Functional adaptation through changes in regional biochemical characteristics during maturation of equine superficial digital flexor tendons.

OBJECTIVE: To quantify and compare biochemical characteristics of the extracellular matrix (ECM) of specimens harvested from tensional and compressive regions of the superficial digital flexor tendon (SDFT) of horses in age classes that include neonates to mature horses. SAMPLE POPULATION: Tendon specimens were collected on postmortem examination from 40 juvenile horses (0, 5, 12, and 36 months old) without macroscopically visible signs of tendonitis. PROCEDURE: Central core specimens of the SDFT were obtained with a 4-mm-diameter biopsy punch from 2 loaded sites, the central part of the mid-metacarpal region and the central part of the mid-sesamoid region. Biochemical characteristics of the collagenous ECM content (ie, collagen, hydroxylysylpyridinoline crosslink, and pentosidine crosslink concentrations and percentage of degraded collagen) and noncollagenous ECM content (percentage of water and glycosaminoglycans, DNA, and hyaluronic acid concentrations) were measured. RESULTS: The biochemical composition of equine SDFT was not homogeneous at birth with respect to DNA, glycosaminoglycans, and pentosidine concentrations. For most biochemical variables, the amounts present at birth were dissimilar to those found in mature horses. Fast and substantial changes in all components of the matrix occurred in the period of growth and development after birth. CONCLUSIONS AND CLINICAL RELEVANCE: Unlike cartilage, tendon tissue is not biochemically blank (ie, homogeneous) at birth. However, a process of functional adaptation occurs during maturation that changes the composition of equine SDFT from birth to maturity. Understanding of the maturation process of the juvenile equine SDFT may be useful in developing exercise programs that minimize tendon injuries later in life that result from overuse.

Biochemical analysis of the articular cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis.

OBJECTIVE: To assess whether site-related changes in biochemical composition are present in the cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis. SAMPLE POPULATION: Right metacarpophalangeal joints from 59 mature warmblood horses. PROCEDURE: Biochemical data (cross-link, amino acid, DNA, and ash contents; denatured collagen and glycosaminoglycan [GAG] concentrations; bone mineral density; and mineral composition) were obtained from 2 differently loaded sites of phalanx I cartilage and subchondral and trabecular bone samples; data were compared with previously published values from nonosteoarthritic equine joints. RESULTS: Compared with findings in nonosteoarthritic joints, GAG concentration was lower in cartilage from osteoarthritic joints and there was a loss of site differences in cellularity and lysylpyridinoline (LP) cross-link content. In subchondral bone, LP cross-link content was decreased overall and there was a loss of site differences in osteoarthritic joints; ash content was higher in the osteoarthritic joints. Hydroxyproline content in trabecular bone from osteoarthritic joints was greater than that in nonosteoarthritic trabecular bone. In all 3 layers and at both sites, the linear increase of the pentosidine cross-link content with age had diminished or was not apparent in the horses with osteoarthritic joints. CONCLUSIONS AND CLINICAL RELEVANCE: In equine metacarpophalangeal joints with early osteoarthritis, distinct biochemical changes were detected in the cartilage and subchondral and trabecular bone. The dissimilarity in response of the different tissues and differences between the sites that are affected may be related to differences in biomechanical loading and transmission and dissipation of force.

An integral biochemical analysis of the main constituents of articular cartilage, subchondral and trabecular bone.

OBJECTIVE: In articular joints, the forces generated by locomotion are absorbed by the whole of cartilage, subchondral bone and underlying trabecular bone. The objective of this study is to test the hypothesis that regional differences in joint loading are related to clear and interrelated differences in the composition of the extracellular matrix (ECM) of all three weight-bearing constituents. METHOD: Cartilage, subchondral- and trabecular bone samples from two differently loaded sites (site 1, dorsal joint margin; site 2, central area) of the proximal articular surface of 30 macroscopically normal equine first phalanxes were collected. Collagen content, cross-linking (pentosidine, hydroxylysylpyridinoline (HP), lysylpyridinoline (LP)) hydroxylation, and denaturation, as well as glycosaminoglycan (GAG) and DNA content were measured in all three tissues. In addition, bone mineral density (BMD), the percentage of ash and the mineral composition (calcium, magnesium and phosphorus) were determined in the bony samples. RESULTS: For pentosidine cross-links there was an expected correlation with age. Denatured collagen content was significantly higher in cartilage at site 1 than at site 2 and was higher in trabecular bone compared to subchondral bone, with no site differences. There were significant site differences in hydroxylysine (Hyl) concentration and HP cross-links in cartilage that were paralleled in one or both of the bony layers. In subchondral bone there was a positive correlation between total (HP+LP) cross-links and Ca content. For Ca and other minerals there were corresponding site differences in both bony layers. CONCLUSIONS: It is concluded that there are distinct differences in distribution of the major biochemical components over both sites in all three layers. These differences show similar patterns in cartilage, subchondral bone and trabecular bone, stressing the functional unity of these tissues. Overall, differences could be interpreted as adaptations to a considerably higher cumulative loading over time at site 2, requiring stiffer tissue. Turnover is higher in trabecular bone than in subchondral bone. In cartilage, the dorsal site 1 appears to suffer more tissue damage.

Assessment of the effects of age and joint disease on hydroxyproline and glycosaminoglycan concentrations in synovial fluid from the metacarpophalangeal joint of horses.

OBJECTIVE: To assess the effects of age and joint disease on hydroxyproline and glycosaminoglycan (GAG) concentrations in synovial fluid from the metacarpophalangeal joint of horses and evaluate the association of those concentrations with severity of osteoarthritis and general matrix metalloproteinase (MMP) activity. SAMPLE POPULATION: Synovial fluid was collected from the metacarpophalangeal joints of foals at birth (n = 10), 5-month-old foals (10), 11-month-old foals (5), and adult horses (73). PROCEDURE: Hydroxyproline and GAG concentrations were determined in synovial fluid samples. The severity of osteoarthritis in adult joints was quantified by use of a cartilage degeneration index (CDI) and assessment of general MMP-activity via a fluorogenic assay. RESULTS: Hydroxyproline and GAG concentrations in synovial fluid were highest in neonates and decreased with age. Concentrations reached a plateau in adults by 4 years and remained constant in healthy joints. In synovial fluid from osteoarthritic joints, hydroxyproline and GAG concentrations were not increased, compared with unaffected joints, but hydroxyproline were significantly correlated with the CDI and general MMP activity. There was no significant correlation between GAG concentration and CDI value or MMP activity. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in hydroxyproline concentration in synovial fluid appeared to indicate damage to collagen of the articular cartilage. In joints with osteoarthritis, the lack of high GAG concentration in synovial fluid and the absence of a significant correlation between GAG concentration and CDI values or MMP activity may severely limit the usefulness of this marker for monitoring equine joint disease.

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics.

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.