Longitudinal Profiling of Articular Cartilage Degradation in Osteoarthritis by High-Resolution Magic Angle Spinning 1H NMR Spectroscopy: Experimental Study in the Meniscectomized Guinea Pig Model Michele Borel,*,† Philippe Pastoureau,‡ Janine Papon,† Jean Claude Madelmont,† Nicole Moins,† Jean Maublant,† and Elisabeth Miot-Noirault† EA 4231, Univ d’Auvergne, Clermont-Ferrand, F-63001 France, INSERM UMR 484, Clermont-Ferrand F-63005 France, Centre Jean Perrin, Clermont-Ferrand, F-63001 France, and Institut de Recherches Servier, 92150 Suresnes, France Received November 17, 2008
Abstract: This study assessed the 1H HRMAS NMR spectroscopic profile of articular cartilage in both physiological and osteoarthitic situations. One-dimensional and two-dimensional 1H HRMAS NMR spectra were obtained from the tibial plateau cartilage of healthy and operated (unilateral medial meniscectomy and sham surgery) guinea pigs at different stages of disease, over a 6-month period. The major osteoarthritis-induced 1H HRMAS NMR changes were an increase of the N-acetyl peak of proteoglycans (at day 20 after meniscectomy) and a decrease after day 60 as the pathology evolved. These proteoglycan changes revealed by 1H HRMAS NMR analysis were validated by proteoglycan biochemistry assays. 1H HRMAS NMR analysis also evidenced a sharp increase in methylene resonances of chondrocyte membrane lipids from day 90 as a marker of apoptosis. There was an increase of the mobile methyl group of collagen at day 120, which was associated with collagen breakdown. 1H HRMAS NMR analysis provided a multifactorial and sequential picture of cartilage degradation at the extracellular matrix and chondrocyte levels. Keywords: 1HRMAS NMR spectroscopy • cartilage • tibial plateau • proteoglycans • collagen • chondrocytes • osteoarthritis • meniscectomy • guinea pig
Introduction The rapidly increasing fraction of aging population worldwide has meant that osteoarticular pathologies, especially osteoarthritis (OA), have become a major public health issue in western countries, affecting more than 50% of people over age 65. This equates to approximately 10% of the French population being diagnosed with OA.1 OA is a heterogeneous and multifactorial degenerative joint disease characterized by gradual loss of articular cartilage and associated joint remodel* Corresponding Author: Dr. Michele Borel, EA 4231 INSERM UMR 484, Rue Montalembert, BP 184, 63005 Clermont-Ferrand Ce´dex, France. Tel: +33 (0)4 73 15 08 00. Fax: +33 (0)4 73 15 08 01. E-mail:
[email protected]. † Univ d’Auvergne. ‡ Institut de Recherches Servier.
2594 Journal of Proteome Research 2009, 8, 2594–2600 Published on Web 03/26/2009
ing, that is, concomitant hypertrophic reactions in the bone, including osteophyte formation at the joint margins and inflammation of the synovium.2-5 Articular cartilage is an avascular tissue mainly composed of a single type of cells, chondrocytes (2-5% of total tissue), and an extracellular matrix (ECM) characterized by a tight meshwork of both type II collagen and proteoglycans (PG). Homeostasis of the highly complex ECM is regulated by chondrocytes through the synthesis/degradation of macromolecular components, that is, proteoglycans, collagens, and noncollagenous proteins.6 Chondrocytes are also responsible for producing and releasing proteolytic enzymes, both proteoglycanases and collagenases, in order to maintain a balance between the anabolic and catabolic processes of the biochemical constituents of cartilage.6,7 It is generally accepted that an imbalance between anabolic and catabolic pathways in ECM turnover contributes significantly to OA.2,4-8 Since one of the central and earliest-occurring pathophysiological features contributing to cartilage erosion during joint degeneration is PG loss and alteration, many authors consider PG as the appropriate target for studying OA and understanding and monitoring the pathological process.9-12 The relative importance of macromolecular ECM compounds involved in cartilage alteration and degradation has been characterized by a number of nuclear magnetic resonance (NMR) spectroscopic analyses of both normal and osteoarthritic synovial fluids.13-15 Clinicians looking to obtain in vivo data on the molecular composition and organization of cartilage prefer biochemical characterization of native cartilage tissues over the other analytic assays available, all of which requiring cartilage extraction. Standard 1H NMR spectroscopic analysis of cartilage tissue is made unfeasible by the fact that dipolar coupling and chemical shift anisotropy generate broad resonances due to motionally constrained species. These limitations can be partially overcome by high-resolution magic-angle spinning (HRMAS) spectroscopy of articular cartilage tissue samples. 1H HRMAS NMR spectroscopy is able to attenuate most of the effects of restricted molecular motion-associated line-broadening, chemical shift anisotropy, dipolar coupling and field inhomogeneity by high-rate spinning the sample at the θ ) 54.7° ‘magic angle’.16-18 1H HRMAS NMR spectroscopy has been previously demonstrated as a powerful tool for 10.1021/pr8009963 CCC: $40.75
2009 American Chemical Society
Profiling of Articular Cartilage Degradation in Osteoarthritis characterizing the composition of various intact biologic tissues,19-24 including bovine nasal cartilage and intervertebral disk, and in both physiological and pathological settings.25-28 One of the main advantages of this approach is that it provides detailed quantitative molecular information on small intact tissue samples, and generates a molecular signature that potentially encodes latent biological or pathological information. This paper reports the use of 1H HRMAS NMR spectroscopy to establish the metabolic profile of tibial plateau articular cartilage of (i) adult healthy guinea pigs, (ii) meniscectomized (MNX) animals at different stages of the disease, and (iii) shamoperated (SHAM) animals. To our knowledge, this technique developed as a new diagnostic tool for studying cartilage degradation in a model of OA has not been previously reported.
Experimental Methods Animals. This study used male Hartley albino guinea pigs (Charles River, L’Arbresle, France) that were handled and cared for in accordance with National Research Council guidelines on the care and use of laboratory animals (1996) and European Directive 86/809/EEC. They were maintained at 21 °C with a 12/12 h light/dark cycle, fed a certified guinea pig breeding diet (UAR, Villemoisson, France), and given ad libitum access to water. At the beginning of the study, the animals were 12 weeks of age and weighed about 650 g. Protocols were performed under the authorization of the French Veterinary Services Directorate (authorization No. C63-113-10) and conducted under the supervision of authorized investigators in accordance with the institution’s recommendations on the use of laboratory animals. Forty-five guinea pigs were studied, consisting in 5 healthy animals, 20 MNX animals and 20 SHAM animals. OA Model of Unilateral Partial Medial Meniscectomy. Unilateral medial meniscectomy was performed on the left knee of 20 guinea pigs anesthetized by inhalation of 5% isoflurane (Fore`ne, Abbott Park, IL) in a mixture of 50% oxygen and 50% nitrous oxide. The technical aspect of the operation was carried out in accordance with previously published surgical procedures.29-31 Briefly, after preparing the left femorotibial joint for surgery by clipping the hair and cleansing the skin with povidone (Betadine, Asta Medica, Frankfurt, Germany), a vertical skin incision of approximately 1 cm was made at the site of the medial collateral ligament. The joint capsule was opened, the medial collateral ligament was transected, and the medial meniscus was grasped with a fine-toothed hemostat and transected with a scalpel. The joint capsule was then sutured and the skin was closed with two wound clips. Twenty animals underwent sham surgery, in which the joint capsule was opened by transecting the medial collateral ligament, while the meniscus was left intact. After the operation, the collateral ligament was sutured and the skin incision closed with two wound clips, then disinfected with povidone. Cartilage Sampling. After sacrificing both MNX and SHAM animals at selected time-points after surgery (day 20, 40, 60, 90, 120, 150, 180), posterior femorotibial joints were removed and immediately dissected and the tibia and femur were separated, with any excess soft tissue being removed by careful dissection under a binocular loupe. Specimens were then fixed in 96% ethanol solution for 24 h, and then decalcified in 20% formic acid before cartilage sampling. For cartilage sampling of each femorotibial joint, two consecutive full-depth cartilage disks (1.5 mm diameter) in close proximity were removed from “middle-inner” locations
technical notes
on the medial and lateral tibial plateaus using a biopsy punch (2 mm in diameter) and a scalpel blade under binocular loupe (we used a grid as a guide). After drying, one disk was taken for NMR analysis and one for biochemical assessment. 1 H HRMAS NMR Analysis of Cartilage Samples. 1H NMR spectroscopy was performed on a small-bore Bruker DRX 500 magnet (Bruker, Karlsruhe, Germany) equipped with an HRMAS probe. All experiments were done on cartilage samples (1-2 mg) that were set into 2 mm diameter, 18 µL free volume ZrO2 rotor tubes with upper spacer. A total of 3 µL of D2O containing 1.0 wt % of sodium 2,2-dimethyl-2-silapentane-5sulfonate (DSS Sigma) was added to the rotor tubes to lock the spectrometer; then, the rotors were spun at 277K at 4 KHz to keep the rotation sidebands out of the acquisition window. The HRMAS technique consists of spinning the sample at a rate
