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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 61, NO. 5, MAY 2014
Hydrogels as Carriers for Stem Cell Transplantation Melissa Alvarado-Velez, S. Balakrishna Pai, and Ravi V. Bellamkonda∗
Abstract—Stem cells are a promising source for cell replacement therapy for several degenerative conditions. However, a number of limitations such as low cell survival, uncontrolled and/or low differentiation, induction of host immune response, and the risk of teratoma formation remain as challenges. In this review, we explore the utility of hydrogels as carriers for stem cell delivery and their potential to overcome some of the current limitations in stem cell therapy. We focus on in situ gelling hydrogels, and also discuss other strategies to modulate the immune response to promote controlled stem cell differentiation. Immunomodulatory hydrogels and gels designed to promote cell survival and integration into the host site will likely have a significant effect on enhancing the efficacy of stem cell transplantation as a therapy for debilitating degenerative diseases. Index Terms—Immunomodulation, injectable hydrogels, stem cell transplantation.
I. STEM CELL THERAPY TEM cell transplantation has emerged as a promising therapy for degenerative diseases. The self-renewal capacity of stem cells and their ability to differentiate into a wide range of specialized tissues make them a promising cell source for cell therapy [1]. The therapeutic effect of stem cells has been explored in myocardial infarction, neurodegenerative diseases, and bone defect repair among others [2]–[4]. However, there are limitations that need to be overcome before wide spread clinical use becomes prevalent. Some of these limitations include low cell survival, uncontrolled, and/or low differentiation into desired phenotypes, induction of the host immune response, and the risk of teratoma formation [1]. In addition to transplanted stem cell state, integration of transplanted cells into host remains challenging. Natural or synthetic hydrogel-based scaffolds have the potential to mimic the extracellular matrix (ECM) and serve to organize the cells into a 3-D architecture [5], [6].
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II. GENERAL CHARACTERISTICS OF HYDROGELS Hydrogels are cross-linked network of polymers with high water content, which allow them to promote cell viability [7].
Manuscript received October 2, 2013; revised December 14, 2013; accepted January 22, 2014. Date of publication February 11, 2014; date of current version April 17, 2014. The work of R. Bellamkonda supported in part by The National Institutes of Health under Grant 1-R01-NS079739-01. Asterisk indicates corresponding author. M. Alvarado-Velez and S. B. Pai are with the Neurological Biomaterials and Cancer Therapeutics Laboratory, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University School of Medicine, Atlanta, GA 30332 USA (e-mail: [email protected]; [email protected]). ∗ R. V. Bellamkonda is with the Neurological Biomaterials and Cancer Therapeutics Laboratory, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University School of Medicine, Atlanta, GA 30332-0535 USA (e-mail: [email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TBME.2014.2305753
The high water content promotes the exchange of ions, nutrients, and metabolites with the fluids of the surrounding tissue, thus helping to maintain the viability of the transplanted cells [8]. The favorable diffusion of biomolecules through the hydrogels also facilitates the communication between the host tissue cells and the transplanted cells, which influence the cell behavior of both the host and the transplanted cells. For example, transplanted mesenchymal stem cells (MSCs) can modulate the immune response at the transplantation site by their interaction with T-cells, B-cells, and macrophages [9], [10]. Hydrogels can also be used to deliver bioactive molecules, by diffusion or chemical conjugation, in order to modulate cell behavior [11]. In addition to the delivery of bioactive molecules, the spatial presentation of the environmental cues may play an important role in stem cell behavior. Hydrogels can be patterned to create protein gradients or regions that can potentially have a positive effect on stem cell survival, migration, and differentiation. For example, the incorporation of hydrogel microparticles within 3-D stem cell aggregates has been shown to modulate the gene expression of various differentiation markers, which may be due to the spatial control of ECM cues [12]. Lastly, the mechanical properties of hydrogels can be tuned to mimic soft tissues by changing the hydrogel precursor or crosslinker concentrations. Various studies have shown that stem cell fate can be strongly influenced by the stiffness or rigidity of the ECM [13]. Engler et al. studied the effect of substrate stiffness on the differentiation of myoblast cells and showed that these cells differentiated on gels with stiffness similar to a normal muscle, but not in significantly softer or stiffer gels [14]. Also, Banerjee et al. have shown that neural stem cell differentiation into neurons was favored within hydrogels with an elastic modulus similar to brain tissue [15]. All these properties make hydrogels suitable to be used as scaffolds for cell delivery and provide a valuable tool to enhance stem cell therapy. III. In situ GELLING HYDROGELS FOR STEM CELL DELIVERY In several injury situations, the injury site receiving stem cell transplantation has an irregular shape due to the direct consequence of injury and the subsequent degenerative process. Therefore, it is not preferable to use premade hydrogels that do not fill the space completely leaving gaps between the hydrogels and the host tissue. Different variables such as temperature or pH can be used to control the gelling process allowing the formation of the hydrogel in situ. The in situ gelling process results in the conformational filling of the transplantation site, which increases the hydrogel-tissue interface thereby improving the hydrogel integration with the host tissue. The variety of hydrogels that can be gelled in situ depending on their chemical and physical properties has been reviewed elsewhere [16]–[18].
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TABLE I In Situ GELLING HYDROGELS FOR STEM CELL DELIVERY in vivo
In situ gelling thermoresponsive and photopolymerizable hydrogels with emphasis on their current application in stem cell delivery are discussed here in Table I. A. Thermoresponsive Hydrogels Thermoresponsive hydrogels have the ability to undergo reversible soluble-gelling transitions due to changes in temperature. Hydrogels exhibit a soluble-gel transition temperature. At this soluble-gel transition temperature, the physical forces (e.g., hydrogen bond, ionic bond) between the polymer and the solvent are modified resulting in the gelation or solubilization of the polymer. An example of this process is the aggregation of the gelatin helices, which are in a random coil conformation at high temperatures but undergo aggregation with a decrease in temperature [16]. Hydrogels with transition temperatures similar to the human body may be good candidates as injectable scaffolds for stem cell delivery. Natural polymers, such as agarose, and alginate have been used for 3-D stem cell culture and in vivo stem cell delivery [19], [20]. These natural polymers have shown good biocompatibility, and also promote cell survival and migration. Agarose, for example, is a polysaccharide of D-galactose and 3,6- anhydroL-galactopyranose derived from red algae. Agarose needs to be heated in order to solubilize the powder in aqueous solutions and then gels at lower temperatures [20]. This heating-cooling process may represent a challenge to preserve the function of conjugated protein(s) and/or to maintain the cell viability of the transplanted cells. However, there are a variety of polymers
that exhibit reverse thermogelation. These polymers are soluble at cold temperatures and the gel formation results due to an increase in temperature [16]. Reversed thermogelation can be very useful for the delivery of proteins and/or stem cell scaffolds, in which the low temperature is important to maintain the protein function and cell viability. Collagen is an ideal candidate for in situ gelling due to its reverse thermogelation property and high biocompatibility, as it is an abundant component of the ECM [20]. Despite the wide use of collagen for 3-D cell culture in vitro, only a few studies have focused on its in situ gelling capacity. For example, collagen-based scaffolds containing adsorbed fibronectin or laminin have been used to deliver neural stem cells into the injured brain, enhancing the cell survival of the transplanted cells and functional recovery [21]. One of the limitations that might be impeding the use of collagen hydrogels in vivo is its long gelation time (∼30 min) versus a favorable gelation time of 5 min or less. Another well-studied polymer with reverse thermogelation properties is the combination of chitosan, a chitin derivative with glycerol phosphate (chitosan-GP) [22]. These hydrogels have been effectively used for stem cell delivery in models of acute kidney injury (AKI) and myocardial infarction [22]–[24]. In the AKI model, the chitosan-GP hydrogels enhanced the survival of adipose-derived mesenchymal stem cells (ADMSCs) improving their therapeutic effect [23]. Lastly, the biocompatibility of the cellulose derivativemethylcellulose (MC) has been studied for in situ gelling applications. MC hydrogels have been used for the culture of
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neuronal and astrocytic cells in vitro. In addition, Tate et al. reported that MC hydrogels were biocompatible when implanted in the injured brain, suggesting its potential use for stem cell delivery after brain injury [25]. There are a great variety of thermoreversible hydrogels that have not been evaluated as carriers for cell delivery and that may offer specific advantages for various applications. For example, the degradation of different materials can be evaluated in order to tune the degradation of the scaffold with the production of ECM by the transplanted cells. In addition, many polymers that exhibit reversed thermogelation are relatively inert materials with low cell-material interactions and/or lack chemical groups that would allow functionalization. Currently, researchers are attempting to design hydrogels with a goal to improve biocompatibility, gelation properties, cell interaction, and functionalization of biomaterials.
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photopolymerization without the use of photoiniator, which may potentially improve the biocompatibility of the transplants, and pave the way for the use of photopolymerization in deep tissues in a minimally invasive way [34]. IV. BIOACTIVE HYDROGELS Hydrogels can be used to deliver bioactive molecules by diffusion or chemical crosslinking. In addition, small peptide sequences derived from ECM proteins (e.g., IKVAV from laminin) can be incorporated into the hydrogels in order to promote cell adhesion and migration. In this review, we will focus on the use of signaling molecules to modulate the immune system to improve cell survival, and to direct stem cell differentiation into desired phenotypes.
B. Photopolymerizable Hydrogels
A. Immunomodulatory Hydrogels for Improving Stem Cell Survival
Photopolymerizable hydrogels are formed due to the polymerization of soluble macromolecular monomers after exposure to UV light in the presence of a photoinitiator. A variety of materials have been used for the synthesis of photopolymerizable hydrogels, methacrylated hyaluronic acid (MeHA) and poly(ethylene glycol) diacrylate (PEGDA) being the ones that are widely used [26]–[29]. During the photopolymerization process, UV light disassociates photoinitiator molecules into free radicals that induce the polymerization of the hydrogel network [26]. The photopolymerization process allows the spatial and temporal control of the hydrogel formation using fast curing rates with low heat production [30]. One of the risks of the photopolymerization process is that free radicals can also react with cellular components such as cell membranes, proteins, and DNA, which may result in low cell viability or malignant transformation of the exposed cells [26], [31]. However, it has been shown that the photoinitiator molecules in the presence of the hydrogel precursors do not significantly reduce the viability of various stem cell types [26], [28], [31]. Despite the extensive research performed using photopolymerizable hydrogels in vitro, very few studies have been translated to in vivo scenarios. Sharma et al. transplanted MSCs subcutaneously into nude mice using the photocrosslinkable polymer blend of poly(ethylene oxide) diacrylate (PEODA) and hyaluronic acid (HA). The hydrogel blend was also mixed with TGFβ3 to promote the MSCs differentiation into chondrocytes, and crosslinked using transdermal photopolymerization [32]. The transplanted MSCs produced high quality cartilage according to the expression of cartilage-specific genes, and the production of proteoglycan and collagen II [32]. Another study using photopolymerizable hydrogels in vivo was reported by Rossi et al., wherein they delivered Satellite cells (SCs) using an in situ photocrosslinkable HA-based hydrogel in order to restore a partially ablated tibialis anterior (TA) muscle in mice [33]. The HA hydrogels improved the muscle structure and the number of new myofibers promoting functional recovery in the animals [33]. The positive reports using photopolymerizable hydrogels in vivo have set a precedent for similar future applications. Current research is exploring the use of X-rays to induce
During stem cell transplantation, the host immune response is induced by factors such as an involuntary injury during the delivery process, and the immune rejection of the biomaterial(s) and/or the transplanted cells. Therefore, development of biomaterials that can modulate the immune response in a localized manner is of interest. Numerous examples of endogenous immunosuppression are extant in the human body. The eye and the testis induce localized immunosuppression by the release of soluble cytokines like TGF-β2 and IL-10 among others [35]–[37]. In addition, various types of stem cells such as MSCs and retinal progenitor cells (RPCs) exhibit immunomodulatory properties. Although the mechanisms for MSCs-mediated immunomodulation are not fully understood, it is known that MSCs express low levels of human MHC class I and lack human MHC class II, two important molecules in the antigen presentation pathways [10], [38]. Likewise, RPCs transplanted into a kidney pouch model using poly (lactic-co-glycolic acid) (PLGA) polymers showed an enhanced survival even in the presence of interferon γ (IFNγ), a pro-inflammatory cytokine [39]. The survival of the RPCs was due to the production of immunesuppressive factors such as TGF-β2, and Fas ligand [39]. All these examples found in nature serve as inspiration for the development of immunomodulatory biomaterials. During the process of elicitation of immune response there exist various checkpoints that can be targeted to generate the desired effect (see Fig. 1). For instance, an important part of the immune response is the presentation of antigens to the T-cells by the antigen presenting cells (APCs) such as macrophages and dendritic cells. The stimulation of the T-cells to maintain a tolerogenic state can be achieved by the presentation of the antigen in the presence of stimulatory molecules such as TGF-β1 and interleukin-10 (IL-10), whereas presenting the antigens in the presence of cytokines like interleukin-6 (IL-6) and interleukin-23 (IL-23) leads to immunogenic T-cells [40]. Hume et al. demonstrated that functionalized poly(ethylene glycol) hydrogels with immobilized TGF-β1 and IL-10 decreased activation markers on dendritic cells and reduced their ability to activate T cells in vitro [41] (see Table II). Thus, the integration of cytokines that promote a tolerogenic response into hydrogels
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Fig. 1. Checkpoints during the immune response that can be used to suppress inflammation and to enhance stem cell survival. An important step during the immune response is the activation of antigen presenting cells, such as macrophages and dendritic cells, and the subsequent antigen presentation to T-cells. Different cytokines can be used to shift the macrophage phenotypes from a pro-inflammatory (classically activated, M1) to an anti-inflammatory (alternative activated, M2) phenotype. In addition, blocking of toll-like receptors is associated with a reduction of dendritic cell maturation, which promotes the formation of regulatory T-cells. These T-cells play an important role in suppressing effector T-cells (cytotoxic) in order to avoid an exacerbated immune reaction that may damage healthy tissue. Another approach to obtain a localized immune response is to create an immune-barrier around the transplanted cells. For example, hydrogels coated with FasL can induce the apoptosis of effector T-cells at the transplantation site ( [40], [42], [62]–[64]). TABLE II EFFECTS OF BIOACTIVE MOLECULES ON STEM CELL DIFFERENTIATION
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could potentially lead to a localized immunosuppression at the implantation site thus resulting in an increased survival of the transplanted stem cells. Another strategy that has been explored is designing immunebarriers to induce the apoptosis of T-cells. Hume et al. designed immunoactive polymer coatings on poly(ethylene glycol) (PEG) hydrogels for the encapsulation of cells. In this study, a bifunctional coating was synthesized using Anti-Fas antibody and the cell adhesion molecule ICAM-1, which resulted in the apoptosis of Jurkat T-cells without affecting the viability of the encapsulated cells [4], [42]. Despite the successful development of immunomodulatory hydrogels, there are few reports of their use in vivo. It is critical to elucidate the short- and long-term effects of complexes with immunomodulatory hydrogels in the context of the in vivo environment that include a variety of immune and nonimmune cells. The development of a localized immune suppression may potentially reduce or eliminate the current systemic effects of the immune-suppressive treatments during transplantation therapies. B. Bioactive Hydrogels for Controlled Stem Cell Differentiation During the embryogenesis process, embryonic stem cells (ESCs) are exposed to different chemical cues or growth factors that favor specific cell lineages, among many others. For instance, vascular endothelial growth factor (VEGF) is associated with the differentiation of ESCs into endothelial cells and cardiomyocytes, whereas nerve growth factor (NGF) is associated with the differentiation of ESCs into neurons [43]–[46]. It is important to recognize that different stem cells might have distinctive differentiation properties. ESCs are pluripotent stem cells that can differentiate into all cell types. Unlike ESCs, neural stem cells are multipotent stem cells that can only differentiate to specific lineages: neurons, astrocytes, and oligodendrocytes. Growth factors can have varied effects depending on the stem cell lineage, which needs to be determined experimentally (see Table II for details). Several studies have investigated the use of bioactive hydrogels to direct stem cell differentiation into endothelial cells, osteoblasts, and neurons among others. Ferreira et al. prepared VEGF loaded microparticles and encapsulated them along with ESCs into dextran-based hydrogels. The encapsulated ESCs expressed lower neuronal and hepatic markers, whereas increased expression of KDR/Flk-1 an indicator of vascular differentiation was observed [47]. Another study explored conditions that would commit neural stem/progenitor cells (NPCs) to neuronal differentiation. Leipzig et al. synthesized a photocrosslinked methacrylamide-chitosan hydrogel scaffold with immobilized proneurogenic rat interferon-γ (rIFN-γ). This scaffold increased the differentiation of NPCs and favored a neuronal lineage instead of glial lineage [48]. Usually, the delivery of bioactive molecules by diffusion is achieved during a short time period. Also, the long-term delivery of bioactive molecules using chemical crosslinking methods can be challenging due to the denaturing of the protein during the conjugation process. An alternative
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method is the incorporation of active domains into the hydrogels that can naturally interact with the growth factors, thereby slowing down the diffusion process. Butterfield et al. incorporated the glycosaminoglycan Chondroitin Sulfate (CS), which naturally interacts with NGF, into a poly (ethylene glycol) hydrogel in order to promote the controlled release of NGF [49]. In addition to the delivery of single growth factors, hydrogels can be modified with multiple proteins creating a more complex cellular environment. Furthermore, the chemical crosslinking of proteins can be controlled to create protein gradients or drug release profiles that can alter cellular behavior [50]. V. ELECTROCONDUCTIVE HYDROGELS The development of electroconductive hydrogels (ECHs) is a novel approach motivated by the fact that several body tissues such as heart, nerve, and muscle respond to electrical stimulus. In addition, electromagnetic fields are involved in various biological processes including: angiogenesis, wound healing, and cell signaling among others [51], [52]. Studies suggest that endogenous electrical fields play an important role during the embryonic development [53]. Moreover, the application of an electrical field to embryonic bodies derived from embryonic stem cells (ESCs) resulted in a marked increase of cardiomyocyte differentiation [53]. Wen et al. in an experiment using cardiac myocytes and MSCs, demonstrated that the enhancement of cardiogenesis correlated with an increase in the expression of the protein S100A4 (an early cardiogenesis promoter) [54]. Yamada et al. performed a similar experiment using milder voltage conditions and observed an enhanced differentiation of embryonic bodies into neurons [55]. Therefore, it is important to determine the appropriate combinations of electrical, chemical, and topographical cues that direct stem cell fate to generate a specific phenotype. ECHs combine the conductive properties of electroactive polymers with the high hydration and biocompatibility of hydrogels [56]. The synthesis of ECHs can be achieved by mixing a hydrogel network with a conductive polymer. For example, the hydrogel precursor poly(ethylene glycol) methacrylate (PEGMA) can be supplemented with the conductive polymer polypyrrole (PPy) and photopolymerized to yield a ECHs [56]. The biocompatibility of ECHs based PEGMA and PPy was tested using human muscle fibroblasts and rat pheochromocytoma cells showing no cytotoxicity and supporting cell proliferation [57]. However, the use of ECHs for stem cell culture in vitro and for stem cell delivery in vivo has not been explored. It still needs to be determined if the synthesis process of ECHs can support cell encapsulation and the ability of the hydrogel to gel in situ. VI. CONCLUSION AND FUTURE OUTLOOK Hydrogels offer several advantages to overcome the current limitations during stem cell transplantation. Their biocompatibility, mechanical properties comparable to soft tissue that minimizes foreign material/tissue mismatch, gelling properties, and potential to be chemically modified make them ideal candidates as cell delivery carriers. Despite its potential, there is still a
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need to optimize the conditions that favor directed stem cell differentiation, and minimize the host immune response. Better understanding of the signaling pathways that contribute to the survival of the stem cells as well as their contribution to directed differentiation would greatly aid in the development of stem cell therapy. This information on the stem cell “behavior” coupled to the refinement in designing appropriate hydrogel technology is critical for the advancement of this field. In the near future, developments in the various disciplines in the biomedical field such as biomaterials, stem cell biology, and immunology should help in the formulation of hydrogel-based cell replacement technology. Improved hydrogel-encapsulated stem cell methodology will go a long way in the much needed treatment of a number of ailments including neurodegenerative diseases.
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[59] K. H. Park and K. Na, “Effect of growth factors on chondrogenic differentiation of rabbit mesenchymal cells embedded in injectable hydrogels,” J. Biosci. Bioeng., vol. 106, pp. 74–79, Jul. 2008. [60] B. G. Ballios, M. J. Cooke, D. van der Kooy, and M. S. Shoichet, “A hydrogel-based stem cell delivery system to treat retinal degenerative diseases,” Biomaterials, vol. 31, pp. 2555–2564, Mar. 2010. [61] C. Fuoco, M. L. Salvatori, A. Biondo, K. Shapira-Schweitzer, S. Santoleri, S. Antonini, S. Bernardini, F. S. Tedesco, S. Cannata, D. Seliktar, G. Cossu, and C. Gargioli, “Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration,” Skeletal Muscle, vol. 2, p. 24, 2012. [62] A. Michelucci, T. Heurtaux, L. Grandbarbe, E. Morga, and P. Heuschling, “Characterization of the microglial phenotype under specific proinflammatory and anti-inflammatory conditions: Effects of oligomeric and fibrillar amyloid-beta,” J. Neuroimmunol., vol. 210, pp. 3–12, May 29, 2009. [63] N. Mokarram, A. Merchant, V. Mukhatyar, G. Patel, and R. V. Bellamkonda, “Effect of modulating macrophage phenotype on peripheral nerve repair,” Biomaterials, vol. 33, pp. 8793–801, Dec. 2012. [64] S. Rutella, S. Danese, and G. Leone, “Tolerogenic dendritic cells: Cytokine modulation comes of age,” Blood, vol. 108, pp. 1435–1440, Sep. 1, 2006. [65] C. Dani, A. G. Smith, S. Dessolin, P. Leroy, L. Staccini, P. Villageois, C. Darimont, and G. Ailhaud, “Differentiation of embryonic stem cells into adipocytes in vitro,” J. Cell Sci., vol. 110, pp. 1279–1285, Jun. 1997. [66] E. Dupin and N. M. Le Douarin, “Retinoic acid promotes the differentiation of adrenergic cells and melanocytes in quail neural crest cultures,” Developmental Biol., vol. 168, pp. 529–548, Apr. 1995. [67] M. Schuldiner, O. Yanuka, J. Itskovitz-Eldor, D. A. Melton, and N. Benvenisty, “Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells,” Proc. Nat. Acad. Sci. USA, vol. 97, pp. 11307–11312, Oct. 10, 2000. [68] G. Chamberlain, J. Fox, B. Ashton, and J. Middleton, “Concise review: Mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing,” Stem Cells, vol. 25, pp. 2739–2749, Nov. 2007. [69] N. D. Leipzig, C. Xu, T. Zahir, and M. S. Shoichet, “Functional immobilization of interferon-gamma induces neuronal differentiation of neural stem cells,” J. Biomed. Mater. Res. A, vol. 93, pp. 625–633, May 2010. [70] S. Ahmed, B. A. Reynolds, and S. Weiss, “BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors,” J. Neurosci., vol. 15, pp. 5765–5778, Aug. 1995. [71] R. E. Allen and L. K. Boxhorn, “Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor,” J. Cell Physiol., vol. 138, pp. 311–315, Feb. 1989.
Melissa Alvarado-Velez received the B.S. degree in industrial biotechnology from the University of Puerto Rico-Mayaguez, Puerto Rico. She is a graduate student in the Biomedical Engineering joint program between the Georgia Institute of Technology, Atlanta, USA and Emory University, Atlanta. Along her academic career, she has received various recognitions including the Goizueta Foundation Fellowship and the President’s Fellowship award from the Georgia Institute of Technology. Her research interests include the development of biomaterials that enhance stem cell therapy for traumatic brain injury or other neurodegenerative diseases.
ALVARADO-VELEZ et al.: HYDROGELS AS CARRIERS FOR STEM CELL TRANSPLANTATION
S. Balakrishna Pai received the Ph.D. degree from the Indian Institute of Science, India. He is a Senior Research Scientist in the Neurological Biomaterials and Cancer Therapeutics lab at the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, USA and Emory University School of Medicine, Atlanta. He was a Fogarty Visiting fellow at the National Institutes of Health, USA. He continued his research at the University of Calgary, Canada and at the Yale University School of Medicine, CT, USA. His current research interests include understanding molecular mechanisms in cells including stem cells and developing biomaterial-based solutions to nervous system deficiencies/diseases including cancer.
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Ravi V. Bellamkonda received the B.S. degree in biomedical engineering from Osmania University, Andhra Pradesh, India and the Ph.D. degree in the medical sciences from Brown University, RI, USA. He is the Wallace H. Coulter Chair and Professor in biomedical engineering and a Georgia Research Alliance Distinguished Scholar at Georgia Institute of Technology, Atlanta, USA and Emory University School of Medicine, Atlanta. He is the Director of the Neurological Biomaterials and Cancer Therapeutics Lab at the Coulter Department of Biomedical Engineering. He completed his Postdoctoral Fellowship in the Department of Brain and Cognitive Sciences at Massachusetts Institute of Technology, USA. Prior to joining Georgia Tech, he was an Associate Professor with tenure at Case Western Reserve University, OH, USA. His research interests include the application of principles of regenerative medicine for the repair and regeneration of neural tissue, development of novel electrode designs for neural interfacing, and rational design of nanocarriers for pediatric cancer diagnosis and therapy. Dr. Bellamkonda has received numerous awards including EUREKA Award from National Cancer Institute, CAREER award from National Science Foundation and Best Professor award at GT/BME. He is a Fellow of the Institute of Physics, and is the President-elect and Fellow of AIMBE.
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Hydrogels as Carriers for Stem Cell Transplantation Melissa Alvarado-Velez, S. Balakrishna Pai, and Ravi V. Bellamkonda∗
Abstract—Stem cells are a promising source for cell replacement therapy for several degenerative conditions. However, a number of limitations such as low cell survival, uncontrolled and/or low differentiation, induction of host immune response, and the risk of teratoma formation remain as challenges. In this review, we explore the utility of hydrogels as carriers for stem cell delivery and their potential to overcome some of the current limitations in stem cell therapy. We focus on in situ gelling hydrogels, and also discuss other strategies to modulate the immune response to promote controlled stem cell differentiation. Immunomodulatory hydrogels and gels designed to promote cell survival and integration into the host site will likely have a significant effect on enhancing the efficacy of stem cell transplantation as a therapy for debilitating degenerative diseases. Index Terms—Immunomodulation, injectable hydrogels, stem cell transplantation.
I. STEM CELL THERAPY TEM cell transplantation has emerged as a promising therapy for degenerative diseases. The self-renewal capacity of stem cells and their ability to differentiate into a wide range of specialized tissues make them a promising cell source for cell therapy [1]. The therapeutic effect of stem cells has been explored in myocardial infarction, neurodegenerative diseases, and bone defect repair among others [2]–[4]. However, there are limitations that need to be overcome before wide spread clinical use becomes prevalent. Some of these limitations include low cell survival, uncontrolled, and/or low differentiation into desired phenotypes, induction of the host immune response, and the risk of teratoma formation [1]. In addition to transplanted stem cell state, integration of transplanted cells into host remains challenging. Natural or synthetic hydrogel-based scaffolds have the potential to mimic the extracellular matrix (ECM) and serve to organize the cells into a 3-D architecture [5], [6].
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II. GENERAL CHARACTERISTICS OF HYDROGELS Hydrogels are cross-linked network of polymers with high water content, which allow them to promote cell viability [7].
Manuscript received October 2, 2013; revised December 14, 2013; accepted January 22, 2014. Date of publication February 11, 2014; date of current version April 17, 2014. The work of R. Bellamkonda supported in part by The National Institutes of Health under Grant 1-R01-NS079739-01. Asterisk indicates corresponding author. M. Alvarado-Velez and S. B. Pai are with the Neurological Biomaterials and Cancer Therapeutics Laboratory, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University School of Medicine, Atlanta, GA 30332 USA (e-mail: [email protected]; [email protected]). ∗ R. V. Bellamkonda is with the Neurological Biomaterials and Cancer Therapeutics Laboratory, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University School of Medicine, Atlanta, GA 30332-0535 USA (e-mail: [email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TBME.2014.2305753
The high water content promotes the exchange of ions, nutrients, and metabolites with the fluids of the surrounding tissue, thus helping to maintain the viability of the transplanted cells [8]. The favorable diffusion of biomolecules through the hydrogels also facilitates the communication between the host tissue cells and the transplanted cells, which influence the cell behavior of both the host and the transplanted cells. For example, transplanted mesenchymal stem cells (MSCs) can modulate the immune response at the transplantation site by their interaction with T-cells, B-cells, and macrophages [9], [10]. Hydrogels can also be used to deliver bioactive molecules, by diffusion or chemical conjugation, in order to modulate cell behavior [11]. In addition to the delivery of bioactive molecules, the spatial presentation of the environmental cues may play an important role in stem cell behavior. Hydrogels can be patterned to create protein gradients or regions that can potentially have a positive effect on stem cell survival, migration, and differentiation. For example, the incorporation of hydrogel microparticles within 3-D stem cell aggregates has been shown to modulate the gene expression of various differentiation markers, which may be due to the spatial control of ECM cues [12]. Lastly, the mechanical properties of hydrogels can be tuned to mimic soft tissues by changing the hydrogel precursor or crosslinker concentrations. Various studies have shown that stem cell fate can be strongly influenced by the stiffness or rigidity of the ECM [13]. Engler et al. studied the effect of substrate stiffness on the differentiation of myoblast cells and showed that these cells differentiated on gels with stiffness similar to a normal muscle, but not in significantly softer or stiffer gels [14]. Also, Banerjee et al. have shown that neural stem cell differentiation into neurons was favored within hydrogels with an elastic modulus similar to brain tissue [15]. All these properties make hydrogels suitable to be used as scaffolds for cell delivery and provide a valuable tool to enhance stem cell therapy. III. In situ GELLING HYDROGELS FOR STEM CELL DELIVERY In several injury situations, the injury site receiving stem cell transplantation has an irregular shape due to the direct consequence of injury and the subsequent degenerative process. Therefore, it is not preferable to use premade hydrogels that do not fill the space completely leaving gaps between the hydrogels and the host tissue. Different variables such as temperature or pH can be used to control the gelling process allowing the formation of the hydrogel in situ. The in situ gelling process results in the conformational filling of the transplantation site, which increases the hydrogel-tissue interface thereby improving the hydrogel integration with the host tissue. The variety of hydrogels that can be gelled in situ depending on their chemical and physical properties has been reviewed elsewhere [16]–[18].
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TABLE I In Situ GELLING HYDROGELS FOR STEM CELL DELIVERY in vivo
In situ gelling thermoresponsive and photopolymerizable hydrogels with emphasis on their current application in stem cell delivery are discussed here in Table I. A. Thermoresponsive Hydrogels Thermoresponsive hydrogels have the ability to undergo reversible soluble-gelling transitions due to changes in temperature. Hydrogels exhibit a soluble-gel transition temperature. At this soluble-gel transition temperature, the physical forces (e.g., hydrogen bond, ionic bond) between the polymer and the solvent are modified resulting in the gelation or solubilization of the polymer. An example of this process is the aggregation of the gelatin helices, which are in a random coil conformation at high temperatures but undergo aggregation with a decrease in temperature [16]. Hydrogels with transition temperatures similar to the human body may be good candidates as injectable scaffolds for stem cell delivery. Natural polymers, such as agarose, and alginate have been used for 3-D stem cell culture and in vivo stem cell delivery [19], [20]. These natural polymers have shown good biocompatibility, and also promote cell survival and migration. Agarose, for example, is a polysaccharide of D-galactose and 3,6- anhydroL-galactopyranose derived from red algae. Agarose needs to be heated in order to solubilize the powder in aqueous solutions and then gels at lower temperatures [20]. This heating-cooling process may represent a challenge to preserve the function of conjugated protein(s) and/or to maintain the cell viability of the transplanted cells. However, there are a variety of polymers
that exhibit reverse thermogelation. These polymers are soluble at cold temperatures and the gel formation results due to an increase in temperature [16]. Reversed thermogelation can be very useful for the delivery of proteins and/or stem cell scaffolds, in which the low temperature is important to maintain the protein function and cell viability. Collagen is an ideal candidate for in situ gelling due to its reverse thermogelation property and high biocompatibility, as it is an abundant component of the ECM [20]. Despite the wide use of collagen for 3-D cell culture in vitro, only a few studies have focused on its in situ gelling capacity. For example, collagen-based scaffolds containing adsorbed fibronectin or laminin have been used to deliver neural stem cells into the injured brain, enhancing the cell survival of the transplanted cells and functional recovery [21]. One of the limitations that might be impeding the use of collagen hydrogels in vivo is its long gelation time (∼30 min) versus a favorable gelation time of 5 min or less. Another well-studied polymer with reverse thermogelation properties is the combination of chitosan, a chitin derivative with glycerol phosphate (chitosan-GP) [22]. These hydrogels have been effectively used for stem cell delivery in models of acute kidney injury (AKI) and myocardial infarction [22]–[24]. In the AKI model, the chitosan-GP hydrogels enhanced the survival of adipose-derived mesenchymal stem cells (ADMSCs) improving their therapeutic effect [23]. Lastly, the biocompatibility of the cellulose derivativemethylcellulose (MC) has been studied for in situ gelling applications. MC hydrogels have been used for the culture of
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neuronal and astrocytic cells in vitro. In addition, Tate et al. reported that MC hydrogels were biocompatible when implanted in the injured brain, suggesting its potential use for stem cell delivery after brain injury [25]. There are a great variety of thermoreversible hydrogels that have not been evaluated as carriers for cell delivery and that may offer specific advantages for various applications. For example, the degradation of different materials can be evaluated in order to tune the degradation of the scaffold with the production of ECM by the transplanted cells. In addition, many polymers that exhibit reversed thermogelation are relatively inert materials with low cell-material interactions and/or lack chemical groups that would allow functionalization. Currently, researchers are attempting to design hydrogels with a goal to improve biocompatibility, gelation properties, cell interaction, and functionalization of biomaterials.
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photopolymerization without the use of photoiniator, which may potentially improve the biocompatibility of the transplants, and pave the way for the use of photopolymerization in deep tissues in a minimally invasive way [34]. IV. BIOACTIVE HYDROGELS Hydrogels can be used to deliver bioactive molecules by diffusion or chemical crosslinking. In addition, small peptide sequences derived from ECM proteins (e.g., IKVAV from laminin) can be incorporated into the hydrogels in order to promote cell adhesion and migration. In this review, we will focus on the use of signaling molecules to modulate the immune system to improve cell survival, and to direct stem cell differentiation into desired phenotypes.
B. Photopolymerizable Hydrogels
A. Immunomodulatory Hydrogels for Improving Stem Cell Survival
Photopolymerizable hydrogels are formed due to the polymerization of soluble macromolecular monomers after exposure to UV light in the presence of a photoinitiator. A variety of materials have been used for the synthesis of photopolymerizable hydrogels, methacrylated hyaluronic acid (MeHA) and poly(ethylene glycol) diacrylate (PEGDA) being the ones that are widely used [26]–[29]. During the photopolymerization process, UV light disassociates photoinitiator molecules into free radicals that induce the polymerization of the hydrogel network [26]. The photopolymerization process allows the spatial and temporal control of the hydrogel formation using fast curing rates with low heat production [30]. One of the risks of the photopolymerization process is that free radicals can also react with cellular components such as cell membranes, proteins, and DNA, which may result in low cell viability or malignant transformation of the exposed cells [26], [31]. However, it has been shown that the photoinitiator molecules in the presence of the hydrogel precursors do not significantly reduce the viability of various stem cell types [26], [28], [31]. Despite the extensive research performed using photopolymerizable hydrogels in vitro, very few studies have been translated to in vivo scenarios. Sharma et al. transplanted MSCs subcutaneously into nude mice using the photocrosslinkable polymer blend of poly(ethylene oxide) diacrylate (PEODA) and hyaluronic acid (HA). The hydrogel blend was also mixed with TGFβ3 to promote the MSCs differentiation into chondrocytes, and crosslinked using transdermal photopolymerization [32]. The transplanted MSCs produced high quality cartilage according to the expression of cartilage-specific genes, and the production of proteoglycan and collagen II [32]. Another study using photopolymerizable hydrogels in vivo was reported by Rossi et al., wherein they delivered Satellite cells (SCs) using an in situ photocrosslinkable HA-based hydrogel in order to restore a partially ablated tibialis anterior (TA) muscle in mice [33]. The HA hydrogels improved the muscle structure and the number of new myofibers promoting functional recovery in the animals [33]. The positive reports using photopolymerizable hydrogels in vivo have set a precedent for similar future applications. Current research is exploring the use of X-rays to induce
During stem cell transplantation, the host immune response is induced by factors such as an involuntary injury during the delivery process, and the immune rejection of the biomaterial(s) and/or the transplanted cells. Therefore, development of biomaterials that can modulate the immune response in a localized manner is of interest. Numerous examples of endogenous immunosuppression are extant in the human body. The eye and the testis induce localized immunosuppression by the release of soluble cytokines like TGF-β2 and IL-10 among others [35]–[37]. In addition, various types of stem cells such as MSCs and retinal progenitor cells (RPCs) exhibit immunomodulatory properties. Although the mechanisms for MSCs-mediated immunomodulation are not fully understood, it is known that MSCs express low levels of human MHC class I and lack human MHC class II, two important molecules in the antigen presentation pathways [10], [38]. Likewise, RPCs transplanted into a kidney pouch model using poly (lactic-co-glycolic acid) (PLGA) polymers showed an enhanced survival even in the presence of interferon γ (IFNγ), a pro-inflammatory cytokine [39]. The survival of the RPCs was due to the production of immunesuppressive factors such as TGF-β2, and Fas ligand [39]. All these examples found in nature serve as inspiration for the development of immunomodulatory biomaterials. During the process of elicitation of immune response there exist various checkpoints that can be targeted to generate the desired effect (see Fig. 1). For instance, an important part of the immune response is the presentation of antigens to the T-cells by the antigen presenting cells (APCs) such as macrophages and dendritic cells. The stimulation of the T-cells to maintain a tolerogenic state can be achieved by the presentation of the antigen in the presence of stimulatory molecules such as TGF-β1 and interleukin-10 (IL-10), whereas presenting the antigens in the presence of cytokines like interleukin-6 (IL-6) and interleukin-23 (IL-23) leads to immunogenic T-cells [40]. Hume et al. demonstrated that functionalized poly(ethylene glycol) hydrogels with immobilized TGF-β1 and IL-10 decreased activation markers on dendritic cells and reduced their ability to activate T cells in vitro [41] (see Table II). Thus, the integration of cytokines that promote a tolerogenic response into hydrogels
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Fig. 1. Checkpoints during the immune response that can be used to suppress inflammation and to enhance stem cell survival. An important step during the immune response is the activation of antigen presenting cells, such as macrophages and dendritic cells, and the subsequent antigen presentation to T-cells. Different cytokines can be used to shift the macrophage phenotypes from a pro-inflammatory (classically activated, M1) to an anti-inflammatory (alternative activated, M2) phenotype. In addition, blocking of toll-like receptors is associated with a reduction of dendritic cell maturation, which promotes the formation of regulatory T-cells. These T-cells play an important role in suppressing effector T-cells (cytotoxic) in order to avoid an exacerbated immune reaction that may damage healthy tissue. Another approach to obtain a localized immune response is to create an immune-barrier around the transplanted cells. For example, hydrogels coated with FasL can induce the apoptosis of effector T-cells at the transplantation site ( [40], [42], [62]–[64]). TABLE II EFFECTS OF BIOACTIVE MOLECULES ON STEM CELL DIFFERENTIATION
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could potentially lead to a localized immunosuppression at the implantation site thus resulting in an increased survival of the transplanted stem cells. Another strategy that has been explored is designing immunebarriers to induce the apoptosis of T-cells. Hume et al. designed immunoactive polymer coatings on poly(ethylene glycol) (PEG) hydrogels for the encapsulation of cells. In this study, a bifunctional coating was synthesized using Anti-Fas antibody and the cell adhesion molecule ICAM-1, which resulted in the apoptosis of Jurkat T-cells without affecting the viability of the encapsulated cells [4], [42]. Despite the successful development of immunomodulatory hydrogels, there are few reports of their use in vivo. It is critical to elucidate the short- and long-term effects of complexes with immunomodulatory hydrogels in the context of the in vivo environment that include a variety of immune and nonimmune cells. The development of a localized immune suppression may potentially reduce or eliminate the current systemic effects of the immune-suppressive treatments during transplantation therapies. B. Bioactive Hydrogels for Controlled Stem Cell Differentiation During the embryogenesis process, embryonic stem cells (ESCs) are exposed to different chemical cues or growth factors that favor specific cell lineages, among many others. For instance, vascular endothelial growth factor (VEGF) is associated with the differentiation of ESCs into endothelial cells and cardiomyocytes, whereas nerve growth factor (NGF) is associated with the differentiation of ESCs into neurons [43]–[46]. It is important to recognize that different stem cells might have distinctive differentiation properties. ESCs are pluripotent stem cells that can differentiate into all cell types. Unlike ESCs, neural stem cells are multipotent stem cells that can only differentiate to specific lineages: neurons, astrocytes, and oligodendrocytes. Growth factors can have varied effects depending on the stem cell lineage, which needs to be determined experimentally (see Table II for details). Several studies have investigated the use of bioactive hydrogels to direct stem cell differentiation into endothelial cells, osteoblasts, and neurons among others. Ferreira et al. prepared VEGF loaded microparticles and encapsulated them along with ESCs into dextran-based hydrogels. The encapsulated ESCs expressed lower neuronal and hepatic markers, whereas increased expression of KDR/Flk-1 an indicator of vascular differentiation was observed [47]. Another study explored conditions that would commit neural stem/progenitor cells (NPCs) to neuronal differentiation. Leipzig et al. synthesized a photocrosslinked methacrylamide-chitosan hydrogel scaffold with immobilized proneurogenic rat interferon-γ (rIFN-γ). This scaffold increased the differentiation of NPCs and favored a neuronal lineage instead of glial lineage [48]. Usually, the delivery of bioactive molecules by diffusion is achieved during a short time period. Also, the long-term delivery of bioactive molecules using chemical crosslinking methods can be challenging due to the denaturing of the protein during the conjugation process. An alternative
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method is the incorporation of active domains into the hydrogels that can naturally interact with the growth factors, thereby slowing down the diffusion process. Butterfield et al. incorporated the glycosaminoglycan Chondroitin Sulfate (CS), which naturally interacts with NGF, into a poly (ethylene glycol) hydrogel in order to promote the controlled release of NGF [49]. In addition to the delivery of single growth factors, hydrogels can be modified with multiple proteins creating a more complex cellular environment. Furthermore, the chemical crosslinking of proteins can be controlled to create protein gradients or drug release profiles that can alter cellular behavior [50]. V. ELECTROCONDUCTIVE HYDROGELS The development of electroconductive hydrogels (ECHs) is a novel approach motivated by the fact that several body tissues such as heart, nerve, and muscle respond to electrical stimulus. In addition, electromagnetic fields are involved in various biological processes including: angiogenesis, wound healing, and cell signaling among others [51], [52]. Studies suggest that endogenous electrical fields play an important role during the embryonic development [53]. Moreover, the application of an electrical field to embryonic bodies derived from embryonic stem cells (ESCs) resulted in a marked increase of cardiomyocyte differentiation [53]. Wen et al. in an experiment using cardiac myocytes and MSCs, demonstrated that the enhancement of cardiogenesis correlated with an increase in the expression of the protein S100A4 (an early cardiogenesis promoter) [54]. Yamada et al. performed a similar experiment using milder voltage conditions and observed an enhanced differentiation of embryonic bodies into neurons [55]. Therefore, it is important to determine the appropriate combinations of electrical, chemical, and topographical cues that direct stem cell fate to generate a specific phenotype. ECHs combine the conductive properties of electroactive polymers with the high hydration and biocompatibility of hydrogels [56]. The synthesis of ECHs can be achieved by mixing a hydrogel network with a conductive polymer. For example, the hydrogel precursor poly(ethylene glycol) methacrylate (PEGMA) can be supplemented with the conductive polymer polypyrrole (PPy) and photopolymerized to yield a ECHs [56]. The biocompatibility of ECHs based PEGMA and PPy was tested using human muscle fibroblasts and rat pheochromocytoma cells showing no cytotoxicity and supporting cell proliferation [57]. However, the use of ECHs for stem cell culture in vitro and for stem cell delivery in vivo has not been explored. It still needs to be determined if the synthesis process of ECHs can support cell encapsulation and the ability of the hydrogel to gel in situ. VI. CONCLUSION AND FUTURE OUTLOOK Hydrogels offer several advantages to overcome the current limitations during stem cell transplantation. Their biocompatibility, mechanical properties comparable to soft tissue that minimizes foreign material/tissue mismatch, gelling properties, and potential to be chemically modified make them ideal candidates as cell delivery carriers. Despite its potential, there is still a
ALVARADO-VELEZ et al.: HYDROGELS AS CARRIERS FOR STEM CELL TRANSPLANTATION
need to optimize the conditions that favor directed stem cell differentiation, and minimize the host immune response. Better understanding of the signaling pathways that contribute to the survival of the stem cells as well as their contribution to directed differentiation would greatly aid in the development of stem cell therapy. This information on the stem cell “behavior” coupled to the refinement in designing appropriate hydrogel technology is critical for the advancement of this field. In the near future, developments in the various disciplines in the biomedical field such as biomaterials, stem cell biology, and immunology should help in the formulation of hydrogel-based cell replacement technology. Improved hydrogel-encapsulated stem cell methodology will go a long way in the much needed treatment of a number of ailments including neurodegenerative diseases.
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Melissa Alvarado-Velez received the B.S. degree in industrial biotechnology from the University of Puerto Rico-Mayaguez, Puerto Rico. She is a graduate student in the Biomedical Engineering joint program between the Georgia Institute of Technology, Atlanta, USA and Emory University, Atlanta. Along her academic career, she has received various recognitions including the Goizueta Foundation Fellowship and the President’s Fellowship award from the Georgia Institute of Technology. Her research interests include the development of biomaterials that enhance stem cell therapy for traumatic brain injury or other neurodegenerative diseases.
ALVARADO-VELEZ et al.: HYDROGELS AS CARRIERS FOR STEM CELL TRANSPLANTATION
S. Balakrishna Pai received the Ph.D. degree from the Indian Institute of Science, India. He is a Senior Research Scientist in the Neurological Biomaterials and Cancer Therapeutics lab at the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, USA and Emory University School of Medicine, Atlanta. He was a Fogarty Visiting fellow at the National Institutes of Health, USA. He continued his research at the University of Calgary, Canada and at the Yale University School of Medicine, CT, USA. His current research interests include understanding molecular mechanisms in cells including stem cells and developing biomaterial-based solutions to nervous system deficiencies/diseases including cancer.
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Ravi V. Bellamkonda received the B.S. degree in biomedical engineering from Osmania University, Andhra Pradesh, India and the Ph.D. degree in the medical sciences from Brown University, RI, USA. He is the Wallace H. Coulter Chair and Professor in biomedical engineering and a Georgia Research Alliance Distinguished Scholar at Georgia Institute of Technology, Atlanta, USA and Emory University School of Medicine, Atlanta. He is the Director of the Neurological Biomaterials and Cancer Therapeutics Lab at the Coulter Department of Biomedical Engineering. He completed his Postdoctoral Fellowship in the Department of Brain and Cognitive Sciences at Massachusetts Institute of Technology, USA. Prior to joining Georgia Tech, he was an Associate Professor with tenure at Case Western Reserve University, OH, USA. His research interests include the application of principles of regenerative medicine for the repair and regeneration of neural tissue, development of novel electrode designs for neural interfacing, and rational design of nanocarriers for pediatric cancer diagnosis and therapy. Dr. Bellamkonda has received numerous awards including EUREKA Award from National Cancer Institute, CAREER award from National Science Foundation and Best Professor award at GT/BME. He is a Fellow of the Institute of Physics, and is the President-elect and Fellow of AIMBE.
