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...

No abstract

No abstract

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.

No abstract

Angiogenesis, which is morphogenesis undertaken by endothelial cells (ECs) during new blood vessel formation, has been traditionally studied on natural extracellular matrix proteins. In this work, we aimed to regulate and guide angiogenesis on synthetic, bioactive poly(ethylene glycol)-diacrylate (PEGDA) hydrogels. PEGDA hydrogel is intrinsically cell nonadhesive and highly resistant to protein adsorption, allowing a high degree of control over presentation of ligands for cell adhesion and signaling. Since these materials are photopolymerizable, a variety of photolithographic technologies may be applied to spatially control presentation of bioactive ligands. To manipulate EC adhesion, migration, and tubulogenesis, the surface of PEGDA hydrogels was micropatterned with a cell adhesive ligand, Arg-Gly-Asp-Ser (RGDS), in desired concentrations and geometries. ECs cultured on these RGDS patterns reorganized their cell bodies into cord-like structures on 50-microm-wide stripes, but not on wider stripes, suggesting that EC morphogenesis can be regulated by geometrical cues. The cords formed by ECs were reminiscent of capillaries with cells participating in the self-assembly and reorganization into multicellular structures. Further, endothelial cord formation was stimulated on intermediate concentration of RGDS at 20 microg/cm(2), whereas it was inhibited at higher concentrations. This work has shown that angiogenic responses can be tightly regulated and guided by micropatterning of bioactive ligands and also demonstrated great potentials of micropatterned PEGDA hydrogels for various applications in tissue engineering, where vascularization prior to implantation is critical.

The collagen content of the human LP is approximately 60% to 70% of that of human dermis. Although canine LP collagen levels are most similar to those of humans, quantitative histology indicates that the collagen distribution of the human LP is best matched by the porcine LP. Collagen types I and III seem to be the dominant LP collagens. Spatial variations in collagen turnover appear to exist that may contribute to normal LP physiology.

Current clinical management of vocal fold (VF) scarring produces inconsistent and often suboptimal results. Researchers are investigating a number of alternative treatments for VF lamina propria (LP) scarring, including designer implant materials for functional LP regeneration. In the present study, we investigate the effects of the initial scaffold elastic modulus and mesh size on encapsulated VF fibroblast (VFF) extracellular matrix (ECM) production toward rational scaffold design. Polyethylene glycol diacrylate (PEGDA) hydrogels were selected for this study since their material properties, including mechanical properties, mesh size, degradation rate and bioactivity, can be tightly controlled and systematically modified. Porcine VFF were encapsulated in four PEGDA hydrogels with degradation half lives of ~25 days and initial elastic compressive moduli ranging from ~30 to 100 kPa and initial mesh sizes ranging from ~9 to 27 nm. After 30 days of static culture, VFF ECM production and phenotype in each formulation was assessed biochemically and histologically. Sulfated glycosaminoglycan synthesis increased in similar degree with both increasing initial modulus and decreasing initial mesh size. In contrast, elastin production decreased with increasing initial modulus but increased with decreasing initial mesh size. Both collagen deposition and the induction of a myofibroblastic phenotype depended strongly on initial mesh size but appeared largely unaffected by variations in initial modulus. The present results indicate that scaffold mesh size warrants further investigation as a critical regulator of VFF ECM synthesis. Furthermore, this study validates a systematic and controlled approach for analyzing VFF response to scaffold properties, which should aid in rational scaffold selection/design.