Publications by authors named "Thomas H Barker"

74 Publications

The Amot/integrin protein complex transmits mechanical forces required for vascular expansion.

Cell Rep 2021 Aug;36(8):109616

Department of Oncology-Pathology, Bioclinicum, Karolinska Institutet, Stockholm 17164, Sweden. Electronic address:

Vascular development is a complex multistep process involving the coordination of cellular functions such as migration, proliferation, and differentiation. How mechanical forces generated by cells and transmission of these physical forces control vascular development is poorly understood. Using an endothelial-specific genetic model in mice, we show that deletion of the scaffold protein Angiomotin (Amot) inhibits migration and expansion of the physiological and pathological vascular network. We further show that Amot is required for tip cell migration and the extension of cellular filopodia. Exploiting in vivo and in vitro molecular approaches, we show that Amot binds Talin and is essential for relaying forces between fibronectin and the cytoskeleton. Finally, we provide evidence that Amot is an important component of the endothelial integrin adhesome and propose that Amot integrates spatial cues from the extracellular matrix to form a functional vascular network.
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http://dx.doi.org/10.1016/j.celrep.2021.109616DOI Listing
August 2021

Extracellular matrix remodeling associated with bleomycin-induced lung injury supports pericyte-to-myofibroblast transition.

Matrix Biol Plus 2021 Jun 30;10:100056. Epub 2020 Dec 30.

Department of Biomedical Engineering, University of Virginia, 415 Lane Road, Charlottesville, VA, United States.

Of the many origins of pulmonary myofibroblasts, microvascular pericytes are a known source. Prior literature has established the ability of pericytes to transition into myofibroblasts, but provide limited insight into molecular cues that drive this process during lung injury repair and fibrosis. Fibronectin and RGD-binding integrins have long been considered pro-fibrotic factors in myofibroblast biology, and here we test the hypothesis that these known myofibroblast cues coordinate pericyte-to-myofibroblast transitions. Specifically, we hypothesized that αvβ3 integrin engagement on fibronectin induces pericyte transition into myofibroblastic phenotypes in the murine bleomycin lung injury model. Myosin Heavy Chain 11 (Myh11)-CreERT2 lineage tracing in transgenic mice allows identification of cells of pericyte origin and provides a robust tool for isolating pericytes from tissues for further evaluation. We used this murine model to track and characterize pericyte behaviors during tissue repair. The majority of Myh11 lineage-positive cells are positive for the pericyte surface markers, PDGFRβ (55%) and CD146 (69%), and display typical pericyte morphology with spatial apposition to microvascular networks. After intratracheal bleomycin treatment of mice, Myh11 lineage-positive cells showed significantly increased contractile and secretory markers, as well as αv integrin expression. According to RNASeq measurements, many disease and tissue-remodeling genesets were upregulated in Myh11 lineage-positive cells in response to bleomycin-induced lung injury. , blocking αvβ3 binding through cycloRGDfK prevented expression of the myofibroblastic marker αSMA relative to controls. In response to RGD-containing provisional matrix proteins present in lung injury, pericytes may alter their integrin profile.
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http://dx.doi.org/10.1016/j.mbplus.2020.100056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233458PMC
June 2021

Synthetic platelet microgels containing fibrin knob B mimetic motifs enhance clotting responses.

Adv Ther (Weinh) 2021 May 18;4(5). Epub 2021 Mar 18.

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Raleigh, NC, USA.

Native platelets are crucial players in wound healing. Key to their role is the ability of their surface receptor GPIIb/IIIa to bind fibrin at injury sites, thereby promoting clotting. When platelet activity is impaired as a result of traumatic injury or certain diseases, uncontrolled bleeding can result. To aid clotting and tissue repair in cases of poor platelet activity, our lab has previously developed synthetic platelet-like particles capable of promoting clotting and improving wound healing responses. These are constructed by functionalizing highly deformable hydrogel microparticles (microgels) with fibrin-binding ligands including a fibrin-specific whole antibody or a single-domain variable fragment. To improve the translational potential of these clotting materials, we explored the use of fibrin-binding peptides as cost-effective, robust, high-specificity alternatives to antibodies. Herein, we present the development and characterization of soft microgels decorated with the peptide AHRPYAAK that mimics fibrin knob 'B' and targets fibrin hole 'b'. These "Fibrin-Affine Microgels with Clotting Yield" (FAMCY) were found to significantly increase clot density and decrease bleeding in a rodent trauma model . These results indicate that FAMCYs are capable of recapitulating the platelet-mimetic properties of previous designs while utilizing a less costly, more translational design.
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http://dx.doi.org/10.1002/adtp.202100010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8171167PMC
May 2021

Decellularized Extracellular Matrix (ECM) as a Model to Study Fibrotic ECM Mechanobiology.

Methods Mol Biol 2021 ;2299:237-261

Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA.

Aberrant deposition of the extracellular matrix (ECM) causes fibrosis and leads to ECM stiffening. This fibrotic ECM provides biological and biophysical stimulations to alter cell activity and drive progression of fibrosis. As an emerging discipline, mechanobiology aims to access the impact of both these cues on cell behavior and relates the reciprocity of mechanical and biological interactions; it incorporates concepts from different fields, like biology and physics, to help study the mechanical and biological facets of fibrosis extensively. A useful experimental platform in mechanobiology is decellularized ECM (dECM), which mimics the native microenvironment more accurately than standard 2D culture techniques as its composition includes similar ECM protein components and stiffness. dECM, therefore, generates more reliable results that better recapitulate in vivo fibrosis.
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http://dx.doi.org/10.1007/978-1-0716-1382-5_18DOI Listing
August 2021

T. gondii infection induces IL-1R dependent chronic cachexia and perivascular fibrosis in the liver and skeletal muscle.

Sci Rep 2020 09 24;10(1):15724. Epub 2020 Sep 24.

Department of Microbiology, Immunology, and Cancer Biology and The Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, VA, USA.

Cachexia is a progressive muscle wasting disease that contributes to death in a wide range of chronic diseases. Currently, the cachexia field lacks animal models that recapitulate the long-term kinetics of clinical disease, which would provide insight into the pathophysiology of chronic cachexia and a tool to test therapeutics for disease reversal. Toxoplasma gondii (T. gondii) is a protozoan parasite that uses conserved mechanisms to infect rodents and human hosts. Infection is lifelong and has been associated with chronic weight loss and muscle atrophy in mice. We have recently shown that T. gondii-induced muscle atrophy meets the clinical definition of cachexia. Here, the longevity of the T. gondii-induced chronic cachexia model revealed that cachectic mice develop perivascular fibrosis in major metabolic organs, including the adipose tissue, skeletal muscle, and liver by 9 weeks post-infection. Development of cachexia, as well as liver and skeletal muscle fibrosis, is dependent on intact signaling through the type I IL-1R receptor. IL-1α is sufficient to activate cultured fibroblasts and primary hepatic stellate cells (myofibroblast precursors in the liver) in vitro, and IL-1α is elevated in the sera and liver of cachectic, suggesting a mechanism by which chronic IL-1R signaling could be leading to cachexia-associated fibrosis.
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http://dx.doi.org/10.1038/s41598-020-72767-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515928PMC
September 2020

Fibroblasts: The arbiters of extracellular matrix remodeling.

Matrix Biol 2020 09 3;91-92:1-7. Epub 2020 Jun 3.

Department of Cellular and Integrative Physiology, Center for Heart and Vascular Research, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA; and Research Service, Nebraska-Western Iowa Health Care System, Omaha, NE 68105; Research Service, Nebraska-Western Iowa Health Care System, Omaha, NE 68105. Electronic address:

Extracellular matrix (ECM) is the foundation on which all cells and organs converge to orchestrate normal physiological functions. In the setting of pathology, the ECM is modified to incorporate additional roles, with modifications including turnover of existing ECM and deposition of new ECM. The fibroblast is center stage in coordinating both normal tissue homeostasis and response to disease. Understanding how fibroblasts work under normal conditions and are activated in response to injury or stress will provide mechanistic insight that triggers discovery of new therapeutic treatments for a wide range of disease. We highlight here fibroblast roles in the cancer, lung, and heart as example systems where fibroblasts are major contributors to homeostasis and pathology.
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http://dx.doi.org/10.1016/j.matbio.2020.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434687PMC
September 2020

Multi-scale models of lung fibrosis.

Matrix Biol 2020 09 11;91-92:35-50. Epub 2020 May 11.

Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

The architectural complexity of the lung is crucial to its ability to function as an organ of gas exchange; the branching tree structure of the airways transforms the tracheal cross-section of only a few square centimeters to a blood-gas barrier with a surface area of tens of square meters and a thickness on the order of a micron or less. Connective tissue comprised largely of collagen and elastic fibers provides structural integrity for this intricate and delicate system. Homeostatic maintenance of this connective tissue, via a balance between catabolic and anabolic enzyme-driven processes, is crucial to life. Accordingly, when homeostasis is disrupted by the excessive production of connective tissue, lung function deteriorates rapidly with grave consequences leading to chronic lung conditions such as pulmonary fibrosis. Understanding how pulmonary fibrosis develops and alters the link between lung structure and function is crucial for diagnosis, prognosis, and therapy. Further information gained could help elaborate how the healing process breaks down leading to chronic disease. Our understanding of fibrotic disease is greatly aided by the intersection of wet lab studies and mathematical and computational modeling. In the present review we will discuss how multi-scale modeling has facilitated our understanding of pulmonary fibrotic disease as well as identified opportunities that remain open and have produced techniques that can be incorporated into this field by borrowing approaches from multi-scale models of fibrosis beyond the lung.
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http://dx.doi.org/10.1016/j.matbio.2020.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434655PMC
September 2020

Spatial-omics: Novel approaches to probe cell heterogeneity and extracellular matrix biology.

Matrix Biol 2020 09 19;91-92:152-166. Epub 2020 May 19.

Department of Biomedical Engineering, 415 Lane Road, University of Virginia, Charlottesville, VA 22903, USA. Electronic address:

Complex intercellular interactions as well as biomolecular and biomechanical cues from the extracellular matrix (ECM) profoundly affect cellular functions. Traditional transcriptomic and proteomic approaches have provided insight into disease progression by identifying discrete cellular subpopulations or microenvironmental signatures characteristic of normal or pathological tissues, however these techniques do not examine how a given cellular state relates to its interactions with neighboring cells or its surrounding ECM with multiparametric characterization (i.e. ECM alignment, mechanical forces, crosslinking, etc.). Emerging spatial-omic techniques can provide high-resolution mapping of expression profiles similar to scRNA-seq and mass spectroscopy directly within tissues. The ability to preserve the spatial context of cells within samples, their cellular geometry, as well as their surrounding ECM gives spatial-omics the opportunity to interrogate previously unexplored signaling modalities, which has the potential to revolutionize ECM research and our understanding of fibrotic diseases. In this review, we present current spatial transcriptomic and proteomic techniques and discuss how they may be applied to investigate cell-ECM interactions.
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http://dx.doi.org/10.1016/j.matbio.2020.04.004DOI Listing
September 2020

Feeling Things Out: Bidirectional Signaling of the Cell-ECM Interface, Implications in the Mechanobiology of Cell Spreading, Migration, Proliferation, and Differentiation.

Adv Healthc Mater 2020 04 9;9(8):e1901445. Epub 2020 Feb 9.

Department of Biomedical Engineering, University of Virginia, 415 Lane Rd. MR5 1225, Charlottesville, VA, 22903, USA.

Biophysical cues stemming from the extracellular environment are rapidly transduced into discernible chemical messages (mechanotransduction) that direct cellular activities-placing the extracellular matrix (ECM) as a potent regulator of cell behavior. Dynamic reciprocity between the cell and its associated matrix is essential to the maintenance of tissue homeostasis and dysregulation of both ECM mechanical signaling, via pathological ECM turnover, and internal mechanotransduction pathways contribute to disease progression. This review covers the current understandings of the key modes of signaling used by both the cell and ECM to coregulate one another. By taking an outside-in approach, the inherent complexities and regulatory processes at each level of signaling (ECM, plasma membrane, focal adhesion, and cytoplasm) are captured to give a comprehensive picture of the internal and external mechanoregulatory environment. Specific emphasis is placed on the focal adhesion complex which acts as a central hub of mechanical signaling, regulating cell spreading, migration, proliferation, and differentiation. In addition, a wealth of available knowledge on mechanotransduction is curated to generate an integrated signaling network encompassing the central components of the focal adhesion, cytoplasm and nucleus that act in concert to promote durotaxis, proliferation, and differentiation in a stiffness-dependent manner.
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http://dx.doi.org/10.1002/adhm.201901445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274903PMC
April 2020

Vendor-specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4 Foxp3 regulatory T cell levels.

Am J Transplant 2019 10 2;19(10):2705-2718. Epub 2019 Aug 2.

Department of Surgery, University of Virginia, Charlottesville, Virginia.

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4 Foxp3 regulatory T cells (T ) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad-spectrum antibiotics decreases T and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3-driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of T in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through T -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period.
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http://dx.doi.org/10.1111/ajt.15523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919421PMC
October 2019

Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion.

Matrix Biol 2019 09 17;82:86-104. Epub 2019 Apr 17.

University of Virginia, Charlottesville, VA, USA. Electronic address:

The extracellular matrix (ECM) microenvironment is increasingly implicated in the instruction of pathologically relevant cell behaviors, from aberrant transdifferentation to invasion and beyond. Indeed, pathologic ECMs possess a panoply of alterations that provide deleterious instructions to resident cells. Here we demonstrate the precise manner in which the ECM protein fibronectin (FN) undergoes the posttranslational modification citrullination in response to peptidyl-arginine deiminase (PAD), an enzyme associated with innate immune cell activity and implicated in systemic ECM-centric diseases, like cancer, fibrosis and rheumatoid arthritis. FN can be citrullinated in at least 24 locations, 5 of which reside in FN's primary cell-binding domain. Citrullination of FN alters integrin clustering and focal adhesion stability with a concomitant enhancement in force-triggered integrin signaling along the FAK-Src and ILK-Parvin pathways within fibroblasts. In vitro migration and in vivo wound healing studies demonstrate the ability of citrullinated FN to support a more migratory/invasive phenotype that enables more rapid wound closure. These findings highlight the potential of ECM, particularly FN, to "record" inflammatory insults via post-translational modification by inflammation-associated enzymes that are subsequently "read" by resident tissue fibroblasts, establishing a direct link between inflammation and tissue homeostasis and pathogenesis through the matrix.
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http://dx.doi.org/10.1016/j.matbio.2019.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168757PMC
September 2019

Thy-1 in Integrin Mediated Mechanotransduction.

Front Cell Dev Biol 2019 25;7:22. Epub 2019 Feb 25.

Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, United States.

The glycosylphosphatidylinositol (GPI) anchored glycoprotein Thy-1 has been prevalently expressed on the surface of various cell types. The biological function of Thy-1 ranges from T cell activation, cell adhesion, neurite growth, differentiation, metastasis and fibrogenesis and has been extensively reviewed elsewhere. However, current discoveries implicate Thy-1 also functions as a key mechanotransduction mediator. In this review, we will be focusing on the role of Thy-1 in translating extracellular mechanic cues into intracellular biological cascades. The mechanotransduction capability of Thy-1 relies on and interaction between Thy-1 and RGD-binding integrins; and will be discussed in depth in the review.
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http://dx.doi.org/10.3389/fcell.2019.00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397864PMC
February 2019

Macrophage and Fibroblast Interactions in Biomaterial-Mediated Fibrosis.

Adv Healthc Mater 2019 02 18;8(4):e1801451. Epub 2019 Jan 18.

School of Biomedical Engineering, Science and Health Systems, Drexel University, 3141 Chestnut Street, Philadelphia, PA, 19104, USA.

Biomaterial-mediated inflammation and fibrosis remain a prominent challenge in designing materials to support tissue repair and regeneration. Despite the many biomaterial technologies that have been designed to evade or suppress inflammation (i.e., delivery of anti-inflammatory drugs, hydrophobic coatings, etc.), many materials are still subject to a foreign body response, resulting in encapsulation of dense, scar-like extracellular matrix. The primary cells involved in biomaterial-mediated fibrosis are macrophages, which modulate inflammation, and fibroblasts, which primarily lay down new extracellular matrix. While macrophages and fibroblasts are implicated in driving biomaterial-mediated fibrosis, the signaling pathways and spatiotemporal crosstalk between these cell types remain loosely defined. In this review, the role of M1 and M2 macrophages (and soluble cues) involved in the fibrous encapsulation of biomaterials in vivo is investigated, with additional focus on fibroblast and macrophage crosstalk in vitro along with in vitro models to study the foreign body response. Lastly, several strategies that have been used to specifically modulate macrophage and fibroblast behavior in vitro and in vivo to control biomaterial-mediated fibrosis are highlighted.
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http://dx.doi.org/10.1002/adhm.201801451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415913PMC
February 2019

αvβ3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis.

JCI Insight 2018 10 18;3(20). Epub 2018 Oct 18.

Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA.

Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvβ3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvβ3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvβ3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvβ3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.
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http://dx.doi.org/10.1172/jci.insight.97597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237458PMC
October 2018

Bacteria-Based Materials for Stem Cell Engineering.

Adv Mater 2018 Oct 12;30(43):e1804310. Epub 2018 Sep 12.

Centre for the Cellular Microenvironment, University of Glasgow, Glasgow, G12 8LT, UK.

Materials can be engineered to deliver specific biological cues that control stem cell growth and differentiation. However, current materials are still limited for stem cell engineering as stem cells are regulated by a complex biological milieu that requires spatiotemporal control. Here a new approach of using materials that incorporate designed bacteria as units that can be engineered to control human mesenchymal stem cells (hMSCs), in a highly dynamic-temporal manner, is presented. Engineered Lactococcus lactis spontaneously colonizes a variety of material surfaces (e.g., polymers, metals, and ceramics) and is able to maintain growth and induce differentiation of hMSCs in 2D/3D surfaces and hydrogels. Controlled, dynamic, expression of fibronectin fragments supports stem cell growth, whereas inducible-temporal regulation of secreted bone morphogenetic protein-2 drives osteogenesis in an on-demand manner. This approach enables stem cell technologies using material systems that host symbiotic interactions between eukaryotic and prokaryotic cells.
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http://dx.doi.org/10.1002/adma.201804310DOI Listing
October 2018

Interdigitated microelectronic bandage augments hemostasis and clot formation at low applied voltage in vitro and in vivo.

Lab Chip 2018 09;18(19):2985-2993

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Hemorrhage or uncontrolled bleeding can arise either due to a medical condition or from a traumatic injury and are typically controlled with the application of a hemostatic agent. Hemostatic agents are currently derived from animal or human products, which carry risks of blood borne infections and immune dysregulation. Therefore, the need exists for novel biomedical therapies not derived from animal or human products to achieve hemostasis. Accordingly, we created an interdigitated microelectronic bandage that applies low voltage electrical stimulation to an injury site, resulting in faster clot formation without excessive heating, accelerated fibrin formation, and hemostasis overall. Our interdigitated microelectronic bandage found fibrin formed 1.5× faster in vitro. In vivo, total cessation of bleeding was 2.5× faster, resulting in 2× less blood loss. Electricity has been used in medical applications such as defibrillation, cauterization, and electrosurgery, but scant research has focused on hemostasis. Here we report a novel surface treatment using an interdigitated microelectronic device that creates rapid hemostasis in both in vitro and in vivo bleeding models with low applied voltages, representing a new and novel class of hemostatic agents that are electrically-based.
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http://dx.doi.org/10.1039/c8lc00573gDOI Listing
September 2018

LEM domain-containing protein 3 antagonizes TGFβ-SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol.

J Biol Chem 2018 10 14;293(41):15867-15886. Epub 2018 Aug 14.

Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908, and

Transforming growth factor-β (TGFβ) signaling through SMAD2/3 is an important driver of pathological fibrosis in multiple organ systems. TGFβ signaling and extracellular matrix (ECM) stiffness form an unvirtuous pathological circuit in which matrix stiffness drives activation of latent TGFβ, and TGFβ signaling then drives cellular stress and ECM synthesis. Moreover, ECM stiffness also appears to sensitize cells to exogenously activated TGFβ through unknown mechanisms. Here, using human fibroblasts, we explored the effect of ECM stiffness on a putative inner nuclear membrane protein, LEM domain-containing protein 3 (LEMD3), which is physically connected to the cell's actin cytoskeleton and inhibits TGFβ signaling. We showed that LEMD3-SMAD2/3 interactions are inversely correlated with ECM stiffness and TGFβ-driven luciferase activity and that LEMD3 expression is correlated with the mechanical response of the TGFβ-driven luciferase reporter. We found that actin polymerization but not cellular stress or LEMD3-nuclear-cytoplasmic couplings were necessary for LEMD3-SMAD2/3 interactions. Intriguingly, LEMD3 and SMAD2/3 frequently interacted in the cytosol, and we discovered LEMD3 was proteolytically cleaved into protein fragments. We confirmed that a consensus C-terminal LEMD3 fragment binds SMAD2/3 in a stiffness-dependent manner throughout the cell and is sufficient for antagonizing SMAD2/3 signaling. Using human lung biopsies, we observed that these nuclear and cytosolic interactions are also present in tissue and found that fibrotic tissues exhibit locally diminished and cytoplasmically shifted LEMD3-SMAD2/3 interactions, as noted Our work reveals novel LEMD3 biology and stiffness-dependent regulation of TGFβ by LEMD3, providing a novel target to antagonize pathological TGFβ signaling.
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http://dx.doi.org/10.1074/jbc.RA118.003658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187619PMC
October 2018

Suppression of TGFβ-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition.

Br J Cancer 2018 05 26;118(10):1359-1368. Epub 2018 Apr 26.

Department of Microbiology, Immunology, and Cancer Biology, The University of Virginia, Charlottesville, VA, 22908, USA.

Background: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy.

Methods: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo.

Results: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFβ-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo.

Conclusions: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.
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http://dx.doi.org/10.1038/s41416-018-0072-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959903PMC
May 2018

Fibroblasts: Diverse Cells Critical to Biomaterials Integration.

ACS Biomater Sci Eng 2018 Apr 13;4(4):1223-1232. Epub 2017 Jun 13.

Department of Biomedical Engineering, University of Virginia, 415 Lane Road, Charlottesville, Virginia 22903, United States.

Fibroblasts are key participants in wound healing and inflammation, and are capable of driving the progression of tissue repair to fully functional tissue or pathologic scar, or fibrosis, depending on the specific mechanical and biochemical cues with which they are presented. Thus, understanding and modulating the fibroblastic response to implanted materials is paramount to achieving desirable outcomes, such as long-term implant function or tissue regeneration. However, fibroblasts are remarkably heterogeneous and can differ vastly in their contributions to regeneration and fibrosis. This heterogeneity exists between tissues and within tissues, down to the level of individual cells. This review will discuss the role of fibroblasts, the pitfalls of describing them as a collective, the specifics of their function, and potential future directions to better understand and organize their highly variable biology.
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http://dx.doi.org/10.1021/acsbiomaterials.7b00244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705602PMC
April 2018

Enhancing clot properties through fibrin-specific self-cross-linked PEG side-chain microgels.

Colloids Surf B Biointerfaces 2018 Jun 2;166:89-97. Epub 2018 Mar 2.

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA; Schmid College of Science and Technology, Chapman University, Orange, CA 92866, USA. Electronic address:

Excessive bleeding and resulting complications are a major cause of death in both trauma and surgical settings. Recently, there have been a number of investigations into the design of synthetic hemostatic agents with platelet-mimicking activity to effectively treat patients suffering from severe hemorrhage. We developed platelet-like particles from microgels composed of polymers carrying polyethylene glycol (PEG) side-chains and fibrin-targeting single domain variable fragment antibodies (PEG-PLPs). Comparable to natural platelets, PEG-PLPs were found to enhance the fibrin network formation in vitro through strong adhesion to the emerging fibrin clot and physical, non-covalent cross-linking of nascent fibrin fibers. Furthermore, the mechanical reinforcement of the fibrin mesh through the incorporation of particles into the network leads to a ∼three-fold decrease of the overall clot permeability as compared to control clots. However, transport of biomolecules through the fibrin clots, such as peptides and larger proteins is not hindered by the presence of PEG-PLPs and the altered microstructure. Compared to control clots with an elastic modulus of 460+/-260 Pa, PEG-PLP-reinforced fibrin clots exhibit higher degrees of stiffness as demonstrated by the significantly increased average Younǵs modulus of 1770 +/±720 Pa, as measured by AFM force spectroscopy. Furthermore, in vitro degradation studies with plasmin demonstrate that fibrin clots formed in presence of PEG-PLPs withstand hydrolysis for 24 h, indicating enhanced stabilization against exogenous fibrinolysis. The entire set of data suggests that the designed platelet-like particles have high potential for use as hemostatic agents in emergency medicine and surgical settings.
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http://dx.doi.org/10.1016/j.colsurfb.2018.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050065PMC
June 2018

The fibrotic tumor stroma.

J Clin Invest 2018 01 2;128(1):16-25. Epub 2018 Jan 2.

Department of Thoracic/Head and Neck Medical Oncology and.

Intratumoral fibrosis results from the deposition of a cross-linked collagen matrix by cancer-associated fibroblasts (CAFs). This type of fibrosis has been shown to exert mechanical forces and create a biochemical milieu that, together, shape intratumoral immunity and influence tumor cell metastatic behavior. In this Review, we present recent evidence that CAFs and tumor cells are regulated by provisional matrix molecules, that metastasis results from a change in the type of stromal collagen cross-link, and that fibrosis and inflammation perpetuate each other through proteolytic and chemotactic mediators released into the tumor stroma. We also discuss aspects of the emerging biology that have potential therapeutic value.
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http://dx.doi.org/10.1172/JCI93554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749516PMC
January 2018

Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability.

Nat Mater 2017 09 7;16(9):953-961. Epub 2017 Aug 7.

Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, California 90095, USA.

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.
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http://dx.doi.org/10.1038/nmat4954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809173PMC
September 2017

Detection of an Integrin-Binding Mechanoswitch within Fibronectin during Tissue Formation and Fibrosis.

ACS Nano 2017 07 12;11(7):7110-7117. Epub 2017 Jul 12.

Department of Biomedical Engineering, University of Virginia , Charlottesville, Virginia 22904, United States.

Fibronectin (Fn) is an extracellular matrix protein that orchestrates complex cell adhesion and signaling through cell surface integrin receptors during tissue development, remodeling, and disease, such as fibrosis. Fn is sensitive to mechanical forces in its tandem type III repeats, resulting in extensive molecular enlongation. As such, it has long been hypothesized that cell- and tissue-derived forces may activate an "integrin switch" within the critical integrin-binding ninth and 10th type III repeats-conferring differential integrin-binding specificity, leading to differential cell responses. Yet, no direct evidence exists to prove the hypothesis nor demonstrate the physiological existence of the switch. We report direct experimental evidence for the Fn integrin switch both in vitro and ex vivo using a scFv engineered to detect the transient, force-induced conformational change, representing an opportunity for detection and targeting of early molecular signatures of cell contractile forces in tissue repair and disease.
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http://dx.doi.org/10.1021/acsnano.7b02755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842356PMC
July 2017

The provisional matrix: setting the stage for tissue repair outcomes.

Matrix Biol 2017 07;60-61:1-4

Department of Bioengineering, Jacob School of Engineering, University of California San Diego, La Jolla, CA. Electronic address:

Since its conceptualization in the 1980s, the provisional matrix has often been characterized as a simple fibrin-containing scaffold for wound healing that supports the nascent blood clot and is functionally distinct from the basement membrane. However subsequent advances have shown that this matrix is far from passive, with distinct compositional differences as the wound matures, and providing an active role for wound remodeling. Here we review the stages of this matrix, provide an update on the state of our understanding of provisional matrix, and present some of the outstanding issues related to the provisional matrix, its components, and their assembly and use in vivo.
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http://dx.doi.org/10.1016/j.matbio.2017.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831186PMC
July 2017

Charge segregation in weakly ionized microgels.

Phys Rev E 2017 Jan 19;95(1-1):012608. Epub 2017 Jan 19.

School of Physics, Georgia Institute of Technology, Atlanta, Georgia 30332-0430, USA.

We investigate microgels synthesized from N-isopropylacrylamide (NIPAM) copolymerized with a large mol% of acrylic acid, finding that when the acid groups are partially ionized at high temperatures, competition between ion-induced swelling and hydrophobic deswelling of poly(NIPAM) chains results in microphase separation. In cross-linked microgels, this manifests as a dramatic decrease in the ratio between the radius of gyration and the hydrodynamic radius to ∼0.2, indicating that almost all the mass of the microgel is concentrated near the particle center. We also observe a concurrent decrease of the polymer network length scale via small-angle neutron scattering, confirming the presence of a dense, deswollen core surrounded by a diffuse, charged periphery. We compare these results to those obtained for a system of charged ultralow-cross-linked microgels; the form factor shows a distinct peak at high q when the temperature exceeds a threshold value. We successfully fit the form factor to theory developed to describe scattering from weakly charged gels in poor solvents, and we tie this behavior to charge segregation in the case of the cross-linked microgels.
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http://dx.doi.org/10.1103/PhysRevE.95.012608DOI Listing
January 2017

Dynamic assembly of ultrasoft colloidal networks enables cell invasion within restrictive fibrillar polymers.

Proc Natl Acad Sci U S A 2017 01 18;114(5):885-890. Epub 2017 Jan 18.

Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908

In regenerative medicine, natural protein-based polymers offer enhanced endogenous bioactivity and potential for seamless integration with tissue, yet form weak hydrogels that lack the physical robustness required for surgical manipulation, making them difficult to apply in practice. The use of higher concentrations of protein, exogenous cross-linkers, and blending synthetic polymers has all been applied to form more mechanically robust networks. Each relies on generating a smaller network mesh size, which increases the elastic modulus and robustness, but critically inhibits cell spreading and migration, hampering tissue regeneration. Here we report two unique observations; first, that colloidal suspensions, at sufficiently high volume fraction (ϕ), dynamically assemble into a fully percolated 3D network within high-concentration protein polymers. Second, cells appear capable of leveraging these unique domains for highly efficient cell migration throughout the composite construct. In contrast to porogens, the particles in our system remain embedded within the bulk polymer, creating a network of particle-filled tunnels. Whereas this would normally physically restrict cell motility, when the particulate network is created using ultralow cross-linked microgels, the colloidal suspension displays viscous behavior on the same timescale as cell spreading and migration and thus enables efficient cell infiltration of the construct through the colloidal-filled tunnels.
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http://dx.doi.org/10.1073/pnas.1607350114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293010PMC
January 2017

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

Acta Biomater 2016 05 4;36:86-98. Epub 2016 Mar 4.

Emory University, Department of Surgery, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA; Atlanta Veterans Affairs Medical Center, Surgical and Research Services, Decatur, GA 30030, USA. Electronic address:

Unlabelled: Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration.

Statement Of Significance: Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.
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http://dx.doi.org/10.1016/j.actbio.2016.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846562PMC
May 2016

Fibrin Network Changes in Neonates after Cardiopulmonary Bypass.

Anesthesiology 2016 05;124(5):1021-31

From the Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, Raleigh, North Carolina (A.C.B.); The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia (R.H., L.H.T., T.H.B.); Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia (L.H.T.); and Department of Anesthesiology, Emory University School of Medicine, Children's Healthcare of Atlanta at Egleston, Atlanta, Georgia (J.D.F., N.A.G.).

Background: Quantitative and qualitative differences in the hemostatic systems exist between neonates and adults, including the presence of "fetal" fibrinogen, a qualitatively dysfunctional form of fibrinogen that exists until 1 yr of age. The consequences of "fetal" fibrinogen on clot structure in neonates, particularly in the context of surgery-associated bleeding, have not been well characterized. Here, the authors examine the sequential changes in clotting components and resultant clot structure in a small sample of neonates undergoing cardiac surgery and cardiopulmonary bypass (CPB).

Methods: Blood samples were collected from neonates (n = 10) before surgery, immediately after CPB, and after the transfusion of cryoprecipitate (i.e., adult fibrinogen component). Clots were formed from patient samples or purified neonatal and adult fibrinogen. Clot structure was analyzed using confocal microscopy.

Results: Clots formed from plasma obtained after CPB and after transfusion were more porous than baseline clots. Analysis of clots formed from purified neonatal and adult fibrinogen demonstrated that at equivalent fibrinogen concentrations, neonatal clots lack three-dimensional structure, whereas adult clots were denser with significant three-dimensional structure. Clots formed from a combination of purified neonatal and adult fibrinogen were less homogenous than those formed from either purified adult or neonatal fibrinogen.

Conclusions: The results of this study confirm that significant differences exist in clot structure between neonates and adults and that neonatal and adult fibrinogen may not integrate well. These findings suggest that differential treatment strategies for neonates should be pursued to reduce the demonstrated morbidity of blood product transfusion.
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http://dx.doi.org/10.1097/ALN.0000000000001058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4837010PMC
May 2016

Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction.

J Cell Biol 2015 Oct;211(1):173-90

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332 Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332

Progressive fibrosis is characterized by excessive deposition of extracellular matrix (ECM), resulting in gross alterations in tissue mechanics. Changes in tissue mechanics can further augment scar deposition through fibroblast mechanotransduction. In idiopathic pulmonary fibrosis, a fatal form of progressive lung fibrosis, previous work has shown that loss of Thy-1 (CD90) expression in fibroblasts correlates with regions of active fibrogenesis, thus representing a pathologically relevant fibroblast subpopulation. We now show that Thy-1 is a regulator of fibroblast rigidity sensing. Thy-1 physically couples to inactive αvβ3 integrins via its RGD-like motif, altering baseline integrin avidity to ECM ligands and also facilitating preadhesion clustering of integrin and membrane rafts via Thy-1's glycophosphatidylinositol tether. Disruption of Thy-1-αvβ3 coupling altered recruitment of Src family kinases to adhesion complexes and impaired mechanosensitive, force-induced Rho signaling, and rigidity sensing. Loss of Thy-1 was sufficient to induce myofibroblast differentiation in soft ECMs and may represent a physiological mechanism important in wound healing and fibrosis.
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http://dx.doi.org/10.1083/jcb.201505007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602038PMC
October 2015

Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells.

Bioconjug Chem 2015 Dec 20;26(12):2336-49. Epub 2015 Oct 20.

Regenerative Bioscience Center, ADS Complex, The University of Georgia , 425 River Road, Athens, Georgia 30602, United States.

Neural stem cells (NSCs) possess great potential for neural tissue repair after traumatic injuries to the central nervous system (CNS). However, poor survival and self-renewal of NSCs after injury severely limits its therapeutic potential. Sulfated chondroitin sulfate glycosaminoglycans (CS-GAGs) linked to CS proteoglycans (CSPGs) in the brain extracellular matrix (ECM) have the ability to bind and potentiate trophic factor efficacy, and promote NSC self-renewal in vivo. In this study, we investigated the potential of CS-GAG hydrogels composed of monosulfated CS-4 (CS-A), CS-6 (CS-C), and disulfated CS-4,6 (CS-E) CS-GAGs as NSC carriers, and their ability to create endogenous niches by enriching specific trophic factors to support NSC self-renewal. We demonstrate that CS-GAG hydrogel scaffolds showed minimal swelling and degradation over a period of 15 days in vitro, absorbing only 6.5 ± 0.019% of their initial weight, and showing no significant loss of mass during this period. Trophic factors FGF-2, BDNF, and IL10 bound with high affinity to CS-GAGs, and were significantly (p < 0.05) enriched in CS-GAG hydrogels when compared to unsulfated hyaluronic acid (HA) hydrogels. Dissociated rat subventricular zone (SVZ) NSCs when encapsulated in CS-GAG hydrogels demonstrated ∼88.5 ± 6.1% cell viability in vitro. Finally, rat neurospheres in CS-GAG hydrogels conditioned with the mitogen FGF-2 demonstrated significantly (p < 0.05) higher self-renewal when compared to neurospheres cultured in unconditioned hydrogels. Taken together, these findings demonstrate the ability of CS-GAG based hydrogels to regulate NSC self-renewal, and facilitate growth factor enrichment locally.
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http://dx.doi.org/10.1021/acs.bioconjchem.5b00397DOI Listing
December 2015
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