Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 18 de 18
Filter
Add more filters










Publication year range
1.
J Exp Zool B Mol Dev Evol ; 320(5): 276-85, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23640850

ABSTRACT

The first mammalian metapodial (MP1) has periodically been argued to actually be a phalanx, because the first ray has one less element than the four posterior rays, and because the MP1 growth plate is proximal like those of all phalanges, rather than distal as in metapodials 2-5. However, growth plates are formed at both ends in non-therian tetrapod metapodials, and phylogenetic analysis demonstrates that growth plate loss is a therian synapomorphy that postdates the establishment of the mammalian phalangeal formula. These data, along with results of developmental and morphological studies, suggest that the MP1 is not a phalanx. The singular, proximal growth plates in MPs 2-5 are likely to be an adaptation to dynamic erect quadrupedal gait which was characterized by conversion of the posterior metapodials into rigid struts with the carpus/tarsus. While the adaptive significance of the reversed ossification of MP1 is less clear, we present three functional/developmental hypotheses.


Subject(s)
Biological Evolution , Finger Phalanges/anatomy & histology , Growth Plate/anatomy & histology , Osteogenesis , Animals , Epiphyses , Phylogeny
2.
Biomacromolecules ; 13(5): 1625-31, 2012 May 14.
Article in English | MEDLINE | ID: mdl-22559049

ABSTRACT

The development of advanced materials that facilitate hyaline cartilage formation and regeneration in aging populations is imperative. Critical to the success of this endeavor is the optimization of ECM production from clinically relevant cells. However, much of the current literature focuses on the investigation of primary bovine chondrocytes from young calves, which differ significantly than osteoarthritic cells from human sources. This study examines the levels of extracellular matrix (ECM) production using various levels of type I collagen and hyaluronic acid in poly(ethylene glycol) dimethacrylate (PEGDM) hydrogels in total knee arthroplasties, compared with the results from bovine chondrocytes. The addition of type 1 collagen in both the presence and absence of low levels of hyaluronic acid increased ECM production and/or retention in scaffolds containing either bovine or human chondrocytes. These findings are supported consistently with colorimetric quantification, whole mount extracellular matrix staining for both cell types, and histological staining for glycoaminoglycans and collagen of human chondrocyte containing samples. While exhibiting similar trends, the relative ECM productions levels for the primary human chondrocytes are significantly less than the bovine chondrocytes which reinforces the need for additional optimization.


Subject(s)
Chondrocytes/metabolism , Collagen Type I/chemistry , Extracellular Matrix/metabolism , Hyaluronic Acid/chemistry , Hydrogels/chemistry , Methacrylates/chemistry , Polyethylene Glycols/chemistry , Animals , Cattle , Cells, Cultured , Chondrocytes/cytology , Extracellular Matrix/chemistry , Humans , Molecular Structure
3.
Biomacromolecules ; 12(5): 1795-9, 2011 May 09.
Article in English | MEDLINE | ID: mdl-21449616

ABSTRACT

This paper is the first report of electrospinning neat polyisobutylene-based thermoplastic elastomers. Two generations of these materials are investigated: a linear poly(styrene-b-isobutylene-b-styrene) (L_SIBS) triblock copolymer and a dendritic poly(isobutylene-b-p-methylstyrene) (D_IB-MS), also a candidate for biomedical applications. Cross-polarized optical microscopy shows birefringence, indicating orientation in the electrospun fibers, which undergo large elongation and shear during electrospinning. In contrast to the circular cross section of L_SIBS fibers, D_IB-MS yields dumbbell-shaped fiber cross sections for the combination of processing conditions, molecular weight, and architecture. Hydrophobic surfaces with a water contact angle as high as 146 ± 3° were obtained with D_IB-MS that had the noncircular fiber cross section and a hierarchical arrangement of nano- to micrometer-sized fibers in the mat. These highly water repellent fiber mats were found to serve as an excellent scaffold for bovine chondrocytes to produce cartilage tissue.


Subject(s)
Microscopy, Electron, Scanning/methods , Polyenes/chemistry , Polymers/chemistry , Animals , Cattle , Cells, Cultured , Chondrocytes/metabolism
4.
J Cell Biochem ; 112(4): 1118-29, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21308740

ABSTRACT

The objective of this study was to test the hypothesis that extracellular matrix (ECM) would alter the endoplasmic reticulum (ER) stress response of chondrocytes. Chondrocytes were isolated from calf knees and maintained in monolayer culture or suspended in collagen I to form spot cultures (SCs). Our laboratory has shown that bovine chondrocytes form cartilage with properties similar to native cartilage after 2-4 weeks in SCs. Monolayer cultures treated with ER stressors glucose withdrawal (-Glu), tunicamycin (TN), or thapsigargin (TG) up-regulated Grp78 and Gadd153, demonstrating a complete ER stress response. SCs were grown at specific times from 1 day to 6 weeks before treatment with ER stressors. Additionally, SCs grown for 1, 2, or 6 weeks were treated with increasing concentrations of TN or TG. Western blotting of SCs for Grp78 indicated that increased ECM accumulation results in delayed expression; however, Grp78 mRNA is up-regulated in response to ER stressors even after 6 weeks in culture. SCs treated with ER stressors did not up-regulate Gadd153, suggesting that the cells experienced ER stress but would not undergo apoptosis. In fact, SCs undergo apoptosis upon ER stress treatment after 0-1 day of growth; however, after 4 days and to 6 weeks, apoptosis in treated samples was not different than controls. Pro-survival molecules Bcl-2 and Bag-1 were up-regulated upon ER stress in SCs. These results suggest that presence of ECM confers protection from ER stressors. Future studies involving chondrocyte physiology should focus on responses in conditions more closely mimicking the in vivo cartilage environment.


Subject(s)
Chondrocytes/metabolism , Endoplasmic Reticulum/metabolism , Extracellular Matrix/metabolism , Stress, Physiological , Animals , Apoptosis/drug effects , Blotting, Western , Cartilage, Articular/cytology , Cattle , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Glucose/pharmacology , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thapsigargin/pharmacology , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Tunicamycin/pharmacology
5.
Tissue Eng Part A ; 16(7): 2183-96, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20136403

ABSTRACT

Osteoarthritis (OA) is a prevalent age-associated disease involving altered chondrocyte homeostasis and cartilage degeneration. The avascular nature of cartilage and the altered chondrocyte phenotype characteristic of OA severely limit the capacity for in vivo tissue regeneration. Cell- and tissue-based repair has the potential to revolutionize treatment of OA, but those approaches have exhibited limited clinical success to date. In this study, we test the hypothesis that bovine and human chondrocytes in a collagen type I scaffold will form hyaline cartilage ex vivo with immunohistochemical, biochemical, and magnetic resonance (MR) endpoints similar to the original native cartilage. Chondrocytes were isolated from 1- to 3-week-old calf knee cartilage or from cartilage obtained from human total knee arthroplasties, suspended in 2.7 mg/mL collagen I, and plated as 300 microL spot cultures with 5 x 10(6) each. Medium formulations were varied, including the amount of serum, the presence or absence of ascorbate, and treatments with cytokines. Bovine chondrocytes generated metachromatic territorial and interstitial matrix and accumulated type II collagen over time. Type VI collagen was confined primarily to the pericellular region. The ex vivo-formed bovine cartilage contained more chondroitin sulfate per dry weight than native cartilage. Human chondrocytes remained viable and generated metachromatic territorial matrix, but were unable to support interstitial matrix accumulation. MR analysis of ex vivo-formed bovine cartilage revealed evidence of progressively maturing matrix, but MR-derived indices of tissue quality did not reach those of native cartilage. We conclude that the collagen-spot culture model supports formation and maturation of three-dimensional hyaline cartilage from active bovine chondrocytes. Future studies will focus on determining the capacity of human chondrocytes to show comparable tissue formation.


Subject(s)
Cartilage, Articular/metabolism , Tissue Engineering/methods , Aged , Animals , Cartilage, Articular/drug effects , Cartilage, Articular/growth & development , Cattle , Cell Proliferation/drug effects , Collagen Type II/metabolism , Culture Media/pharmacology , Cytokines/pharmacology , Glycosaminoglycans/metabolism , Humans , Immunohistochemistry , Magnetic Resonance Spectroscopy , Middle Aged
6.
J Histochem Cytochem ; 57(10): 923-31, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19546472

ABSTRACT

To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo- or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2-associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cell-associated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patient-matched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen.


Subject(s)
Collagen Type VI/biosynthesis , DNA-Binding Proteins/biosynthesis , Endoplasmic Reticulum/metabolism , Heat-Shock Proteins/biosynthesis , Osteoarthritis, Knee/metabolism , Transcription Factors/biosynthesis , Adult , Antigens/biosynthesis , Biomarkers/metabolism , Bone Neoplasms/metabolism , Cartilage, Articular/metabolism , Chondrosarcoma/metabolism , Down-Regulation , Endoplasmic Reticulum Chaperone BiP , Humans , Middle Aged , Osteoarthritis, Knee/physiopathology , Osteosarcoma/metabolism , Proteoglycans/biosynthesis , Up-Regulation
7.
J Cell Biochem ; 102(3): 786-800, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17546604

ABSTRACT

BAG-1 (Bcl-2 associated athanogene-1) is a multifunctional protein, linking cell proliferation, cell death, protein folding, and cell stress. In vivo, BAG-1 is expressed in growth plate and articular cartilage, and the expression of BAG-1 is decreased with aging. Chondrocytes respond to endoplasmic reticulum (ER) stress with decreased expression of extracellular matrix proteins, and prolonged ER stress leads to chondrocyte apoptosis. Here we demonstrate for the first time that BAG-1 is involved in ER stress-induced apoptosis in chondrocytes. Induction of ER stress through multiple mechanisms all resulted in downregulation of BAG-1 expression. In addition, direct suppression of BAG-1 expression resulted in chondrocyte growth arrest and apoptosis, while stable overexpression of BAG-1 delayed the onset of ER stress-mediated apoptosis. In addition to regulating apoptosis, we also observed decreased expression of collagen type II in BAG-1 deficient chondrocytes. In contrast, overexpression of BAG-1 resulted in increased expression of collagen type II. Moreover, under ER stress conditions, the reduced expression of collagen type II was delayed in chondrocytes overexpressing BAG-1. These results suggest a novel role for BAG-1 in supporting viability and matrix expression of chondrocytes.


Subject(s)
Chondrocytes/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Transcription Factors/metabolism , Animals , Apoptosis , Cell Proliferation , Chondrocytes/pathology , Collagen Type II/metabolism , DNA-Binding Proteins/physiology , Endoplasmic Reticulum/metabolism , Models, Biological , Phenotype , RNA Interference , Rats , Subcellular Fractions/metabolism , Time Factors , Transcription Factors/physiology , Transfection
8.
J Exp Zool B Mol Dev Evol ; 308(3): 283-96, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17285637

ABSTRACT

Mammalian metapodials (metacarpals and metatarsals), unlike most long bones, form a single growth plate, and undergo longitudinal growth at only one end. The growth dynamics of non-mammalian tetrapod metapodials have not been systematically examined in order to determine if unidirectional growth is unique to mammals. Here we compare murine metapodial ossification in growth stages that parallel those of embryonic, juvenile and subadult American alligators (Alligator mississippiensis). Safranin O staining was used for qualitative histology, and chondrocyte differentiation and proliferation were assessed via immunohistochemistry for type X collagen and proliferative cell nuclear antigen (PCNA). We establish that growth plates form at both ends of alligator metapodials and are maintained in the subadult. PCNA results show that alligators and mice share common patterns of chondrocyte proliferation during growth plate formation. In addition, while alligators and mice differ initially in the degree of organization and pace of chondrocyte differentiation, these parameters are largely similar in established growth plates. However, the replacement of cartilage by bone is highly irregular throughout growth in the alligator, in contrast to the more uniform process in the mouse. These results indicate that while alligators and mammals share common mechanisms of chondrocyte regulation, they differ substantially in their processes of ossification. Phylogenetic analysis indicates that the direct ossification of one epiphysis and reliance on a single growth plate is a derived character (synapomorphy) in therian mammals and likely indicates an adaptation for erect quadrupedal gait.


Subject(s)
Alligators and Crocodiles/physiology , Bone Development/physiology , Growth Plate/growth & development , Metacarpal Bones/growth & development , Metatarsal Bones/growth & development , Mice/physiology , Animals , Immunohistochemistry , Phenazines , Phylogeny , Proliferating Cell Nuclear Antigen , Species Specificity
9.
Cryobiology ; 54(1): 36-43, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17174945

ABSTRACT

The objective of this study was to investigate the effects of cryopreservation on the components of articular cartilage (AC) matrix by utilizing magnetic resonance imaging (MRI) and biochemical assessments. Porcine AC (10mm osteochondral dowels) was collected into four groups - (1) phosphate buffered saline (PBS) control, (2) PBS snap frozen in liquid nitrogen, (3) slow-cooled in dimethyl sulfoxide (DMSO), and (4) slow cooled in PBS (in absence of DMSO). MRI results demonstrated three distinct zones in the cartilage. After exposure to ice formation during cryopreservation procedures, alterations in MRI determined matrix fixed charged density and magnetization transfer rate were noted. In addition, biochemical assays demonstrated significant alterations in chondroitin sulfate and hydroxyproline content over time without differences in hydration or DNA content. In conclusion, MRI was able to detect some changes in the intact cartilage matrix structure consistent with biochemical assessments after ice formation during cryopreservation of intact porcine AC. Furthermore, biochemical assessments supported some of these findings and changed significantly after incubating the cartilage matrix for 36-72 h in PBS in terms of chondroitin sulfate and hydroxyproline content.


Subject(s)
Cartilage, Articular , Cryopreservation/methods , Freezing , Animals , Cartilage, Articular/anatomy & histology , Cartilage, Articular/metabolism , Chondroitin Sulfates/metabolism , Hydroxyproline/metabolism , Image Processing, Computer-Assisted , Magnetic Resonance Imaging , Swine
10.
Anat Rec A Discov Mol Cell Evol Biol ; 288(1): 104-18, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16342215

ABSTRACT

There is significant diversity in growth plate behavior among sites within an individual skeleton and between skeletons of different species. This variation within wild-type animals is an underutilized resource for studying skeletal development. One bone that potentially exhibits the most diverse behavior is the metatarsal. While one end forms a growth plate with an epiphyseal secondary center of ossification as in other long bones, the opposite end undergoes direct ossification in a manner more similar to short bones. Although descriptions of human metatarsal/metacarpal ossification are available, a detailed comparative analysis has yet to be conducted in an animal model amenable to biomolecular analysis. Here we report an analysis of proximal and distal ossification in an age series of mouse metatarsals. Safranin O staining was used for qualitative and quantitative histology, and chondrocyte differentiation and proliferation were analyzed using immunohistochemistry for type X collagen and proliferative cell nuclear antigen expression. We establish that, as in the human, both growth plate formation and direct ossification occur in the mouse metatarsal, with chondrocyte populations showing distinct differentiation patterns at opposite ends of the bone. In addition, growth plate formation is characterized by a peak of proliferation in reserve zone chondrocytes that distinguishes it from both established growth plates and direct ossification. Our analysis demonstrates that the mouse metatarsal is a productive model for investigating natural variation in ossification that can further understanding of vertebrate skeletal development and evolution.


Subject(s)
Growth Plate/physiology , Metatarsal Bones/physiology , Osteogenesis/physiology , Animals , Cell Proliferation , Chondrocytes/physiology , Collagen Type X/metabolism , Diaphyses/anatomy & histology , Epiphyses/anatomy & histology , Female , Immunohistochemistry , Male , Metatarsal Bones/anatomy & histology , Metatarsal Bones/cytology , Metatarsal Bones/growth & development , Mice , Mice, Inbred C57BL
11.
J Biomed Opt ; 10(3): 031105, 2005.
Article in English | MEDLINE | ID: mdl-16229630

ABSTRACT

The composition of cartilage is predictive of its in vivo performance. Therefore, the ability to assess its primary macromolecular components, proteoglycan (PG) and collagen, is of great importance. In the current study, we hypothesized that PG content and distribution in tissue engineered cartilage could be determined using Fourier-transform infrared imaging spectroscopy (FT-IRIS). The cartilage was grown from chondrocytes within a hollow fiber bioreactor (HFBR) system previously used extensively to study cartilage development. FT-IRIS analysis showed a gradient of PG content, with the highest content in the center near the nutritive fibers and the lowest near the interior surface of the HFBR. Further, we found significantly greater PG content in the region near culture medium inflow (45.0%) as compared to the outflow region (24.7%) (p<0.001). This difference paralleled the biochemically determined glycosaminoglycan difference of 42.6% versus 27.8%. In addition, FT-IRIS-determined PG content at specific positions within the tissue sections correlated with histologically determined PG content (R=0.73, p=0.007). In summary, FT-IRIS determination of PG correlates with histological determination of PG and yields quantitatively similar results to biochemical determination of glycosaminoglycan in developing cartilage.


Subject(s)
Cartilage/metabolism , Chondrocytes/metabolism , Collagen/metabolism , Proteoglycans/metabolism , Spectroscopy, Fourier Transform Infrared/methods , Tissue Engineering/methods , Animals , Cartilage/cytology , Cartilage/growth & development , Cell Culture Techniques/methods , Cells, Cultured , Chickens , Chondrocytes/cytology , Collagen/analysis , Proteoglycans/analysis , Statistics as Topic
12.
J Biol Chem ; 280(35): 31156-65, 2005 Sep 02.
Article in English | MEDLINE | ID: mdl-16000304

ABSTRACT

The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1beta or tumor necrosis factor alpha induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.


Subject(s)
Apoptosis/physiology , Cell Differentiation/physiology , Chondrocytes/physiology , Endoplasmic Reticulum/metabolism , Signal Transduction/physiology , Animals , Annexin A5/metabolism , Anti-Bacterial Agents/metabolism , Biomarkers , CCAAT-Enhancer-Binding Proteins/metabolism , Caspase 12 , Caspases/metabolism , Cells, Cultured , Chondrocytes/cytology , Collagen Type II/metabolism , DNA Fragmentation , Extracellular Matrix/metabolism , Gene Expression Regulation , Glucose/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Rats , Thapsigargin/metabolism , Transcription Factor CHOP , Transcription Factors/metabolism , Tunicamycin/metabolism
13.
J Pediatr Orthop ; 25(4): 440-4, 2005.
Article in English | MEDLINE | ID: mdl-15958891

ABSTRACT

Ultrastructural studies of slipped capital femoral epiphysis (SCFE) growth plates have shown diminished cellularity and marked distortion of the architecture in the proliferative and hypertrophic zones. Chondrocyte degeneration and death were noted at all levels of the hypertrophic and proliferative zones, suggesting an accelerated disturbance in the life-to-death cycle of the chondrocytes. The current study examines the mechanism responsible for the diminished cell number and whether increased programmed cell death (apoptosis) or necrosis was operative. Proximal femoral growth plates from patients with SCFE (three patients) were prepared and sectioned for histochemistry, in situ detection of apoptosis, and immunohistochemistry. The results showed that the diminished cell number is due to an abnormal frequency and distribution of chondrocytes undergoing apoptosis. Although it is unclear whether the increased apoptosis is occurring early or late in the disease, it is highly likely that it is directly linked to pathogenesis.


Subject(s)
Apoptosis , Chondrocytes/ultrastructure , Epiphyses, Slipped/pathology , Femur/ultrastructure , Growth Plate/ultrastructure , Adolescent , Child , Chondrocytes/metabolism , Collagen Type II/immunology , Collagen Type II/metabolism , DNA/analysis , Female , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Male , Proteoglycans/immunology , Proteoglycans/metabolism
14.
J Biol Chem ; 280(34): 30517-25, 2005 Aug 26.
Article in English | MEDLINE | ID: mdl-15975921

ABSTRACT

Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase C alpha and NFkappaB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype.


Subject(s)
Chondrocytes/metabolism , Gene Expression Regulation , High Mobility Group Proteins/metabolism , MAP Kinase Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Transcription Factors/metabolism , Adenoviridae/genetics , Animals , Apoptosis , Blotting, Western , Butadienes/pharmacology , Caspase Inhibitors , Cell Differentiation , Cell Line , Collagen Type II/metabolism , Down-Regulation , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , Lac Operon , Luciferases/metabolism , Microscopy, Fluorescence , NF-kappa B/metabolism , Nitriles/pharmacology , Phenotype , Phosphorylation , Promoter Regions, Genetic , Protein Kinase C/antagonists & inhibitors , Protein Kinase C-alpha , Proteoglycans/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor , Signal Transduction , Time Factors , Transcription, Genetic , Transfection , beta-Galactosidase/metabolism
15.
J Orthop Res ; 23(5): 1128-38, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15936918

ABSTRACT

Osteoarthritis (OA) is a degenerative cartilage disease with varying degrees of severity within a given joint. The purpose of this study was to define a sampling procedure for comparing human minimal and advanced OA cartilage in the same patient and to determine basic patterns of gene expression in these regions. A specific hypothesis under study was that the expression level of Bcl-2 would correlate with Sox9 and aggrecan mRNA expression in vivo as has been demonstrated in vitro. Femoral condylar advanced OA cartilage was located within 1cm of overt lesions, and minimal cartilage was taken from areas with no obvious surface defects. Histological sections were scored for disease severity and chondroitin sulfate and hydroxyproline content was determined. The expression level of nine specific genes (aggrecan, collagen type II, Bcl-2, Sox9, Link protein, osteopontin, and MMP-13, -3, and -9) was determined by quantitative real time PCR. The scores for fibrillation, chondrocyte cloning, and proteoglycan depletion were significantly different between advanced and minimal OA cartilage. The advanced OA cartilage had significantly less chondroitin sulfate than the minimal OA cartilage. Osteopontin mRNA expression showed a 3.6-fold increase in advanced compared to minimal OA cartilage. In contrast, the level of mRNA coding for aggrecan, link protein, Bcl-2, Sox9 and MMP-3, -9, -13 were all decreased in advanced compared to minimal cartilage in the majority of the patients studied. Collagen type II mRNA expression displayed a wide-range of variation. A statistically significant correlation was observed both between Bcl-2 and Sox9 mRNA level, and between Bcl-2 and aggrecan mRNA expression. The patient matched comparison of minimal and advanced OA cartilage revealed differences in cellular and tissue characteristics, and changes in gene expression that may be involved in OA progression. In addition, Bcl-2 may also play a role in regulating the expression of aggrecan through Sox9 in vivo as well as in vitro.


Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Gene Expression Profiling , Osteoarthritis/metabolism , Aged , Aged, 80 and over , Collagen Type II/genetics , Glycosaminoglycans/analysis , High Mobility Group Proteins/genetics , Humans , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 9/genetics , Phenotype , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/analysis , SOX9 Transcription Factor , Transcription Factors/genetics
16.
Anat Rec A Discov Mol Cell Evol Biol ; 279(2): 720-8, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15278942

ABSTRACT

Aging cartilage displays increased chondrocyte apoptosis and decreased responsiveness of chondrocytes to growth factors. The molecular mechanisms responsible for these changes have not been identified. Bag-1 is a Bcl-2-binding protein that promotes cell survival, interacts with a diverse group of cellular proteins, and may integrate multiple pathways involved in controlling cell survival, growth, and phenotype. Bcl-2 is important for maintaining chondrocyte phenotype and delaying terminal differentiation and apoptosis of chondrocytes. Comparatively little is known about the role of Bag-1 in cartilage. Here we show that both growth plate and articular chondrocytes in the mouse express the Bag-1 protein. In the growth plate, Bag-1 expression is prominent in the late proliferative and prehypertrophic chondrocytes, displaying a pattern similar to what has been reported for Bcl-2. Further, the expression of both Bcl-2 and Bag-1 declines with age in the articular cartilage. Growth assays demonstrate that knocking down Bag-1 expression causes a decrease in growth rate. These results suggest that Bag-1 is involved in the regulation of chondrocyte phenotype and cartilage aging.


Subject(s)
Aging/metabolism , Carrier Proteins/metabolism , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Growth Plate/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Carrier Proteins/genetics , Cartilage, Articular/cytology , Cell Division/physiology , Cells, Cultured , Chondrocytes/cytology , DNA-Binding Proteins , Gene Expression Regulation , Mice , Mice, Inbred C57BL , Tissue Distribution , Transcription Factors
17.
Arthritis Rheum ; 48(4): 1047-56, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12687548

ABSTRACT

OBJECTIVE: To use noninvasive magnetic resonance imaging (MRI), biochemical analyses, and mechanical testing of engineered neocartilage grown in a hollow- fiber bioreactor (HFBR) to establish tissue properties, and to test the hypothesis that MRI can be used to monitor biochemical and biomechanical properties of neocartilage. METHODS: Chondrocytes from day 16 embryonic chick sterna were inoculated into an HFBR and maintained for up to 4 weeks with and without exposure to chondroitinase ABC. The fixed-charge density (FCD) of the cartilage was determined using the MRI gadolinium exclusion method. The sulfated glycosaminoglycan (S-GAG), hydroxyproline, and DNA contents were determined using biochemical procedures, while dynamic and equilibrium moduli were determined from mechanical indentation tests. RESULTS: S-GAG content, tissue cross-sectional area, and equilibrium modulus of the neocartilage increased with development time. There was a gradient of S-GAG content across the length of control neocartilage at the 4-week time point, with higher values being found toward the inflow region. Exposure to chondroitinase ABC resulted in a decrease in tissue area, negative FCD, proteoglycan content, and equilibrium and dynamic moduli. The treated bioreactors displayed a lengthwise variation in S-GAG content, with higher values toward the outflow end. Linear correlations were established among FCD, proteoglycan content, and biomechanical properties. CONCLUSION: HFBR-derived neocartilage showed regional variation in S-GAG content under control conditions, and in the decrease of S-GAG in response to enzyme treatment. In addition, the results support the hypothesis that tissue parameters derived from MRI can be used to noninvasively monitor focal neocartilage formation and biochemical and biomechanical properties.


Subject(s)
Bioreactors , Cartilage/chemistry , Magnetic Resonance Imaging/methods , Animals , Biomechanical Phenomena , Cartilage/drug effects , Cartilage/physiology , Chick Embryo , Chondrocytes/chemistry , Chondrocytes/cytology , Chondrocytes/physiology , Chondroitin ABC Lyase/pharmacology , Culture Techniques , DNA/analysis , Extracellular Matrix , Glycosaminoglycans/analysis , Hydroxyproline/analysis , Static Electricity , Sternum
18.
J Cell Biochem ; 88(5): 941-53, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12616533

ABSTRACT

The anti-apoptotic protein Bcl-2 has been shown to function in roles unrelated to apoptosis in a variety of cell types. We have previously reported that loss of Bcl-2 expression alters chondrocyte morphology and modulates aggrecan expression via an apoptosis-independent pathway. Here we show that Bcl-2 is required for chondrocytes to maintain expression of a variety of cartilage-specific matrix proteins. Using quantitative, real-time PCR, we demonstrate that Bcl-2-deficient chondrocytes coordinately down-regulate genes coding for hyaline cartilage matrix proteins including collagen II, collagen IX, aggrecan, and link protein. The decrease in steady-state level of these mRNA transcripts results, in part, from decreased mRNA stability in Bcl-2-deficient chondrocytes. Transcriptional regulation is also likely involved because chondrocytes with decreased Bcl-2 levels show decreased expression of SOX9, a transcription factor necessary for expressing the major cartilage matrix proteins. In contrast, chondrocytes constitutively expressing Bcl-2 have a stable phenotype when subjected to loss of serum factor signaling. These cells maintain high levels of SOX9, as well as the SOX9 targets collagen II and aggrecan. These results suggest that Bcl-2 is involved in a pathway important for maintaining a stable chondrocyte phenotype.


Subject(s)
Chondrocytes/physiology , Extracellular Matrix Proteins/metabolism , Glycoproteins/metabolism , High Mobility Group Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/physiology , Transcription Factors/metabolism , Aggrecans , Animals , Cell Line , Collagen Type II/genetics , Collagen Type II/metabolism , Dactinomycin , Down-Regulation , Extracellular Matrix Proteins/analysis , Extracellular Matrix Proteins/genetics , Gene Expression Regulation , Glycoproteins/analysis , Glycoproteins/genetics , High Mobility Group Proteins/genetics , Lectins, C-Type , Matrilin Proteins , Polymerase Chain Reaction/methods , Proteoglycans/genetics , Proteoglycans/metabolism , Proto-Oncogene Proteins c-bcl-2/deficiency , Proto-Oncogene Proteins c-bcl-2/genetics , RNA Stability , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Rats , SOX9 Transcription Factor , Signal Transduction , Transcription Factors/genetics , Transfection
SELECTION OF CITATIONS
SEARCH DETAIL
...