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Find video protocols related to scientific articles indexed in Pubmed.
Comparing variable-length polyglutamate domains to anchor an osteoinductive collagen-mimetic Peptide to diverse bone grafting materials.
Int J Oral Maxillofac Implants
PUBLISHED: 11-15-2014
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Allografts, xenografts, and alloplasts are commonly used in craniofacial medicine as alternatives to autogenous bone grafts; however, these materials lack important bone-inducing proteins. A method for enhancing the osteoinductive potential of these commercially available materials would provide a major clinical advance. In this study, a calcium-binding domain, polyglutamate, was added to an osteoinductive peptide derived from collagen type I, Asp-Gly-Glu-Ala (DGEA), to anchor the peptide onto four different materials: freeze-dried bone allograft (FDBA); anorganic bovine bone (ABB); ?-tricalcium phosphate (?-TCP); and a calcium sulfate bone cement (CaSO4). The authors also examined whether peptide binding and retention could be tuned by altering the number of glutamate residues within the polyglutamate domain.
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Microporous dermal-like electrospun scaffolds promote accelerated skin regeneration.
Tissue Eng Part A
PUBLISHED: 03-31-2014
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The goal of this study was to synthesize skin substitutes that blend native extracellular matrix (ECM) molecules with synthetic polymers which have favorable mechanical properties. To this end, scaffolds were electrospun from collagen I (col) and poly(?-caprolactone) (PCL), and then pores were introduced mechanically to promote fibroblast infiltration, and subsequent filling of the pores with ECM. A 70:30 col/PCL ratio was determined to provide optimal support for dermal fibroblast growth, and a pore diameter, 160 ?m, was identified that enabled fibroblasts to infiltrate and fill pores with native matrix molecules, including fibronectin and collagen I. Mechanical testing of 70:30 col/PCL scaffolds with 160 ?m pores revealed a tensile strength of 1.4 MPa, and the scaffolds also exhibited a low rate of contraction (<19%). Upon implantation, scaffolds should support epidermal regeneration; we, therefore, evaluated keratinocyte growth on fibroblast-embedded scaffolds with matrix-filled pores. Keratinocytes formed a stratified layer on the surface of fibroblast-remodeled scaffolds, and staining for cytokeratin 10 revealed terminally differentiated keratinocytes at the apical surface. When implanted, 70:30 col/PCL scaffolds degraded within 3-4 weeks, an optimal time frame for degradation in vivo. Finally, 70:30 col/PCL scaffolds with or without 160 ?m pores were implanted into full-thickness critical-sized skin defects. Relative to nonporous scaffolds or sham wounds, scaffolds with 160 ?m pores induced accelerated wound closure, and stimulated regeneration of healthy dermal tissue, evidenced by a more normal-appearing matrix architecture, blood vessel in-growth, and hair follicle development. Collectively, these results suggest that microporous electrospun scaffolds are effective substrates for skin regeneration.
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Targeting protein kinase CK2 suppresses prosurvival signaling pathways and growth of glioblastoma.
Clin. Cancer Res.
PUBLISHED: 09-13-2013
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Gliomas are the most frequently occurring primary malignancies in the brain, and glioblastoma is the most aggressive of these tumors. Protein kinase CK2 is composed of two catalytic subunits (? and/or ?) and two ? regulatory subunits. CK2 suppresses apoptosis, promotes neoangiogenesis, and enhances activation of the JAK/STAT, NF-?B, PI3K/AKT, Hsp90, Wnt, and Hedgehog pathways. Aberrant activation of the NF-?B, PI3K/AKT, and JAK/STAT-3 pathways is implicated in glioblastoma progression. As CK2 is involved in their activation, the expression and function of CK2 in glioblastoma was evaluated. Experimental Design and
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Tunable delivery of bioactive peptides from hydroxyapatite biomaterials and allograft bone using variable-length polyglutamate domains.
J Biomed Mater Res A
PUBLISHED: 04-02-2013
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Hydroxyapatite (HA) biomaterials and allograft bone are common alternatives to autogenous grafts; however, these materials lack the strong osteoinductive potential of autologous bone. Previous studies have established that polyglutamate domains, which bind selectively to HA, can be engineered onto bioactive peptides as a mechanism for coupling osteoinductive signals onto HA and allograft. In the current investigation, we adapted the polyglutamate approach to tailor delivery of a model collagen-derived peptide, Asp-Gly-Glu-Ala (DGEA), by manipulating the number of glutamates in the HA binding domain. Specifically, DGEA was modified with diglutamate (E2-DGEA), tetraglutamate (E4-DGEA), or heptaglutamate (E7-DGEA), and it was found that initial peptide binding to HA and allograft was significantly enhanced as the number of glutamates increased. We also determined that the rate of release of polyglutamate-DGEA from substrates over a 5-day interval increased proportionally as the number of glutamate residues was decreased. Additionally, we tuned the peptide release rate by creating mixtures of E2-DGEA, E4-DGEA, and E7-DGEA, and observed that release kinetics of the mixtures were distinct from pure solutions of each respective peptide. These collective results suggest that variable-length polyglutamate domains provide an effective mechanism for controlled delivery of osteoregenerative peptides on HA-containing bone graft materials. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
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ST6Gal-I sialyltransferase confers cisplatin resistance in ovarian tumor cells.
J Ovarian Res
PUBLISHED: 02-11-2013
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Platinum drugs, including cisplatin, are a frontline therapeutic in ovarian cancer treatment and acquired resistance to these agents is a major contributor to ovarian cancer morbidity and mortality. In this study a novel glycosylation-dependent mechanism for cisplatin resistance is described. Specifically, cisplatin-induced cell death is blocked by the activity of the ST6Gal-I sialyltransferase. ST6Gal-I modifies specific receptors by adding a negatively charged sialic acid sugar which influences diverse receptor functions. Overexpression of ST6Gal-I is a hallmark of ovarian and other cancers and its expression has been correlated to metastasis and poor prognosis.
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ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines.
Cancer Res.
PUBLISHED: 01-28-2013
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The ST6Gal-I sialyltransferase adds an ?2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here, we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations, we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations.
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Engineering nanocages with polyglutamate domains for coupling to hydroxyapatite biomaterials and allograft bone.
Biomaterials
PUBLISHED: 01-11-2013
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Hydroxyapatite (HA) is the principal constituent of bone mineral, and synthetic HA is widely used as a biomaterial for bone repair. Previous work has shown that polyglutamate domains bind selectively to HA and that these domains can be utilized to couple bioactive peptides onto many different HA-containing materials. In the current study we have adapted this technology to engineer polyglutamate domains into cargo-loaded nanocage structures derived from the P22 bacteriophage. P22 nanocages have demonstrated significant potential as a drug delivery system due to their stability, large capacity for loading with a diversity of proteins and other types of cargo, and ability to resist degradation by proteases. Site-directed mutagenesis was used to modify the primary coding sequence of the P22 coat protein to incorporate glutamate-rich regions. Relative to wild-type P22, the polyglutamate-modified nanocages (E2-P22) exhibited increased binding to ceramic HA disks, particulate HA and allograft bone. Furthermore, E2-P22 binding was HA selective, as evidenced by negligible binding of the nanocages to non-HA materials including polystyrene, agarose, and polycaprolactone (PCL). Taken together these results establish a new mechanism for the directed coupling of nanocage drug delivery systems to a variety of HA-containing materials commonly used in diverse bone therapies.
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ST6Gal-I regulates macrophage apoptosis via ?2-6 sialylation of the TNFR1 death receptor.
J. Biol. Chem.
PUBLISHED: 09-19-2011
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Macrophages play a central role in innate immunity, however mechanisms regulating macrophage survival are not fully understood. Herein we describe a novel apoptotic pathway involving ?2-6 sialylation of the TNFR1 death receptor by the ST6Gal-I sialyltransferase. Variant glycosylation of TNFR1 has not previously been implicated in TNFR1 function, and little is known regarding the TNFR1 glycan composition. To study sialylation in macrophages, we treated U937 monocytic cells with PMA, which stimulates both macrophage differentiation and apoptosis. Interestingly, macrophage differentiation induces ST6Gal-I down-regulation, leading to reduced ?2-6 sialylation of selected receptors. To prevent loss of ?2-6 sialylation, we forced constitutive expression of ST6Gal-I, and found that this strongly inhibited PMA-induced apoptosis. Given that PMA-mediated apoptosis is thought to result from up-regulation of TNF?, which then activates TNFR1, we next evaluated the ?2-6 sialylation of TNFR1. U937 cells with forced ST6Gal-I displayed TNFR1 with elevated ?2-6 sialylation, and this was associated with diminished TNF?-stimulated apoptosis. Correspondingly, removal of ?2-6 sialylation from TNFR1 through either neuraminidase treatment or expression of ST6Gal-I shRNA markedly enhanced TNF?-mediated apoptosis. To confirm the physiologic importance of TNFR1 sialylation, we generated overexpressing ST6Gal-I transgenic mice. Peritoneal macrophages from transgenic lines displayed TNFR1 with elevated ?2-6 sialylation, and these cells were significantly protected against TNF?-stimulated apoptosis. Moreover, greater numbers of thioglycollate-induced peritoneal cells were observed in transgenic mice. These collective results highlight a new mechanism of TNFR1 regulation, and further intimate that loss of ?2-6 sialylation during macrophage differentiation may limit macrophage lifespan by sensitizing cells to TNF?-stimulated apoptosis.
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Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration.
Biomaterials
PUBLISHED: 09-07-2011
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Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.
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Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells.
J. Biol. Chem.
PUBLISHED: 05-05-2011
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The glycosyltransferase, ST6Gal-I, adds sialic acid in an ?2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that ?2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that ?2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that ?2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, ?2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.
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Engineering an antiplatelet adhesion layer on an electrospun scaffold using porcine endothelial progenitor cells.
J Biomed Mater Res A
PUBLISHED: 03-02-2011
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In coronary artery bypass graft interventions, luminal thrombosis is one of the greatest challenges for polymeric grafts with a luminal diameter less than 4 mm. Previously, we reported the fabrication of a highly porous micro/nanofibrous electrospun scaffold and demonstrated the excellent biocompatibility of the scaffolding materials with human endothelial cells. In this study, we explored the engineering of an antithrombotic layer on the scaffolds lumen within 48 h, using peripheral blood-derived porcine endothelial progentior cells (EPC). Flow cytometric results showed that they were CD31(-) but highly CD34(+) and CD105(+) , suggesting the primitive nature of these freshly isolated EPC. The vast majority of EPC readily took up low density lipoprotein, confirming their endothelial phenotype. These EPC also exhibited a strong proliferation capacity on the scaffold for up to 11 days. A confluent layer could be easily engineered within 24 h, which successfully prevented platelet adhesion, a critical step in the cascade of thrombotic events. We concluded that this scaffold could afford a convenient means for the regeneration of a functional cardiovascular endothelium shortly after implantation. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
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Advantages of RGD peptides for directing cell association with biomaterials.
Biomaterials
PUBLISHED: 01-28-2011
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Despite many years of in vitro research confirming the effectiveness of RGD in promoting cell attachment to a wide variety of biomaterials, animal studies evaluating tissue responses to implanted RGD-functionalized substrates have yielded more variable results. The goals of this report are to present some of the reasons why cell culture studies may not always reliably predict in vivo responses, and more importantly, to highlight potential applications that may benefit from the use of RGD peptides.
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Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite.
PLoS ONE
PUBLISHED: 01-14-2011
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The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.
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Emerging role of alpha2,6-sialic acid as a negative regulator of galectin binding and function.
J. Biol. Chem.
PUBLISHED: 12-20-2010
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Galectins are ?-galactoside-binding lectins that regulate diverse cell behaviors, including adhesion, migration, proliferation, and apoptosis. Galectins can be expressed both intracellularly and extracellularly, and extracellular galectins mediate their effects by associating with cell-surface oligosaccharides. Despite intensive current interest in galectins, strikingly few studies have focused on a key enzyme that acts to inhibit galectin signaling, namely ?-galactoside ?2,6-sialyltransferase (ST6Gal-I). ST6Gal-I adds an ?2,6-linked sialic acid to the terminal galactose of N-linked glycans, and this modification blocks galectin binding to ?-galactosides. This minireview summarizes the evidence suggesting that ST6Gal-I activity serves as an "off switch" for galectin function.
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Enhancement of peptide coupling to hydroxyapatite and implant osseointegration through collagen mimetic peptide modified with a polyglutamate domain.
Biomaterials
PUBLISHED: 07-21-2010
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Hydroxyapatite (HA) is a widely-used biomaterial for bone repair due to its high degree of osteoconductivity. However, strategies for improving HA performance by functionalizing surfaces with bioactive factors are limited. In this study, we explored the use of a HA-binding domain (heptaglutamate, "E7") to facilitate coupling of the collagen mimetic peptide, DGEA, to two types of HA-containing materials, solid HA disks and electrospun polycaprolactone matrices incorporating nanoparticulate HA. We found that the E7 domain directed significantly more peptide to the surface of HA and enhanced peptide retention on both materials in vitro. Moreover, E7-modified peptides were retained in vivo for at least two months, highlighting the potential of this mechanism as a sustained delivery system for bioactive peptides. Most importantly, E7-DGEA-coupled HA, as compared with DGEA-HA, enhanced the adhesion and osteoblastic differentiation of mesenchymal stem cells, and also increased new bone formation and direct bone-implant contact on HA disks implanted into rat tibiae. Collectively, these results support the use of E7-DGEA peptides to promote osteogenesis on HA substrates, and further suggest that the E7 domain can serve as a universal tool for anchoring a wide variety of bone regenerative molecules to any type of HA-containing material.
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Aligned bioactive multi-component nanofibrous nanocomposite scaffolds for bone tissue engineering.
Macromol Biosci
PUBLISHED: 01-30-2010
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The ability to mimic the chemical, physical and mechanical properties of the natural extra-cellular matrix is a key requirement for tissue engineering scaffolds to be successful. In this study, we successfully fabricated aligned nanofibrous multi-component scaffolds for bone tissue engineering using electrospinning. The chemical features were mimicked by using the natural components of bone: collagen and nano-hydroxyapatite along with poly[(D,L-lactide)-co-glycolide] as the major component. Anisotropic features were mimicked by aligning the nanofibers using a rotating mandrel collector. We evaluated the effect of incorporation of nano-HA particles to the system. The morphology and mechanical properties revealed that,at low concentrations, nano-HA acted as a reinforcement. However, at higher nano-HA loadings, it was difficult to disrupt aggregations and, hence, a detrimental effect was observed on the overall scaffold properties. Thermal analysis showed that there were slight interactions between the individual components even though the polymers existed as a two-phase system. Preliminary in vitro cell-culture studies revealed that the scaffold supported cell adhesion and spreading. The cells assumed a highly aligned morphology along the direction of fiber orientation. Protein adsorption experiments revealed that the synergistic effect of increased surface area and the presence of nano-HA in the polymer matrix enhanced total protein adsorption. Crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride resulted in improved mechanical properties of the scaffolds and improved degradation stability, under physiological conditions.
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An in vitro regenerated functional human endothelium on a nanofibrous electrospun scaffold.
Biomaterials
PUBLISHED: 01-20-2010
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The capacity of the luminal layer of an electrospun bi-layer scaffold composed of gelatin, elastin, polycaprolactone (PCL), and poliglecaprone (PGC) to promote endothelial regeneration was investigated using human aortic endothelial cells (HAECs). HAECs of different densities were cultured on a thin film of the luminal layer of the scaffold mounted on a cell crown for desired periods. Fluorescent images showed that HAECs formed a mono-layer within 24 h after having successfully adhered to the scaffolds surface. Scanning electron microscopy (SEM) revealed a satisfactory coverage by the HAECs. Death rates of HAECs populations determined by fluorescent staining were below 5% within the initial 3 days while the profile of proliferation exhibited an exponential increase within 11 days as determined by the 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide (MTT) assay. The functionalities of the endothelial mono-layer were probed by ZO-1 staining for tight junction formation, by 6-keto-PGF(1alpha) assay for prostacyclin (PGI(2)) secretion, and by human platelets for its anti-thrombotic capability. The results indicated that the regenerated endothelium possessed normal functions associated with native endothelium. This study suggests that this electrospun bi-layer scaffold is a promising candidate for cardiovascular grafting for its capability of promoting the regeneration of a functional endothelium to prevent blood clotting in small diameter grafts.
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Osteogenic differentiation of human mesenchymal stem cells directed by extracellular matrix-mimicking ligands in a biomimetic self-assembled peptide amphiphile nanomatrix.
Biomacromolecules
PUBLISHED: 09-15-2009
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This study investigated the ability of nanoscale, biomimetic peptide amphiphile (PA) scaffolds inscribed with specific cellular adhesive ligands to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs) without osteogenic supplements. PA sequences were synthesized to mimic the native bone extracellular matrix (ECM), expressing different isolated ligands (i.e., RGDS, DGEA, KRSR). All PAs were presented as self-assembled two-dimensional coatings for the seeded hMSCs. Initial attachment results demonstrated that the different PAs could be individually recognized based on the incorporated adhesive ligands. Long-term studies assessed osteogenic differentiation up to 35 days. The RGDS-containing PA nanomatrix expressed significantly greater alkaline phosphatase activity, indicating the early promotion of osteogenic differentiation. A progressive shift toward osteogenic morphology and positive staining for mineral deposition provided further confirmation of the RGDS-containing PA nanomatrix. Overall, the PA nanomatrix clearly has great promise for directing the osteogenic differentiation of hMSCs without the aid of supplements by mimicking the native ECM, providing an adaptable environment that allows for different adhesive ligands to control cellular behaviors. This research model establishes the beginnings of a new versatile approach to regenerate bone tissues by closely following the principles of natural tissue formation.
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The effect of collagen I mimetic peptides on mesenchymal stem cell adhesion and differentiation, and on bone formation at hydroxyapatite surfaces.
Biomaterials
PUBLISHED: 01-20-2009
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Integrin-binding peptides increase cell adhesion to naive hydroxyapatite (HA), however, in the body, HA becomes rapidly modified by protein adsorption. Previously we reported that, when combined with an adsorbed protein layer, RGD peptides interfered with cell adhesion to HA. In the current study we evaluated mesenchymal stem cell (MSC) interactions with HA disks coated with the collagen-mimetic peptides, DGEA, P15 and GFOGER. MSCs adhered equally well to disks coated with DGEA, P15, or collagen I, and all three substrates, but not GFOGER, supported greater cell adhesion than uncoated HA. When peptide-coated disks were overcoated with proteins from serum or the tibial microenvironment, collagen mimetics did not inhibit MSC adhesion, as was observed with RGD, however neither did they enhance adhesion. Given that activation of collagen-selective integrins stimulates osteoblastic differentiation, we monitored osteocalcin secretion and alkaline phosphatase activity from MSCs adherent to DGEA or P15-coated disks. Both of these osteoblastic markers were upregulated by DGEA and P15, in the presence and absence of differentiation-inducing media. Finally, bone formation on HA tibial implants was increased by the collagen mimetics. Collectively these results suggest that collagen-mimetic peptides improve osseointegration of HA, most probably by stimulating osteoblastic differentiation, rather than adhesion, of MSCs.
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Polyglutamate directed coupling of bioactive peptides for the delivery of osteoinductive signals on allograft bone.
Biomaterials
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Allograft bone is commonly used as an alternative to autograft, however allograft lacks many osteoinductive factors present in autologous bone due to processing. In this study, we investigated a method to reconstitute allograft with osteoregenerative factors. Specifically, an osteoinductive peptide from collagen I, DGEA, was engineered to express a heptaglutamate (E7) domain, which binds the hydroxyapatite within bone mineral. Addition of E7 to DGEA resulted in 9× greater peptide loading on allograft, and significantly greater retention after a 5-day interval with extensive washing. When factoring together greater initial loading and retention, the E7 domain directed a 45-fold enhancement of peptide density on the allograft surface. Peptide-coated allograft was also implanted subcutaneously into rats and it was found that E7DGEA was retained in vivo for at least 3 months. Interestingly, E7DGEA peptides injected intravenously accumulated within bone tissue, implicating a potential role for E7 domains in drug delivery to bone. Finally, we determined that, as with DGEA, the E7 modification enhanced coupling of a bioactive BMP2-derived peptide on allograft. These results suggest that E7 domains are useful for coupling many types of bone-regenerative molecules to the surface of allograft to reintroduce osteoinductive signals and potentially advance allograft treatments.
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Delivery of platelet-derived growth factor as a chemotactic factor for mesenchymal stem cells by bone-mimetic electrospun scaffolds.
PLoS ONE
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The recruitment of mesenchymal stem cells (MSCs) is a vital step in the bone healing process, and hence the functionalization of osteogenic biomaterials with chemotactic factors constitutes an important effort in the tissue engineering field. Previously we determined that bone-mimetic electrospun scaffolds composed of polycaprolactone, collagen I and nanohydroxyapatite (PCL/col/HA) supported greater MSC adhesion, proliferation and activation of integrin-related signaling cascades than scaffolds composed of PCL or collagen I alone. In the current study we investigated the capacity of bone-mimetic scaffolds to serve as carriers for delivery of an MSC chemotactic factor. In initial studies, we compared MSC chemotaxis toward a variety of molecules including PDGF-AB, PDGF-BB, BMP2, and a mixture of the chemokines SDF-1?, CXCL16, MIP-1?, MIP-1?, and RANTES. Transwell migration assays indicated that, of these factors, PDGF-BB was the most effective in stimulating MSC migration. We next evaluated the capacity of PCL/col/HA scaffolds, compared with PCL scaffolds, to adsorb and release PDGF-BB. We found that significantly more PDGF- BB was adsorbed to, and subsequently released from, PCL/col/HA scaffolds, with sustained release extending over an 8-week interval. The PDGF-BB released was chemotactically active in transwell migration assays, indicating that bioactivity was not diminished by adsorption to the biomaterial. Complementing these studies, we developed a new type of migration assay in which the PDGF-BB-coated bone-mimetic substrates were placed 1.5 cm away from the cell migration front. These experiments confirmed the ability of PDGF-BB-coated PCL/col/HA scaffolds to induce significant MSC chemotaxis under more stringent conditions than standard types of migration assays. Our collective results substantiate the efficacy of PDGF-BB in stimulating MSC recruitment, and further show that the incorporation of native bone molecules, collagen I and nanoHA, into electrospun scaffolds not only enhances MSC adhesion and proliferation, but also increases the amount of PDGF-BB that can be delivered from scaffolds.
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Regulation of the metastatic cell phenotype by sialylated glycans.
Cancer Metastasis Rev.
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Tumor cells exhibit striking changes in cell surface glycosylation as a consequence of dysregulated glycosyltransferases and glycosidases. In particular, an increase in the expression of certain sialylated glycans is a prominent feature of many transformed cells. Altered sialylation has long been associated with metastatic cell behaviors including invasion and enhanced cell survival; however, there is limited information regarding the molecular details of how distinct sialylated structures or sialylated carrier proteins regulate cell signaling to control responses such as adhesion/migration or resistance to specific apoptotic pathways. The goal of this review is to highlight selected examples of sialylated glycans for which there is some knowledge of molecular mechanisms linking aberrant sialylation to critical processes involved in metastasis.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.