## 3 Results  
Ultrasound Exposure Produces Changes in Collagen-Fibronectin Fiber Structure that Support Accelerated Microtissue Formation  
In previous work, we demonstrated that ultrasound exposure during collagen hydrogel formation can produce functional changes in the organization of collagen fibers [28]. However, in healing dermal wounds, fibronectin often co-localizes with collagen fibers [33] and collagen fibril formation and remodeling by cells requires fibronectin-collagen interactions [15; 20]. Thus, composite hydrogels, containing both collagen and fibronectin, were fabricated in the presence and absence of ultrasound, and then imaged by multiphoton microscopy. As observed previously [28], ultrasound-exposed collagen gels contained regions of dense, radially aligned collagen fibers (Figure 1, 8.2 W/cm\({}^{2}\), SHG). Co-staining for fibronectin revealed diffuse co-localization with collagen fibrils (Figure 1, Merge); individual fibronectin fibrils were not detected (Figure 1, 8.2 W/cm\({}^{2}\), AF546-FN). Composite hydrogels polymerized under sham conditions contained short, randomly oriented collagen fibers (Figure 1, Sham, SHG) and exhibited some, albeit limited fibronectin staining (Figure 1, Sham, AF546-FN). No differences were observed in the overall intensity of fibronectin staining on ultrasound-exposed compared to sham-polymerized hydrogels (Figure 1, AF546). However, changes in fibronectin organization were observed that were consistent with the corresponding collagen fiber structures. Fibronectin and collagen co-regulate a variety of cell behaviors involved in tissue formation, including migration, proliferation, and matrix contraction [17]. Previous work characterizing the behavior of mesenchymal cells in collagen- and fibronectin-containing cell culture systems demonstrated that on native collagen gels and in the presence of fibronectin, FN-null MEFs [18] and dermal fibroblasts [34] can self-assemble to form 3D multicellular spheroids or \"microtissues\", by a process that requires fibronectin fibril assembly. Microtissue formation is further influenced by collagen-fibronectin interactions [18], substrate adhesive properties, and cellular contractility [35]. As such, cohesion of FN-null MEFs into 3D multicellular structures is a useful tool for assessing the ability of fibronectin/collagen-based hydrogels to support a coordinated cellular response. Cells cultured on sham-exposed, collagen-fibronectin hydrogels formed tightly adherent, well-spread monolayers across the gel surface within the first 24 h of culture (Figure 2A, Sham, 24 h). As expected [18], after 3 days of culture, cells had coalesced into 3D multicellular spheroids (Figure 2A, Sham, 3 d), termed \"microtissues\". At 24 h, the morphology of cells cultured on collagen-fibronectin gels fabricated in the presence of 8.8-MHz ultrasound at the lower intensity of 3.8 W/cm\({}^{2}\) was similar to that of cells on sham-exposed gels (Figure 2A, 3.8 W/cm\({}^{2}\), 24 h). In contrast, cells cultured on the surface of collagen-fibronectin gels fabricated using 8.8-MHz ultrasound at the higher intensity of 8.2 W/cm\({}^{2}\) formed numerous small microtissues within the first 24 h of culture (Figure 2A,B, 8.2 W/cm\({}^{2}\)). By day 3, significantly more microtissues had formed on ultrasound-fabricated versus sham-exposed gels (Figure 2A,B, 3 d). Between days 3 and 6, the number of microtissues on ultrasound-exposed gels decreased as some of the microtissues coalesced; however the total number of microtissues on gels fabricated using ultrasound at 8.2 W/cm\({}^{2}\) remained significantly greater than sham-exposed gels (Figure 2B, 6 d). As a second measure of cellular self-assembly, the area of the gel surface occupied by cells was quantified. Cells were maximally spread on sham-exposed gels at 24 h post-seeding, occupying \(\sim\)35% of the gel surface (Figure 2C, Sham, 24 h). As microtissues formed on sham-exposed gels over the course of 6 days, cell-occupied area decreased (Figure 2C, Sham, 3 and 6 d).