Three‐Dimensional Biochemical and Biomechanical Patterning of Hydrogels for Guiding Cell Behavior

Abstract

The ability to tailor the biochemical and biomechanical properties of 3D materials at the microscale is important for a range of biotechnology applications, including the engineering of complex tissues, the development of biosensors, the elucidation of cell-cell and cell-material interactions, and the guidance of cellular differentiation. [1,2] To this end, techniques have emerged for the fabrication of 3D microcontrolled materials, including conventional photolithographic patterning, [3,4] electrochemical deposition, [5] 3D printing, [6] and soft-lithographic approaches. [7][8][9][10] To create internally complex 3D materials, these methods are repeated in a layer-by-layer fashion until a scaffold of the desired dimensionality is achieved. However, an alternate approach to the fabrication of internally complex 3D scaffolds, that is, the patterning of bioactivity into preformed materials of the desired final dimensions, has not been similarly examined. [11][12][13] Here, we develop a new paradigm for generating 3D microcontrolled materials using two-photon absorption (TPA) photolithography to pattern bioactivity into existing photoactive materials. We demonstrate the ability to spatially tailor material biomechanical and biochemical properties at the microscale and to create freeform 3D patterns and gradients. Furthermore, to illustrate the power of this approach for guiding cell behavior, proteolytically degradable hydrogels were patterned in 3D with the cell adhesive peptide arginine-glycine-aspartic acid-serine (RGDS), and cells were shown to invade and migrate into only the RGDS-containing regions.In the present study, we first establish the feasibility of patterning bioactive features into optically transparent, photoactive materials using an adaptation of conventional photolithography, that is, single-photon absorption (SPA) photolithography. Although, as previously mentioned, SPA photolithography has been used to create topographical microstructures on surfaces, [3] it has not, to the best of our knowledge, been developed for the internal modification of preformed materials. We show that SPA photolithography allows for rapid and inexpensive biochemical and biomechanical patterning of existing photoactive materials in three dimensions. However, pattern complexity is limited to features of axially uniform cross section, since light passes vertically through the entire sample. Thus, we went on to develop TPA photolithography for creating axially complex, freeform 3D biochemical and biomechanical patterns and gradients in existing photoactive materials. TPA has enabled the development of 3D fluorescence imaging, [14] 3D lithographic microfabrication, [15,16] and new approaches to 3D optical data storage.[15] Each of these applications takes advantage of the fact that, by tightly focusing an excitation beam, the region of TPA can be confined to a focal volume roughly half the excitation wavelength in dimension. [16] Any subsequent process, such as a photoinitiated or radical-based polymerization, is also locali...

Microstructured extracellular matrices in tissue engineering and development

“…While early efforts generated patterns by UV exposure through a simple mask, 21 recent work has become far more elaborate. For instance, Hahn et al 16 showed that two-photon activation can generate three-dimensional patterns of acrylated PEG with microscale resolution throughout the bulk of the gel.…”

Bioartificial matrices for therapeutic vascularization

“…PEG has a well-established chemistry and long history of safety in vivo. Addition of acrylate groups flanking the PEG chain allows for photo or chemical cross-linking of a PEGDA macromer solution, as well as incorporation of biomolecules such as protease-degradable sites, adhesive ligands, and growth factors (14)(15)(16). A major strength of this strategy is the modular "plugand-play" design of the base hydrogel system, which allows the tailoring of the biochemical and mechanical properties of the delivery vehicle.…”

Volume Holography: Novel Materials, Methods and Applications

“…Furthermore, hydrogels can also be structured photolithographically, taking advantage of diffusion processes. Three-dimensional structuring at the microscale results in the ability to spatially tailor biomechanical and biochemical material properties [52].…”