The propensity of individual trabeculae to fracture (microfracture) may be important clinically since it could be indicative of bone fragility. Whether or not an overloaded trabecula fractures is determined in part by its structural ductility, a mechanical property that describes how much deformation a trabecula can sustain. The overall goal of this study was to determine the structural ductility of individual trabeculae and the degree to which it is influenced by pyridinium and non-enzymatic collagen cross-links. Vertically oriented rod-like trabeculae were taken from the thoracic vertebral bodies of 32 cadavers (16 male and 16 female, 54-94 years of age). A total of 221 trabeculae (4-9 per donor) were tested to failure in tension using a micro-tensile loading device. A subset of 76 samples was analyzed to determine the concentration of hydroxylysyl-pyridinoline (HP) and lysylpyridinoline (LP) cross-links as well as pentosidine, a marker of non-enzymatic glycation. Structural ductility (defined as the ultimate strain of the whole trabecula) ranged from 1.8-20.2% strain (8.8 ± 3.7%, mean ± S.D.) and did not depend on age (p=0.39), sex (p=0.57) or thickness of the sample at the point of failure (p = 0.36). Pentosidine was the only marker of collagen cross-linking measured that was found to be correlated with structural ductility (p = 0.01) and explained about 9% of the observed variance. We conclude that the ductility of individual trabeculae varies tremendously, can be substantial, and is weakly influenced by non-enzymatic glycation.

During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1-A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol x (g of protein)(-1) x year(-1). Using previously reported protein turnover rate constants (k(T)) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (k(F)) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009-13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81+/-0.25 compared with 3.71+/-0.26 micromol of pentosidine x (mol of lysine)(-1) x year(-1) respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year(-1) compared with 0.134 year(-1) for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective k(F) values of 1.81+/-0.25 and 3.18+/-0.37 micromol of pentosidine.(mol of lysine)(-1) x year(-1). We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding.

Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We measured the ratio of the D-and L-isomers in collagen extracted from these tissues as a function of age between 16 and 77 years. For collagen taken from healthy discs, the fractional increase of D-Asp was found to be 6.74 ؋ 10 ؊4 /year; for degenerate discs, the corresponding rate was 5.18 ؋ 10 ؊4 /year. Using the racemization rate found previously for the stable population of collagen molecules in dentin, we found that the rate of collagen turnover (k T ) in discs is not constant but rather a decreasing function of age. The average turnover rate in normal disc between the ages of 20 and 40 is 0.00728 ؎ 0.00275/year, and that between the ages of 50 and 80 is 0.00323 ؎ 0.000947/year, which correspond to average half-lives of 95 and 215 years, respectively. Turnover of collagen from degenerate discs may be more rapid than that found for normal discs; however, statistical analysis leaves this point uncertain. The finding of a similar correlation between the accumulation of D-Asp and that of pentosidine for three normal collagenous tissues further supports the idea that the accumulation of pentosidine in a particular tissue can, along with the racemization of aspartic acid, be used as a reliable measure of protein turnover.The intervertebral disc (IVD), 2 the largest avascular cartilaginous structure (1, 2), plays a primary mechanical role in transmitting loads through the spine and providing flexibility to the spinal column. The disc has a complex structure and contains very few cells embedded in an extracellular matrix. These cells have the essential function of maintaining and repairing the matrix by synthesizing matrix macromolecules and by producing degradative enzymes, including metalloproteinases and their inhibitors (TIMPs (tissue inhibitors of metalloproteinase)), which are all involved in tissue metabolism and turnover. Thus, normal disc function is dependent on a balance between synthesis and matrix breakdown.The fine balance between synthesis and degradation determines the concentrations of tissue components and hence the composition of the tissue. In vivo (3, 4) and in vitro (5) environmental factors such as mechanical stress (6 -8) and nutrient levels have been found to affect matrix composition, presumably by modulating rates of macromolecular biosynthesis and degradation. Normally, when a balance is maintained, damaged tissue can be restored by cellular repair responses; otherwise, the matrix composition and organization are altered, and the cellular repair responses become inadequate. Hence, the degraded matrix can no longer carry loads effectively, which leads to the degeneration of the disc.During aging, many changes involving the proportions and biochemical properties of the matrix occur. These proces...

We have used the racemization of aspartic acid as a marker for the "molecular age" of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the D/L Asp ratio of the various aggrecan species as a function of age and using the values of the racemization constant, k i , found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. The intervertebral disc (IVD)2 is the largest avascular cartilaginous structure. It lies between the vertebral bodies, anchoring them together. IVD plays a primarily mechanical role in transmitting loads through the spine and providing flexibility to the spinal column. The IVD is highly bradytrophic; it is avascular and nourished by diffusion. Although the outer annulus fibrosus (AF) possesses blood vessels in early childhood, the inner nucleus pulposus (NP) remains avascular for the entire life of the organism (1, 2). The disc has a complex structure and contains very few cells embedded in an extracellular matrix. These cells have the essential function of maintaining and repairing the matrix by synthesizing matrix macromolecules and by producing proteinases for matrix breakdown. Thus, disc function is dependent on a balance between synthesis and matrix breakdown. Normally, when this balance is maintained, damaged tissue can be restored by cellular repair responses. In pathology, when there is imbalance between matrix synthesis and breakdown, the matrix composition and organization are altered, and the cellular repair responses are inadequate. Hence, the degraded matrix can no longer carry loads effectively, which leads to the degeneration of the disc.During aging, many changes involving the proportions and biochemical properties of aggrecan occur. In particular, the structure and composition of aggrecan changes both with aging and with degeneration. These changes involve an increase in the relative contents of keratan sulfate and protein and a decrease in the molecular weight of aggrecan (3). The important question of whether these changes in composition of the aggrecan with aging represent changes in biosynthesis or are due to the accumulation of degraded aggrecan fragments can be addressed by measuring the accumulation of the D-aspartic acid isomer and hence the residence time or the "molecular age" of these molecules. In nature, amino acids are synthesized as L-isomers. Spontaneous racemization slowly converts the L-form of amino acids into a racemic mixture of Land D-forms. Aspartic acid is one of the most rapidly racemizing amino acids (4, 5), allowing the measurement of the concentration of D-isomers in living subjects in proteins that are not renewed or that slowly turn over. It is well known that an age-dependent racemization occurs in various human and animal tissues containing metabolically stable, longlived proteins, e.g. in enamel and dent...

Objective. Obesity is associated with systemic inflammation and is a risk factor for osteoarthritis (OA) development. We undertook this study to test the hypothesis that metabolic stress-induced inflammation, and not mechanical overload, is responsible for the development of high-fat diet-induced OA in mice.Methods. Human C-reactive protein (CRP)-transgenic mice received a high-fat diet without or with 0.005% (weight/weight) rosuvastatin or 0.018% (w/w) rosiglitazone, 2 different drugs with antiinflammatory properties. Mice fed chow were included as controls. After 42 weeks, mice were killed and histologic OA grading of the knees was performed. To monitor the overall inflammation state, systemic human CRP levels were determined.Results. Male mice on a high-fat diet had significantly higher OA grades than mice on chow and showed no correlation between OA severity and body weight. In male mice, high-fat diet-induced OA was significantly inhibited by rosuvastatin or rosiglitazone to OA grades observed in control mice. Both treatments resulted in reduced human CRP levels. Furthermore, a positive correlation was found between the relative individual induction of human CRP evoked by a high-fat diet on day 3 and OA grade at end point.Conclusion. High-fat diet-induced OA in mice is due to low-grade inflammation and not to mechanical overload, since no relationship between body weight and OA grade was observed. Moreover, the OA process was inhibited to a great extent by treatment with 2 drugs with antiinflammatory properties. The inflammatory response to a metabolic high-fat challenge may predict individual susceptibility to developing OA later in life. The use of statins or peroxisome proliferator-activated receptor ␥ agonists (e.g., rosiglitazone) could be a strategy for interfering with the progression of OA.Osteoarthritis (OA) is a chronic degenerative joint disease with large consequences for the quality of life of patients. It is now generally accepted that OA is not only a disease of articular cartilage, but in fact involves the entire joint, including Hoffa's fat pad, synovium, subchondral bone, menisci, and ligaments. Insight into the different underlying processes leading to the clinical and pathologic outcomes of OA is crucial in the search for new therapies (1,2).Obesity is a risk factor for the development of OA and is classically seen as a biomechanical factor, suggesting that the increase of loading forces causes cartilage damage. However, from the association between obesity and OA of non-load bearing joints it is hypothesized that systemic factors induced by obesity contribute considerably to the initiation and progression of OA (3). Obesity is associated with a mild chronic inflammation, and adipokines secreted by adipocytes and macrophages within adipose tissue are suggested to be a metabolic link between obesity and OA (4,5). However, the relative contribution of these processes in the onset and progression of OA remains unclear.The association between obesity and the develSupported by Top Institute P...