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  • Anti-apoptotic treatments prevent cartilage degradation after acute trauma to human ankle cartilage. 19332178

    OBJECTIVES: To investigate the effect of anti-apoptotic agents on cartilage degradation after a single impact to ankle cartilage. DESIGN: Ten human normal tali were impacted with the impulse of 1 Ns generating peak forces in the range of 600 N using a 4 mm diameter indenter. Eight millimeter cartilage plugs containing the 4 mm diameter impacted core and a 4 mm adjacent ring were removed and cultured with or without P188 surfactant (8 mg/ml), caspase-3 (10 uM), or caspase-9 (2 uM) inhibitors for 48 h. Results were assessed in the superficial and middle-deep layers immediately after injury at day 0 and at 2, 7 and 14 days after injury by live/dead cell and Tunel assays and by histology with Safranin O/fast green staining. RESULTS: A single impact to human articular cartilage ex vivo resulted in cell death, cartilage degeneration, and radial progression of apoptosis to the areas immediately adjacent to the impact. The P188 was more effective in preventing cell death than the inhibitors of caspases. It reduced cell death by more than 2-fold (P0.05) in the core and by about 30% in the ring in comparison with the impacted untreated control at all time points. P188 also prevented radial expansion of apoptosis in the ring region especially in the first 7 days post-impaction (7.5% Tunel-positive cells vs 46% in the untreated control; P0.01). Inhibitors of caspase-3 or -9 were effective in reducing cell death in the impacted core only at early time points, but were ineffective in doing so in the ring. Mankin score was significantly improved in the P188 and caspase-3 treated groups. CONCLUSIONS: Early intervention with the P188 and caspase-3 inhibitor may have therapeutic potential in the treatment of cartilage defects immediately after injury.
    Document Type:
    Reference
    Product Catalog Number:
    S7101
    Product Catalog Name:
    ApopTag® Plus Peroxidase In Situ Apoptosis Kit

  • Chondrogenesis of synovium-derived mesenchymal stem cells in photopolymerizing hydrogel scaffolds. 20537247

    Recently, tissues adjacent to the wound sites are regarded as a promising therapeutic cell source for curing and repairing purpose. Specifically, therapeutic stem cells have been identified in synovial tissue, a tissue adjacent to articular cartilage. The purpose of this study was to explore therapeutic chondrogenesis with rabbit synovium-derived mesenchymal stem cells (SMSCs) encapsulated in photopolymerized hydrogels. A non-degradable poly(ethylene glycol) diacrylate (PEGDA)-based hydrogel and biodegradable phosphoester-poly(ethylene glycol) (PhosPEG)-based hydrogel were both applied as 3-D scaffolds mediating SMSC chondrogenesis in vitro. The viability of SMSCs in both hydrogels was assessed by fluorescent Live/Dead assay and WST-1 assay. Levels of genes and proteins specific to SMSC chondrogenesis were evaluated by real-time RT-PCR, biochemical analysis and immunohistochemical analysis, respectively. The results demonstrated that SMSCs continue to have a high viability when encapsulated in the hydrogel. By treatment with transforming growth factor (TGF)-beta1 or TGF-beta3, positive SMSC chondrogenesis was successfully achieved in both gels, with the best outcome in the PEGDA system. It can be concluded that both PEGDA and PhosPEG hydrogels are appropriate cell-delivery vehicles for SMSC chondrogenesis. Especially as a biodegradable material, PhosPEG hydrogel displayed great potentials in future applications for articular cartilage regeneration coupling with SMSCs.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8887
    Product Catalog Name:
    Anti-Collagen Type II Antibody, clone 6B3

  • Scaffolds with covalently immobilized VEGF and Angiopoietin-1 for vascularization of engineered tissues. 19800684

    The aim of this study was to engineer a biomaterial capable of supporting vascularization in vitro and in vivo. We covalently immobilized vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang1) onto three-dimensional porous collagen scaffolds using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) chemistry. Over both 3 and 7 days in vitro, seeded endothelial cells (ECs) had increased proliferation on scaffolds with immobilized VEGF and/or Ang1 compared to unmodified scaffolds and soluble growth factor controls. Notably, the group with co-immobilized VEGF and Ang1 showed significantly higher cell number (P=0.0079), higher overall lactate production rate (P=0.0044) and higher overall glucose consumption rate (P=0.0034) at Day 3, compared to its corresponding soluble control for which growth factors were added to culture medium. By Day 7, hematoxylin and eosin, live/dead, CD31, and von Willebrand factor staining all showed improved tube formation by ECs when cultivated on scaffolds with co-immobilized growth factors. Interestingly, scaffolds with co-immobilized VEGF and Ang1 showed increased EC infiltration in the chorioallantoic membrane (CAM) assay, compared to scaffolds with independently immobilized VEGF/Ang1. This study presents an alternative method for promoting the formation of vascular structures, via covalent immobilization of angiogenic growth factors that are more stable than soluble ones and have a localized effect.
    Document Type:
    Reference
    Product Catalog Number:
    ECM595

  • Vibratome sectioning mouse retina to prepare photoreceptor cultures. 25548881

    The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer's solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco's Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10(5) cells/cm(2). The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5316
    Product Catalog Name:
    Anti-Rhodopsin Antibody, clone RET-P1

  • Protective effects of dispersive viscoelastics on corneal endothelial damage in a toxic anterior segment syndrome animal model. 22899758

    Purpose. We evaluated whether viscoelastics have protective effects on the corneal endothelial cell damage in a toxic anterior segment syndrome (TASS) animal model depending on the types of viscoelastics. Methods. A TASS animal model was established with an injection of 0.1 mL o-phthaldehyde solution (0.14%) into the anterior chamber of New Zealand white rabbits. One of two different viscoelastics, 1% sodium hyaluronate (cohesive group) or a 1:3 mixture of 4% chondroitin sulfate and 3% sodium hyaluronate (dispersive group), was injected into the anterior chamber. After five minutes, it was removed using a manual I/A instrument, and then 0.1 mL of o-phthaldehyde solution (0.14%) was injected into the anterior chamber. Damage to corneal endothelial cells was compared between the two groups. Results. The corneal thickness increased quickly in both groups after the disinfectant injection. However, the dispersive group showed relatively mild corneal edema compared to the cohesive group. The mean corneal haze score in the dispersive group also was lower than that of the cohesive group. These partial protective effects of the dispersive viscoelastic were demonstrated by the different findings of a live/dead cell assay, TUNEL staining, and scanning electron microscopy between the two groups. Conclusions. The TASS animal model seems to be a useful means to evaluate corneal endothelial cell damage caused by toxic substances to find ways to protect or reduce endothelial cell damage. Dispersive viscoelastics were shown to have partial protective effects against corneal endothelial cell damage caused by a toxic disinfectant.
    Document Type:
    Reference
    Product Catalog Number:
    S7165
    Product Catalog Name:
    ApopTag® Red In Situ Apoptosis Detection Kit

  • Life and death decisions: secondary complexes and lipid rafts in TNF receptor family signal transduction. 15485624

    Signaling by receptors in the TNF receptor (TNFR) superfamily mediate biological outcomes ranging from inflammation to apoptosis and other forms of programmed cell death. How receptor signaling mediates these divergent responses is just beginning to be understood. Here, we discuss how receptor submembrane localization and the formation of alternate signaling complexes can alter the fate of cells stimulated through TNFRs with a death domain, also known as "death receptors."
    Document Type:
    Reference
    Product Catalog Number:
    16-217

  • A novel gellan gel-based microcarrier for anchorage-dependent cell delivery. 18434266

    Competent vehicles are highly sought after as a means to transplant cells for tissue regeneration. In this study, novel hydrogel-based microspherical cell carriers are designed and developed with an FDA-approved natural polysaccharide, gellan gum. The bulk fabrication of these microspheres is performed via a water-in-oil (W/O) emulsion process followed by a series of redox (oxidation-reduction) crosslinking treatments; this enables the microspherical dimensions to be precisely manipulated in terms of injectability, and simultaneously ensures the structural stability. To acquire adhesion affinity with anchorage-dependent cells (ADCs), a covalent coating of gelatin is further applied on the microspherical surfaces. The final product is constructed as a variety of gelatin-grafted-gellan microspherical cell carriers, abbreviated as TriG microcarriers. The cell-loading tests are conducted, respectively, with human dermal fibroblasts (HDFs) and human fetal osteoblasts (hFOBs). Morphological observation from optical microscopy and field emission scanning electron microscopy indicates that the HDFs spread well and populate rapidly on surfaces of TriG microcarriers. Immunofluorescent staining reveals the activation of focal adhesion and subsequent organization of F-actin from the attached cell surfaces, which suggests the TriG microspherical substrate is favorable to ADC adhesion and therefore capable of promoting HDF proliferation to achieve confluence by turning over three times within 10 days. The hFOBs are also cultivated on the TriG carriers, where ideal viability and clear potentials for osteogenesis are demonstrated by fluorescent Live/Dead screening and specific histobiochemical indications. All these findings suggest that the TriG microcarriers are suitable to provide open platforms for therapeutic ADC proliferation and differentiation.
    Document Type:
    Reference
    Product Catalog Number:
    FAK100
    Product Catalog Name:
    Actin Cytoskeleton / Focal Adhesion Staining Kit

  • Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing. 21724858

    After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.
    Document Type:
    Reference
    Product Catalog Number:
    FAK100
    Product Catalog Name:
    Actin Cytoskeleton / Focal Adhesion Staining Kit

  • Noradrenaline activation of neurotrophic pathways protects against neuronal amyloid toxicity. 20132474

    Degeneration of locus coeruleus (LC) noradrenergic forebrain projection neurons is an early feature of Alzheimer's disease. The physiological consequences of this phenomenon are unclear, but observations correlating LC neuron loss with increased Alzheimer's disease pathology in LC projection sites suggest that noradrenaline (NA) is neuroprotective. To investigate this hypothesis, we determined that NA protected both hNT human neuronal cultures and rat primary hippocampal neurons from amyloid-beta (Abeta(1-42) and Abeta(25-35)) toxicity. The noradrenergic co-transmitter galanin was also effective at preventing Abeta-induced cell death. NA inhibited Abeta(25-35)-mediated increases in intracellular reactive oxygen species, mitochondrial membrane depolarization, and caspase activation in hNT neurons. NA exerted its neuroprotective effects in these cells by stimulating canonical beta(1) and beta(2) adrenergic receptor signaling pathways involving the activation of cAMP response element binding protein and the induction of endogenous nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Treatment with functional blocking antibodies for either NGF or BDNF blocked NA's protective actions against Abeta(1-42) and Abeta(25-35) toxicity in primary hippocampal and hNT neurons, respectively. Taken together, these data suggest that the neuroprotective effects of noradrenergic LC afferents result from stimulating neurotrophic NGF and BDNF autocrine or paracrine loops via beta adrenoceptor activation of the cAMP response element binding protein pathway.
    Document Type:
    Reference
    Product Catalog Number:
    AB1779SP
    Product Catalog Name:
    Anti-Brain Derived Neurotrophic Factor Antibody

  • An islet-stabilizing implant constructed using a preformed vasculature. 18333795

    Islet transplantation for the purpose of treating insulin-sensitive diabetes is currently limited by several factors, including islet survival posttransplantation. In the current study, a tissue-engineered prevascularized pancreatic encapsulating device (PPED) was developed. Isolated islets were placed in collagen gels, and they exhibited fourfold more insulin release than islets not in collagen. The insulin released by beta-cells in islets encapsulated in collagen exhibited unobstructed diffusion within the collagen gels. Subsequent studies evaluated the ability to create a sandwich comprised of two layers of prevascularized collagen gels around a central collagen gel containing islets. In vitro characterization of the islets showed that islets are functional and responded to glucose stimulation. The PPEDs were implanted subcutaneously into severe combined immunodeficient mice. Islet survival was assessed after 7, 14, and 28 days. Immunohistochemical analysis was performed on the implants to detect insulin and the presence of intraislet endothelial cells. At all time points, insulin was localized in association with intact and partially dissociated islets. Moreover, cells that exhibited insulin staining were colocalized with intraislet endothelial cells. These data indicate that the PPED enhances islet survival by supporting islet viability and maintaining intraislet endothelial cell structures.
    Document Type:
    Reference
    Product Catalog Number:
    RI-13K
    Product Catalog Name:
    Rat Insulin RIA