April M. Kloxin

Photodegradable hydrogels for dynamic tuning of physical and chemical properties

We report a strategy to create photodegradable poly(ethylene glycol)–based hydrogels through rapid polymerization of cytocompatible macromers for remote manipulation of gel properties in situ. Postgelation control of the gel properties was demonstrated to introduce temporal changes, creation of arbitrarily shaped features, and on-demand pendant functionality release. Channels photodegraded within a hydrogel containing encapsulated cells allow cell migration. Temporal variation of the biochemical gel composition was used to influence chondrogenic differentiation of encapsulated stem cells. Photodegradable gels that allow real-time manipulation of material properties or chemistry provide dynamic environments with the scope to answer fundamental questions about material regulation of live cell function and may affect an array of applications from design of drug delivery vehicles to tissue engineering systems.

In situ elasticity modulation with dynamic substrates to direct cell phenotype

Microenvironment elasticity influences critical cell functions such as differentiation, cytoskeletal organization, and process extension. Unfortunately, few materials allow elasticity modulation in real time to probe its direct effect on these dynamic cellular processes. Here, a new approach is presented for the photochemical modulation of elasticity within the cells microenvironment at any point in time. A photodegradable hydrogel was irradiated and degraded under cytocompatible conditions to generate a wide range of elastic moduli similar to soft tissues and characterized using rheometry and atomic force microscopy (AFM). The effect of the elastic modulus on valvular interstitial cell (VIC) activation into myofibroblasts was explored. In these studies, gradient samples were used to identify moduli that either promote or suppress VIC myofibroblastic activation. With this knowledge, VICs were cultured on a high modulus, activating hydrogel substrate, and uniquely, results show that decreasing the substrate modulus with irradiation reverses this activation, demonstrating that myofibroblasts can be de-activated solely by changing the modulus of the underlying substrate. This finding is important for the rational design of biomaterials for tissue regeneration and offers insight into fibrotic disease progression. These photodegradable hydrogels demonstrate the capability to both probe and direct cell function through dynamic changes in substrate elasticity.

Mechanical Properties of Cellularly Responsive Hydrogels and Their Experimental Determination

Hydrogels are increasingly employed as multidimensional cell culture platforms often with a necessity that they respond to or control the cellular environment. Specifically, synthetic hydrogels, such as poly(ethylene glycol) (PEG)-based gels, are frequently utilized for probing the microenvironments influence on cell function, as the gel properties can be precisely controlled in space and time. Synthetically tunable parameters, such as monomer structure and concentration, facilitate initial gel property control, while incorporation of responsive degradable units enables cell- and/or user-directed degradation. Such responsive gel systems are complex with dynamic changes occurring over multiple time-scales, and cells encapsulated in these synthetic hydrogels often experience and dictate local property changes profoundly different from those in the bulk material. Consequently, advances in bulk and local measurement techniques are needed to monitor property evolution quantatively and understand its effect on cell function. Here, recent progress in cell-responsive PEG hydrogel synthesis and mechanical property characterization is reviewed.

Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms

We describe a detailed procedure to create photolabile, polyethylene glycol (PEG)-based hydrogels and manipulate material properties in situ. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in two-dimensional (2D) or 3D cell culture. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. In this study, we provide coupling procedures to PEG-bis-amine to form a photodegradable cross-linker or to the fibronectin-derived peptide RGDS to form a photoreleasable tether. Hydrogels are synthesized with the photodegradable cross-linker in the presence or absence of cells, allowing direct encapsulation or seeding on surfaces. Cell-material interactions can be probed in 2D or 3D by spatiotemporally controlling the gel microenvironment, which allows unique experiments to be performed to monitor cell response to changes in their niche. Degradation is readily achieved with cytocompatible wavelengths of low-intensity flood irradiation (365–420 nm) in minutes or with high-intensity laser irradiation (405 nm) in seconds. In this protocol, synthesis and purification of photodegradable monomers take approximately 2 weeks, but the process can be substantially shortened by purchasing the o-nitrobenzylether precursor. Preparation of sterile solutions for hydrogel fabrication takes hours, whereas the reaction to form the final hydrogel is complete in minutes. Hydrogel degradation occurs on demand, in seconds to minutes, with user-directed light exposure. This comprehensive protocol is useful for controlling peptide presentation and substrate modulus during cell culture on or within an elastic matrix. These PEG-based materials are useful for probing the dynamic influence of cell-cell and cell-material interactions on cell function in 2D or 3D. Although other protocols are available for controlling peptide presentation or modulus, few allow manipulation of material properties in situ and in the presence of cells down to the micrometer scale.

Tunable Hydrogels for External Manipulation of Cellular Microenvironments through Controlled Photodegradation

Hydrogels provide a unique environment for three-dimensionalcell culture, but typically, the material properties are fixed uponformation. Given the growing interest in understanding howmaterialmicroenvironmentsinfluencecellularfunctions,numer-ous approaches have emerged to control not only the initialbiochemicalandbiophysicalpropertiesofgels,butalsohowtheseproperties change with degradation. While these strategies allowthe synthesis of hydrogels with predictable degradation profilesand property changes, a material system that allows externalmanipulation of the properties of a cell-laden gel at any point intime or space would fill a unique niche. For example, such a cellculture system would allow real-time manipulation of theextracellular microenvironment and simultaneous monitoringof cellular processes in three-dimensional culture. In thiscontribution, photocleavable linkers were integrated into thecrosslinks of a poly(ethylene glycol)-based (PEG) hydrogel,allowing the network structure to be tuned exogeneously andpredictably with irradiation under cytocompatible conditions.Such a material system will enable new opportunities to testhypotheses about how precise variations in the local gelenvironment direct important cellular functions, such as processextension, migration, and mechanotransduction.There is a growing interest in the development of hydrogels asa platform for encapsulating cells and their application in fieldsranging from tissue engineering to three-dimensional cellculture.

Redirecting Valvular Myofibroblasts into Dormant Fibroblasts through Light-mediated Reduction in Substrate Modulus

Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young’s modulus of the substrate was reduced from ∼32 kPa, mimicking pre-calcified diseased tissue, to ∼7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in α-smooth muscle actin (α-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including α-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-β1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.

Mechanical Properties and Degradation of Chain and Step Polymerized Photodegradable Hydrogels.

The relationship between polymeric hydrogel microstructure and macroscopic properties is of specific interest to the materials science and polymer science communities for the rational design of materials for targeted applications. Specifically, research has focused on elucidating the role of network formation and connectivity on mechanical integrity and degradation behavior. Here, we compared the mechanical properties of chain- and step-polymerized, photodegradable hydrogels. Increased ductility, tensile toughness, and shear strain to yield were observed in step-polymerized hydrogels, as compared to the chain-polymerized gels, indicating that increased homogeneity and network cooperativity in the gel backbone improves mechanical integrity. Furthermore, the ability to degrade the hydrogels in a controlled fashion with light was exploited to explore how hydrogel microstructure influences photodegradation and erosion. Here, the decreased network connectivity at the junction points in the step-polymerized gels resulted in more rapid erosion. Finally, a relationship between the reverse gelation threshold and erosion rate was developed for the general class of photodegradable hydrogels. In all, these studies further elucidate the relationship between hydrogel formation and microarchitecture with macroscale behavior to facilitate the future design of polymer networks and degradable hydrogels, as well as photoresponsive materials such as cell culture templates, drug delivery vehicles, responsive coatings, and anisotropic materials.

Controlled two-photon photodegradation of PEG hydrogels to study and manipulate subcellular interactions on soft materials.

Cell adhesion and detachment to and from the extracellular matrix (ECM) are critical regulators of cell function and fate due to the exchange of mechanical signals between the cell and its microenvironment. To study this cell mechanobiology, researchers have developed several innovative methods to investigate cell adhesion in vitro; however, most of these culture platforms are unnaturally stiff or static. To better capture the soft, dynamic nature of the ECM, we present a PEG-based hydrogel in which the context and geometry of the extracellular space can be precisely controlled in situ via two-photon induced erosion. Here, we characterize the two-photon erosion process, demonstrate its efficacy in the presence of cells, and subsequently exploit it to induce subcellular detachment from soft hydrogels. A working space was established for a range of laser powers required to induce complete erosion of the gel, and these data are plotted with model predictions. From this working space, two-photon irradiation parameters were selected for complete erosion in the presence of cells. Micron-scale features were eroded on and within a gel to demonstrate the resolution of patterning with these irradiation conditions. Lastly, two-photon irradiation was used to erode the material at the cell-gel interface to remove cell adhesion sites selectively, and cell retraction was monitored to quantify the mesenchymal stem cell (MSC) response to subcellular detachment from soft materials.

Metastatic melanoma – A review of current and future treatment options

Despite advances in treatment and surveillance, melanoma continues to claim approximately 9,000 lives in the US annually (SEER 2013). The National Comprehensive Cancer Network currently recommends ipilumumab, vemurafenib, dabrafenib, and high-dose IL-2 as first line agents for Stage IV melanoma. Little data exists to guide management of cutaneous and subcutaneous metastases despite the fact that they are relatively common. Existing options include intralesional Bacillus Calmette-Guérin, isolated limb perfusion/infusion, interferon-α, topical imiquimod, cryotherapy, radiation therapy, interferon therapy, and intratumoral interleukin-2 injections. Newly emerging treatments include the anti-programmed cell death 1 receptor agents (nivolumab and pembrolizumab), anti-programmed death-ligand 1 agents, and oncolytic vaccines (talimogene laherparepevec). Available treatments for select sites include adoptive T cell therapies and dendritic cell vaccines. In addition to reviewing the above agents and their mechanisms of action, this review will also focus on combination therapy as these strategies have shown promising results in clinical trials for metastatic melanoma treatment.

Hydrogel scaffolds as in vitro models to study fibroblast activation in wound healing and disease

Hydrogels offer controllable and well-defined in vitro platforms to study the role of the fibroblast in wound healing and fibrosis.