Shyni Varghese

Rapid self-healing hydrogels

Synthetic materials that are capable of autonomous healing upon damage are being developed at a rapid pace because of their many potential applications. Despite these advancements, achieving self-healing in permanently cross-linked hydrogels has remained elusive because of the presence of water and irreversible cross-links. Here, we demonstrate that permanently cross-linked hydrogels can be engineered to exhibit self-healing in an aqueous environment. We achieve this feature by arming the hydrogel network with flexible-pendant side chains carrying an optimal balance of hydrophilic and hydrophobic moieties that allows the side chains to mediate hydrogen bonds across the hydrogel interfaces with minimal steric hindrance and hydrophobic collapse. The self-healing reported here is rapid, occurring within seconds of the insertion of a crack into the hydrogel or juxtaposition of two separate hydrogel pieces. The healing is reversible and can be switched on and off via changes in pH, allowing external control over the healing process. Moreover, the hydrogels can sustain multiple cycles of healing and separation without compromising their mechanical properties and healing kinetics. Beyond revealing how secondary interactions could be harnessed to introduce new functions to chemically cross-linked polymeric systems, we also demonstrate various potential applications of such easy-to-synthesize, smart, self-healing hydrogels.

In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells

Development of clinically relevant regenerative medicine therapies using human embryonic stem cells (hESCs) requires production of a simple and readily expandable cell population that can be directed to form functional 3D tissue in an in vivo environment. We describe an efficient derivation method and characterization of mesenchymal stem cells (MSCs) from hESCs (hESCd-MSCs) that have multilineage differentiation potential and are capable of producing fat, cartilage, and bone in vitro. Furthermore, we highlight their in vivo survival and commitment to the chondrogenic lineage in a microenvironment comprising chondrocyte-secreted morphogenetic factors and hydrogels. Normal cartilage architecture was established in rat osteochondral defects after treatment with chondrogenically-committed hESCd-MSCs. In view of the limited available cell sources for tissue engineering applications, these embryonic-derived cells show significant potential in musculoskeletal tissue regeneration applications.

Engineering the cell-material interface for controlling stem cell adhesion, migration, and differentiation.

The effective utilization of stem cells in regenerative medicine critically relies upon our understanding of the intricate interactions between cells and their extracellular environment. While bulk mechanical and chemical properties of the matrix have been shown to influence various cellular functions, the role of matrix interfacial properties on stem cell behavior is unclear. Here, we report the striking effect of matrix interfacial hydrophobicity on stem cell adhesion, motility, cytoskeletal organization, and differentiation. This is achieved through the development of tunable, synthetic matrices with control over their hydrophobicity without altering the chemical and mechanical properties of the matrix. The observed cellular responses are explained in terms of hydrophobicity-driven conformational changes of the pendant side chains at the interface leading to differential binding of proteins. These results demonstrate that the hydrophobicity of the extracellular matrix could play a considerably larger role in dictating cellular behaviors than previously anticipated. Additionally, these tunable matrices, which introduce a new control feature for regulating various cellular functions offer a platform for studying proliferation and differentiation of stem cells in a controlled manner and would have applications in regenerative medicine.

Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces

Realization of the full potential of human pluripotent stem cells (hPSCs) in regenerative medicine requires the development of well-defined culture conditions for their long-term growth and directed differentiation. Current practices for maintaining hPSCs generally utilize empirically determined combinations of feeder cells and other animal-based products, which are expensive, difficult to isolate, subject to batch-to-batch variations, and unsuitable for cell-based therapies. Using a high-throughput screening approach, we identified several polymers that can support self-renewal of hPSCs. While most of these polymers provide support for only a short period of time, we identified a synthetic polymer poly(methyl vinyl ether-alt-maleic anhydride) (PMVE-alt-MA) that supported the long-term attachment, proliferation and self-renewal of HUES1, HUES9, and iPSCs. The hPSCs cultured on PMVE-alt-MA maintained their characteristic morphology, expressed high levels of markers of pluripotency, and retained a normal karyotype. Such cost-effective, polymer-based matrices that support long-term self-renewal and proliferation of hPSCs will not only help to accelerate the translational perspectives of hPSCs, but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation.

Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

Significance A mechanistic understanding of how calcium phosphate (CaP) minerals contribute to osteogenic commitment of stem cells and bone tissue formation is a necessary requirement for developing efficient CaP-based synthetic matrices to treat bone defects. This study unravels a previously unknown mechanism, phosphate-ATP-adenosine metabolic signaling, by which the CaP-rich mineral environment in bone tissues promotes osteogenic differentiation of human mesenchymal stem cells. In addition to a mechanical perspective on how biomaterials can influence stem cell differentiation through metabolic pathways, this discovery opens up new avenues for treating critical bone defects and bone metabolic disorders. Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

PEG/clay nanocomposite hydrogel: a mechanically robust tissue engineering scaffold

Nanocomposite hydrogels with enhanced mechanical properties could have tremendous biomedical applications. Here we describe synthesis and characterizations of biocompatible poly(ethylene glycol) diacrylate (PEGDA)/Laponite nanocomposite (NC) hydrogels that can support both two- and three-dimensional (2D and 3D) cell cultures. The PEGDA/Laponite NC hydrogels with enhanced mechanical properties were developed by harnessing the ability of PEGDA oligomers to simultaneously form chemically crosslinked networks while interacting with Laponite nanoparticles through secondary interactions. Incorporation of Laponite nanoparticles significantly enhanced both the compressive and tensile properties of PEGDA hydrogels, which were dependent on both the molecular weight of PEG, and concentrations of Laponite nanoparticles. Unlike PEGDA hydrogels, PEGDA-NC hydrogels supported cell adhesion and their subsequent spreading in a 2D culture. In addition to supporting the 2D cell growth, the PEGDA NC hydrogels supported 3D cell encapsulation similar to that of widely used PEGDA hydrogel systems. Such nanocomposite hydrogels with enhanced mechanical properties could have potential applications as 3D scaffolds for tissue engineering. Additionally, the ability of PEGDA NC hydrogels to support 3D culture of encapsulated cells makes them an ideal injectable system with minimally invasive strategies for in vivo applications.

Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well‐defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte‐conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte‐secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte‐conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo. J. Cell. Physiol. 212: 281–284, 2007.

Derivation of Chondrogenically-Committed Cells from Human Embryonic Cells for Cartilage Tissue Regeneration

Background Heterogeneous and uncontrolled differentiation of human embryonic stem cells (hESCs) in embryoid bodies (EBs) limits the potential use of hESCs for cell-based therapies. More efficient strategies are needed for the commitment and differentiation of hESCs to produce a homogeneous population of specific cell types for tissue regeneration applications. Methodology/Principal Findings We report here that significant chondrocytic commitment of feeder-free cultured human embryonic stem cells (FF-hESCs), as determined by gene expression and immunostaining analysis, was induced by co-culture with primary chondrocytes. Furthermore, a dynamic expression profile of chondrocyte-specific genes was observed during monolayer expansion of the chondrogenically-committed cells. Chondrogenically-committed cells synergistically responded to transforming growth factor-β1 (TGF-β1) and β1-integrin activating antibody by increasing tissue mass in pellet culture. In addition, when encapsulated in hydrogels, these cells formed cartilage tissue both in vitro and in vivo. In contrast, the absence of chondrocyte co-culture did not result in an expandable cell population from FF-hESCs. Conclusions/Significance The direct chondrocytic commitment of FF-hESCs can be induced by morphogenetic factors from chondrocytes without EB formation and homogenous cartilage tissue can be formed in vitro and in vivo.

Enhanced Chondrogenesis of Mesenchymal Stem Cells in Collagen Mimetic Peptide-Mediated Microenvironment

A new type of synthetic hydrogel scaffold that mimics certain aspects of structure and function of natural extracellular matrix (ECM) has been developed. We previously reported the conjugation of collagen mimetic peptide (CMP) to poly(ethylene oxide) diacrylate (PEODA) to create a polymer-peptide hybrid scaffold for a suitable cell microenvironment. In this study, we showed that the CMP-mediated microenvironment enhances the chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were harvested and photo-encapsulated in CMP-conjugated PEODA (CMP/PEODA). After 3 weeks, the histological and biochemical analysis of the CMP/PEODA gel revealed twice as much glycosaminoglycan and collagen contents as in control PEODA hydrogels. Moreover, MSCs cultured in CMP/PEODA hydrogel exhibited a lower level of hypertrophic markers, core binding factor alpha 1, and type X collagen than MSCs in PEODA hydrogel as revealed by gene expression and immunohistochemisty. These results indicate that CMP/PEODA hydrogel provides a favorable microenvironment for encapsulated MSCs and regulates their downstream chondrogenic differentiation.

RESPONSE OF ZONAL CHONDROCYTES TO EXTRACELLULAR MATRIX-HYDROGELS

We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)‐based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS‐based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen‐based hydrogels demonstrated zonal‐dependent cellular responses.