Bok 03-06.indb Increased elasticity of synovial membrane after immobilization 303 Received 17 February 2006 Accepted 21 March 2006 Increased Elasticity of Capsule After Immobilization in a Rat Knee Experimental Model Assessed by Scanning Acoustic Microscopy Yoshihiro Hagiwara1*, Yoshifumi Saijo2, Eiichi Chimoto1, Hirotoshi Akita3, Yasuyuki Sasano3, Fujio Matsumoto1, Shoichi Kokubun1 1Department of Orthopaedic Surgery, Tohoku University Graduate School of Medicine, Sendai, 980-8574, Japan 2Department of Medical Engineering and Cardiology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan 3Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, Sendai, Japan Abstract Objectives: The mechanical property of immobilized joints is not well understood. The present study was designed to investigate the tissue elasticity of the anterior and posterior synovial membrane (SM) in a rat immobilized knee model using scanning acoustic microscopy (SAM). Moreover, the structural characteristics of the SM after immobilization were examined by transmission electron microscopy (TEM). Methods: Thirty rats had their knee joints immobilized with a plate and metal screws. The rats were fixed at 1, 2, 4, 8 and 16 weeks after surgery and the knee joints were sectioned sagittally for SAM. Selected specimens were processed for TEM. A new concept SAM using a single pulsed wave instead of continuous waves was ap- plied to measure the sound speed of the anterior and posterior SM, comparing it with the corresponding light microscopic images. Results: The sound speed of the posterior SM increased significantly in the 8- and 16-week experimental group compared with that in the control group. The sound speed of the anterior SM showed no statistical difference between the experimental and the control groups at any period of immobilization. The posterior SM of the ex- perimental group was different from that of the control group in the ultrastructural characteristics of extracellular matrices. Conclusions: Our data suggest that the increased elasticity and structural changes of the posterior SM are one of the main causes of limited extension after a long period of immobilization in flexion using SAM, which is a powerful tool for evaluating the elasticity of targeted tissues. Introduction Joint contracture is defined as a decrease in both active and passive ranges of mo- tion (ROM) after immobilization. The decreased ROM limits the activity of daily living in various aspects. Immobilization, which is a major cause of joint contrac- ture, is beneficial for decreasing pain caused by trauma and preventing the joint from damage in the acute phase of arthritis such as pyogenic and rheumatoid arthri- tis [1–3]. Even by extensive rehabilitation or surgical treatment, however, it is dif- ficult to regain the full ROM in an established joint contracture after a long period of immobilization [4,5]. Upsala J Med Sci 111 (3): 303–313, 2006 304 Yoshihiro Hagiwara et al. The components of joint contracture after immobilization are classified into ar- throgenic and myogenic ones. The arthrogenic components are lesions of bone, ligaments, capsule and synovial membrane (SM), while the myogenic components are lesions of muscle, tendon and fascia [6,7]. Some investigators have attributed contracture to myogenic causes (8), while others attributed it to arthrogenic causes [4,7,9–13]. It is difficult to evaluate such contradictory reports because differ- ent animal species and methods were used in their immobilization experiments. Among the arthrogenic components, the stiffness of the capsule and SM through synovial atrophy, retraction, fibrosis, and adhesion may contribute to the limited ROM [1,7,8,10,14–17]. Though increased elasticity of the capsule or SM has been suggested to be a cause of joint contracture [18], it is not known how the elasticity and the structural characteristics of extracellular matrices are affected by immobi- lization. A scanning acoustic microscopy (SAM) using continuous waves characterize biological tissues by determining the elastic parameters based on the sound speed [19]. Recent studies on infarcted myocardium [20], atherosclerosis of aorta [21] and carotid arterial plaques [22] have shown that the acoustic properties reflect the collagen types. In the present study, we applied a new concept SAM using single pulsed wave, which can make total time for calculation significantly shorter, to examine the elasticity of the anterior and posterior SM (synovial intima and subin- tima) in the course of knee joint immobilization in a rat experimental model. Material and Methods Animals. The protocol for this experiment was approved by the Animal Research Committee of Tohoku University. Adult male Sprague-Dawley rats weighing from 380 to 400 g were used. Their knee joints were immobilized at 145° in flexion by rigid internal but extra-articular fixation for various periods (1, 2, 4, 8 and 16 weeks) according to a previously described method (1). The left and right hind legs were immobilized alternately to avoid potential systematic side differences. The surgery was performed under anesthesia with sodium pentobarbital (50 mg/kg) ad- ministered intraperitoneally. A rigid plastic plate (POM-N, Senko Med. Co., Japan) implanted subcutaneously joined the proximal femur and the distal tibia away from the knee joint and was solidly held in place with one metal screw (Stainless Steel, Morris, J. I., Co., USA) at each end. The knee joint capsule and the joint itself were untouched. Postoperative analgesia with buprenorphine (0.05 mg/kg) was injected subcutaneously. Sham operated animals had holes drilled in the femur and tibia and screws inserted but none of them were plated. The animals were allowed unlim- ited activity and free access to water and food. The immobilized animals and the sham operated animals made up the experimental groups and the control groups, respectively. Thirty rats (1, 2, 4, 8 and 16 weeks) were prepared. Each group was composed of 6 experimental and 5 control animals. Increased elasticity of synovial membrane after immobilization 305 Tissue preparation. The rats were anesthetized and fixed with 4% paraformalde- hyde in 0.1M phosphate-buffer, pH7.4 by perfusion through the aorta. The knee joints were resected and kept in the same fixative overnight at 4C°. The fixed speci- mens were decalcified in 10% EDTA in 0.01M phosphate-buffer, pH7.4 for 1–2 months at 4C°. After dehydration through a graded series of ethanol solutions, the specimens were embedded in paraffin. The embedded tissue was cut into 5-μm thick sagittal sections from the medial to the lateral side of the joint. Standardized serial sections of the medial midcondylar region of the knee were made. The serial sections were prepared for hematoxylin-eosin stain to observe the histological appearance of SM after immobilization. Scanning acoustic microscopy. Our SAM consists of five parts: 1) ultrasonic trans- ducer, 2) pulse generator, 3) digital oscilloscope with PC, 4) microcomputer board and 5) display unit (Figure 1). A single pulsed ultrasound with 5 ns pulse width was emitted and received by the same transducer above the specimen. The aperture diameter of the transducer was 1.2 mm and the focal length was 1.5 mm. The cen- tral frequency was 80 MHz and the pulse repetition rate was 10 kHz. Considering the focal distance and the sectional area of the transducer, the diameter of the focal spot was estimated as 20 m at 80 MHz. Distilled water was used as the coupling medium between the transducer and the specimen. The reflections from the tissue surface and from interface between the tissue and the glass were received by the transducer and were introduced into a digital oscilloscope (Tektronics TDS 5052, Figure 1. Schematic illustration of a new concept scanning acoustic microscopy (SAM). A new con- cept SAM can make total time for calculation significantly shorter than a conventional SAM by using a single pulsed wave instead of continuous waves. 306 Yoshihiro Hagiwara et al. USA). The frequency range was 300 MHz and the sampling rate was 2.5 GS/s. Four pulse responses at the same point were averaged in the oscilloscope in order to reduce random noise. The transducer was mounted on an X–Y stage with a microcomputer board that was driven by the computer installed in the digital oscilloscope through an RS- 232C. The X-scan was driven by a linear servo-motor and the Y-scan was driven by a stepping motor. Finally, two-dimensional distributions of the ultrasonic intensity, sound speed and thickness of the 2.4 by 2.4 mm specimen area were visualized with 300 by 300 pixels. The total scanning time was 121 sec. Signal analysis. The reflected waveform comprises two reflections at the surface and the interface between the tissue and the glass. The thickness and sound speed were calculated by Fourier-transforming the waveform [19]. Image analysis. Normal light microscopic images corresponding to the stored acoustic images were captured (DMLB 100 HC light microscope, LEICA Wetzlar, Germany). A region of analysis by SAM was set in the anterior and posterior SM each in each section (Figure 2). In the region, the sound speed of SM, excluding meniscus, bone and cartilage, was calculated with a gray scale SAM images using commercially available image analysis software (PhotoShop 6.0, Adobe Systems Inc., San Jose, CA) (Figure 4). SAM images with a gradation color scale were also produced for clear visualization of the sound speed. The optical and acoustic im- ages were compared to ensure morphological congruence in the analysis. Transmission electron microscopy. The posterior SM of 8-week experimental and control groups were fixed with a mixture of 0.04% glutaraldehyde and 4.0% para- formaldehyde in 0.1M phosphate-buffered saline, pH 7.4, at 4C° into the intra- articular space for rapid fixation. The skin around the knee was excised and the posterior SM was immersed with the same fixative for 1h at 4C°. After washed thoroughly with Dulbecco’s PBS to remove the fixative, the tissue was cut with a razor blade into pieces and post-fixed with 2% buffered osmium tetroxide. The tis- sues were stained en bloc in aqueous uranyl acetate solution, dehydrated through a graded series of ethanol solutions and embedded in EPON 812 resin (TAAB Labo- ratory Equipment Ltd). Ultrathin resin sections of the specimens were mounted on copper, counterstained with uranyl acetate and Reynold’s lead citrate solution, then observed with a Hitachi H-9000 electron microscope [23]. Statistics. All data were expressed as the mean ± SD. The statistical significance of difference in the results was evaluated by unpaired analysis of variance, and P values were calculated by Tukey’s method. A P value less than 0.05 was considered statistically significant. Increased elasticity of synovial membrane after immobilization 307 Results SAM examination. The gradation color images of the posterior SM in the experi- mental group differed from those in the control group (Figure 3). The posterior SM was composed of low sound speed areas (black to blue) in 2-week immobiliza- tion (Figure 3A). The low sound speed area decreased and high sound speed areas (yellow to red) gradually increased in the posterior SM of the experimental group Figure 2. Microphotograph of a sagittal section in the medial midcondylar region of a rat knee. Squares indicate regions of analysis by scanning acoustic microscopy in the anterior( ) and posterior( ) synovial membrane. (Original magnification × 10, hematoxylin-eosin stain) 308 Yoshihiro Hagiwara et al. with time (Figure 3B). The posterior SM remained same in all the control groups (Figure 3C). The anterior SM was similar in all the experimental and control groups irrespec- tive of immobilization periods (Figure 3D). The sound speed of the posterior SM is shown in Figure 4. There was no statisti- cal difference between the experimental and the control groups in 1-, 2- or 4-week immobilization (1w: 1560 m/s 18.7 m/s vs. 1543 m/s 16.3 m/s; p = 0.152, 2w: 1552 m/s 26.0m/s vs. 1535 m/s 8.17 m/s; p = 0.207, 4w: 1551 m/s 4.01 m/s vs. 1553 m/s 13.3 m/s; p = 0.698). In 8- and 16-week immobilization, however, the sound speed in the experimental group was significantly higher than that in the control group (8w: 1546 m/s 18.7 m/s vs. 1646 m/s 11.8 m/s; p = 6.69 × 10–6, 16w: 1568 m/s 26.5 m/s vs. 1677 m/s 32.8 m/s; p = 1.06 × 10–4) (Figure 4A). There was no statistical difference in the anterior SM in all the experimental and the control groups at any period of immobilization (1w: 1563 m/s 22.7 m/s vs. 1556 m/s 13.8 m/s; p = 0.545, 2w: 1562 m/s 12.4 m/s vs. 1565 m/s 11.4 m/s; p = 0.74, 4w: 1559 m/s 10.1 m/s vs. 1554 m/s 30.4 m/s; p = 0.745, 8w: 1550m/ Figure 3. Gradation color images of scanning acoustic microscopy in the posterior and the anterior synovial membrane (SM). A, 2-week immobilization group (posterior). B, 16-week immobilization group (posterior). C, a representative of the control groups (posterior, 16-week). D, a representative of the control group (anterior, 16-week). E, gradation color table. Most low sound speed areas (black to blue) were replaced by high sound speed area (yellow to red) over time in the posterior experimental group. The posterior SM in the control groups remained entirely black to blue throughout the duration. The anterior SM was similar in the experimental and control group irrespective of the immobilization periods. Regions enclosed with a dotted line indicate the SM for calculation. Increased elasticity of synovial membrane after immobilization 309 s 20.5 m/s vs. 1564 m/s 15.3 m/s; p = 0.263, 16w: 1556 m/s 14.1 m/s vs. 1558 m/s 22.6 m/s; p = 0.846) (Figure 4B). TEM examination. In the experimental group, the space among collagen bundles and cells were occupied with the high density matrix, which fills the interspace of collagen microfibrils within the collagen bundle. In contrast, the high density ma- trix surrounding cells, collagen bundles and fibrils were scarce in the control group (Figure 5). Discussion The arthrogenic component has been considered as an important factor of joint con- tracture after immobilization (1,7,8,10,14–18). In a study using a rabbit knee con- tracture model, the mechanical characteristics were quantified by a torque-angular displacement diagram [18]. Knees in 9-week immobilization in flexion showed a significantly larger torque in extension in the experimental group than in the control Figure 4. Sound speed changes of the posterior and the anterior synovial membrane (SM). A, the posterior SM. B, the anterior SM. In the posterior SM, significant difference of sound speed is seen in 8- and 16-week immobilization. There was no statistical difference at any period of immobilization in the anterior SM. solid bars = experimental groups, shaded bars = control groups. Values are the mean ± SD. * = P < 0.005 versus control, by Tukey’s method. 310 Yoshihiro Hagiwara et al. group even after total extra-articular myotomies. In the same rat model as ours in the present study but immobilized up to 32 weeks, ROM in extension still remained restricted even after total extra-articular myotomies [7]. In canine glenohumeral joint immobilized up to 16 weeks, the intra-articular pressure rose higher by injec- tion of Hypaque contrast medium and the filling volume was smaller compared with the control group at a rupture of the capsule [24]. These studies suggest that among the arthogenic components, the capsule and SM may mostly contribute to production of joint contracture. Connective tissue proliferation in the SM and its adhesion to articular cartilage in the intra-articular space has been considered as pathological features of con- tracture after immobilization [25–29]. But conflicting studies with it have been reported. No intra-articular connective tissue proliferation occurred after immobi- lization [1,30–32]. No contact between the connective tissue and articular cartilage was observed [32]. In the same rat model as ours in the present study but immo- bilized up to 32 weeks, the decrease in the synovial intima length was observed after 4-week immobilization [1]. This study concluded that mutual adhesions of synovial villi rather than the connective tissue proliferation were the major patho- physiological changes leading to contracture. Further, the decrease of the synovial intima length was reported to be greater in the posterior SM than in the anterior SM in the same model as ours in the present study [1]. It may be explained as earlier mutual adhesion of synovial villi in the posterior SM under less tension with the knee immobilized in flexion. Connective tissue response after immobilization is important to understand the mechanism of the increased elasticity of the posterior SM. Some suggestions con- Figure 5. Structural characteristics of the posterior synovial membrane (SM). A, 8-week experimen- tal group. B, 8-week control group. Compared with the control group, the space among collagen bundles and cells were occupied with the high density matrix, which fills the interspace of collagen microfibrils within the collagen bundle in the experimental group. C, cell; MF, microfibrils. (Original magnification × 360,000, scale bar = 0.5 μm). Increased elasticity of synovial membrane after immobilization 311 cern changes in the biochemical composition of periarticular fibrous connective tis- sue (e.g. patellar tendon, ligament and joint capsule) after immobilization. The no- table change was a reduction of water and glycosaminoglycans without decreased collagen mass [9,18,32,33]. These changes were expected to alter plasticity and pliability of connective tissue matrices and to reduce lubrication efficiency [32]. In the same rat model as ours in the present study but immobilized up to 32 weeks, the posterior subintimal area of the experimental groups was smaller than that of the control groups through all the immobilization periods [1]. This result may reflect the decreased water and glycosaminoglycans of the SM. Our TEM observation showed that the space among collagen bundles and microfibrils was occupied with dense matrices in the experimental group, which may reflect the synovial atrophy due to decreased water content but not increased extracellular matrices. Further, adhesions of collagen bundles may limit lubrication and increase elasticity. Previous studies analyzed the elasticity of the joint as a whole including liga- ment, capsule and SM with or without muscles [7,15,18,24]. But it was impossible to evaluate the elasticity of the individual arthrogenic components, especially of capsule and SM in those studies. The present study is the first that measured the elasticity of SM in situ by SAM in rat immobilized knees and revealed the in- creased elasticity of the posterior SM, subsequent to the inhibition of extension to cause the joint contracture. One reason why the elasticity is different between the anterior and the posterior SM of 8- and 16-week experimental group may be that compared with the posterior SM immobilized rigidly, the anterior SM keeps motion to a larger extent after immobilization with patella while being active. The present study suggested that the increased elasticity and structural changes of the posterior SM are one of the main causes of limited extension after immobilized in flexion. 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Akeson WH, Woo SL, Amiel D, Coutts RD, Daniel D (1973) The connective tissue response to immobility: biochemical changes in periarticular connective tissue of the immobilized rabbit knee. Clin Orthop 93:356–362. 33. Akeson WH, Amiel D and La Violette D (1968) The connective tissue response to immobility: An accelerated aging response? Exp Gerotol 3:289–301. Corresponding author: Yoshihiro Hagiwara 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan e-mail: hagi@mail.tains.tohoku.ac.jp Tel: +81-22-717-7245 Fax: +81-22-717-7248