Chondroprotective Effect of High-Dose Zoledronic Acid: An Experimental Study in a Rabbit Model of Osteoarthritis Kalliopi Lampropoulou-Adamidou,1,2 Ismene Dontas,1 Ioannis P. Stathopoulos,1,2 Lubna Khaldi,1,3 Pavlos Lelovas,1 John Vlamis,2 Ioannis K. Triantafillopoulos,1 Nikolaos A. Papaioannou1 1
Laboratory for Research of the Musculoskeletal System “Theodoros Garofalidis”, University of Athens, KAT General Hospital, Athens, Greece, Third Orthopaedic Department, University of Athens, KAT General Hospital, Athens, Greece, 3Department of Pathology, Amalia Fleming General Hospital, Athens, Greece 2
Received 3 May 2014; accepted 14 July 2014 Published online 13 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22712
ABSTRACT: To address the need to impact the subchondral bone-articular cartilage interaction for the treatment of degenerative osteoarthritis (OA), bisphosphonates may be used as a means to inhibit the subchondral bone resorption. The purpose of the present study is to evaluate the chondroprotective effect of zoledronic acid (ZOL) in a model of OA. Eighteen adult male rabbits underwent an anterior cruciate ligament transection and were separated into two groups: ZOL group (n ¼ 10) received 0.6 mg/kg intravenous injection of ZOL on day 1, 15, and 29 and placebo group (n ¼ 8) received saline. The animals were euthanized at 8 weeks. Macroscopically, the ZOL group had significantly milder ulcerations, cartilage softening and fibrillation compared to the placebo group. Microscopically, morphology of the articular cartilage was better in the ZOL treated group compared with the placebo group, without complete disorganization in any section of the ZOL group. Furthermore, the chondrocytes in the ZOL treated group were mainly cloning, indicating cartilage repairing and regeneration process, while in the placebo group hypocellularity predominated. Additionally, subchondral necrosis was evident in some specimens of the placebo group. Zoledronic acid, in a high-dose regimen, proved to be chondroprotective in a well-established animal model of OA. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1646–1651, 2014. Keywords: osteoarthritis; animal model; zoledronic acid; subchondral bone; cartilage
Degenerative osteoarthritis (OA) is a disorder of synovial joints characterized by progressive loss of cartilage and subchondral bone alterations. It is the most common joint disease and millions of people suffer from this musculoskeletal disorder, that may lead to disability, deterioration of quality of life and a heavy socioeconomic impact on every health system.1 The treatment of end stage OA is surgical, when patients’ pain and disability significantly alter their quality of life. Before the end stage, non-pharmacological and pharmacological conservative treatment can only reduce symptoms and is limited to control pain and inflammation, but cannot modify the course of the disease. The aim of recent pharmacological studies is the development of conservative treatment of OA with chondroprotective and/or chondrotherapeutic agents. For decades, OA has been considered a disease of the articular cartilage. However, the subchondral bone plays a key role in cartilage changes with the exact pathophysiological mechanism remaining unclear.2,3 Bisphosphonates inhibit bone resorption and are widely used for the treatment of osteoporosis, Paget’s disease, metastatic bone disease, multiple myeloma and hypercalcaemia of malignancy.4 Recent preclinical and limited clinical studies have shown promising results concerning their chondroprotective role.5–13
Conflict of Interest: None. Grant sponsor: Hellenic Society for the Study of Bone Metabolism; Grant Sponsor: Novartis Pharma AG (Basel) Novartis (Hellas) SACI. Correspondence to: Kalliopi Lampropoulou-Adamidou, (T: þ306984229202; F: 00302132086765; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
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Furthermore, there is evidence that bisphosphonates may act directly on both chondrocytes and subchondral bone influencing the cartilage repair, on immune system cells affecting the course of chronic inflammatory arthritis, and have direct analgesic action, however possibly these effects are not dominant in the influence of bisphosphonates in the cartilage repair.14,11 The purpose of the present study is to evaluate the chondroprotective effect after macroscopic and histological examination of articular cartilage of a potent bisphosphonate, zoledronic acid (ZOL), in a high-dose regimen in a well-established animal model of OA.15
MATERIALS AND METHODS The experimental protocol was approved by the Veterinary Directorate, according to the Greek Presidential Decree 160/ 1991, which conforms to the EEC Directive 609/1986 for the “protection of vertebrate animals used for experimental or other scientific purposes”. Eighteen adult male New Zealand White rabbits (Oryctolagus cuniculus), conventionally bred, were used. The animals were housed singly in steel cages of 45 cm 30 cm 60 cm dimensions (IFFA CREDO, BP 010969592, L’Arbresle, France) in the Laboratory for Research of the Musculoskeletal System “Th. Garofalidis” conventional animal house. Their mean age was 25 weeks (24–26) and their mean weight was 4 kg (3.5–4.6) at the time of surgery. The temperature therein ranged between 18 and 21˚C, relative humidity 50–60%, the light/dark cycle was from 06:00 to 18:00 and there were 15 air changes/hour. They had free access to standard rabbit pellets (14% protein, 7% fat, 15% cellulose, 1–1.2% calcium and phosphorus) (ELVIZ Hellenic Feedstuffs Ind. SA, Plati, Imathia, Greece) and tap water. All animals were operated on day 0. After induction of general anesthesia (ketamine 25 mg/kg and xylazine 5 mg/kg intramuscularly), the right stifle was shaved and the skin prepared. Under aseptic conditions, through a medial
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parapatellar incision, the anterior cruciate ligament (ACL) was transected with a no 11 blade. After transection, anterior instability was manually confirmed by the anterior drawer test. The incision did not disrupt the patellar apparatus. The articular capsule and the medial retinaculum were closed with absorbable sutures and the skin with metal staples. After recovery, rabbits received paracetamol suppository (Depon supp. bebe, 1/3 supp ¼ 50 mg) for pain relief and were free to move in their cages without any external immobilization. They also received paracetamol syrup (Depon sir. 1 ml ¼ 25 mg) twice a day the next postoperative day. We refrained from using non-steroidal anti-inflammatory drugs, in order to avoid their interference with the development of the OA model. The animals were checked daily (activity, body weight, food consumption, rectal temperature, wound healing) for signs of ill health. The animals were separated in two groups. The first group (ZOL treated group) included ten rabbits that received intravenously 0.6 mg/kg injection of ZOL, lasting at least 15 min, on day 1, 15, and 29. This dose was considered as high as the corresponding usual dose in human for the treatment of osteoporosis and the dose regimens used in the other corresponding experimental studies.8,11,12 The second group (placebo group) included eight rabbits that received saline on the same days. Euthanasia was carried out 8 weeks postoperatively by ketamine/midazolam premedication administered intramuscularly, followed by slow intravenous administration of sodium thiopental (20–30 mg/kg until cessation of cardiac function) in normal saline drip. After euthanasia, all specimens were prepared with excision of the skin, osteotomy 3 cm above and below the stifle, removal of soft tissues and ligaments, and underwent macroscopic evaluation. Grossly, the specimens were examined for cartilage erosions (ulcerations, fissures) and loss of cartilage luster (softening, fibrillation). Both parameters of cartilage lesions were assessed as absent, mild or severe and were scored from 0 to 2. Total macroscopic score was measured as the sum of the two parameters (from 0 to 4). Macroscopic evaluation also included digital photographing of the articular surfaces. The articular surfaces of the stifles were divided in five specimens in order to define if there are areas with highest incidence of osteoarthritic alterations and standardize the size of histological sections. Site I was defined as the femoral trochlea, site II as the middle of the medial femoral condyle, site III as the middle of the lateral femoral condyle, site IV as the anterior part of the medial and lateral tibial plateau, and site V as the posterior part of the medial and lateral tibial plateau. The specimens were then fixed in 10% formaldehyde solution for 24–36 h in room temperature and decalcified in 10% nitric acid for 3–5 days. After incubation in buffers with gradually increasing concentration of alcohol and xylol, the samples were embedded in paraffin, cut in 2 mm sections and were stained with hematoxylin/eosin solution. The histological sections were examined in the abovementioned sites, except site I because osteophytes predominated and osteoarthritic lesions were mild. For the microscopic evaluation, morphology of the articular cartilage, arrangement of chondrocytes into the hyaline cartilage, tidemark integrity, subchondral necrosis, and subchondral cysts were taken into consideration. In order to establish a more objective demonstration of the histological parameters, a scoring system was used based on a modified Mankin’s grading system (Table 1).16,17
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Table 1. The Histological Evaluation Was Based on Five Parameters. The Modified Mankin’s Score Was Measured as The Sum of Articular Cartilage Structure, Arrangement of Chondrocytes and Tidemark Integrity Articular cartilage structure Normal Surface irregularities Pannus and surface irregularities Clefts to transitional zone Clefts to radial zone Clefts to calcified zone Complete disorganisation Arrangement of chondrocytes Normal Diffuse hypercellularity Cloning Hypocellularity Tidemark integrity Intact Crossed by blood vessels Subchondral bone morphology Normal Cyst development Subchondral necrosis Absent Present
Grade 0 1 2 3 4 5 6 0 1 2 3 0 1 0 1 0 1
The Pearson’s x-squared test was used to evaluate differences between categorical values and the non-parametric Wilcoxon test between the ordinal data. The results are presented as the mean standard deviation (SD). A value of p < 0.05 was selected to indicate statistical significance. All statistical tests were performed using SPSS 20 statistical software (SPSS Inc., Chicago, IL).
RESULTS Osteoarthritic lesions were present in all specimens. In site I (femoral trochlea), the osteophytes were the predominant finding. Concerning the other femoral areas, in site II, the ulceration and loss of cartilage luster were more severe in comparison with site III. As for the tibia, in sites IV and V equally mild ulcerations and osteophytes were observed. Macroscopically, the ZOL treated group had milder ulcerations, cartilage softening, and fibrillation compared to the placebo group (mean total macroscopic score, 1.3 1.2 versus 3.1 0.9, p < 0.001) (Figs. 1 and 2). Both groups presented formation of osteophytes. Microscopically, morphology of the articular cartilage was better in the ZOL treated group compared with the placebo group (mean articular cartilage structure score, 2.2 1.5 versus 2.9 1.5, p ¼ 0.041) (Fig. 3a). In the ZOL treated group, there was no complete disorganization in any section. Furthermore, the chondrocytes in the first group were mainly cloning, while in the second hypocellularity predominated (mean arrangement of chondrocytes score, 2 0.2 versus 2.3 0.5, p ¼ 0.001) (Fig. 3b). Additionally, subchondral necrosis was evident
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Figure 1. Right rabbit stifle of the ZOL treated group (a-c) and the placebo group (d-f) after euthanasia. The ACL is transected (a, d). The removal of soft tissues and ligaments allows the gross examination of the articular surfaces. In site II (medial femoral condyle) the ulceration and loss of cartilage luster is more severe in comparison with site III (lateral femoral condyle) (b, e). The ZOL treated group has milder ulceration and cartilage softening (b) compared to the placebo group (e). As for the tibia, the ZOL treated group has milder ulcerations (c) in comparison with the placebo group (f).
only in some sections of the placebo group (p ¼ 0.021) (Fig. 3c). In all sections of both groups, subchondral cysts were observed and tidemark integrity was crossed by blood vessels. The mean modified Mankin’s score was 5.2 1.4 in the ZOL treated group and 6.2 1.8 in the placebo group (p ¼ 0.027) (Fig. 3d and 3e–j). Zoledronic acid was well-tolerated without causing any apparent adverse effect.
Figure 2. Mean total macroscopic score differs significantly between the two groups. The ZOL treated group had significantly milder ulcerations, cartilage softening and fibrillation compared to the placebo group. JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2014
DISCUSSION Numerous studies focus on the interaction between the articular cartilage and subchondral bone in the development of OA. However, inhomogeneous data have been recorded in the literature about the alterations occurring in the subchondral bone during progression of OA.18–23 An explanation could be that subchondral bone changes depend on the stage of the disease and differ in cortical, trabecular and subchondral bone. Initially, there is increased subchondral bone remodelling leading to bone resorption and in the later stages bone accretion is predominant, resulting in sclerosis of the tissue.24 The suspicion of the articular cartilagesubchondral bone interaction engendered by the observation that acute subchondral bone injury, osteochondritis dissecans and osteonecrosis may lead to the development of OA and OA leads to increased turnover of subchondral bone.3 Mechanically, the subchondral bone supports the overlying cartilage and absorbs the forces transmitted by the joint. Alterations of subchondral bone mineralization may lead to changes in stress distribution and morphology of the articular surface.2,3 Biologically, articular vascular plexus connects the two different tissues, extending from the subchondral bone to the calcified cartilage in normal joints and to the tidemark in osteoarthritic joints. This plexus indicates that there is also a metabolic interaction between bone and cartilage. However, whether the development of OA originates from cartilage or subchondral bone alterations remains unknown.2,3
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Figure 3. Mean scores of histological parameters of articular cartilage structure (a), arrangement of chondrocytes (b), and subchondral necrosis (c) were significantly lower in the ZOL group. Mean modified Mankin’s score was also significantly lower compared to the placebo group (d). Histological findings in site II (e, h), III (f, i) and IV (g, j) of the articular cartilage of the ZOL treated group (e-g) and the placebo group (h-j) are presented. In the ZOL treated group, the cartilage structure has some surface irregularities in all sites (e-g) with hypocellularity in femoral sections (e, f) and diffuse hypercellularity in site IV (g). In the placebo group, the cartilage structure has clefts to calcified zone in femoral sections (h, i) and clefts to transitional zone in tibial section (j) with chondrocyte cloning in site II (h) and hypocellularity in site III and IV (i, j). In the present case, sections of both groups present subchondral cysts without subchondral necrosis.
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To address the need to impact the subchondral bone-articular cartilage interaction, bone anticatabolic agents such as bisphosphonates and calcitonin,25 may be used as a means to inhibit the subchondral bone resorption. Interestingly, there are in vivo and in vitro studies highlighting the effect of bisphosphonates on the immune system cells, through an influence on the production of pro- and anti-inflammatory cytokines. Thus, they may present positive effect in chronic inflammatory arthritis.14 Most experimental studies have shown positive effect of bisphosphonates on subchondral bone and reduction of progression of OA. The majority of such studies have been performed with the administration of alendronate and have shown that its chondroprotective effect is through a decrease of subchondral bone resorption.7,9,13 We thus consider that a more potent bisphosphonate, such as ZOL, may have a better chondroprotective effect.6 In particular, Strassle et al. showed that ZOL increased subchondral BMD, prevented cartilage degeneration and inhibited pain in a dose-depended manner with a greater efficacy when administered in the early stages of OA in a monosodium iodoacetate (MIA) rat model (chondroprotective effect).11 Another study confirmed that ZOL had protective effect on cartilage and subchondral bone in a dose-dependent manner with greater efficacy when administered early from the induction of OA (immediately after surgery or at one month, chondroprotective effect) in a medial meniscal tear rat model.12 Also, ZOL has demonstrated chondroprotective effect by decreasing the bone resorption and protecting the cartilage degeneration without achieving to reduce the proteoglycan loss in a chymopapain rabbit model.8 However, clinical trials investigating the effect of bisphosphonates on OA have not presented such encouraging results. A 1-year clinical study showed that risedronate improved patients’ symptoms and physical activity, and significantly reduced markers of cartilage degradation and bone resorption.10 In another 2-year trial, risedronate decreased biochemical markers of cartilage degeneration but did not decrease symptoms or radiographic progression of OA.5 A crosssectional study had shown that older women who were receiving alendronate had significantly fewer OArelated subchondral bone abnormalities in the knee and had less knee pain, compared with women who were not taking any bone antiresorptive drugs, but there was no association of alendronate use with changes in cartilage lesion and volume on MRI.26 In the aforementioned experimental studies, bisphosphonates were effective in the early stages of OA but in humans there is no study evaluating a selected sample of patients with early OA. Radiographs, which are the usual means for the investigation of OA in humans, cannot detect the early stage of the disease. The direct study of degenerative OA in humans presents major difficulties, such as the slow JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2014
progression of the disease requiring long-term studies, the variety of causes and environmental influences (such as heredity, history of trauma, lifestyle and underlying conditions), the variety of symptoms, location and radiological depiction (sclerosis or cysts of subchondral bone, narrowing of joint space and osteophytes) leading to disparate data difficult to interpret, the absence of symptoms in the early stages of the disease, the absence of exact correlation between progression of OA and severity of symptoms, the lack of sensitive imaging techniques and the inability to perform histological studies by harvesting articular tissue due to ethical reasons.15 We considered the ACL transection rabbit model as the best model for the study of the chondroprotective effect of ZOL, which is a well-established and reproducible animal model of OA. The period between 6 and 8 weeks after sectioning the ACL is sufficient for the development of OA in rabbits.27,28 We administered a high dose of ZOL (0.6 mg/kg intravenous injection three times in a 2 month period) to clearly see its effect on cartilage and subchondral bone. It was well-tolerated without causing any apparent adverse effect. Grossly, osteoarthritic lesions and formation of osteophytes were present in all animals, but the ZOL treated group had significantly milder ulcerations, cartilage softening and fibrillation compared to the placebo group. Microscopically, morphology of the articular cartilage was better in the ZOL treated group compared with the placebo group, without complete disorganization in any section of the first group. Furthermore, the chondrocytes in the ZOL treated group were mainly cloning, indicating cartilage repairing and regeneration process,29,30 while in the placebo group hypocellularity predominated. Additionally, subchondral necrosis was evident only in some sections of the placebo group. A similar positive effect on cartilage has been reported with calcitonin, another antiresorptive drug, in a comparable study using the same animal model and experimental protocol in our institution.25 A limitation of the present study can be considered the fact that osteoarthritic lesions, in our experimental model, occurred after an acute traumatic event that mimics posttraumatic OA in humans and not degenerative OA. However, certain stages of the progression of OA are similar with degenerative OA. Additionally, there are differences in anatomy and mechanics between animals and between animal and human knees, thus we have to be cautious when comparing results of studies performed with different species. Furthermore, a non-operated control group was not employed, because no degenerative changes were observed in any of the joints during surgery and also rabbits have very little spontaneous degeneration in their stifles.31 In conclusion, zoledronic acid, in a high-dose regimen, was proved to be chondroprotective in a wellestablished animal model of OA.
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ACKNOWLEDGMENTS The authors would like to thank the Hellenic Society for the Study of Bone Metabolism, the Novartis Pharma AG (Basel) and the Novartis (Hellas) SACI for the financial support of this study, and Novartis Pharma AG (Basel) and Novartis (Hellas) SACI for the drug supply.The authors would also like to thank Mr. Antonios Galanos MSc, PhD, for his contribution to the statistical analysis, Ms. Chrysavgi Kapsi and Ms. Helen Kostakioti for their expertise in veterinary nursing, Ms. Maria Tsipra for the histological specimen preparation, and Mr. Panagiotis Prokopis for the animal husbandry.
14. 15.
16.
17.
REFERENCES 1. Gore M, Tai KS, Sadosky A, et al. 2011. Clinical comorbidities, treatment patterns, and direct medical costs of patients with osteoarthritis in usual care: a retrospective claims database analysis. J Med Econ 14:497–507. 2. Baker-LePain JC, Lane NE. 2012. Role of bone architecture and anatomy in osteoarthritis. Bone 51:197–203. 3. Madry H, van Dijk CN, Mueller-Gerbl M. 2010. The basic science of the subchondral bone. Knee Surg Sports Traumatol Arthrosc 18:419–433. 4. Papapetrou PD. 2009. Bisphosphonate-associated adverse events. Hormones (Athens) 8:96–110. 5. Bingham CO, Buckland-Wright JC, Garnero P, et al. 2006. Risedronate decreases biochemical markers of cartilage degradation but does not decrease symptoms or slow radiographic progression in patients with medial compartment osteoarthritis of the knee: results of the two-year multinational knee osteoarthritis structural arthritis study. Arthritis Rheum 54:3494–3507. 6. Hadji P, Gamerdinger D, Spieler W, et al. 2012. Rapid Onset and Sustained Efficacy (ROSE) study: results of a randomised, multicentre trial comparing the effect of zoledronic acid or alendronate on bone metabolism in postmenopausal women with low bone mass. Osteoporos Int 23:625–633. 7. Mohan G, Perilli E, Parkinson IH, et al. 2013. Pre-emptive, early, and delayed alendronate treatment in a rat model of knee osteoarthritis: effect on subchondral trabecular bone microarchitecture and cartilage degradation of the tibia, bone/cartilage turnover, and joint discomfort. Osteoarthritis Cartilage 21:1595–1604. 8. Muehleman C, Green J, Williams JM, et al. 2002. The effect of bone remodeling inhibition by zoledronic acid in an animal model of cartilage matrix damage. Osteoarthritis Cartilage 10:226–233. 9. Shirai T, Kobayashi M, Nishitani K, et al. 2011. Chondroprotective effect of alendronate in a rabbit model of osteoarthritis. J Orthop Res 29:1572–1577. 10. Spector TD, Conaghan PG, Buckland-Wright JC, et al. 2005. Effect of risedronate on joint structure and symptoms of knee osteoarthritis: results of the BRISK randomized, controlled trial [ISRCTN01928173]. Arthritis Res Ther 7: R625–633. 11. Strassle BW, Mark L, Leventhal L, et al. 2010. Inhibition of osteoclasts prevents cartilage loss and pain in a rat model of degenerative joint disease. Osteoarthritis Cartilage 18: 1319–1328. 12. Yu DG, Yu B, Mao YQ, et al. 2012. Efficacy of zoledronic acid in treatment of teoarthritis is dependent on the disease progression stage in rat medial meniscal tear model. Acta Pharmacol Sin 33:924–934. 13. Zhang L, Hu H, Tian F, et al. 2011. Enhancement of subchondral bone quality by alendronate administration for
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the reduction of cartilage degeneration in the early phase of experimental osteoarthritis. Clin Exp Med 11:235–243. Corrado A, Santoro N, Cantatore FP. 2007. Extraskeletal effects of bisphosphonates. Joint Bone Spine 74:32–38. Lampropoulou-Adamidou K, Lelovas P, Karadimas EV, et al. 2014. Useful animal models for the research of osteoarthritis. Eur J Orthop Surg Traumatol 24:263–271. Mankin HJ, Dorfman H, Lippiello L, Zarins A. 1971. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 53:523–537. Pearson RG, Kurien T, Shu KS, Scammell BE. 2011. Histopathology grading systems for characterisation of human knee osteoarthritis–reproducibility, variability, reliability, correlation, and validity. Osteoarthritis Cartilage 19: 324–331. Bettica P, Cline G, Hart DJ, et al. 2002. Evidence for increased bone resorption in patients with progressive knee osteoarthritis: longitudinal results from the Chingford study. Arthritis Rheum 46:3178–3184. Beuf O, Ghosh S, Newitt DC, et al. 2002. Magnetic resonance imaging of normal and osteoarthritic trabecular bone structure in the human knee. Arthritis Rheum 46:385– 393. Calvo E, Castaneda S, Largo R, et al. 2007. Osteoporosis increases the severity of cartilage damage in an experimental model of osteoarthritis in rabbits. Osteoarthritis Cartilage 15:69–77. Dedrick DK, Goldstein SA, Brandt KD, et al. 1993. A longitudinal study of subchondral plate and trabecular bone in cruciate-deficient dogs with osteoarthritis followed up for 54 months. Arthritis Rheum 36:1460–1467. Dore D, Quinn S, Ding C, et al. 2009. Correlates of subchondral BMD: a cross-sectional study. J Bone Miner Res 24:2007–2015. Radin EL, Rose RM. 1986. Role of subchondral bone in the initiation and progression of cartilage damage. Clin Orthop Relat Res 34–40. Kwan Tat S, Lajeunesse D, Pelletier JP, Martel-Pelletier J. 2010. Targeting subchondral bone for treating osteoarthritis: what is the evidence? Best Pract Res Clin Rheumatol 24: 51–70. Papaioannou NA, Triantafillopoulos IK, Khaldi L, et al. 2007. Effect of calcitonin in early and late stages of experimentally induced osteoarthritis. A histomorphometric study. Osteoarthritis Cartilage 15:386–395. Carbone LD, Nevitt MC, Wildy K, et al. 2004. The relationship of antiresorptive drug use to structural findings and symptoms of knee osteoarthritis. Arthritis Rheum 50:3516– 3525. Papaioannou N, Krallis N, Triantafillopoulos I, et al. 2004. Optimal timing of research after anterior cruciate ligament resection in rabbits. Contemp Top Lab Anim Sci 43:22–27 quiz 58. Yoshioka M, Coutts RD, Amiel D, Hacker SA. 1996. Characterization of a model of osteoarthritis in the rabbit knee. Osteoarthritis Cartilage 4:87–98. Poole CA. 1997. Articular cartilage chondrons: form, function and failure. J Anat 191:1–13. Quintavalla J, Kumar C, Daouti S, et al. 2005. Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis. J Cell Physiol 204:560–566. Bendele AM. 2001. Animal models of osteoarthritis. J Musculoskelet Neuronal Interact 1:363–376.
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Laboratory for Research of the Musculoskeletal System “Theodoros Garofalidis”, University of Athens, KAT General Hospital, Athens, Greece, Third Orthopaedic Department, University of Athens, KAT General Hospital, Athens, Greece, 3Department of Pathology, Amalia Fleming General Hospital, Athens, Greece 2
Received 3 May 2014; accepted 14 July 2014 Published online 13 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22712
ABSTRACT: To address the need to impact the subchondral bone-articular cartilage interaction for the treatment of degenerative osteoarthritis (OA), bisphosphonates may be used as a means to inhibit the subchondral bone resorption. The purpose of the present study is to evaluate the chondroprotective effect of zoledronic acid (ZOL) in a model of OA. Eighteen adult male rabbits underwent an anterior cruciate ligament transection and were separated into two groups: ZOL group (n ¼ 10) received 0.6 mg/kg intravenous injection of ZOL on day 1, 15, and 29 and placebo group (n ¼ 8) received saline. The animals were euthanized at 8 weeks. Macroscopically, the ZOL group had significantly milder ulcerations, cartilage softening and fibrillation compared to the placebo group. Microscopically, morphology of the articular cartilage was better in the ZOL treated group compared with the placebo group, without complete disorganization in any section of the ZOL group. Furthermore, the chondrocytes in the ZOL treated group were mainly cloning, indicating cartilage repairing and regeneration process, while in the placebo group hypocellularity predominated. Additionally, subchondral necrosis was evident in some specimens of the placebo group. Zoledronic acid, in a high-dose regimen, proved to be chondroprotective in a well-established animal model of OA. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1646–1651, 2014. Keywords: osteoarthritis; animal model; zoledronic acid; subchondral bone; cartilage
Degenerative osteoarthritis (OA) is a disorder of synovial joints characterized by progressive loss of cartilage and subchondral bone alterations. It is the most common joint disease and millions of people suffer from this musculoskeletal disorder, that may lead to disability, deterioration of quality of life and a heavy socioeconomic impact on every health system.1 The treatment of end stage OA is surgical, when patients’ pain and disability significantly alter their quality of life. Before the end stage, non-pharmacological and pharmacological conservative treatment can only reduce symptoms and is limited to control pain and inflammation, but cannot modify the course of the disease. The aim of recent pharmacological studies is the development of conservative treatment of OA with chondroprotective and/or chondrotherapeutic agents. For decades, OA has been considered a disease of the articular cartilage. However, the subchondral bone plays a key role in cartilage changes with the exact pathophysiological mechanism remaining unclear.2,3 Bisphosphonates inhibit bone resorption and are widely used for the treatment of osteoporosis, Paget’s disease, metastatic bone disease, multiple myeloma and hypercalcaemia of malignancy.4 Recent preclinical and limited clinical studies have shown promising results concerning their chondroprotective role.5–13
Conflict of Interest: None. Grant sponsor: Hellenic Society for the Study of Bone Metabolism; Grant Sponsor: Novartis Pharma AG (Basel) Novartis (Hellas) SACI. Correspondence to: Kalliopi Lampropoulou-Adamidou, (T: þ306984229202; F: 00302132086765; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
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Furthermore, there is evidence that bisphosphonates may act directly on both chondrocytes and subchondral bone influencing the cartilage repair, on immune system cells affecting the course of chronic inflammatory arthritis, and have direct analgesic action, however possibly these effects are not dominant in the influence of bisphosphonates in the cartilage repair.14,11 The purpose of the present study is to evaluate the chondroprotective effect after macroscopic and histological examination of articular cartilage of a potent bisphosphonate, zoledronic acid (ZOL), in a high-dose regimen in a well-established animal model of OA.15
MATERIALS AND METHODS The experimental protocol was approved by the Veterinary Directorate, according to the Greek Presidential Decree 160/ 1991, which conforms to the EEC Directive 609/1986 for the “protection of vertebrate animals used for experimental or other scientific purposes”. Eighteen adult male New Zealand White rabbits (Oryctolagus cuniculus), conventionally bred, were used. The animals were housed singly in steel cages of 45 cm 30 cm 60 cm dimensions (IFFA CREDO, BP 010969592, L’Arbresle, France) in the Laboratory for Research of the Musculoskeletal System “Th. Garofalidis” conventional animal house. Their mean age was 25 weeks (24–26) and their mean weight was 4 kg (3.5–4.6) at the time of surgery. The temperature therein ranged between 18 and 21˚C, relative humidity 50–60%, the light/dark cycle was from 06:00 to 18:00 and there were 15 air changes/hour. They had free access to standard rabbit pellets (14% protein, 7% fat, 15% cellulose, 1–1.2% calcium and phosphorus) (ELVIZ Hellenic Feedstuffs Ind. SA, Plati, Imathia, Greece) and tap water. All animals were operated on day 0. After induction of general anesthesia (ketamine 25 mg/kg and xylazine 5 mg/kg intramuscularly), the right stifle was shaved and the skin prepared. Under aseptic conditions, through a medial
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parapatellar incision, the anterior cruciate ligament (ACL) was transected with a no 11 blade. After transection, anterior instability was manually confirmed by the anterior drawer test. The incision did not disrupt the patellar apparatus. The articular capsule and the medial retinaculum were closed with absorbable sutures and the skin with metal staples. After recovery, rabbits received paracetamol suppository (Depon supp. bebe, 1/3 supp ¼ 50 mg) for pain relief and were free to move in their cages without any external immobilization. They also received paracetamol syrup (Depon sir. 1 ml ¼ 25 mg) twice a day the next postoperative day. We refrained from using non-steroidal anti-inflammatory drugs, in order to avoid their interference with the development of the OA model. The animals were checked daily (activity, body weight, food consumption, rectal temperature, wound healing) for signs of ill health. The animals were separated in two groups. The first group (ZOL treated group) included ten rabbits that received intravenously 0.6 mg/kg injection of ZOL, lasting at least 15 min, on day 1, 15, and 29. This dose was considered as high as the corresponding usual dose in human for the treatment of osteoporosis and the dose regimens used in the other corresponding experimental studies.8,11,12 The second group (placebo group) included eight rabbits that received saline on the same days. Euthanasia was carried out 8 weeks postoperatively by ketamine/midazolam premedication administered intramuscularly, followed by slow intravenous administration of sodium thiopental (20–30 mg/kg until cessation of cardiac function) in normal saline drip. After euthanasia, all specimens were prepared with excision of the skin, osteotomy 3 cm above and below the stifle, removal of soft tissues and ligaments, and underwent macroscopic evaluation. Grossly, the specimens were examined for cartilage erosions (ulcerations, fissures) and loss of cartilage luster (softening, fibrillation). Both parameters of cartilage lesions were assessed as absent, mild or severe and were scored from 0 to 2. Total macroscopic score was measured as the sum of the two parameters (from 0 to 4). Macroscopic evaluation also included digital photographing of the articular surfaces. The articular surfaces of the stifles were divided in five specimens in order to define if there are areas with highest incidence of osteoarthritic alterations and standardize the size of histological sections. Site I was defined as the femoral trochlea, site II as the middle of the medial femoral condyle, site III as the middle of the lateral femoral condyle, site IV as the anterior part of the medial and lateral tibial plateau, and site V as the posterior part of the medial and lateral tibial plateau. The specimens were then fixed in 10% formaldehyde solution for 24–36 h in room temperature and decalcified in 10% nitric acid for 3–5 days. After incubation in buffers with gradually increasing concentration of alcohol and xylol, the samples were embedded in paraffin, cut in 2 mm sections and were stained with hematoxylin/eosin solution. The histological sections were examined in the abovementioned sites, except site I because osteophytes predominated and osteoarthritic lesions were mild. For the microscopic evaluation, morphology of the articular cartilage, arrangement of chondrocytes into the hyaline cartilage, tidemark integrity, subchondral necrosis, and subchondral cysts were taken into consideration. In order to establish a more objective demonstration of the histological parameters, a scoring system was used based on a modified Mankin’s grading system (Table 1).16,17
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Table 1. The Histological Evaluation Was Based on Five Parameters. The Modified Mankin’s Score Was Measured as The Sum of Articular Cartilage Structure, Arrangement of Chondrocytes and Tidemark Integrity Articular cartilage structure Normal Surface irregularities Pannus and surface irregularities Clefts to transitional zone Clefts to radial zone Clefts to calcified zone Complete disorganisation Arrangement of chondrocytes Normal Diffuse hypercellularity Cloning Hypocellularity Tidemark integrity Intact Crossed by blood vessels Subchondral bone morphology Normal Cyst development Subchondral necrosis Absent Present
Grade 0 1 2 3 4 5 6 0 1 2 3 0 1 0 1 0 1
The Pearson’s x-squared test was used to evaluate differences between categorical values and the non-parametric Wilcoxon test between the ordinal data. The results are presented as the mean standard deviation (SD). A value of p < 0.05 was selected to indicate statistical significance. All statistical tests were performed using SPSS 20 statistical software (SPSS Inc., Chicago, IL).
RESULTS Osteoarthritic lesions were present in all specimens. In site I (femoral trochlea), the osteophytes were the predominant finding. Concerning the other femoral areas, in site II, the ulceration and loss of cartilage luster were more severe in comparison with site III. As for the tibia, in sites IV and V equally mild ulcerations and osteophytes were observed. Macroscopically, the ZOL treated group had milder ulcerations, cartilage softening, and fibrillation compared to the placebo group (mean total macroscopic score, 1.3 1.2 versus 3.1 0.9, p < 0.001) (Figs. 1 and 2). Both groups presented formation of osteophytes. Microscopically, morphology of the articular cartilage was better in the ZOL treated group compared with the placebo group (mean articular cartilage structure score, 2.2 1.5 versus 2.9 1.5, p ¼ 0.041) (Fig. 3a). In the ZOL treated group, there was no complete disorganization in any section. Furthermore, the chondrocytes in the first group were mainly cloning, while in the second hypocellularity predominated (mean arrangement of chondrocytes score, 2 0.2 versus 2.3 0.5, p ¼ 0.001) (Fig. 3b). Additionally, subchondral necrosis was evident
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Figure 1. Right rabbit stifle of the ZOL treated group (a-c) and the placebo group (d-f) after euthanasia. The ACL is transected (a, d). The removal of soft tissues and ligaments allows the gross examination of the articular surfaces. In site II (medial femoral condyle) the ulceration and loss of cartilage luster is more severe in comparison with site III (lateral femoral condyle) (b, e). The ZOL treated group has milder ulceration and cartilage softening (b) compared to the placebo group (e). As for the tibia, the ZOL treated group has milder ulcerations (c) in comparison with the placebo group (f).
only in some sections of the placebo group (p ¼ 0.021) (Fig. 3c). In all sections of both groups, subchondral cysts were observed and tidemark integrity was crossed by blood vessels. The mean modified Mankin’s score was 5.2 1.4 in the ZOL treated group and 6.2 1.8 in the placebo group (p ¼ 0.027) (Fig. 3d and 3e–j). Zoledronic acid was well-tolerated without causing any apparent adverse effect.
Figure 2. Mean total macroscopic score differs significantly between the two groups. The ZOL treated group had significantly milder ulcerations, cartilage softening and fibrillation compared to the placebo group. JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2014
DISCUSSION Numerous studies focus on the interaction between the articular cartilage and subchondral bone in the development of OA. However, inhomogeneous data have been recorded in the literature about the alterations occurring in the subchondral bone during progression of OA.18–23 An explanation could be that subchondral bone changes depend on the stage of the disease and differ in cortical, trabecular and subchondral bone. Initially, there is increased subchondral bone remodelling leading to bone resorption and in the later stages bone accretion is predominant, resulting in sclerosis of the tissue.24 The suspicion of the articular cartilagesubchondral bone interaction engendered by the observation that acute subchondral bone injury, osteochondritis dissecans and osteonecrosis may lead to the development of OA and OA leads to increased turnover of subchondral bone.3 Mechanically, the subchondral bone supports the overlying cartilage and absorbs the forces transmitted by the joint. Alterations of subchondral bone mineralization may lead to changes in stress distribution and morphology of the articular surface.2,3 Biologically, articular vascular plexus connects the two different tissues, extending from the subchondral bone to the calcified cartilage in normal joints and to the tidemark in osteoarthritic joints. This plexus indicates that there is also a metabolic interaction between bone and cartilage. However, whether the development of OA originates from cartilage or subchondral bone alterations remains unknown.2,3
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Figure 3. Mean scores of histological parameters of articular cartilage structure (a), arrangement of chondrocytes (b), and subchondral necrosis (c) were significantly lower in the ZOL group. Mean modified Mankin’s score was also significantly lower compared to the placebo group (d). Histological findings in site II (e, h), III (f, i) and IV (g, j) of the articular cartilage of the ZOL treated group (e-g) and the placebo group (h-j) are presented. In the ZOL treated group, the cartilage structure has some surface irregularities in all sites (e-g) with hypocellularity in femoral sections (e, f) and diffuse hypercellularity in site IV (g). In the placebo group, the cartilage structure has clefts to calcified zone in femoral sections (h, i) and clefts to transitional zone in tibial section (j) with chondrocyte cloning in site II (h) and hypocellularity in site III and IV (i, j). In the present case, sections of both groups present subchondral cysts without subchondral necrosis.
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To address the need to impact the subchondral bone-articular cartilage interaction, bone anticatabolic agents such as bisphosphonates and calcitonin,25 may be used as a means to inhibit the subchondral bone resorption. Interestingly, there are in vivo and in vitro studies highlighting the effect of bisphosphonates on the immune system cells, through an influence on the production of pro- and anti-inflammatory cytokines. Thus, they may present positive effect in chronic inflammatory arthritis.14 Most experimental studies have shown positive effect of bisphosphonates on subchondral bone and reduction of progression of OA. The majority of such studies have been performed with the administration of alendronate and have shown that its chondroprotective effect is through a decrease of subchondral bone resorption.7,9,13 We thus consider that a more potent bisphosphonate, such as ZOL, may have a better chondroprotective effect.6 In particular, Strassle et al. showed that ZOL increased subchondral BMD, prevented cartilage degeneration and inhibited pain in a dose-depended manner with a greater efficacy when administered in the early stages of OA in a monosodium iodoacetate (MIA) rat model (chondroprotective effect).11 Another study confirmed that ZOL had protective effect on cartilage and subchondral bone in a dose-dependent manner with greater efficacy when administered early from the induction of OA (immediately after surgery or at one month, chondroprotective effect) in a medial meniscal tear rat model.12 Also, ZOL has demonstrated chondroprotective effect by decreasing the bone resorption and protecting the cartilage degeneration without achieving to reduce the proteoglycan loss in a chymopapain rabbit model.8 However, clinical trials investigating the effect of bisphosphonates on OA have not presented such encouraging results. A 1-year clinical study showed that risedronate improved patients’ symptoms and physical activity, and significantly reduced markers of cartilage degradation and bone resorption.10 In another 2-year trial, risedronate decreased biochemical markers of cartilage degeneration but did not decrease symptoms or radiographic progression of OA.5 A crosssectional study had shown that older women who were receiving alendronate had significantly fewer OArelated subchondral bone abnormalities in the knee and had less knee pain, compared with women who were not taking any bone antiresorptive drugs, but there was no association of alendronate use with changes in cartilage lesion and volume on MRI.26 In the aforementioned experimental studies, bisphosphonates were effective in the early stages of OA but in humans there is no study evaluating a selected sample of patients with early OA. Radiographs, which are the usual means for the investigation of OA in humans, cannot detect the early stage of the disease. The direct study of degenerative OA in humans presents major difficulties, such as the slow JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2014
progression of the disease requiring long-term studies, the variety of causes and environmental influences (such as heredity, history of trauma, lifestyle and underlying conditions), the variety of symptoms, location and radiological depiction (sclerosis or cysts of subchondral bone, narrowing of joint space and osteophytes) leading to disparate data difficult to interpret, the absence of symptoms in the early stages of the disease, the absence of exact correlation between progression of OA and severity of symptoms, the lack of sensitive imaging techniques and the inability to perform histological studies by harvesting articular tissue due to ethical reasons.15 We considered the ACL transection rabbit model as the best model for the study of the chondroprotective effect of ZOL, which is a well-established and reproducible animal model of OA. The period between 6 and 8 weeks after sectioning the ACL is sufficient for the development of OA in rabbits.27,28 We administered a high dose of ZOL (0.6 mg/kg intravenous injection three times in a 2 month period) to clearly see its effect on cartilage and subchondral bone. It was well-tolerated without causing any apparent adverse effect. Grossly, osteoarthritic lesions and formation of osteophytes were present in all animals, but the ZOL treated group had significantly milder ulcerations, cartilage softening and fibrillation compared to the placebo group. Microscopically, morphology of the articular cartilage was better in the ZOL treated group compared with the placebo group, without complete disorganization in any section of the first group. Furthermore, the chondrocytes in the ZOL treated group were mainly cloning, indicating cartilage repairing and regeneration process,29,30 while in the placebo group hypocellularity predominated. Additionally, subchondral necrosis was evident only in some sections of the placebo group. A similar positive effect on cartilage has been reported with calcitonin, another antiresorptive drug, in a comparable study using the same animal model and experimental protocol in our institution.25 A limitation of the present study can be considered the fact that osteoarthritic lesions, in our experimental model, occurred after an acute traumatic event that mimics posttraumatic OA in humans and not degenerative OA. However, certain stages of the progression of OA are similar with degenerative OA. Additionally, there are differences in anatomy and mechanics between animals and between animal and human knees, thus we have to be cautious when comparing results of studies performed with different species. Furthermore, a non-operated control group was not employed, because no degenerative changes were observed in any of the joints during surgery and also rabbits have very little spontaneous degeneration in their stifles.31 In conclusion, zoledronic acid, in a high-dose regimen, was proved to be chondroprotective in a wellestablished animal model of OA.
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ACKNOWLEDGMENTS The authors would like to thank the Hellenic Society for the Study of Bone Metabolism, the Novartis Pharma AG (Basel) and the Novartis (Hellas) SACI for the financial support of this study, and Novartis Pharma AG (Basel) and Novartis (Hellas) SACI for the drug supply.The authors would also like to thank Mr. Antonios Galanos MSc, PhD, for his contribution to the statistical analysis, Ms. Chrysavgi Kapsi and Ms. Helen Kostakioti for their expertise in veterinary nursing, Ms. Maria Tsipra for the histological specimen preparation, and Mr. Panagiotis Prokopis for the animal husbandry.
14. 15.
16.
17.
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