Yoshihiro Hagiwara

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.

Imaging features of foreign body granuloma in the lower extremities mimicking a soft tissue neoplasm

Foreign body granuloma is a tissue reaction for retained foreign bodies after skin-penetrating trauma. Detection of retained foreign bodies can be extremely difficult when the patients present with non-specific symptoms such as pain and/or swelling without recognizing a previous trauma. We report three patients of foreign body granulomas in the lower extremities with emphasis placed on their unique clinical and radiological features. The involved sites were the foot, posterior thigh, and posterior lower leg, with wooden splinters in two patients and a fragment of tile in one. Plain radiographs could not reveal the existence of foreign bodies. Magnetic resonance imaging (MRI) showed foreign bodies as low intensities on both T1- and T2-weighted images in two patients, and the surrounding reactive lesion as low to iso intensities on T1- and high intensities on T2-weighted images in all the patients. The peripheral areas of the lesion were strongly enhanced after gadolinium injection. Ultrasound sonography could clearly visualize a foreign body as an echogenic area with posterior acoustic shadowing in one patient. The surrounding ring-like reactive lesion is easily mistaken for a soft tissue neoplasm when foreign bodies are not identified. The key to arriving at the correct diagnosis is to clarify the previous trauma and to identify foreign bodies with low signal intensities on both T1- and T2-weighted images and/or the characteristic ring-like enhancement on MRI. It is also necessary to rule out a foreign body granuloma whenever we see patients with a soft tissue tumor in the extremities, irrespective of their previous trauma history.

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.

Eosinophilic granuloma arising from the pelvis in children: A report of three cases

Eosinophilic granuloma (EG) is a benign tumor-like condition which is characterized by a clonal proliferation of Langerhans-type histiocytes and defined as a local form of Langerhans cell histiocytosis (LCH). The radiographic appearances of EG are quite different depending on the phase of the disease and the site of involvement. A status of EG in the bone is divided into acute and chronic phases. Radiologically acute phase of EG is difficult to differentiate from a malignant bone tumor such as Ewings sarcoma or acute osteomyelitis. Chronic phase of EG may mimic a chronic osteomyelitis or a benign bone tumor. We report 3 childrens cases of EG in the pelvis which showed quite different radiological features and clinical courses. A 6-year-old boy (Case 1) had an osteolytic lesion with slightly defined margins in the right acetabulum. A 4-year-old boy (Case 2) had a radiologically similar-looking lesion in the left acetabulum. These lesions resembled radiologically chronic osteomyelitis (Brodies abscess) or a benign bone tumor and healed spontaneously after biopsy. A 2-year-old boy (Case 3) had an osteolytic lesion with ill-defined margin in the ilium. It was difficult to differentiate from a malignant tumor such as Ewings sarcoma, or acute osteomyelitis. The lesion became enlarged after needle biopsy. In spite of an additional curettage, the osteolytic lesion remained in the left pelvis in 1 year. Treatment for EG is controversial. Curettage of the affected site and bone grafting is usually accomplished. However, some EG heal spontaneously. It is of great importance to understand the natural course of EG and this knowledge will give us the opportunity to avoid unnecessary treatment. EG with poor osteolytic margins may progress further after biopsy. EG with well-defined margins may heal spontaneously after biopsy only.

Joint Immobilization Induced Hypoxic and Inflammatory Conditions in Rat Knee Joints

The purpose of this study was to examine the hypoxic and inflammatory conditions after immobilization in the joint capsule of rat knees. The unilateral knee joints of adult male rats were immobilized with an internal fixator (Im group) for 1 day, 3 days, and 1, 2, 4, 8, and 16 weeks. Sham-operated animals had holes drilled in the femur and tibia and screws inserted without a plate (control group). The number of cells and blood vessels in the capsule were histologically examined. The hypoxic condition in the capsule was histologically examined with a Hypoxyprobe™-1. The gene expressions related to the hypoxic (hypoxia inducible factor-1α, vascular endothelial growth factor, and fibroblast growth factor 2) and inflammatory conditions [interleukin-6 (IL-6), IL-1α, IL-1β, tumor necrosis factor-α, and tumor necrosis factor-β] were evaluated by quantitative reverse transcription polymerase chain reaction. The number of cells was unchanged at 1 day in the two groups; however, the number significantly increased at 3 days in the Im group. The number of blood vessels in the Im group gradually decreased. Strong immunostaining of Hypoxyprobe™-1 around the blood vessels was observed in the Im group. The gene expressions of hypoxia inducible factor-1α and fibroblast growth factor 2 were significantly higher in the Im group compared with those in the control group. The gene expressions of IL-6, IL-1α, IL-1β, and tumor necrosis factor-β were significantly higher in the Im group compared with those in the control group. These data indicated that joint immobilization induced hypoxic and inflammatory conditions in the joint capsule, which might be an initiating factor for joint contracture.

Chondrogenic and fibrotic process in the ligamentum flavum of patients with lumbar spinal canal stenosis.

Study Design. A histological, biological, and immunohisto-chemical study of human lumbar ligamentum flavum. Objective. To analyze changes in the hypertrophied ligamentum flavum and clarify their etiology. Summary of Background Data. Hypertrophy of the ligamentum flavum has been considered a major contributor to the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported some factors related to ligamentum flavum hypertrophy, its etiology is still unclear. Methods. Ligamentum flavum samples were collected from 20 patients with LSCS (LSCS group) and 10 patients with lumbar disc herniation (LDH group) as a control. The thickness of the ligamentum flavum was measured histologically. The amounts of elastic fibers and proteoglycans were assessed by Elastica-Masson staining and alcian blue staining, respectively. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative reverse transcription–polymerase chain reaction and immunohistochemistry. The total genes of the 2 groups were compared by DNA microarray analysis. Results. The ligamentum flavum was significantly thicker in the LSCS group, which had a smaller amount of elastic fibers and a larger amount of proteoglycans. The gene expression related to fibrosis was significantly higher in the LSCS group; however, the immunoreactivities of collagen types I and III were weaker on the dorsal side of the ligamentum flavum in the LSCS group. The gene expression related to chondrogenesis and proteoglycan synthesis was significantly higher in the LSCS group. There was no significant difference in the gene expression related to inflammation between the 2 groups. Conclusion. Synthesis of the collagenous fibers and degradation of the elastic and collagenous fibers are both accelerated in the ligamentum flavum of patient with LSCS, which may be the reason for hypertrophy of the tissue. In addition, chondrogenesis and proteoglycan synthesis may have critical roles in the pathogenesis of the ligamentum flavum hypertrophy. Level of Evidence: 5

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.