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Objective: The collagen network in human articular cartilage experiences a large number of stress cycles during life as it shows hardly any turnover after adolescence. We hypothesized that, to withstand fatigue failure, the physical condition of the collagen network laid down at adolescence is of crucial importance for the age of onset of osteoarthritis (OA). Methods: We have compared the lysyl hydroxylation level and pyridinoline cross-link level of the collagen network of degenerated (DG) cartilage of the femoral knee condyle (representing a preclinical early stage of OA) with that of normal cartilage from the contralateral knee. The biological age of the collagen network was determined by means of pentosidine levels. For each donor, collagen modifications of normal cartilage were compared with DG cartilage that showed no significant remodeling of the collagen network (as evidenced by identical pentosidine levels). Results: DG cartilage contained significantly more hydroxylysine residues per collagen molecule in comparison with healthy cartilage from the same donor, both in the upper and lower half (the region near the articular surface and adjacent to bone, respectively). In addition, a significantly higher level of pyridinoline cross-linking was observed in the upper half of DG cartilage. Considering the biological age of the collagen network, the changes observed in DG cartilage must have been present several decades before cartilage became degenerated. Conclusions: The data suggest that high levels of lysyl hydroxylation and pyridinoline cross-linking result in a collagen network that fails mechanically in long term loading. Areas containing collagen with low hydroxylysine and pyridinoline levels are less prone to degeneration. As such, this study indicates that post-translational modifications of collagen molecules synthesized during adolescence are causally involved in the pathogenesis of OA. © 2002 OsteoArthritis Research Society International. Chemicals/CAS: Amino Acids; Arginine, 74-79-3; Collagen, 9007-34-5; Cross-Linking Reagents; Hydroxylysine, 28902-93-4; Lysine, 56-87-1; pentosidine, 124505-87-9; pyridinoline, 63800-01-1

Collagen deposits in fibrotic lesions often display elevated levels of hydroxyallysine (pyridinoline) cross-links. The relation between the occurrence of pyridinoline cross-links and the irreversibility of fibrosis suggests that these cross-links contribute to the aberrant accumulation of collagen. Based on its inhibitory effect on lysyl hydroxylase activity minoxidil has been postulated to possess anti-fibrotic properties by limiting the hydroxylysine supply for hydroxyallysine cross-linking. However, to interfere with hydroxyallysine cross-linking specifically lysyl hydroxylation of the collagen telopeptide should be inhibited, a reaction predominantly catalysed by lysyl hydroxylase (LH) 2b. In this study, we demonstrate that minoxidil treatment of cultured fibroblasts reduces LH1>>LH2b>LH3 mRNA levels dose-and time-dependently, but has essentially no effect on the total number of pyridinoline cross-links in the collagen matrix. Still the collagen produced in the presence of minoxidil displays some remarkable features: hydroxylation of triple helical lysine residues is reduced to 50% and lysylpyridinoline cross-linking is increased at the expense of hydroxylysylpyridinoline cross-linking. These observations can be explained by our finding that LH1 mRNA levels are the most sensitive to minoxidil treatment, corroborating that LH1 has a preference for triple helical lysine residues as substrate. In addition, the non-proportional increase in cross-links (20-fold) with respect to the decrease in lysyl hydroxylation state of the triple helix (2-fold) even suggests that LH1 preferentially hydroxylates triple helical lysine residues at the cross-link positions. We conclude that minoxidil is unlikely to serve as an anti-fibroticum, but confers features to the collagen matrix, which provide insight into the substrate specificity of LH1. © 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Chemicals / CAS: collagen, 9007-34-5; minoxidil, 38304-91-5; procollagen lysine 2 oxoglutarate 5 dioxygenase, 9059-25-0; pyridinoline, 63800-01-1; 2-Aminoadipic Acid, 542-32-5; Collagen, 9007-34-5; hydroxyallysine, 30382-02-6; lysyl hydroxylase 1, human, EC 1.14.11.4; lysyl hydroxylase 3, human, EC 1.14.11.-; Minoxidil, 38304-91-5; PLOD2 protein, human, EC 1.14.11.4; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase, EC 1.14.11.4

Although >80% of the mineral in mammalian bone is present in the collagen fibrils, limited information is available about factors that determine a proper deposition of mineral. This study investigates whether a specific collagen matrix is required for fibril mineralization. Calcifying callus from dog tibias was obtained at various times (3-21 weeks) after fracturing. At 3 weeks, hydroxylysine (Hyl) levels were almost twice as high as in control bone, gradually reaching normal levels at 21 weeks. The decrease in Hyl levels can only be the result of the formation of a new collagen network at the expense of the old one. The sum of the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) in callus matched that of bone at all stages of maturation. However, the ratio HP/LP was 2.5-4.5 times higher in callus at 3-7 weeks than in normal bone and was normalized at 21 weeks. Some 40% of the collagen was nonmineralized at the early stages of healing, reaching control bone values (≃10%) at 21 weeks. In contrast, only a small increase in callus mineral content from 20.0 to 22.6 (% of dry tissue weight) from week 3 to 21 was seen, indicating that initially a large proportion of the mineral was deposited between, and not within, the fibrils. A strong relationship (r = 0.80) was found between the ratio HP/LP and fibril mineralization; the lower the HP/LP ratio, the more mineralized the fibrils were. Because the HP/LP ratio is believed to be the result of a specific packing of intrafibrillar collagen molecules, this study implies that mineralization of fibrils is facilitated by a specific orientation of collagen molecules in the fibrils.

Objectives. Exploration of bone metabolism changes at different levels of disease activity, both with and without oral corticosteroid therapy, and prediction of changes in joint damage and bone density from the observed changes in markers of bone turnover. Methods. Data analysis from a randomized clinical trial with 155 rheumatoid arthritis (RA) patients; median age 50 yr, early and active disease (diagnosis <2 yr); one group treated with a combination of sulphasalazine (SSZ; 2000 mg/day), methotrexate (MTX; 7.5 mg/week) and prednisolone (initially 60 mg/day, tapered in six weekly steps to 7.5 mg/day), the other group with SSZ alone. Prednisolone and MTX were tapered and stopped after weeks 28 and 40, respectively, while SSZ was continued. Urine and serum samples were collected at baseline and weeks 16, 28, 40 and 56. Measurements of urinary pyridinoline (PYD) and deoxypyridinoline (DPD) and serum alkaline phosphatase (tAP) and osteocalcin (OC) were performed, as well as standard clinimetry and bone densitometry. Results. Over time and in both treatment groups, bone formation and bone resorption markers showed a pattern similar to erythrocyte sedimentation rate (ESR): a significant decrease compared with baseline and a larger decrease with combined treatment at weeks 16 and 28. PYD excretion, tAP, OC, and joint damage scores were significantly lower in the combined treatment group. Changes in bone density (of spine and hips) did not significantly differ between treatment groups. Mainly cumulative ESR explained progression of joint damage. Conclusions. Prednisolone and disease-modifying anti-rheumatic drug therapy in patients with early and active RA are both independently associated with decreased levels of urinary excretion of bone collagen resorption markers PYD and DPD. Markers of bone formation and resorption closely followed changes in ESR in both treatment groups. Reduced bone resorption together with reduced bone formation-initially at a somewhat faster pace-resulted in less bone turnover and explain the observed (non-significant and partially reversible) extra bone loss in the lumbar spine associated with prednisolone (combined treatment).

Context: Molecules that are released into biological fluids during matrix metabolism of articular cartilage, subchondral bone, and synovial tissue could serve as biochemical markers of the process of osteoarthritis (OA). Unfortunately, actual breakthroughs in the biochemical OA marker field are limited so far. Objective: By reviewing the status of commercially available biochemical OA markers according to the "Burden of disease, Investigative, Prognostic, Efficacy of intervention, and Diagnostic" ("BIPED") classification, future use of this "BIPED" classification is encouraged and more efficient biochemical OA marker research stimulated. Data sources: Three electronic databases [PubMed, Scopus, EMBASE (1997-May 2009)] were searched for publications on blood and urinary biochemical markers in human primary knee and hip-OA. Study selection: Stepwise selection of original English publications describing human studies on blood or urinary biochemical markers in primary knee or hip-OA was performed. Selected articles were fully read to determine whether biochemical markers were investigated on performance within any of the "BIPED" categories. Eighty-four relevant publications were identified. Data extraction: Data from relevant publications were tabulated according to the "BIPED" classification. Individual analyses within a publication were summarized in general "BIPED" scores. Data synthesis: An uneven distribution of scores on biochemical marker performance and heterogeneity among the publications complicated direct comparison of individual biochemical markers. Comparison of categories of biochemical markers was therefore performed instead. In general, biochemical markers of cartilage degradation were investigated most extensively and performed well in comparison with other categories of biochemical markers. Biochemical markers of bone metabolism performed less adequately. Biochemical markers of synovial tissue metabolism were not investigated extensively, but performed quite well. Conclusions: Specific biochemical markers and categories of biochemical markers as well as their nature, origin and metabolism, need further investigation. International standardization of future investigations should be pursued to obtain more high-quality, homogenous data on the full spectrum of biochemical OA markers. © 2010 Osteoarthritis Research Society International.

The aim was to identify suspect collagen cross-links in dentine, eluting close to known cross-links in ion-exchange HPLC. Bovine tooth roots as source of dentine were powdered, demineralised, reduced, and acid-hydrolysed. Cross-linking amino acids were isolated from the acid hydrolysate by size exclusion, adsorption, and sequential ion exchange chromatography. In addition to dihydroxylysinonorleucine and hydroxylysylpyridinoline, an unknown cross-link was isolated (V-2). The ultraviolet, mass, and nuclear magnetic resonance spectra support the proposed structure of V-2, a trimeric amino acid with a pyrroleninone nucleus. Copyright (C) 1998 Elsevier Science B.V.

Biochemical heterogeneity of cartilage within a joint is well known in mature individuals. It has recently been reported that heterogeneity for proteoglycan content and chondrocyte metabolism in sheep develops postnatally under the influence of loading. No data exist on the collagen network in general or on the specific situation in the horse. The objective of this study was to investigate the alterations in equine articular cartilage biochemistry that occur from birth up to age one year, testing the hypothesis that the molecular composition of equine cartilage matrix is uniform at birth and biochemical heterogeneity is formed postnatally. Water content, DNA content, glycosaminoglycan content (GAG) and biochemical characteristics of the collagen network (collagen content, hydroxylysine content and hydroxylysylpyridinoline [HP] crosslinks) were measured in immature articular cartilage of neonatal (n = 16), 5-month-old foals (n = 16) and yearlings (n = 16) at 2 predefined differently loaded sites within the metacarpophalangeal joint. Statistical differences between sites were analysed by ANOVA (P<0.01), and age correlation was tested by Pearson's product moment correlation analysis (P<0.01). In neonatal cartilage no significant site differences were found for any of the measured biochemical parameters. This revealed that the horse has a biochemically uniform joint (i.e. the cartilage) at birth. In the 5-month-old foals and yearlings, significant site differences, comparable to those in the mature horse, were found for DNA, GAG, collagen content and hydroxylysine content. This indicates that functional adaptation of articular cartilage to weight bearing for these biochemical parameters takes place during the first months postpartum. Water content and HP crosslinks showed no difference between the 2 sites from neonatal horses, 5-month-old animals and yearlings. At both sites water, DNA and GAG decreased during maturation while collagen content, hydroxylysine content and HP crosslinks increased. We propose that a foal is born with a uniform biochemical composition of cartilage in which the functional adaptation to weight bearing takes place early in life. This adaptation results in biochemical and therefore biomechanical heterogeneity and is thought to be essential to resist the different loading conditions to which articular cartilage is subjected during later life. As collagen turnover is extremely low at mature age, an undisturbed functional adaptation of the collagen network of articular cartilage at a young age may be of significant importance for future strength and resistance to injury.

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross- links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.

The aim of this study was to evaluate topographical differences in the biochemical composition of the extracellular matrix of articular cartilage of the normal equine fetlock joint. Water content, DNA content, glycosaminoglycan (GAG) content and a number of characteristics of the collagen network (total collagen content, levels of hydroxylysine- (Hyl) and the crosslink hydroxylysylpyridinoline, (HP) of articular cartilage in the proximal 1st phalanx (P1), distal 3rd metacarpal bone (MC), and proximal sesamoid bones (PSB) were determined in the left and right fetlock joint of 6 mature horses (age 5-9 years). Twenty-eight sites were sampled per joint, which included the clinically important areas often associated with pathology. Biochemical differences were evaluated between sampling sites and related with the predisposition for osteochondral injury and type of loading. Significant regional differences in the composition of the extracellular matrix existed within the joint. Furthermore, left and right joints exhibited biochemical differences. Typical topographic distribution patterns were observed for each parameter. In P1 the dorsal and palmar articular margin showed a significantly lower GAG content than the more centrally located sites. Collagen content and HP crosslinks were higher at the joint margins than in the central area. Also, in the MC, GAG content was significantly lower at the (dorsal) articular margin compared with the central area. Consistent with findings in P1, collagen and HP crosslinks were significantly lower in the central area compared to the (dorsal) articular margin. Biochemical and biomechanical heterogeneity of articular cartilage is supposed to reflect the different functional demands made at different sites. In the present study, GAG content was highest in the constantly loaded central areas of the joint surfaces. In contrast, collagen content and HP crosslinks were higher in areas intermittently subjected to peak loading which suggests that the response to a certain type of loading of the various components of the extracellular matrix of articular cartilage are different. The differences in biochemical characteristics between the various sites may help to explain the site specificity of osteochondral lesions commonly found in the equine fetlock joint. Finally, these findings emphasise that the choice of sampling sites may profoundly influence the outcome of biochemical studies of articular cartilage.

The plasminogen activation system is one of the enzyme systems held responsible for bone and cartilage degradation in rheumatoid arthritis (RA). In this study, we evaluated the effect of tranexamic acid (TEA), an inhibitor of plasminogen activation, on urinary collagen cross-link excretion and radiological joint damage in rat adjuvant arthritis (AA) and on urinary collagen cross-link excretion in patients with RA. In the animal study, adjuvant arthritis was induced in male Lewis rats. From day 7 onward, high-dose TEA (500 mg/kg body weight, once daily) or placebo was administered orally. Study groups consisted of TEA-treated normal rats (C + TEA), placebo-treated normal rats (C + plac), AA rats treated with TEA (AA + TEA) or with placebo (AA + plac). To monitor joint destruction, urinary collagen cross-link excretion (pyridinoline, HP: deoxypyridinoline, LP) was measured by high-performance liquid chromatography at days 14 and 21. Radiological evaluation of joints was performed at day 21. In the patient study, TEA was administered to nine patients with RA as adjuvant medication (~20 mg/kg body weight, three times daily) for 12 weeks. Urinary HP and LP excretion levels were measured before and during TEA treatment, and 4 weeks after the cessation of TEA treatment. In AA + TEA rats, a significant reduction of HP and a tendency towards a reduction of LP excretion were found compared with AA + plac rats (P < 0.05), at day 14, whereas the HP/LP ratio did not change. No difference was observed in HP, LP excretion, HP/LP ratio and radiological damage score between the TEA- and placebo-treated AA rats at day 21. In RA patients, a significant reduction of HP and LP excretion was found during the TEA treatment period (P < 0.05). After the cessation of TEA treatment, HP and LP excretion increased towards baseline levels. No effect on disease activity was observed. The plasmin antagonist TEA reduced the excretion of collagen pyridinoline cross-links in both experimental and rheumatoid arthritis. As such, this study not only supports the involvement of the plasminogen activation system in the destructive phase of arthritis, but also suggests a beneficial effect of therapeutic strategies directed against inhibition of matrix proteolysis.

A biomechanical failure of the collagen network is postulated in many hypotheses of the development of osteoarthritis with advancing age. Here we investigate the accumulation of non-enzymatic glycation (NEG) products in healthy human articular cartilage, its relation to tissue remodelling and its role in tissue stiffening. Pentosidine levels were low up to age 20 years, and increased linearly after this age. This indicates extensive tissue remodelling at young age, and slow turnover of collagen after maturity has been reached. The slow remodelling is supported by the finding that enzymatic modifications of collagen (hydroxylysine, hydroxylysylpyridinoline, and lysylpyridinoline) were not related to age. The high remodelling is supported by levels of the crosslink lysylpyridinoline (LP) as a function of distance from the articular surface. LP was highest at the surface in mature cartilage (> 20 years), whereas in young cartilage (< 10 years) the opposite was seen; highest levels were close to the bone. LP levels in cartilage sections at age 14 years are high at the surface and close to the bone, but they are low in the middle region. This indicates that maturation of cartilage in the second decade of life starts in the upper half of the tissue, and occurs last in the tissue close to the bone. The effect of NEG products on instantaneous deformation of cartilage was investigated as a functional of topographical variations in pentosidine levels in vivo and in relation to in vitro induced NEG. Consistently, higher pentosidine levels were associated with a stiffer collagen network. A stiffer and more crosslinked collagen network may become more brittle and more prone to fatigue.

The objective of this study was to analyse parameters in rhesus monkey collagen-induced arthritis (CIA) with which the inflammation and destruction of the joints can be described in quantitative terms. CIA was induced in genetically susceptible and resistant monkeys, which can be distinguished on the basis of the dominant resistance marker Mamu-A26. The disease course was monitored daily using a semiquantitative scoring system. Plasma samples were collected once or twice weekly and analysed for C-reactive protein (CRP). Urines were collected overnight once a week and analysed for excretion rates of the collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP). The results show that periods of active CIA are characterized by substantial weight loss and increased plasma CRP levels, followed shortly thereafter by increased excretion rates of the collagen cross-links HP and LP. Remission of the disease can be recognized by a decline in plasma CRP levels and especially an increase in body weight. The highest CRP levels were found in the most severely arthritic monkeys, indicating a possible relationship of the absolute plasma CRP levels to the severity of inflammation. During periods of active arthritis, increased excretion rates of collagen cross-links HP and LP in the urine were found. In particular, the major collagen crosslink in articular cartilage, HP, showed a strong increase (9- to 15-fold). The excretion rates of LP, which is considered as a bone-specific degradation marker, only increased 4- to 6-fold, thus indicating predominant destruction of cartilage and less of bone. In conclusion, the severity of CIA can be monitored in a quantitative manner using plasma CRP levels, urinary excretion rates of HP and LP, and body weights, superimposed on semiquantitative clinical scores. The parameters also facilitate a more objective assessment of the effect of anti-arthritic drugs in the model than with the clinical scores alone.

The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically. Predominantly type H collagen was present in the alginate bead, as reflected by the unique extent of lysyl hydroxylation, glycosylation, and pyridinoline crosslink formation measured. Collagen crosslinks, predominantly hydroxylysylpyridinoline (>93%), were observed after 7 to 11 days of culture and their formation was effectively blocked by β-aminopropionitrile (BAPN). Unexpectedly, BAPN treatment resulted in a 100% increase of collagen levels, without influencing cell proliferation and proteoglycan levels. In control cultures 90% of the synthesized collagen was retained in the cell-associated matrix, while in BAPN-treated cultures half of the collagen was found in the interterritorial matrix compartment further removed from the cells. This suggests that impaired crosslinking of collagen interferes with pericellular collagen deposition, causing upregulation of collagen synthesis by impaired cell-matrix interactions. Integrins are likely to be involved in this feedback inhibition by extracellular collagen since the cyclic RGD-containing peptide CGRGDSPC downregulated collagen synthesis by 37%. Chemicals/CAS: Alginates; alginic acid, 9005-32-7; Amino Acids; Aminopropionitrile, 151-18-8; arginyl-glycyl-aspartic acid, 99896-85-2; Collagen, 9007-34-5; DNA, 9007-49-2; Glucuronic Acid, 576-37-4; glycyl-arginyl-glycyl-aspartyl-seryl-proline, 91037-75-1; Hexuronic Acids; Oligopeptides; Proteoglycans; pyridinoline, 63800-01-1

The hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-β induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I α2 (COL1A2). The TGF-β isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-α. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines. © 2004 Elsevier B.V. All rights reserved. Chemicals / CAS: activin A, 104625-48-1; collagen, 9007-34-5; procollagen lysine 2 oxoglutarate 5 dioxygenase, 9059-25-0; protein lysine 6 oxidase, 99676-44-5; activin A; Activins, 104625-48-1; Amino Acids; Collagen Type I; Cross-Linking Reagents; Cytokines; Inhibin-beta Subunits, 93443-12-0; Interleukin-4, 207137-56-2; PLOD2 protein, human, EC 1.14.11.4; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase, EC 1.14.11.4; Protein Isoforms; pyridinoline, 63800-01-1; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Collagen and elastin are the primary determinants of vascular integrity, with elastin hypothesized to be the major contributor to aortic compliance and type I collagen the major contributor to aortic strength and stiffness. Type I collagen is normally heterotrimeric composed of two α1(I) and one α2(I) collagen chains, α1(I)2α2(I). Recent investigations have reported that patients with recessively inherited forms of Ehlers Danlos syndrome that fail to synthesize proα2(I) chains have increased risks of cardiovascular complications. To assess the role of α2(I) collagen in aortic integrity, we used the osteogenesis imperfecta model (oim) mouse. Oim mice, homozygous for a COL1A2 mutation, synthesize only homotrimeric type I collagen, α1(I)3. We evaluated thoracic aortas from 3-month-old oim, heterozygote, and wildtype mice biomechanically for circumferential breaking strength (Fmax) and stiffness (IEM), histologically for morphological differences, and biochemically for collagen content and crosslinking. Circumferential biomechanics of oim and heterozygote descending thoracic aortas demonstrated the anticipated reduced Fmax and IEM relative to wildtype mice. Histological analyses of oim descending aortas demonstrated reduced collagen staining relative to wildtype aortas suggesting decreased collagen content, which hydroxyproline analyses of ascending and descending oim aortas confirmed. These findings suggest the reduced oim thoracic aortic integrity correlates with the absence of the α2(I)collagen chains and in part with reduced collagen content. However, oim ascending aortas also demonstrated a significant increase in pyridinoline crosslinks/collagen molecule as compared to wildtype ascending aortas. The role of increased collagen crosslinks is uncertain; increased crosslinking may represent a compensatory mechanism for the decreased integrity. © 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Chemicals / CAS: hydroxyproline, 51-35-4, 6912-67-0; pyridinoline, 63800-01-1; Collagen Type I; Collagen, 9007-34-5

Consumption of fructo-oligosaccharides (FOS) has been shown to improve mineral absorption in the short term, but no long-term effects were studied in girls with a low calcium intake. Therefore, we hypothesized that short- and long-term consumption of short-chain FOS (sc-FOS) improves calcium and magnesium absorption in girls with a low habitual calcium intake. Fourteen girls aged between 12 and 14 years received, for 36 days, 10 g sc-FOS (sc-FOS) or maltodextrin (placebo). Short-chain FOS were taken daily for 8 days followed by an intermittent intake mode on 28 random days to mimic noncontinuous intake. In a crossover design, true calcium and magnesium absorption was computed on the 8th and 36th day of each treatment period from isotope enrichment in urine. In addition, the parathyroid hormone and vitamin D in serum and markers of bone resorption in urine (pyridinoline and deoxypyridinoline) were determined. Short-chain FOS increased magnesium absorption by 18% after 36 days (30.1% ± 9.1% vs 35.4% ± 12.8%). Magnesium absorption did not change after the initial 8 days of long-term sc-FOS intake. Short-chain FOS did not affect calcium absorption. In addition, sc-FOS did not affect vitamin D, parathyroid hormone, or markers of bone resorption. It is concluded that consumption of sc-FOS for 36 days stimulated magnesium absorption in girls but did not affect calcium absorption. © 2009 Elsevier Inc. All rights reserved.

Study Design. Caprine lumbar intervertebral discs (IVD) were collected from previous studies and categorized as normal, mildly, or severely degenerated. The biochemical composition and the RNA profiles present in both the nucleus pulposus (NP) and the anulus fibrosus (AF) were analyzed. Objective. To determine the molecular changes occurring in a disc degeneration model, evaluating the mechanism through which the degeneration develops in this model. Summart of Background Data. Recently we described an IVD degeneration model in the goat by injecting chondroitinase ABC. This results in mild progressive disc degeneration. Methods. One hundred nine caprine IVDs were assigned to 3 classes: no degeneration, mild, or severe degeneration. Collagen content, collagen cross-links (hydroxylysyl pyridinoline) and the ratio between the glycosaminoglycans (GAGs) and hydroxyprolines (Hyp) (GAG/Hyp ratio) in the NP and AF samples were studied. Furthermore, the gene expression of collagen type I, type II, and aggrecan as well as a desintegrin and metalloproteinase with thrombospondin motifs (ADAMTIS)-2, ADAMTS-14, and matrix metalloproteinases-13 were studied. Results. Collagen content was increased in severely degenerated NPs and decreased in severely degenerated AFs. Collagen cross-links were decreased in the severely degenerated NPs indicating de novo deposition of immature, reducible cross-linked collagens. The GAG/Hyp ratio found in none-degenerate goat discs was comparable to human ratios and decreased in degenerated discs, similar as in humans. The ADAMTS genes were increasingly detectable in the degenerated discs. The matrix metalloproteinases-13 gene increased significantly in degenerated discs. The expression of collagen type I increased in degenerated discs while aggrecan decreased. Conclusion. Changes in the GAG/Hyp ratio of chemically induced degeneration in goat IVD resemble the changes seen in humans. Gene expression profiles match the pattern of degeneration, suggesting that the injection of chondroitinase ABC might mimic the onset of human disc degeneration. © 2008 Lippincott Williams & Wilkins.

Subchondral bone provides structural support to the overlying articular cartilage, and plays an important role in osteochondral diseases. There is growing insight that the mechanical features of bone are related to the biochemistry of the collagen network and the mineral content. In the present study, part of the normal developmental process and the influence of physical activity on biochemical composition of subchondral bone was studied. Water content, calcium content and characteristics of the collagen network (collagen, hydroxylysine, lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) crosslinking) of subchondral bone were measured in newborn foals, 5-month-old foals (pasture-grown and box-confined) and 11-month-old foals at 2 differently loaded sites of the proximal articular surface of the first phalanx. During the first 5 months postpartum, water and hydroxylysine content decreased significantly while calcium and collagen content and the amount of HP and LP crosslinks increased significantly. The withholding of physical activity during this developmental phase affected the biochemical characteristics of subchondral bone only at the site that is loaded during physical exercise. At this site, calcium content and both HP and LP crosslink levels increased significantly less than in pasture-raised animals. During development from 5-11 months, measured parameters remained essentially constant, except for water content, which decreased further. It is concluded that substantial changes, presumed to be largely exercise-driven, take place during the normal process of development in the biochemical composition of equine subchondral bone. Normal development of subchondral bone is presumably important for the normal functional adaptation of this bone to the loading conditions it is subjected to and therefore essential to resist the future biomechanical challenges the horse will encounter during its athletic career. The findings from this study and the assumed important role of subchondral bone quality in the pathogenesis of osteochondral disease merit more attention to the role of the collagen network in subchondral bone.

Cleft lip and palate is a common craniofacial malformation in man. The aetiology is multifactorial and not known. Since collagen is a major structural component of the developing palate, we studied its composition and metabolism during palate shelf formation and elevation in the rat. Palatal shelves were harvested at embryonic days (E) 15, 16 and 17 as well as post-partum. Palatal collagen increased threefold from E15 to E17 and tenfold from E17 to 5-day-old pups. Palatal calcification was seen in the main, post-partum. Collagen cross-linking, which may be important in shelf elevation and union, varied. The concentration of hydroxylysyl-pyridinolone cross-links was greatest prior to shelf elevation, declining thereafter. Similarly, the highest concentration of dihydroxylysinononorleucine was seen at E16 and this supports the concept of a compliant mesenchymal shelf responding to an intrinsic elevating force. We then determined if enzymes responsible for matrix degradation, matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMPs) altered over the same time periods. MMP-2, and TIMP-1 and TIMP-2 were identified by gelatin zymography and reverse zymography, respectively. MMP-3 activity was determined with a fluorogenic substrate assay. TIMP-1, TIMP-2 and MMP-3 levels remained constant from E15 to E17. The MMP-2 levels showed a significant elevation from E15 to E16 and E16 to E17. This suggests the regulation of extracellular matrix is likely to be of importance in palate morphogenesis. Copyright (C) 2000 Elsevier Science Ireland Ltd.

Objective. To investigate the effects of doxycycline on disease activity and joint destruction in patients with rheumatoid arthritis (RA). Methods. A 36 week double blind, placebo controlled crossover trial was conducted. Patients (n = 66) received 50 mg doxycycline or placebo twice a day during 12, 24, or 36 weeks. Patient assessments were performed before the treatment was administered, at 6, 12, 24 and 36 weeks of treatment, and finally at 4 weeks after cessation of treatment. Patient assessments, swollen and tender joint counts, duration of morning stiffness, erythrocyte sedimentation rate, and Modified Disease Activity Score were used as measures of disease activity. Effects on joint destruction were assessed by urinary excretion of the pyridinolines hydroxylysylpyridinoline and lysylpyridinoline and by scoring radiographic damage of hands and feet before and after treatment. Results. The changes of clinical or laboratory disease activity measures, pyridinoline excretion, or progression of radiographic joint damage during doxycycline or placebo treatment did not differ significantly. Conclusion. The results indicate that 50 mg doxycycline twice a day provided no therapeutic benefit for patients with RA.