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 = 10) received 0.6 mg/kg intravenous injection of ZOL on day 1, 15, and 29 and placebo group (n = 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. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1646–1651, 2014.
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. 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. Recent preclinical and limited clinical studies have shown promising results concerning their chondroprotective role.[5-13] 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.[11, 14]
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.
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 0109-69592, 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 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 = 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 = 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 µm 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]
|Articular cartilage structure||Grade|
|Pannus and surface irregularities||2|
|Clefts to transitional zone||3|
|Clefts to radial zone||4|
|Clefts to calcified zone||5|
|Arrangement of chondrocytes|
|Crossed by blood vessels||1|
|Subchondral bone morphology|
The Pearson's χ-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).
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 = 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 = 0.001) (Fig. 3b). Additionally, subchondral necrosis was evident only in some sections of the placebo group (p = 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 = 0.027) (Fig. 3d and 3e–j).
Zoledronic acid was well-tolerated without causing any apparent adverse effect.
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. The suspicion of the articular cartilage-subchondral 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. 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]
To address the need to impact the subchondral bone-articular cartilage interaction, bone anticatabolic agents such as bisphosphonates and calcitonin, 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.
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. 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). 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. 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.
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. In another 2-year trial, risedronate decreased biochemical markers of cartilage degeneration but did not decrease symptoms or radiographic progression of OA. A cross-sectional study had shown that older women who were receiving alendronate had significantly fewer OA-related 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.
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 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.
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.
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.
In conclusion, zoledronic acid, in a high-dose regimen, was proved to be chondroprotective in a well-established animal model of OA.
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.