Materials that support the assembly of new vasculature are critical for regenerative medicine. Controlling the scaffold’s mechanical properties may help to optimize neovascularization within implanted biomaterials. However, reducing the stiffness of synthetic hydrogels usually requires decreasing polymer densities or increasing chain lengths, both of which accelerate degradation. We synthesized enzymatically-degradable poly(ethylene glycol) hydrogels with compressive moduli from 2 to 18 kPa at constant polymer density, chain length, and proteolytic degradability by inserting an allyloxycarbonyl functionality into the polymer backbone. This group competes with acrylates during photopolymerization to alter the crosslink network structure and reduce the hydrogel’s stiffness. Hydrogels that incorporated (soft) or lacked (stiff) this group were implanted subcutaneously in rats to investigate the role of stiffness on host tissue interactions. Changes in tissue integration were quantified after 4 weeks via the hydrogel area replaced by native tissue (tissue area fraction), yielding 0.136 for softer vs. 0.062 for stiffer hydrogels. Including soluble FGF-2 and PDGF-BB improved these responses to 0.164 and 0.089, respectively. Softer gels exhibited greater vascularization with 8.6 microvessels mm−2 compared to stiffer gels at 2.4 microvessels mm−2. Growth factors improved this to 11.2 and 4.9 microvessels mm−2, respectively. Softer hydrogels tended to display more sustained responses, promoting neovascularization and tissue integration in synthetic scaffolds.

Knowledge of how extracellular matrix (ECM) binding impacts valve interstitial cells (VICs) is critical not only to better understanding the etiology of valvular diseases but also to constructing living valve substitutes that can grow and remodel. Use of ECM-mimicking adhesive peptides with specific affinity to different receptors provides insights into adhesion-mediated cell signaling and downstream outcomes. Expression of adhesion receptors by VICs was assessed by flow cytometry and used to guide the choice of peptides studied. The peptide RGDS with affinity to multiple integrin receptors, and specific receptor-targeting peptides DGEA (integrin α2β1), YIGSR (67 kDa laminin/elastin receptor; 67LR), and VAPG (67LR) were incorporated into hydrogels to investigate their effects on VICs. DGEA, YIGSR, and VAPG alone were insufficient to induce stable VIC adhesion. As a result, these peptides were studied in combination with 1 mM RGDS. For VICs cultured on two-dimensional hydrogel surfaces, YIGSR and VAPG down-regulated the expression of smooth muscle α-actin (myofibroblast activation marker); DGEA promoted VIC adhesion and VIC-mediated ECM deposition and inhibited the activity of alkaline phosphatase (osteogenic differentiation marker). Further, YIGSR and DGEA in combination promoted ECM deposition while inhibiting both myofibroblastic and osteogenic differentiation. However, VICs behaved differently to adhesive ligands when cultured within three-dimensional hydrogels, with most VICs assuming a healthy, quiescent phenotype under all peptide conditions tested. DGEA promoted ECM deposition by VICs within hydrogels. Overall, we demonstrate that the presentation of defined peptides targeting specific adhesion receptors can be used to regulate VIC adhesion, phenotype and ECM synthesis.

The effects of mechanical cues on cell behaviors in 3D remain difficult to characterize as the ability to tune hydrogel mechanics often requires changes in the polymer density, potentially altering the material’s biochemical and physical characteristics. Additionally, with most PEG diacrylate (PEGDA) hydrogels, forming materials with compressive moduli less than ∼10 kPa has been virtually impossible. Here, we present a new method of controlling the mechanical properties of PEGDA hydrogels independent of polymer chain density through the incorporation of additional vinyl group moieties that interfere with the cross-linking of the network. This modification can tune hydrogel mechanics in a concentration dependent manner from <1 to 17 kPa, a more physiologically relevant range than previously possible with PEG-based hydrogels, without altering the hydrogel’s degradation and permeability. Across this range of mechanical properties, endothelial cells (ECs) encapsulated within MMP-2/MMP-9 degradable hydrogels with RGDS adhesive peptides revealed increased cell spreading as hydrogel stiffness decreased in contrast to behavior typically observed for cells on 2D surfaces. EC-pericyte cocultures exhibited vessel-like networks within 3 days in highly compliant hydrogels as compared to a week in stiffer hydrogels. These vessel networks persisted for at least 4 weeks and deposited laminin and collagen IV perivascularly. These results indicate that EC morphogenesis can be regulated using mechanical cues in 3D. Furthermore, controlling hydrogel compliance independent of density allows for the attainment of highly compliant mechanical regimes in materials that can act as customizable cell microenvironments.Keywords: biomimetic material; PEG hydrogel; tissue engineering; vasculogenesis

Heterogeneity of cell populations can confound population-averaged measurements and obscure important findings or foster inaccurate conclusions. The ability to generate a homogeneous cell population, at least with respect to a chosen trait, could significantly aid basic biological research and development of high-throughput assays. Accordingly, we developed a high-resolution, image-based patterning strategy to produce arrays of single-cell patterns derived from the morphology or adhesion site arrangement of user-chosen cells of interest (COIs). Cells cultured on both cell-derived patterns displayed a cellular architecture defined by their morphology, adhesive state, cytoskeletal organization, and nuclear properties that quantitatively recapitulated the COIs that defined the patterns. Furthermore, slight modifications to pattern design allowed for suppression of specific actin stress fibers and direct modulation of adhesion site dynamics. This approach to patterning provides a strategy to produce a more homogeneous cell population, decouple the influences of cytoskeletal structure, adhesion dynamics, and intracellular tension on mechanotransduction-mediated processes, and a platform for high-throughput cellular assays.Keywords: actin cytoskeleton; biomimetic; cell adhesion; cell arrays; cell engineering; cell patterning; cell population heterogeneity; cell population homogeneity; cell-derived patterning; image-guided patterning; laser scanning lithography; mechanotransduction; nanopatterning; self-assembled monolayers;

Nanoparticle drug delivery carriers that can modulate drug release based on an exogenous signal, such as light, are of great interest, especially for improving cancer therapy. A light-activated delivery vehicle was fabricated by synthesizing a thin, thermally responsive poly(N-isopropylacrylamide-co-acrylamide) hydrogel coating directly onto the surfaces of individual near-infrared (NIR) absorbing gold-silica nanoshells. This hydrogel was designed to be in a swollen state under physiological conditions and expel large amounts of water, along with any entrapped drug, at elevated temperatures. The required temperature change can be achieved via NIR absorption by the nanoshell, allowing the hydrogel phase change to be triggered by light, which was observed by monitoring changes in particle sizes as water was expelled from the hydrogel network. The phase change was reversible and repeatable. As a model drug, the chemotherapeutic doxorubicin was loaded into this delivery vehicle, and rapid release of doxorubicin occurred upon NIR exposure. Further, colon carcinoma cells exposed to the irradiated platform displayed nearly 3 times as much doxorubicin uptake as cells exposed to nonirradiated particles or free drug, which in turn resulted in a higher loss of cell viability. We hypothesize these effects are because the NIR-mediated heating results in a transient increase in cell membrane permeability, thus aiding in cellular uptake of the drug.Keywords: atom transfer radical polymerization (ATRP); drug delivery; gold nanoshells; n-isopropylacrylamide; optically triggered; thermally responsive

In order to independently study the numerous variables that influence cell movement, it will be necessary to employ novel tools and materials that allow for exquisite control of the cellular microenvironment. In this work, we have applied advanced 3D micropatterning technology, known as two-photon laser scanning lithography (TP-LSL), to poly(ethylene glycol) (PEG) hydrogels modified with bioactive peptides in order to fabricate precisely designed microenvironments to guide and quantitatively investigate cell migration. Specifically, TP-LSL was used to fabricate cell adhesive PEG-RGDS micropatterns on the surface of non-degradable PEG-based hydrogels (2D) and in the interior of proteolytically degradable PEG-based hydrogels (3D). HT1080 cell migration was guided down these adhesive micropatterns in both 2D and 3D, as observed via time-lapse microscopy. Differences in cell speed, cell persistence, and cell shape were observed based on variation of adhesive ligand, hydrogel composition, and patterned area for both 2D and 3D migration. Results indicated that HT1080s migrate faster and with lower persistence on 2D surfaces, while HT1080s migrating in 3D were smaller and more elongated. Further, cell migration was shown to have a biphasic dependence on PEG-RGDS concentration and cells moving within PEG-RGDS micropatterns were seen to move faster and with more persistence over time. Importantly, the work presented here begins to elucidate the multiple complex factors involved in cell migration, with typical confounding factors being independently controlled. The development of this unique platform will allow researchers to probe how cells behave within increasingly complex 3D microenvironments that begin to mimic specifically chosen aspects of the in vivo landscape.

Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor–matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor–ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis.

Tumor angiogenesis is a hallmark of cancer that has been identified as a critical component of cancer progression, facilitating rapid tumor growth and metastasis. Anti-angiogenic therapies have exhibited only modest clinical success, highlighting a need for better models that can be used to gain a more thorough understanding of tumor angiogenesis and screen potential therapeutics more accurately. This review explores how recent progress in in vitro cancer and vascular models individually can be applied to the development of in vitro tumor angiogenesis models. Current in vitro tumor angiogenesis models are also discussed, with a focus on aspects of the process that have been successfully recapitulated and opportunities for applying new technologies to expand model complexity to better represent the tumor microenvironment. Continued advances in vascularized tumor models will provide tools to identify novel therapeutic targets and validate their therapeutic benefit.Download high-res image (217KB)Download full-size image