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  • Fibroblast growth factor-2-overexpressing myoblasts encapsulated in alginate spheres increase proliferation, reduce apoptosis, induce adipogenesis, and enhance regenerati ... 21815803

    The fibroblast growth factor 2 (FGF-2) is known as pleiotropic cytokine with myoblast proliferative properties. In the present study, we tested the hypothesis that gene transfer of human FGF-2 via transplantation of genetically modified L8-myoblast encapsulated in alginate modulates the skeletal muscle recovery after crush injury in Wistar rats. Therefore, we performed a crush injury to the soleus muscle and transplanted alginate spheres containing myoblasts genetically modified to overexpress human FGF-2 (FGF-2) or a luciferase (LUC) cDNA at the site of injury. Animals that underwent muscle injury without transplantation of alginate spheres served as control (control). At day 4 after trauma the FGF-2 group showed significant higher mean values of cell proliferation (bromodeoxyuridine immunohistochemistry) and significant lower values of cell apoptosis (terminal deoxynucleotidyl transferase nick end labeling histology) compared to animals receiving luciferase-overexpressing myoblasts. At the same time point adiponectin expression (ACRP30 immunohistochemistry) was increased in the FGF-2 group exclusively. The p75(NTR) expression (p75(NTR) immunohistochemistry) significantly improved in both the FGF-2 and LUC group compared to the control group. Functional analysis of the injured muscle did not reveal a significant increase of the muscle force in the FGF-2 group compared to the control and LUC group 14 days after injury. In vitro analysis for 14 days of the FGF-2-modified spheres demonstrated at day 7 and day 14 a significant increase of the relative cell count as well as of the relative viable cell count in the FGF-2 myoblast spheres compared to luciferase myoblast spheres. Additionally, the expression of FGF-2 (enzyme-linked immunosorbent assay analysis) and luciferase (chemiluminescence analysis) persisted in vitro for 4 and 14 days, respectively. These results demonstrate that FGF-2-overexpressing myoblasts cannot considerably improve muscle strength but are able to modulate the proliferation as well as the apoptosis of injured muscle tissue mainly by conducting adipogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    S7100
    Product Catalog Name:
    ApopTag® Peroxidase In Situ Apoptosis Detection Kit

  • Assembly of multilayer PSS/PAH membrane on coherent alginate/PLO microcapsule for long-term graft transplantation. 18286625

    Conventional alginate/poly-L-ornithine (AP) membranes used to immunoisolate foreign tissue transplants fail in long-term transplantations of immortal cell lines. We have developed a novel layer-by-layer (LbL) membrane using polystyrene sulfonate and polyallylamine hydrochloride (PSS/PAH) on top of the coherent AP membrane. Assembly of the LbL membrane was followed by electrophoresis, and the surface morphologies and structure were characterized and examined by cryo-scanning electron microscope and transmission electron microscopy. Unlike the standard AP membrane, the LbL membrane withstood the internal pressure generated by continuous cell proliferation of microencapsulated HEK-293 and Min-6 cells. The new membrane did not affect insulin secretion or diffusion by Min-6 cells.
    Document Type:
    Reference
    Product Catalog Number:
    SRI-13K
    Product Catalog Name:
    Sensitive Rat Insulin RIA

  • Neocartilage from human mesenchymal stem cells in alginate: implied timing of transplantation. 16013059

    Previous reports have demonstrated the suitability of alginate microencapsulation for chondrogenesis of human mesenchymal stem cells (MSCs) in vitro. This study examined the MSCs-alginate constructs that were transplanted beneath the dorsal skin of nude mice for 8 weeks after a variety of in vitro culture periods. The in vitro culture had great effects on gross morphology and histological characteristics of transplants. The integrity of alginate of transplants increased as the in vitro culture period increased. Transplants were characterized by an opaque and yellowish color, fair burnish, a firm to elastic texture, but without any evidence of calcification spots. Histological findings agreed with the clinical determination of hyaline cartilage, characterized by isolated cells with basophilic ground substance positive in Safranin-O staining and collagen type II immunohistochemistry. Transplants with exposure to TGF-beta1 for more than 2 weeks before transplantation, lost burnish, were flexible in texture, and had an increased formation of calcification spots. Accordingly, 1-week exposure to TGF-beta1 in vitro before transplantation is appropriate for neocartilage formation of human MSCs in alginate. These findings suggested that regeneration using cell therapy or tissue engineering should assist in ascertaining the optimal timing of transplantation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1330
    Product Catalog Name:
    Anti-Collagen Type II Antibody, clone COLL-II

  • Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in self-gelling alginate discs reveals novel chondrogenic signature gene clusters. 21087199

    We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants.
    Document Type:
    Reference
    Product Catalog Number:
    AB5535
    Product Catalog Name:
    Anti-Sox9 Antibody

  • Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. 12522814

    Mesenchymal stem cells (MSCs) have the capacity for self-renewal and can form bone, fat, and cartilage. Alginate forms a viscous solution when dissolved in 0.9% saline and gels on contact with divalent cations. The viability and phenotype maintenance of chondrocytes in alginate beads have been well documented. However, little is known about the effect of microencapsulation in alginate on chondrogenesis of MSCs. In this study, human MSCs encapsulated in alginate beads were cultured in serum-free medium with the addition of transforming growth factor (TGF)beta1 (10 ng/mL), dexamethasone (10(-7) M), and ascorbate 2-phosphate (50 microg/mL). The MSCs in alginate assumed a rounded morphology with lacunae around them after 1 week in culture. Cell aggregates were observed at 2 weeks or longer in culture. Histological findings agreed with the clinical determination of hyaline cartilage, characterized by isolated cells with ground substance positive in Safranin-O staining and immunohistochemistry for collagen type II at the periphery of cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the expression of COL2A1 and COL10A1, marker of chondrocytes and hypertrophy chondrocytes, respectively. These results indicate MSCs in alginate can form cartilage and the MSCs-alginate system represents a relevant model for the study of the molecular mechanisms involved in the chondrogenesis and endochondral ossification.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1330
    Product Catalog Name:
    Anti-Collagen Type II Antibody, clone COLL-II

  • Hydrogel effects on bone marrow stromal cell response to chondrogenic growth factors. 17257670

    The aim of this study was to investigate the effects of alginate and agarose on the response of bone marrow stromal cells (BMSCs) to chondrogenic stimuli. Rat BMSCs were expanded in monolayer culture with or without FGF-2 supplementation. Cells were then seeded in 2% alginate and agarose gels and cultured in media with or without TGF-beta1 or dexamethasone (Dex). Sulfated glycosaminoglycans (sGAGs), collagen type II, and aggrecan were expressed in all groups that received TGF-beta1 treatment during hydrogel culture. Expansion of rat BMSCs in the presence of FGF-2 increased production of sGAG in TGF-beta1-treated groups over those cultures that were treated with TGF-beta1 alone in alginate cultures. However, in agarose, cells exposed to FGF-2 during expansion produced less sGAG within TGF-beta1-supplemented groups over those cultures treated with TGF-beta1 alone. Dex was required for optimal matrix synthesis in both hydrogels, but was found to decrease cell viability in agarose constructs. These results indicate that the response of BMSCs to a chondrogenic growth factor regimen is scaffold dependent.
    Document Type:
    Reference
    Product Catalog Number:
    AB1031
    Product Catalog Name:
    Anti-Aggrecan Antibody

  • A new method of selecting Schwann cells from adult mouse sciatic nerve. 15970332

    We describe a method of using laminin for the selection and purification of Schwann cells in vitro. We also studied the viability of the selected cells suspended in alginate beads both in vitro and in vivo. We observed that the homogeneity of the Schwann cell culture increased with each round of laminin selection and reached 85-90% after five passages. The viability of cells after incubation within an alginate bead in vivo was between 73 and 76% compared with greater than 90% viability for cells that were maintained in monolayer culture. This new method of serial selection using laminin-coated surfaces has optimized the purification of a Schwann cell culture expanded from cells harvested from the adult sciatic nerve of a mouse. This method has the advantage of being technically easier than other methods described and results in a Schwann cell culture that is 80-90% homogenous.
    Document Type:
    Reference
    Product Catalog Number:
    PP40
    Product Catalog Name:
    IgG, Goat

  • Preservation of the chondrocyte's pericellular matrix improves cell-induced cartilage formation. 20213765

    The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real-time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross-linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>>50-fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell-induced cartilage production.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1330
    Product Catalog Name:
    Anti-Collagen Type II Antibody, clone COLL-II

  • Tailoring secretion of proteoglycan 4 (PRG4) in tissue-engineered cartilage. 16846341

    Articular cartilage provides a low-friction surface for joint articulation, with boundary lubrication facilitated by proteoglycan 4 (PRG4), which is secreted by chondrocytes of the superficial zone. Chondrocytes from different zones are phenotypically distinct, and their phenotypes in vitro are influenced by the system in which they are cultured. We hypothesized that culturing cells from the superficial (S) zone in two-dimensional monolayer or three-dimensional alginate would affect their synthesis of PRG4, and that subsequently seeding them atop alginate-recovered cells from the middle/ deep (M) zone in various proportions would result in tissue-engineered constructs with varying levels of PRG4 secretion and matrix accumulation. During monolayer culture, S cells retained their PRG4-secreting phenotype, whereas in alginate culture the percentage of cells secreting PRG4 decreased with time. Constructs formed with increasing percentages of S cells decreased in thickness and matrix accumulation, depending on both the culture conditions before construct formation and the S-cell density. PRG4-secreting cells were localized to the S-cell seeded construct surface, with secretion rates of 0.1-4 pg/cell/day or 0.1-1 pg/cell/day for constructs formed with monolayer-recovered or alginate-recovered S cells, respectively. Tailoring secretion of PRG4 in cartilage constructs may be useful for enhancing low-friction properties at the articular surface, while maintaining other surfaces free of PRG4 for enhancing integration with surrounding tissues.
    Document Type:
    Reference
    Product Catalog Number:
    AB2200
    Product Catalog Name:
    Anti-PRG4 Antibody

  • Resveratrol and N-acetylcysteine influence redox balance in equine articular chondrocytes under acidic and very low oxygen conditions. 25998424

    Mature articular cartilage is an avascular tissue characterized by a low oxygen environment. In joint disease, acidosis and further reductions in oxygen levels occur, compromising cartilage integrity.This study investigated how acidosis and very low oxygen levels affect components of the cellular redox system in equine articular chondrocytesand whether the antioxidants resveratrol and N-acetylcysteine could modulate this system. We used articular chondrocytes isolated from nondiseased equine joints and cultured them in a 3-D alginate bead system for 48h in less than 1, 2, 5, and 21% O2 at pH 7.2 or 6.2 in the absence or presence of the proinflammatory cytokine, interleukin-1β (10ng/ml).In addition, chondrocytes were cultured with resveratrol (10µM) or N-acetylcysteine (NAC) (2mM).Cell viability, glycosaminoglycan (GAG) release, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), GSH:GSSG ratio, and SOD1 and SOD2 protein expression were measured. Very low levels of oxygen (less than 1%), acidosis (pH 6.2), and exposure to IL-1β led to reductions in cell viability, increased GAG release, alterations in ΔΨm and ROS levels, and reduced GSH:GSSG ratio. In addition, SOD1 and SOD2 protein expressions were reduced. Both resveratrol and NAC partially restored ΔΨm and ROS levels and prevented GAG release and cell loss and normalized SOD1 and SOD2 protein expression. In particular NAC was highly effective at restoring the GSH:GSSG ratio.These results show that the antioxidants resveratrol and N-acetylcysteine can counteract the redox imbalance in articular chondrocytes induced by low oxygen and acidic conditions.
    Document Type:
    Reference
    Product Catalog Number:
    AB5535
    Product Catalog Name:
    Anti-Sox9 Antibody