The Effect of Adenosine on Extracellular Matrix Production in Porcine Intervertebral Disc Cells

2018 ◽  
Vol 206 (1-2) ◽  
pp. 73-81 ◽  
Author(s):  
Xue Yin ◽  
Silvia  Gonzales ◽  
Somya Sha ◽  
Howard Levene ◽  
Chun-Yuh Huang
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Intervertebral Disc ◽  
Adenosine Receptors ◽  
Type Ii Collagen ◽  
Underlying Mechanism ◽  
Receptor Activation ◽  
Type Ii ◽  
Atp Production ◽  
Intracellular Atp

Compressive loading promotes adenosine triphosphate (ATP) production and release by intervertebral disc (IVD) cells. Extracellular ATP can be rapidly hydrolyzed by ectonucleotidases. Adenosine, one of the adenine derivatives of ATP hydrolysis, can modulate diverse cellular actions via adenosine receptors. The objectives of this study were to investigate the effects of exogenous adenosine on the production of extracellular matrix (ECM; i.e., collagen type II and aggrecan) and ATP of IVD cells and explore the underlying mechanism of action. It was found that adenosine treatment significantly upregulated aggrecan and type II collagen gene expression and the ATP level in IVD cells. Dipyridamole, an adenosine transport blocker, completely suppressed the effects of adenosine on the ATP production and ECM gene expression of the IVD cells, whereas antagonists of adenosine receptors did not significantly affect adenosine-treated IVD cells. The findings suggested that elevated intracellular ATP and upregulation of ECM gene expression by adenosine treatment are mainly due to adenosine uptake rather than receptor activation. Since ECM biosynthesis is a high ATP demanding process, supplementing adenosine could be beneficial as IVD cells are able to utilize it to replenish intracellular ATP and sequentially promote ECM production, which is constantly suppressed by limited nutrition supply due to the avascular nature of the IVD.

scholarly journals Combining Innovative Bioink and Low Cell Density for the Production of 3D-Bioprinted Cartilage Substitutes: A Pilot Study

2020 ◽  
Vol 2020 ◽  
pp. 1-16 ◽  
Author(s):  
Christel Henrionnet ◽  
Léa Pourchet ◽  
Paul Neybecker ◽  
Océane Messaoudi ◽  
Pierre Gillet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Cell Encapsulation ◽  
Type Ii Collagen ◽  
Mitochondrial Activity ◽  
Type Ii ◽  
3D Bioprinting ◽  
Matrix Synthesis ◽  
M Cell ◽  
Low Concentration

3D bioprinting offers interesting opportunities for 3D tissue printing by providing living cells with appropriate scaffolds with a dedicated structure. Biological advances in bioinks are currently promising for cell encapsulation, particularly that of mesenchymal stem cells (MSCs). We present herein the development of cartilage implants by 3D bioprinting that deliver MSCs encapsulated in an original bioink at low concentration. 3D-bioprinted constructs (10×10×4 mm) were printed using alginate/gelatin/fibrinogen bioink mixed with human bone marrow MSCs. The influence of the bioprinting process and chondrogenic differentiation on MSC metabolism, gene profiles, and extracellular matrix (ECM) production at two different MSC concentrations (1 million or 2 million cells/mL) was assessed on day 28 (D28) by using MTT tests, real-time RT-PCR, and histology and immunohistochemistry, respectively. Then, the effect of the environment (growth factors such as TGF-β1/3 and/or BMP2 and oxygen tension) on chondrogenicity was evaluated at a 1 M cell/mL concentration on D28 and D56 by measuring mitochondrial activity, chondrogenic gene expression, and the quality of cartilaginous matrix synthesis. We confirmed the safety of bioextrusion and gelation at concentrations of 1 million and 2 million MSC/mL in terms of cellular metabolism. The chondrogenic effect of TGF-β1 was verified within the substitute on D28 by measuring chondrogenic gene expression and ECM synthesis (glycosaminoglycans and type II collagen) on D28. The 1 M concentration represented the best compromise. We then evaluated the influence of various environmental factors on the substitutes on D28 (differentiation) and D56 (synthesis). Chondrogenic gene expression was maximal on D28 under the influence of TGF-β1 or TGF-β3 either alone or in combination with BMP-2. Hypoxia suppressed the expression of hypertrophic and osteogenic genes. ECM synthesis was maximal on D56 for both glycosaminoglycans and type II collagen, particularly in the presence of a combination of TGF-β1 and BMP-2. Continuous hypoxia did not influence matrix synthesis but significantly reduced the appearance of microcalcifications within the extracellular matrix. The described strategy is very promising for 3D bioprinting by the bioextrusion of an original bioink containing a low concentration of MSCs followed by the culture of the substitutes in hypoxic conditions under the combined influence of TGF-β1 and BMP-2.

scholarly journals Spatial distribution of type II collagen gene expression in the mouse intervertebral disc

JOR Spine ◽  
2019 ◽  
Vol 2 (4) ◽  
Author(s):  
Yulong Wei ◽  
Robert J. Tower ◽  
Zuozhen Tian ◽  
Bhavana Mohanraj ◽  
Robert L. Mauck ◽  
...  
Keyword(s):  
Gene Expression ◽  
Spatial Distribution ◽  
Intervertebral Disc ◽  
Type Ii Collagen ◽  
Collagen Gene ◽  
Type Ii ◽  
Collagen Gene Expression

Type-II collagen gene expression is transiently upregulated in experimentally induced degeneration of rabbit intervertebral disc

1997 ◽  
Vol 15 (4) ◽  
pp. 528-538 ◽  
Author(s):  
Hironari Takaishi ◽  
Osamu Nemoto ◽  
Masanobu Shiota ◽  
Toshiyuki Kikuchi ◽  
Harumoto Yamada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intervertebral Disc ◽  
Type Ii Collagen ◽  
Collagen Gene ◽  
Type Ii ◽  
Collagen Gene Expression ◽  
Experimentally Induced

Down-regulation of chondrocyte aggrecan and type-II Collagen gene expression correlates with increases in static compression magnitude and duration

1999 ◽  
Vol 17 (6) ◽  
pp. 836-842 ◽  
Author(s):  
Paula M. Ragan ◽  
Alison M. Badger ◽  
Michael Cook ◽  
Vicki I. Chin ◽  
Maxine Gowen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type Ii Collagen ◽  
Collagen Gene ◽  
Type Ii ◽  
Down Regulation ◽  
Static Compression ◽  
Collagen Gene Expression

scholarly journals Elevated lymphotoxin-α (TNFβ) is associated with intervertebral disc degeneration

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhu Guo ◽  
Chensheng Qiu ◽  
Christina Mecca ◽  
Yang Zhang ◽  
Jiang Bian ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Cell Viability ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Caspase 3 ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Optimal Conditions ◽  
Cell Degeneration ◽  
Lymphotoxin Α

Abstract Background Intervertebral disc degeneration (IVDD) is a primary cause of degenerative disc diseases; however, the mechanisms underlying the degeneration remain unclear. The immunoinflammatory response plays an important role in IVDD progression. The inflammatory cytokine lymphotoxin-α (LTα), formerly known as TNFβ, is associated with various pathological conditions, while its role in the pathogenesis of IVDD remains elusive. Methods Real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and enzyme-linked immunosorbent assays were used to assess the levels of LTα in human nucleus pulposus (NP) tissues between degeneration and control groups. The plasma concentrations of LTα and C-reactive protein (CRP) were compared between healthy and IVDD patients. Rat primary NP cells were cultured and identified via immunofluorescence. Methyl-thiazolyl-tetrazolium assays and flow cytometry were used to evaluate the effects of LTα on rat NP cell viability. After NP cells were treated with LTα, degeneration-related molecules (Caspase-3, Caspase-1, matrix metalloproteinase (MMP) -3, aggrecan and type II collagen) were measured via RT-qPCR and WB. Results The levels of both the mRNA and protein of LTα in human degenerated NP tissue significantly increased. Plasma LTα and CRP did not differ between healthy controls and IVDD patients. Rat primary NP cells were cultured, and the purity of primary NP cells was > 90%. Cell experiments showed inversely proportional relationships among the LTα dose, treatment time, and cell viability. The optimal conditions (dose and time) for LTα treatment to induce rat NP cell degeneration were 5 μg/ml and 48 ~ 72 h. The apoptosis rate and the levels of Caspase-3, Caspase-1, and MMP-3 significantly increased after LTα treatment, while the levels of type II collagen and aggrecan were decreased, and the protein expression levels were consistent with their mRNA expression levels. Conclusions This study demonstrated that elevated LTα is closely associated with IVDD and that LTα may induce NP cell apoptosis and reduce important extracellular matrix (ECM) proteins, which cause adverse effects on IVDD progress. Moreover, the optimal conditions for LTα treatment to induce NP cell degeneration were determined.

scholarly journals Type II collagen-hyaluronan hydrogel – a step towards a scaffold for intervertebral disc tissue engineering

2010 ◽  
Vol 20 ◽  
pp. 134-148 ◽  
Author(s):  
L Calderon ◽  
◽  
E Collin ◽  
D Velasco-Bayon ◽  
M Murphy ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Intervertebral Disc ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Disc Tissue

scholarly journals Effects of Extracellular Matrix on the Morphology and Behaviour of Rabbit Auricular Chondrocytes in Culture

2005 ◽  
Vol 2005 (4) ◽  
pp. 364-373 ◽  
Author(s):  
Vega Villar-Suárez ◽  
B. Colaço ◽  
I. Calles-Venal ◽  
I. G. Bravo ◽  
J. G. Fernández-Álvarez ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type Ii Collagen ◽  
Cell Phenotype ◽  
Nodule Formation ◽  
Type Ii ◽  
Protein Extract ◽  
Positive Effects ◽  
Reticular Pattern ◽  
Chondrocyte Cultures ◽  
Plastic Surfaces

Isolated chondrocytes dedifferentiate to a fibroblast-like shape on plastic substrata and proliferate extensively, but rarely form nodules. However, when dissociation is not complete and some cartilage remnants are included in the culture, proliferation decreases and cells grow in a reticular pattern with numerous nodules, which occasionally form small cartilage-like fragments. In an attempt to reproduce this stable chondrogenic state, we added a cartilage protein extract, a sugar extract, and hyaluronan to the medium of previously dedifferentiated chondrocytes. When protein extract was added, many cartilaginous nodules appeared. Hyaluronan produced changes in cell phenotype and behaviour, but not nodule formation. Protein extract has positive effects on the differentiation of previously proliferated chondrocytes and permits nodule formation and the extensive production of type-II collagen. A comparison with incompletely dissociated chondrocyte cultures suggests that the presence of some living cells anchored to their natural extracellular matrix provides some important additional factors for the phenotypical stability of chondrocytes on plastic surfaces. In order to elucidate if it is possible that the incidence of apoptosis is related to the results, we also characterized the molecular traits of apoptosis.

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