Y. Onoda

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.

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.

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.

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.

Immobilization induced prolonged inflammatory conditions in rat knee capsule

Purpose: Joint immobilization often induces joint stiffness, however, it is performed in daily examination in orthopaedics. Recent studies suggested that the joint capsule was one of the main sources of joint stiffness. In our previous reports, joint immobilization in rat knee joints induced increased stiffness of the capsule, and up-regulated gene and protein expressions in the capsule of fibrotic factors, such as TGF-b1 and CTGF. Though the precise etiology of joint stiffness has been still unknown, prolonged inflammation is one of the possible causes. In this study, we examined the histological changes of the capsule and expression of inflammatory cytokines by quantitative PCR and immunohistochemistry. Methods: Experimental Design: Unilateral knee joints were immobilized at 150 of flexionwith a plastic plate andmetal screws for1, 3 days,1, 2, 4, 8, 16 weeks. Only screws were inserted for sham-operated rats. The immobilized rats and sham-operated rats made up the immobilized group and the control group, respectively. One hundred and twelve ratswereprepared for histological analysis (n 1⁄4 8/each period). Thirty rats were prepared for quantitative PCR (1 day, 1 week, and 4 weeks; n 1⁄4 5/each period). Histology & Immunohistochemistry: The rats were fixed with 4% PFA in 0.1M PBS and the resected knee joints were decalcified in 10% EDTA in 0.01M PBS and embedded in paraffin. Five-mm sections were obtained at the medial mid-condylar region of the knee in the sagittal plane. The sections were stained with Elastica-Masson and the number of cells per unit in the capsule was counted. The sections were stained with rabbit anti-rat IL-6 antibody (Abcam, ab6672) and rabbit anti-rat IL-1b (Ab Frontier, LF-PA50204), and the expression patterns in the capsule were examined. Quantitative PCR: The anterior and posterior capsule were harvested and immediately placed in 1 ml QIAzol (Qiagen). The tissue was homogenized, the total RNAwas purified (RNeasy Lipid Tissue Kit, Qiagen), and the cDNA was synthesized (cDNA Synthesis Kit, Invitrogen). The relative expression levels of IL-6, IL-1a, IL-1b, TNF-a, and TNF-b as a function of EF1a1 was calculated. Results: Histology: The number of cells in the control groups was almost unchanged throughout the experimental periods. The number of cells at 1 daywasnot changed, however, thenumberat 3 dayswas significantly higher in the immobilized group. The number of cells in the immobilized groups peaked at 3 days and then gradually decreased after 1 week. Immunocytochemistry: The staining intensity of IL-6 in the capsule was weak at 1 day, however, the intensitywas stronger at 3 days in the two groups. The staining intensity in the control groups peaked at 3 days, and decreased after 1week. The strong staining intensity in the immobilized groups was kept until 4 weeks, and then became weaker at 8 weeks. The staining intensity of IL-1b was similar fashion to IL-6. Quantitative PCR: Increased expression of IL-6 was observed at 1 day in the two groups. The expression levels in the control groups were normalized after 1 week, whereas, the expression levels in the immobilized groups were kept higher level at 1 week. Conclusions: The prolonged inflammatory conditions were kept in the joint capsule after immobilization. Increased number of cells in the immobilized group may support the results. It was indicated that prolonged inflammation and subsequent fibrosis of the joint capsule was a main pathology of the joint contracture after immobilization.

135 HYPOXIC CONDITIONS IN THE CAPSULE AFTER JOINT IMMOBILIZATION

Suda, H; +Hagiwara, Y; Ando, A; Onoda, Y; Chimoto, E; Hatori, K; Itoi, E Tohoku University, Sendai, Japan, Takeda General Hospital, Aizuwakamatsu, Japan, Funabashi Orthopaedic Clinic, Funabashi, Japan hagi@mail.tains.tohoku.ac.jp Introduction: Joint immobilization is a useful and commonly performed treatment modality in orthopaedics. However, it also causes unfavorable outcomes such as joint contracture, periarticular osteoporosis, and cartilage degeneration. Once joint contracture is established, it is extremely difficult to regain a full range of motion (ROM) with vigorous and extensive rehabilitation, or even with surgical treatment. In our previous reports, ROM increased after the posterior capsular release in a rat knee flexion contracture model, which indicated the capsule was one of the main causes of joint contracture (1). Further, angiogenesis factors of transforming growth factor-β1 and connective tissue growth factor were increased in the capsule after prolonged immobilization (2). This result might indicate presence of hypoxia in the capsule after immobilization. The purpose of this study was to elucidate changes in the number of blood vessels in the capsule and presence of hypoxia by hypoxyprobe-1 (HP-1) stain in the rat knee contracture model.