Importance of reference gene selection for articular cartilage mechanobiology studies.

OBJECTIVE: Identification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively. DESIGN: The expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder. RESULTS: Appropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions. CONCLUSION: This study demonstrates that commonly used 'reference genes' may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation.

Protein kinase R plays a pivotal role in oncostatin M and interleukin-1 signalling in bovine articular cartilage chondrocytes.

This study investigated whether treatment of articular cartilage chondrocytes with a combination of oncostatin M (OSM) and interleukin-1 (IL-1) could induce a degradative phenotype that was mediated through the protein kinase R (PKR) signalling pathway. High-density monolayer cultures of full depth, bovine chondrocytes were treated with a combination of OSM and IL-1 (OSM+IL-1) for 7 days. To inhibit the activation of PKR, a pharmacological inhibitor of PKR was added to duplicate cultures. Pro- and active matrix metalloproteinase-9 (MMP9) and MMP9 mRNA were significantly upregulated by OSM+IL-1 through a PKR dependent mechanism. ADAMTS4 and ADAMTS5 mRNA were also upregulated by OSM+IL-1. The upregulation of ADAMTS4 and ADAMTS5 were, in part, mediated through PKR. OSM+IL-1 resulted in a loss of type II collagen, which could not be rescued by PKR inhibition. OSM+IL-1 reduced the expression of COL2A1 (type II collagen), COL9A1 (type IX collagen), COL11A1 (type XI collagen), and ACAN (aggrecan) mRNAs. Expression of type II and XI collagen and aggrecan was reduced further when PKR was inhibited. OSM+IL-1 resulted in an 11-fold increase in TNFa mRNA which was, in part, mediated through the PKR pathway. This study demonstrates, for the first time, that a number of catabolic and pro-inflammatory effects known to be important in human arthritis and induced by OSM and IL-1, are mediated by the PKR signalling pathway.

The effects of cyclic tensile strain on the organisation and expression of cytoskeletal elements in bovine intervertebral disc cells: an in vitro study.

It is still relatively unclear how intervertebral disc (IVD) cells sense a mechanical stimulus and convert this signal into a biochemical response. Previous studies demonstrated that the cytoskeletal elements are mechano-responsive in many cell types and may contribute to mechano-signalling pathways. The objective of this study was to determine the response of cells from the outer annulus fibrosus (OAF) to physiological levels of cyclic tensile strain; further, cells from the nucleus pulposus (NP) were also subjected to an identical loading regime to compare biological responses across the IVD populations. We determined whether the organisation and expression of the major cytoskeletal elements and their associated accessory proteins are responsive to mechanical stimulation in these cells, and whether these changes correlated with either a catabolic or anabolic phenotype. OAF and NP cells from immature bovine IVD were seeded onto Flexcell® type I collagen coated plates. Cells were subjected to cyclic tensile strain (10 %, 1 Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy, RNA extracted for quantitative PCR and protein extracted for Western blotting analysis. F-actin reorganisation was evident in OAF and NP cells subjected to tensile strain; strain induced ß-actin at the transcriptional and translational level in OAF cells. ß-tubulin mRNA and protein synthesis increased in strained OAF cells, but vimentin expression was significantly inhibited. Cytoskeletal element organisation and expression were less responsive to strain in NP cells. Tensile strain increased type I collagen and differentially regulated extracellular matrix (ECM)-degrading enzymes' mRNA levels in OAF cells. Strain induced type II collagen transcription in NP cells, but had no effect on the transcription of any other genes analysed. Tensile strain induces different mechano-responses in the organisation and/or expression of cytoskeletal elements and on markers of IVD metabolism. Differential mechano-regulation of anabolic and catabolic ECM components in the OAF and NP populations reflects their respective mechanical environments in situ.

Interleukin-1ß enhances cartilage-to-cartilage integration.

The failure of cartilages to fuse, particularly in the case of articular cartilage under conditions of repair is due to morphological and structural constraints of the tissue. Factors that impede integration include, non-vascularisation, low cellularity, and proteoglycan in the surrounding extracellular matrix acting as a natural barrier to cellular migration. We hypothesised that brief activation of a catabolic cascade by cytokines followed by culture under anabolic conditions would promote tissue fusion in a ring-disk model of cartilage integration. Our results show that transient exposure to 10 ng mL(-1) interleukin-1ß, followed by two weeks post-culture under anabolic conditions, enhanced cartilage-cartilage integration compared to untreated explants. Quantitative PCR analysis of catabolism-related genes ADAMTS4 and MMP13 showed both were transiently upregulated and these findings correlated with evidence of extracellular matrix remodelling. At the level of histology, we observed chondrocytes readily populated the interfacial matrix between fused explants in interleukin-1ß treated explants, whereas in control explants this region was relatively acellular in comparison. Catabolic cytokine treated explants exhibited 29-fold greater adhesive strength (0.859 MPa versus 0.028 MPa, P 〈 0.05) than untreated counterparts. Collectively, our results demonstrate that a single short catabolic pulse followed by an anabolic response is sufficient to generate mechanically robust, integrative cartilage repair.

Amyloid P-component is a constituent of normal human glomerular basement membrane.

Glomerular and other vascular basement membranes were found to contain an antigen that was immunochemically indistinguishable from serum amyloid P-component. There was no immunological cross-reactivity between antisera to serum amyloid P-component and to collagen types I, III, IV, or V. The amyloid P-component antigen was confined to the endothelial aspect, the lamina rara interna, of glomerular basement membrane. It could not be eluted by high-ionic-strength saline, EDTA, dithiothreitol, or either polar or nonpolar detergents, but was released into solution when isolated glomerular basement membrane was digested by highly purified bacterial collagenase. Most of these P-component molecules and their constituent polypeptide chains were of higher molecular weight and lower isoelectric point than serum amyloid P-component. These findings indicate that, as well as being a normal plasma protein and a universal constituent of amyloid deposits, P-component is also a normal matrix glycoprotein of basement membrane in which it is covalently linked to collagen and/or other matrix proteins. This may be relevant both to the pathogenesis of amyloidosis and to other aspects of physiology and pathology of basement membranes.

Cartilage integration: evaluation of the reasons for failure of integration during cartilage repair. A review.

Articular cartilage is a challenging tissue to reconstruct or replace principally because of its avascular nature; large chondral lesions in the tissue do not spontaneously heal. Where lesions do penetrate the bony subchondral plate, formation of hematomas and the migration of mesenchymal stem cells provide an inferior and transient fibrocartilagenous replacement for hyaline cartilage. To circumvent the poor intrinsic reparative response of articular cartilage several surgical techniques based on tissue transplantation have emerged. One characteristic shared by intrinsic reparative processes and the new surgical therapies is an apparent lack of lateral integration of repair or graft tissue with the host cartilage that can lead to poor prognosis. Many factors have been cited as impeding cartilage:cartilage integration including; chondrocyte cell death, chondrocyte dedifferentiation, the nature of the collagenous and proteoglycan networks that constitute the extracellular matrix, the type of biomaterial scaffold employed in repair and the origin of the cells used to repopulate the defect or lesion. This review addresses the principal intrinsic and extrinsic factors that impede integration and describe how manipulation of these factors using a host of strategies can positively influence cartilage integration.

Increased expression of promatrix metalloproteinase-9 and neutrophil elastase in canine dilated cardiomyopathy.

OBJECTIVE: Canine dilated cardiomyopathy, commonly affecting Doberman pinschers, results in extracellular matrix remodelling within the myocardium. The aim of this study was to examine the proteolytic activity in myocardium from Doberman pinschers with dilated cardiomyopathy. METHODS: Samples of myocardium, obtained rapidly post mortem from the left ventricular free wall of Dobermans with dilated cardiomyopathy, clinically normal Dobermans and control dogs (non-Dobermans), were examined for proteolytic activity using substrate gel zymography. Gels were analysed by scanning densitometry. RESULTS: Promatrix metalloproteinase-9 activity was significantly increased in all Doberman myocardium when compared to controls. A significant increase in an enzyme, identified to be neutrophil elastase by inhibition of its activity by Elastatinal and Western blotting, was also detected in all Dobermans when compared to controls. CONCLUSIONS: The results indicate that promatrix metalloproteinase-9 and neutrophil elastase, both of which are implicated in inflammatory responses, are present in significantly elevated levels in Doberman dilated cardiomyopathy and are raised in clinically normal Dobermans. Both proteolytic enzymes degrade a wide variety of connective tissue components and thus the increased levels found may play an important role in the structural remodelling seen in the myocardium and subsequent heart failure. Increased proteolytic enzyme levels in clinically normal Dobermans may be indicative of the predisposition of the breed to dilated cardiomyopathy.

Type IX collagen: a possible function in articular cartilage.

The effect of type IX on in vitro fibrillogenesis of type II collagen indicated that, while not preventing fibrillogenesis, the presence of type IX collagen reduced the size of the type II fibre aggregates. This observation is consistent with the in vivo localisation studies of type IX collagen. Using the immunogold labelling technique, type IX collagen was shown to be located evenly on small fibrils which occur at higher concentration closer to the cell. Therefore type IX collagen may function as a regulator of fibre diameter in articular cartilage.

Susceptibility of the cartilage collagens types II, IX and XI to degradation by the cysteine proteinases, cathepsins B and L.

We have investigated the susceptibility of both the helical and non-helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37 degrees C and pH values from 3.5 to 7.0. Cleavage occurs only within the non-helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

Immunolocalization of collagen types II and III in single fibrils of human articular cartilage.

Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmium-acetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several noncollagenous components of the extracellular matrix.

Immunolocalization of type III collagen in human articular cartilage prepared by high-pressure cryofixation, freeze-substitution, and low-temperature embedding.

We localized Type III collagen by immunogold electron microscopy in resin sections of intact normal and osteoarthritic human articular cartilage. Comparisons of antibody staining between tissue prepared by high-pressure cryofixation and freeze-substitution without fixatives and that exposed to conventional mild chemical fixation with paraformaldehyde showed that dedicated cryotechniques yielded superior preservation of epitopes that are modified by chemical fixation, and simultaneously provided good ultrastructural preservation. Type III collagen was detected with two polyclonal antibodies, one against the triple-helical domain of the molecule and a second against the more antigenic, globular amino pro-peptide domain, which in this collagen is retained in the extracellular matrix after secretion. Positive labeling was seen in association with the major interstitial fibrils, suggesting co-polymerization of Types III and II collagen in cartilage. Type III collagen could not be detected in aldehyde-fixed normal cartilage. In fixed osteoarthritic cartilage, Type III was detectable only when the antibody to the amino pro-peptide was employed. In contrast, high-pressure cryofixation and freeze-substitution preserved epitopes for both antibodies, permitting immunodetection of Type III collagen in normal and osteoarthritic cartilage. Cryotechniques offer exciting possibilities for significantly improving the immunolocalization of collagens and other fixative-sensitive antigens in situ.

Elevated levels of proteolytic enzymes in the aging human vitreous.

PURPOSE: To identify whether aging of human vitreous is accompanied by an elevation in degradative enzymes within the tissue. METHODS: Human vitreous samples from donors aged 10 to 88 years were placed in two groups based on donor age of less than or more than 50 years. Homogenized samples were analyzed by gelatin substrate zymography for matrix metalloproteinases (MMP). Serine proteinases were detected by casein substrate zymography, and a specific antibody was used to confirm the identity of, and to quantify, the serine proteinase, plasmin. RESULTS: Progelatinase A (ProMMP-2) was present in all the vitreous samples but did not show an age-related increase. MMP-2 was also present at low levels. Progelatinase B (ProMMP-9) was found in approximately 80% of samples analyzed, but neither its presence nor level of activity was age dependent. Of the serine proteinase activities detected, an enzyme of approximately 80 kDa was identified by Western blot analysis as plasmin(ogen). Quantitative analysis revealed a significant increase in plasmin(ogen) with age. CONCLUSIONS: This study shows there is an age-related increase in potential degradative activity in human vitreous that may be responsible for degenerative changes such as vitreous liquefaction. The data suggest that increased levels of these enzymes precede, or are indicative of, underlying ocular disease in some individuals.

Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta.

Confocal and conventional indirect immunofluorescence and immunogold electron microscopic methods were applied to examine the distribution of extracellular matrix constituents (collagens types III and IV) in the villi of immature and term human placentae. The immunofluorescence study revealed that collagen type III is more distinct in the villous stroma of term placenta as compared with that of the first trimester. Collagen type IV was detected mainly in endothelial and epithelial basement membranes and interestingly also to a certain extent in the stroma. Results obtained using immunoelectron microscopy support the proposal that collagen types III and IV are characteristic of stromal and basement membranes, respectively. Stromal collagen type IV is apparently localized in association with the interstitial types of collagen (I and III), in the villous stroma of term placenta.

Collagen types in neuromuscular diseases.

The striking proliferation of connective tissue characteristic of the muscular dystrophies can be attributed predominantly to an increase in endomysial and perimysial type III collagen. Carriers of muscular dystrophy occasionally revealed a slight increase in anti-type III collagen fluorescence, but no abnormalities in collagen disposition were observed in foetuses "at risk" for DMD. In contrast, the proportion of collagen types in neurogenic atrophies appeared normal although anti-type IV and V staining, which delineated the basement membrane, was very intense around atrophied fibres, as was also the case in small fibres in myopathic diseases. The detection of staining with anti-type III, IV and V collagens in splits which are sometimes observed in hypertrophied fibres in the muscular dystrophies supports the suggestion that abnormalities in collagen production, perhaps involving a defective modulation of myoblast-fibroblast expression, may be involved in the pathogenesis of these diseases.

Age related changes in the non-collagenous components of the extracellular matrix of the human lamina cribrosa.

AIMS: To investigate age related alterations in the non-collagenous components of the human lamina cribrosa. METHODS: Fibronectin, elastin, and glial fibrillary acidic protein (GFAP) staining were assessed in young and old laminae cribrosae. An age range (7 days to 96 years) of human laminae cribrosae were analysed for lipid content (n=9), cellularity (n=28), total sulphated glycosaminoglycans (n=28), elastin content (n=9), and water content (n=56), using chloroform-methanol extraction, fluorimetry, the dimethylmethylene blue assay, and ion exchange chromatography, respectively. RESULTS: Qualitatively, an increase in elastin and a decrease in fibronectin and GFAP were demonstrated when young tissue was compared with the elderly. Biochemical analysis of the ageing human lamina cribrosa demonstrated that elastin content increased from 8% to 28% dry tissue weight, total sulphated glycosaminoglycans decreased, and lipid content decreased from 45% to 25%. There were no significant changes in total cellularity or water content. CONCLUSION: These alterations in composition may be indicative of the metabolic state of the lamina cribrosa as it ages, and may contribute to changes in mechanical integrity. Such changes may be implicated in the susceptibility of the elderly lamina cribrosa and also its response to glaucomatous optic neuropathy.

Changes in the collagenous matrix of the aging human lamina cribrosa.

AIMS: The age-related changes in the biochemical composition of the collagenous matrix of the human lamina cribrosa were investigated. METHODS: An age range (3 weeks to 92 years old) of human laminae cribrosae, dissected free of any surrounding structures which contained collagen, were analysed for collagen solubility (n = 58) total collagen content (n = 46), proportion of collagen types (n = 38), and collagen cross linking (n = 30), using hydroxyproline analysis, scanning densitometry of peptides after cyanogen bromide digestion, and high performance liquid chromatography, respectively. RESULTS: Age-related changes included an increase in total collagen and a decrease in the proportion of type III collagen within the lamina cribrosa. The collagen cross link pyridinoline was present at low levels, but demonstrated no trend with age. An age-related increase was found in pentosidine, an advanced glycation product. CONCLUSION: These changes in collagen composition imply that the mechanical properties of the lamina cribrosa are altered, resulting in a stiffer, less resilient structure with age. Such alterations in structure may contribute to the increased susceptibility of the elderly to axonal damage in chronic open angle glaucoma.

Preparation of Bruch's membrane and analysis of the age-related changes in the structural collagens.

AIMS/BACKGROUND: The morphological changes in Bruch's membrane and its constituent collagen seen during aging have been studied extensively but the chemical nature of the collagen and any aging changes have not previously been evaluated. METHODS: A method for preparing purified Bruch's membrane from human cadaver eyes by dissection preceded by trypsin digestion was developed. Following pepsin digestion, the constituent collagens were analysed by SDS-PAGE and by immunoblotting. Cyanogen bromide digestion was used to ascertain the solubility of the collagen and the proportion of type I to type III collagen. After hydrolysis of Bruch's membrane samples the constituent amino acids and collagen crosslinks were measured. RESULTS: The presence of collagen types I, III, IV, and V in Bruch's membrane was confirmed. The proportion of type III collagen as a percentage of total fibrous collagens was calculated as being between 35% and 39%, with no significant difference between different macular and peripheral sites or with age. There was a highly significant decline in the solubility of Bruch's membrane collagen with age, from near 100% in the first decade of life to 40-50% in the ninth decade at both macular and peripheral sites. There was no significant change in the amount of enzymatically formed collagen crosslinks with age. Amino acid analysis indicated a significant increase in the amount of non-collagen protein with age in macular but not peripheral sites. CONCLUSION: Changes in the constituent collagens may contribute to the accumulation of debris in Bruch's membrane with age and interfere with the function of the retinal pigment epithelium, with subsequent consequences for the overlying photoreceptors.

Polypeptide composition of the mammalian tectorial membrane.

The effects of the enzymes collagenase, pepsin, chondroitinase ABC and keratanase on the polypeptide composition of the mammalian tectorial membrane have been analysed using one dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After reduction at least ten polypeptides can be consistently and clearly recognized in SDS gels with molecular weights relative to globular protein standards of 245, 235, 190, 165, 155, 145, 100, 93, 60-73 and 35-49 kDa. With the exception of the 60-73 and 35-49 kDa bands all these polypeptides are sensitive to digestion with bacterial collagenase. The 235, 165, 155, 145 and 93 kDa bands also resist degradation by cold, acidic pepsin. Amino acid analysis of whole tectorial membranes demonstrates that glycine accounts for nearly 25% of the total amino acid content, that proline, hydroxyproline and hydroxylysine are present and that amine sugars can be detected in fairly high concentrations. Estimates based on hydroxyproline content suggest that collagens account for 25-50% of the total tectorial membrane protein. Immunoblotting techniques demonstrate the presence of polypeptides cross reacting with antisera to Type II collagen, Type IX collagen and Type V collagen. Results from immunohistochemical studies confirm that these polypeptides are present in the tectorial membrane and are not contaminants of the isolation procedure. Collagenase treatment of tectorial membranes reveals the presence of an additional non-collagenous polypeptide with an apparent molecular weight of 173 kDa on 7.5% polyacrylamide gels, and polydisperse high molecular weight material spreading over a broad range at the top of the gels. This high molecular weight material and the 173, 60-73 and 35-49 kDa non-collagenous polypeptides are pepsin sensitive and all bind wheat germ agglutinin (WGA) suggesting that they contain N-acetyl glucosamine. The 173 kDa band also binds soybean agglutinin (SBA) suggesting the presence of N-acetyl galactosamine. In the absence of reducing agent the 173 and 60-73 kDa bands are no longer observed and high molecular weight material forming a broad band at the top of the separating gel is seen. The electrophoretic behaviour of this non-collagenous, glycosylated, disulphide bonded, high molecular weight material is altered by treatment with keratanase but not by chondroitinase ABC. The results of this study indicate the tectorial membrane contains at least three different collagen types and, in addition to these collagenous proteins, several non-collagenous, glycosylated polypeptides that may account for as much as 50% of the total tectorial membrane protein.

Immunofluorescence localization of type-M collagen in articular cartilage.

Normal mammalian articular cartilage has been found to contain several collagenous peptide chains in addition to type-II collagen. We now report the distribution in adult pig cartilage of one of these new peptides, type-M collagen, using type-specific antibodies. Type M is primarily located in the pericellular environment of the cells in the deeper zones of the articular cartilage. The distribution is shown to be distinct from that of type-II collagen. This finding suggests that type-M collagen may play an important role in the metabolism of articular cartilage.

Identification of two further collagenous fractions from articular cartilage.

Salt fractionation of pepsin-solubilized human and porcine articular cartilage has revealed the presence of two further collagenous fractions, CF1 and CF2, at high salt concentration following the precipitation of Type-II, 1 alpha, 2 alpha, 3 alpha, and Type-M collagens. Both fractions precipitate at 2.0 M NaCl, but higher yields of CF1 are obtained at 3.0 M NaCl. CF1 and CF2 can be separated in the native form using carboxymethyl-cellulose chromatography. Under denaturing conditions, CF1 has an apparent molecular weight of 25 000 and is unaffected by mercaptoethanol, whereas CF2 has a molecular weight of 35 000 before and 12 000 after reduction by mercaptoethanol. These fractions are probably fragments derived from larger collagen molecules, although the cyanogen bromide digest studies suggest that they are derived from a collagenous type other than one of those previously identified in cartilage.