4D printing of self-folding and cell-encapsulating 3D microstructures as scaffolds for tissue-engineering applications.

Technology of tissue-engineering advanced rapidly in the last decade and motivated numerous studies in cell-engineering and biofabrication. Three-dimensional (3D) tissue-engineering scaffolds play a critical role in this field, as the scaffolds provide the biomimetic microenvironments that could stimulate desired cell behaviors for regeneration. However, despite many achievements, the fabrication of 3D scaffold remains challenging due to the difficulty of encapsulating cells in 3D scaffolds, controlling cell-cell organization in 3D, and being adapted by users unfamiliar with 3D biofabrication. In this study, we circumvent these obstacles by creating a four-dimensional (4D) inkjet-printing platform. This platform produces micropatterns that self-fold into a 3D scaffold. Seeding live cells uniformly onto the micropatterns before self-folding leads to cell-encapsulating 3D scaffolds with layer-wise cell-cell organization. Photo-crosslinkable biomaterial-inks of distinct swelling rates were synthesized from gelatin, and the biomaterial-inks were patterned by a customized high-precision inkjet-printer into bilayer micropatterns that were capable of self-folding into 3D microstructures. A mathematical model was developed to help design self-folding and to aid the understanding of the self-folding mechanism. Human umbilical vein endothelial cells (HUVECs) were embedded in self-folded microtubes to mimic microvessels. HUVECs in the microtube spread, proliferated, showed high cell viability, and engrafted on the microtube's inner wall mimicking the native endothelial cells. For physician and biologist end-users, this 4D printing method provides an easy-to-use platform that supports standard two-dimensional cell-seeding protocol while enabling the users to customize 3D cellularized scaffold as desired. This work demonstrated 4D printing as a promising tool for tissue-engineering applications.

Mediation of Cartilage Matrix Degeneration and Fibrillation by Decorin in Post-traumatic Osteoarthritis.

OBJECTIVE: To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). METHODS: Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (Dcni KO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1ß (IL-1ß) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1ß and release of sGAGs were quantified. RESULTS: In both Dcn-/- and Dcni KO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1ß, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). CONCLUSION: In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Role of Surfactant in Evaporation and Deposition of Bisolvent Biopolymer Droplets.

Inkjet printing of biopolymer droplets is gaining popularity because of its potential applications in regenerative medicine, particularly the fabrication of tissue-regenerative scaffolds. The quality of bioprinting, which affects cellular behaviors and the subsequent tissue formation, is determined by the solvent evaporation and deposition processes of biopolymer droplets, during which instantaneous local viscosity and surface tension changes occur because of the redistribution of the biopolymer inside the drop. Such dynamics is complex and not well understood. Most biopolymer inks also contain multiple solvents of distinct evaporation rates, further complicating the system dynamics. Using high-speed interferometry, we directly observe in real time the instantaneous drop shape of inkjet-printed picoliter gelatin drops containing glycerol and water. It is observed that, for bisolvent gelatin drops with surfactants, highly viscous gelatin and glycerol accumulated near the pinned contact line at an early stage suppress the evaporation-driven outward flow and create a stagnation zone near the contact line region. Lower surface tension at the contact line, because of its high local surfactant concentration, as compared to the drop apex induces a strong Marangoni recirculation, which in conjunction with a stagnation zone in the contact line region causes the instantaneous drop shape to transition from a spherical cap to a volcano shape during evaporation and resulting in a volcano-like deposition profile. In contrast, the suppressed evaporation outward flow together with a weak Marangoni flow leads to a domelike deposition for the case without surfactant. The role of surfactant in polymer drop deposition with water-only solvent is also investigated and compared against that of bisolvent drops. For the single-solvent case, the deposition profile is found to shift from a coffee-eye shape in the presence of surfactant to a uniform deposition without surfactant. The results reveal new insight into the complex role surfactant plays during polymer drop evaporation and deposition processes.

Gelatin-Based Microribbon Hydrogels Accelerate Cartilage Formation by Mesenchymal Stem Cells in Three Dimensions.

Hydrogels (HGs) are attractive matrices for cell-based cartilage tissue regeneration given their injectability and ability to fill defects with irregular shapes. However, most HGs developed to date often lack cell scale macroporosity, which restrains the encapsulated cells, leading to delayed new extracellular matrix deposition restricted to pericellular regions. Furthermore, tissue-engineered cartilage using conventional HGs generally suffers from poor mechanical property and fails to restore the load-bearing property of articular cartilage. The goal of this study was to evaluate the potential of macroporous gelatin-based microribbon (µRB) HGs as novel 3D matrices for accelerating chondrogenesis and new cartilage formation by human mesenchymal stem cells (MSCs) in 3D with improved mechanical properties. Unlike conventional HGs, these µRB HGs are inherently macroporous and exhibit cartilage-mimicking shock-absorbing mechanical property. After 21 days of culture, MSC-seeded µRB scaffolds exhibit a 20-fold increase in compressive modulus to 225 kPa, a range that is approaching the level of native cartilage. In contrast, HGs only resulted in a modest increase in compressive modulus of 65 kPa. Compared with conventional HGs, macroporous µRB scaffolds significantly increased the total amount of neocartilage produced by MSCs in 3D, with improved interconnectivity and mechanical strength. Altogether, these results validate gelatin-based µRBs as promising scaffolds for enhancing and accelerating MSC-based cartilage regeneration and may be used to enhance cartilage regeneration using other cell types as well.

Modeling Physiological Events in 2D vs. 3D Cell Culture.

Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research.

Winner of the Young Investigator Award of the Society for Biomaterials at the 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Microribbon-based hydrogels accelerate stem cell-based bone regeneration in a mouse critical-size cranial defect model.

Stem cell-based therapies hold great promise for enhancing tissue regeneration. However, the majority of cells die shortly after transplantation, which greatly diminishes the efficacy of stem cell-based therapies. Poor cell engraftment and survival remain a major bottleneck to fully exploiting the power of stem cells for regenerative medicine. Biomaterials such as hydrogels can serve as artificial matrices to protect cells during delivery and guide desirable cell fates. However, conventional hydrogels often lack macroporosity, which restricts cell proliferation and delays matrix deposition. Here we report the use of injectable, macroporous microribbon (µRB) hydrogels as stem cell carriers for bone repair, which supports direct cell encapsulation into a macroporous scaffold with rapid spreading. When transplanted in a critical-sized, mouse cranial defect model, µRB-based hydrogels significantly enhanced the survival of transplanted adipose-derived stromal cells (ADSCs) (81%) and enabled up to three-fold cell proliferation after 7 days. In contrast, conventional hydrogels only led to 27% cell survival, which continued to decrease over time. MicroCT imaging showed µRBs enhanced and accelerated mineralized bone repair compared to hydrogels (61% vs. 34% by week 6), and stem cells were required for bone repair to occur. These results suggest that paracrine signaling of transplanted stem cells are responsible for the observed bone repair, and enhancing cell survival and proliferation using µRBs further promoted the paracrine-signaling effects of ADSCs for stimulating endogenous bone repair. We envision µRB-based scaffolds can be broadly useful as a novel scaffold for enhancing stem cell survival and regeneration of other tissue types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1321-1331, 2016.

Winner of the Young Investigator Award of the Society for Biomaterials (USA) for 2016, 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Aligned microribbon-like hydrogels for guiding three-dimensional smooth muscle tissue regeneration.

Smooth muscle tissue is characterized by aligned structures, which is critical for its contractile functions. Smooth muscle injury is common and can be caused by various diseases and degenerative processes, and there remains a strong need to develop effective therapies for smooth muscle tissue regeneration with restored structures. To guide cell alignment, previously cells were cultured on 2D nano/microgrooved substrates, but such method is limited to fabricating 2D aligned cell sheets only. Alternatively, aligned electrospun nanofiber has been employed as 3D scaffold for cell alignment, but cells can only be seeded post fabrication, and nanoporosity of electrospun fiber meshes often leads to poor cell distribution. To overcome these limitations, we report aligned gelatin-based microribbons (µRBs) as macroporous hydrogels for guiding smooth muscle alignment in 3D. We developed aligned µRB-like hydrogels using wet spinning, which allows easy fabrication of tissue-scale (cm) macroporous matrices with alignment cues and supports direct cell encapsulation. The macroporosity within µRB-based hydrogels facilitated cell proliferation, new matrix deposition, and nutrient diffusion. In aligned µRB scaffold, smooth muscle cells showed high viability, rapid adhesion, and alignment following µRB direction. Aligned µRB scaffolds supported retention of smooth muscle contractile phenotype, and accelerated uniaxial deposition of new matrix (collagen I/IV) along the µRB. In contrast, cells encapsulated in conventional gelatin hydrogels remained round with matrix deposition limited to pericellular regions only. We envision such aligned µRB scaffold can be broadly applicable in growing other anisotropic tissues including tendon, nerves and blood vessel.

Modulating stem cell-chondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels.

Stem cells can contribute to cartilage repair either directly through chondrogenic differentiation or indirectly through paracrine signaling. Using a 3D co-culture model, we have recently reported that adipose-derived stem cells (ADSCs) can catalyze cartilage formation by neonatal chondrocytes (NChons) when mixed co-cultured in biomimetic hydrogels. However, how matrix cues influence such catalyzed cartilage formation remains unknown. To answer this question, ADSCs and NChons were co-encapsulated in 39 combinatorial hydrogel compositions with decoupled biochemical and mechanical properties. Methacrylated extracellular matrix (ECM) molecules including chondroitin sulfate, hyaluronic acid and heparan sulfate were incorporated at varying concentrations (0.5%, 1.25%, 2.5% and 5%) (w/v). Mechanical testing confirmed that hydrogel stiffness was largely decoupled from ECM cues (15 kPa, 40 kPa and 100 kPa). The biochemical assay and histology results showed that the type of ECM cue played a dominant role in modulating catalyzed cartilage formation, while varying hydrogel stiffness and doses of ECM led to more modest changes. Both chondroitin sulfate and hyaluronic acid led to robust articular cartilage matrix deposition, as shown by the intense staining of aggrecan and type II collagen. In soft hydrogels (15 kPa), chondroitin sulfate led to the highest amount of sulfated glycosaminoglycan deposition and increased compressive moduli. In contrast, heparan sulfate promoted type I collagen deposition, an undesirable fibrocartilage phenotype, and increasing heparan sulfate decreased cell proliferation and ECM deposition. Findings from the present study may guide the optimal scaffold design to maximize the synergistic cartilage formation using mixed cell populations.

Microfluidic Synthesis of Biodegradable Polyethylene-Glycol Microspheres for Controlled Delivery of Proteins and DNA Nanoparticles.

Polymeric microspheres represent an injectable platform for controlling the release of a variety of biologics; microspheres may be combined in a modular fashion to achieve temporal release of two or more biomolecules. Microfluidics offers a versatile platform for synthesizing uniform polymeric microspheres harboring a variety of biologics under relatively mild conditions. Poly(ethylene glycol) (PEG) is a bioinert polymer that can be easily tailored to encapsulate and control the release of biologics. In this study, we report the microfluidic synthesis of biodegradable PEG-based microparticles for controlled release of growth factors or DNA nanoparticles. Simple changes in microfluidic design increased the rate of microparticle formation and controlled the size of the microspheres. Mesh size and degradation rate were controlled by varying the PEG polymer weight percent from 7.5 to 15% (w/v), thus tuning the release of growth factors and DNA nanoparticles, which retained their bioactivity in assays of cell proliferation and DNA transfection, respectively. This platform may provide a useful tool for synthesizing microspheres for use as injectable carriers to achieve coordinated growth-factor or DNA nanoparticle release in therapeutic applications.

Chondrogenic differentiation of adipose-derived stromal cells in combinatorial hydrogels containing cartilage matrix proteins with decoupled mechanical stiffness.

Adipose-derived stromal cells (ADSCs) are attractive autologous cell sources for cartilage repair given their relative abundance and ease of isolation. Previous studies have demonstrated the potential of extracellular matrix (ECM) molecules as three-dimensional (3D) scaffolds for promoting chondrogenesis. However, few studies have compared the effects of varying types or doses of ECM molecules on chondrogenesis of ADSCs in 3D. Furthermore, increasing ECM molecule concentrations often result in simultaneous changes in the matrix stiffness, which makes it difficult to elucidate the relative contribution of biochemical cues or matrix stiffness on stem cell fate. Here we report the development of an ECM-containing hydrogel platform with largely decoupled biochemical and mechanical cues by modulating the degree of methacrylation of ECM molecules. Specifically, we incorporated three types of ECM molecules that are commonly found in the cartilage matrix, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). To elucidate the effects of interactive biochemical and mechanical signaling on chondrogenesis, ADSCs were encapsulated in 39 combinatorial hydrogel compositions with independently tunable ECM types (CS, HA, and HS), concentrations (0.5%, 1.25%, 2.5%, and 5% [w/v]), and matrix stiffness (3, 30, and 90 kPa). Our results show that the effect of ECM composition on chondrogenesis is dependent on the matrix stiffness of hydrogels, suggesting that matrix stiffness and biochemical cues interact in a nonlinear manner to regulate chondrogenesis of ADSCs in 3D. In soft hydrogels (~3 kPa), increasing HA concentrations resulted in substantial upregulation of aggrecan and collagen type II expression in a dose-dependent manner. This trend was reversed in HA-containing hydrogels with higher stiffness (~90 kPa). The platform reported herein could provide a useful tool for elucidating how ECM biochemical cues and matrix stiffness interact together to regulate stem cell fate, and for rapidly optimizing ECM-containing scaffolds to support stem cell differentiation and tissue regeneration.

Co-release of cells and polymeric nanoparticles from sacrificial microfibers enhances nonviral gene delivery inside 3D hydrogels.

Hydrogels can promote desirable cellular phenotype by mimicking tissue-like stiffness or serving as a gene delivery depot. However, nonviral gene delivery inside three-dimensional (3D) hydrogels remains a great challenge, and increasing hydrogel stiffness generally results in further decrease in gene delivery efficiency. Here we report a method to enhance nonviral gene delivery efficiency inside 3D hydrogels across a broad range of stiffness using sacrificial microfibers for co-releasing cells and polymeric nanoparticles (NPs). We fabricated hydrolytically degradable alginate as sacrificial microfibers, and optimized the degradation profile of alginate by varying the degree of oxidization. Scanning electron microscopy confirmed degradation of alginate microfibers inside hydrogels, leaving behind microchannel-like structures within 3D hydrogels. Sacrificial microfibers also serve as a delivery vehicle for co-releasing encapsulated cells and NPs, allowing cell attachment and spreading within the microchannel surface upon microfiber degradation. To examine the effects of sacrificial microfibers on nonviral gene delivery inside 3D hydrogels, alginate microfibers containing human embryonic kidney 293 cells and polymeric NPs were encapsulated within 3D hydrogel scaffolds with varying stiffness (9, 58, and 197 kPa). Compared with cells encapsulated in bulk hydrogels, we observed up to 15-fold increase in gene delivery efficiency using sacrificial microfibers, and gene delivery efficiency increased as hydrogel stiffness increased. The platform reported herein provides a strategy for enhancing nonviral gene delivery inside 3D hydrogels across a broad range of stiffness, and may aid tissue regeneration by engaging both mechanotransduction and nonviral gene delivery.

A facile method to fabricate hydrogels with microchannel-like porosity for tissue engineering.

Hydrogels are widely used as three-dimensional (3D) tissue engineering scaffolds due to their tissue-like water content, as well as their tunable physical and chemical properties. Hydrogel-based scaffolds are generally associated with nanoscale porosity, whereas macroporosity is highly desirable to facilitate nutrient transfer, vascularization, cell proliferation and matrix deposition. Diverse techniques have been developed for introducing macroporosity into hydrogel-based scaffolds. However, most of these methods involve harsh fabrication conditions that are not cell friendly, result in spherical pore structure, and are not amenable for dynamic pore formation. Human tissues contain abundant microchannel-like structures, such as microvascular network and nerve bundles, yet fabricating hydrogels containing microchannel-like pore structures remains a great challenge. To overcome these limitations, here we aim to develop a facile, cell-friendly method for engineering hydrogels with microchannel-like porosity using stimuli-responsive microfibers as porogens. Microfibers with sizes ranging 150-200 µm were fabricated using a coaxial flow of alginate and calcium chloride solution. Microfibers containing human embryonic kidney (HEK) cells were encapsulated within a 3D gelatin hydrogel, and then exposed to ethylenediaminetetraacetic acid (EDTA) solution at varying doses and duration. Scanning electron microscopy confirmed effective dissolution of alginate microfibers after EDTA treatment, leaving well-defined, interconnected microchannel structures within the 3D hydrogels. Upon release from the alginate fibers, HEK cells showed high viability and enhanced colony formation along the luminal surfaces of the microchannels. In contrast, HEK cells in non-EDTA treated control exhibited isolated cells, which remained entrapped in alginate microfibers. Together, our results showed a facile, cell-friendly process for dynamic microchannel formation within hydrogels, which may simultaneously release cells in 3D hydrogels in a spatiotemporally controlled manner. This platform may be adapted to include other cell-friendly stimuli for porogen removal, such as Matrix metalloproteinase-sensitive peptides or photodegradable gels. While we used HEK cells in this study as proof of principle, the concept described in this study may also be used for releasing clinically relevant cell types, such as smooth muscle and endothelial cells that are useful for repairing tissues involving tubular structures.

Photo-crosslinkable PEG-based microribbons for forming 3D macroporous scaffolds with decoupled niche properties.

PEG-based microribbons are designed and fabricated as building blocks for constructing a 3D cell niche with independently tunable biochemical, mechanical, and topographical cues. This platform supports direct cell encapsulation, allows spatial patterning of biochemical cues, and may provide a valuable tool for facilitating the analyses of how interactive niche signaling regulates cell fate in three dimensions.

Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness.

Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.

Dynamic tissue engineering scaffolds with stimuli-responsive macroporosity formation.

Macropores in tissue engineering scaffolds provide space for vascularization, cell-proliferation and cellular interactions, and is crucial for successful tissue regeneration. Modulating the size and density of macropores may promote desirable cellular processes at different stages of tissue development. Most current techniques for fabricating macroporous scaffolds produce fixed macroporosity and do not allow the control of porosity during cell culture. Most macropore-forming techniques also involve non-physiological conditions, such that cells can only be seeded in a post-fabrication process, which often leads to low cell seeding efficiency and uneven cell distribution. Here we report a process to create dynamic hydrogels as tissue engineering scaffolds with tunable macroporosity using stimuli-responsive porogens of gelatin, alginate and hyaluronic acid, which degrade in response to specific stimuli including temperature, chelating and enzymatic digestion, respectively. SEM imaging confirmed sequential pore formation in response to sequential stimulations: 37 °C on day 0, EDTA on day 7, and hyaluronidase on day 14. Bovine chondrocytes were encapsulated in the Alg porogen, which served as cell-delivery vehicles, and changes in cell viability, proliferation and tissue formation during sequential stimuli treatments were evaluated. Our results showed effective cell release from Alg porogen with high cell viability and markedly increased cell proliferation and spreading throughout the 3D hydrogels. Dynamic pore formation also led to significantly enhanced type II and X collagen production by chondrocytes. This platform provides a valuable tool to create stimuli-responsive scaffolds with dynamic macroporosity for a broad range of tissue engineering applications, and may also be used for fundamental studies to examine cell responses to dynamic niche properties.

The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels.

Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often leads to simultaneous changes in mechanical properties, which makes it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell-material interactions have emerged as a tool to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on two-dimensional environments. Here we developed three-dimensional combinatorial hydrogels with independent control of biochemical and mechanical properties to facilitate analysis of interactive biochemical and mechanical signaling on adipose-derived stem cell osteogenesis in three dimensions. Our results suggest that scaffold biochemical and mechanical signals synergize only at specific combinations to promote bone differentiation. Leading compositions were identified to have intermediate stiffness (∼55kPa) and low concentration of fibronectin (10µg ml(-1)), which led to an increase in osteocalcin gene expression of over 130-fold. Our results suggest that scaffolds with independently tunable niche cues could provide a powerful tool for conducting mechanistic studies to decipher how complex niche cues regulate stem cell fate in three dimensions, and facilitate rapid identification of optimal niche cues that promote desirable cellular processes or tissue regeneration.

Three-Dimensional Polymer Constructs Exhibiting a Tunable Negative Poisson's Ratio.

Young's modulus and Poisson's ratio of a porous polymeric construct (scaffold) quantitatively describe how it supports and transmits external stresses to its surroundings. While Young's modulus is always non-negative and highly tunable in magnitude, Poisson's ratio can, indeed, take on negative values despite the fact that it is non-negative for virtually every naturally occurring and artificial material. In some applications, a construct having a tunable negative Poisson's ratio (an auxetic construct) may be more suitable for supporting the external forces imposed upon it by its environment. Here, three-dimensional polyethylene glycol scaffolds with tunable negative Poisson's ratios are fabricated. Digital micromirror device projection printing (DMD-PP) is used to print single-layer constructs composed of cellular structures (pores) with special geometries, arrangements, and deformation mechanisms. The presence of the unit-cellular structures tunes the magnitude and polarity (positive or negative) of Poisson's ratio. Multilayer constructs are fabricated with DMD-PP by stacking the single-layer constructs with alternating layers of vertical connecting posts. The Poisson's ratios of the single- and multilayer constructs are determined from strain experiments, which show (1) that the Poisson's ratios of the constructs are accurately predicted by analytical deformation models and (2) that no slipping occurrs between layers in the multilayer constructs and the addition of new layers does not affect Poisson's ratio.

Solid freeform fabrication of designer scaffolds of hyaluronic acid for nerve tissue engineering.

The field of tissue engineering and regenerative medicine will tremendously benefit from the development of three dimensional scaffolds with defined micro- and macro-architecture that replicate the geometry and chemical composition of native tissues. The current report describes a freeform fabrication technique that permits the development of nerve regeneration scaffolds with precisely engineered architecture that mimics that of native nerve, using the native extracellular matrix component hyaluronic acid (HA). To demonstrate the flexibility of the fabrication system, scaffolds exhibiting different geometries with varying pore shapes, sizes and controlled degradability were fabricated in a layer-by-layer fashion. To promote cell adhesion, scaffolds were covalently functionalized with laminin. This approach offers tremendous spatio-temporal flexibility to create architecturally complex structures such as scaffolds with branched tubes to mimic branched nerves at a plexus. We further demonstrate the ability to create bidirectional gradients within the microfabricated nerve conduits. We believe that combining the biological properties of HA with precise three dimensional micro-architecture could offer a useful platform for the development of a wide range of bioartificial organs.

Fabrication of three-dimensional scaffolds for heterogeneous tissue engineering.

The development of biomedical scaffolds mimicking a heterogeneous cellular microenvironment for a specified regulation of cell-fates is very promising for tissue engineering. In this study, three-dimensional scaffolds with heterogeneous microstructure were developed using a DMD-PP apparatus. During the fabrication process, this apparatus can efficiently switch monomers to form microstructures with localized, different material properties; the resolution in the arrangement of material properties is comparable to the characteristic size of functional subunits in living organs, namely, a hundred microns. The effectiveness of this DMD-PP apparatus is demonstrated by a woodpile microstructure with heterogeneous fluorescence and also by a microporous cell-culturing scaffold with selected sites for protein adhesion. Cell-cultivation experiment was performed with the microporous scaffold, in which selective cell adhesion was observed.

Fluorinated colloidal emulsion of photochangeable rheological behavior as a sacrificial agent to fabricate organic, three-dimensional microstructures.

Three-dimensional organic microfabrication, an emerging technology, faces the challenge of lacking a sacrificial agent (SA) to temporarily support the formation of microscale geometries, which can be removed after a microstructure is constructed. In this study, an ultradense oil-in-organofluorine colloidal emulsion with photopolymerizable submicrometer droplets (diameter approximately 500 nm) was prepared and used as the required SA. Upon exposure to light, the colloidal emulsion undergoes a significant rheological change, which hardens the emulsion and presents the molding/protecting function that an SA must have. Importantly, the emulsion includes a synthesized fluorophilic/fluorophobic block copolymer surfactant to stabilize the droplet compartments, facilitating the dissolution of the postexposure SA. Two successfully built, complex, organic 3D microstructures show the effectiveness of using this novel SA material.