Interleukin-1 is essential for systemic inflammatory bone loss.

OBJECTIVES: Chronic inflammation is a major risk factor for systemic bone loss leading to osteoporotic fracture and substantial morbidity and mortality. Inflammatory cytokines, particularly tumour necrosis factor (TNF) and interleukin-1 (IL1), are thought to play a key role in the pathogenesis of inflammation-induced bone loss, but their exact roles are yet to be determined. METHODS: To determine whether TNF directly triggers bone loss or requires IL1, human TNFalpha mice (hTNFtg) were crossed with mice lacking IL1alpha and IL1beta (IL1(-/-)hTNFtg). Systemic bone architecture was evaluated using CT scanning, static and dynamic bone histomorphometry and serum markers of bone metabolism. RESULTS: hTNFtg mice developed severe bone loss accompanied by a severe distortion of bone microarchitecture. Bone trabeculae were thinner and decreased in numbers, resulting in increased trabecular separation. Histomorphometric analyses revealed strongly increased bone resorption in hTNFtg mice compared with wild-type mice. In contrast, IL1(-/-)hTNFtg mice were fully protected from systemic bone loss despite still developing inflammation in their joints. Lack of IL1 completely reversed increased osteoclast formation and bone resorption in hTNFtg mice and the increased levels of RANKL in these mice. Structural parameters and osteoclast and osteoblast numbers were indistinguishable from wild-type mice. CONCLUSIONS: These data indicate that IL1 is essential for TNF-mediated bone loss. Despite TNF-mediated inflammatory arthritis, systemic bone is fully protected by the absence of IL1, which suggests that IL1 is an essential mediator of inflammatory osteopenia.

Cartilage biomarkers in hemodialysis patients and the effect of beta2-microglobulin on articular chondrocytes.

OBJECTIVE: Dialysis-related amyloidosis (DRA) is a severe complication of maintenance hemodialysis (HD). Given the predominant deposition of beta(2)-microglobulin (beta2m) fibrils on articular cartilage in early DRA, we investigated the significance of beta2m and its relationship to distinct cartilage biomarkers in early DRA diagnosis in HD patients. Furthermore, we assessed the effects of beta2m on articular chondrocytes in vitro. METHODS: Serum samples from 133 patients were collected before and after HD. Type II collagen cleavage product (C2C), procollagen II c-propeptide (CPII), aggrecan chondroitin sulfate 846 epitope (CS-486) and cartilage oligomeric matrix protein (COMP) levels were determined by enzyme-linked immunosorbent assay. Primary bovine articular chondrocytes were cultured as monolayers and incubated with beta2m at 1.5mg/l and 20mg/l. Cartilage glucosaminoglycan synthesis was measured by [(35)S]sulfate incorporation. mRNA expression of interleukin (IL)-1beta, matrix metalloproteinases (MMPs)-3 and -9 was measured by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Incubation with beta2m at 20mg/l significantly decreased matrix biosynthesis. PCR analysis revealed an increase of IL-1beta, as well as MMPs-3 and -9 on the mRNA level. C2C/CPII, CS-486 and COMP levels were increased only in a subset of patients without a significant correlation with beta2m concentrations. A subgroup analysis elucidated an increase in type II collagen degradation during the first years of HD, as shown by the elevation of C2C/CPII ratio. CONCLUSION: beta2m exerted anti-anabolic effects on articular chondrocytes in vitro and might be involved in cartilage degradation in HD patients. beta2m serum levels, however, did not reflect cartilage degradation in DRA. The assessment of C2C/CPII, CS-486 or COMP concentrations apparently has minor relevance in DRA diagnosis in HD patients. However, the increased type II collagen breakdown within 5 years after HD onset possibly mirrors the early stages of DRA. Thus, the C2C/CPII ratio could be employed in longitudinal studies, since it may reflect a risk for DRA related arthropathy development in a subset of patients.

Toll-like receptors and chondrocytes: the lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7.

OBJECTIVE: To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)-induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes. METHODS: TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with interleukin-1beta (IL-1beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by (35)S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1beta was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis. RESULTS: TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1beta gene expression and the negative effects of LPS on matrix biosynthesis. CONCLUSION: These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.

Effect of pulsed electromagnetic fields on proteoglycan biosynthesis of articular cartilage is age dependent.

OBJECTIVE: To investigate the effects of a pulsed electromagnetic field (EMF) on articular cartilage matrix biosynthesis with regard to age and cartilage damage using a matrix depleted cartilage explant model. METHODS: Cartilage explants were obtained from metacarpophalangeal joints of calves and adult cows. After depletion of the extracellular matrix by trypsin digestion, samples were maintained in serum-free basal medium with and without the addition of interleukin 1beta (IL1beta). Half the samples were subjected to an EMF for 24 minutes daily; the other half were left untreated. Undigested and untreated explants served as negative controls. After 7 days, biosynthesis of matrix macromolecules was assessed by [35S]sulphate incorporation and values were normalised to hydroxyproline content. RESULTS: The EMF increased matrix macromolecule synthesis in undigested, untreated explants (p<0.009). In matrix depleted samples the EMF had no stimulatory effect on proteoglycan biosynthesis. IL1beta significantly decreased the de novo synthesis of matrix macromolecules (p<0.00004) in young and adult samples, but an EMF partly counteracted this inhibitory effect in cartilage samples from young, but not old animals. CONCLUSION: EMF promoted matrix macromolecule biosynthesis in intact tissue explants but had no stimulatory effect on damaged articular cartilage. The supressive effects of IL1beta were partially counteracted by EMF exposure, exclusively in cartilage derived from young animals. An EMF has age dependent chondroprotective but not structure modifying properties when cartilage integrity is compromised.

Stimulatory effects of distinct members of the bone morphogenetic protein family on ligament fibroblasts.

OBJECTIVE: To investigate effects of cartilage derived morphogenetic protein-1 and -2 (CDMP-1, CDMP-2), bone morphogenetic protein (BMP)-7 and BMP-6 on metabolism of ligament fibroblasts and their osteogenic or chondrogenic differentiation potential. METHODS: Ligament fibroblasts were obtained from 3 month old calves, plated as monolayers or micromass cultures, and incubated with or without CDMP-1, CDMP-2, BMP-7, and BMP-6. Expression of the indicated growth factors was assessed by RT-PCR and western immunoblotting. The presence of their respective type I and II receptors, and lineage related markers, was investigated in stimulated and unstimulated cells by RT-PCR and northern blotting. Biosynthesis of matrix proteoglycans was assessed by [(35)S]sulphate incorporation in monolayers. Alcian blue and toluidine blue staining was done in micromass cultures. RESULTS: CDMP-1, CDMP-2, BMP-7, and BMP-6 were detected on mRNA and on the protein level. Type I and II receptors were endogenously expressed in unstimulated ligament fibroblasts. The growth factors significantly stimulated total proteoglycan synthesis as assessed by [(35)S]sulphate incorporation. Toluidine blue staining showed cartilage-specific metachromasia in the growth factor treated micromass cultures. Transcription analysis of stimulated ligament fibroblasts demonstrated coexpression of chondrocyte markers but no up regulation of osteogenic markers. CONCLUSION: CDMP-1, CDMP-2, BMP-7, and BMP-6 and their receptors were expressed in ligament tissue. These growth factors induced matrix synthesis in fibroblasts derived from bovine ligament. The preferential expression of cartilage markers in vitro suggests that CDMP-1, CDMP-2, BMP-7, and BMP-6 have the potential to induce differentiation towards a chondrogenic phenotype in ligament fibroblasts. Thus, fibroblasts from ligaments may serve as a source for chondrogenesis and tissue repair.

Chondrocyte number and proteoglycan synthesis in the aging and osteoarthritic human articular cartilage.

OBJECTIVE: To correlate the number of chondrocytes in healthy and osteoarthritic human articular cartilage with age, and to evaluate the influence of donor age on total proteoglycan synthesis. METHODS: Chondrocytes were isolated from human articular cartilage derived from hip joints with and without osteoarthritic lesions. The cell number was normalised to cartilage sample wet weight. In addition, the influence of age on chondrocyte numbers was assessed histomorphometrically. Chondrocytes were grown as monolayer cultures for seven days in a chemically defined serum-free basal medium. Total proteoglycan synthesis was measured by [(35)S]sulphate incorporation into newly synthesised macromolecules. RESULTS: Chondrocyte numbers in healthy cartilage decreased significantly with advancing age (r = -0.69, p<0.0001). In contrast to healthy specimens, chondrocyte numbers were decreased in osteoarthritic cartilage irrespective of and unrelated to age, and differed markedly, by an average of 38%, from the cell numbers found in healthy individuals (p<0.0001). Regarding synthesis of matrix macromolecules, no dependence on patients' age, either in healthy or in osteoarthritic specimens, could be observed. CONCLUSIONS: Under the experimental conditions employed, chondrocytes from healthy and osteoarthritic joints synthesised comparable amounts of cartilage macromolecules, independent of age or underlying osteoarthritic disease. Thus the decrease in chondrocyte number in aging and osteoarthritic joints could be a crucial factor in limiting tissue replenishment.

Expression of bone morphogenetic protein 6 in healthy and osteoarthritic human articular chondrocytes and stimulation of matrix synthesis in vitro.

OBJECTIVE: To elucidate the role of bone morphogenetic protein 6 (BMP-6) in human articular cartilage, we investigated whether BMP-6 is expressed in adult human articular chondrocytes and analyzed the potential stimulatory effects of BMP-6 on these cells. In addition, we investigated whether osteoarthritic (OA) and normal cartilage chondrocytes behave differently. METHODS: Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. BMP-6 protein was detected by Western immunoblotting. Chondrocytes were grown as monolayer cultures for 7 days in a chemically defined serum-free medium, in the absence or presence of recombinant BMP-6. Proteoglycan (PG) synthesis was measured by (35)S-sulfate incorporation into newly synthesized macromolecules. Cell proliferation was assessed by (3)H-thymidine incorporation. RESULTS: BMP-6 was expressed in both healthy and OA chondrocytes at the messenger RNA and protein levels. Total PG synthesis was significantly increased after BMP-6 stimulation of healthy (mean +/- SEM 191 +/- 11%; P < 0.001) and OA (150 +/- 25%; P < 0.03) chondrocyte cultures. A direct comparison between healthy and OA samples revealed no significant difference. The proliferation rates of normal and OA chondrocytes were not affected by BMP-6 treatment. CONCLUSION: BMP-6 is endogenously expressed in chondrocytes obtained from OA and normal adult human articular cartilage. Furthermore, BMP-6 has the potential to stimulate total PG synthesis in human articular chondrocytes derived from normal as well as OA joints. We conclude that the presence of BMP-6 in adult human articular cartilage indicates a functional role for this growth factor in the maintenance of joint integrity.

Is pro-matrix metalloproteinase-3 a marker for posttraumatic cartilage degradation?

OBJECTIVE: Since the development of posttraumatic osteoarthritis (OA) is a relatively slow process, estimation of OA risk would be of value with regard to chondroprotective measures and medication. In this study we investigated the significance of pro-matrixmetalloproteinase-3 (proMMP-3) for this purpose. DESIGN: Synovial fluid (SF) and serum samples were collected from 259 patients of our trauma clinic at the time of arthroscopy. The extent of cartilage damage was assessed according to the Outerbridge-score. ProMMP-3 levels in SF and serum were determined by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody. Additionally we determined SF and serum levels of total MMP-3 and COMP levels as well as TIMP-1 and -2 concentrations in 40 randomly selected patients by ELISA. RESULTS: Serum proMMP-3 levels of the total cohort were markedly increased compared to healthy controls (P<0.007). The comparison of serum and SF lavage proMMP-3 concentrations showed a significant correlation (r(s)=0.41, P<0.0001), however, only 26% of the investigated samples were increased above normal ranges. The grade of cartilage damage did not correlate with enzyme concentration neither in patients' serum nor in SF samples. ProMMP-3 SF concentration was increased early after trauma. Furthermore, proMMP-3 correlated significantly with total MMP-3 serum and SF levels as well as COMP SF levels. CONCLUSIONS: The measurement of proMMP-3 in serum or SF did not reflect the present cartilage damage and thus appears to have only minor potential for clinical use, but it should be considered for longitudinal studies, since it may reflect a risk for cartilage degradation in a subset of patients.

Cartilage-derived morphogenetic protein-1 and -2 are endogenously expressed in healthy and osteoarthritic human articular chondrocytes and stimulate matrix synthesis.

OBJECTIVE: We investigated whether chondrocytes derived from osteoarthritic cartilage may lose their responsiveness to cartilage-derived morphogenetic protein-1, -2 (CDMP-1, -2) and osteogenic protein-1 (OP-1) compared with healthy cells, thus leading to an impaired maintenance of matrix integrity. DESIGN: Chondrocytes were isolated from articular cartilage from patients with and without osteoarthritic lesions. Cells were grown as monolayer cultures for 7 days in a chemically defined serum-free basal medium (BM) in the presence of recombinant CDMP-1, -2, and OP-1. Glycosaminoglycan synthesis was measured by [35S]Sulfate incorporation into newly synthesized macromolecules. Cell proliferation was investigated by [3H]Thymidine incorporation. The endogenous gene expression of CDMPs/OP-1 and their respective type I and type II receptors was examined using RT-PCR. The presence of CDMP proteins in tissue and cultured cells was detected by Western immunoblots. RESULTS: mRNAs coding for CDMPs and their respective receptors are endogenously expressed not only in healthy, but also in osteoarthritic cartilage. CDMP proteins are present in both normal and osteoarthritic articular cartilage and cultured chondrocytes. CDMP-1, CDMP-2 and OP-1 markedly increased glycosaminoglycan synthesis in both healthy (P< 0.01) and osteoarthritic (P< 0.05) human articular chondrocytes. A comparison of the glycosaminoglycan biosynthetic activity between healthy and osteoarthritic samples revealed no detectable difference, neither in stimulated nor in unstimulated cultures. [(3)H]Thymidine incorporation showed that CDMPs/OP-1 did not affect cell proliferation in vitro. CONCLUSION: CDMPs and OP-1 exert their anabolic effects on both healthy and osteoarthritic chondrocytes indicating no loss in responsiveness to these growth factors in OA. The endogenous expression of CDMPs/OP-1 and their receptors suggest an important role in cartilage homeostasis.

[The effect of Condrosulf (sodium chondroitin sulfate) on proteoglycan synthesis by human osteoarthritic an bovine juvenile articular cartilage chondrocytes--an in vitro study]. / Der Einfluss von Condrosulf (Natriumchondroitinsulfat) auf die Proteoglykansynthese und Zellproliferation osteoarthrotischer humaner und juveniler boviner Gelenksknorpelzellen--eine In-vitro-Untersuchung.

BACKGROUND: Sodiumchondroitinsulfate, Condrosulf, is used in osteoarthritis therapy and belongs to the group of symptomatic slow-acting drugs for osteoarthritis. The aim of this study was to investigate the effects of Condrosulf on total proteoglycan synthesis and cell proliferation in human osteoarthritis and healthy juvenile bovine chondrocytes in vitro. METHODS: Chondrocytes were grown as monolayers and stimulated for 7 (human cartilage), or 4, 8 and 12 days (bovine cartilage) with different concentrations of Condrosulf (100 micrograms/ml, 500 micrograms/ml, 1000 micrograms/ml, 2500 micrograms/ml and 5000 micrograms/ml). Proteoglycan synthesis was measured by [35S]Sulfate incorporation. The cell proliferation rate was determined using a [3H]Thymidin assay. The expression of the cartilage markers aggrecan and collagen type II was assessed by Northern blot analysis. RESULTS: We show that the incubation with Condrosulf did not affect proteoglycan synthesis neither in osteoarthritis, nor in healthy chondrocytes under the present culture conditions. Cell proliferation rate was also not increased by Condrosulf stimulation. The results of the Northern blot assays demonstrated a dose-dependent down regulation of aggrecan expression on mRNA level. CONCLUSIONS: These data indicate a lack of direct anabolic effects of Condrosulf on the biosynthetic activity of cultured articular chondrocytes. The well known ease of clinical symptoms, such as pain or swelling under Condrosulf medication may be interpreted by an interaction with pro-inflammatory cytokines.

Effects of cartilage-derived morphogenetic proteins and osteogenic protein-1 on osteochondrogenic differentiation of periosteum-derived cells.

Localization studies and genetic evidence have implicated cartilage-derived morphogenetic proteins-1, -2 (CDMP-1 and CDMP-2), and osteogenic protein-1 (OP-1) in the osteochondrogenic differentiation of mesenchymal progenitor cells during embryonic development and in postnatal life. Based on their expression pattern and the evidence that periosteum contains mesenchymal cells in the cambium layer that can undergo bone and cartilage formation, we hypothesized that CDMPs and OP-1 may be involved in long bone development and fracture healing. To test this hypothesis, periosteum-derived cells from young calves were cultured as monolayers under serum-free conditions with and without the addition of recombinant CDMP-1, CDMP-2 and OP-1. Phenotypic analysis indicate that periosteum-derived cell populations prepared, expanded, and cultured under the conditions described below, constitutively express messenger RNAs for the bone markers osteocalcin, osteopontin and collagen type I, and the chondrogenic markers collagen type II and aggrecan as determined by RT-PCR. Moreover, histologic examinations showed positive staining for alcian blue and alkaline phosphatase (AP). Treatment of periosteum-derived cells with CDMPs and OP-1 resulted in a dose-dependent increase of cell proliferation; CDMP-2 was less active in this regard. Furthermore, all growth factors enhanced osteogenic differentiation as assessed by a time- and dose-dependent stimulation of AP activity and OP-1 increased messenger RNA expression for osteocalcin and collagen type I. We further examined the effects of CDMPs and OP-1 on chondrogenic differentiation of periosteum-derived cells. Both CDMPs and OP-1 stimulated (35)S-sulfate incorporation into newly synthesized macromolecules with OP-1 having a more pronounced stimulatory effect when compared with CDMP-1 and CDMP-2. Our results indicate that distinct members of the BMP-family increase the mitotic and metabolic activity of periosteum-derived cells. The enhancement of both the chondrogenic and osteogenic differentiation suggests that these growth factors might contribute to the local regulation of bone formation and fracture repair.