Eiichi Chimoto

Changes of articular cartilage after immobilization in a rat knee contracture model

The objective was to determine the changes of articular cartilage of the knee joint during immobilization in a rat model. The knee joints of adult male rats were immobilized at 150° of flexion using an internal fixator for 3 days, and 1, 2, 4, 8, and 16 weeks. The articular cartilage from the medial midcondylar region of the knee was obtained, divided into three areas (non‐contact area, transitional area, contact area), and in each area, a degree of degeneration was evaluated by gross observation, histomorphometric grading, and measurements of thickness and number of chondrocytes. Elasticity of the articular cartilage was estimated by measuring the sound speed with use of scanning acoustic microscopy. Degeneration of the articular cartilage was mainly observed in the contact and transitional areas. Matrix staining intensity by safranin‐O and number of chondrocytes were decreased in these two areas. The thickness of the articular cartilage in the non‐contact and contact areas was unchanged, but it was increased in the transitional area. Decrease in sound speed was observed in the transitional area of both the femoral and tibial cartilage, indicating the softening of the articular cartilage. The changes of articular cartilage became obvious as early as 1 week after immobilization. These changes may be due to a lack of mechanical stress or a lack of joint fluid circulation during immobilization. Although we do not know the reversibility of these changes of articular cartilage, early mobilization is preferable to avoid these cartilage changes.

Increased Elasticity of Capsule After Immobilization in a Rat Knee Experimental Model Assessed by Scanning Acoustic Microscopy

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 applied 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 experimental 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.

Coexistence of fibrotic and chondrogenic process in the capsule of idiopathic frozen shoulders

OBJECTIVE To analyze changes in the capsule from idiopathic frozen shoulders and clarify their etiology. MATERIALS AND METHODS Samples (the rotator interval capsule, middle glenohumeral ligament (MGHL), and inferior glenohumeral ligament (IGHL)) were collected from 12 idiopathic frozen shoulders with severe stiffness and 18 shoulders with rotator cuff tears as a control. The number of cells was counted and the tissue elasticity of the samples was calculated by scanning acoustic microscopy (SAM). The amount of glycosaminoglycan content was assessed by alcian blue staining. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Furthermore, the total genes of the two groups were compared by DNA microarray analysis. RESULTS The number of cells was significantly higher and the capsular tissue was significantly stiffer in idiopathic frozen shoulders compared with shoulders with rotator cuff tears. Staining intensity of alcian blue was significantly stronger in idiopathic frozen shoulders. Gene expressions related to fibrosis, inflammation, and chondrogenesis were significantly higher in idiopathic frozen shoulders compared with shoulders with rotator cuff tears assessed by both qPCR and DNA microarray analysis. CONCLUSION In addition to fibrosis and inflammation, which used to be considered the main pathology of frozen shoulders, chondrogenesis is likely to have a critical role in pathogenesis of idiopathic frozen shoulders.

Progression of an arthrogenic motion restriction after immobilization in a rat experimental knee model

Background. Contracture is defined as a decrease in both active and passive ranges of motion after immobilization. A fibrotic change of a capsule is suggested to be one of the main causes of the joint contracture. The goal of this study is to determine the effect of capsule on limiting the range of motion after immobilization. Materials and Methods. We immobilized the knee joint of 35 rats with an internal fixator with the knee joint flexed at 150 degrees. The rats were sacrificed at 1, 2, 4, 6, 8, 12, and 16 weeks after surgery and the lower extremities were disarticulated at the hip joint. After extra-articular myotomies around the tibia and femur, x-rays were taken to measure the angles of extension of the knee joint under 3 different torques. The measurements were repeated after releasing the posterior capsule in order to observe their effects on knee motion. Results. Joint contracture was rapidly progressed until 8 weeks and advanced slowly after 8 weeks. After releasing the posterior capsule, both the immobilized and the control groups gained the angle of knee extension. The acquired angle in the immobilized group was significantly greater than in the control group after 4 weeks and became plateau after 8 weeks. Conclusion. Joint contracture develops at the early stage of immobilization and progresses over time. The posterior capsule significantly contributes to the limitation in extension.

Expression of transforming growth factor-β1 and connective tissue growth factor in the capsule in a rat immobilized knee model

Background: Contracture is a very common complication of joint immobilization in daily examination, but its cause is still unknown. A fibrotic change of the capsule is suggested to be one of the main causes of the joint contracture. The goal of this study was to analyze the expression pattern of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), which are implicated in fibrosis in the capsule of a rat immobilized knee model. Materials and Methods: We immobilized the unilateral knee joints of 66 rats in 150 degrees of flexion using a plastic plate and metal screws. Sham operated knee joints of 66 rats had holes drilled and screws inserted but none of them were plated. The capsule from the anterior and posterior portion of the knee joints was harvested at 3 days, 1, 2, 4, 8 and 16 weeks after immobilization and the expression patterns of TGF-β1 and CTGF were characterized using in situ hybridization and immunohistochemistry. Results: The in situ hybridization demonstrated that the mRNAs of both TGF-β1 and CTGF increased continuously during the first 2 weeks after immobilization and then decreased. The response was relatively higher in the posterior capsule than in the anterior one. In contrast, the immunoreactivity of both TGF-β1 and CTGF increased gradually with time. The response was much stronger in the posterior capsule than in the anterior one. Conclusions: The capsule has a potency to produce TGF-β1 and CTGF after immobilization. CTGF may play a role in causing and maintaining capsular fibrosis in collaboration with TGF-β1. The fibrotic change in the posterior capsule may have resulted in limited motion in extension in this immobilized knee model in rats. It may be possible to prevent joint contractures by somehow blocking the fibrotic process.

Expression patterns of collagen types I and III in the capsule of a rat knee contracture model

Our objective was to determine the changes in expression of collagen types I and III in the capsule of a rat knee contracture model. The unilateral knee joints of adult male rats were rigidly immobilized at 150° of flexion using a rigid plastic plate and screws for 3 days, 1, 2, 4, 8, and 16 weeks (immobilized group). Sham‐operated animals had holes drilled in the femur and tibia with screws inserted without a plate (control group). The expression patterns of collagen types I and III in the anterior and posterior capsule were evaluated by in situ hybridization (ISH), quantitative real‐time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Western blotting (WB). Expressions of collagen types I and III were decreased after immobilization compared to the control group by ISH and qPCR. The expression was not changed after immobilization compared to the control group by IHC and WB. The expression of mRNA and protein levels of collagen types I and III were not increased after immobilization, which indicated that accumulation of the two types of collagen was not the etiology of joint contracture. Another process, such as capsule and synovial adhesions, may be one possible cause of joint contracture.

Expression of collagen types I and II on articular cartilage in a rat knee contracture model.

The purpose of our study was to clarify the expression patterns of collagen types I and II on articular cartilage after immobilization in a rat knee contracture model in 3 specific areas (noncontact area, transitional area, contact area). The unilateral knee joints of adult male rats were rigidly immobilized at 150° of flexion using screws and a rigid plastic plate. Sham-operated animals had holes drilled in the femur and the tibia and screws inserted but were not plated. The expression patterns of collagen types I and II in each area were evaluated by in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative real-time polymerase chain reaction (qPCR). The expression of collagen type II in the noncontact area was decreased by ISH but appeared unchanged when examined by IHC. In the transitional and contact areas, the expression of collagen type II was initially shown to have decreased and then increased at the hypertrophic chondrocytes by ISH but appeared decreased by IHC. Quantitative PCR revealed the decreased expression of type II collagen in the contact area. Immunostaining of collagen type I was increased at the noncontact area and transitional areas. Alterations of collagen types I and II expression may also affect the degeneration of articular cartilage after immobilization and the changes were different in the three areas.

Expression of type I collagen in the capsule of a contracture knee in a rat model

Contracture is a very common complication in daily examination and a fibrotic change of a capsule is suggested to be a one of the main causes. Type I collagen is a major component of a synovial capsule and also has been implicated in tissue elasticity of other organs. We immobilized the knee joints of 66 rats in 150 degrees of flexion using a plastic plate and metal screws. Sham operated knee joints had holes drilled and screws inserted but none of them were plated. The expression patterns of type I collagen were characterized using in situ hybridization and immunohistochemistry. The in situ hybridization demonstrated that the mRNA of type I collagen decreased rapidly after immobilization. However, the immunoreactivity of the capsule was not changed in the immobilized and the control groups at any time points. Other processes might be considered to evaluate the contracture capsule.

Joint haemorrhage partly accelerated immobilization-induced synovial adhesions and capsular shortening in rats

AbstractPurpose To elucidate the effects of intra-articular haemorrhage on the joint capsule of immobilized knees in rats.Methods The unilateral knee joints were immobilized using a plastic plate and screws. Sham operated rats had only screws inserted. A single injection of fresh autologous blood was given postoperatively into the knee joints of the immobilized blood injection (Im-B) and the Sham blood injection (Sm-B) groups. Normal saline was administered for the immobilized-saline injection (Im-S) group. Sagittal sections were prepared from the medial midcondylar region of the knee and assessed with histological, histomorphometric, and immunohistochemical methods. The range of motion (ROM) was measured, and the mechanical property of the capsule was assessed by scanning acoustic microscope.ResultsAbsorption of the injected blood was delayed and made severe adhesions in the Im-B group. The length of the synovial membrane in the Im-B group was significantly shorter than that of the other groups. The ROM was significantly restricted in the Im-B group compared with the other groups. The elasticity of the posterior capsule in the Im-B group was significantly lower than that in the Sm-B group. Iron deposition was observed in the Im-B and Sm-B groups. Strong immunoreactivities of CD68, TGF-β1, and α-SMA were observed in the adhesion area of the Im-B group. Joint immobilization with blood injection caused severe capsular adhesion and limited range of motion. Immunostaining related to fibrosis increased with joint haemorrhage.ConclusionIntra-articular haemorrhage with joint immobilization might be an accelerated risk factor for joint contracture. It is likely that leaving a haematoma inside an immobilized joint should be avoided.

Comparison of articular cartilage images assessed by high-frequency ultrasound microscope and scanning acoustic microscope

PurposeThe purpose of this study was to compare images of a newly developed high-frequency ultrasound imaging system (HFUIS) and scanning acoustic microscope (SAM) and to calculate their Pearson product moment correlations with a view to applying HFUIS for clinical use.MethodsCylindrical cartilage–bone complexes from adult male Sprague-Dawley rats were obtained. The specimens were immersed in normal saline and scanned by HFUIS. Intensity by HFUIS was normalised by reflection from a steel plate at the same distance. After the scanning, specimens were fixed with paraformaldehyde, decalcified and embedded in paraffin. Thinly sliced tissues were prepared for SAM evaluation. After the scanning, three layers of articular cartilage (superficial, middle and deep) were independently evaluated and their relationships calculated.ResultsThe superficial and deep layers indicated high relative intensity, whereas the middle layer showed nonhomogeneous relative intensity by HFUIS. A high relative intensity by HFUIS and high sound speed area by SAM had strong correlations (Pearson product moment correlation, superficial layer 0.704, middle layer 0.731).ConclusionsHFUIS produced high-resolution images of the articular cartilage and its intensity was strongly correlated with sound speed by SAM.