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Dr. Moghe's research is focused on three major areas, described further in the narrative below. (1) Cellular Bioengineering; (2) Nanobiomaterials and Nanobiotechnology; and (3) Cell-Biomaterial Interactions.
- Collaborators: Dr. Fredrick Kauffman (Pharmacology, Rutgers), Dr. Ronald Hart (Cell Biology, Rutgers), Dr. Surendra Batra (Integra LifeSciences), Dr. Mehmet Toner (Harvard), Dr. Lionel Larue (Institut Curie, France), Dr. Malcolm Steinberg (Princeton)
- Funding Sources: NSF, NIH, Johnson & Johnson, Merck, Biocure, Integra LifeSciences
Dr. Moghe's research in the area of cell and tissue engineering has addressed the role of matrix, substrate, and growth factor cues on the phenotypic and differentiation behavior of liver cells, hepatocytes, and embryonic stem cells. At Rutgers, Dr. Moghe and his graduate students have reported that the morphogenesis and differentiation of anchorage-sensitive hepatocytes can be acutely governed by changes in the substrate microscale topography as well as the nature of adsorbed matrix ligands. Dr. Moghe's studies (supported by the NSF CAREER Program) examined how hepatocyte differentiation can be engineered through the interplay of substrate topography and matrix/mechanochemical stimulation. Dr. Moghe's papers with his graduate students and postdocs in Tissue Engineering, Biomaterials, and J. Biomedical Materials Research, have been frequently cited by investigators in the field. For example, a recent review in Science (Assender et al., 2002) cites the publication on cooperative effects on cell motility dynamics of ligand adsorption and material microstructure (Ranucci and Moghe, 2001).
Using differentially compliant hydrogels, Dr. Moghe reported that increased growth factor stimulation and growth factor pulsing can be used as a dual strategy to promote differentiation or cell growth (Semler and Moghe, 2001; Semler et al., 2000). This pioneering work has been widely cited, ranging from tissue engineering papers such as Dr. Robert Langer's review in Developmental Biology (Levenberg and Langer, 2004), Dr. Paul Janmey's review in J. Appl. Physiology (Georges and Janmey, 2005), all the way to leading cell/molecular biology journal such as J. Cell Science (Haouzi et al., 2005). Advancing these studies further using a new system of polyacrylamide substrates functionalized with matrix ligand, fibronectin, Dr. Moghe and graduate student, Eric Semler, showed using hydrogels of intermediate compliance that hepatocytes can be made to differentiate with highest sensitivity when exposed to increased ligand concentration (Semler et al., 2005). This recent study was cited in a prominent review in Science focussed on cellular engineering via rigid/compliant substrates by Dr. Dennis Discher and colleagues at U. Pennsylvania (Discher et al., 2005). Dr. Linda Griffith of MIT also cites this work in her 2006 Nature Reviews- Molecular & Cell Biology (Griffith and Swartz, 2006).
In the area of hepatocellular engineering, Dr. Moghe has pioneered the approach of incorporation of adhesive and signaling cell-cell adhesion molecule, the cadherins, for controlled differentiation of hepatocytes. Five prominent publications from his laboratory have by now documented that (a) cadherin based cell-cell adhesion between liver cells and other cell types can significantly promote cell differentiation (Brieva and Moghe, 2001) (b) the mode of cadherin display can engineer the differentiation-proliferation balance (Brieva and Moghe, 2004) (c) acellular fragments of E-cadherin can used on artificial substrates to promote hepatocyte differentiation (Semler et al., 2005); and, recently, that (d) E-cadherins can promote embryonic stem cell differentiation in conjunction with growth factor stimulation (Dasgupta et al., 2005). Current advances in the Moghe laboratory, using approaches utilizing microfabrication, immunocytochemistry, and DNA microarray technology, show that E-cadherin engineered embryonic stem cells can be more effectively primed to mature to differentiated hepatocyte-like cells when transplanted within liver-like environments in vitro (Hughey et al., 2006). This is the first report in the field that probes the maturation potential of ES cells in the presence of adult hepatocytes. These studies are expanding the investigation to include several new questions in the area: (a) Can cadherin-engineered ES cells be further genetically modified via SiRNA technology? (b) Can cadherin-based wnt signaling and other growth factor signaling be used cooperatively to promote the kinetics of ES cell maturation? (c) Can cadherin engineering insights learned from murine ES cells be applied to human ES cells (being addressed through a current collaboration between Dr. Moghe, and Drs. Larue of France and Cellartis, Inc. of Sweden).
Nanobiomaterials and Nanobiotechnology
- Collaborators: Dr. Brian Aneskievich (UMDNJ/U. Connecticut); Dr. Jean Schwarzbauer (Princeton); Dr. Kathryn Uhrich (Rutgers, Chemistry); Dr. David Talaga (Rutgers, Chemistry); Dr. Gary Nackman (UMDNJ, Surgery); Dr. Joanna Aizenberg (Lucent Technologies/Bell Labs); Dr. Thomas Tsakalakos (Rutgers Materials Science & Eng); Dr. Aric Menon (Tech. U, Denmark)
- Funding Sources: NSF, NIH, Johnson & Johnson, Integra LifeSciences
Two major federally funded research projects in the Moghe laboratory pursue studies on the design and elucidation of nanoscale particles for controlled interactions with cells. These are discussed below.
Nanoparticles for Engineered Cell Biodynamics in Cell/Matrix Engineering
Cell adhesion to the extracellular matrix is followed by a series of cellular events, such as cell spreading, cell migration or differentiation or proliferation. Given the complexity and dynamic nature of cell interactions with the matrix ligands, biointerfacial/biomaterials scientists and bioengineers are now trying to recapitulate the biodynamic nature of the cell-ligand interface. Dr. Moghe has made a major contribution to this area of research over the past nine years. Having recognized the importance of cytointernalizable matrix ligands for the motility processes in skin cells, his laboratory published the first series of systematic reports on a model system to quantitatively describe cell-nanoparticle interactions. Using both experimental and modeling approaches focused on a collagen-colloidal gold particle system, Dr. Moghe and his graduate student, Jane Tjia, published four papers from 1999-2000 (Tjia et al., 1999; Tjia and Moghe, 2002; Tjia and Moghe, 2002; Tjia and Moghe, 2002), which highlighted the following findings: (1) Nano/submicroscale particles presenting adhesive matrix ligands significantly activated a motile cell phenotype in terms of cell filopodial morphology and cytoskeletal organization; and consequently enhanced cytointernalization-coupled cell migration rates relative to controls (nanoparticles alone; ligand alone) in a ligand concentration dependent manner (2) The enhancement in cell migration depended critically on cell-engendered mobility and cytointernalization of the nanoparticles (3) The rate of cell migration is correlated to the rate of ligand sampling, in turn, related to the rate of cell-ligand binding and rate of ligand internalization (4) Matrix cytointernalization-coupled cell motility is regulated by growth factor stimulation and reciprocity with cell-secreted matrix (fibronectin).
Given the limitations of the gold system for long-term applications, since 2003, the Moghe laboratory developed a new platform based on albumin nanoparticles, which can be easily metabolized once internalized, can be fabricated to various sizes; and is easily derivatized with organic ligands. In 2006, Dr. Moghe's team published the first paper using 100 nm albumin nanoparticles showing that the albumin nanoparticle core exposes the cell-adhesion domain of III9-10 fibronectin fragments (Sharma et al., 2006). Keratinocyte cell adhesion to substrate deposited ligand-nanoparticles resulted in a prominent F-actin filopodial organization, and cell motility was significantly enhanced relative to substrate controls with comparable levels of ligand alone. A subsequent manuscript focuses on the role of variation of nanoparticle size in induction of ligand/nanoparticle cytointernalization and migration (Nature Nanotechnology, Submission 7/06). Smaller nanoparticles (30-50 nm) further promote cytointernalization dynamics, and thus promote cell migration kinetics (Sharma et al., 2006).
Separately, the Moghe laboratory investigated the potential for manipulating dynamic nanoparticle interactions with integrins, the cell adhesion receptors. Using dermal fibroblasts, fibronectin fragment presentation from albumin nanoparticles was found to (1) markedly promote cell elongation and a contractile phenotype in a ligand concentration dependent manner (Tissue Engineering, in review) (2) promote fibrillogenesis/assembly of cell-secreted fibronectin (3) accelerate the centripetal translocation of beta1 integrins bound to ligand-nanoparticles, suggesting that the nanoparticle-induced receptor dynamics correlates with increased cell-assembly of fibronectin (Pereira et al., 2006). Notably, the nanoparticle effects were abolished if the nanoparticles were merely presented via solution or if Rac/Rho inhibitors were incorporated, indicating the importance of cell adhesion and contractile signaling mechanisms for the biodynamics. A subsequent manuscript investigates the role of nanoparticle size on the cell contractility and cell matrix assembly. A combination of the nanoscale particle effects on cell motility and matrix assembly processes is being presented in a manuscript (Sharma et al., 2006). The key finding of this paper is that two scale regimens were found: for smaller ligand-particles, the biodynamics of internalization was accelerated, while for larger ligand-particles, biodynamics of membrane clustering, contraction and assembly was accelerated.
Nanoparticles for Controlled Cellular Uptake of Lipoproteins
A prime example of nanoscale interactions of cells and matrix is initiated within the vascular circulation (blood vessels). Low density lipoproteins trapped within the vascular intima are progressively oxidized and modified. The oxidized LDL is rapidly internalized within blood immune cells such as macrophages, which transform into foamy cells, secrete cytokines that trigger the excessive proliferation of smooth muscle cells, and ultimately undergo apoptosis. These inflammatory events, in conjunction with thrombosis, escalate the development of atherosclerotic plaques, and pose a major risk factor for plaque growth, plaque destabilization, and narrowing of blood vessels (stenosis).
The Moghe laboratory, in collaboration with Professor Uhrich (Chemistry, Rutgers), has designed nanoscale self-assembled particles whose backbone chemistry and architecture can be systematically varied to exhibit controlled amphiphilicity and anionic groups. The particles are self-assembled from unimers comprised of acylated derivatives of biocompatible mucic acid conjugated with poly(ethylene glycol), where anionic functional groups can be derivatized to either terminus. In a first publication, such nanoparticles were shown to complex with unoxidized LDL but not with oxidized LDL (Chnari et al., 2005). A coarse grain and molecular dynamics simulation study is ongoing to describe the lipoprotein retentivity of the nanoparticles (Li et al., 2006). Subsequent studies in the Moghe laboratory report that the anionic nanoparticles reduce the kinetics of unoxidized LDL by complexation with LDL but reduce the kinetics of oxidized LDL by binding to scavenger receptors (Chnari et al., 2006). Recent efforts have identified SRA and CD36 to be the key scavenger receptor targets for the anionic nanoparticles; consequently, blockage of scavenger receptors by the nanoparticles reduced cytokine secretion, foam cell formation, and cholesterol accumulation (Chnari et al., 2006). Certain characteristics of the nanoparticles were found to be requisite to maximal scavenger receptor binding. For example, positioning of anionic groups on hydrophobic termini were found to reduce oxLDL uptake but not if the anionic groups were displayed from hydrophobic termini. Recent studies implicate the size as well as anionic charge density to be important variables that further accentuate the ability of nanoparticles to inhibit oxLDL uptake (Wang et al., 2006). These studies are promising as nanotechnology affords a possible avenue to effectively alter the dynamics of lipoportein matrix retention within the intima. Systematic studies of binding affinities between the nanoparticles and major scavenger receptors are being pursued using surface plasmon resonance. Further studies are proposed to elucidate how the nanoparticle structure influences the receptor binding, cross-linking, and possible conformational changes leading to receptor internalization. The goals are to identify nanoparticle structures that maximally occupy scavenger receptors with minimal degree of receptor internalization. Animal models are currently being tested to examine the potential for the nanoparticles to reduce inflammation and atherogenesis. The advances in this project have been highlighted recently by news release from the American Chemical Society and Nanobiotechnology News, and industrial partnering is envisioned for successful translation of this project for further development and possible testing for therapeutic potential.
Characterization of Cell-Biomaterials Interactions
- Collaborators: Dr. Joachim Kohn (Chemistry, Rutgers), Dr. Matthew Becker (NIST), Dr. Sangeeta Bhatia (MIT), Dr. Peter Ma (U. Michigan), Dr. Treena Arinzeh (NJIT), Dr. Gary Nackman (UMDNJ), Dr. Andres Garcia (Georgia Tech)
- Funding: NIH, NJCST, Rutgers Academic Excellence Fund, Whitaker Foundation
Between 1996 and 2001, Moghe's group has examined various interactions between white blood cells and prosthetic materials. Five publications have appeared quantifying leukocyte adhesive and motility responsiveness in situ to plasma proteins, flow exposure, and molecular variations in cell adhesion to materials (e.g. CD43) (Chang et al., 1999; Chang et al., 2000; Chang et al., 2000; Rosenson-Schloss et al., 2002; Rosenson-Schloss et al., 1999). Since 1997, Moghe's group and collaborators from the laboratory of Joachim Kohn at Rutgers have jointly investigated the cellular interactions with substrates made from the family tyrosine-derived polycarbonates. These materials can be tailored with different levels of hydrophobicity, degradation rates, and protein adsorptivity. In the first major report, Moghe et al., showed that increased levels, within limits, of incorporation of poly(ethylene glycol) in the polymer backbone weakened cell adhesion strength and promoted cell motility kinetics (Tziampazis et al., 2000). Subsequent studies that equalized levels of proteins adsorbed on polycarbonates with different levels of PEG showed that increased levels of PEG (a) promote cell-mediated ligand remodeling and exposure of cell adhesive epitopes on the ligand; (b) enhance cell migration kinetics (Sharma et al., 2004).
Recent reports from the Moghe laboratory comparing two members of the polyarylate family of polymers with similar levels of hydrophobicity and glass transition temperatures, but differing in the presence of a single atom substitution of a carbon by an oxygen, showed that cell adhesion and motility behavior on the substrates are identical at all ligand levels except at intermediate ligand concentrations, where cellular responses were significantly different (Bae et al., 2006). Studies on a library of combinatorially designed polycarbonate polymers are currently exploring how the incorporation of PEG, DT (acid), and iodine (for radio-opacity) modulate cell adhesivity and motility responsiveness (Johnson et al., 2006). To this end, a library of biodegradable, tyrosine-derived polycarbonates was selected with tunable protein/cell adsorption, X-ray visibility, and degradability. Three chemical components were selectively varied through copolymerization: 1) iodine to achieve X-ray visibility; 2) poly(ethylene glycol) (PEG) to decrease protein adsorption and cell adhesivity; and 3) DT to increase the degradation rate. In a rapid screening format, the complex interplay of the chemical components on smooth muscle cell attachment, motility and proliferation, was investigated. For the base polymer, poly(DTE carbonate) the progressive incorporation of PEG reduced cell attachment. However, upon the inclusion of iodine in the tyrosine ring, the PEG effect was significantly reduced. Furthermore, following copolymerization with 10% of the DT monomeric derivative containing a free carboxylic acid, the PEG effect was negated for the iodine containing polymers and reversed for the non-iodinated polymers. Cross-functional analysis of motility and proliferation revealed substrate chemistry related cell response regimes. For instance, with the base poly(DTE carbonate) polymers, increasing PEG levels increased smooth muscle cell motility at the expense of proliferation. In contrast, for poly(DTE carbonate) with 10% DT, increasing PEG levels increased cell adhesion, motility, and proliferation. These studies provide an example of multidimensional, quantitative cross-functional profiling of cells on biomaterials-- further correlations are sought for other cell functions in strategic cell types for cell differentiation and apoptosis.
Moghe's laboratory has actively employed a variety of high-resolution optical microscopy approaches to elucidate cell-material interactions, including confocal laser-scanning microscopy (CLSM), atomic force microscopy (AFM), and recently, multi-photon microscopy (MPM). In earlier reports, Moghe et al, quantified polymer scaffold microstructure using fluorescence and reflection mode CLSM both pre- and post-degradation (Semler et al., 1997; Tjia and Moghe, 1998), while more recently, they have authored a manuscript focused on microstructure analysis of scaffolds of blends of poly(DTE carbonates) and poly(DTO carbonates), that shows the increased signal-to-noise ratio, biorelevant cell characterization, and quantitative capabilities of MPM relative to CLSM (Liu et al., 2006). The imaging methods are also being applied to fluorescently engineered cells on biomaterials, as described next.
Moghe's studies on cell profiling on biomaterials are integral to the NIH program grant on Integrated Resources for Polymeric Materials. Two major toolboxes are being assembled under Moghe's directorship and in conjunction with collaborators toward the building of a cell-biomaterials interactome. The first is a suite of imaging and image analysis modalities assembled on the multiphoton microscope. The imaging platform is currently a 100-grid chamber assembled in collaboration with NIST using a optically active adhesive glue to create a chamber where polymers can be deposited and cell dynamics studies using real-time MPM. The imaging modality consists of generation of genetically engineered fluororeporter cell lines, which express cell morphologic, cytoskeletal, and differentiation markers. A manuscript under preparation for submission to Nature Methods summarizes for over one-hundred compositions on gradients of polymer blends, the reporter profiling for several successful cell lines, which respond sensitively to changes in biomaterial chemistry (Treiser et al., 2006). The advantage of such fluororeporters is that for each fluororeporter, up to hundred cell descriptors can be quantified using fluorescent imaging and image analysis. A major endeavor is currently underway to identify the descriptors that discern these biomaterial features: an example is the identification of descriptors that resolve incorporation of PEG and DT, both singly and together, in relation to poly(DTE carbonates). Once such descriptors are identified, they are rank ordered via decision tree analysis in relation to cell functions, such as cell proliferation, motility, and apoptosis. Thus, a family of key biomaterial-responsive cell descriptors can be identified for each strategic cell function. Cross co-relations between such descriptors can be examined for the selective amplification or suppression of certain functions. These ideas, in conjunction with systems biology concepts, are being investigated further within the paradigm of modeling of cell-material interactome (Kohn and Moghe, 2006).