Cartilage Macromolecules Research Articles

Modern approaches to the treatment of osteoarthritis

The data given in the article is in accordance with the latest recommendations of ESCEO years 2016, 2019 in regard to possibilities of the sympthom-modifying and structure-modifying action of chondroitin sulfate in the treatment of patients with osteoarthritis. There is the data on the therapy effect when treating patients with osteoarthritis and with periarticular tissues damage with chondroitin sulfate injections (Alflutop, Biotehnos) having strong evidence base, including randomized placebo-controlled studies. In the randomized study it is shown reliably high effectiveness of Alflutop against placebo in regard to main clinical manifestations of the desease as well as possibility to slow down progress of the destructive processes at osteoarthritis of the knee-joint. The data given on estimation of effectiveness and safety of alternate introduction of Alflutop at osteoarthritis 2 ml intramuscularly every other day (10 injections) compared with a standard mode of use of Alflutop 1 ml intramuscularly every day for 20 days. Equal effectiveness of the two modes of Alflutop introduction is demonstrated subject to good tolerance of the drug. Ability of Alflutop to supress the mediators of inflammation, such as interleukins 6 and 8, and degradations at osteoarthritis, as well as to stimulate the proliferation and renewal of chondrocytes is shown according to the results of an international experimental study, explaining its ability to cause a quick analgesic and anti-inflammatory effect. Alflutop inhibits in vitro extracellular release of the pro-inflammatory cytokine interleukin 6 (IL-6) for 16 per cent and chemokine IL-8 for 35 per cent, inhibits pro-angiogenic factor VEGF for 56 per cent, has an antioxidant effect, which contributes to the synthesis of cartilage macromolecules, a decrease in the activity of matrix metalloproteases (MMPs), a decrease in the concentration of prostaglandins and leukotrienes, and a decrease in chondrocyte apoptosis. The data on the effectiveness of the Alflutop injection course in case of damage to the periaticular tissues in the shoulder, elbow, knee joint is given. The data on the gastroprotective effect of the drug, demonstrated in experimental and clinical studies is given.

Anisotropic Diffusivity Tensor in Articular Cartilage: Effective Medium Approach.

Due to the avascular nature of articular cartilage, molecular transport occurs via interstitial fluid flow as well as via diffusion. Diffusion in cartilage has been studied experimentally, but no mathematical models have been developed to interpret the experimental results and the observed isotropy or anisotropy in the different cartilage zones. Here, we propose a model for the determination of the diffusivity tensor of uncharged macromolecules in articular cartilage, accounting for the inhomogeneity and anisotropy arising from fiber arrangement, volumetric fraction, and radius. We study a representative element of volume (REV) comprising a fiber surrounded by fluid-saturated proteoglycan matrix. The REV permeability tensor is evaluated using a previously developed model, while the REV diffusivity tensor is obtained by incorporating the hydrodynamic effect and the steric effect of the fiber-reinforced matrix. Both effects are represented by anisotropic second-order tensors. The overall diffusivity tensor is obtained as the averaging integral of the REV diffusivity, weighted by the probability distribution of fiber orientation. The model's predictions of the trend of the magnitude of the diffusivity of spheroidal macromolecules as a function of molecular radius agree with published experimental results. For large linear macromolecules, the model underestimates the diffusivity magnitude (i.e., the equivalent isotropic diffusivity). The model correctly predicts the anisotropic behavior for linear macromolecules, although it underestimates the numerical value of the diffusivity anisotropy ratio of large linear macromolecules in the superficial zone, and overestimates it in the deep zone. In summary, this model constitutes a first step toward understanding the relation between diffusivity and permeability in articular cartilage.

Safranin O without fast green is the best staining method for testing the degradation of macromolecules in a cartilage extracellular matrix for the determination of the postmortem interval.

Methods for the determination of the postmortem interval (PMI) include methods that monitor the postmortem changes of cells and molecules in different tissues. The rate of pathological degradation of macromolecules in the extracellular matrix (ECM) of hyaline cartilage could be verified by assessing the intensity of collagen and proteoglycan (PG) staining. In the presented in vitro pilot study, this methodology was used for the first time to determine PMI. The osteochondral samples of three donors were stored at 11°C and 35°C and analyzed on day 1, day 12, and day 36 postmortem. The intensity of staining using Masson's trichrome and Sirius red for collagen, and Alcian blue and Safranin O dyes for PG was estimated ten times according to the modified Bern grading scale. Statistical analysis showed that the Safranin O without Fast green method is the most appropriate (raters agreement 0.5541) for up to 36days postmortem, and that the influence of time is more important (p = 0.023) than the influence of temperature (p = 0.061) on the degradation of the ECM macromolecules. The described method, which is simple and can be performed in any histological laboratory, should be verified in corpore conditions, on a large number of donors, and using an objective method for assessing the intensity of cartilage macromolecule staining for PMI determination.

Prospects and limitations of improving skeletal growth in a mouse model of spondyloepiphyseal dysplasia caused by R992C (p.R1192C) substitution in collagen II.

Skeletal dysplasias form a group of skeletal disorders caused by mutations in macromolecules of cartilage and bone. The severity of skeletal dysplasias ranges from precocious arthropathy to perinatal lethality. Although the pathomechanisms of these disorders are generally well defined, the feasibility of repairing established aberrant skeletal tissues that developed in the presence of mutant molecules is currently unknown. Here, we employed a validated mouse model of spondyloepiphyseal dysplasia (SED) that enables temporal control of the production of the R992C (p.R1192C) collagen II mutant that causes this disease. Although in our earlier studies we determined that blocking the expression of this mutant at the early prenatal stages prevents a SED phenotype, the utility of blocking the R992C collagen II at the postnatal stages is not known. Here, by switching off the expression of R992C collagen II at various postnatal stages of skeletal development, we determined that significant improvements of cartilage and bone morphology were achieved only when blocking the production of the mutant molecules was initiated in newborn mice. Our study indicates that future therapies of skeletal dysplasias may require defining a specific time window when interventions should be applied to be successful.

Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage.

BackgroundRheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.MethodsCitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.FindingsSera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.ConclusionsCitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.

Genetically Engineered Mouse Models Reveal the Importance of Proteases as Osteoarthritis Drug Targets

More than two decades of research has revealed a combination of proteases that determine cartilage degradation in osteoarthritis. These include metalloproteinases, which degrade the major macromolecules in cartilage, aggrecan and type II collagen, serine proteases, and cysteine proteases, for example cathepsin K. This review summarizes the function of proteases in osteoarthritis progression, as revealed by studies of genetically engineered mouse models. A brief overview of the biochemical characteristics and features of several important proteases is provided, with the objective of increasing understanding of their function. Published data reveal at least three enzymes to be major targets for osteoarthritis drug development: ADAMTS-5, MMP-13, and cathepsin K. In surgical models of osteoarthritis, mice lacking these enzymes are protected from cartilage damage and, to varying degrees, from bone changes. In-vivo studies targeting these proteases with selective small-molecule inhibitors have been performed for a variety of animal models. Mouse models will provide opportunities for future tests of the therapeutic effect of protease inhibitors, both on progression of structural damage to the joint and on associated pain.

Novel Cartilage‐derived Biomimetic Scaffold for Human Nucleus Pulposus Regeneration: a Promising Therapeutic Strategy for Symptomatic Degenerative Disc Diseases

Because current therapies have not always been successful and effective, the possibility of regenerating the nucleus pulposus (NP) through a tissue-engineered construct offers a novel therapeutic possibility for symptomatic degenerative disc diseases (DDDs). However, more research is necessary to identify the optimal scaffold, cell type and mixture of signal factors needed for NP regeneration. Numerous possible scaffolds for NP regeneration have been investigated; they have many shortcomings in common. Various biological scaffolds derived from decellularized tissue and organs have been successfully used in tissue engineering and received approval for use in humans. Regretfully, harvesting of human NP is difficult and only small amounts can be obtained. The macromolecules of cartilage, which include collagen and proteoglycan aggrecan, are similar to those of the extracellular matrix of immature NP. Recent studies have shown that adipose-derived stem cells (ADSC) can be induced to develop NP-like phenotypes when stimulated by appropriate signals. We thus reasonably postulated that an ideal NP scaffold for tissue engineering could be fabricated from decellularized cartilage matrix (DCM). Furthermore, a combination of ADSCs and DCM-derived biomimetic scaffolds would be advantageous in NP tissue engineering and, in the long run, could become an effective treatment option for symptomatic DDD.

Degradation Improves Tissue Formation in (Un)Loaded Chondrocyte-laden Hydrogels

Photopolymerizable poly(ethylene glycol) (PEG) hydrogels offer a platform to deliver cells in vivo and support three-dimensional cell culture but should be designed to degrade in sync with neotissue development and endure the physiologic environment. We asked whether (1) incorporation of degradation into PEG hydrogels facilitates tissue development comprised of essential cartilage macromolecules; (2) with early loading before pericellular matrix formation, the duration of load affects matrix production; and (3) dynamic loading in general influences macroscopic tissue development. Primary bovine chondrocytes were encapsulated in hydrogels (n = 3 for each condition). The independent variables were hydrogel degradation (nondegrading PEG and degrading oligo(lactic acid)-b-PEG-b-oligo(lactic acid) [PEG-LA]), culture condition (free swelling, unconfined dynamic compressive loading applied intermittently for 1 or 4 weeks), and time (up to 28 days). The dependent variables were neotissue deposition through biochemical contents, immunohistochemistry, and compressive modulus. Degradation led to 2.3- and 2.9-fold greater glycosaminoglycan and collagen contents, respectively; macroscopic cartilage-like tissue formation comprised of aggrecan, collagen II and VI, link protein, and decorin; but decreased moduli. Loading, applied early or throughout culture, did not affect neotissue content in either hydrogel but affected neotissue spatial distribution in degrading hydrogels where 4 weeks of loading appeared to enhance hydrogel degradation resulting in tissue defects. PEG-LA hydrogels led to macroscopic tissue development comprised of key cartilage macromolecules under loading, but hydrogel degradation requires further tuning. PEG-LA hydrogels have potential for delivering chondrocytes in vivo to replace damaged cartilage with a tissue-engineered native equivalent, overcoming many limitations associated with current clinical treatments.

Mapping proteoglycan-bound water in cartilage: Improved specificity of matrix assessment using multiexponential transverse relaxation analysis

Association of MR parameters with cartilage matrix components remains an area of ongoing investigation. Multiexponential analysis of nonlocalized transverse relaxation data has previously been used to quantify water compartments associated with matrix macromolecules in cartilage. We extend this to mapping the proteoglycan (PG)-bound water fraction in cartilage, using mature and young bovine nasal cartilage model systems, toward the goal of matrix component-specific imaging. PG-bound water fraction from mature and young bovine nasal cartilage was 0.31 ± 0.04 and 0.22 ± 0.06, respectively, in agreement with biochemically derived PG content and PG-to-water weight ratios. Fourier transform infrared imaging spectroscopic-derived PG maps normalized by water content (IR-PG(ww) ) showed spatial correspondence with PG-bound water fraction maps. Extensive simulation analysis demonstrated that the accuracy and precision of our determination of PG-bound water fraction was within 2%, which is well-within the observed tissue differences. Our results demonstrate the feasibility of performing imaging-based multiexponential analysis of transverse relaxation data to map PG in cartilage.

Effects of intermittent hydrostatic pressure and BMP‐2 on osteoarthritic human chondrocyte metabolism in vitro

This study examined effects of intermittent hydrostatic pressure (IHP) and a chondrogenic growth factor, bone morphogenetic protein-2 (BMP-2), on anabolic, catabolic, and other metabolic markers in human osteoarthritic (OA) chondrocytes in vitro. Articular chondrocytes, isolated from femoral OA cartilage and maintained in high-density monolayer culture, were examined for effects of BMP-2 and IHP on gene expression of matrix-associated proteins (aggrecan, type II collagen, and SOX9) and catabolic matrix metalloproteinases (MMP-2 and MMP-3) and culture medium levels of the metabolic markers MMP-2, nitric oxide (NO), and glycosaminoglycan (GAG). The results were analyzed using a mixed linear regression model to investigate the effects of load and growth factor concentration. IHP and BMP-2 modulated OA chondrocyte metabolism in accordance with growth factor concentration independently, without evidence of synergism or antagonism. Each type of stimulus acted independently on anabolic matrix gene expression. Type II collagen and SOX9 gene expression were stimulated by both IHP and BMP-2 whereas aggrecan was increased only by BMP-2. IHP exhibited a trend to decrease MMP-2 gene expression as a catabolic marker whereas BMP-2 did not. NO production was increased by addition of BMP-2 and IHP exhibited a trend for increased levels. GAG production was increased by BMP-2. This study confirmed the hypothesis that human OA chondrocytes respond to a specific type of mechanical load, IHP, through enhanced articular cartilage macromolecule gene expression and that IHP, in combination with a chondrogenic growth factor BMP-2, additively enhanced matrix gene expression without interactive effects.

The mobility of chondroitin sulfate in articular and artificial cartilage characterized by 13C magic‐angle spinning NMR spectroscopy

We have studied the molecular dynamics of one of the major macromolecules in articular cartilage, chondroitin sulfate. Applying (13)C high-resolution magic-angle spinning NMR techniques, the NMR signals of all rigid macromolecules in cartilage can be suppressed, allowing the exclusive detection of the highly mobile chondroitin sulfate. The technique is also used to detect the chondroitin sulfate in artificial tissue-engineered cartilage. The tissue-engineered material that is based on matrix producing chondrocytes cultured in a collagen gel should provide properties as close as possible to those of the natural cartilage. Nuclear relaxation times of the chondroitin sulfate were determined for both tissues. Although T(1) relaxation times are rather similar, the T(2) relaxation in tissue-engineered cartilage is significantly shorter. This suggests that the motions of chondroitin sulfate in natural and artificial cartilage are different. The nuclear relaxation times of chondroitin sulfate in natural and tissue-engineered cartilage were modeled using a broad distribution function for the motional correlation times. Although the description of the microscopic molecular dynamics of the chondroitin sulfate in natural and artificial cartilage required the identical broad distribution functions for the correlation times of motion, significant differences in the correlation times of motion that are extracted from the model indicate that the artificial tissue does not fully meet the standards of the natural ideal. This could also be confirmed by macroscopic biomechanical elasticity measurements. Nevertheless, these results suggest that NMR is a useful tool for the investigation of the quality of artificially engineered tissue.

Genetic mouse models for the functional analysis of the perifibrillar components collagen IX, COMP and matrilin-3: Implications for growth cartilage differentiation and endochondral ossification.

The mutual interaction of the two supramolecular compartments, the fibrillar and extrafibrillar matrix is a prerequisite for stability and integrity of the cartilage extracellular matrix. The fibrillar periphery, composed of collagen IX, matrilins and cartilage oligomeric matrix protein (COMP) among other components, constitutes the interface which mediates interactions between the two compartments. Mutations in these peripheral macromolecules cause a broad spectrum of skeletal conditions such as pseudo-achondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), which severely affect the organization and integrity of the cartilage growth matrix in humans. Transgenic and knockout mouse models for collagen IX, matrilin-3 and COMP and combinations thereof display cartilage abnormalities and pathologies of varying severity. Absence of collagen IX appears to cause the most severe growth plate phenotype with a profoundly disturbed morphological organization affecting size and shape of the long bones. Notably, similar growth plate phenotypes, including irregularities in the proteoglycan content, hypocellular central regions, disorganized proliferation columns with atypically shaped and oriented chondrocytes and alterations in the hypertrophic zone are observed in transgenic mice lacking other macromolecules or carrying mutations therein. These include collagens II and XI, integrin subunits, integrin linked kinase (ILK), HIF-1alpha, VEGFalpha and the tumor suppressor PTEN. Notably, mutations in ciliar proteins such as Kif3alpha, polaris or Smo/Gli severely affect the ability of chondrocytes to move and to become arranged in columns. Absence or mutational changes of a variety of different, non-related cartilage macromolecules apparently cause similar pathologies and abnormalities of the growth cartilage, suggesting a limited number of underlying molecular mechanisms.

Advanced Morphological and Biochemical Magnetic Resonance Imaging of Cartilage Repair Procedures in the Knee Joint at 3 Tesla

Morphological and biochemical magnetic resonance imaging (MRI) is due to high field MR systems, advanced coil technology, and sophisticated sequence protocols capable of visualizing articular cartilage in vivo with high resolution in clinical applicable scan time. Several conventional two-dimensional (2D) and three-dimensional (3D) approaches show changes in cartilage structure. Furthermore newer isotropic 3D sequences show great promise in improving cartilage imaging and additionally in diagnosing surrounding pathologies within the knee joint. Functional MR approaches are additionally able to provide a specific measure of the composition of cartilage. Cartilage physiology and ultra-structure can be determined, changes in cartilage macromolecules can be detected, and cartilage repair tissue can thus be assessed and potentially differentiated. In cartilage defects and following nonsurgical and surgical cartilage repair, morphological MRI provides the basis for diagnosis and follow-up evaluation, whereas biochemical MRI provides a deeper insight into the composition of cartilage and cartilage repair tissue. A combination of both, together with clinical evaluation, may represent a desirable multimodal approach in the future, also available in routine clinical use.

Proprotein convertase activation of aggrecanases in cartilage in situ

Proteolytic degradation of the major cartilage macromolecules, aggrecan and type II collagen, is a key pathological event in osteoarthritis (OA). ADAMTS-4 and ADAMTS-5, the primary aggrecanases capable of cartilage aggrecan cleavage, are synthesized as latent enzymes and require prodomain removal for activity. The N-termini of the mature proteases suggest that activation involves a proprotein convertase, but the specific family member responsible for aggrecanase activation in cartilage in situ has not been identified. Here we describe purification of a proprotein convertase activity from human OA cartilage. Through biochemical characterization and the use of siRNA, PACE4 was identified as a proprotein convertase responsible for activation of aggrecanases in osteoarthritic and cytokine-stimulated cartilage. Posttranslational activation of ADAMTS-4 and ADAMTS-5 was observed in the extracellular milieu of cartilage, resulting in aggrecan degradation. These findings suggest that PACE4 represents a novel target for the development of OA therapeutics.

Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells

For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.

Impairment of chondrocyte biosynthetic activity by exposure to 3-tesla high-field magnetic resonance imaging is temporary

The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissues is unknown. We investigated the effects of a 3-tesla electromagnetic field (EMF) on the biosynthetic activity of bovine articular cartilage. Bovine articular cartilage was obtained from juvenile and adult animals. Whole joints or cartilage explants were subjected to a pulsed 3-tesla EMF; controls were left unexposed. Synthesis of sulfated glycosaminoglycans (sGAGs) was measured by using [35S]sulfate incorporation; mRNA encoding the cartilage markers aggrecan and type II collagen, as well as IL-1β, were analyzed by RT–PCR. Furthermore, effects of the 3-tesla EMF were determined over the course of time directly after exposure (day 0) and at days 3 and 6. In addition, the influence of a 1.5-tesla EMF on cartilage sGAG synthesis was evaluated. Chondrocyte cell death was assessed by staining with Annexin V and TdT-mediated dUTP nick end labelling (TUNEL). Exposure to the EMF resulted in a significant decrease in cartilage macromolecule synthesis. Gene expression of both aggrecan and IL-1β, but not of collagen type II, was reduced in comparison with controls. Staining with Annexin V and TUNEL revealed no evidence of cell death. Interestingly, chondrocytes regained their biosynthetic activity within 3 days after exposure, as shown by proteoglycan synthesis rate and mRNA expression levels. Cartilage samples exposed to a 1.5-tesla EMF remained unaffected. Although MRI devices with a field strength of more than 1.5 T provide a better signal-to-noise ratio and thereby higher spatial resolution, their high field strength impairs the biosynthetic activity of articular chondrocytes in vitro. Although this decrease in biosynthetic activity seems to be transient, articular cartilage exposed to high-energy EMF may become vulnerable to damage.

Usurped SLRPs: novel arthritis biomarkers exposed by catabolism of small leucine-rich proteoglycans?

Proteolytic degradation of articular cartilage macromolecules, including the large aggregating cartilage proteoglycan (aggrecan) and small leucine-rich proteoglycans (SLRPs), is a prominent pathophysiological feature of arthritic diseases such as osteoarthritis (OA). Molecular profiling and monitoring of soluble/circulating proteoglycan catabolites that may be released from the cartilage matrix therefore represents an attractive strategy for evaluating OA disease progression and intervention. The recent identification of discrete metalloproteinase-sensitive SLRP cleavage sites, and complementary neoepitope-bearing SLRP catabolites, extends decisive insight into the functional regulation of extracellular matrix integrity, and proffers poignant leads to assist in disclosing and appraising applicable biomarkers of cartilage degeneration during arthritis.

Fourier transform infrared imaging of focal lesions in Human osteoarthritic cartilage

Fourier Transform Infrared Imaging (FTIRI) is a new method for quantitatively assessing the spatial-chemical composition of complex materials. This technique has been applied to examine the feasibility of measuring changes in the composition and distribution of collagen and proteoglycan macromolecules in human osteoarthritic cartilage. Human cartilage was acquired post-operatively from total joint replacement patients. Samples were taken at the site of a focal lesion, adjacent to the lesion, and from relatively healthy cartilage away from the lesion. Sections were prepared for FTIRI and histochemical grading. FTIRI spectral images were acquired for the superficial, intermediate, and deep layers for each sample. Euclidean distance mapping and quantitative partial least squares analysis (PLS) were performed using reference spectra for type-II collagen and chondroitin 6-sulphate (CS6). FTIRI results were correlated to the histology-based Mankin scoring system. PLS analysis found relatively low relative concentrations of collagen (38 +/- 10%) and proteoglycan (22 +/- 9%) in osteoarthritic cartilage. Focal lesions were generally found to contain less CS6 compared to cartilage tissue adjacent to the lesion. Loss of proteoglycan content was well correlated to histological Mankin scores (r=0.69, p<0.0008). The evaluation of biological tissues with FTIRI can provide unique quantitative information on how disease can affect biochemical distribution and composition. This study has demonstrated that FTIRI is useful in quantitatively assessing pathology-related changes in the composition and distribution of primary macromolecular components of human osteoarthritic cartilage.

Effect of glucosamine and chondroitin sulfate on regulation of gene expression of proteolytic enzymes and their inhibitors in interleukin-1–challenged bovine articular cartilage explants

To determine the effects of glucosamine (GLN) and chondroitin sulfate (CS), at concentrations attainable in vivo, on expression of genes encoding proteolytic enzymes, enzyme inhibitors, and macromolecules of articular cartilage in interleukin-1(IL-1)-challenged bovine cartilage explants. Articular cartilage explants harvested from 9 steers. Cartilage explants were exposed to media containing 10% fetal bovine serum (FBS) only, IL-1 (50 ng/mL), IL-1 with GLN (5 microg/mL), IL-1 with CS (20 microg/mL), or IL-1 with GLN and CS for 24 and 48 hours. Cartilage was frozen, and RNA was extracted. Gene expression of matrix metalloproteinases (MMPs)-2, -3, -9, -13, and -14; aggrecanases (Aggs)-1 and -2; tissue inhibitors of metalloproteinases (TIMPs)-1, -2, and -3; and type II collagen and aggrecan were assessed with quantitative real-time polymerase chain reaction. Upregulated MMP-3, MMP-13, and Agg-1 transcripts at 24 hours were repressed by the GLN and CS combination by at least approximately 6-fold. Glucosamine was effective in suppressing IL-1-induced mRNA expression of MMP-13, Agg-1, and Agg-2, whereas CS was effective in decreasing IL-1-induced MMP-13 transcript at 24 hours. At 48 hours, GLN and CS added separately and in combination significantly abrogated Agg-1 and Agg-2 gene induction. The combination also decreased IL-1-stimulated MMP-13 transcript. GLN and CS, at concentrations that are within the range measured in synovial fluid and blood after oral administration, may regulate expression of matrix degrading enzymes and their inhibitors at the transcriptional level, providing a plausible mechanism for their purported chondroprotective properties.

Hypoxia inducible factor-1 and facilitative glucose transporters GLUT1 and GLUT3: putative molecular components of the oxygen and glucose sensing apparatus in articular chondrocytes.

Articular cartilage is an avascular connective tissue in which the availability of oxygen and glucose is significantly lower than synovial fluid and plasma. Glucose is an important metabolic fuel and structural precursor that plays a key role in the synthesis of extracellular matrix macromolecules in articular cartilage. However, glucose concentrations in cartilage can fluctuate depending on age, physical activity and endocrine status. Chondrocytes are glycolytic cells and must be able to sense the quantities of oxygen and glucose available to them in the extracellular matrix and respond appropriately by adjusting cellular metabolism. Consequently chondrocytes must have the capacity to survive in an extracellular matrix with limited nutrients and low oxygen tensions. The molecular mechanisms responsible for allowing chondrocytes to adapt to these harsh environmental conditions are poorly understood. In this article we present a novel "dual" model of oxygen and glucose sensing in chondrocytes based on recent experimental data. This model incorporates the hypoxia-inducible factor alpha (HIF-1alpha) as an oxygen sensor and the hypoxia responsive facilitative glucose transporters, GLUT1 and GLUT3 as putative components of the glucose sensing apparatus in chondrocytes. Recent studies have shown that GLUT1 and GLUT3 are both expressed in chondrocytes and their HIF-1alpha-mediated transcription may be dually stimulated in response to hypoxia and low glucose conditions which in turn promote anaerobic glycolysis in favor of oxidative phosphorylation. This working model provides, for the first time, a unifying hypothesis to explain how chondrocytes might sense and respond to low oxygen tensions and alterations in extracellular glucose.