Collagen type I is the main structural component of numerous cellular environments, ranging from soft tissues, such as skin or tendons, to hard bone tissue. The extraordinary range of collagen functional properties drives the attention of researchers within the biomedical field to improve the bioactivity of collagen-based biomaterials. The ultimate goal is to enhance tissue repair and regeneration or even to engineer complex tissues through manipulating collagen properties and, through a bi-directional interplay, modifying cellular behaviors.

Collagen fiber microstructure within ultrasound-exposed hydrogels
Collagen fiber microstructure within ultrasound-exposed hydrogels

Norris and colleagues at the University of Rochester in New York, USA, are exploiting ultrasound to engineer 3D biomaterials with controlled collagen fiber structure [Norris et al.Materials Today Bio (2019) doi: 10.1016/j.mtbio.2019.100018]. The team previously developed an ultrasound exposure system [Garvin et al. Ultrasound Med. Biol. (2010 doi: 10.1016/j.ultrasmedbio.2010.08.007] to spatially pattern distinct collagen microstructures within a 3D engineered tissue [Garvin et al. J. Acoust. Soc. Am. (2013) doi:10.1121/1.4812868].

Ultrasound fields are applied in a non-invasive and site-specific manner with high precision and can interact with biomaterials through thermal and/or mechanical effects. The researchers have shown the application of ultrasound to control the length and diameter of collagen fibers during hydrogel polymerization [Garvin et al. J. Acoust. Soc. Am. (2013) doi:10.1121/1.4812868]. This ultrasound-based fabrication technology generates local variations in collagen fiber microstructure and organization. In the absence of ultrasound, hydrogels are fabricated with short, randomly oriented and homogeneously distributed collagen fibers. Instead, by exposing soluble collagen to ultrasound waves (7.8 MHz in a range of 3.2-10 W/cm2) during polymerization, hydrogels form with distinct structural features and spatial arrangements. Radially aligned collagen fibers, heterogeneous fibrillar structures and regions of interconnected porosity can be generated by controlling ultrasound intensity. 

Collagen fiber organization. Soluble collagen was exposed to 7.8 MHz ultrasound at intensities of 0 (A), 3.2 (B), 6.8 (C) or 10 (D) W/cm2 for 15 minutes. Images were obtained using second harmonic generation microscopy. Acoustically modified collagen hydrogels exhibit distinctive features, including radial fiber alignment (arrows), increased fiber density (boxes) short fibers (X) and absent/reduced collagen content (asterisks). Scale bar, 500 µm.
Collagen fiber organization. Soluble collagen was exposed to 7.8 MHz ultrasound at intensities of 0 (A), 3.2 (B), 6.8 (C) or 10 (D) W/cm2 for 15 minutes. Images were obtained using second harmonic generation microscopy. Acoustically modified collagen hydrogels exhibit distinctive features, including radial fiber alignment (arrows), increased fiber density (boxes) short fibers (X) and absent/reduced collagen content (asterisks). Scale bar, 500 µm.

The researchers demonstrate that ultrasound modifies the microstructure of collagen, enabling cell-mediated collagen fiber remodeling. Subtle conformational changes in collagen lead to a reduction in cell-substrate attachments and facilitate cell migration. Researchers demonstrate a spatial-specific effect of acoustic modification of collagen gels on cell adhesion given the lower number of fibroblasts that adhered to ultrasound exposed, in comparison to non-exposed hydrogels. Strikingly, cells on ultrasound-exposed regions migrate into radially aligned circular aggregates co-localizing with collagen fiber bundles, a specific arrangement of collagen fibers at the micro-scale. Acoustic modification of collagen structure results from mechanical rather than thermal effects, and sensitizes collagen to cellular remodeling events.

Cellular organization and collagen remodeling on acoustically modified collagen hydrogels. Fibroblasts are shown in green and second harmonic generation images are depicted in white. (A-C) shows ultrasound exposed regions and (D) corresponds to non-exposed collagen hydrogels. Scale bars, 500 µm (A and B), 200 µm (C and D).
Cellular organization and collagen remodeling on acoustically modified collagen hydrogels. Fibroblasts are shown in green and second harmonic generation images are depicted in white. (A-C) shows ultrasound exposed regions and (D) corresponds to non-exposed collagen hydrogels. Scale bars, 500 µm (A and B), 200 µm (C and D).

An ex vivo full-thickness skin explant model demonstrates the ability of different cell types to change their surrounding microenvironment within ultrasound-exposed collagen hydrogels, organizing into multicellular fibrillar structures, as opposed to cells in non-exposed control hydrogels. Therefore, acoustic-based technologies enable the fabrication of a 3D biomaterial with controlled microstructural organization, which can support cell infiltration and microenvironment remodeling cellular activities. Such outcomes have impact toward the development of in vitro engineered tissues with complex geometries and structural features at the micro-scale for wound healing applications.