| Previous investigations demonstrated both overloading and reduced loading may indicate the imbalance of metabolism and therefore result in malnutrition of bone and joint. This may lead to the degeneration of bone and joint and osteoarthritis (OA). However, others indicated that the mechanical loading was not absolutely detrimental to joint, but moderate loading could be beneficial for maintaining the balance of metabolism of joint. In other words, the absolute non-loading could exert a deleterious effect on the articular cartilage. For instance, our previous study demonstrated that excessive treadmill running may lead to cartilage degeneration. Similarly, reduced loading, occurring commonly as a result of joint immobilization, also result in catabolic responses within articular cartilage. Multitudes of studies demonstrated that joint immobilization might change morphological,biochemical and biomechanical characteristics of articular cartilage.As the most common arthritis in human, OA is characterized by the degeneration of articular cartilage which is the pathologic prerequisite for damage of joint structure and function. As a weightbearing joint, knee is susceptible to OA, in particular among the elder which would lead them to lose mobility. Traditionally cartilage degradation has been considered the main hallmark of osteoarthritis (OA). It is currently believed that subchondral bone plays an important role in the pathogenesis of the disease. Nevertheless, it remains controversial whether subchondral bone degeneration proceeds or precedes cartilage damage during evolution of OA. Recent investigations from animal models showed that microstructural subchondral bone damage may occur before, during or after cartilage alteration. Such discrepancy may be associated with differences in species evaluated, OA model used, and stages of the disease.Because there is a tight coupling between cartilage and underlying bone, alteration in articular cartilage integrity should be linked to the remodeling responses in the underlying bone, sufficient evidence shows that joint immobilization may lead to cartilage atrophy, but its effect on subchondral bone or about the role of subchondral bone in the process of cartilage degeneration because of joint immobilization remains to be known. There have been so far only two animal studies investigating alterations in cartilage and subchondral bone subject to joint immobilization. Smith et al. showed that femoral metaphyseal bone density decreased significantly, while the amount of cartilage glycosaminoglycan and hydroxyproline in femoral cartilage stayed unchanged after 3 weeks’knee immobilization in a rabbit model, demonstrating that bone loss and remodeling preceded erosive cartilage degradation. On the contrary, it was reported that a mild level of cartilage degeneration accompanied with no alteration in subchondral bone thickness after the canine knee was cast-immobilized for 4 weeks in a other study. The role of subchondral bone in the process of cartilage change induced by immobilization remains to be unclear because their evaluation were conducted at a single time-point, and the results of the aforementioned two studies were inconsistent. Moreover, they both failed to clarify the intrinsic mechanism by which how subchondral bone influences articular cartilage secondary to immobilization.Therefore, we investigated the spatial and temporal changes in subchondral bone in rats after their knee joints were immobilized for 1,4, and 8 weeks in this study. We also conducted histological and bone geometric analyses to assess the alterations of subchondral bone and their association with the changes of its overlying articular cartilage.1. Objectives1. To investigate the temporal influence of immobilization on subchondral bone.2. To investigate the temporal influence of immobilization on articular cartilage.3. To investigate the potential relationship between subchondral bone and articular cartilage subjected to immobilization.2. Materials2.1. Experimental animal and immobilization method36 male Wistar rats (11-13 weeks old and weighting from 210g to 250g) were randomly and averagely assigned to one of the following three treatment (including either immobilization or no treatment) groups:(1) treatment for 1 week (1W, n=12); (2) treatment for 4 weeks (4W, n= 12); and (3) treatment for 8 weeks (8W, n=12). All of the experimental rats were housed in cages under artificially controlled conditions (12/12h for daytime/night, a constant humidity of 55±5% and constant temperature of 22±1℃), and provided with food and water ad libitum. Before and after experiment, each rat was weighted to achieve their exact weight. The protocol of this investigation was approved by the animal ethics committee of the institute.For each group, knee joints in 6 rats of each groups were randomly and unilaterally immobilized in flexion with internal fixation for 1,4 or 8 weeks respectively, which was named as immobilized knee region (IKR). At the same time their contralateral knees underwent no treatment and served as non-immobilized knee region (non-KIR). Their immobilization method could be stated briefly as follows:under halothane anesthesia, two 5-cm long Kirschner wires were inserted vertically through the proximal femur and distal tibia, respectively. Their ends were bent and connected to form a closed circle to make sure that the knee joint was immobilized and fixed in 150° flexion. Surgical procedure was achieved without disrupting knee joint capsule as well as cartilage. X-rays were taken shortly after the operation. In each group, the remaining 6 rats were allowed free activity and acted as external control knee regions (ECKR).2.2 Specimen collection and experiment protocolsAt the end of 1,4 or 8 weeks’immobilization respectively, all rats were sacrificed. For each animal, femurs at both sides were dissected. After the surrounding soft tissue was removed immediately, the distal part of each femur was examined by Micro-CT. Afterwards, femoral condyle was cut and fixed in 4% pH 7.4 buffered formaldehyde for 24 hours. Then, decalcification was done in 10% EDTA solution before the specimen was embedded in paraffin wax. Thereafter, the femoral specimen was resected into 5-μm sagital sections from medial to lateral for further investigation.2.3 Micro-computed tomography measurementsThe distal femur was scanned via a micro-CT scanner (SkyScan 1076 Micro-CT system, Kontich, Belgium) with isotropic voxel size of 17.33μm. The current was 100μA and the X-ray tube voltage was 88kV, with a 1.0mm aluminum filter. Scanning was performed from the distal growth plate, and the region of interest (ROI) was the area 1.0mm beneath the lower end of the growth plate extending 3.2mm distally. The subchondral trabecular bone region was isolated from the cortex on 2D images by manual contouring. Direct 3D methods were utilized to work out such parameters as trabecular thickness (Tb.Th, mm), bone mess density (BMD, g/mm3), bone volume fraction (BV/TV,%), structure model index (SMI), trabecular separation (Tb.Sp, mm), degree of anisotropy (DA), trabecular number (Tb.N) and trabecular bone pattern factor (Tb.Pf, 1/mm). Similarly parameters including bone mess density (BMD, g/mm3), trabecular thickness (Tb.Th, mm) and porosity (%) of subchondral bone plate were also calculated.2.4 Histomorphometric evaluationEach section was stained with Safranin-O (SO), hematoxylin eosin (HE), and Picrosirius red for histological observation respectively. To better evaluate various morphological changes, structure of subchondral and bone cartilage, number of chondrocytes, pannus formation, cells’ condition, and tidemark were taken into assessment system. Thickness (μm) of total articular cartilage for each section was defined as the mean distance between cartilage surface and osteochondral junction at the middle point and 300-μm around. As the thickness of articular cartilage differed in various areas, a certain range of interest (rectangles of 400μm long and 100μm deep) was set to count the number of chondrocytes. The cells stained blue with hematoxylin were thought to be chondrocytes. Each section was blindly assessed by two observers and then averaged. Nikon-H600L (Nikon, Japan) microscope with an image analyzing system was used to examine the Safranin-O staining sections. Picrosirius red-staining was used to assess the alignment of collagen by fibers Polarizing light microscopy. The sections were examined by optical microscope equipped with a polarizing light coupled to an image analyzing system (Olympus CCD DP71/Olympus Microscope BX-51).3. Results3.1 Micro-CT changes3.1.1 The Micro-CT results of subchondral trabecular boneThe results of micro-CT analyses of subchondral trabecular bone were showed in Table 1. No significant difference were observed between external control knee regions and non-immobilized knee regions regarding each parameter (p>0.05), while some difference were significant when compared immobilized knee regions with non-immobilized knee regions and external control knee regions (p<0.05, Table 2). In general, immobilization could lead to a lower BV/TV and Tb.Th, a higher Tb.Pf and more rod-like trabecular bone. More importantly, such alterations became more obvious with time.3.1.2 The Micro-CT results of subchondral bone plateA. Tb.Th of subchondral bone plateSubchondral bone plate in immobilized knee region was significantly thinner compared with that in external control knee regions at medial side (0.113±0.019 mm vs.0.151±0.016mm) (p=0.004) at 1 week. Significant thinner subchondral bone plate in immobilized knee region (0.109±0.011mm) than non-immobilized knee region (0.143±0.011 mm) (p=0.003) was found at medial side at 4 week. Subchondral bone plate in immobilized knee region (0.109±0.010 mm) was also significantly thinner than that in external control knee region at lateral side (0.138±0.018 mm) (p=0.013) at 4 week. At 8 week, in both medial (0.107±0.017 mm vs.0.134±0.015 mm, p=0.014) and lateral side (0.101±0.014 mm vs.0.139±0.011 mm, p=0.000), subchondral bone plate was thinner in immobilized knee regions than non-immobilized knee region, as well as than external control knee regions (medial: 0.107±0.017 mm vs.0.144±0.021, p=0.007 and lateral:0.101±0.014 mm vs. 0.150±0.024mm,p=0.022).B. porosity of subchondral bone plateA significantly higher porosity of subchondral plate was defined in immobilized knee region than both non-immobilized knee region in lateral side (64.6±7.2% vs. 39.9±5.9%, p=0.027). Similarly significant increase of porosity was found compared with control groups at lateral side (66.8±8.1% vs.45.6±6.2%, p=0.028) and medial side (72.5±8.3% vs.40.3±7.4%, p=0.041) at 4 weeks. Significant difference was also observed when compared with non-immobilized knee region in medial side (72.5±8.2% vs.42.6±7.9%, p=0.022) at 4 week and lateral side (72.1±8.0% vs. 42.4±6.6%, p=0.041) at 8 week. When compared with external control knee regions at 8 week, significant difference was found in medial side (47.83±6.3%, p=0.034) and lateral side (43.8±6.2%, p=0.042).3.2 Morphological changesSO staining and HE staining indicated that striking degenerative changes in subchondral bone and articular cartilage in a time-dependant manner. With HE staining, articular cartilage surface in non-immobilized groups and external control knee region was smooth, chondrocytes were averagely spread in most parts of articular cartilage. In SO-stained images, clear tidemark was observed, osteochondral junction was intact, and vascular invasion was scarcely found to penetrate through subchondral bone plate to reach articular cartilage. On the contrary, histological changes with cell cloning, surface irregularities, and reduction in SO staining were grossly found in immobilized knee regions, and such alteration aggravated as immobilization duration prolonged.In staining with Picrosirius red, it was found that when immobilization was elongated thickness of the collagen fibers was decreasing. Moreover, in the immobilized knee regions collagen fibers seemed to be disorganized at 4 week, and gradually vanished as immobilization continued.3.3 The number of chondrocytes and thickness of articular cartilageThe chondrocyte number of immobilization groups for 1,4,8 weeks respectively was counted, which was 91.83±5.34,93.67±6.53 and 92.5±5.36 in external control knee region,89.83±5.34,88.17±5.49 and 86.83±3.67 in non-immobilized knee regions, and 83.33±4.55,72.17±3.82 and 65.33±3.33 in immobilized knee regions. Moreover, the number of chondrocytes in immobilized knee region was significantly less than that of external immobilized knee region at 8 week, and also significantly less than that non-immobilized knee region at 4 and 8 week.The thickness (u m) of articular cartilage of immobilization groups for 1,4,8 weeks respectively was 204.56±9.92,212.03±10.68 and 209.61±9.19 in external control knee region,207.46±10.03,206.97±8.83 and 203.91±7.79 in non-immobilized knee regions, and 205.28±10.48,186.43±4.43 and 177.53±3.09 in immobilized knee regions. Moreover, the thickness of articular cartilage in immobilized knee region was significantly less than that of external immobilized knee region as well as non-immobilized knee region at both 4 and 8 week.4. ConclusionsOur investigation suggested that under immobilization, the changes of subchondral bone took place within 1 week while the changes of articular cartilage took place within 4 weeks. The alteration of articular cartilage would be influenced by that of underlying subchondral bone. The mechanism by which of it could be related with the penetration of neo-vascular channels from the latter into the former, which spread the catabolic cytokines and further deteriorated the structure of cartilage. Therefore, we suggest it should be cautious when treated the patients using immobilization, and it is better for patients to physiotherapy after immobilization. |
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