enyung Cha

A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity.

Three dimensional (3D) tumor spheroid models are becoming important biomedical tools for both fundamental and applied cancer studies, but current models do not account for different levels of cancer malignancy. Several studies have reported that the mechanical rigidity of a hydrogel plays a significant role in regulating the phenotypes of cancer cells adhered to the gel surface. This finding suggests that matrix rigidity should also modulate the malignancy of 3D tumor spheroids. However, the role of matrix stiffness is often confounded by concurrent changes in 3D matrix permeability. This study reports an advanced strategy to assemble 3D liver tumor spheroids with controlled intercellular organization, phenotypes, and angiogenic activities using hydrogels with controlled stiffness and minimal differences in molecular diffusivity. The elastic moduli of cell-encapsulated collagen gels were increased by stiffening interconnected collagen fibers with varied amounts of poly(ethylene glycol) di-(succinic acid N-hydroxysuccinimidyl ester). Interestingly, hepatocellular carcinoma cells encapsulated in a fat-like, softer hydrogel formed malignant cancer spheroids, while cells cultured in a liver-like, stiffer gel formed compact hepatoids with suppressed malignancy. Overall, both the hydrogel and the 3D tumor spheroids developed in this study will be greatly useful to better understand and regulate the emergent behaviors of various cancer cells.

Decoupled control of stiffness and permeability with a cell-encapsulating poly(ethylene glycol) dimethacrylate hydrogel.

Hydrogels are increasingly used as a cell encapsulation and transplantation device. The successful use of a hydrogel greatly relies on an ability to control hydrogel stiffness which affects structural integrity and regulates cellular phenotypes. However, conventional strategies to increase the gel stiffness lead to decrease in the gel permeability and subsequently deteriorate the viability of cells encapsulated in a gel matrix. This study presents a strategy to decouple the inversed dependency of permeability on the stiffness of a hydrogel by chemically cross-linking methacrylic alginate with poly(ethylene glycol) dimethacrylate (PEGDA). As expected, gel stiffness represented by elastic modulus was tuned over one order of magnitude with the concentration of methacrylic alginate and the degree of substitution of methacrylic groups. In contrast, swelling ratio of the hydrogel indicative of gel permeability was minimally changed because of multiple hydrophilic groups of alginate, similar to function of proteoglycans in a natural extracellular matrix. Furthermore, viability of neural cells encapsulated in a hydrogel of PEGDA and methacrylic alginate rather increased with hydrogel stiffness. Overall, the results of this study demonstrate an advanced biomaterial design paradigm which allows one to culture cells in a 3D matrix of varying rigidity. This study will therefore greatly expedite the use of a hydrogel system in both fundamental studies and clinical settings of cell therapies.

Tuning the dependency between stiffness and permeability of a cell encapsulating hydrogel with hydrophilic pendant chains.

The mechanical stiffness of a hydrogel plays a significant role in regulating the phenotype of cells that adhere to its surface. However, the effect of hydrogel stiffness on cells cultured within its matrix is not well understood, because of the intrinsic inverse dependency between the permeability and stiffness of hydrogels. This study therefore presents an advanced biomaterial design strategy to decrease the inverse dependency between permeability and stiffness of a cell encapsulating hydrogel. Hydrogels were made by cross-linking poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) monoacrylate (PEGMA), with PEGMA acting as a pendant polymer chain. Increasing the mass fraction of PEGMA while keeping the total polymer concentration constant led to a decrease in the elastic modulus (E) of the hydrogel, but caused a minimal increase in the swelling ratio (Q). The size and hydrophobicity of the end groups of pendant PEG chains further fine tuned the dependency between Q and E of the hydrogel. Pure PEGDA hydrogels with varying molecular weights, which show the same range of E but a much greater range of Q, were used as a control. Fibroblasts encapsulated in PEGDA-PEGMA hydrogels displayed more significant biphasic dependencies of cell viability and vascular endothelial growth factor (VEGF) expression on E than those encapsulated in pure PEGDA hydrogels, which were greatly influenced by Q. Overall, the hydrogel design strategy presented in this study will be highly useful to better regulate the phenotype and ultimately improve the therapeutic efficacy of a wide array of cells used in various biology studies and clinical settings.

Integrative design of a poly(ethylene glycol)-poly(propylene glycol)-alginate hydrogel to control three dimensional biomineralization

A mineralized polymeric matrix has been extensively studied to understand biomineralization processes and to further regulate phenotypic functions of various cells involved in osteogenesis and physiological homeostasis. It has been often proposed that several matrix variables including charge density, hydrophobicity, and pore size play vital roles in modulating composition and morphology of minerals formed within a three dimensional (3D) matrix. However, the aspects have not yet been systematically examined because a tool enabling the independent control of the matrix variables is lacking. This study presents an advanced integrative strategy to control morphology and composition of biominerals with matrix properties, by using a hydrogel formulated to independently control charge density, hydrophobicity, and porosity. The hydrogel consists of poly(ethylene glycol) monomethacrylate (PEGmM), poly(propylene glycol) monomethacrylate (PPGmM), and methacrylic alginate (MA), so the charge density and hydrophobicity of the hydrogel can be separately controlled with mass fractions of MA and PPGmM. Also, hydrogels which present only nano-sized pores, termed nanoporous hydrogels, are lyophilized and rehydrated to prepare the hydrogels containing micro-sized pores, termed microporous hydrogels. We find that increasing the mass fractions of MA and PPGmM of the microporous hydrogel promotes the growth of apatite layers because of the increases in the charge density, hydrophobicity and pore size. In contrast, increasing mass fractions of MA and PPGmM of the nanoporous hydrogel enhances the formation of calcium carbonate minerals. The dependency of the mineralization on hydrogel variables is related to the change in supersaturation of mineral ions. Overall, the results of this study will be highly useful to better understand the interplay of matrix variables in biomineralization and to design a wide array of mineralized matrix potentially used in cell therapies and tissue engineering.

Tailoring Hydrogel Adhesion to Polydimethylsiloxane Substrates Using Polysaccharide Glue

With the emergence of microscale biotechnology, such as biomicroelectromechanical systems (“Bio-MEMS”) and microfluidic-based microchips for sensing and diagnostics, polydimethylsiloxane (PDMS)-based elastomers have become very popular materials. PDMS elastomers possess several features that are well suited for these applications: mechanical stability and elasticity, chemical inertness, optical transparency, gas permeability, ease of fabrication, and biocompatibility. However, the extremely hydrophobic nature of PDMS often limits its applicability (e.g. poor aqueous fluid flow and nonspecific adhesion of biomolecules). Various methods have been proposed to modify the PDMS surface to impart hydrophilicity, for example, UV or plasma treatment to oxidize the surface and coating the surface with hydrophilic polymers. However, the treated PDMS surfaces often recover their hydrophobic traits due to the migration of unreacted PDMS oligomers to the surface and the rearrangement of PDMS polymer chains. We suggest that coating PDMS with hydrophilic materials would be more effective than the molecular level modifications. Hydrogels, which are networks of cross-linked polymers taking up large amounts of water, are therefore considered promising materials. Hydrogels can also be designed to present functionalities for specific purposes, such as in vitro cell culture, cell encapsulation, and molecular capture and release. Therefore, PDMS coated with hydrogels with desired properties would significantly enhance the performance of PDMS-based devices. However, it is a significant challenge to attain and sustain the adhesion between hydrogel and PDMS, due to the stark discrepancy between the bulk properties of PDMS substrates and hydrogels. To meet this challenge, we describe a unique approach to tailor hydrogel adhesion to a PDMS substrate. Alginate, a naturally derived polysaccharide, was covalently linked to the PDMS surface. This attached alginate acted as a “glue” to allow the strong, permanent adhesion of the hydrogel onto the PDMS surface by 1) imparting hydrophilicity to improve compatibility with hydrogels, and 2) providing functional groups for the stable conjugation of hydrogels. The resulting hydrogel-coated PDMS substrate was used in the following two applications: 1) it served as an in vitro cell culture platform to study cellular behavior in response to cyclic mechanical strain, and 2) it was used in a microfluidic device with hydrogel-filled channels. The PDMS surface was chemically grafted with alginate following a series of modification steps: step 1: oxidation to present hydroxy groups (OH-PDMS, Figure 1a); step 2: silanization using 3-aminopropyltriethoxysilane to present primary amino groups (NH2-PDMS); and step 3: conjugation of alginate by carbodiimide-mediated amide coupling between amino groups on the PDMS surface and carboxylic acid groups of alginate (alginate-PDMS). The successive modifications of PDMS were confirmed with FTIR spectroscopy (Figure S1 and Table S1 in the Supporting Information). The chemical linkage of alginate to the PDMS surface was further confirmed with fluorescently labeled alginate (Figure S2 in the Supporting Information). The decreased water contact angle of alginate-PDMS also showed that it is more hydrophilic than unmodified PDMS, OH-PDMS, and NH2-PDMS (Figure 1b). Next, alginate hydrogels were fabricated on the alginatePDMS by means of activating a covalent or an ionic crosslinking reaction. We thought that the alginate glue on the PDMS surface would participate in the reaction and hold the alginate hydrogel to the surface (Figure 2). First, an aqueous mixture of alginate and adipic acid dihydrazide (AAD) was placed on the alginate-PDMS, in order to fabricate the hydrogel by carbodiimide-mediated amide coupling. The resulting AAD-alginate hydrogel remained stably attached to alginate-PDMS for several months, regardless of the gel thickness, demonstrating that the alginate glue on the PDMS participated in the crosslinking reaction. No interfacial failure was observed between the bulk hydrogel and alginate-PDMS even when the construct was bent (Figure 3a). Alginate hydrogel crosslinked with calcium ions could also be prepared on alginate-PDMS. The resulting calciumalginate hydrogel also remained stably attached to the alginate-PDMS for several months. No interfacial failure [*] M. Lee, J. H. Jeong, Prof. H. Kong Department of Chemical and Biomolecular Engineering University of Illinois, Urbana, IL 61801 (USA) E-mail: hjkong06@illinois.edu

Tuning responsiveness and structural integrity of a pH responsive hydrogel using a poly(ethylene glycol) cross-linker

Stimulus responsive hydrogels have received considerable attention due to their ability to exhibit reversible volumetric changes in response to external stimuli. The shrinkage or expansion of a hydrogel often results in irreversible structural failure, but limited efforts have been made to resolve the challenge of hydrogel fracture. This study presents that the number (FM) of cross-linkers and the molecular weight (Mw) of the cross-linkers play critical roles determining the responsiveness and the structural integrity of a hydrogel exposed to external stimuli. A pH-responsive hydrogel was prepared by cross-linking poly(acrylamide-co-acrylic acid) with bis-acrylamide (Bis) or poly(ethylene glycol) diacrylate (PEGDA) of varying Mw. The deswelling rate of the hydrogel incubated in an acidic medium was solely controlled by the number of elastically effective cross-links (N) which was increased with FM and Mw of the cross-linker. The structural integrity of the hydrogel during a volumetric change was dependent on the work to fracture and the deswelling rate, which was tuned with the Mw of the cross-linker. These critical roles of cross-linkers in mediating the elastic response and the structural integrity of the hydrogel were further related to the ability of cross-linkers of different Mw to self-organize. Ultimately, the results of this study will be highly useful to tuning performance of a wide array of stimulus responsive hydrogels.

Tuning the non-equilibrium state of a drug-encapsulated poly(ethylene glycol) hydrogel for stem and progenitor cell mobilization

Injectable and biodegradable hydrogels have been increasingly studied for sustained drug delivery in various molecular therapies. However, it remains a challenge to attain desired delivery rate at injection sites due to local tissue pressures exerted on the soft hydrogels. Furthermore, there is often limited controllability of stiffness and degradation rates, which are key factors required for achieving desired drug release rate and therapeutic efficacy. This study presents a stiff and metastable poly(ethylene glycol) diacrylate (PEGDA)-poly(ethylene imine) (PEI) hydrogel which exhibits an elastic modulus equivalent to bulk plastic materials, and controllable degradation rate independent of its initial elastic modulus. Such unique stiffness was attained from the highly branched architecture of PEI, and the decoupled controllability of degradation rate was achieved by tuning the non-equilibrium swelling of the hydrogel. Furthermore, a single intramuscular administration of granulocyte colony stimulating factor (GCSF)-encapsulated PEGDA-PEI hydrogel extended the mobilization of mononuclear cells to four days. A larger yield of expanded CD34+ and CD31+ endothelial progenitor cells (EPCs) was also obtained as compared to the daily bolus administration. Overall, the hydrogel created in this study will be useful for the controlled and sustained delivery of a wide array of drug molecules.

Tuning structural durability of yeast‐encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross‐linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross‐links in an ionically cross‐linked alginate gel bead significantly enhances the beads structural durability. The interpenetrating network (IPN) was prepared by first cross‐linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross‐linking reaction. The resulting methacrylic alginate gel beads (IPN‐MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross‐linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN‐MA gel beads remained more metabolically active in ethanol production than those in calcium cross‐linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes. Biotechnol. Bioeng. 2012;109: 63–73.

Top-down synthesis of versatile polyaspartamide linkers for single-step protein conjugation to materials

Materials used in various biological applications are often modified with proteins to regulate biomolecular and cellular adhesion. Conventional strategies of protein conjugation accompany monovalent bifunctional protein linkers, which present several limitations in molecular synthesis and protein conjugation. Herein, we present a new strategy of preparing multivalent polyaspartamide linkers in a simple top-down manner, and also demonstrate that the resulting polymer linkers allow us to readily conjugate proteins to both organic and inorganic materials. The top-down synthesis of polyaspartamide linkers was performed by partially opening succinimidyl ring moieties of polysuccinimide (PSI) with the controlled number of nucleophiles reactive to photo-cross-linked hydrogel or gold-coated inorganic materials: (1) Poly(2-hydroxyethyl-co-2-methacryloxyethyl aspartamide) (PHMAA) presenting methacrylate was used to micropattern fibronectin or collagen on a hydrogel in order to regulate cell adhesion and growth area on a micrometer scale. (2) Poly(2-hydroxyethyl-co-2-mercaptoethyl aspartamide) (PHMCA) presenting thiol functional groups was used to link fibronectin to a gold-coated silicon microelectromechanical probe designed to measure cell traction force. Overall, these multivalent polyaspartamide protein linkers will greatly assist efforts to analyze and regulate the cellular adhesion to and phenotypic activities of a wide array of substrates and devices.

Tailoring the dependency between rigidity and water uptake of a microfabricated hydrogel with the conformational rigidity of a polymer cross-linker

Many diverse applications utilize hydrogels as carriers, sensors, and actuators, and these applications rely on the refined control of physical properties of the hydrogel, such as elastic modulus and degree of swelling. Often, hydrogel properties are interdependent; for example, when elastic modulus is increased, degree of swelling is decreased. Controlling these inverse dependencies remains a major barrier for broader hydrogel applications. We hypothesized that polymer cross-linkers with varied chain flexibility would allow us to tune the inverse dependency between the elastic modulus and the degree of swelling of the hydrogels. We examined this hypothesis by using alginate and poly(acrylic acid) (PAA) modified with a controlled number of methacrylic groups as model inflexible and flexible cross-linkers, respectively. Interestingly, the polyacrylamide hydrogel cross-linked by the inflexible alginate methacrylates exhibited less dependency between the degree of swelling and the elastic modulus than the hydrogel cross-linked by flexible PAA methacrylates. This critical role of the cross-linkers inflexibility was related to the difference of the degree of hydrophobic association between polymer cross-linkers, as confirmed with pyrene probes added in pregel solutions. Furthermore, hydrogels cross-linked with alginate methacrylates could tune the projection area of adhered cells by solely altering elastic moduli. In contrast, gels cross-linked with PAA methacrylates failed to modulate the cellular adhesion morphology due to a lower, and smaller, elastic modulus range to be controlled. Overall, the results of this study will significantly advance the controllability of hydrogel properties and greatly enhance the performance of hydrogels in various biological applications.