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A R Poole - One of the best experts on this subject based on the ideXlab platform.
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chondrogenesis in periosteal explants an organ culture model for in vitro study
Journal of Bone and Joint Surgery American Volume, 1994Co-Authors: Shawn W Odriscoll, A D Recklies, A R PooleAbstract:ABSTRACT Periosteal grafts have chondrogenic potential and have been used to repair defects in articular cartilage. We studied the effects of the culture conditions and of transforming growth factor-beta 1 on chondrogenesis in rabbit periosteal explants that were cultured in vitro. A total of 390 periosteal explants were obtained from the anteromedial sides of the proximal parts of the tibiae of eleven rabbits that were two weeks, two months, or six months old.The culture medium (alpha minimum essential medium or Dulbecco minimum essential medium) contained fetal calf serum, with or without transforming growth factor-beta 1, at a concentration of one or ten nanograms per milliliter for the first two weeks of culture. Three hundred and twenty-one explants were submerged in liquid medium and sixty-nine were suspended in an agarose gel; they were then evaluated histochemically, histomorphometrically, and by collagen-typing. In the media without agarose, in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter, chondrogenesis was commonly seen after two to four weeks with use of safranin-O staining and histomorphometry. In the agarose gels, chondrogenesis from the periosteum was observed at four and six weeks and was enhanced by the presence of one or ten nanograms of transforming growth factor-beta 1 per milliliter. The combination of agarose with transforming growth factor-beta 1 most favored the formation of cartilage, which was maximum at six weeks in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter. Under these conditions, chondrogenesis occurred in almost every explant, with 50 +- 30 per cent of the tissue being composed of cartilage. Type-II collagen was present in the explants that had undergone chondrogenesis. The finding that transforming growth factor-beta 1 can induce chondrogenesis in the periosteum, particularly when the explant had been cultured in an agarose gel, confirms the potential of the periosteum to form cartilage. With use of in vitro methodology to define the optimum conditions that enhance chondrogenesis, it should be possible to elicit chondrogenesis when periosteal grafts are used to repair cartilage defects in vivo.
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chondrogenesis in periosteal explants an organ culture model for in vitro study
Journal of Bone and Joint Surgery American Volume, 1994Co-Authors: Shawn W Odriscoll, A D Recklies, A R PooleAbstract:Periosteal grafts have chondrogenic potential and have been used to repair defects in articular cartilage. We studied the effects of the culture conditions and of transforming growth factor-beta 1 on chondrogenesis in rabbit periosteal explants that were cultured in vitro. A total of 390 periosteal explants were obtained from the anteromedial sides of the proximal parts of the tibiae of eleven rabbits that were two weeks, two months, or six months old. The culture medium (alpha minimum essential medium or Dulbecco minimum essential medium) contained fetal calf serum, with or without transforming growth factor-beta 1, at a concentration of one or ten nanograms per milliliter for the first two weeks of culture. Three hundred and twenty-one explants were submerged in liquid medium and sixty-nine were suspended in an agarose gel; they were then evaluated histochemically, histomorphometrically, and by collagen-typing. In the media without agarose, in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter, chondrogenesis was commonly seen after two to four weeks with use of safranin-O staining and histomorphometry. In the agarose gels, chondrogenesis from the periosteum was observed at four and six weeks and was enhanced by the presence of one or ten nanograms of transforming growth factor-beta 1 per milliliter. The combination of agarose with transforming growth factor-beta 1 most favored the formation of cartilage, which was maximum at six weeks in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter. Under these conditions, chondrogenesis occurred in almost every explant, with 50 +/- 30 per cent of the tissue being composed of cartilage. Type-II collagen was present in the explants that had undergone chondrogenesis.
Fernando F Costa - One of the best experts on this subject based on the ideXlab platform.
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beta spectrin campinas a novel shortened beta chain variant associated with skipping of exon 30 and hereditary elliptocytosis
British Journal of Haematology, 1997Co-Authors: Daniela S Basseres, Fernando F Costa, Patricia Pranke, Tereza Sueko Ide Sales, Sara T O SaadAbstract:Beta-Spectrin Campinas is a novel spectrin variant associated with a shortened beta-chain in a kindred with hereditary elliptocytosis (HE). The propositus and her mother exhibited increased amounts of spectrin dimers and an increase in the alphaI 74 kD fragment from the alpha-chain after partial tryptic digestion of spectrin. The shortened beta-chain appeared as an additional band of approximately 200 kD on SDS-PAGE. In order to delineate the molecular defect of this abnormality at the gene level, reticulocyte mRNA was transcribed into cDNA and the last four exons of the beta-spectrin gene were amplified. Agarose gel of the amplification product of the propositus revealed the expected band of 487 bp as well as a shortened band of approximately 300 bp (size determined on gel). This shortened cDNA amplification product was cloned and nucleotide sequencing revealed the absence of the entire exon 30. In order to determine the underlying mutation responsible for this abnormal splicing, a genomic DNA fragment containing exons 30 and 31 was amplified and nucleotide sequencing revealed a G-->A substitution at the 5' donor splice site consensus sequence of intron 30 (nt + 1 IVS30). The skip splicing observed in this study results in a frameshift, creating a new stop codon and causing a deletion of 129 amino acids at the very COOH-terminus of the protein, thus impairing spectrin dimers self-association. We classified this HE as spherocytic HE because the propositus presented a few spherocytes in addition to many elliptocytes in the blood smear, whereas her mother, who was splenectomized, showed many schizocytes, poikilocytes and spherocytes.
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a splice site mutation of the beta spectrin gene causing exon skipping in hereditary elliptocytosis associated with a truncated beta spectrin chain
Journal of Biological Chemistry, 1991Co-Authors: Patrick G Gallagher, William T Tse, Fernando F Costa, Alphonse L Scarpa, Pierre Boivin, J Delaunay, Bernard G ForgetAbstract:Abstract We studied a French kindred with hereditary elliptocytosis associated with a spectrin variant (spectrin LePuy) containing a beta-spectrin chain that is truncated at its C terminus (Dhermy, D., Lecomte, M., Garbarz, M., Bournier, O., Galand, C., Gautero, H., Feo, C., Alloisio, N., Delaunay, J., and Boivin, P. (1982) J. Clin. Invest. 70, 707-715). The structure of the 3' end of the beta-spectrin gene, the region encoding the C terminus of beta-spectrin, was determined. Nucleotide sequencing of amplified genomic DNA revealed a mutation at position +4 (A----G) of the 5' donor consensus splice site of the intron following the third-to-last exon (exon X) in one beta-spectrin allele of a heterozygous patient. Agarose gel electrophoresis of polymerase chain reaction-amplified cDNA revealed an extra band of lower molecular weight, suggesting that the shortened beta-spectrin chain of spectrin LePuy arises from aberrant mRNA splicing. Nucleotide sequencing of the shorter cDNA amplification product revealed that the sequences encoding exon X were absent. Southern blotting of cDNA amplification products confirmed this result. The skipping of exon X causes a shift in the normal reading frame resulting in the encoding of a new amino acid sequence at the C terminus of the mutant beta-spectrin chain. A new in-frame stop codon is encountered following a single residue of this novel sequence.
Bernard G Forget - One of the best experts on this subject based on the ideXlab platform.
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a splice site mutation of the beta spectrin gene causing exon skipping in hereditary elliptocytosis associated with a truncated beta spectrin chain
Journal of Biological Chemistry, 1991Co-Authors: Patrick G Gallagher, William T Tse, Fernando F Costa, Alphonse L Scarpa, Pierre Boivin, J Delaunay, Bernard G ForgetAbstract:Abstract We studied a French kindred with hereditary elliptocytosis associated with a spectrin variant (spectrin LePuy) containing a beta-spectrin chain that is truncated at its C terminus (Dhermy, D., Lecomte, M., Garbarz, M., Bournier, O., Galand, C., Gautero, H., Feo, C., Alloisio, N., Delaunay, J., and Boivin, P. (1982) J. Clin. Invest. 70, 707-715). The structure of the 3' end of the beta-spectrin gene, the region encoding the C terminus of beta-spectrin, was determined. Nucleotide sequencing of amplified genomic DNA revealed a mutation at position +4 (A----G) of the 5' donor consensus splice site of the intron following the third-to-last exon (exon X) in one beta-spectrin allele of a heterozygous patient. Agarose gel electrophoresis of polymerase chain reaction-amplified cDNA revealed an extra band of lower molecular weight, suggesting that the shortened beta-spectrin chain of spectrin LePuy arises from aberrant mRNA splicing. Nucleotide sequencing of the shorter cDNA amplification product revealed that the sequences encoding exon X were absent. Southern blotting of cDNA amplification products confirmed this result. The skipping of exon X causes a shift in the normal reading frame resulting in the encoding of a new amino acid sequence at the C terminus of the mutant beta-spectrin chain. A new in-frame stop codon is encountered following a single residue of this novel sequence.
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spectrin rouen beta 220 218 a novel shortened beta chain variant in a kindred with hereditary elliptocytosis characterization of the molecular defect as exon skipping due to a splice site mutation
Journal of Clinical Investigation, 1991Co-Authors: M Garbarz, Patrick G Gallagher, William T Tse, C Picat, Marie Christine Lecomte, Francis Galibert, Didier Dhermy, Bernard G ForgetAbstract:The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.
Robert L Mauck - One of the best experts on this subject based on the ideXlab platform.
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nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long term agarose culture
European Cells & Materials, 2011Co-Authors: Lachlan J Smith, Joseph A Chiaro, Nandan L Nerurkar, Daniel H Cortes, Sarena D Horava, Nader M Hebela, Robert L Mauck, George R Dodge, Dawn M ElliottAbstract:Intervertebral disc degeneration is characterized by a cascade of cellular, biochemical and structural changes that may lead to functional impairment and low back pain. Interleukin-1 beta (IL-1β) is strongly implicated in the etiology of disc degeneration, however there is currently no direct evidence linking IL-1β upregulation to downstream biomechanical changes. The objective of this study was to evaluate long-term agarose culture of nucleus pulposus (NP) cells as a potential in vitro model system to investigate this. Bovine NP cells were cultured in agarose for 49 days in a defined medium containing transforming growth factor-beta 3, after which both mechanical properties and composition were evaluated and compared to native NP. The mRNA levels of NP cell markers were compared to those of freshly isolated NP cells. Glycosaminoglycan (GAG) content, aggregate modulus and hydraulic permeability of mature constructs were similar to native NP, and aggrecan and SOX9 mRNA levels were not signifi cantly different from freshly isolated cells. To investigate direct links between IL-1β and biomechanical changes, mature agarose constructs were treated with IL-1β, and effects on biomechanical properties, extracellular matrix composition and mRNA levels were quantifi ed. IL-1β treatment resulted in upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 4, matrix metalloproteinase-13 and inducible nitric oxide sythase, decreased GAG and modulus, and increased permeability. To evaluate the model as a test platform for therapeutic intervention, cotreatment with IL-1β and IL-1 receptor antagonist (IL-1ra) was evaluated. IL-1ra signifi cantly attenuated degradative changes induced by IL-1β. These results suggest that this in vitro model represents a reliable and cost-effective platform for evaluating new therapies for disc degeneration.
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transient exposure to transforming growth factor beta 3 improves the mechanical properties of mesenchymal stem cell laden cartilage constructs in a density dependent manner
Tissue Engineering Part A, 2009Co-Authors: Alice H Huang, Ashley Stein, Rocky S Tuan, Robert L MauckAbstract:Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering and regenerative medicine. However, the use of these cells has been limited by their reduced ability to form functional tissue compared to chondrocytes when placed in three-dimensional culture systems. To optimize MSC functional chondrogenesis, we examined the effects of increasing seeding density and transient application of transforming growth factor beta 3 (TGF-β3), two factors previously shown to improve growth of chondrocyte-based constructs. Chondrocytes seeded in agarose at 20 million cells/mL and MSCs seeded at 20 or 60 million cells/mL agarose were cultured for 7 weeks under continuous or transient application of TGF-β3. In the transient group, cell-laden constructs were exposed to TGF-β3 for the initial 3 weeks, followed by 4 weeks of culture in medium without TGF-β3. Compressive properties, biochemical content, and gene expression were assessed at 3, 5, and 7 weeks. Matrix distribution and collagen type...
Shawn W Odriscoll - One of the best experts on this subject based on the ideXlab platform.
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chondrogenesis in periosteal explants an organ culture model for in vitro study
Journal of Bone and Joint Surgery American Volume, 1994Co-Authors: Shawn W Odriscoll, A D Recklies, A R PooleAbstract:ABSTRACT Periosteal grafts have chondrogenic potential and have been used to repair defects in articular cartilage. We studied the effects of the culture conditions and of transforming growth factor-beta 1 on chondrogenesis in rabbit periosteal explants that were cultured in vitro. A total of 390 periosteal explants were obtained from the anteromedial sides of the proximal parts of the tibiae of eleven rabbits that were two weeks, two months, or six months old.The culture medium (alpha minimum essential medium or Dulbecco minimum essential medium) contained fetal calf serum, with or without transforming growth factor-beta 1, at a concentration of one or ten nanograms per milliliter for the first two weeks of culture. Three hundred and twenty-one explants were submerged in liquid medium and sixty-nine were suspended in an agarose gel; they were then evaluated histochemically, histomorphometrically, and by collagen-typing. In the media without agarose, in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter, chondrogenesis was commonly seen after two to four weeks with use of safranin-O staining and histomorphometry. In the agarose gels, chondrogenesis from the periosteum was observed at four and six weeks and was enhanced by the presence of one or ten nanograms of transforming growth factor-beta 1 per milliliter. The combination of agarose with transforming growth factor-beta 1 most favored the formation of cartilage, which was maximum at six weeks in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter. Under these conditions, chondrogenesis occurred in almost every explant, with 50 +- 30 per cent of the tissue being composed of cartilage. Type-II collagen was present in the explants that had undergone chondrogenesis. The finding that transforming growth factor-beta 1 can induce chondrogenesis in the periosteum, particularly when the explant had been cultured in an agarose gel, confirms the potential of the periosteum to form cartilage. With use of in vitro methodology to define the optimum conditions that enhance chondrogenesis, it should be possible to elicit chondrogenesis when periosteal grafts are used to repair cartilage defects in vivo.
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chondrogenesis in periosteal explants an organ culture model for in vitro study
Journal of Bone and Joint Surgery American Volume, 1994Co-Authors: Shawn W Odriscoll, A D Recklies, A R PooleAbstract:Periosteal grafts have chondrogenic potential and have been used to repair defects in articular cartilage. We studied the effects of the culture conditions and of transforming growth factor-beta 1 on chondrogenesis in rabbit periosteal explants that were cultured in vitro. A total of 390 periosteal explants were obtained from the anteromedial sides of the proximal parts of the tibiae of eleven rabbits that were two weeks, two months, or six months old. The culture medium (alpha minimum essential medium or Dulbecco minimum essential medium) contained fetal calf serum, with or without transforming growth factor-beta 1, at a concentration of one or ten nanograms per milliliter for the first two weeks of culture. Three hundred and twenty-one explants were submerged in liquid medium and sixty-nine were suspended in an agarose gel; they were then evaluated histochemically, histomorphometrically, and by collagen-typing. In the media without agarose, in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter, chondrogenesis was commonly seen after two to four weeks with use of safranin-O staining and histomorphometry. In the agarose gels, chondrogenesis from the periosteum was observed at four and six weeks and was enhanced by the presence of one or ten nanograms of transforming growth factor-beta 1 per milliliter. The combination of agarose with transforming growth factor-beta 1 most favored the formation of cartilage, which was maximum at six weeks in the presence of ten nanograms of transforming growth factor-beta 1 per milliliter. Under these conditions, chondrogenesis occurred in almost every explant, with 50 +/- 30 per cent of the tissue being composed of cartilage. Type-II collagen was present in the explants that had undergone chondrogenesis.