Induction of apoptosis of articular chondrocytes and suppression of articular cartilage proteoglycan synthesis by heat shock

We investigated cellular and matrix responses of articular cartilage to heat shock. Rat articular cartilage was pretreated at 37 degrees C for 24 h before being exposed to 48 degrees C for 10 min and subsequently incubated at 37 degrees C for 1, 2, 4, 7, 10, and 14 days. Following heat shock, a terminal deoxynucleotidyl transferase nick end labeling assay showed that articular chondrocyte apoptosis appeared at day 1, peaked at day 7, and declined by day 14. Analysis by transmission electron microscopy confirmed that the chondrocytes had characteristic morphological features of apoptosis; immunohistochemical staining revealed that caspase-3 activity in chondrocytes increased, 3-B-3-positive articular chondrocytes decreased in number, and the expression of 3-B-3 native epitope in articular chondrocytes was reduced. Safranin-O staining revealed that depletion of proteoglycans in the matrix was not found in any group. Morphological and biochemical evidence from this study suggested that heat shock at 48 degrees C induced articular chondrocyte apoptosis and suppressed proteoglycan synthesis of articular cartilage in vitro. This study thus provides evidence of the onset of osteoarthritis induced by heat shock and a basis for choosing a temperature at which malignant bone tumor cells can be killed with minimal damage to articular cartilage.     

Keratan sulfate content and articular cartilage maturation during postnatal rabbit growth

This article describes the macromolecular changes in keratan sulfate and proteoglycan that occur in rabbit articular cartilage during postnatal development. Articular cartilage glycosaminoglycans from femoral condyles and the tibial plateaus of rabbits at 8, 12, 18, and 26 weeks and 2 years of age were extracted, fractionated, and quantified. The predominant glycosaminoglycan present in articular cartilage at 8 weeks was chondroitin sulfate. During subsequent maturation the relative proportions of keratan sulfate and chondroitin sulfate varied inversely. The greatest increase in the amount of keratan sulfate present in cartilage was observed between 12 and 26 weeks of age. Hyaluronic acid content was measurable at 12 weeks; afterward the amount remained relatively constant with age. Proteoglycans, extracted from 6-, 12-, and 22-week-old rabbit femoral and tibial cartilage in the presence of protease inhibitors, were analyzed on columns of Sepharose CL-2B. Cartilage proteoglycans decreased in hydrodynamic size between 12 and 22 weeks, corresponding to the period of maximal change in content of keratan and chondroitin sulfate.     

Clusterin expression in adult human normal and osteoarthritic articular cartilage

OBJECTIVE: To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage. METHODS: Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization. RESULTS: Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. CONCLUSION: The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.     

The effects of glucosamine derivatives on equine articular cartilage degradation in explant culture

OBJECTIVE: To determine whether glucosamine-3-sulfate, glucose-3-sulfate (control) and N-acetyl glucosamine inhibit experimentally induced degradation of equine articular cartilage explants similar to glucosamine HCl. DESIGN: Articular cartilage was obtained from the antebrachio-carpal and middle joints of horses (2-8 years old) killed for reasons unrelated to lameness. Cartilage discs were harvested from the weight-bearing region of the articular surface and cultured. Media were exchanged daily and the recovered media stored at 4 degrees C. On days 1 and 2 lipopolysaccharide (LPS, 10 microg/ml) was added to induce cartilage degradation. To evaluate the effects of different sources of glucosamine (on an equal molar basis), varying concentrations of glucosamine HCl (0.25, 2.5, or 25 mg/ml), glucosamine-3-sulfate (0.304, 3.04, or 30.4 mg/ml), or N-acetyl-glucosamine (0.256, 2.56, or 25.6 mg/ml) were added to the cultures. The glucose-3-sulfate control was added at 0.3075, 3.075 or 30.75 mg/ml. Nitric oxide and proteoglycan released into conditioned media and tissue proteoglycan synthesis and total tissue PG content were measured as indicators of cartilage metabolism. RESULTS: Glucosamine-3-sulfate consistently inhibited cartilage degradation in a manner similar to glucosamine HCl, while the effects of N-acetyl-glucosamine were highly variable and did not inhibit cartilage degradation. Glucose-3-sulfate did not inhibit cartilage degradation. CONCLUSION: Our results indicate that glucosamine sulfate also has the potential to prevent or reduce articular cartilage degradation similar to glucosamine HCl in vitro. The amine group at the carbon-2 position appears important for the effectiveness of the glucosamine derivative. The therapeutic value of N-acetyl-glucosamine remains questionable.     

Growth hormone stimulates insulin-like growth factor I actions on adult articular chondrocytes

We report effects of adding insulin-like growth factor I (IGF-I) and methionyl human growth hormone (GH), alone or in combination, to adult bovine articular chondrocytes plated at high density. Purified human and synthetic IGF-I stimulated chondrocyte DNA and proteoglycan synthesis. GH had no effect on either process. However, GH added in combination with IGF-I increased proteoglycan, cell-associated proteoglycan, and keratan sulfate synthesis over levels observed with IGF-I alone. IGF-I and GH did not alter the hydrodynamic size of proteoglycans or synthesis of collagen. Our results show that GH and IGF-I act together to stimulate adult chondrocyte extracellular matrix synthesis.     

Effects of hyaluronan on proteoglycan content of osteoarthritic chondrocytes in vitro.

PURPOSE: To investigate the effect of hyaluronic acid (HA) on proteoglycan (PG) concentration in alginate cultures of human osteoarthritic chondrocytes and to analyze whether HA exhibit anti-degradative effects in the presence of the cytokine IL-1beta. METHODS: Cartilage samples from ten patients with osteoarthritis of the knee were harvested and chondrocytes were cultivated in alginate beads. Four groups were cultured: control group with and without IL-1beta (500 pg/ml) and HA group (100 microg/ml) with and without IL-1beta (500 pg/ml). PG concentration was estimated by a dimethylmethylene blue assay. To assess cell proliferation, we measured DNA content fluorometrically. RESULTS: The proliferation rate (DNA) was unchanged in all culture groups. In the control-group PG/DNA (ng/ng) concentration was 27.1 +/- 7.2. Supplementation of the medium with HA decreased PG concentration to 25.3 +/- 6.9 (p < 0.05). After administration of IL-1beta PG/DNA concentration dropped to 23.1 +/- 6.0 (p < 0.01). By contrast HA treatment of IL-1beta stimulated chondrocytes did not further decrease PG concentration (23.9 +/- 6.1). In fact the negative effect of isolated HA application was inverted if HA was given with IL-1beta (p < 0.05). CONCLUSIONS: In osteoarthritic chondrocytes cultured phenotypically stable, HA could exhibit protective effects only in the presence of the degradative cytokine IL-1beta. Thus, the reported anti-inflammatory effects of HA to cartilage matrix seem to be more indirect by blocking degradative effects of cytokines to the matrix.     

关节软骨源性微载体与软骨细胞的体外相容性

目的评估人关节软骨源性微载体与人软骨细胞的体外粘附性。方法切取人关节软骨,对之冷冻干燥后,经粉碎机粉碎,筛取150～200μm大小的软骨粒,经0.25%的胰酶在37℃消化24h,再经1%的化学去污剂TritonX-100震荡72h,蒸馏水清洗48h后,在冻干机内再次冻干12h,经钴60灭菌后,与第6代人软骨细胞共同培养,分别在倒置显微镜下和电镜下观察即刻、2h、8h、30h的粘附情况。结果倒置显微镜下见软骨粒变为绒球状或毛刷状,将此微载体加入培养基后,即见有呈圆球形的软骨细胞与微载体相粘附,2h见微载体上粘附了大量软骨细胞,仍呈圆球形,8h见微载体上粘附的大量软骨细胞仍呈圆球形,而瓶底贴附的软骨细胞已呈现为成纤维细胞样形状,30h见软骨细胞-微载体复合体已沉降贴附于培养瓶底部,软骨细胞增殖明显并向周围伸展,而电镜下观察见粘附于微载体上的软骨细胞仍维持了软骨细胞的形状。整个观察期间均见软骨细胞增殖良好,提示制备的关节软骨源性微载体对软骨细胞无毒害作用。结论关节软骨源性微载体与软骨细胞的体外相容性良好。     

In vitro inhibition of aggrecanase activity by tetracyclines and proteoglycan loss from osteoarthritic human articular cartilage

Tetracyclines were reported to slow down the progression of cartilage damage both in an animal model of osteoarthritis (OA) and in humans. In search for the underlying mechanisms we examined whether tetracyclines possess an inhibitory potential on the activity of aggrecanases and inflammatory mediators and can thus prevent proteoglycan (PG) loss from human articular cartilage. In vitro activity of aggrecanase‐1 and ‐2 was recorded in the presence of 1–100 µM tetracycline, minocycline, or doxycyline. Human knee articular cartilage explants were sorted according to the degree of OA and treated for 10 days with tetracycline derivatives in the presence of interleukin‐1 (IL‐1β). Synthesis and loss of PGs, nitric oxide (NO), and prostaglandin E₂ (PGE₂), as well as the viability were determined. Tetracyclines derivatives dose‐dependently inhibited the activities of both aggrecanases in vitro, whereas no inhibitory effect of tetracyclines on any proteoglycanolytic activities within IL‐1β‐treated human cartilage explants were found. Tetracyclines can significantly modulate NO and PGE₂ levels, but have no effect on PG synthesis and loss within the same human cartilage explant cultures. Altogether, our data show that tetracyclines have no inhibitory potential on any proteoglycanolytic activities within mild or moderately affected human OA cartilage at therapeutic achievable plasma levels. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:828–833, 2010     

Centrifugal characterization of proteoglycans from various depth layers and weight-bearing areas of normal and abnormal human articular cartilage

Ultracentrifugal polydispersity differential [g(S)] distributions were determined for the proteoglycans of various postmortem human articular cartilage samples extracted from six lateral patellar grooves in nondissociative conditions after mild collagenase digestion of the tissue. The samples consisted of 53 slices (250 microns thick), from normal, mildly fibrillated, and extensively ulcerated knee joints. When statistically analyzed in various subgroupings, the obtained average sedimentation coefficients and polydispersity profiles supported the following conclusions: (a) loss of proteoglycan aggregation and sedimentability is confirmed to be a primary sign of cartilage matrix degradation; (b) higher S values for proteoglycans of the high weight (HW)-bearing areas and lower values for those of the low weight (LW)-bearing areas were a typical finding in normal cartilage samples; (c) inversion of this pattern was indicative of matrix degradation, suggesting that the HW regions are more affected than the LW-bearing areas; (d) the average S value distribution across cartilage thickness tended to resemble the corresponding proteoglycan content versus distance from articular surface; and (e) the deepest cartilage layer had, in most cases, the smallest amount of aggregates while the highest average sedimentability was observed at the middle zone of the normal samples. In the discussion, a role of proteoglycan aggregation for providing a means to "pack" more proteoglycans within the collagen meshwork and to control the generation of osmotic pressure gradients is suggested.     

Local stimulation of proteoglycan synthesis in articular cartilage explants by dynamic compression in vitro.

Cultured bovine articular cartilage was subjected to 50 ms, 0.5-1.0 MPa compressions repeated at intervals of 2-60 s for 1.5 h and simultaneously labeled with 35SO4. The compression was delivered with a 4-mm-diameter nonporous loading head on an 8-mm-diameter cartilage explant. This method created directly compressed (central) and uncompressed (border) areas within the tissue. Analysis of the whole explant under a 0.5 MPa load showed significantly increased 35SO4 incorporation by compression repeated at 2- and 4-s but not at 20- and 60-s intervals. When the incorporation was studied separately in the border and central areas, a statistically significant stimulation was noticed in the central area with a 4-s cycle, while the border area was stimulated with a 2-s cycle. Autoradiography of the central area showed that the stimulation with 0.5 MPa and a 4-s cycle occurred through the whole depth of the cartilage, while raising the pressure to 1 MPa or the frequency to 2 s reduced the stimulation, particularly in the superficial cartilage. In the border area the stimulation with 0.5 MPa and a 2-s cycle was noted in the superficial zone only. The stimulation of proteoglycan synthesis is thus limited to certain loading frequencies and pressures and occurs in specific areas under and around the loaded site. Its rapid appearance suggests enhanced glycosylation or sulfation of core proteins or enhanced speed of posttranslational processing.     

Immunological analysis of proteoglycan structural changes in the early stage of experimental osteoarthritic canine cartilage lesions.

Specific modifications of the proteoglycan (PG) structure of osteoarthritic (OA) dog cartilage in relation to endogenous metalloprotease activity were examined using murine anti-proteoglycan monoclonal antibodies (MoAbs). OA lesions were induced over a period of 8 weeks in crossbred dogs (Pond-Nuki model). The articular cartilage was removed and homogenized in a Tris buffer, pH 7.5, and then divided into four groups: direct PG extraction, no addition, presence of 1 mM p-aminophenyl mercuric acetate (APMA), and presence of 1 mM APMA and 10 mM o-phenanthroline, incubated for 42 h at 37 degrees C followed by PG extraction. MoAbs reactive with PG protein and carbohydrate epitopes included 1C6, 3B3, 5D4, D1B2, and m4D6. The results showed marked alterations induced by APMA activation of the endogenous metalloproteases. PG changes were apparent at at least three sites: one was either in the hyaluronic acid-binding region or between the hyaluronic-binding region and the G2 globular domain, another was between the keratan-sulfate-rich domain and the chondroitin sulfate-attachment domain, and a third was in the chondroitin sulfate-attachment domain. Constitutive metalloprotease activity resulted in less marked PG alterations with preservation of functional PG aggregation to hyaluronan.     

硫酸氨基葡萄糖联合依托考昔治疗膝关节骨性关节炎的临床研究

目的研究硫酸氨基葡萄糖联合依托考昔治疗膝关节骨性关节炎的疗效以及安全性。方法将2013年9月至2014年9月于我院骨科门诊确诊为膝关节骨性关节炎的150例患者按随机数字表法分成依托考昔组、硫酸氨基葡萄糖组和联合组,每组50例,分别给予依托考昔、硫酸氨基葡萄糖、两种药物联合用药治疗6周。于用药2周和6周时采用西安大略和麦克马斯特大学（the Western Ontario and McMaster Universities, WOMAC）骨关节炎指数评估疗效并观察不良反应。结果11例因不良反应退出试验,6例失访,最终133例完成试验且随访资料完整,其中依托考昔组44例,硫酸氨基葡萄糖组43例,联合组46例。治疗2周后,依托考昔组和联合组的WOMAC指数较治疗前有显著改善,其中联合组的改善优于依托考昔组（P<0.05）;依托考昔组、硫酸氨基葡萄糖组、联合组的不良反应例数分别为2例、1例和2例。治疗6周后,3组患者的WOMAC指数均优于治疗前（均P<0.05）,其中联合组优于依托考昔组和硫酸氨基葡萄糖组（P<0.05）,硫酸氨基葡萄糖组优于依托考昔组（P<0.05）;3组各发生2例不良反应。结论硫酸氨基葡萄糖联合依托考昔治疗膝关节骨性关节炎比单独使用依托考昔或硫酸氨基葡萄糖的疗效好、起效快,值得临床推广。     

Homeostasis of the extracellular matrix of normal and osteoarthritic human articular cartilage chondrocytes in vitro

OBJECTIVE: In normal articular cartilage cells, the IGFRI/insulin-like growth factor 1 (IGF-1) autocrine pathway was shown to overrule the catabolic effects of the IL-1/IL-1RI pathway by up-regulation of the IL-1RII decoy receptor. The activity of the IGF-1/IGFR1 and IL-1/IL-1R pathways, and of the IL-1RII control mechanism in the synthesis and turnover of the extracellular matrix (ECM) by chondrocytes from normal and osteoarthritic (OA) articular cartilage was compared in order to identify possible therapeutic targets of this disease. METHODS: Phenotypically stable human articular cartilage cells were obtained from normal and OA cartilage of the same knee showing focal OA. The cells were cultured in alginate beads over 1 week to re-establish the intracellular cytokine and growth factors, to reexpress the respective plasma membrane receptors and to reach equilibrium in accumulated cell-associated matrix (CAM) compounds. Following liberation of the cells from the alginate beads, the levels of cell-associated matrix (CAM) aggrecan, type II collagen and fibronectin, of intracellular IGF-1, IL-1alpha and beta and of their respective plasma membrane-bound receptors, IGFR1, IL-1RI and the decoy receptor IL-1RII, were assayed using flow cytometry. RESULTS: Coordinated production and accumulation of CAM aggrecan and type II collagen under the effect of the IGFR1/IGF-1 autocrine pathway-as documented for chondrocytes from healthy controls-was absent when the chondrocytes had been obtained from OA joints. When compared with cells obtained from normal tissues, chondrocytes from fibrillated OA cartilage expressed significantly higher intracellular IGF-1 levels and plasma membrane-bound IGFR1. At the same time, significantly higher intracellular IL-1alpha and beta levels and upregulated plasma membrane-bound IL-1RI were observed. Plasma membrane-bound IL-1RII decoy receptor was downregulated in OA chondrocytes. The levels of CAM aggrecan, type II collagen and fibronectin were significantly reduced in the chondrocytes obtained from pathological tissue. CONCLUSION: Paired analysis of normal and OA chondrocytes from the same knee joint has shown an enhanced capacity of chondrocytes from OA cartilage to produce ECM macromolecules. However, the same cells have increased catabolic signalling pathways. As a consequence of this increased IL-1 activity and the reduced amounts of IL-1RII decoy receptor, less of the produced ECM macromolecules may persist in the CAM of the OA chondrocytes.     

Glucosamine oral bioavailability and plasma pharmacokinetics after increasing doses of crystalline glucosamine sulfate in man

OBJECTIVE: Pharmacokinetic data on glucosamine are scant, limiting the understanding of glucosamine sulfate mechanism of action in support of its treatment effects in osteoarthritis. This study investigated the oral pharmacokinetics and dose-proportionality of glucosamine after administration of the patented crystalline glucosamine sulfate in man. METHODS: Twelve healthy volunteers received three consecutive once-daily oral administrations of glucosamine sulfate soluble powder at the doses of 750, 1,500, and 3,000 mg, in an open, randomised, cross-over fashion. Glucosamine was determined in plasma collected up to 48 h after the last dose by a validated Liquid Chromatography method with Mass Spectrometry detection. Pharmacokinetic parameters were calculated at steady state. RESULTS: Endogenous plasma levels of glucosamine were detected (10.4-204 ng/ml, with low intra-subject variability). Glucosamine was rapidly absorbed after oral administration and its pharmacokinetics were linear in the dose range 750-1,500 mg, but not at 3,000 mg, where the plasma concentration-time profiles were less than expected based on dose-proportionality. Plasma levels increased over 30-folds from baseline and peaked at about 10 microM with the standard 1,500 mg once-daily dosage. Glucosamine distributed to extravascular compartments and its plasma concentrations were still above baseline up to the last collection time. Glucosamine elimination half-life was only tentatively estimated to average 15 h. CONCLUSIONS: Glucosamine is bioavailable after oral administration of crystalline glucosamine sulfate, persists in circulation, and its pharmacokinetics support once-daily dosage. Steady state peak concentrations at the therapeutic dose of 1,500 mg were in line with those found to be effective in selected in vitro mechanistic studies. This is the only glucosamine formulation for which pharmacokinetic, efficacy and safety data are now available.     

Electron microscopic analysis of articular cartilage proteoglycan degradation by growth plate enzymes

To assess the effect of intracellular growth plate chondrocyte enzymes on proteoglycan structure, we examined enzyme-treated articular cartilage proteoglycans and untreated articular cartilage proteoglycans with the electron microscopic monolayer technique. The untreated proteoglycan monomers ranged in length from less than 20 nm to more than 700 nm, with a mean length of 224.5 +/- 101.6 nm in one experiment and 224.6 +/- 95.7 nm in a second experiment. Incubation with growth plate enzymes reduced proteoglycan monomers to fragments with lengths that varied from less than 5 nm to 143 nm, increased the variability in monomer length, and destroyed proteoglycan aggregates. The enzyme treated monomers had an average length of 29.5 +/- 17.9 nm in one experiment and 35.2 +/- 17.0 nm in a second experiment. The smallest common fragments were 15 nm long and would be expected to contain about 15 glycosaminoglycan chains. This experiment demonstrates that enzymes extracted from growth plate chondrocytes can degrade the chondroitin sulfate-rich region of proteoglycan monomer core proteins, produce a range of monomer fragment sizes with less than 2% of the fragments shorter than 5 nm or longer than 100 nm, increase the variability in monomer length, and degrade proteoglycan aggregates.     

Alpha and epsilon isozymes of protein kinase C in the chondrocytes in normal and early osteoarthritic articular cartilage

Protein kinase C (PKC) isozymes (α, βI, βII, γ and, ∈-PKC) were examined immunocytochemically in control and mechanically induced osteoarthritic knee joints in rats. βI, βII, and γ PKC-positive cells were not observed in sham-operated or osteoarthritic knee joints. α-PKC, which was observed only in the cells in the subchondral bony layer in the controls, appeared in the chondrocytes in the superficial and columnar layers of the osteoarthritic knees. ∈-PKC was observed in the control chondrocytes in the superficial portion of the columnar layers. In early osteoarthritic joints, however, ∈-PKC-positive chondrocytes disappeared from the superficial portion of columnar layer and increased in number in the middle columnar layers. The appearance and changes in distribution of these PKC-positive chondrocytes, either under normal or osteoarthritic conditions, have not been reported previously. The role of the redistribution of PKC isozymes is still unclear as to whether they are involved in initiating destructive processes, reflect attempted cell repair mechanisms, or are simply a consequence of the cellular changes.     

Cyclic loading is harmful to articular cartilage from which proteoglycans have been partially depleted by retinoic acid

We studied whether cyclic loading is harmful to degraded cartilage. Sets of four cartilage-bearing sesamoid bones were dissected from 5-year-old cows. One bone from each set was cultured for 17 h in control medium to serve as an ex vivo control. The three others were cultured for 1 week in control medium to which 0, 10 or 300 ng/ml retinoic acid (RAc), which depletes the cartilage matrix of proteoglycans, had been added. Two were then cultured for another week in control medium. During the last week, one of the two was subjected to a cyclic load (1 MPa, 0.2 Hz). Following treatment with RAc, glycosaminoglycan content and synthesis were significantly decreased, as confirmed by safranin O staining and autoradiography. They were further diminished by loading during the second week of culture. Increased amounts of 3-B-3(−) epitope were found in cartilage that had been treated with 300 ng/ml RAc and then loaded. While loading cartilage matrix that was only slightly degraded proved to be damaging, loading severely degraded cartilage matrix apparently induced osteoarthritic-like changes.