H. Richard

Cartilage matrix changes in the developing epiphysis: early events on the pathway to equine osteochondrosis?

REASONS FOR PERFORMING STUDY The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth. OBJECTIVE To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions. METHODS Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix. RESULTS Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur. CONCLUSION Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.

Comparison of pharmacokinetics of glucosamine and synovial fluid levels following administration of glucosamine sulphate or glucosamine hydrochloride

OBJECTIVE To compare the pharmacokinetics of glucosamine and the synovial fluid levels attained following treatment with glucosamine sulphate or glucosamine hydrochloride in a large animal model at clinically relevant doses. METHODS Eight adult female horses were used. Crystalline glucosamine sulphate (Dona) or glucosamine hydrochloride was administered at a dose of 20 mg/kg by either intravenous (i.v.) injection or nasogastric (n.g.) intubation. Plasma samples were collected before dosing and at 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid samples were collected from the radiocarpal joints within 48 h before dosing and at 1, 6 and 12 h post-dosing. Glucosamine was assayed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). RESULTS Plasma concentrations reached approximately 50 microg/mL after i.v. injection and approximately 1 microg/mL after n.g. administration of both types of glucosamine. The median oral bioavailability was 9.4% for glucosamine sulphate and 6.1% for glucosamine hydrochloride. Synovial fluid concentrations were significantly higher at 1 and 6 h following oral treatment with glucosamine sulphate compared to glucosamine hydrochloride. Twelve hours following oral administration, glucosamine levels in the plasma and the synovial fluid were still significantly higher than baseline for the glucosamine sulphate preparation, but not for the hydrochloride preparation. CONCLUSION Following oral administration of a clinically recommended dose of glucosamine sulphate (Dona), significantly higher synovial fluid concentrations of glucosamine are attained, when compared to an equivalent dose of glucosamine hydrochloride. Whether this difference is translated into a therapeutic effect on the joint tissues remains to be elucidated.

Evidence to suggest that cathepsin K degrades articular cartilage in naturally occurring equine osteoarthritis

OBJECTIVE The mechanisms leading to degeneration of articular cartilage in osteoarthritis (OA) are complex and not yet fully understood. Cathepsin K (CK) is a cysteine protease which can also cleave the triple helix of type II collagen. This exposes a neoepitope that can now be identified by specific antibodies. The aim of this study was to obtain evidence suggesting a role for CK in naturally occurring equine OA in both lesional and peri-lesional regions. METHODS Articular cartilages (n=12 horses; 5 healthy, 7 OA) were harvested from animals postmortem. A gross macroscopic examination, histologic (Safranin O-Fast Green and Picrosirius red staining) and immunohistochemical evaluation were performed. Samples were divided into normal appearing cartilage, peri-lesional and lesional cartilage. Cartilage degradation in the samples was graded histologically and immunohistochemically. CK and possible CK cleavage were detected immunohistochemically with specific anti-protein and anti-neoepitope antibodies, respectively. A comparison of CK neoepitope (C2K) production with the collagenase-generated neoepitope produced by matrix metalloproteinases (MMP)-1, 8 and 13 (C2C) was also assessed immunohistochemically. RESULTS CK and CK cleavage were significantly more abundant in OA cartilage (both peri-lesional and lesional) when compared to remote cartilage within the sample joint or cartilage from healthy joints. The immunohistochemical pattern observed for CK degradation (C2K) was similar to that of collagenase degradation (C2C). Macroscopic cartilage changes and histologic findings were significantly correlated with immunohistochemistry results. CONCLUSION The data generated suggests that CK may be involved in cartilage collagen degradation in naturally occurring osteoarthritis.

Joint inflammation increases glucosamine levels attained in synovial fluid following oral administration of glucosamine hydrochloride

OBJECTIVE To compare synovial glucosamine levels in normal and inflamed equine joints following oral glucosamine administration and to determine whether single dose administration alters standard synovial parameters of inflammation. METHODS Eight adult horses were studied. On weeks 1 and 2, all horses received 20mg/kg glucosamine hydrochloride by nasogastric (NG) intubation or intravenous injection. On weeks 3 and 4, 12h after injection of both radiocarpal joints with 0.25 ng Escherichia coli lipopolysaccharide (LPS) to induce inflammation, glucosamine hydrochloride or a placebo was administered by NG intubation. Plasma samples were collected at baseline and 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid (SF) samples were collected within 48 h before dosing and 1, 6 and 12h post-dosing. Glucosamine was analyzed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). Clinicopathological evaluation of SF parameters included white blood cell (WBC) count and total protein (TP) analyses. RESULTS No significant differences between groups were observed in SF baseline levels of WBC and TP at any stage of the study. SF WBC and TP significantly increased following IA LPS. The mean (+/-SD) maximal SF glucosamine levels (422.3+/-244.8 ng/mL) were significantly higher (>fourfold) in inflamed joints when compared to healthy joints (92.7+/-34.9 ng/mL). Glucosamine did not have any effect on standard SF parameters of inflammation. CONCLUSION Synovial inflammation leads to significantly higher synovial glucosamine concentrations compared to levels attained in healthy joints following oral administration of glucosamine hydrochloride. Whether these higher levels are translated into a therapeutic effect on the joint tissues remains to be elucidated.

Use of routine clinical multimodality imaging in a rabbit model of osteoarthritis – part II: bone mineral density assessment

OBJECTIVE To assess bone mineral density (BMD) at different depths from the articular surface in vivo and temporally in a rabbit model of osteoarthritis (OA) using clinical computed tomography (CT) equipment. METHODS The knee joints of rabbits (N=10 with Anterior cruciate ligament transection (ACLT) and contralateral sham joints, and N=6 unoperated controls) were scanned in a transverse image plane with a single-slice helical CT scanner. BMD was calculated at defined depths from the articular surface to the growth plate in the lateral femoral condyle (LFC), medial femoral condyle (MFC), lateral tibial plateau (LTP) and medial tibial plateau (MTP). Baseline BMD was measured at 2 weeks before surgery, and then repeated at weeks 2, 4 and 8 post-surgery in all 10 operated rabbits, and again at week 12 in five of the operated rabbits and at weeks -2 and 8 in the six control rabbits. RESULTS In the control joints, BMD decreased with increasing distance into the epiphysis and remained stable over time within each depth. A significant reduction in BMD was observed at week 2 post-operatively in three compartments (LFC, MFC and MTP) in the ACLT joints and persisted to week 12. A modest reduction in BMD occurred in the LTP and MTP of the sham joints at week 12 alone. CONCLUSION Clinical CT equipment permitted rapid, repeated, in vivo, non-invasive BMD assessment in a rabbit model of OA. A marked BMD reduction was measured with progression of OA until the end point at 12 weeks.

In Vivo Effects of a Single Intra-Articular Injection of 2% Lidocaine or 0.5% Bupivacaine on Articular Cartilage of Normal Horses

OBJECTIVE To assess the effects of an intra-articular (IA) lidocaine or bupivacaine injection on synovial fluid (SF) biomarkers of cartilage metabolism. STUDY DESIGN Experimental. ANIMALS Horses (n = 6). METHODS The study had 2 components, each with an identical design: the first arm assessed the effects of bupivacaine (0.5%) IA in the intercarpal joints and, after a 2-week washout period, the second was conducted to evaluate the effects of IA lidocaine (2%) in the tarsocrural joints. The mares were randomly assigned to receive the test local anesthetic in the target joint or the placebo (0.9% NaCl) in the contralateral joint. After a 2-week washout period, treatments were reversed, yielding 6 joints for each treatment. SF samples were collected for measurement of biomarkers of cartilage metabolism. RESULTS IA bupivacaine or lidocaine induced a significant increase in both markers of cartilage matrix synthesis (CS846-aggrecan and CPII-type II collagen) suggesting an anabolic effect. Bupivacaine also resulted in an unanticipated decrease in both collagen degradation markers (C2C and C1,2C). CONCLUSIONS These results reveal an anabolic effect of single IA injections of local anesthetics on cartilage metabolism. Although collagen degradation biomarker results were not increased, it is possible that the anabolic response is because of an undetected cartilage insult, and requires further investigation.

Use of routine clinical multimodality imaging in a rabbit model of osteoarthritis – part I

OBJECTIVE To evaluate in vivo the evolution of osteoarthritis (OA) lesions temporally in a rabbit model of OA with clinically available imaging modalities: computed radiography (CR), helical single-slice computed tomography (CT), and 1.5 tesla (T) magnetic resonance imaging (MRI). METHODS Imaging was performed on knees of anesthetized rabbits [10 anterior cruciate ligament transection (ACLT) and contralateral sham joints and six control rabbits] at baseline and at intervals up to 12 weeks post-surgery. Osteophytosis, subchondral bone sclerosis, bone marrow lesions (BMLs), femoropatellar effusion and articular cartilage were assessed. RESULTS CT had the highest sensitivity (90%) and specificity (91%) to detect osteophytes. A significant increase in total joint osteophyte score occurred at all time-points post-operatively in the ACLT group alone. BMLs were identified and occurred most commonly in the lateral femoral condyle of the ACLT joints and were not identified in the tibia. A significant increase in joint effusion was present in the ACLT joints until 8 weeks after surgery. Bone sclerosis or cartilage defects were not reliably assessed with the selected imaging modalities. CONCLUSION Combined, clinically available CT and 1.5 T MRI allowed the assessment of most of the characteristic lesions of OA and at early time-points in the development of the disease. However, the selected 1.5 T MRI sequences and acquisition times did not permit the detection of cartilage lesions in this rabbit OA model.

Osteoclasts are recruited to the subchondral bone in naturally occurring post-traumatic equine carpal osteoarthritis and may contribute to cartilage degradation

UNLABELLED The role of osteoclasts in osteochondral degeneration in osteoarthritis (OA) has rarely been investigated in spontaneous disease or animal models of OA. OBJECTIVE The objectives of the current study were to investigate osteoclast density and location in post-traumatic OA (PTOA) and control specimens from racehorses. METHOD Cores were harvested from a site in the equine third carpal bone, that undergoes repetitive, high intensity loading. Histological and immunohistochemical (Cathepsin K and Receptor-activator of Nuclear Factor kappa-β ligand (RANKL)) stained sections were scored (global and subregional) and the osteoclast density calculated. The cartilage histological scores were compared with osteoclast density and RANKL scores. RESULTS There was a greater density of osteoclasts in PTOA samples and they were preferentially located in the subchondral bone plate. RANKL scores positively correlated to the scores of cartilage degeneration and the osteoclast density. The relationship between hyaline articular cartilage RANKL score and osteoclast density was stronger than that of the subchondral bone RANKL score suggesting that cartilage RANKL may have a role in recruiting osteoclasts. The RANKL score in the articular calcified cartilage correlated with the number of microcracks also suggesting that osteoclasts recruited by RANKL may contribute to calcified cartilage degeneration in PTOA. CONCLUSION Our results support the hypothesis that osteoclasts are recruited during the progression of spontaneous equine carpal PTOA by cartilage RANKL, contributing to calcified cartilage microcracks and focal subchondral bone loss.

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.

Computed tomography and magnetic resonance imaging in the study of joint development in the equine pelvic limb.

Osteochondrosis dissecans (OCD) is a focal failure of endochondral ossification of the epiphysis characterized by the presence of cartilage flaps and osteochondral fragments. The objective of this study was to image epiphyseal development in the equine pelvic limb to determine whether there was a variation in site maturation that could be a predisposing factor for OCD. Pelvic limbs (fetuses and foals) were studied post-mortem. The epiphyses of the distal femur, tibia and talus were scanned with computed tomography (CT) and 1.5T magnetic resonance imaging (MRI) to investigate the degree and pattern of ossification, the regularity of the ossification front and cartilage percentage (articular epiphyseal cartilage thickness as a percentage of total epiphyseal diameter) at predetermined sites. The secondary ossification centers (SOCs) were first identified in the femoral epiphyses at 7months, and both tibia and talus at 8months of gestation (MOG). At ⩾8 MOG the cartilage percentage was higher at the majority of OCD-susceptible sites when compared to control sites. At 8-9 MOG the lateral trochlear ridge of the femur, medial malleolus of the tibia (MM), cranial part of the distal intermediate ridge of the tibia (DIRT(Cr)), all OCD susceptible sites, had the greatest cartilage percentage compared to all other sites assessed. Post-partum, the cartilage percentage of the MM and DIRT(Cr), common sites of OCD, remained high. CT and MRI images illustrate equine epiphyseal development and provide additional evidence that greater cartilage thickness at specific joint sites could play a role in the development of OCD.