Subchondral Bone Volume Research Articles

Sclerostin expression in the subchondral bone of patients with knee osteoarthritis.

The aim of this study was to assess the expression of β-catenin, transcription factor-4 (TCF-4) and sclerostin in the subchondral bone of patients with primary knee osteoarthritis (OA). Tibial plateau specimens from patients with OA who underwent total knee arthroplasty were classified into the early stage (n=15), intermediate stage (n=13) and late stage (n=17) groups using the Mankin score. Structural parameters, including total articular cartilage (TAC), subchondral bone plate (SCP) thickness and trabecular bone volume (BV/TV), were assessed using Image-Pro Plus 6.0 analysis software. Subsequently, β-catenin and sclerostin expression levels in subchondral bone were determined by immunohistochemistry. In addition, the mRNA and protein levels of β-catenin, TCF-4 and sclerostin were evaluated by RT-qPCR and western blot analysis, respectively. As regards the cartilage and subchondral bone structural parameters, TAC was reduced, while SCP thickness and BV/TV were increased due to OA, with significant differences observed among the different stages (all P<0.05). The results of immunohistochemistry revealed that the β-catenin levels in the intermediate- and late-stage samples were significantly increased, while the levels of sclerostin were markedly decreased compared with the values in the early-stage samples (all P<0.05). Compared with the intermediate-stage samples, the sclerostin levels were decreased, and SCP thickness and the β-catenin levels were increased in the late-stage samples (all P<0.05). The results of RT-qPCR and western blot analysis revealed that the β-catenin and TCF-4 mRNA and protein levels in the intermediate- and late-stage samples were significantly increased, while sclerostin expression was significantly decreased compared with the early-stage samples; a similar trend was observed between the intermediate- and late-stage samples (all P<0.05). Finally, the β-catenin and TCF-4 levels positively correlated with the Mankin scores, while there was a negative correlation with sclerostin expression. Our findings demonstrate that sclerostin expression is closely associated with the degree of joint damage in patients with OA, confirming its involvement in the development of OA.

Long term bone alterations in aged rats suffering type 1 diabetes

Increasing duration of type 1 diabetes mellitus alters bone metabolism. Clinical studies and experimental studies in long bones of rats with experimentally induced diabetes have reported a decrease in bone density. Few studies have explored this diabetes related alteration in the maxillae. Given that this finding could indicate the possible development of osteopenia in the maxilla in the long term, the present study sought to analyze alterations in alveolar bone in aged rats, 12, 18, and 24weeks after inducing diabetes, and compare alveolar bone response to that of tibial subchondral bone at the same experimental times. Thirty-six male Wistar rats, 130g body weight, were divided into 2 groups: an experimental group (E) receiving a single i.p. 60mg/kg dose of streptozotocin, and a control group (C). Both the control and experimental groups were divided into 3 sub-sets, according to the time of euthanasia: 12, 18 and 24weeks. The alveolar bone and tibiae were examined histologically and histomorphometrically. The results were analyzed using Student's t-test; a value of p<0.05 was considered statistically significant. Results: Subchondral bone volume and bone activity/remodeling, mainly bone rest, were significantly lower in diabetic animals compared to controls, at both 12 and 18weeks. No differences in alveolar bone parameters were observed between diabetic and control animals at either of the experimental times. Animals surviving at 24weeks showed few trabeculae at rest and severe destruction of dental and periodontal tissues. The results of the present study show that diabetic osteopenia is evident in the tibia at 12 and at 18weeks, whereas its effects on the maxilla can be seen at 24weeks, with substantial destruction of alveolar bone and of the remaining periodontal and dental tissues. All the above observations highlight the need for preventive oral care in diabetic patients, before irreversible damage to dental and periodontal tissues occurs.

Subchondral bone scan uptake correlates with articular cartilage degeneration in osteoarthritic knees.

The aim of this study was to analyze subchondral bone scan uptake in osteoarthritic knees with reference to subchondral bone microstructure and articular cartilage histology. This cross-sectional, laboratory study evaluated 123 human distal femoral condyle specimens of 67 patients after joint replacement surgery. All patients were preoperatively examined with bone scan of the knee joint. Specimens were evaluated for cartilage histology and micro-computed tomography analysis of subchondral bone. Data between bone scan, histology and micro-computed tomography were statistically analyzed using either coefficient of correlation, Student's t-test or one-way analysis of variance with Tukey post hoc test. Bone scan grading and histological articular cartilage degeneration scores showed significant correlation (r = 0.812, P < 0.001). Both bone scan positive and histologically confirmed osteoarthritis samples showed increase in subchondral trabecular bone volume and thickness, reflected in micro-computed tomography. Overall, positive predictive value (%) of bone scan for osteoarthritic cartilage lesions was 91.9%, and the sensitivity and specificity were 88.3% and 60%, respectively. Histology showed that bone scan has both a high positive predictive and a low negative predictive value for detection of osteoarthritic cartilage lesions. Bone scan uptake correlated with articular cartilage degeneration in osteoarthritic knees. Bone scan may be a useful diagnostic tool that reflects pathologic changes of cartilage in osteoarthritis.

Effect of cadmium on bone tissue in growing animals

Accumulation of cadmium (Cd), an extremely toxic metal, can cause renal failure, decreased vitamin D synthesis, and consequently osteoporosis. The aim of this work was to evaluate the effect of Cd on two types of bone in growing Wistar rats. Sixteen 21-day-old male Wistar rats were assigned to one of two groups. The Cd group subcutaneously received 0.5mg/kg of CdCl2 5 times weekly for 3 months. The control group similarly received bidistilled water. Following euthanasia, the mandibles and tibiae were resected, fixed, decalcified and processed histologically to obtain sections for H&E and tartrate-resistant acid phosphatase (TRAP) staining. Photomicrographs were used to determine bone volume (BV/TV%), total growth cartilage width (GPC.Wi) hypertrophic cartilage width (HpZ.Wi), percentage of yellow bone marrow (%YBM), megakaryocyte number (N.Mks/mm2), and TRAP+osteoclast number (N.TRAP+Ocl/mm2). Results were statistically analyzed using Student’s t test. Cd exposed animals showed a significant decrease in subchondral bone volume and a significant increase in TRAP+ osteoclast number and percentage of yellow bone marrow in the tibia, and an increase in megakaryocyte number in mandibular interradicular bone. No significant differences were observed in the remaining parameters. The results obtained with this experimental design show that Cd would seemingly have a different effect on subchondral and interradicular bone. The decrease in bone volume and increase in tibial yellow bone marrow suggest that cadmium inhibits differentiation of mesenchymal cells to osteoblasts, favoring differentiation into adipocytes. The different effects of Cd on interradicular bone might be due to the protective effect of the mastication forces.

Subchondral and epiphyseal bone remodeling following surgical transection and noninvasive rupture of the anterior cruciate ligament as models of post-traumatic osteoarthritis

Animal models are frequently used to study post-traumatic osteoarthritis (PTOA). A common anterior cruciate ligament (ACL) injury model is surgical transection, which may introduce confounding factors from surgery. Noninvasive models could model human injury more closely. The purpose of this study was to compare subchondral and epiphyseal trabecular bone remodeling after surgical transection and noninvasive rupture of the ACL. Thirty-six rats were randomized to an uninjured control, surgical transection (Transection), or noninvasive rupture (Rupture). Animals were randomized to 4 or 10 week time points (n=6 per group). Micro computed tomography (μCT) imaging was performed with an isotropic voxel size of 12μm. Subchondral and epiphyseal bone was segmented semi-automatically, and morphometric analysis was performed. Transection caused a greater decrease in subchondral bone volume fraction (BV/TV) than Rupture in the femur and tibia. Rupture had greater subchondral bone tissue mineral density (TMD) at 4 and 10 weeks in the femur and tibia. Subchondral bone thickness (SCB.Th) was decreased in the femur in Transection only. Epiphyseal BV/TV was decreased in Transection only, and Rupture exhibited increased femoral epiphyseal TMD compared to both Control and Transection. Rupture exhibited greater femoral epiphyseal trabecular thickness (Tb.Th.) compared to Control and Transection at 4 weeks, and both Rupture and Transection had increased femoral epiphyseal Tb.Th. at 10 weeks. Epiphyseal trabecular number (Tb.N) was decreased in both injury groups at both time points. Femoral and tibial epiphyseal structure model index (SMI) increased in both groups. The two injury models cause differences in post-injury bone morphometry, and surgical transection may be introducing confounding factors that affect downstream bony remodeling.

Histomorphometry of the tibia and mandible of healthy female Wistar rats at different stages of growth.

Female Wistar rats are frequently used in experimental models to study hormone and bone pathologies and treatments. Most experimental studies involving histomorphometric evaluation assessed long bones, and few reports also studied mandibular bone. The aim of this work was to clarify and distinguish the age-related histomorphometric changes that occur in the tibia (subchondral bone) and in the mandible (interradicular bone), and thus obtain reference histomorphometric data of healthy female Wistar rats at different growth stages. Three groups of 8 healthy female Wistar rats were euthanized at 6 (GI), 10 (GII), and 14 (GIII) weeks. The tibiae and mandible were resected and histologically processed to obtain H&E stained sections of the tibia and the lower first molar to analyze the following histomorphometric parameters: Bone volume, trabecular width, trabecular number (Th.N)(1/mm), growth cartilage width, hypertrophic cartilage width and number of osteoclasts per area in the tibiae, and bone volume and number of osteoclasts per area N.Oc/mm2 in the interradicular bone of the first lower molar. A significant decrease in subchondral bone volume as a result of a decrease in trabecular number and growth cartilage width was observed in 14-week-old rats. Conversely, interradicular bone volume was found to increase with age. The results highlight the importance of analyzing both types of bone to better understand the response of two different trabecular bones, contributing in turn to decision making regarding treatment strategies and disease management.

Iron overload in a murine model of hereditary hemochromatosis is associated with accelerated progression of osteoarthritis under mechanical stress

Hereditary hemochromatosis (HH) is a disease caused by mutations in the Hfe gene characterised by systemic iron overload and associated with an increased prevalence of osteoarthritis (OA) but the role of iron overload in the development of OA is still undefined. To further understand the molecular mechanisms involved we have used a murine model of HH and studied the progression of experimental OA under mechanical stress. OA was surgically induced in the knee joints of 10-week-old C57BL6 (wild-type) mice and Hfe-KO mice. OA progression was assessed using histology, micro CT, gene expression and immunohistochemistry at 8 weeks after surgery. Hfe-KO mice showed a systemic iron overload and an increased iron accumulation in the knee synovial membrane following surgery. The histological OA score was significantly higher in the Hfe-KO mice at 8 weeks after surgery. Micro CT study of the proximal tibia revealed increased subchondral bone volume and increased trabecular thickness. Gene expression and immunohistochemical analysis showed a significant increase in the expression of matrix metallopeptidase 3 (MMP-3) in the joints of Hfe-KO mice compared with control mice at 8 weeks after surgery. HH was associated with an accelerated development of OA in mice. Our findings suggest that synovial iron overload has a definite role in the progression of HH-related OA.

Identifying Patient-Specific Pathology in Osteoarthritis Development Based on MicroCT Analysis of Subchondral Trabecular Bone

The goal of this study was to identify alternative mechanisms of osteoarthritis pathology by analyzing subchondral bone. Femoral condyle samples were collected from post-menopausal female patients with knee osteoarthritis undegoing total knee arthroplasty. In the majority of patients, subchondral trabecular bone volume doubled under a region of the medial femoral condyle with full-thickness cartilage deterioration. However, in a subset of patients the bone volume in this region remained constant. This subset also had larger areas of vascular penetration in the calcified cartilage of the lateral condyle concurrent with increased vascular endothelial growth factor expression. Subtyping by subchondral bone characteristics identified a unique population, which lacked the sclerotic bone characteristic of late-stage osteoarthritis. Identification of subtypes within the osteoarthritis population allows investigation of alternate disease pathologies.

Prenatal Exposure to Continuous Constant Light Alters Endochondral Ossification of the Tibiae of Rat Pups

Clinical and experimental studies suggest that prenatal exposure to stress can impact the growth and development of offspring. The effect of prenatal exposure to constant light, applied as a chronic stressor, on endochondral ossification of the tibiae of 3-day-old and 15-day-old pups was histomorphometrically evaluated. Pregnant rats were divided into 2 groups: mothers chronically exposed to a 12:12-hour light/light cycle (LL) and control mothers maintained on a 12:12-hour light/dark cycle on days 10-20 of pregnancy. On postnatal days 3 and 15, the pups were weighed and euthanized. The tibiae were resected and histologically processed to obtain sections for hematoxylin and eosin staining (HE) and tartrate-resistant acid phosphatase (TRAP) histochemistry, in order to perform histomorphometric determinations. The data were statistically analyzed. A significant decrease in hypertrophic cartilage thickness was observed in the tibiae of the 3-day-old (LL: 0.134 ± 0.02 vs. controls: 0.209 ± 0.023 mm; p < 0.01) and 15-day-old (LL: 23.32 ± 3.98 vs. controls: 22.96 ± 1.93 mm; p < 0.05) prenatally stressed pups. The subchondral bone volume was significantly lower in the tibiae of the 3-day-old LL pups (38.83 ± 6.14%) than in the controls (62.83 ± 10.67%; p < 0.01). The decrease in subchondral bone volume and hypertrophic cartilage thickness shows that the normal growth process of the tibia is impaired in prenatally stressed pups.

Oestrogen receptor beta mediates decreased occlusal loading induced inhibition of chondrocyte maturation in female mice

ObjectiveTemporomandibular joint (TMJ) disorders predominantly afflict women, suggesting that estrogen may play a role in the disease process. Defects in mechanical loading-induced TMJ remodelling are believed to be a major etiological factor in TMJ degenerative disease. Previously, we found that, decreased occlusal loading caused a significant decrease in early chondrocyte maturation markers (Sox9 and Col 2) in female, but not male, C57BL/6 wild type mice (1). The goal of this study was to examine the role of Estrogen Receptor (ER) beta in mediating these effects. Design21-day-old male (n=24) and female (n=25) ER beta KO mice were exposed to decreased occlusal loading (soft diet administration and incisor trimming) for 4 weeks. At 49 days of age the mice were sacrificed. Proliferation, gene expression, Col 2 immunohistochemistry and micro-CT analysis were performed on the mandibular condyles. ResultsDecreased occlusal loading triggered similar effects in male and female ER beta KO mice; specifically, significant decreases in Col 10 expression, subchondral total volume, bone volume, and trabecular number. ConclusionDecreased occlusal loading induced inhibition of chondrocyte maturation markers (Sox9 and Col 2) did not occur in female ER beta deficient mice.

OP0130 Dickkopf-Related Protein 1 Produced by Bone Decreases Osteoarthritis through VEGF Inhibition

BackgroundOsteoarthritis (OA) is characterized by cartilage and subchondral bone dammage. The Wnt family is involved in joint homeostasis. We have shown that the Wnt/β-catenin pathway is activated in bone during...

SAT0551 Location Dependence of the Microstructure of Cartilage and of Subchondral Cortical and Trabecular Bone Microstructure in Knees with NO or Mild OA Assessed by High Resolution Micro-Computed Tomography

BackgroundLoss of articular cartilage and alterations in subchondral bone architecture with the thickening of the cortical subchondral plate and trabeculae are important characteristics of osteoarthritis (OA). The local heterogeneity of...

Chip is a critical regulator of traf6 degradation and osteoclast formation

Chip is a critical regulator of traf6 degradation and osteoclast formation

Reducing dietary loading decreases mouse temporomandibular joint degradation induced by anterior crossbite prosthesis

Purpose: Loading on temporomandibular joint (TMJ) originates from the contraction of jaw-closing muscles. Variations in food properties, such as volume or hardness, will initiate different levels of masticatory muscle contraction via periodontal-muscular reflex, leading to differences in levels of occlusal force and TMJ loading. Soft diet will cause growing rats a thinning cartilage, enhanced cartilage catabolic activity and decreased subchondral bone volume fraction in condyles. Aberrant biomechanical stimulation from abnormal dental occlusion plays an important role in TMJ osteoarthritis (OA) development. Incisors of rodent animals function more than other teeth because when a rodent catches or cuts foods they used to move the mandible in the sagittal direction following the incisal guidance. Unilateral anterior crossbite prosthesis will heavily interfere with this function because the totally opposite guidances will be provided by the normal incisor pair and the crossbite incisor pair while the TMJ can support only one pattern of them at the same time. A small-size diet may diminish the requirement of the cutting function of incisors, and thus may reduce the harmful biomechanical stimulation from the anterior crossbite prosthesis. Hence, in the present study, we exposed the mice that received the unilateral anterior crossbite prosthesis to a small-size versus a large-size diet. The aim was to test whether mice receiving soft diet have a decreased TMJ degradation induced by the unilateral anterior crossbite prosthesis using our reported methods. Methods: Forty 6-week-old female C57BL/6J mice were randomly divided into two experimental and two control groups (n = 10), one experimental and one control group received small-size diet, less than 3mm thick and the other two groups received large-size diet, about 12.5mm in diameter and 15-20mm in length. For experimental groups, metal tubes made of pinheads were bonded onto the mouse left maxillary and mandibular incisors to create unilateral anterior crossbite relationship. For the control groups, the mice underwent the similar procedures but no metal tubes were bonded. No differences in degrading changes were found between the left side and right side TMJs in the experimental mice as we reported. Left side TMJ tissue blocks from six mice of each group were fixed, decalcified and embedded in paraffin. Sagittal sections were prepared for Hematoxylin & Eosin (H-E) staining, toluidine blue staining, Safranin O staining, tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemical staining of Collagen II, OPG and RANKL. For each group, the condylar cartilages and subchondral bones of the other 14 TMJs were respectively separated and preserved at -80°C. Four to five condyles from 3 or 4 different mice were pooled to create a single sample of the cartilage or subchondral bone respectively for RNA extraction to compare the mRNA expression levels of Col2a1, Aggrecan, ADAMTS-5, RANK, OPG, RANKL. Differences were set at 0.05 level (α=0.05). Results: Thinner and degraded cartilage, reduced cartilage cellular density, decrease in toluidine blue staining (reflecting reduced proteoglycan levels) in cartilage, decreased expression levels of Collagen II and Aggrecan, loss of subchondral bone assessed by histomorphometry, and enhanced osteoclast activity were observed in both experimental groups. However, the cartilage degradation phenotype was less severe and cartilage ADAMTS-5 mRNA was lower while OPG/RANKL ratio in both cartilage and subchondral bone was higher in the small-size diet than the large-size experimental group. In contrast, no differences of histomorphology and OPG/RNAKL expression were found in the two control groups. Conclusions: The current findings suggest that a lower level of functional loading by providing small-size diet could reduce TMJ degradation induced by the biomechanical stimulation from abnormal occlusion.

Cartilage repair and subchondral bone migration using 3D printing osteochondral composites: a one-year-period study in rabbit trochlea.

Increasing evidences show that subchondral bone may play a significant role in the repair or progression of cartilage damage in situ. However, the exact change of subchondral bone during osteochondral repair is still poorly understood. In this paper, biphasic osteochondral composite scaffolds were fabricated by 3D printing technology using PEG hydrogel and β-TCP ceramic and then implanted in rabbit trochlea within a critical size defect model. Animals were euthanized at 1, 2, 4, 8, 16, 24, and 52 weeks after implantation. Histological results showed that hyaline-like cartilage formed along with white smooth surface and invisible margin at 24 weeks postoperatively, typical tidemark formation at 52 weeks. The repaired subchondral bone formed from 16 to 52 weeks in a “flow like” manner from surrounding bone to the defect center gradually. Statistical analysis illustrated that both subchondral bone volume and migration area percentage were highly correlated with the gross appearance Wayne score of repaired cartilage. Therefore, subchondral bone migration is related to cartilage repair for critical size osteochondral defects. Furthermore, the subchondral bone remodeling proceeds in a “flow like” manner and repaired cartilage with tidemark implies that the biphasic PEG/β-TCP composites fabricated by 3D printing provides a feasible strategy for osteochondral tissue engineering application.

Comparison between subchondral bone change and cartilage degeneration in collagenase- and DMM- induced osteoarthritis (OA) models in mice

Osteoarthritis (OA) is a multifactorial disease affecting both the cartilage and the subchondral bone. However, animal models of OA cannot represent both of the changes and show variations depending on induction methods and animal species/strains. This study first investigated subchondral bone changes and its correlation with cartilage degeneration in two different OA models in C57Bl/6 mice. Experimental OA was produced by type II collagenase-induced osteoarthritis (CIOA) and destabilization of the medial meniscus (DMM). The subchondral plate and trabecular bone of the tibia were analyzed by micro-computed tomography and cartilage degeneration was analyzed histologically after safranin-O staining at 2, 4, 6 and 8 weeks. In the DMM model, cartilage degeneration was induced reproducibly and progressively with time. Progressive increase in subchondral bone volume, but not in bone thickness, was also observed in both subchondral bone plate and subchondral trabecular bone in the medial tibial area. The changes in subchondral bone volume correlated well with the histological cartilage degeneration (R2=0.7870). In contrast, in the CIOA model, cartilage degeneration was relatively unstable, increasing only until 4 weeks and decreasing thereafter. No significant changes in subchondral bone were observed in all areas at all time points. These results suggest that the DMM OA mouse model is more reliable and useful than the CIOA model by virtue of its better representation of cartilage degeneration and subchondral bone change with high correlation coefficient.

1,25(OH)2D deficiency induces temporomandibular joint osteoarthritis via secretion of senescence-associated inflammatory cytokines

1,25-Dihydroxyvitamin D [1,25(OH)2D] insufficiency appears to be associated with several age-related diseases. Insufficient levels of serum 25-hydroxyvitamin D has been shown to lead to the progression of osteoarthritis (OA) while underlying biological mechanisms remain largely unknown. In this study, we sought to determine whether 1,25(OH)2D deficiency has a direct effect on the process of murine temporomandibular joint (TMJ) OA in 25-hydroxyvitamin D 1α-hydroxylase knockout [1α(OH)ase−/−] mice that had been fed a rescue diet (high calcium, phosphate, and lactose) from weaning until 6 or 18months of age. Our results showed that the bone mineral density and subchondral bone volume were reduced in mandibular condyles, articular surfaces were collapsed, the thickness of articular cartilage and cartilage matrix protein abundance were progressively decreased and eventually led to an erosion of articular cartilage of mandibular condyles. We also found that DNA damage, cellular senescence and the production of senescence-associated inflammatory cytokines were increased significantly in 1α(OH)ase−/− mice. This study demonstrates that 1,25(OH)2D deficiency causes an erosive TMJ OA phenotype by inducing DNA damage, cellular senescence and the production of senescence-associated inflammatory cytokines. Our results indicate that 1,25(OH)2D plays an important role in preventing the development and progression of OA.

Inhibition of cathepsin K reduces cartilage degeneration in the anterior cruciate ligament transection rabbit and murine models of osteoarthritis

ObjectiveTo investigate the disease modifying effects of cathepsin K (CatK) inhibitor L-006235 compared to alendronate (ALN) in two preclinical models of osteoarthritis (OA). MethodsSkeletally mature rabbits underwent sham or anterior cruciate ligament transection (ACLT)-surgery and were treated with L-006235 (L-235, 10mg/kg or 50mg/kg, PO, daily) or ALN (0.6mg/kg, s.c., weekly) for 8-weeks. ACLT joint instability was also induced in CatK−/− versus wild type (wt) mice and treated for 16-weeks. Changes in cartilage degeneration, subchondral bone volume and osteophyte area were determined by histology and μ-CT. Collagen type I helical peptide (HP-I), a bone resorption marker and collagen type II C-telopeptide (CTX-II), a cartilage degradation marker were measured. ResultsL-235 (50mg/kg) and ALN treatment resulted in significant chondroprotective effects, reducing CTX-II by 60% and the histological Mankin score for cartilage damage by 46% in the ACLT-rabbits. Both doses of L-235 were more potent than ALN in protecting against focal subchondral bone loss, and reducing HP-I by 70% compared to vehicle. L-235 (50mg/kg) and ALN significantly reduced osteophyte formation in histomorphometric analysis by 55%. The Mankin score in ACLT-CatK−/− mice was ~2.5-fold lower than the ACLT-wt mice and was not different from sham-CatK−/−. Osteophyte development was not different among the groups. ConclusionInhibition of CatK provides significant benefits in ACLT-model of OA, including: 1) protection of subchondral bone integrity, 2) protection against cartilage degradation and 3) reduced osteophytosis. Preclinical evidence supports the role of CatK as a potential therapeutic target for the treatment of OA.

Chondroprotective effect of alendronate in a rabbit model of osteoarthritis

Purpose: It is generally recognized that osteoarthritis (OA) is a complex multifactorial disease process involving articular cartilage, subchondral bone, tendons, and synovium. Alendronate (ALN) inhibits bone loss and increases bone mineralization. The aim of this study was to examine whether ALN altered cartilage degeneration and periarticular bone quality in the rabbit anterior cruciate ligament transection (ACLT) model of OA. Moreover, the mechanism responsible for ALN effects on cartilage was investigated using immunohistochemical analysis. Methods: Skeletally matured, thirty female Japanese white rabbits underwent an ACLT on the left knee and a sham operation on the right knee. Fifteen rabbits then received weekly subcutaneous injections of ALN (0.14 mg/kg) beginning on the day of surgery and 15 rabbits received subcutaneous injections of saline according to the same schedule. Five animals from each group were sacrificed 2, 4 or 12 weeks after surgery. Sixty operated knees of 30 rabbits were divided into four groups. The right and left knees of rabbits that received saline treatment (control [cont] group) and those of rabbits that received ALN treatment were designated cont/sham, cont/ACLT, ALN/sham and ALN/ACLT, respectively. Assays for serum N-telopeptide of type I collagen (NTX) level were performed before treatment of ALN and after sacrificed. The distal femur and the proximal tibia were scanned by micro-computed tomography (mCT) to quantify the bone volume ratio. Macroscopic view, histological score, tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemical index for matrix metalloproteinase 13 (MMP13), interleukin-1b (IL-1b), type X collagen (COLX), vascular endothelial growth factor (VEGF), receptor activator of nuclear factor kB ligand (RANKL) were evaluated for each group. Results: Serum NTX level was suppressed by ALN at 4 weeks post-surgery. Twelve weeks after the surgery, the cont/ACLT group had more periarticular trabecular bone loss and osteophyte formation than the cont/ sham group. However, the ALN/ACLT group maintained bone formation in a manner similar to that of the ALN/sham group. The expression of TRAPpositive osteoclasts in subchondral bone was prevented by ALN. Macroscopic and histological evaluation revealed that the cont/ACLT group had significantly worse cartilage damage than the cont/sham group 12 weeks after the surgery, but the ALN/ACLT group had mild cartilage degeneration compared with that of the ALN/sham group. Protein expression levels of MMP13, IL-1b, COLX, VEGF and RANKL in the cartilage were suppressed in the ALN/ACLT group compared with the cont/ACLT group. Conclusions: The effect of ALN as a potent inhibitor of osteoclastic bone resorption was verified by suppressed serum NTX level, increased subchondral bone volume and inhibited TRAP-positive osteoclasts expression. Macroscopically and histologically, ALN had a chondroprotective effect in our experimental rabbit model of OA. Immunohistochemical analysis showed that ALN suppressed the expression of MMP13, IL-1b, COLX, VEGF and RANKL in OA cartilage. We propose that ALN protects normal joint loading by preserving subchondral bone quality and thereby conserves articular cartilage by inhibiting the expression of MMP13, IL-1b, COLX, VEGF and RANKL. Our results suggest therapeutic roles for ALN in inhibiting subchondral bone loss in the early stage of OA, in protecting cartilage against degeneration and in preserving long-term joint function.

Decreased bone tissue mineralization can partly explain subchondral sclerosis observed in osteoarthritis

For many years, pharmaceutical therapies for osteoarthritis (OA) were focused on cartilage. However, it has been theorized that bone changes such as increased bone volume fraction and decreased bone matrix mineralization may play an important role in the initiation and pathogenesis of OA as well. The mechanisms behind the bone changes are subject of debate, and a better understanding may help in the development of bone-targeting OA therapies. In the literature, the increase in bone volume fraction has been hypothesized to result from mechanoregulated bone adaptation in response to decreased mineralization. Furthermore, both changes in bone volume fraction and mineralization have been reported to be highest close to the cartilage, and bone volume fraction has been reported to be correlated with cartilage degeneration. These data indicate that cartilage degeneration, bone volume fraction, and bone matrix mineralization may be related in OA. In the current study, we aimed to investigate the relationships between cartilage degeneration, bone matrix mineralization and bone volume fraction at a local level. With microCT, we determined bone matrix mineralization and bone volume fraction as a function of distance from the cartilage in osteochondral plugs from human OA tibia plateaus with varying degrees of cartilage degeneration. In addition, we evaluated whether mechanoregulated bone adaptation in response to decreased bone matrix mineralization may be responsible for the increase in bone volume fraction observed in OA. For this purpose, we used the experimentally obtained mineralization data as input for bone adaptation simulations. We simulated the effect of mechanoregulated bone adaptation in response to different degrees of mineralization, and compared the simulation results to the experimental data. We found that local changes in subchondral bone mineralization and bone volume fraction only occurred underneath severely degenerated cartilage, indicating that bone mineralization and volume fraction are related to cartilage degeneration at a local level. In addition, both the experimental data and the simulations indicated that a depth-dependent increase in bone volume fraction could be caused by decreased bone matrix mineralization. However, a quantitative comparison showed that decreased mineralization can only explain part of the subchondral sclerosis observed in OA.