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...

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Mechanical conditioning represents a potential means to enhance the biochemical and biomechanical properties of tissue engineered vascular grafts (TEVGs). A pulsatile flow bioreactor was developed to allow shear and pulsatile stimulation of TEVGs. Physiological 120 mmHg/80 mmHg peak-to-trough pressure waveforms can be produced at both fetal and adult heart rates. Flow rates of 2 mL/sec, representative of flow through small diameter blood vessels, can be generated, resulting in a mean wall shear stress of approximately 6 dynes/cm(2) within the 3 mm ID constructs. When combined with non-thrombogenic poly(ethylene glycol) (PEG)-based hydrogels, which have tunable mechanical properties and tailorable biofunctionality, the bioreactor represents a flexible platform for exploring the impact of controlled biochemical and biomechanical stimuli on vascular graft cells. In the present study, the utility of this combined approach for improving TEVG outcome was investigated by encapsulating 10T-1/2 mouse smooth muscle progenitor cells within PEG-based hydrogels containing an adhesive ligand (RGDS) and a collagenase degradable sequence (LGPA). Constructs subjected to 7 weeks of biomechanical conditioning had significantly higher collagen levels and improved moduli relative to those grown under static conditions.