure of collagen via distinct thermal [27] and non-thermal mechanisms [28]. Here, acoustically modified hydrogels were manufactured under two sets of temperature conditions to assess the effects of ultrasound on collagen and fibronectin structure and function. Results show that exposing solutions of collagen and fibronectin to ultrasound during co-polymerization at temperatures greater than 30 \({}^{\circ}\)C produced composite hydrogels that supported accelerated microtissue formation and reduced cell adhesion. Similar effects were observed with composite hydrogels polymerized under temperature-matched sham conditions, indicating that the structural changes responsible for both accelerated microtissue formation and reduced adhesion are mediated by a thermal acoustic mechanism. Figure 6: Mechanical effects of ultrasound trigger fibronectin fibril formation under permissive temperature conditions. Neutralized solutions of type I collagen (2 mg/mL) and fibronectin (25 mg/mL, 10% AF546-labeled) were co-exposed to either 8.8-MHz ultrasound at an intensity of 8.2 W/cm\({}^{2}\) in an 18 \({}^{\circ}\)C water bath or sham-exposed in a 25 \({}^{\circ}\)C water bath such that the peak temperature achieved within each sample during exposure was 24–25 \({}^{\circ}\)C. Unbound fibronectin was removed by media exchange prior to fixation and multiphoton imaging of collagen (white) and fibronectin (red) and fiber structures. Maximum intensity z-projection images through a depth of 70 \(\upmu\)m (5-\(\upmu\)m z-steps) at the gel surface were assembled using FIJI software. Representative images from 1 of 3 samples per condition fabricated on independent days. Scale bar = 100 \(\upmu\)m. Arrows indicate the presence of fibronectin fibrils. Acoustically modified composite hydrogels fabricated in the presence of ultrasound under temperature conditions below 30 \({}^{\circ}\)C also supported microtissue formation, but this was not observed on corresponding temperature-matched sham hydrogels (Figure 5). Multiphoton imaging of composite hydrogels polymerized under these reduced-temperature conditions revealed the formation of fibronectin fibrils at the hydrogel surface. The formation of fibronectin fibers was observed only on ultrasound-exposed gels polymerized under low-temperature conditions. In contrast, low-intensity fibronectin staining that co-localized diffusely with collagen fibers was observed under ultrasound exposure conditions in which temperature exceeded 30 \({}^{\circ}\)C (Figure 1) as well as temperature-matched sham gels (Figure 6). These data suggest that under permissive temperature conditions, ultrasound can influence fibronectin structure and function via a non-thermal acoustic mechanism. Fibronectin fibril assembly by cells is a highly regulated process requiring both integrin ligation and cell-derived mechanical forces [13]. Several acellular techniques to produce artificial fibronectin fibrils have been reported, typically via the application of mechanical force at a fluid-air interface [24; 38; 39; 40]. To the best of our knowledge, the importance of a fluid-air interface in acellular models of fibronectin assembly has not been investigated systematically. However, application of mechanical forces that lead to exposure of cryptic fibronectin self-association sites is an essential step in the formation of both cell-derived and artificial fibronectin matrices [41]. Of note, the fibronectin fibers observed in the present study were primarily at or near the hydrogel surface boundary. As such, we speculate that ultrasound-driven fluid streaming at the fluid-air interface may be a mechanism to induce fibronectin self-association. Potential contributions of other non-thermal effects of ultrasound, such as acoustic cavitation, may also play a role in fibril formation by producing localized mechanical forces on collagen, fibronectin, or both proteins to trigger fibril formation.