Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing

Organ printing, a novel approach in tissue engineering, applies layered computer-driven deposition of cells and gels to create complex 3-dimensional cell-laden structures. It shows great promise in regenerative medicine, because it may help to solve the problem of limited donor grafts for tissue and organ repair. The technique enables anatomical cell arrangement using incorporation of cells and growth factors at predefined locations in the printed hydrogel scaffolds. This way, 3-dimensional biological structures, such as blood vessels, are already constructed. Organ printing is developing fast, and there are exciting new possibilities in this area. Hydrogels are highly hydrated polymer networks used as scaffolding materials in organ printing. These hydrogel matrices are natural or synthetic polymers that provide a supportive environment for cells to attach to and proliferate and differentiate in. Successful cell embedding requires hydrogels that are complemented with biomimetic and extracellular matrix components, to provide biological cues to elicit specific cellular responses and direct new tissue formation. This review surveys the use of hydrogels in organ printing and provides an evaluation of the recent advances in the development of hydrogels that are promising for use in skeletal regenerative medicine. Special emphasis is put on survival, proliferation and differentiation of skeletal connective tissue cells inside various hydrogel matrices.     

Inverse opal hydrogel-collagen composite scaffolds as a supportive microenvironment for immune cell migration

Immunotherapies harness the inherent potential of the body to destroy foreign or infected cells, and are currently being investigated as treatments for cancer. One way to boost native immune responses might be to engineer ectopic lymphoid tissue, providing a supportive microenvironment for immune cell priming, and/or bringing together immune cells at a desired location (e.g., solid tumor sites). Here we describe the development and in vitro testing of composite macroporous poly(ethylene glycol) (PEG) hydrogel scaffolds infused with collagen as a tissue engineering platform for immunotherapy. The PEG hydrogel with ordered, interconnected pores provided mechanical stability and the potential to depot supporting cytokines/chemokines, while an infused collagen matrix supported intra-scaffold migration of loaded T cells and dendritic cells. Rapid, nearly unconstrained T cell migration through scaffolds was achieved by using inverse opal supporting structures with 80 microm macropores. In addition, we demonstrated that the lymphoid tissue chemokine CCL21 could be bound to the inverse opal gel walls of these scaffolds, to provide motility-inducing cues for T cells within these structures. This hybrid scaffold approach combines the strengths of the synthetic and biopolymer hydrogels used in a highly synergistic fashion, allowing each material to compensate for limiting properties of its partner.     

MMP-2 sensitive, VEGF-bearing bioactive hydrogels for promotion of vascular healing

We sought to develop bioactive hydrogels to facilitate arterial healing, e.g., after balloon angioplasty. Toward this end, we developed a new class of proteolytically sensitive, biologically active polyethylene glycol (PEG)-peptide hydrogels that can be formed in situ to temporarily protect the arterial injury from blood contact. Furthermore, we incorporated endothelial cell-specific biological signals with the goal of enhancing arterial reendothelialization. Here we demonstrate efficient endothelial cell anchorage and activation on PEG hydrogel matrices modified by conjugation with both the cell adhesive peptide motif RGD and an engineered variant of vascular endothelial growth factor (VEGF). By crosslinking peptide sequences for cleavage by MMP-2 into the polymer backbone, the hydrogels became sensitive to proteolytic degradation by cell-derived matrix metalloproteinases (MMPs). Analysis of molecular hallmarks associated with endothelial cell activation by VEGF-RGD hydrogel matrices revealed a 70% increase in production of the latent MMP-2 zymogen compared with PEG-peptide hydrogels lacking VEGF. By additional provision of transforming growth factor beta1 (TGF-beta1) within the PEG-peptide hydrogel, conversion of the latent MMP zymogen into its active form was demonstrated. As a result of MMP-2 activation, strongly enhanced hydrogel degradation by activated endothelial cells was observed. Our data illustrate the critical importance of growth factor activities for remodeling of synthetic biomaterials into native tissue, as it is desired in many applications of regenerative medicine. Functionalized PEG-peptide hydrogels could help restore the native vessel wall and improve the performance of angioplasty procedures.     

Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment

Micropatterning of hydrogels is potentially useful for a variety of applications, including tissue engineering, fundamental biological studies, diagnostics, and high-throughput screening. Although synthetic polymers have been developed for these applications, natural polymers such as polysaccharides may have advantages for biological samples and cell-based devices because they are natural components of the in vivo microenvironment. In this study, we synthesized and used hyaluronic acid (HA) modified with photoreactive methacrylates to fabricate microstructures as functional components of microfabricated devices. To demonstrate the universality of this approach, two types of microstructures were formed. In the first approach, HA microstructures were fabricated and used as docking templates to enable the subsequent formation of cell microarrays within low shear stress regions of the patterns. Cells within these microwells remained viable, could generate spheroids, and could be retrieved using mechanical disruption. In the second approach, cells were encapsulated directly within the HA hydrogels. Arrays of viable embryonic stem (ES) cells or fibroblasts were encapsulated within HA hydrogels and could later be recovered using enzymatic digestion of the microstructures. These results demonstrate that it is possible to incorporate photocrosslinkable HA, a natural, versatile, degradable, and biocompatible biopolymer, into micro-electromechanical systems.     

Dense fibrillar collagen matrices to analyse extracellular matrix receptor function

Aim

The goal of this study was to understand whether dense fibrillar collagen matrices, with a hierarchical structure resembling native collagen matrices, could be useful to study collagen receptor function, in a more physiological context. The receptor analysed here was integrin α11β1, already shown to be involved in cell attachment and migration on collagen-coated plastic, and also in contraction of loose fibrillar collagen hydrogels.

Materials and methods

Collagen matrices prepared here corresponded to dense fibrillar hydrogels concentrated at 5 mg/ml. The behaviour of α11β1 deficient fibroblasts seeded on these concentrated matrices was assessed in terms of adhesion, morphology and migration, then compared to that observed on classical hydrogels at 1 mg/ml, corresponding to loose collagen matrices.

Results

Short-term attachment assays showed disturbed interactions between α11β1 deficient cells and collagen matrices in a concentration-dependent manner. Long-term assays revealed reduced cell spreading of alpha 11^−/− cells on the dense collagen matrices, associated with a disturbed cytoskeleton network. Moreover, anoikis was observed when alpha 11^−/− cells were seeded on 5 mg/ml matrices, and not on looser 1 mg/ml matrices. In scratch wound in vitro assays, carried out with cells on 5 mg/ml fibrillar collagen matrices, alpha 11^−/− cells migrated much better than their wild-type counterparts. In contrast, no significant difference was observed between wild and knock-out cells seeded on plastic.

Conclusions

The present study demonstrates the validity of in vivo-like dense fibrillar collagen matrices to evaluate cell receptor functions more significantly than with 2D cell cultures or loose hydrogels.     

A novel method to understand tumor cell invasion: integrating extracellular matrix mimicking layers in microfluidic chips by “selective curing”

A major challenge in studying tumor cell invasion into its surrounding tissue is to identify the contribution of individual factors in the tumor microenvironment (TME) to the process. One of the important elements of the TME is the fibrous extracellular matrix (ECM) which is known to influence cancer cell invasion, but exactly how remains unclear. Therefore, there is a need for new models to unravel mechanisms behind the tumor-ECM interaction. In this article, we present a new microfabrication method, called selective curing, to integrate ECM-mimicking layers between two microfluidic channels. This method enables us to study the effect of 3D matrices with controlled architecture, beyond the conventionally used hydrogels, on cancer invasion in a controlled environment. As a proof of principle, we have integrated two electrospun Polycaprolactone (PCL) matrices with different fiber diameters in one chip. We then studied the 3D migration of MDA-MB-231 breast cancer cells into the matrices under the influence of a chemotactic gradient. The results show that neither the invasion distance nor the general cell morphology is affected significantly by the difference in fiber size of these matrices. The cells however do produce longer and more protrusions in the matrix with smaller fiber size. This microfluidic system enables us to study the influence of other factors in the TME on cancer development as well as other biological applications as it provides a controlled compartmentalized environment compatible with cell culturing.     

Micropatterning of poly(ethylene glycol) diacrylate hydrogels with biomolecules to regulate and guide endothelial morphogenesis

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.     

An adaptable hydrogel array format for 3-dimensional cell culture and analysis

Hydrogels have been commonly used as model systems for 3-dimensional (3-D) cell biology, as they have material properties that resemble natural extracellular matrices (ECMs), and their cell-interactive properties can be readily adapted in order to address a particular hypothesis. Natural and synthetic hydrogels have been used to gain fundamental insights into virtually all aspects of cell behavior, including cell adhesion, migration, and differentiated function. However, cell responses to complex 3-D environments are difficult to adequately explore due to the large number of variables that must be controlled simultaneously. Here we describe an adaptable, automated approach for 3-D cell culture within hydrogel arrays. Our initial results demonstrate that the hydrogel network chemistry (both natural and synthetic), cell type, cell density, cell adhesion ligand density, and degradability within each array spot can be systematically varied to screen for environments that promote cell viability in a 3-D context. In a test-bed application we then demonstrate that a hydrogel array format can be used to identify environments that promote viability of HL-1 cardiomyocytes, a cell line that has not been cultured previously in 3-D hydrogel matrices. Results demonstrate that the fibronectin-derived cell adhesion ligand RGDSP improves HL-1 viability in a dose-dependent manner, and that the effect of RGDSP is particularly pronounced in degrading hydrogel arrays. Importantly, in the presence of 70mum RGDSP, HL-1 cardiomyocyte viability does not decrease even after 7 days of culture in PEG hydrogels. Taken together, our results indicate that the adaptable, array-based format developed in this study may be useful as an enhanced throughput platform for 3-D culture of a variety of cell types.     

Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels

Graft survival and integration are major factors that limit the efficacy of cell therapies for the treatment of disease and injury in the central nervous system. Efforts to improve cell survival and integration have focused in part on the development of biocompatible scaffolds that support neural cell growth and function. Here we photoencapsulate neural cells within degradable hydrogels and use confocal microscopy to non-invasively monitor these key cell functions over time. By directly imaging fluorescently labeled cells we show that neural cells cultured within three-dimensional polymer networks create their own cellular microenvironment to survive, proliferate and differentiate and form neurons and glia that are electrophysiologically responsive to neurotransmitter. By changing the degradation rate of the polymer network, the time-scale over which neural cells extend processes throughout the hydrogel could be tuned on a time-scale that ranged from 1-3 weeks. These studies were carried out in the absence of serum and extracellular matrix molecules that can be immunogenic and identify degradable PEG hydrogels as suitable synthetic cell carriers for neural transplantation.     

Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration

Micropatterning techniques that control three-dimensional (3D) arrangement of biomolecules and cells at the microscale will allow development of clinically relevant tissues composed of multiple cell types in complex architecture. Although there have been significant developments to regulate spatial and temporal distribution of biomolecules in various materials, most micropatterning techniques are applicable only to two-dimensional patterning. We report here the use of two-photon laser scanning (TPLS) photolithographic technique to micropattern cell adhesive ligand (RGDS) in hydrogels to guide cell migration along pre-defined 3D pathways. The TPLS photolithographic technique regulates photo-reactive processes in microscale focal volumes to generate complex, free from microscale patterns with control over spatial presentation and concentration of biomolecules within hydrogel scaffolds. The TPLS photolithographic technique was used to dictate the precise location of RGDS in collagenase-sensitive poly(ethylene glycol-co-peptide) diacrylate hydrogels, and the amount of immobilized RGDS was evaluated using fluorescein-tagged RGDS. When human dermal fibroblasts cultured in fibrin clusters were encapsulated within the micropatterned collagenase-sensitive hydrogels, the cells underwent guided 3D migration only into the RGDS-patterned regions of the hydrogels. These results demonstrate the prospect of guiding tissue regeneration at the microscale in 3D scaffolds by providing appropriate bioactive cues in highly defined geometries.     

On-chip cell migration assay using microfluidic channels

Cell migration plays a crucial role in various biological processes including embryogenesis, wound healing, immune response, and tissue development. Conventional cell migration assays for screening of chemo-attractants or -repellants are initiated by physical scraping of a portion of confluent cells on normal culture surfaces. However, this protocol requires both a large number of cells and an increased amount of reagents. Additionally, these methods are not suitable for scaling-up for high-throughput screening. Here, we show an on-chip cell migration assay utilizing microfluidic channels. Laminar flow of trypsin solution in microfluidic channels achieved well-controlled cell detachment of a portion of confluent cell monolayers, which could effectively pattern wound edges to mimic biological wounding in vivo. Trypsin laminar flow in precisely fabricated microfluidic devices enables accurate and reliable cell migration assay with limited amounts of reagents to either promote or inhibit cell migration.     

Protein-polymer conjugates for forming photopolymerizable biomimetic hydrogels for tissue engineering

Collagen, fibrin and albumin are popular proteins for making biological scaffolds for tissue engineering because of their biocompatibility, biodegradability, and availability. A major drawback of biological protein-based biomaterials is the limited control over their physical and biodegradation properties. Our laboratory has been developing new protein-based biomaterials with tunable properties without the use of cytotoxic protein cross-linking techniques. We describe the formation and assembly of photopolymerizable biomimetic hydrogel scaffolds made from protein-polymer conjugates of poly(ethylene glycol) (PEG) and collagen, fibrin or albumin. The conjugation of PEG to these proteins (PEGylation) was verified by SDS-PAGE and the polymerization reaction into a hydrogel network was confirmed by shear rheometry. The differences in rheology and swelling characteristics of the three hydrogel materials underscore the importance of the molecular relationship between the PEG and the protein constituent in this protein-polymer arrangement. The biofunctionality of the PEGylated collagen and fibrinogen hydrogels sustained both cell adhesion and proteolytic degradation that enabled 3-D cell spreading and migration within the hydrogel network. PEG-albumin hydrogels exhibited poor cell spreading and migration by virtue of the fact that the albumin backbone lacks any known cell adhesion sites. Despite differences in the biological and structural composition of the PEGylated fibrinogen and collagen hydrogels, the rate of cellular migration within each material was not significantly different.     

Assessment of the cytotoxicity of photocrosslinked dextran and hyaluronan-based hydrogels to vascular smooth muscle cells

The cytotoxicity of polysaccharide-based hydrogels and solutions was studied in vitro after 48h of indirect exposure of the materials with vascular smooth muscle cells. Dextran and/or hyaluronan were derivatized using glycidyl methacrylate, and hydrogels were formed in the presence of photoinitiators and ultraviolet radiation in multiwell inserts to avoid direct contact with cell monolayers. Observation of cell morphology indicated that dextran hydrogels, a blend of non-derivatized hyaluronan into dextran hydrogel. and a hyaluronan solution were highly cytocompatible. However, hydrogels made of derivatized hyaluronan were cytotoxic when compared to unexposed sham controls that contained multiwell inserts but no hydrogels. Results from quantitative assays for proliferation and viability corroborated the qualitative observations, and scrape wound assays revealed a significant increase in smooth muscles cell migration/proliferation after indirect exposure to several of the polysaccharide-based materials. Results from this study demonstrate that hydrogels made of dextran and hyaluronan solution show good cytocompatibility in vitro. making these degradable matrices interesting candidates for drug delivery purposes.     

Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes

After entry into lymph nodes (LNs), B cells migrate to follicles, whereas T cells remain in the paracortex, with each lymphocyte type showing apparently random migration within these distinct areas. Other than chemokines, the factors contributing to this spatial segregation and to the observed patterns of lymphocyte movement are poorly characterized. By combining confocal, electron, and intravital microscopy, we showed that the fibroblastic reticular cell network regulated naive T cell access to the paracortex and also supported and defined the limits of T cell movement within this domain, whereas a distinct follicular dendritic cell network similarly served as the substratum for movement of follicular B cells. These results highlight the central role of stromal microanatomy in orchestrating cell migration within the LN.     

Sculpting the blank slate: How fibrin’s support of vascularization can inspire biomaterial design

Fibrin is the primary extracellular constituent of blood clots, and plays an important role as a provisional matrix during wound healing and tissue remodeling. Fibrin-based biomaterials have proven their utility as hemostatic therapies, scaffolds for tissue engineering, vehicles for controlled release, and platforms for culturing and studying cells in three dimensions. Nevertheless, fibrin presents a complex milieu of signals to embedded cells, many of which are not well understood. Synthetic extracellular matrices (ECMs) provide a blank slate that can ostensibly be populated with specific bioactive cues, including growth factors, growth factor binding motifs, adhesive peptides and peptide crosslinks susceptible to proteases, thereby enabling a degree of customization for specific applications. However, the continued evolution and improvement of synthetic ECMs requires parallel efforts to deconstruct native ECMs and decipher the cues they provide to constituent cells. The objective of this review is to reintroduce fibrin, a protein with a well-characterized structure and biochemistry, and its ability to support angiogenesis specifically. Although fibrin’s structure–function relationships have been studied for decades, opportunities to engineer new and improved synthetic hydrogels can be realized by further exploiting fibrin’s inspiring design.     

Fibronectin-hyaluronic acid composite hydrogels for three-dimensional endothelial cell culture

Biomaterials that actively promote both wound healing and angiogenesis are of critical importance for many biomedical applications, including tissue engineering. In particular, hyaluronic acid (HA) is an important player that has multiple roles throughout the angiogenic process in the body. Previously, our laboratory has developed photocrosslinkable HA-based scaffolds that promote angiogenesis when implanted in vivo. This paper reports the incorporation of a photocrosslinkable fibronectin (FN) conjugate into three-dimensional (3-D) HA hydrogel networks to enhance endothelial cell adhesion and angiogenesis. The results demonstrate significantly better retention of FN that was photocrosslinked within HA hydrogels compared to FN that was physically adsorbed within HA hydrogels. Increased viability of endothelial cells cultured in 3-D HA hydrogels with photoimmobilized FN, compared to adsorbed FN, was also observed. Endothelial cells were cultured within hydrogels for up to 6 days, a period over which cell proliferation, migration and an angiogenic phenotype were influenced by varying the concentration of incorporated FN. The results demonstrate the potential of these composite hydrogels as biomaterial scaffolds capable of promoting wound healing and angiogenesis.     

Engineering of synthetic cellular microenvironments: Implications for immunity

In this article, we discuss novel synthetic approaches for studying the interactions of cells with their microenvironment. Notably, critical cellular processes such as growth, differentiation, migration, and fate determination, are tightly regulated by interactions with neighboring cells, and the surrounding extracellular matrix. Given the huge complexity of natural cellular environments, and their rich molecular and physical diversity, the mission of understanding “environmental signaling” at a molecular-mechanistic level appears to be extremely challenging. To meet these challenges, attempts have been made in recent years to design synthetic matrices with defined chemical and physical properties, which, artificial though they may be, could reveal basic “design principles” underlying the physiological processes. Here, we summarize recent developments in the characterization of the chemical and physical properties of cell sensing and adhesion, as well as the design and use of engineered, micro- to nanoscale patterned and confined environments, for systematic, comprehensive modulation of the cells' environment. The power of these biomimetic surfaces to highlight environmental signaling events in cells, and in immune cells in particular, will be discussed.     

Increasing capillary diameter and the incorporation of gelatin enhance axon outgrowth in alginate-based anisotropic hydrogels

Substantial recovery of function following peripheral and central nervous system (CNS) injury critically depends on longitudinally directed axon regeneration across the injury site, which requires a mechanical guidance providing scaffold. We have previously shown that anisotropic alginate-based hydrogels with a defined capillary diameter (25 μm), which form via a self-organizing process driven by unidirectional diffusion of divalent cations into sodium alginate sols, promoted longitudinally oriented elongation of CNS axons in vitro and in vivo. In the present study the influence of various capillary diameters and the incorporation of gelatin to promote directed axon outgrowth and Schwann cell migration were assessed in a dorsal root ganglion outgrowth assay in vitro. Superimposing an alginate sol with Cu(2+), Sr(2+), or Zn(2+) ion containing solutions allowed the creation of hydrogels with capillaries 18, 25 and 55 μm in diameter, respectively. Axon outgrowth and Schwann cell migration were analyzed in terms of axon length/density and Schwann cell density within the capillary structures. Axon ingrowth into capillary hydrogels, which was always accompanied by Schwann cells, was enhanced with increasing capillary diameter. The incorporation of gelatin did not influence overall axon density, but promoted the length of axon outgrowth within the hydrogels. The longitudinal orientation of axons decreased in wider capillaries, which suggests that medium-sized capillaries are the optimal substrate to elicit substantial axon growth and longitudinal orientation after axon injury.     

Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters

Understanding the influence of a controlled spatial distribution of biological cues on cell activities can be useful to design “cell instructive” materials, able to control and guide the formation of engineered tissues in vivo and in vitro. To this purpose, biochemical and mechanical properties of the resulting biomaterial must be carefully designed and controlled. In this work, the effect of covalently immobilized RGD peptide gradients on poly(ethylene glycol) diacrylate hydrogels on cell behaviour was studied. We set up a mechanical device generating gradients based on a fluidic chamber. Cell response to RGD gradients with different slope (0.7, 1 and 2 mM cm^?1) was qualitatively and quantitatively assessed by evaluating cell adhesion and, in particular, cell migration, compared to cells seeded on hydrogels with uniform distribution of RGD peptides. To evaluate the influence of RGD gradient and to exclude any concentration effect on cell response, all analyses were carried out in a specific region of the gradients which displayed the same average concentration of RGD (1.5 mM). Results suggest that cells recognize the RGD gradient and adhere onto it assuming a stretched shape. Moreover, cells tend to migrate in the direction of the gradient, as their speed is higher than that of cells migrating on hydrogels with a uniform distribution of RGD and increases by increasing RGD gradient steepness. This increment is due to an augmentation of bias speed component of the mean squared speed, that is, the drift of the cell population migrating on the anisotropic surface provided by the RGD gradient.     

Magnetically-guided self-assembly of fibrin matrices with ordered nano-scale structure for tissue engineering

The development of effective biological scaffold materials for tissue engineering and regenerative medicine applications hinges on the ability to present precise environmental cues to specific cell populations to guide their position and function. Natural extracellular matrices have an ordered nano-scale structure that can modulate cell behaviors critical for developmental control, including directional cell motility. Here we describe a method for fabricating fibrin gels with defined architecture on the nanometer scale in which magnetic forces are used to position thrombin-coated magnetic micro-beads in a defined 2-dimensional array and thereby guide the self-assembly of fibrin fibrils through catalytic cleavage of soluble fibrinogen substrate. Time-lapse and confocal microscopy confirmed that fibrin fibrils nucleate near the surface of the thrombin-coated beads and extend out in a radial direction to form these gels. When controlled magnetic fields were used to position the beads in hexagonal arrays, the fibrin nano-fibrils that polymerized from the beads oriented preferentially along the bead--bead axes in a geodesic (minimal path) pattern. These biocompatible scaffolds supported adhesion and spreading of human microvascular endothelial cells, which exhibited co-alignment of internal actin stress fibers with underlying fibrin nano-fibrils within some membrane extensions at the cell periphery. This magnetically-guided, biologically-inspired microfabrication system is unique in that large scaffolds may be formed with little starting material, and thus it may be useful for in vivo tissue engineering applications in the future.