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Hydrogels are widely used in stem cell therapy due to their extensive tunability and resemblance to the extracellular matrix (ECM), which has a three-dimensional (3D) structure. These features enable various applications that enhance stem cell maintenance and function. However, fast and simple hydrogel fabrication methods are desirable for stem cells for efficient encapsulation and to reduce adverse effects on the cells. In this study, we present a one-pot double-crosslinked hydrogel consisting of polyethylene glycol (PEG) and collagen, which can be prepared without the multi-step sequential synthesis of each network, by using bio-orthogonal chemistry. To enhance the adipogenic differentiation efficiency of adipose-derived stem cells (ADSCs), we added degradable components within the hydrogel to regulate matrix stiffness through cell-mediated degradation. Bio-orthogonal reactions used for hydrogel gelation allow rapid gel formation for efficient cell encapsulation without toxic by-products. Furthermore, the hybrid network of synthetic (PEG) and natural (collagen) components demonstrated adequate mechanical strength and higher cell adhesiveness. Therefore, ADSCs grown within this hybrid hydrogel proliferated and functioned better than those grown in the single-crosslinked hydrogel. The degradable elements further improved adipogenesis in ADSCs with dynamic changes in modulus during culture and enabled the retrieval of differentiated cells for potential future applications. [ABSTRACT FROM AUTHOR]

The fabrication of mammalian cell-containing poly(ethylene glycol) (PEG) hydrogel microstructures on glass and silicon substrates is described. Using photoreaction injection molding in poly(dimethylsiloxane) microfluidic channels, three-dimensional hydrogel microstructures encapsulating cells (fibroblasts, hepatocytes, macrophage) were fabricated with cells uniformly distributed to each hydrogel microstructure, and the number of cells in each hydrogel microstructure was controlled by changing the cell density of the precursor solution. PEG hydrogels were modified using an Arg-Gly-Asp (RGD) peptide sequence, with the incorporation of RGD into the hydrogel matrix promoting the spreading of encapsulated fibroblasts over a 24-h period in culture. Cells remained viable encapsulated in these hydrogel microstructures for a period in excess of 1 week in culture. Arrays of hydrogel microstructures encapsulating two or more phenotypes on a single substrate were successfully fabricated using multimicrofluidic channels, creating the potential for multiphenotype cell-based biosensors.

The available treatment options include corneal transplantation for significant corneal defects and opacity. However, shortage of donor corneas and safety issues in performing corneal transplantation are the main limitations. Accordingly, we adopted the injectable in situ-forming hydrogels of collagen type I crosslinked via multifunctional polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS) for treatment and evaluated in vivo biocompatibility. The New Zealand White rabbits (N = 20) were randomly grouped into the keratectomy-only and keratectomy with PEG-collagen hydrogel-treated groups. Samples were processed for immunohistochemical evaluation. In both clinical and histologic observations, epithelial cells were able to migrate and form multilayers over the PEG-collagen hydrogels at the site of the corneal stromal defect. There was no evidence of inflammatory or immunological reactions or increased IOP for PEG-collagen hydrogel-treated corneas during the four weeks of observation. Immunohistochemistry revealed the presence of α-smooth muscle actin (α-SMA) in the superior corneal stroma of the keratectomy-only group (indicative of fibrotic healing), whereas low stromal α-SMA expression was detected in the keratectomy with PEG-collagen hydrogel-treated group. Taken together, we suggest that PEG-collagen may be used as a safe and effective alternative in treating corneal defect in clinical setting. [ABSTRACT FROM AUTHOR]

To enhance the mechanical strength of poly(ethylene glycol)(PEG) gels and to provide functional groups for surface modification, we prepared interpenetrating (IPN) hydrogels by incorporating poly(2-hydroxyethyl methacrylate)(PHEMA) inside PEG hydrogels. Formation of IPN hydrogels was confirmed by measuring the weight percent gain of the hydrogels after incorporation of PHEMA, as well as by ATR/FTIR analysis. Synthesis of IPN hydrogels with a high PHEMA content resulted in optically transparent and extensively crosslinked hydrogels with a lower water content and a 6 ∼ 8-fold improvement in mechanical properties than PEG hydrogels. Incorporation of less than 90 wt % PHEMA resulted in opaque hydrogels due to phase separation between water and PHEMA. To overcome the poor cell adhesion properties of the IPN hydrogels, collagen was covalently grafted to the surface of IPN hydrogels via carbamate linkages to hydroxyl groups in PHEMA. Resultant IPN hydrogels were proven to be noncytotoxic and cell adhesion study revealed that collagen immobilization resulted in a significant improvement of cell adhesion and spreading on the IPN hydrogel surfaces. The resultant IPN hydrogels were noncytotoxic, and a cell adhesion study revealed that collagen immobilization improved cell adhesion and spreading on the IPN hydrogel surfaces significantly. These results indicate that PEG/PHEMA IPN hydrogels are highly promising biomaterials that can be used in artificial corneas and a variety of other load-bearing tissue engineering applications. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011 [ABSTRACT FROM AUTHOR]

A non-positional (or suspension) cell microarray was developed using shape-coded SU-8 photoresist microboards for potential application in multiplex and high-throughput cell-based assays. A conventional photolithography process on glass slides produced various shapes of SU-8 micropatterns that had a lateral dimension of 200 μm and a thickness of 40 μm. The resultant micropatterns were detached from the slides by sonication and named 'microboards' due to the fact that had a much larger lateral dimension than thickness. The surfaces of the SU-8 microboards were modified with collagen to promote cell adhesion, and it was confirmed that collagen-coated SU-8 microboards supported cell adhesion and proliferation. Seeding of cells into poly(ethylene glycol)(PEG) hydrogel-coated well plates containing collagen-modified microboards resulted in selective cell adhesion onto the microboards due to the non-adhesiveness of PEG hydrogel toward cells, thereby creating non-positional arrays of microboards carrying cells. Finally, two different cell types (fibroblasts and HeLa cells) were separately cultured on different shapes of microboards and subsequently mixed together to create a non-positional cell microarray consisting of multiple cell types where each cell could be easily identified by the shape of the microboard to which they had adhered. Because numerous unique shapes of microboards can be fabricated using this method by simply changing the photomask designs, high throughput and multiplex cell-based assays would be easily achieved with this system in the future. [ABSTRACT FROM AUTHOR]

This paper describes the development of multi-functional nanofiber scaffolds consisting of multiple layers of nanofiber scaffolds and nanofiber-incorporated poly(ethylene glycol) (PEG) hydrogels. As a proof-of-concept demonstration, we fabricated micropatterned polymeric nanofiber scaffolds that were capable of simultaneously generating cellular micropatterns within a biomimetic environment and detecting cellular metabolic products within well-defined microdomains. To achieve this goal, we designed nanofiber scaffolds with both vertical and lateral microdomains. Vertically heterogeneous structures that were responsible for multi-functionality were realized by preparing double-layered nanofiber scaffolds consisting of an antibody-immobilized bottom layer of nanofibers and an upper layer of bare polystyrene (PS) nanofibers by a two-step sequential electrospinning process. Photopatterning of poly(ethylene glycol) (PEG) hydrogel on the electrospun nanofibers produced laterally heterogeneous micropatterned nanofiber scaffolds made of hydrogel microwells filled with a nanofibrous region, which is capable of generating cell and protein micropatterns due to the different interactions that cells and proteins have with PEG hydrogels and nanofibers. When HepG2 cells were seeded into resultant nanofiber scaffolds, cells selectively adhered within the 200 μm × 200 μm PS fiber microdomain and formed 180.2 ± 6.7 μm spheroids after 5 days of culture in the upper layer. Furthermore, immobilized anti-albumin in the bottom layer detected albumin secreted by micropatterned HepG2 cells with higher sensitivity than flat PS substrates, demonstrating successful accomplishment of dual functions using micropatterned double-layered nanofiber scaffolds. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, we describe a simple method for fabricating micropatterned nanoporous substrates that are capable of controlling the spatial positioning of mammalian cells. Micropatterned substrates were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on alumina membranes with 200nm nanopores using photolithography. Because hydrogel precursor solution could infiltrate and become crosslinked within the nanopores, the resultant hydrogel micropatterns were firmly anchored on the substrate without the use of adhesion-promoting monolayers, thereby allow tailoring of the surface properties of unpatterned nanoporous areas. For mammalian cell patterning, arrays of microwells of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding nanoporous bottoms that were modified with cell-adhesive Arg–Gly–Asp (RGD) peptides. Because the PEG hydrogel was non-adhesive towards proteins and cells, cells adhered selectively and remained viable within the RGD-modified nanoporous regions, thereby creating cellular micropatterns. Although the morphology of cell clusters and the number of cells inside one microwell were dependent on the lateral dimension of the microwells, adhered cells that were in direct contact with nanopores were able to penetrate into the nanopores by small extensions (filopodia) for all the different sizes of microwells evaluated. [Copyright &y& Elsevier]

Abstract: This paper describes a multiplexed enzyme-based assay within a microfluidic device using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles. A microfluidic device was constructed by serially connecting two patterning chambers and a microfilter-integrated detection chamber through a Y-shaped microchannel. Various shapes and sizes of hydrogel microparticles entrapping different enzymes were fabricated in the patterning chamber by photolithography and collected in the detection chamber by pressure-driven flow. Due to the action of the microfilter, hydrogel microparticles were perfectly retained inside the detection chamber without passing through it. The sequential bienzymatic reactions of glucose oxidase (GOX) and peroxidase (POD) or alcohol oxidase (AOX) and POD were successfully investigated using Amplex Red within the hydrogel microparticles. Furthermore, by entrapping GOX and AOX into different shapes of hydrogel microparticles, each enzyme-catalyzed reaction was easily identified, and therefore, simultaneous detection of glucose and ethanol was possible in the concentration range from 1.0 to 10.0mM without cross-talk by using same fluorescence indicator in a single detection chamber. [Copyright &y& Elsevier]

Poly(ethylene glycol)(PEG)-based interpenetrating polymeric network (IPN) hydrogels were prepared for the application of enzyme immobilization. Poly(acrylamide)(PAAm) was chosen as the other network of IPN hydrogel and different concentration of PAAm networks were incorporated inside the PEG hydrogel to improve the mechanical strength and provide functional groups that covalently bind the enzyme. Formation of IPN hydrogels was confirmed by observing the weight per cent gain of hydrogel after incorporation of PAAm network and by attenuated total reflectance/Fourier transform infrared (ATR/FTIR) analysis. Synthesis of IPN hydrogels with higher PAAm content produced more crosslinked hydrogels with lower water content (WC), smaller Mc and mesh size, which resulted in enhanced mechanical properties compared to the PEG hydrogel. The IPN hydrogels exhibited tensile strength between 0.2 and 1.2 MPa while retaining high levels of hydration (70-81% water). For enzyme immobilization, glucose oxidase (GOX) was immobilized to PEG and IPN hydrogel beads. Enzyme activity studies revealed that although all the hydrogels initially had similar enzymatic activity, enzyme-immobilizing PEG hydrogels lost most of the enzymatic activity within 2 days due to enzyme leaching while IPN hydrogels maintained a maximum 80% of the initial enzymatic activity over a week due to the covalent linkage between the enzyme and amine groups of PAAm. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

This study presents a novel in vitro bilayer 3D co‐culture platform designed to obtain cancer‐associated fibroblasts (CAFs)‐like cells. The platform consists of a bilayer hydrogel structure with a collagen/polyethylene glycol (PEG) hydrogel for fibroblasts as the upper layer and an alginate hydrogel for tumor cells as the lower layer. The platform enabled paracrine interactions between fibroblasts and cancer cells, which allowed for selective retrieval of activated fibroblasts through collagenase treatment for further study. Fibroblasts remained viable throughout the culture periods and showed enhanced proliferation when co‐cultured with cancer cells. Morphological changes in the co‐cultured fibroblasts resembling CAFs were observed, especially in the 3D microenvironment. The mRNA expression levels of CAF‐related markers were significantly upregulated in 3D, but not in 2D co‐culture. Proteomic analysis identified upregulated proteins associated with CAFs, further confirming the transformation of normal fibroblasts into CAF within the proposed 3D co‐culture platform. Moreover, co‐culture with CAF induced radio‐ and chemoresistance in pancreatic cancer cells (PANC‐1). Survival rate of cancer cells post‐irradiation and gemcitabine resistance increased significantly in the co‐culture setting, highlighting the role of CAFs in promoting cancer cell survival and therapeutic resistance. These findings would contribute to understanding molecular and phenotypic changes associated with CAF activation and provide insights into potential therapeutic strategies targeting the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we developed a novel microfluidic device for enzyme-based assays that incorporates heterogeneous reaction and microarray-based detection systems. The microfluidic device had two serial chambers connected through microchannels; one for the reaction between analytes and enzymes, and the other for the quantitative detection of analytes. The reaction chamber was filled with glass microbeads covalently bound to enzymes via aminopropyltriethoxysilane (APTES). Enzyme-immobilized microbeads 70μm in diameter were retained within the reaction chamber using a microfilter composed of micropillars with 30μm interspaces. In the detection chamber, a poly(ethylene glycol)-based hydrogel microarray was fabricated using photolithography, which could immobilize other protein molecules or fluorescent dyes for the optical analysis of enzyme-catalyzed reaction. Different concentrations of glucose were detected within the microfluidic system where the reaction chamber was filled with glucose oxidase (GOX)-immobilizing glass microbeads, and a horseradish peroxidase (POD)-entrapping hydrogel microarray was placed in the detection chamber. A sequential bienzymatic reaction resulted in the conversion of non-fluorescent Amplex Red into fluorescent resorufin within the hydrogel microarray and glucose concentrations ranging from 1 to 10mM were successfully detected by measuring the change of emission intensity of resorufin inside the hydrogel microarray. [Copyright &y& Elsevier]

Abstract: For the first step in the development of a microanalysis device to detect organophosphorus compounds, PEG hydrogel microparticles containing conjugates of enzyme (AChE) and pH-sensitive fluorophore (SNAFL-1) as an optical biosensor were fabricated by a dispersion photopolymerization. Using the dual emission property of pH-sensitive SNAFL-1, changes in the microenvironment pH due to enzyme-catalyzed reactions with the analyte of interest were determined fluorescently and used to detect the analyte. The activity and sensitivity of the enzyme and pH-sensitive fluorophore were maintained when they were conjugated and encapsulated within the PEG hydrogel microparticles. The fluorescent response of the PEG microparticles containing AChE-SNAFL conjugates was affected by the concentration of a model organophosphorus compound (paraoxon) in the range of 1–20μmol. These results indicate that the PEG microparticles containing AChE-SNAFL conjugates prepared in this study have a possibility to be used as an optical biosensor to detect organophosphorus compounds. [Copyright &y& Elsevier]

Ocular prostheses are custom-made polymeric inserts that can be placed in anophthalmic sockets for cosmetic rehabilitation. Prosthetic eye wearers have reduced tear amount, and they often experience dry eye symptoms including dryness, irritation, discomfort, and discharge. Most modern ocular prostheses are made of poly(methyl methacrylate) (PMMA), which is highly hydrophobic. Previous research has shown that improving the wettability of contact lens materials decreases its wearers discomfort by increasing lubrication. Therefore, hydrophilic modification of PMMA-based ocular prostheses might also improve patient discomfort by improving lubrication. We modified the surfaces of PMMA-based ocular prostheses using poly(ethylene glycol) (PEG), which is hydrophilic. To do this, we used two strategies. One was a “grafting from” method, whereby PEG was polymerized from the PMMA surface. The other was a “grafting to” method, which involved PEG being covalently bonded to an amine-functionalized PMMA surface. Assessments involving the water contact angle, ellipsometry, and X-ray photoelectron spectroscopy indicated that PEG was successfully introduced to the PMMA surfaces using both strategies. Scanning electron microscopy and atomic force microscopy images revealed that neither strategy caused clinically significant alterations in the PMMA surface morphology. In vitro bacterial adhesion assessments showed that the hydrophilic modifications effectively reduced bacterial adhesion without inducing cytotoxicity. These results imply that hydrophilic surface modifications of conventional ocular prostheses may decrease patient discomfort and ocular prosthesis-related infections. [ABSTRACT FROM AUTHOR]

A PEG-based hydrogel with a high conductivity and water content was synthesized using a two-step sequential polymerization process by in-situ polymerizing poly(3,4-ethylenedioxythiophene) (PEDOT) within poly(4-styrenesulfonic acid) (PSS)-containing poly(ethylene glycol) diacrylate (PEG-DA) hydrogel matrix. A small amount of diluted sulfuric acid (H 2 SO 4 ) was added as an accelerator to increase the conductivity via reduced polymerization time. Among the various molecular weights (MW) of PEG, PEG-DA with MW 3400 was used for first hydrogel due to its mechanical property and water content. Incorporation of PSS within the PEG hydrogel facilitated the in-situ synthesis of PEDOT within the hydrogel, producing a hydrogel with a higher conductivity, which was further enhanced by H 2 SO 4 treatment. The resultant semi-interpenetrating network hydrogel scaffolds were shown to consist of up to more than 80 wt% of water with a compressive modulus of 21 kPa and an electrical conductivity of 1.69 × 10 − 2 S cm − 1 . The surface of the resultant conductive hydrogel could be modified via photochemical fixation for cell adhesion with negligible conductivity change. In vitro studies using electro-responsive H9C2 myocytes showed that the developed hydrogels not only did not exhibit any cytotoxicity but also supported cell adhesion and proliferation. This work demonstrates that the architectural design of the conductive hydrogel scaffolds and growth mechanism of PEDOT in the hydrogel platform play a pivotal role in determining the properties of the resulting conductive hydrogel. The attractive performance of these hybrid hydrogels will open a new approach for the further research on electrically conductive tissue engineering scaffolds. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, we describe a simple method for fabricating micropatterned polymeric fibrous scaffolds that are capable of controlling the spatial positioning of mammalian cells. Micropatterned scaffolds were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on electrospun poly(styrene) (PS) fiber matrices, and produced as free-standing and bidirectionally-porous sheets. Clearly defined hydrogel micropatterns incorporating PS fibers were created using photolithography without any residual hydrogel precursor solution remaining in the PS fiber region. Most of the PS fibers were inserted through the side walls of the hydrogel microstructures, leaving no fiber residue on the top surfaces of hydrogel micropatterns. Since the PEG hydrogel showed non-adhesiveness toward proteins and cells, cells selectively adhered and remained viable within the PS fiber region, thereby creating cellular micropatterns. Finally, possible application of this system to cell-based biosensor was demonstrated using cytotoxicity assay. [Copyright &y& Elsevier]

Abstract: In this study, hydrogel microparticles containing enzyme-linked magnetic nanoparticles (MNPs) were prepared. Peroxidase (POD), a model enzyme, was covalently immobilized on the surface of MNPs using 3-aminopropyltriethoxysilane (APTES), and the resultant POD-linked MNPs were entrapped within various shapes of hydrogel microparticles using photopatterning. Pre-immobilizing POD on the MNP surface made it possible to use hydrogels prepared from high molecular weight PEG without enzyme leaching, which enhanced the reaction rate of entrapped enzymes by reducing resistance to mass transport. Quantitative assays showed a linear correspondence between fluorescence intensity and H2O2 concentrations below 15mM. [Copyright &y& Elsevier]