scholarly journals Three-Dimensional In Vitro Effects of Compression and Time in Culture on Aggregate Modulus and on Gene Expression and Protein Content of Collagen Type II in Murine Chondrocytes

2009 ◽  
Vol 15 (10) ◽  
pp. 2807-2816 ◽  
Author(s):  
Kumar Chokalingam ◽  
Shawn Hunter ◽  
Cynthia Gooch ◽  
Chris Frede ◽  
Jane Florer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Content ◽  
Collagen Type ◽  
Three Dimensional ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Aggregate Modulus ◽  
In Vitro Effects

scholarly journals Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity

2019 ◽  
Vol 20 (4) ◽  
pp. 795 ◽  
Author(s):  
Ufuk Tan Timur ◽  
Marjolein Caron ◽  
Guus van den Akker ◽  
Anna van der Windt ◽  
Jenny Visser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Protein Secretion ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Reporter Assay

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.

Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity

2007 ◽  
Vol 20 (03) ◽  
pp. 185-191 ◽  
Author(s):  
A. O. Oshin ◽  
E. Caporali ◽  
C. R. Byron ◽  
A. A. Stewart ◽  
M. C. Stewart
Keyword(s):  
Articular Cartilage ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Soluble Form ◽  
Mrna Levels ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Endogenous Expression ◽  
Chondrocytic Phenotype

SummaryArticular chondrocytes are phenotypically unique cells that are responsible for the maintenance of articular cartilage. The articular chondrocytic phenotype is influenced by a range of soluble factors. In particular, members of the bone morphogenetic protein (BMP) family support the articular chondrocytic phenotype and stimulate synthesis of cartilaginous matrix. This study was carried out to determine the importance of BMPs in supporting the differentiated phenotype of articular chondrocytes in vitro. Exogenous BMP-2 supported expression of collagen type II and aggrecan in monolayer chondrocyte cultures, slowing the dedifferentiation process that occurs under these conditions. In contrast, BMP-2 had little effect on expression of these genes in three-dimensional aggregate cultures. Endogenous BMP-2 expression was lost in monolayer cultures, coincident with the down-regulation of collagen type II and aggrecan mRNAs, whereas BMP-2 mRNA levels were stable in aggregate cultures. Antagonism of endogenous BMP activity in aggregate cultures by Noggin or a soluble form of the BMP receptor resulted in reduced expression of collagen type II and aggrecan mRNAs, reduced collagen type II protein and sulfated glycosaminoglycan (GAG) deposition into the aggregate matrices and reduced secretion of GAGs into the culture media. These results indicate that endogenous BMPs are required for maintenance of the differentiated articular chondrocytic phenotype in vitro. These findings are of importance to cell-based strategies designed to repair articular cartilage. Articular chondrocytes require conditions that will support endogenous expression of BMPs to maintain the specialized phenotype of these cells.

scholarly journals Expression of the Heparan Sulfate Proteoglycan, Perlecan, during Mouse Embryogenesis and Perlecan Chondrogenic Activity In Vitro

1999 ◽  
Vol 145 (5) ◽  
pp. 1103-1115 ◽  
Author(s):  
M.M. French ◽  
S.E. Smith ◽  
K. Akanbi ◽  
T. Sanford ◽  
J. Hecht ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Collagen Type ◽  
Heparan Sulfate Proteoglycan ◽  
Surrounding Tissue ◽  
Collagen Type Iv ◽  
Type Ii ◽  
Sulfate Proteoglycan ◽  
Collagen Type Ii ◽  
Murine Embryonic Fibroblast

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.

Selective and transient in vitro effects of heat shock on alveolar type II cell gene expression

1997 ◽  
Vol 272 (1) ◽  
pp. L132-L138 ◽  
Author(s):  
H. R. Wong ◽  
M. Ryan ◽  
S. Gebb ◽  
J. R. Wispe
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Alveolar Type ◽  
Type Ii ◽  
Alveolar Type Ii Cell ◽  
Cell Gene Expression ◽  
Type Ii Cell ◽  
In Vitro Effects ◽  
Cell Gene

The heat shock response is a highly conserved stress response that can transiently inhibit non-heat shock protein gene expression. Although heat shock protects against acute lung injury, its effects on lung cell gene expression are not known. We studied the in vitro effects of heat shock on the expression of several genes important to alveolar type II cells. Prior induction of heat shock transiently inhibited cytokine-mediated inducible nitric oxide synthase gene expression and cytokine-mediated manganese-superoxide dismutase mRNA expression in murine lung epithelium. In contrast, heat shock had no effect on expression of surfactant protein (SP) A or B mRNA, or SP-B peptide synthesis. Cell survival studies indicated that the inhibitory effects were not secondary to cytotoxicity. Previous heat shock also modestly enhanced the ability of cells to withstand oxidant stress. We conclude that in vitro heat shock has selective and transient inhibitory effects on alveolar type II cell gene expression. Transient inhibition of cytokine-inducible genes, with concomitant conservation of genes required for normal respiratory function (SP) may explain, in part, the mechanism by which heat shock protects during acute lung injury.

Variable Effects of 3 Different Chondroitin Sulfate Compounds on Human Osteoarthritic Cartilage/Chondrocytes: Relevance of Purity and Production Process

2010 ◽  
Vol 37 (3) ◽  
pp. 656-664 ◽  
Author(s):  
STEEVE KWAN TAT ◽  
JEAN-PIERRE PELLETIER ◽  
FRANÇOIS MINEAU ◽  
NICOLAS DUVAL ◽  
JOHANNE MARTEL-PELLETIER
Keyword(s):  
Gene Expression ◽  
Chondroitin Sulfate ◽  
Collagen Type ◽  
Cartilage Explants ◽  
Gene Expression Level ◽  
Expression Level ◽  
Prostaglandin E ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Catabolic Pathways

Objective.During osteoarthritis (OA), the altered metabolism of cartilage involves proinflammatory factors and matrix metalloprotease (MMP) activity. Studies showed that chondroitin sulfate (CS) may exert a positive effect on the cartilage. Because of differences in CS in terms of purity and the production/purification process, we compared the effects of 3 different types of CS on human OA cartilage.Methods.Three types of CS were tested: CS1 (porcine, purity 90.4%), CS2 (bovine, purity 96.2%), and CS3 (bovine, purity 99.9%). Treatment with CS at 200 and 1000 μg/ml was performed on human OA cartilage explants in the presence/absence of interleukin 1ß (IL-1ß), and the protein modulations of factors including prostaglandin E2 (PGE2), IL-6, and MMP-1 measured by ELISA. The CS effect on the expression of collagen type II was also investigated on OA chondrocytes using quantitative polymerase chain reaction.Results.In the presence of IL-1ß, CS2 at 1000 μg/ml significantly inhibited IL-6 and PGE2 production, and CS3 at 200 μg/ml markedly reduced the level of IL-6. CS1 was much less efficient at reducing the catabolic markers and in the absence of IL-1ß, it significantly increased IL-6 and MMP-1. IL-1ß significantly inhibited the gene expression level of collagen type II; only CS3 was able to limit this inhibition. CS1, in the presence or absence of IL-1ß, further markedly decreased collagen type II expression.Conclusion.Our data indicate that among the 3 tested CS, CS1 increased production of some catabolic pathways and inhibited the gene expression level of collagen type II. Our study provides new information in the context of prescribing CS for alleviating OA symptoms, as the purity and/or production/purification of the CS compound could orient the current OA disease process toward increased catabolic pathways.

Hypoxia promotes nucleus pulposus phenotype in 3D scaffolds in vitro and in vivo

2014 ◽  
Vol 21 (2) ◽  
pp. 303-309 ◽  
Author(s):  
Ganjun Feng ◽  
Li Li ◽  
Ying Hong ◽  
Hao Liu ◽  
Yueming Song ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Collagen Type ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Low Oxygen ◽  
Nucleus Pulposus Cells ◽  
Experimental Conditions ◽  
3D Scaffolds

Object The role of oxygen in disc metabolism remains a matter of debate. Whether the effect of hypoxic priming on the nucleus pulposus phenotype can be maintained in vivo is not clear. The goal of the present study was to test the hypothesis that priming in a low oxygen tension in vitro could promote a nucleus pulposus phenotype in vivo. Methods Bovine nucleus pulposus cells were seeded in 3D scaffolds and subjected to varying oxygen tensions (2% and 20%) for 3 weeks. The constructs were then implanted subcutaneously for 8 weeks. Changes in the extracellular matrix were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, glycosaminoglycan (GAG) assay, DNA assay, collagen quantification, and histological and immunohistological analyses. Results Hypoxia resulted in greater production of sulfated glycosaminoglycan and higher levels of gene expression for collagen Type II, aggrecan, and SOX-9. Furthermore, after hypoxic priming, the subcutaneously implanted constructs maintained the nucleus pulposus phenotype, which was indicated by a significantly higher amount of glycosaminoglycan and collagen Type II. Conclusions Hypoxia enhanced the nucleus pulposus phenotype under experimental conditions both in vitro and in vivo. When used in combination with appropriate scaffold material, nucleus pulposus cells could be regenerated for tissue-engineering applications.

Cartilage-Specific Matrix and Integrin Expression in Three-Dimensional Articular Chondrocyte Cultures Overexpressing Human Interleukin-10

2008 ◽  
Vol 1 ◽  
pp. CMAMD.S560
Author(s):  
Riccarda D. Müller ◽  
Thilo John ◽  
Benjamin Kohl ◽  
Anja Feldner ◽  
Hala Zreiqat ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Three Dimensional ◽  
3D Culture ◽  
Type I ◽  
Β1 Integrin ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Chondrocyte Cultures

Interleukin (IL)-10 overexpression inhibits joint inflammation, however the effect of high local concentrations of IL-10 on chondrocyte homeostasis remains unclear. The aim of this study was to determine the effects of IL-10 overexpression on cartilage matrix production in three-dimensional (3D) chondrocyte cultures. Human articular chondrocytes were transduced with adenoviral vectors alone (adv/empty) or by vectors either overexpressing enhanced green fluorescence protein (adv/EGFP) or human IL-10 (adv/hIL-10) before their transfer to a 3D culture system. Non-transduced chondrocytes were used as controls. The expression of IL-10 or EGFP was confirmed using ELISA or flow cytometry. Chondrocytes synthesis of collagen types II and I, aggrecan, fibronectin and β1-integrin was determined over a period of 14 days post transduction using flow cytometry or immunohistochemistry. adv/EGFP or adv/IL-10 transduced chondrocytes expressed EGFP or secreted IL-10 detectable over the 2 weeks culture period. No suppression of collagen type II, aggrecan or β1-integrin synthesis by IL-10 overexpression was found and the deposition of collagen type I and fibronectin remained unaffected compared to the controls. IL-10 overexpression does not impair key features of chondrocytes differentiated phenotype (e.g. collagen type II and aggrecan expression) suggesting the potential use of IL-10 for gene therapeutic approaches in the joint.

scholarly journals Effect of Decellularized Cartilage Bovine Scaffold and Hypoxic Condition on Stem Cell Differentiation to Chondrocyte: An In Vitro Study

2018 ◽  
Vol 35 ◽  
pp. 67-76
Author(s):  
Ferdiansyah Mahyudin ◽  
Dwikora Novembri Utomo ◽  
Tri Wahyu Martanto ◽  
Aries Rakhmat Hidayat ◽  
Linta Meyla Putri
Keyword(s):  
Tissue Engineering ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Collagen Type ◽  
Hypoxic Condition ◽  
Stem Cell Differentiation ◽  
Type Ii ◽  
Collagen Type Ii

Autologous Chondrocyte Implantation (ACI) has been established for years to treat cartilage defect. Application of tissue engineering has advantages over ACI as tissue engineering requires simpler procedures without leaving morbidity at the donor site. Decellularized bovine cartilage scaffold and hypoxic stem cell differentiation were used in this in vitro experimental study. Comparative test was done between three study groups using bone marrow mesenchymal stem cells treated in three different conditions: growth factor-rich chondrogenic medium, scaffold without growth factor, and combination of both. Each group was given two oxygen tension conditions of normoxia and hypoxic within phase of stem cell differentiation. Immunohistochemical examinations on SOX9, RUNX2, and collagen type II were done for evaluation. After 5-week treatment, the result showed that the highest expression SOX9 and collagen type II were found within the group that used the combination of both scaffold and chondrogenic medium in hypoxic condition. Collagen type II expression in scaffold without additional growth factor showed no statistically significant difference compared with the combination group in hypoxic condition. Cartilage tissue engineering has proven its effectiveness for cartilage regeneration. Decellularized biomaterial scaffold limited the use of growth factor resulting in better cost and resource efficiency.

scholarly journals Different phenotypes and chondrogenic responses of human menstrual blood and bone marrow mesenchymal stem cells to activin A and TGF-β3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ilona Uzieliene ◽  
Edvardas Bagdonas ◽  
Kazuto Hoshi ◽  
Tomoaki Sakamoto ◽  
Atsuhiko Hikita ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Collagen Type ◽  
Activin A ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Type Ii ◽  
Collagen Type Ii

Abstract Background Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A. Methods MenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-β3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture. Results MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro. Conclusion These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

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