Biomineralized materials as bone ECM mimetics: From understanding molecular mechanisms to new therapeutic interventions

Bone extracellular matrix is a heterogeneous composite material consisting, of an inorganic (or mineral) phase, an organic phase and water. In an effort to mimic the mineral environment of bone tissue, we recently employed biomineralization and created mineralized biomaterials (1). Our studies showed that these biomineralized materials induced osteogenic differentiation of stem cells, including human pluripotent stem cells (which includes both embryonic stem cells and induced pluripotent stem cells), even in growth medium devoid of any osteogenic inducing molecules (2,3). By employing these mineralized materials, we have studied the molecular mechanism through which calcium phosphate minerals support osteogenesis with an emphasis on phosphate metabolism (4). Our studies show that extracellular phosphate (resulting from the dissolution of calcium phosphate minerals) 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 intra-mitochondrial phosphate and ATP synthesis. Our studies further show that the phosphate-ATP-adenosine signaling axis not only promotes osteogenic differentiation of multipotent progenitor cells but also inhibits their adipogenic differentiation (5). When implanted in vivo, these acellular mineralized biomaterials recruited endogenous cells and induced their differentiation to form vascularized bone tissues (6) and also supported donor bone marrow transplantation (7). Leveraging these understandings, we are currently studying the pivotal role of Adenosine A2b receptor, a G-protein coupled receptor on the cell membrane, in regulating bone-specific cells and treating bone metabolic disorders. In this talk, I will discuss these results and the identification of possible drug targets for osteoporosis. REFERENCES [1] Phadke A, Zhang C, Hwang YS, Vecchio K, Varghese S. “Templated mineralization of synthetic hydrogels for bone like composite material: Role of matrix hydrophobicity”, Biomacromolecules. 2010; 11, 2060 [2] Phadke A, Shig Y-R, Varghese, S. “Mineralized synthetic matrices as an instructive microenvironment for osteogenic differentiation of human mesenchymal stem cells” Macromol Biosci. 2012, 12, 1022 [3] Kang H, Shih Y-RV, Hwang YS, Wen C, Rao V, Seo T, and Varghese S. “Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells” Acta Biomater. 2014, 10, 4961 [4] Shih Y-RV, Hwang YS, Phadke A, Kang H, Hwang NS, Caro EJ, Nguyen S, Siu M, Theodorakis EA, Gianneschi NC, Vecchio KS, Chien S, Lee OK and Varghese S. “Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling” Proc. Natl. Acad. Sci. 2014, 111, 990. [5] Kang H, Shih Y-RV, Varghese S. “Biomineralized matrices dominate soluble cues to direct osteogenic differentiation of human mesenchymal stem cells through adenosine signaling” Biomacromolecules 2015, 16, 1050 [6] Shih Y-RV, Phadke A, Yamaguchi T, Kang H, Inoue N, Masuda K and Varghese S. “Synthetic bone mimetic matrix-mediated in situ bone tissue formation through host cell recruitment” Acta Biomater. 2015, 19, 1. [7] Shih Y-RV, Kang H, Rao V, Chu Y-J, Kown SK, and Varghese S. “In vivo engineering of bone organoids with hematopoietic functions” Proc. Natl. Acad. Sci, 2017, 114, 5419

Cellular senescence in neuroinflammation

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Metal-ion-mediated healing of gels

Several researchers have demonstrated the biomimicking attributes of stimuli responsive synthetic hydrogels such as sensitivity, selectivity, mobility, and memory. In this work we demonstrate yet another attribute, namely healing in hydrogels. We show that certain hydrogels having a flexible aliphatic side chain with a terminal carboxyl group can show healing in the presence of transition metal ions. On bringing two initially dried gel pieces into contact with each other in a dilute copper chloride solution the pieces were found to weld and the strength of the weld-line was found to increase gradually with time. The welded gel pieces could be separated by leaching out the metal ions and the separated gel pieces can be welded again by subsequent treatment with the metal ions

In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications

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 coculture 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-b1 (TGF-b1) and b1-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 factor

Scalability Study of the Hybrid ZigBee Multipath Routing Protocol [1]

ABSTRACT-Multipath routing is an efficient technique to route data in wireless sensor networks (WSNs) because it can provide reliability, security and load balance, which are particularly critical in the resource constrained system such as WSNs. Multipath routing protocol helps ZigBee network to improve the global throughput. For finding multiple paths this Hybrid Multipath routing protocol uses both hierarchical tree routing and AODV. The first path is always discovered using the TR routing. Successive path are calculated using combination of TR and neighbor table and AODV. Scalability is a main aspect in designing an efficient routing protocol for wireless sensor networks. This paper checks the scalability of hybrid multipath routing protocol for ZigBee e networks

Role of hydrophobicity on structure of polymer-metal complexes

Metal complexation of a series of polymeric gels, with different degrees of ionization, prepared from acrylic acid and acryloyl amino acid monomers (CH2=CHCONH(CH2)nCOOH, where n = 4, 6 and 8), were investigated. The binding of Cu(II) ions to the gels was studied by means of swelling, quantitative determination of the amount of "bound" or complexed Cu(II), and EPR spectroscopy of Cu(II) complexes. Both the amount of Cu(II) and the structure of polymer-Cu(II) complex were influenced by the length of the pendent chain, i.e., "hydrophobicity" of the polymer gels. The metal uptake by the gels increases with increasing "hydrophobicity". Two types of polymer-Cu(II) complexes (monomer and dimer, respectively) were identified by EPR spectroscopy, their concentrations were found to be a function of hydrophilic-hydrophobic balance of the polymer gels

Effect of polymer-metal complexation on the phase transition of thermoreversible copolymer gels

We have investigated the effect of transition metal complexation on the swelling behavior in water of a new terpolymer gel made from N-tert-butylacrylamide (N-t-BAm) as the hydrophobic monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) as the hydrophilic monomer, and N,N'-methylenebisacrylamide (BisAm) as the cross-linker. At a critical balance of hydrophilicity and hydrophobicity, the terpolymer exhibited a sharp volume transition at 15 °C. A shift in the transition temperature of the terpolymer was observed after complexation with trace metal ions. Electron paramagnetic resonance spectroscopy (EPR) suggested a tetrahedral structure for the complexes. We propose that the shift in volume transition temperature of the gel is due to subtle changes in the hydrophilic-hydrophobic balance resulting from the complexation of hydrophilic -SO3H groups of the gel with trace amounts of the metal ions

Matrix Topographical Cue-Mediated Myogenic Differentiation of Human Embryonic Stem Cell Derivatives

Biomaterials varying in physical properties, chemical composition and biofunctionalities can be used as powerful tools to regulate skeletal muscle-specific cellular behaviors, including myogenic differentiation of progenitor cells. Biomaterials with defined topographical cues (e.g., patterned substrates) can mediate cellular alignment of progenitor cells and improve myogenic differentiation. In this study, we employed soft lithography techniques to create substrates with microtopographical cues and used these substrates to study the effect of matrix topographical cues on myogenic differentiation of human embryonic stem cell (hESC)-derived mesodermal progenitor cells expressing platelet-derived growth factor receptor alpha (PDGFRA). Our results show that the majority (>80%) of PDGFRA+ cells on micropatterned polydimethylsiloxane (PDMS) substrates were aligned along the direction of the microgrooves and underwent robust myogenic differentiation compared to those on non-patterned surfaces. Matrix topography-mediated alignment of the mononucleated cells promoted their fusion resulting in mainly (~86%–93%) multinucleated myotube formation. Furthermore, when implanted, the cells on the micropatterned substrates showed enhanced in vivo survival (>5–7 times) and engraftment (>4–6 times) in cardiotoxin-injured tibialis anterior (TA) muscles of NOD/SCID mice compared to cells cultured on corresponding non-patterned substrates