scholarly journals Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture

1999 ◽  
Vol 194 (3) ◽  
pp. 343-353
Author(s):  
L. A. DURRANT ◽  
C. W. ARCHER ◽  
M. BENJAMIN ◽  
J. R. RALPHS
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Cell Surface ◽  
Organ Culture ◽  
Rapid Change ◽  
Cartilage Explants ◽  
Deep Zone ◽  
Joint Movement ◽  
Free Swelling ◽  
In Cells

Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical deformation and recovery. This loading is essential for chondrocytes to perform their normal function of maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional (3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected by culture. Further experiments were performed to test the effects of load on the cytoskeleton. Explants were placed in culture and immediately subjected to static uniaxial radially unconfined compressive loads of 0.5, 1, 2 or 4 MPa for 1 h using a pneumatic loading device. Loads greater than 0.5 MPa maintained the vimentin organisation over the culture period. At 0.5 MPa, the chondrocytes within the explant behaved as in free-swelling culture. The rapid change in vimentin organisation probably relates to rapid swelling of the explants—under free-swelling conditions, these reached their maximum swollen size in just 15 min of culture. The chondrocytes' response to change in tissue dimensions, and thus to their relationship to their immediate environment, was to disassemble their vimentin networks. Loading probably counteracts the swelling pressure of the tissue. Overall, this work suggests that chondrocytes maintain their actin cytoskeleton and modify their vimentin cytoskeleton in response to changing mechanical conditions.

Ameliorating Glycosaminoglycan (GAG) Loss and Cell Death in Articular Cartilage Following Single-Impact Loading

2007 ◽  
Author(s):  
Roman M. Natoli ◽  
Kyriacos A. Athanasiou
Keyword(s):  
Extracellular Matrix ◽  
Cell Death ◽  
Articular Cartilage ◽  
Impact Loading ◽  
Biomechanical Properties ◽  
Cartilage Explants ◽  
Single Impact ◽  
Igf I ◽  
Post Traumatic ◽  
Bioactive Agents

Impact loading of articular cartilage leads to post-traumatic osteoarthritis (OA) through its effects on the cells and extracellular matrix (ECM) of the tissue. Studies have shown the level of impact or injurious compression correlates with increased cell death, degradation of the ECM, and detrimental changes in biomechanical properties [1]. Recently, several bioactive agents, such as P188 and IGF-I, have shown promising results by reducing cell death following injurious compression of cartilage explants [2, 3].

scholarly journals Localization of hyaluronic acid in human articular cartilage.

1994 ◽  
Vol 42 (4) ◽  
pp. 513-522 ◽  
Author(s):  
A Asari ◽  
S Miyauchi ◽  
S Kuriyama ◽  
A Machida ◽  
K Kohno ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Dermatan Sulfate ◽  
Keratan Sulfate ◽  
Human Articular Cartilage ◽  
Middle Zone ◽  
Deep Zone ◽  
Weak Staining ◽  
Pre Treatment

To demonstrate localization of hyaluronic acid (HA) in articular cartilage of the human femur, biotinylated HA-binding region, which specifically binds HA molecules, was applied to the tissue. In sections fixed by 2% paraformaldehyde-2% glutaraldehyde, HA staining was detected in lamina splendens and chondrocytes in the middle zone. By pretreatment with trypsin, intense HA staining appeared in the extracellular matrix of the deep zone and weak staining in the superficial and middle zones. Moreover, pre-treatment with chondroitinase ABC (CHase ABC) intensely enhanced the stainability for HA in the superficial and middle zones and weakly in the deeper zone. Combined pre-treatment of trypsin with CHase ABC abolished intra- and extracellular staining for HA in all zones. By microbiochemical study, the concentrations of HA and dermatan sulfate were high in the middle zone, whereas those of chondroitin sulfate and keratan sulfate were high in the deep zone. These results suggest that HA is abundantly synthesized in and secreted from the chondrocytes, particularly in the middle zone, whereas it is largely masked by proteoglycan constituents in the extracellular matrix.

Effects of interleukin-1β on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants

2003 ◽  
Vol 57 (7) ◽  
pp. 314-319 ◽  
Author(s):  
Giordano Stabellini ◽  
Monica De Mattei ◽  
Carla Calastrini ◽  
Nicoletta Gagliano ◽  
Claudia Moscheni ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Cartilage Explants ◽  
Interleukin 1Β ◽  
Bovine Articular Cartilage

Matrix metalloproteinase 17 is necessary for cartilage aggrecan degradation in an inflammatory environment

2011 ◽  
Vol 70 (4) ◽  
pp. 683-689 ◽  
Author(s):  
Kristen M Clements ◽  
Jo K Flannelly ◽  
Jonathan Tart ◽  
Sarah M V Brockbank ◽  
John Wardale ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Synovial Fluid ◽  
Matrix Metalloproteinase ◽  
Cartilage Damage ◽  
Cartilage Explants ◽  
Human Cartilage ◽  
Cartilage Extracellular Matrix ◽  
The Media ◽  
Meniscotibial Ligament

ObjectiveAggrecan is a critical component of cartilage extracellular matrix. Several members of the ‘a disintegrin and metalloproteinase with thrombospondin motifs’ (ADAMTS) family have been characterised as aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1β (IL-1β) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated.MethodsMMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1β in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1β or meniscotibial ligament transaction was carried out in MMP17 null mice.ResultsThe data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1β mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor.IAI of IL-1β into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17.ConclusionThese data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.

scholarly journals Genipin crosslinked chitosan/PEO nanofibrous scaffolds exhibiting an improved microenvironment for the regeneration of articular cartilage

2021 ◽  
pp. 088532822110020
Author(s):  
Kuan Yong Ching ◽  
Orestis Andriotis ◽  
Bram Sengers ◽  
Martin Stolz
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Articular Chondrocytes ◽  
Immunohistochemical Analysis ◽  
Chitosan Solution ◽  
Deep Zone ◽  
Superficial Zone ◽  
Fibril Structure ◽  
Nanofibrous Scaffolds ◽  
Poly Ethylene

Towards optimizing the growth of extracellular matrix to produce repair cartilage for healing articular cartilage (AC) defects in joints, scaffold-based tissue engineering approaches have recently become a focus of clinical research. Scaffold-based approaches by electrospinning aim to support the differentiation of chondrocytes by providing an ultrastructure similar to the fibrillar meshwork in native cartilage. In a first step, we demonstrate how the blending of chitosan with poly(ethylene oxide) (PEO) allows concentrated chitosan solution to become electrospinnable. The chitosan-based scaffolds share the chemical structure and characteristics of glycosaminoglycans, which are important structural components of the cartilage extracellular matrix. Electrospinning produced nanofibrils of ∼100 nm thickness that are closely mimicking the size of collagen fibrils in human AC. The polymer scaffolds were stabilized in physiological conditions and their stiffness was tuned by introducing the biocompatible natural crosslinker genipin. We produced scaffolds that were crosslinked with 1.0% genipin to obtain values of stiffness that were in between the stiffness of the superficial zone human AC of 600 ± 150 kPa and deep zone AC of 1854 ± 483 kPa, whereas the stiffness of 1.5% genipin crosslinked scaffold was similar to the stiffness of deep zone AC. The scaffolds were degradable, which was indicated by changes in the fibril structure and a decrease in the scaffold stiffness after seven months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes (HACs) showed a cell viability of over 90% on the scaffolds and new extracellular matrix deposited on the scaffolds.

scholarly journals Osteoarthritic Infrapatellar Fat Pad Aggravates Cartilage Degradation via Activation of p38MAPK and ERK1/2 Pathways

2020 ◽  
Author(s):  
Zuoqing Zhou ◽  
Su'an Tang ◽  
Xiaoyu Nie ◽  
Yiqun Zhang ◽  
Delong Li ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Cartilage Degradation ◽  
Cartilage Explants ◽  
Infrapatellar Fat Pad ◽  
Fat Pad ◽  
Primary Chondrocytes ◽  
Tnf Α ◽  
And Cartilage ◽  
Human Primary Chondrocytes

Abstract Background: Although existing studies have suggested the involvement of the infrapatellar fat pad (IPFP) during the development of knee osteoarthritis (OA), the role of IPFP is still controversial. This study aimed to investigate the biochemical effects of osteoarthritic IPFP on cartilage and the underlying mechanisms.Methods: Human IPFP and articular cartilage were collected from end-stage OA patients during total knee arthroplasty. IPFP derived fat-conditioned medium (FCM) was used to stimulate human primary chondrocytes and cartilage explants. CCK8 was used to detect the viability of human chondrocyte. qRT-PCR and western blotting was performed to evaluate the balance of extracellular matrix (ECM) catabolism and anabolism in human chondrocytes with FCM stimulation. Functional effect of osteoarthritic IPFP was also demonstrated in human articular cartilage by ex vivo assay. Activation of relative pathways and its effects on chondrocytes were assessed through immunoblotting and inhibition experiments, respectively. Neutralization test was performed to identify the main factors and their associated pathways responsible for the effects of IPFP. Results: Osteoarthritic IPFP-derived FCM significantly induced extracellular matrix (ECM) degradation in both human primary chondrocytes and cartilage explants. Several pathways, such as NF-κB, mTORC1, p38MAPK, JNK, and ERK1/2 signaling were significantly activated in human chondrocytes with osteoarthritic IPFP-derived FCM stimulation. Interestingly, inhibition of p38MAPK and ERK1/2 signaling pathway could alleviate the detrimental effects of FCM on chondrocytes while inhibition of other signaling pathways had no similar results. In addition, IL-1β and TNF-α instead of IL-6 in osteoarthritic IPFP-derived FCM played a key role in cartilage degradation via activating p38MAPK rather than ERK1/2 signaling pathway.Conclusions: Osteoarthritic IPFP induces the degradation and inflammation of cartilage via activation of p38MAPK and ERK1/2 pathways, in which IL-1β and TNF-α act as the key factors. Our study suggests that modulating the effects of IPFP on cartilage may be a promising strategy for knee OA intervention.

Depth Dependent Diffusivity Profile in Bovine Articular Cartilage: Comparing Transverse and Axial Diffusivities

2007 ◽  
Author(s):  
Onyi N. Irrechukwu ◽  
Marc E. Levenston
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Articular Surface ◽  
Deep Zone ◽  
Superficial Zone ◽  
Transport Pathway ◽  
Bovine Articular Cartilage ◽  
Extracellular Matrix Components ◽  
Matrix Components ◽  
Oriented Parallel

As articular cartilage is avascular, diffusion at a tissue length scale is the primary mode of solute and nutrient transport to its cells. The major extracellular matrix components are water (70–80%), chondrocytes, collagen (10–20%) and proteoglycans (5–10%) bearing sulfated glycosaminoglycans (GAG) [1]. Electron microscopy studies have shown that articular cartilage can be regarded as having three separate structural zones — superficial, middle and deep. The proportions of the various matrix components vary from the surface to the deep zone in any given joint and the greatest variations in content occur in the GAG content [2]. In addition the collagen fiber alignment varies, with fibers oriented parallel to the articular surface in the superficial zone, randomly oriented in the middle zone and oriented perpendicular to the surface in the deep zone. To a large extent, it is the spatially inhomogeneous composition of articular cartilage and microstructural orientation of its extracellular matrix components that determines the tortuosity of the transport pathway [3]. We therefore hypothesized that the diffusivity profile of a solute through the cartilage depth is inversely related to the GAG content and that the ratio between the axial and lateral diffusivities within each cartilage zone is related to the degree of anisotropy within the zone.

scholarly journals A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage

2018 ◽  
Vol 7 (3) ◽  
pp. 205-212 ◽  
Author(s):  
Y-C. Lin ◽  
A. C. Hall ◽  
A. H. R. W. Simpson
Keyword(s):  
Articular Cartilage ◽  
Organ Culture ◽  
Analysis Of Variance ◽  
Dynamic Load ◽  
Joint Movement ◽  
Chondrocyte Viability ◽  
Dynamic Movement ◽  
Culture Model ◽  
Custom Made ◽  
Static Conditions

Objectives The purpose of this study was to create a novel ex vivo organ culture model for evaluating the effects of static and dynamic load on cartilage. Methods The metatarsophalangeal joints of 12 fresh cadaveric bovine feet were skinned and dissected aseptically, and cultured for up to four weeks. Dynamic movement was applied using a custom-made machine on six joints, with the others cultured under static conditions. Chondrocyte viability and matrix glycosaminoglycan (GAG) content were evaluated by the cell viability probes, 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), and dimethylmethylene blue (DMMB) assay, respectively. Results Chondrocyte viability in the static model decreased significantly from 89.9% (sd 2.5%) (Day 0) to 66.5% (sd 13.1%) (Day 28), 94.7% (sd 1.1%) to 80. 9% (sd 5.8%) and 80.1% (sd 3.0%) to 46.9% (sd 8.5%) in the superficial quarter, central half and deep quarter of cartilage, respectively (p < 0.001 in each zone; one-way analysis of variance). The GAG content decreased significantly from 6.01 μg/mg (sd 0.06) (Day 0) to 4.71 μg/mg (sd 0.06) (Day 28) (p < 0.001; one-way analysis of variance). However, with dynamic movement, chondrocyte viability and GAG content were maintained at the Day 0 level over the four-week period without a significant change (chondrocyte viability: 92.0% (sd 4.0%) (Day 0) to 89.9% (sd 0.2%) (Day 28), 93.1% (sd 1.5%) to 93.8% (sd 0.9%) and 85.6% (sd 0.8%) to 84.0% (sd 2.9%) in the three corresponding zones; GAG content: 6.18 μg/mg (sd 0.15) (Day 0) to 6.06 μg/mg (sd 0.09) (Day 28)). Conclusion Dynamic joint movement maintained chondrocyte viability and cartilage GAG content. This long-term whole joint culture model could be of value in providing a more natural and controlled platform for investigating the influence of joint movement on articular cartilage, and for evaluating novel therapies for cartilage repair. Cite this article: Y-C. Lin, A. C. Hall, A. H. R. W. Simpson. A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage. Bone Joint Res 2018;7:205–212. DOI: 10.1302/2046-3758.73.BJR-2017-0320.

scholarly journals Functional grading of pericellular matrix surrounding chondrocytes: potential roles in signaling and fluid transport

2018 ◽  
Author(s):  
F. Saadat ◽  
M.J. Lagieski ◽  
V. Birman ◽  
S. Thomopoulos ◽  
G.M. Genin
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Cell Surface ◽  
Fluid Transport ◽  
Pericellular Matrix ◽  
Mechanical Environment ◽  
Spatial Gradients ◽  
Linearized Model ◽  
Mechanical Microenvironment

AbstractThe extracellular matrix surrounding chondrocytes within cartilage and fibrocartilage has spatial gradients in mechanical properties. Although the function of these gradients is unknown, the potential exists for cells to tailor their mechanical microenvironment through these gradients. We hypothesized that these gradients enhance fluid transport around the cell during the slow loading cycles that occur over the course of a day, and that this enhancement changes the nature of the mechanical signals received at the surface of the cell. To test this hypothesis, we studied the effect of these gradients on the mechanical environment around a chondrocyte using a closed form, linearized model. Results demonstrated that functional grading of the character observed around chondrocytes in articular cartilage enhances fluid transport, and furthermore inverts compressive radial strains to provide tensile signals at the cell surface. The results point to several potentially important roles for functional grading of the pericellular matrix.

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