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1.  Vasculogenic bio-synthetic hydrogel for enhancement of pancreatic islet engraftment and function in type 1 diabetes 
Biomaterials  2013;34(19):4602-4611.
Type 1 diabetes (T1DM) affects one in every 400 children and adolescents in the US. Due to the limitations of exogenous insulin therapy and whole pancreas transplantation, pancreatic islet transplantation has emerged as a promising therapy for T1DM. However, this therapy is severely limited by donor islet availability and poor islet engraftment and function. We engineered an injectable bio-synthetic, polyethylene glycol-maleimide hydrogel to enhance vascularization and engraftment of transplanted pancreatic islets in a mouse model of T1DM. Controlled presentation of VEGF-A and cell adhesive peptides within this engineered material significantly improved the vascularization and function of islets delivered to the small bowel mesentery, a metabolically relevant site for insulin release. Diabetic mice receiving islets transplanted in proteolytically degradable hydrogels incorporating VEGF-A exhibited complete reversal of diabetic hyperglycemia with a 40% reduction in the number of islets required. Furthermore, hydrogel-delivered islets significantly improved weight gain, regulation of a glucose challenge, and intra-islet vascularization and engraftment compared to the clinical standard of islet infusion through the hepatic portal vein. This study establishes a simple biomaterial strategy for islet transplantation to promote enhanced islet engraftment and function.
doi:10.1016/j.biomaterials.2013.03.012
PMCID: PMC3628538  PMID: 23541111
2.  Adhesive Signature-based, Label-free Isolation of Human Pluripotent Stem Cells 
Nature methods  2013;10(5):438-444.
The ability to efficiently isolate undifferentiated human induced pluripotent stem cells (UD-hiPSCs) as colonies from contaminating non-pluripotent cells is a crucial step in the stem cell field to maintain hiPSC survival, purity, and karyotype stability. Here we demonstrate significant differences in ‘adhesive signature’ among UD-hiPSCs, parental cells, partially reprogrammed cells, and differentiated progeny. The distinct adhesive signature of hiPSCs was exploited to rapidly (~10 min) and efficiently isolate fully reprogrammed bona fide hiPSCs as intact colonies from heterogeneous reprogramming cultures and differentiated progeny using microfluidics. hiPSCs were isolated in a label-free fashion and enriched to > 95–99% purity and survival without adversely affecting the transcriptional profile, differentiation potential or karyotype of the pluripotent cells. This rapid and label-free strategy is applicable to isolate UD-hPSCs (hiPSCs, hESCs) from heterogeneous cultures during reprogramming and routine cultures and can be expanded to purify stem cells of specific lineages, such as neurons and cardiomyocytes.
doi:10.1038/nmeth.2437
PMCID: PMC3641175  PMID: 23563795
reprogramming; cell adhesion; fibronectin; integrin; focal adhesion; microfluidics
3.  Minimally Invasive, Longitudinal Monitoring of Biomaterial-Associated Inflammation by Fluorescence Imaging 
Biomaterials  2011;32(31):7785-7792.
Implant-associated inflammation is a major cause for the reduced performance/lifetime and failure of numerous medical devices. Therefore, the ability to non-invasively and quantitatively monitor implant-associated inflammation is critically important. Here we show that implant-associated inflammation can be imaged via fluorescence imaging using near-infrared hydrocyanine dyes delivered either locally or intravenously in living mice. This imaging strategy allowed quantitative longitudinal monitoring of inflammation by detecting reactive oxygen species (ROS) released by inflammatory cells in response to implanted poly(ethylene terephthalate) (PET) disks or injected poly (lactic-co-glycolic acid) (PLGA) microparticles, and exhibited a strong correlation to conventional analysis of inflammation. Furthermore, modulation of inflammatory responses via controlled release of the anti-inflammatory agent dexamethasone was detected using this sensitive imaging approach. Thus, hydrocyanine-based fluorescence imaging of ROS could serve as a surrogate measure for monitoring implant-associated inflammation as well as evaluating the efficacy of therapeutic approaches to modulate host responses to implanted medical devices.
doi:10.1016/j.biomaterials.2011.07.020
PMCID: PMC3159805  PMID: 21813173
Biocompatibility; Foreign body response; Inflammation; Free radical; Superoxide
4.  Runx2 Overexpression in Bone Marrow Stromal Cells Accelerates Bone Formation in Critical-Sized Femoral Defects 
Tissue Engineering. Part A  2010;16(9):2795-2808.
The repair of large nonunions in long bones remains a significant clinical problem due to high failure rates and limited tissue availability for auto- and allografts. Many cell-based strategies for healing bone defects deliver bone marrow stromal cells (BMSCs) to the defect site to take advantage of the inherent osteogenic capacity of this cell type. However, many factors, including donor age and ex vivo expansion of the cells, cause BMSCs to lose their differentiation ability. To overcome these limitations, we have genetically engineered BMSCs to constitutively overexpress the osteoblast-specific transcription factor Runx2. In the present study, we examined Runx2-modified BMSCs, delivered via polycaprolactone scaffolds loaded with type I collagen meshes, in critical-sized segmental defects in rats compared to unmodified cells, cell-free scaffolds, and empty defects. Runx2 expression in BMSCs accelerated healing of critical-sized defects compared to unmodified BMSCs and defects receiving cell-free treatments. These findings provide an accelerated method for healing large bone defects, which may reduce recovery time and the need for external fixation of critical-sized defects.
doi:10.1089/ten.tea.2010.0025
PMCID: PMC2928046  PMID: 20412027
5.  Poly(dimethylsiloxane) Elastomers with Tethered Peptide Ligands for Cell Adhesion Studies 
Acta biomaterialia  2010;6(8):2898-2902.
Poly(dimethylsiloxane) (PDMS) is the choice of material for a wide range of bio- and non-biological applications because of its chemical inertness, non-toxicity, ease of handling, and commercial availability. However, PDMS exhibits uncontrolled protein adsorption and cell adhesion, and it has proven difficult to functionalize to present bioactive ligands. We present a facile strategy for functional surface modification of PDMS using commercial reagents to engineer polymer brushes of oligo(ethylene glycol) methacrylate that prevent cell adhesion and can be functionalized to display bioadhesive ligands. The polymer brushes resist biofouling and prevent cell adhesion, and bioadhesive peptides can be tethered either uniformly or constrained to micropatterned domains using standard peptide chemistry approaches. This approach is relevant to various biomedical and biotechnological applications.
doi:10.1016/j.actbio.2010.02.025
PMCID: PMC2883028  PMID: 20176151
deformable substrate; fibronectin; cell mechanics; focal adhesion
6.  CHRONIC INFLAMMATORY RESPONSES TO MICROGEL-BASED IMPLANT COATINGS 
Inflammatory responses to implanted biomedical devices elicit a foreign body fibrotic reaction that limits device integration and performance in various biomedical applications. We examined chronic inflammatory responses to microgel conformal coatings consisting of thin films of poly(N-isopropylacrylamide) hydrogel microparticles cross-linked with poly(ethylene glycol) diacrylate deposited on poly(ethylene terephthalate) (PET). Unmodified and microgel-coated PET disks were implanted subcutaneously in rats for 4 weeks and explants were analyzed by histology and immunohistochemistry. Microgel coatings reduced chronic inflammation and resulted in a more mature/organized fibrous capsule. Microgel-coated samples exhibited 22% thinner fibrous capsules that contained 40% fewer cells compared to unmodified PET disks. Furthermore, microgel-coated samples contained significantly higher levels of macrophages (40%) than unmodified PET controls. These results demonstrate that microgel coatings reduce chronic inflammation to implanted biomaterials.
doi:10.1002/jbm.a.32669
PMCID: PMC2875296  PMID: 20166218
foreign body response; macrophage; hydrogel; polyethylene terephthalate; fibrous capsule
7.  Near-Infrared Fluorescence Imaging as an Alternative to Bioluminescent Bacteria to Monitor Biomaterial-Associated Infections 
Acta biomaterialia  2014;10(7):2935-2944.
Biomaterial-associated infection is one of the most common complications related with the implantation of any biomedical device. Several in vivo imaging platforms have emerged as powerful diagnostic tools to longitudinally monitor biomaterial-associated infections in small animal models. In this study, we directly compared two imaging approaches: bacteria engineered to produce luciferase to generate bioluminescence and reactive oxygen species (ROS) imaging of the inflammatory response associated with the infected implant. We performed longitudinal imaging of bioluminescence associated with bacteria strains expressing plasmid-integrated luciferase driven by different promoters or a strain with the luciferase gene integrated into the chromosome. These luminescent strains provided adequate signal for acute (0–4 days) monitoring of the infection, but the bioluminescence signal decreased over time and leveled off by 7 days post-implantation. This loss in bioluminescence signal was attributed to changes in the metabolic activity of the bacteria. In contrast, near-infrared fluorescence imaging of ROS associated with inflammation to the implant provided sensitive and dose-dependent signals of biomaterial-associated bacteria. ROS imaging exhibited higher sensitivity than the bioluminescence imaging and was independent of the bacteria strain. Near-infrared fluorescence imaging of inflammatory responses represents a powerful alternative to bioluminescence imaging for monitoring biomaterial-associated bacterial infections.
doi:10.1016/j.actbio.2014.03.005
PMCID: PMC4041799  PMID: 24632360
Biomaterial-associated infection; Bioluminescence; Near infrared Fluorescence; Noninvasive monitoring; Staphylococcus aureus
8.  Bone Regeneration using an Alpha 2 Beta 1 Integrin-Specific Hydrogel as a BMP-2 Delivery Vehicle 
Biomaterials  2014;35(21):5453-5461.
Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.
doi:10.1016/j.biomaterials.2014.03.055
PMCID: PMC4033404  PMID: 24726536
Integrin-specific; Hydrogel; Polyethylene glycol; Bone healing; BMP-2; Protein delivery
9.  Contractility Modulates Cell Adhesion Strengthening Through Focal Adhesion Kinase and Assembly of Vinculin-Containing Focal Adhesions 
Journal of cellular physiology  2010;223(3):746-756.
Actin-myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum-free cultures. Inhibition of myosin light chain kinase or Rho-kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum-induced enhancements in strengthening. Blebbistatin-induced inhibition of myosin II reduced serum-induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin-null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin-dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum-induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK-dependent, vinculin-containing focal adhesion assembly.
doi:10.1002/jcp.22084
PMCID: PMC2874193  PMID: 20205236
actin-myosin; focal adhesion kinase (FAK); vinculin; contractility
10.  Human mesenchymal stem cell differentiation on self-assembled monolayers presenting different surface chemistries 
Acta biomaterialia  2009;6(1):12-20.
Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression, and function during hMSC commitment along osteogenic, chondrogenic, and adipogenic lineages. Using self-assembled monolayers of ω-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long term functional differentiation of adult stem cells and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.
doi:10.1016/j.actbio.2009.07.023
PMCID: PMC2787851  PMID: 19632360
11.  PROFILES OF CARBOHYDRATE LIGANDS ASSOCIATED WITH ADSORBED PROTEINS ON SELF-ASSEMBLED MONOLAYERS OF DEFINED CHEMISTRIES 
Conserved protein-carbohydrate-lipid Pathogen Associated Molecular Patterns (PAMPs) interact with cells of the innate immune system to mediate antigen recognition and internalization and activation of immune cells. We examined if analogous ‘biomaterial associated molecular patterns’ composed of proteins, specifically their carbohydrate modifications, existed on biomaterials which can play a role in mediating the innate immune response to biomaterials. To probe for these carbohydrates in the adsorbed protein layer, as directed by the underlying biomaterial chemistry, self-assembled monolayers (SAMs) presenting –CH3, -OH, -COOH or –NH2 were pre-incubated with serum/plasma, and the presence of carbohydrate ligands of C-type Lectin Receptors (CLRs) was investigated using lectin probes in an Enzyme-linked Lectin Assays (ELLA). Presentation of CLR ligands was detected on control tissue culture polystyrene (TCPS). Absorbances of mannose or N-acetylglucosamine increased with decreasing incubating serum concentration; absorbances of sialylated epitopes or fucose remained unchanged. Absorbances of α-galactose or N-acetylgalactosamine decreased with decreasing incubating serum concentration; β-galactose was undetectable. Among SAM endgroups, pre-incubation with 10% serum resulted in differential presentation of CLR ligands; higher α-galactose on COOH SAMs than NH2 or CH3 SAMs, highest complex mannose on NH2 SAMs and higher complex mannose on OH SAMs than CH3 SAMs. Least sialylated groups were detected on CH3 SAMs. In summary, biomaterial chemistry may regulate protein adsorption and hence unique presentation of associated carbohydrates. The ultimate goal is to identify the effects of protein glycosylations associated with biomaterials in stimulating innate immune responses.
doi:10.1002/jbm.a.32457
PMCID: PMC4453762  PMID: 19353560
self-assembled monolayers; pattern recognition receptors; glycoprotein; enzyme linked lectin assay; innate immune response
12.  Micropatterned Surfaces with Controlled Ligand Tethering 
Microcontact printing (μ-CP) is a facile, cost-effective, and versatile soft-lithography technique to create 2-dimensional patterns of domains with distinct functionalities that provides a robust platform to generate micropatterned biotechnological arrays and cell culture substrates. Current μ-CP approaches rely on non-specific immobilization of biological ligands, either by direct printing or adsorption from solution, onto micropatterned domains surrounded by a non-fouling background. This technique is limited by insufficient control over ligand density. We present a modified μ-CP protocol involving stamping mixed ratios of carboxyl- and tri(ethylene glycol)-terminated alkanethiols that provides for precise covalent tethering of single or multiple ligands to prescribed micropatterns via standard peptide chemistry. Processing parameters were optimized to identify conditions that control relevant endpoint pattern characteristics. This technique provides a facile method to generate micropatterned arrays with tailorable and controlled presentation of biological ligands for biotechnological applications and analyses of cell-material interactions.
doi:10.1002/jbm.a.32140
PMCID: PMC2717189  PMID: 18570314
micropatterning; lithography; microcontact printing; self-assembled monolayers
13.  Simple application of fibronectin-mimetic coating enhances osseointegration of titanium implants 
Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of fibronectin-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically-translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed RGD peptides, serum proteins, and full-length FN. Differences in biological responses correlated with integrin binding specificity and signaling among surface coatings. This work validates a simple, clinically-translatable, surface biofunctionalization strategy to enhance biomedical device integration.
doi:10.1111/j.1582-4934.2008.00476.x
PMCID: PMC2819599  PMID: 18752639
fibronectin; osseointegration; coating; integrins; biomimetic; implant
14.  Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation 
Cell and islet microencapsulation in synthetic hydrogels provide an immunoprotective and cell-supportive microenvironment. A microfluidic strategy for the genaration of biofunctionalized, synthetic microgel particles with precise control over particle size and molecular permeability for cell and protein delivery is presented. These engineered capsules support high cell viability and function of encapsulated human stem cells and islets.
doi:10.1002/adma.201304880
PMCID: PMC4058833  PMID: 24615922
microfluidics; cell microencapsulation; synthetic microgel; pancreatic islets; hydrogels
15.  Vinculin-dependent actin bundling regulates cell migration and traction forces 
The Biochemical journal  2015;465(3):383-393.
Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been limited . In this study, we found mutation (R1049E) of the vinculin tail impairs its ability to bind F-actin, stimulate actin polymerization, and bundle F-actin in vitro. Further , mutant (R1049E) vinculin expressing cells are altered in cell migration, which is accompanied by changes in cell adhesion, cell spreading, and cell generation of traction forces, providing direct evidence for the critical role of vinculin in mechanotransduction at adhesion sites. Lastly, we herein discuss the viability of models detailing the F-actin-binding surface on vinculin in context of our mutational analysis.
doi:10.1042/BJ20140872
PMCID: PMC4418518  PMID: 25358683
vinculin; actin; focal adhesion; force; adhesion
16.  Reduced Acute Inflammatory Responses to Microgel Conformal Coatings 
Biomaterials  2008;29(35):4605-4615.
Implantation of synthetic materials into the body elicits inflammatory host responses that limit medical device integration and biological performance. This inflammatory cascade involves protein adsorption, leukocyte recruitment and activation, cytokine release, and fibrous encapsulation of the implant. We present a coating strategy based on thin films of poly(N-isopropylacrylamide) hydrogel microparticles (i.e. microgels) cross-linked with poly(ethylene glycol) diacrylate. These particles were grafted onto a clinically relevant polymeric material to generate conformal coatings that significantly reduced in vitro fibrinogen adsorption and primary human monocytes/macrophage adhesion and spreading. These microgel coatings also reduced leukocyte adhesion and expression of pro-inflammatory cytokines (TNF-α, IL-1β, MCP-1) in response to materials implanted acutely in the murine intraperitoneal space. These microgel coatings can be applied to biomedical implants as a protective coating to attenuate biofouling, leukocyte adhesion and activation, and adverse host responses for biomedical and biotechnological applications.
doi:10.1016/j.biomaterials.2008.08.015
PMCID: PMC2585524  PMID: 18804859
cell adhesion; cytokine; foreign body response; hydrogel; macrophage; polyethylene terephthalate
17.  Anti-Inflammatory Polymeric Coatings for Implantable Biomaterials and Devices 
Synthetic polymer coatings are used extensively in modern medical devices and implants because of their material versatility and processability. These coatings are designed for specific applications by controlling composition and physical and chemical properties, and they can be formed into a variety of complex structures and shapes. However, implantation of these materials into the body elicits a strong inflammatory host response that significantly limits the integration and biological performance of devices. Biomaterial-mediated inflammation is a complex reaction involving protein adsorption, leukocyte recruitment and activation, secretion of inflammatory mediators, and fibrous encapsulation of the implant. Significant research efforts have focused on modifying material properties using various anti-inflammatory polymeric surface coatings to generate more biocompatible implants. This minireview provides a brief background on the events of biomaterial-mediated inflammation and highlights various approaches used for modifying material surfaces to modulate inflammatory responses. These include both passive and active strategies, such as nonfouling surface treatments and delivery of anti-inflammatory agents, respectively. Novel approaches will be needed to extend the in vivo lifetime and performance of devices and reduce the need for multiple implantation surgeries.
PMCID: PMC2769825  PMID: 19885288
biomaterial; polymer; coating; host response; implant; anti-inflammatory
18.  Mixed Extracellular Matrix Ligands Synergistically Modulate Integrin Adhesion and Signaling 
Journal of cellular physiology  2008;217(2):450-458.
Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling.
doi:10.1002/jcp.21512
PMCID: PMC2574797  PMID: 18613064
collagen; fibronectin; cell adhesion; focal adhesion; integrin
19.  Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation 
Molecular Biology of the Cell  2009;20(9):2508-2519.
Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.
doi:10.1091/mbc.E08-01-0076
PMCID: PMC2675629  PMID: 19297531
20.  The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration 
Biomaterials  2008;29(19):2849-2857.
Implant osseointegration, defined as bone apposition and functional fixation, is a requisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening. Modification of implants to present bioactive motifs such as the RGD cell-adhesive sequence from fibronectin (FN) represents a promising approach in regenerative medicine. However, these biomimetic strategies have yielded only marginal enhancements in tissue healing in vivo. In this study, clinical-grade titanium implants were grafted with a non-fouling oligo(ethylene glycol)-substituted polymer coating functionalized with controlled densities of ligands of varying specificity for target integrin receptors. Biomaterials presenting the α5β1-integrin-specific FN fragment FNIII7–10 enhanced osteoblastic differentiation in bone marrow stromal cells compared to unmodified titanium and RGD-presenting surfaces. Importantly, FNIII7–10-functionalized titanium significantly improved functional implant osseointegration compared to RGD-functionalized and unmodified titanium in vivo. This study demonstrates that bioactive coatings that promote integrin binding specificity regulate marrow-derived progenitor osteoblastic differentiation and enhance healing responses and functional integration of biomedical implants. This work identifies an innovative strategy for the rational design of biomaterials for regenerative medicine.
doi:10.1016/j.biomaterials.2008.03.036
PMCID: PMC2397448  PMID: 18406458
21.  Cadherin-Mediated Cell–Cell Contact Regulates Keratinocyte Differentiation 
Cell–extracellular matrix (ECM) and cell–cell interactions regulate keratinocyte cell fate and differentiation. In the present analysis, we examined the differentiation of primary human keratinocytes cultured on micropatterned substrates that varied the extent of cell–cell contact while maintaining constant cell–ECM areas. Bowtie-shaped micropatterned areas (75–1600 µm2) were engineered to either permit or prevent cell–cell contact for pairs of adherent keratinocytes. Cell pairs with direct cell–cell contact exhibited enhanced expression of the differentiation markers involucrin and keratin 10 compared to cells with no cell–cell contact. In contrast, available cell-spreading area, as regulated by pattern size, did not alter keratinocyte involucrin expression. Disruption of E-cadherin binding by either antibody blocking or expression of a dominant-negative receptor diminished the ability of micropattern-regulated cell–cell contact to modulate involucrin expression. These results demonstrate that cadherin-mediated cell–cell contact regulates early keratinocyte differentiation independently from changes in cell shape.
doi:10.1038/jid.2008.265
PMCID: PMC2693873  PMID: 18754040
22.  Bioadhesive Hydrogel Microenvironments to Modulate Epithelial Morphogenesis 
Biomaterials  2008;29(17):2637-2645.
Epithelial cells polarize and differentiate into organotypic cell aggregates in response to cell-cell and cell-matrix interactions. For example, Madin-Darby Canine Kidney (MDCK) cells form spherical cell aggregates (cysts) with distinct apical and basolateral polarity when cultured 3-dimensionally (embedded) in type I collagen gels. To investigate the effects of individual extracellular factors on epithelial morphogenesis, we engineered fast degrading protease-responsive polyethylene glycol (PEG) hydrogels functionalized with controlled densities of various bioligands (RGD peptide, laminin-1 (LN)) to allow 3-D culturing of MDCK cells, cyst expansion, and morphogenesis/polarization. Cysts formed after 15 days culture in these hydrogels were analyzed with multiphoton fluorescence microscopy for markers of apical and basolateral membrane domains. Epithelial cysts formed in bioadhesive ligand-functionalized PEG gels exhibited a higher frequency of central lumen and interior apical pole formation as well as basolateral polarization compared to unmodified PEG hydrogels. These results demonstrate that incorporation of specific bioadhesive motifs into synthetic hydrogels provides 3-dimensional culture environments that support epithelial morphogenesis. These microenvironments provide a flexible and controlled system for systematic investigations into normal and pathologic morphogenic behaviors as well as synthetic environments for promoting tissue morphogenesis for regenerative medicine applications.
doi:10.1016/j.biomaterials.2008.03.008
PMCID: PMC2408686  PMID: 18377982
cell adhesion; extracellular matrix; differentiation; laminin; RGD
23.  Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding 
Scientific Reports  2015;5:9533.
The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.
doi:10.1038/srep09533
PMCID: PMC4379501  PMID: 25825012
24.  Biomaterial-Mediated Retroviral Gene Transfer Using Self-Assembled Monolayers 
Biomaterials  2007;28(34):5121-5127.
Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH2-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH3-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH2-SAM was further characterized as a function of coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-L-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer.
doi:10.1016/j.biomaterials.2007.07.047
PMCID: PMC2078532  PMID: 17698189
fibroblast; genetic engineering; gene therapy; gene transfer; self-assembly; alkanethiol
25.  Role of Plasma Fibronectin in the Foreign Body Response to Biomaterials 
Biomaterials  2007;28(25):3626-3631.
Host responses to biomaterials control the biological performance of implanted medical devices. Upon implantation, synthetic materials adsorb biomolecules which trigger an inflammatory cascade comprising coagulation, leukocyte recruitment/adhesion, and foreign body reaction. The foreign body reaction and ensuing fibrous encapsulation severely limit the in vivo performance of numerous biomedical devices. While it is well established that plasma fibrinogen and secreted cytokines modulate leukocyte recruitment and maturation into foreign body giant cells, mediators of chronic inflammation and fibrous encapsulation of implanted biomaterials remain poorly understood. Using plasma fibronectin conditional knock-out mice, we demonstrate that plasma fibronectin modulates the foreign body response to polyethylene terephthalate discs implanted subcutaneously. Fibrous collagenous capsules were two-fold thicker in mice depleted of plasma fibronectin compared to controls. In contrast, deletion of plasma fibronectin did not alter acute leukocyte recruitment to the biomaterial, indicating that plasma fibronectin modulates chronic fibrotic responses. The number of foreign body giant cells associated with the implant was three times higher in the absence of plasma fibronectin while macrophage numbers were not different, suggesting that plasma fibronectin regulates the formation of biomaterial-associated foreign body giant cells. Interestingly, cellular fibronectin was present in the capsules of both normal and plasma fibronectin-depleted mice, suggesting that cellular fibronectin could not compensate for the loss of plasma fibronectin. These results implicate plasma fibronectin in the host response to implanted materials and identify a potential target for therapeutic intervention to enhance the biological performance of biomedical devices.
doi:10.1016/j.biomaterials.2007.04.035
PMCID: PMC1950471  PMID: 17521718

Results 1-25 (57)