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Localization of collagen type II in bovine articular cartilage. Cartilage sections were obtained form non-cultured explants and incubated with an antibody against type II collagen which was followed by a FITC-labeled second antibody. The sections were treated or not with hyaluronidase for 55 h. a without Hyaluronidase treatment (-hyal). Type II collagen label is present in a dot-like
Source publication
The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1alpha (IL-1alpha). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses re...
Contexts in source publication
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... low level of positively stained type II collagen was found in sections of non-cultured articular cartilage localized in the direct vicinity of the cells. In the interterritorial matrix between the chondrons it was present in a punctate pattern (Fig. 1a). Treatment of the sections with hyaluronidase resulted in a more intense staining of type II collagen around the chondron (Fig. ...
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... type II collagen was found in sections of non-cultured articular cartilage localized in the direct vicinity of the cells. In the interterritorial matrix between the chondrons it was present in a punctate pattern (Fig. 1a). Treatment of the sections with hyaluronidase resulted in a more intense staining of type II collagen around the chondron (Fig. ...
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... the intensity of staining proved to differ con- siderably. In all sections of articular cartilage a more pro- nounced labeling of type VI collagen was apparent (compare Fig. 7 with Fig. 6). Hardly any gelatinolytic activity was detectable in media obtained from articular cartilage explants that were cultured for up to 4 weeks without IL-1a (Fig. 8a, lanes 1-4). Also in cultures with IL-1a during the first 2 weeks gelatinolytic activity was not increased (Fig. 8a, lanes 1, 2 with IL-1a). At the 3 and 4 week time points, however, activity of MMP-2 and -9 became apparent (Fig. 8a, lanes 3, 4 with IL-1a). In particular the level of pro-MMP-9 was ...
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... nounced labeling of type VI collagen was apparent (compare Fig. 7 with Fig. 6). Hardly any gelatinolytic activity was detectable in media obtained from articular cartilage explants that were cultured for up to 4 weeks without IL-1a (Fig. 8a, lanes 1-4). Also in cultures with IL-1a during the first 2 weeks gelatinolytic activity was not increased (Fig. 8a, lanes 1, 2 with IL-1a). At the 3 and 4 week time points, however, activity of MMP-2 and -9 became apparent (Fig. 8a, lanes 3, 4 with IL-1a). In particular the level of pro-MMP-9 was ...
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... detectable in media obtained from articular cartilage explants that were cultured for up to 4 weeks without IL-1a (Fig. 8a, lanes 1-4). Also in cultures with IL-1a during the first 2 weeks gelatinolytic activity was not increased (Fig. 8a, lanes 1, 2 with IL-1a). At the 3 and 4 week time points, however, activity of MMP-2 and -9 became apparent (Fig. 8a, lanes 3, 4 with IL-1a). In particular the level of pro-MMP-9 was ...
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... contrast to articular cartilage, zymographic analysis of conditioned media obtained from nasal cartilage cultured in the absence of IL-1a revealed that at all culture time intervals studied MMP-2 and -9 were detectable (Fig. 8b, lanes 1-4). Only the proform of these enzymes proved to be ...
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... the presence of IL-1a (Fig. 8b, lanes 1-4 with IL-1a), not only the proform but also the active form of the enzymes could be detected. This was particularly apparent after 3 and 4 weeks of culture. At all time intervals more gelatinolytic activity was present in the media of nasal cartilage than in that of articular ...
Citations
... Importantly, the increases in Col2 1 and Acan, as the main ECM proteins in matured cartilage, and decreases in MMP13 and Col1a2, as important indicators for the differentiation of mature chondrocytes toward hypertrophy, illustrated that chondrocytes better maintained their phenotype on the soft micropillar substrate. In addition, the increase of Col5 l, that plays a vital role in mediating the progenitor cell niche of cartilage, [45] and Col6 1, that locates at the direct vicinity of chondrocytes and protects cartilage from degradation via proteolysis resistance, [46] further elucidated the phenotype maintenance on soft micropillar substrates, although the basic expression levels of these two proteins cannot be compared to Col2 1 at all. [47,48] We then performed qPCR and confirmed these gene changes of Col2a1, Acan, MMP13, and Col1a2 ( Figure 2i). ...
It is recognized that the changes in the physical properties of extracellular matrix (ECM) result in fine‐tuned cell responses including cell morphology, proliferation and differentiation. In this study, a novel patterned equidistant micropillar substrate based on polydimethylsiloxane (PDMS) is designed to mimic the collagen fiber‐like network of the cartilage matrix. By changing the component of the curing agent to an oligomeric base, micropillar substrates with the same topology but different stiffnesses are obtained and it is found that chondrocytes seeded onto the soft micropillar substrate maintain their phenotype by gathering type II collagen and aggrecan more effectively than those seeded onto the stiff micropillar substrate. Moreover, chondrocytes sense and respond to micropillar substrates with different stiffnesses by altering the ECM‐cytoskeleton‐focal adhesion axis. Further, it is found that the soft substrate‐preserved chondrocyte phenotype is dependent on the activation of Wnt/β‐catenin signaling. Finally, it is indicated that the changes in osteoid‐like region formation and cartilage phenotype loss in the stiffened sclerotic area of osteoarthritis cartilage to validate the changes triggered by micropillar substrates with different stiffnesses. This study provides the cell behavior changes that are more similar to those of real chondrocytes at tissue level during the transition from a normal state to a state of osteoarthritis.
... In articular cartilage, type VI collagen is present in small amounts (1-2%) forming a network that anchors the chondrocytes to the PCM through its interaction with a wide variety of ECM proteins, including type II collagen, type XIV collagen, cartilage matrix protein or matrilin-1, hyaluronan, decorin, and fibronectin; which implies the attachment and integrity of chondrocytes [13,95,102]. ...
... Therefore, based on the information reported in several studies [3,[12][13][14]24,50,51,56,[74][75][76]79,82,90,95,102,[106][107][108][113][114][115][116][117][118][119]127,128,[130][131][132][133], the composition of main and minor collagen types in ECM to consider the quality of a repaired cartilage as hyaline-type cartilage should be: • Absence of type I collagen. ...
Several collagen subtypes have been identified in hyaline articular cartilage. The main and most abundant collagens are type II, IX and XI collagens. The minor and less abundant collagens are type III, IV, V, VI, X, XII, XIV, XVI, XXII, and XXVII collagens. All these collagens have been found to play a key role in healthy cartilage, regardless of whether they are more or less abundant. Additionally, an exhaustive evaluation of collagen fibrils in a repaired cartilage tissue after a chondral lesion is necessary to determine the quality of the repaired tissue and even whether or not this repaired tissue is considered hyaline cartilage. Therefore, this review aims to describe in depth all the collagen types found in the normal articular cartilage structure, and based on this, establish the parameters that allow one to consider a repaired cartilage tissue as a hyaline cartilage.
... 30,33 Tissue engineering has been recognized as an alternative approach to existing regenerative cartilage therapies. 11,19,21 It can be combined with different chondrocyte sources (eg, articular, nasal, auricular) that require specific cell culture conditions 10 and possess different collagen 13 and proteoglycan 15 compositions, which may influence the long-term quality of regeneration. Unconventional cell sources such as nasal chondrocytes (NCs) display significant advantages in clinical practice and efficacy over traditional sources such as articular chondrocytes. ...
Background:
Bipolar or "kissing" cartilage lesions formed on 2 opposite articular surfaces of the knee joint are commonly listed as exclusion criteria for advanced cartilage therapies.
Purpose:
To test, in a pilot large-animal study, whether autologous nasal chondrocyte (NC)-based tissue engineering, recently introduced for the treatment of focal cartilage injuries, could provide a solution for challenging kissing lesions.
Study design:
Controlled laboratory study.
Methods:
Osteochondral kissing lesions were freshly introduced into the knee joints of 26 sheep and covered with NC-based grafts with a low or high hyaline-like extracellular matrix; a control group was treated with a cell-free scaffold collagen membrane (SCA). The cartilage repair site was assessed at 6 weeks and 6 months after implantation by histology, immunohistochemistry, and magnetic resonance imaging evaluation.
Results:
NC-based grafts, independently of their composition, induced partial hyaline cartilage repair with stable integrity in surrounding healthy tissue at 6 months after treatment. The SCA repaired cartilage to a similar degree to that of NC-based grafts.
Conclusion:
Kissing lesion repair, as evidenced in this sheep study, demonstrated the feasibility of the treatment of complex cartilage injuries with advanced biological methods. However, the potential advantages of an NC-based approach over a cell-free approach warrant further investigations in a more relevant preclinical model.
Clinical relevance:
NC-based grafts currently undergoing phase II clinical trials have a high potential to replace existing cartilage therapies that show significant limitations in the quality and reproducibility of the repair method. We have brought this innovative concept to the next level by addressing a new clinical indication.
... The stimulation of BNC with IL-lα represents a reproducible model of the cartilage destruction that is prevalent in the joint diseases [6, 16, 17]. In previous studies, Kozaci et al. [6] and Jansen et al. [19] have used IL-1α (50 ng/ml), and BNC was almost completely degraded within 21 and 18 days, respectively. In the present investigation, the lower concentration of IL-lα (10 ng/ml) was used for 28 days as used for other groups [16, 20] . ...
Background:
Previous studies have shown that some cytokines have protective effects on cartilage in joint diseases. In the current study, effects of IL-4 against morphological changes and tissue degradation induced by IL-1α on bovine nasal cartilage (BNC) explants were investigated.
Methods:
Fresh BNC samples were prepared from a slaughterhouse under sterile conditions. BNC explants culture was treated with both IL-lα (10 ng/ml) and IL-4 (50 ng/ml) at the same time for 28 days. The morphological characteristics of explants were assessed by using histology techniques and invert microscopy. Matrix metalloproteinase-1 (MMP-1) production was assessed within different days by using Western blotting.
Results:
IL-lα induced prominent cartilage morphology degradation. The pro and active form of MMP-1 band substantially increased at day 21 of culture. In the presence of both IL-lα and IL-4, chondrocytes preserved their ordinary normal phenotype with intact extracellular matrix. In addition, a significant reduction in pro-MMP-1and inhibition of active MMP-1 was seen.
Conclusion:
In conclusion, IL-4 could be regarded as a potential candidate in cartilage protecting against the degradation changes of IL-lα. It seems that the preservation effect of IL-4 is associated with significant reduction of MMP-1.
... Degenerative joint disorders such as rheumatoid
arthritis (RA) and osteoarthritis (OA) are characterized
by the destruction of articular cartilage (1,
2). Articular cartilage is composed of extracellular
matrix (ECM) and chondrocytes, which are sparsely
distributed throughout the matrix and appear to
play an important role in the pathogenesis of joint
diseases (3, 4). ...
A study of the histological events under interleukin-1α (IL-lα) induction of bovine nasal cartilage (BNC) could result in useful data to better understand the mechanisms involved in tissue breakdown in joint diseases. The aim of this study was to investigate the effects of IL-lα on chondrocyte phenotype and extracellular matrix (ECM) changes in BNC explants.
In this experimental study, samples were divided into two groups. Group I (control group) BNC explants were cultured only in Dulbecco's modified Eagle's medium (DMEM). In group II, BNC explants were treated with IL-lα (10 ng/ ml) for 28 days. Then, samples were harvested on culture days 3, 7, 14, 21 and 28 and chondrocyte morphology and ECM alterations were assessed by invert microscopy and histology by hematoxylin and eosin (H&E) and Alcian blue. Cell viability was evaluated by the lactate dehydrogenase (LDH) assay test. Data were analyzed by the t test and p<0.05 was considered significant.
IL-lα induced significant morphological changes in cartilage. In the presence of IL-lα, most chondrocytes transformed into a fibroblast-like morphology with a granular black point appearance. An increase in the cell: matrix ratio was observed and there were decreased numbers of chondrocytes.IL-lα induced breakdown of ECM. We observed partial degradation of ECM between days 7-14 and complete degradation occurred between days 21-28 of culture. The LDH levels increased.
IL-1α induced morphological changes in chondrocytes and increased destruction of cartilage ECM. There was a parallel correlation between proteoglycan degradation and changes in chondrocyte morpholgy.
... Increased levels of matrix metalloproteinases (MMP) result in degradation of cartilage ECM components, destruction of bone, and joint abnormalities. Three mammalian collagenases include interstitial collagenase (MMP-1), neutrophil collagenase (MMP- 8), and collagenase 3 (MMP-13) can specifically cleave triple helical collagen56789. Previous studies have reported that MMP-1 is one of the predominant enzymes that mediate fibrillar collagen degradation in bovine nasal cartilage (BNC) explant culture system [4, 6]. ...
... Increased MMP production results in degradation of cartilage ECM components56789. In the presence IL-1α + IL-4, MMP-1 band was clearly decreased that was in agreement with Cleaver et al. study [ study, BNC explants were treated with IL-1α and oncostatin M (10 ng/ml), and collagenolytic activities were determined by bioassay and blot analysis. ...
Current treatments for joint diseases are moderately successful, but unfortunately are associated with significant side effects. This study was undertaken to investigate the combination effects of IL-4 and prednisolone on tissue characteristics and production of matrix metalloproteinase-1(MMP-1) in IL-lα-treated bovine nasal cartilage (BNC) explants.
BNC explants were cultured in DMEM with IL-lα (10 ng/ml), IL-4 (50 ng/ml) and prednisolone (1 or 1,000 nM) at the same time for 28 days. At days 3, 7, 14, 21 and 28, the media were collected and replaced with fresh media, and the removed media were stored at -20°C. The alterations of tissue characteristics were assessed by using histology techniques. Western-blot method was used to determine the effects of IL-4 and prednisolone combination on MMP-1 production. The cell viability was evaluated by using lactate dehydrogenase assay test.
In the presence of IL-lα alone, most chondrocytes were transformed into fibroblast-like morphology with pyknotic nuclei at day 28. In addition, a clear band of MMP-1 and extracellular matrix (ECM) degradation were observed. In combination of IL-4 and prednisolone, chondrocytes preserved their ordinary normal features. MMP-1 band formation was completely inhibited and ECM absolutely showed normal characteristics. IL-4 and prednisolone did not show cytotoxicity effects on BNC explant culture.
This combination can strongly preserve cartilage from degradation features and the data possibly suggest that the combination of IL-4 and prednisolone could be a candidate for alternative therapy in joint diseases.
Chondrocyte apoptosis plays an important role in the developmental dysplasia of the hip (DDH) development. It has been found that WNT1 inducible signaling pathway protein 2 (WISP‐2) and peroxisome proliferator‐activated receptor γ (PPARγ) are involved in cell apoptosis. In this study, we performed the straight‐leg swaddling DDH rat model and we found that cartilage degradation and chondrocyte apoptosis were remarkably increased in DDH rats in vivo. Moreover, we found that WISP‐2 was upregulated in hip acetabular cartilage of DDH rats compared to control rats. Next, the effects of WISP‐2 on chondrocyte apoptosis and its possible underlying mechanism were examined in vitro. The lentivirus‐mediated gain‐ and loss‐of‐function experiments of WISP‐2 and peroxisome proliferator‐activated receptor γ (PPARγ) for cell viability and apoptosis were performed in primary rat chondrocytes. The results showed that the overexpression of WISP‐2 induced chondrocyte apoptosis, and knockdown of WISP‐2 could suppress the chondrocyte apoptosis induced by advanced glycation end products (AGE). Additionally, WISP‐2 could negatively regulate the expression of PPARγ in chondrocytes. Moreover, the knockdown of PPARγ promoted chondrocyte apoptosis and overexpression of PPARγ abated the increased apoptosis and decreased cell viability of chondrocytes induced by WISP‐2. This study demonstrated that WISP‐2 might contribute to chondrocyte apoptosis of hip acetabular cartilage through regulating PPARγ expression and activation, which may play an important role in the development of DDH.
Pathological changes, such as articular cartilage degeneration, destruction, and hyperosteogeny, are regarded as the main features of osteoarthritis (OA). Sinomenine (SIN) is a monomeric component purified from the plant Sinomenium acutum which has been found to have anti-inflammatory effects, however, the mechanism of action of SIN on OA is not clear. In this study, we evaluated whether SIN could regulate the inflammatory response induced by interleukin (IL)-1β and improve outcomes in the instability model of OA (medial meniscus mice (DMM)) by acting on the Nrf2/HO-1 and NF-κ B signaling pathways in chondrocytes. From our experiments, which include Griess reaction, ELISA, Western blot, and immunofluorescence, we found that SIN not only down-regulated the expression of pro-inflammatory factors induced by IL-1β, including; inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nitricoxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), but also decreased the production of IL-1β-induced cartilage matrix catabolic enzymes including; ADAMTS-5 and MMPs, in mouse chondrocytes. In addition, the degradation of aggrecan and type II collagen protein in the extracellular matrix (ECM) stimulated by IL-1β was reversed. Most importantly, we have revealed for the first time that in OA, SIN inhibited the inflammatory response and ECM degradation by activating the Nrf2/HO-1 signaling pathways and inhibiting NF-κB activity in mouse-cartilage cells. In in vivo experiments, SIN treatment helped to improve the cartilage destruction in OA model mice. In conclusion, this study has demonstrated that SIN inhibits the IL-1β-induced inflammatory response and cartilage destruction by activating the Nrf2/HO-1 signaling pathway and inhibiting the NF-κB signaling pathway in mouse chondrocytes, suggesting a new use for SIN in the treatment of OA.
