Debanjan Sarkar

Engineered cell homing

One of the greatest challenges in cell therapy is to minimally invasively deliver a large quantity of viable cells to a tissue of interest with high engraftment efficiency. Low and inefficient homing of systemically delivered mesenchymal stem cells (MSCs), for example, is thought to be a major limitation of existing MSC-based therapeutic approaches, caused predominantly by inadequate expression of cell surface adhesion receptors. Using a platform approach that preserves the MSC phenotype and does not require genetic manipulation, we modified the surface of MSCs with a nanometer-scale polymer construct containing sialyl Lewis(x) (sLe(x)) that is found on the surface of leukocytes and mediates cell rolling within inflamed tissue. The sLe(x) engineered MSCs exhibited a robust rolling response on inflamed endothelium in vivo and homed to inflamed tissue with higher efficiency compared with native MSCs. The modular approach described herein offers a simple method to potentially target any cell type to specific tissues via the circulation.

Chemical Engineering of Mesenchymal Stem Cells to Induce a Cell Rolling Response

Covalently conjugated sialyl Lewis X (SLeX) on the mesenchymal stem cell (MSC) surface through a biotin-streptavidin bridge imparts leukocyte-like rolling characteristics without altering the cell phenotype and the multilineage differentiation potential. We demonstrate that the conjugation of SLeX on the MSC surface is stable, versatile, and induces a robust rolling response on P-selectin coated substrates. These results indicate the potential to increase the targeting efficiency of any cell type to specific tissue.

Synthesis and characterization of L-tyrosine based polyurethanes for biomaterial applications

The use of amino acid based polymers for biomaterial applications enhance biocompatibility and ensure biodegradability. Two polyurethanes based on L-tyrosine based diphenolic dipeptide, desaminotyrosyl tyrosine hexyl ester as chain extender are synthesized with polyethylene glycol (PEG) and polycaprolactone diol (PCL) as soft segment and hexamethylene diisocyanate as diisocyanate. The chemical structure and molecular characteristics of the polymers were studied by 1H NMR, FTIR, and gel permeation chromatography. Results of DSC and TGA analysis were used for examining the thermal behavior of the polyurethanes. In addition, DSC results were used to analyze the morphology of the polymers, which shows characteristic microphase behavior of the polyurethanes. The tensile properties of the polyurethanes are primarily controlled by the soft segment and are higher in PCL based polymers. Contact angle, water vapor permeation, release of model drug, and water absorption characteristics of the polymers were studied and analyzed in terms of structure of the polyurethanes. In vitro degradation studies show that PEG based polyurethane is more degradable than PCL based polyurethane. The difference in the soft segment structure offers significant variation in the properties of the polyurethanes. These polyurethanes show the potential for use in a variety of biomaterial applications including tissue engineering.

BMP2 Genetically Engineered MSCs and EPCs Promote Vascularized Bone Regeneration in Rat Critical-Sized Calvarial Bone Defects

Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.

A Highly Tunable Biocompatible and Multifunctional Biodegradable Elastomer

Biodegradable elastomers have emerged as promising materials for their potential to mimic the viscoelastic properties of several tissues and exhibit compliance with dynamic environments without damaging the surrounding tissue.[1, 2] Several elastomers have been recently proposed;[3–8] however, the development of highly tunable biodegradable elastomers that can effectively and controllably present biological and physical signals and withstand repeated cycles of physiologic loads, has remained elusive. Such materials should be useful for a broad range of clinically-relevant applications, such as cardiac therapy. For example, following myocardial infarction, the local controlled delivery of bioactive cues[9] or the physical support of the left ventricle wall[10] have been shown to improve cardiac function. The synergistic therapeutic effect of biochemical and biophysical cues has not yet been explored using degradable materials given the absence of materials that can simultaneously deliver bioactive cues and maintain mechanical integrity in a dynamic environment such as the beating heart. Here, we describe a novel biocompatible and mechanically tunable elastomer, poly(glycerol sebacate urethane) (PGSU), suitable for efficient encapsulation and controlled delivery of bioactive macromolecules and with the potential to be applied to cardiac drug delivery.

Cellular and extracellular programming of cell fate through engineered intracrine-, paracrine-, and endocrine-like mechanisms

A cells fate is tightly controlled by its microenvironment. Key factors contributing to this microenvironment include physical contacts with the extracellular matrix and neighboring cells, in addition to soluble factors produced locally or distally. Alterations to these cues can drive homeostatic processes, such as tissue regeneration/wound healing, or may lead to pathologic tissue dysfunction. In vitro models of cell and tissue microenvironments are desirable for enhanced understanding of the biology and ultimately for improved treatment. However, mechanisms to exert specific control over cellular microenvironments remains a significant challenge. Genetic modification has been used but is limited to products that can be manufactured by cells and release kinetics of therapeutics cannot easily be controlled. Herein we describe a non-genetic approach to engineer cells with an intracellular depot of phenotype altering agent/s that can be used for altering cell fate via intracrine-, paracrine-, and endocrine-like mechanisms. Specifically, we show that human mesenchymal stem cells (MSCs) can be engineered with poly lactide-co-glycolic acid (PLGA) particles containing dexamethasone, which acts on cytoplasmic receptors. The controlled release properties of these particles allowed for sustained intracellular and extracellular delivery of agent to promote differentiation of particle-carrying cells, as well as neighboring cells and distant cells that do not contain particles.

Mimicking the inflammatory cell adhesion cascade by nucleic acid aptamer programmed cell-cell interactions

Nature has evolved effective cell adhesion mechanisms to deliver inflammatory cells to inflamed tissue; however, many culture‐expanded therapeutic cells are incapable of targeting diseased tissues following systemic infusion, which represents a great challenge in cell therapy. Our aim was to develop simple approaches to program cell‐cell interactions that would otherwise not exist toward cell targeting and understanding the complex biology of cell‐cell interactions. We employed a chemistry approach to engineer P‐ or L‐selectin binding nucleic acid aptamers onto mesenchymal stem cells (MSCs) to enable them to engage inflamed endothelial cells and leukocytes, respectively. We show for the first time that engineered cells with a single artificial adhesion ligand can recapitulate 3 critical cell interactions in the inflammatory cell adhesion cascade under dynamic flow conditions. Aptamer‐engineered MSCs adhered on respective selectin surfaces under static conditions >10 times more efficiently than controls including scrambled‐DNA modified MSCs. Significantly, engineered MSCs can be directly captured from the flow stream by selectin surfaces or selectin‐expressing cells under flow conditions (≤2dyn/cm2). The simple chemistry approach and the versatility of aptamers permit the concept of engineered cell‐cell interactions to be generically applicable for targeting cells to diseased tissues and elucidating the biology of cell‐cell interactions.—Zhao, W., Loh, W., Droujinine, I. A., Teo, W., Kumar, N., Schafer, S., Cui, C. H., Zhang, L., Sarkar, D., Karnik, R., Karp, J. M. Mimicking the inflammatory cell adhesion cascade by nucleic acid aptamer programmed cell‐cell interactions. FASEB J. 25, 3045–3056 (2011). www.fasebj.org

Cell–material interactions on biphasic polyurethane matrix†

Cell-matrix interaction is a key regulator for controlling stem cell fate in regenerative tissue engineering. These interactions are induced and controlled by the nanoscale features of extracellular matrix and are mimicked on synthetic matrices to control cell structure and functions. Recent studies have shown that nanostructured matrices can modulate stem cell behavior and exert specific role in tissue regeneration. In this study, we have demonstrated that nanostructured phase morphology of synthetic matrix can control adhesion, proliferation, organization and migration of human mesenchymal stem cells (MSCs). Nanostructured biodegradable polyurethanes (PU) with segmental composition exhibit biphasic morphology at nanoscale dimensions and can control cellular features of MSCs. Biodegradable PU with polyester soft segment and hard segment composed of aliphatic diisocyanates and dipeptide chain extender were designed to examine the effect polyurethane phase morphology. By altering the polyurethane composition, morphological architecture of PU was modulated and its effect was examined on MSC. Results show that MSCs can sense the nanoscale morphology of biphasic polyurethane matrix to exhibit distinct cellular features and, thus, signifies the relevance of matrix phase morphology. The role of nanostructured phases of a synthetic matrix in controlling cell-matrix interaction provides important insights for regulation of cell behavior on synthetic matrix and, therefore, is an important tool for engineering tissue regeneration.

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell-matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell-cell and cell-matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell-matrix interaction, and cell-cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration.

Deletion of IFT20 in early stage T lymphocyte differentiation inhibits the development of collagen-induced arthritis.

IFT20 is the smallest member of the intraflagellar transport protein (IFT) complex B. It is involved in cilia formation. Studies of IFT20 have been confined to ciliated cells. Recently, IFT20 was found to be also expressed in non-ciliated T cells and have functions in immune synapse formation and signaling in vitro. However, how IFT20 regulates T-cell development and activation in vivo is still unknown. We deleted the IFT20 gene in early and later stages of T-cell development by crossing IFT20flox/flox (IFT20f/f) mice with Lck-Cre and CD4-Cre transgenic mice, and investigated the role of IFT20 in T-cell maturation and in the development of T cell-mediated collagen-induced arthritis (CIA). We found that both Lck-Cre/IFT20f/f and CD4-Cre/IFT20f/f mice were indistinguishable from their wild-type littermates in body size, as well as in the morphology and weight of the spleen and thymus. However, the number of CD4- and CD8-positive cells was significantly lower in thymus and spleen in Lck-Cre/IFT20f/f mice. Meanwhile, the incidence and severity of CIA symptoms were significantly decreased, and inflammation in the paw was significantly inhibited in Lck-Cre/IFT20f/f mice compared to Lck-Cre/IFT20+/+ littermates. Deletion IFT20 in more mature T cells of CD4-Cre/IFT20f/f mice had only mild effects on the development of T cells and CIA. The expression of IL-1β, IL-6 and TGF-β1 were significantly downregulated in the paw of Lck-Cre/IFT20f/f mice, but just slight decreased in CD4-Cre/IFT20f/f mice. These results demonstrate that deletion of IFT20 in the early stage of T-cell development inhibited CIA development through regulating T-cell development and the expression of critical cytokines.