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Dynamic analysis of inflammatory cells in synovial fluid after index anterior cruciate ligament reconstruction surgery
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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
557 DYNAMIC ANALYSIS OF INFLAMMATORY CELLS IN SYNOVIAL FLUID AFTER INDEX ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION SURGERY K. Nakamura y, K. Tsui z, H. Katagiri y, M. Inoue y, M. Ojima y, I. Sekiya x, T. Muneta y. y Dept. of Joint Surgery and Sports Med., Tokyo Med. and Dental Univ., Tokyo, Japan; z Dept. of Cartilage Regeneration, Tokyo Med. and Dental Univ., Tokyo, Japan; x Ctr. for Stem Cell and Regenerative Med., Tokyo Med. and Dental Univ., Tokyo, Japan Purpose: Synovial fluid is an interstitial fluid secreted by fibroblastic cells in the synovial membrane. The physiological functions of synovial fluid may include reduction of friction, shock absorption, nutrient, and waste transportation in the joint. In addition, previous studies showed various types of cells reside in synovial fluid. Those are inflammatory cells such as macrophages and T cells, and mesenchymal stem cells (MSCs), those are considered to contribute the tissue homeostasis and regeneration. We have reported that pathological conditions such as joint injury or joint inflammation increased both cellular components and inflammatory cytokine levels in synovial fluid. We predict that both of them may play important roles during the recovery process after joint injury or surgery. In this study, we aimed to analyze the dynamic changes of cellular components in synovial fluid after anterior cruciate ligament reconstruction surgery (ACL-R) and compared them with the biochemical parameters such as inflammatory cytokine levels in synovial fluid from each patient. Here we report that the population of CD66b-positive Granulocytes surged in the early stage after ACL-R and positively correlated with IL1 beta and IL8 levels in synovial fluid. Methods: This study was approved by the Ethics Committee of this institute. All patients included in this study gave their full, written, informed consent for participation prior to the operative procedure. Synovial fluid was obtained from patients who underwent ACL-R from February 2013 till March 2014 in our University’s Hospital (just before surgery, day 3e4, day 19e21, and day 35 post surgery: n¼58). Cellular components in synovial fluid were analyzed by flow cytometry (BD FACS Verse). In this study, we quantitated the population of T-cells (CD3, CD4, CD8), Granulocytes (CD66b), Macrophages (CD11b, CD14), and MSCs (CD73, CD90, CD105, CD44) in each time point. Inflammatory cytokine levels in synovial fluid (IL1 beta, IL2, IL6, IL8, IL10, TNF alpha, and IFN gamma) were quantitated by ELISA. Kruskal-Wallis test and Peasons correlation analysis were employed for statistical analysis and probability values less than 0.05 were considered as significant. Results: Flow cytometric analyses detected T cells, Granulocytes, Macrophages, and MSCs in synovial fluid. Dynamic analysis indicated that T cell and Macrophage populations in synovial fluid surged at day 19e21 post surgery (1.3-fold and 7.8-fold respectively if compared with that before surgery). Population of MSC gradually increased with time post surgery (1.2-fold at day 35; compared to the population before surgery). Granulocyte population significantly increased at day 3e4 post surgery. We observed the highest individual variation in Granulocyte population between the patients (minimum 0.1% and maximum 96.5% at day 3e4). To examine if the individual variation of Granulocyte population correlates clinical conditions of each patient, we compared it with cytokine levels in synovial fluid at day 3e4 post ACL-R. Among the cytokines tested, we found that Granulocyte population in synovial fluid significantly correlated with the levels of an inflammatory cytokine, IL1 beta (r¼0.55, p<0.01) and chemokine, IL8 (r¼0.52, p<0.01). Conclusions: Dynamic analyses of cellar components in synovial fluid depicted the time sequence of inflammatory cells migrated post ACL-R. And severity of joint inflammation post ACL-R was significantly correlated with the Granulocyte population in synovial fluid. 558 SITE SPECIFIC INFLUENCE OF WEIGHT OR NON-WEIGHT BEARING CONDITION ON THE STIFFNESS OF THE CARTILAGE DURING SHORTTERM IMMOBILIZATION INTERVENTION ON RAT KNEE JOINT M. Nagai y, H. Iijima z, A. Ito x, J. Tajino z, S. Yamaguchi z, X. Zhang z, W. Kiyan z, T. Aoyama z, H. Kuroki z. y Congenital Anomaly Res. Cent., Graduate Sch. of Med., Kyoto Univ., Kyoto, Japan; z Physical Therapy, Human Hlth. Sci.s, Graduate Sch. of Med., Kyoto Univ., Kyoto, Japan; x Orthop Surg, Graduate Sch. of Med., Kyoto Univ., Kyoto, Japan Purpose: Joint immobilization has various influences on joint structure. Previous reports using rat immobilization model which maintained
weight-bearing condition indicated that joint immobilization causes various degeneration of articular cartilage such as thinning, softening and reducing proteoglycan content. These degeneration were different according to the assessment regions because of the different patterns of mechanical stress. Mechanical loading affects synthesis of cartilage extracellular matrix and is important for maintaining of cartilage structure. We reported that degenerated cartilage after immobilization was aggravated by re-mobilization only at around the contact region (Nagai et al., 2014 World Congress on Osteoarthritis). According to the reports, it was thought that focal degradation concerned the characteristic of cartilage region due to weight-bearing condition. However, it is still unknown how does the load condition influence and how cartilage stiffness is different on assessment regions during immobilization period. The purpose of this study was to examine the alteration of cartilage stiffness at each regions respectively, when the knee joint was immobilized with weight or non-weight bearing condition. Understanding of the differences in the cartilage stiffness is necessary to develop strategies to prevent and treat joint disease and cartilage degeneration after immobilization. Methods: The knee joint of Wistar rats aged 12-week old was immobilized at 140 ± 5 degrees of knee flexion with K-wire and resin for 1-, 2, 4-weeks. The experimental rats were separated two groups; weightbearing group (WG: n¼4 legs/ period) and non-weight-bearing group (NWG: n¼4 legs/ period). The rats in the WG were maintained in normal gages. The rats in the NWG were applied hind limb suspension intervention. Control group did not apply intervention and maintained in normal gage for same period as that of the experimental rats (n¼4 legs/ period). The rats were sacrificed at the end of experimental period. The knees were extracted and observed macroscopically. Thereafter, the extracted knee were stored in PBS and trimmed for micro indentation test. The two regions (contact region and transitional region) of cartilage at the medial mid-tibia plateau segment were analyzed. A cartilage elasticity (N/mm2) and displacement (um) were obtained from the micro-indentation test. Results: In macroscopic observation, there was no apparent degeneration of the cartilage in the control group throughout experimental periods. However, in the both of the WG and the NWG, the surface irregularity was observed. The surface irregularity of whole region in the NWG was more severe than that of the WG. Besides, about the color of cartilage, a pale red got reduced slightly in the NWG. The surface of the contact region was concave and the transitional regions clearly encircled at the contact region in the WG compared with that of the NWG. The results of micro indentation test, increased cartilage elasticity and decreased displacement at contact region were confirmed in WG over time (P < 0.05). On the other hand, decreased cartilage elasticity and increased displacement at transitional region were confirmed in the WG and the NWG compare with control group. However, at transitional region, there were not significant different between WG and NWG. Conclusions: Current results showed that the cartilage stiffness increased at contact region with weight bearing condition throughout the experimental period in rat immobilization model. However, the cartilage stiffness decreased at transitional region regardless of weightbearing condition. It means that the cartilage stiffness was different due to weight-bearing condition and assessment region after short-term immobilization period. It might suggest that the intervention such as joint remobilization after immobilization would cause of different alteration on cartilage due to weight-bearing condition or the difference of cartilage region. 559 A SYNOVIOCYTES AND SYNOVIAL FLUID INTERACTION MODEL FOR OSTEOARTHRITIS STUDY: MODULATION OF ANABOLIC AND CATABOLIC MARKERS S. Koppikar y, P. Kulkarni y, S. Deshpande z, D. Shinde x, D. Ingale y, A. Harsulkar y. y Interactive Res. Sch. for Hlth.Affairs (IRSHA), Pune, India; z Dept. of Orthopedics, Bharati Hosp., Pune, India; x Dept. of Kayachikisa, Bharati Ayurvedic Hosp., Pune, India Purpose: Osteoarthritis (OA) is the most common degenerative disease characterized by cartilage loss. A pre-requisite to maintain cartilage integrity is the balance of anabolic and catabolic processes, which are largely compromised in OA to the extent that tissue degradation predominates over the capacity of tissue repair. Where most of research targets cartilage to study anabolism and catabolism, studies on synovial
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
557 DYNAMIC ANALYSIS OF INFLAMMATORY CELLS IN SYNOVIAL FLUID AFTER INDEX ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION SURGERY K. Nakamura y, K. Tsui z, H. Katagiri y, M. Inoue y, M. Ojima y, I. Sekiya x, T. Muneta y. y Dept. of Joint Surgery and Sports Med., Tokyo Med. and Dental Univ., Tokyo, Japan; z Dept. of Cartilage Regeneration, Tokyo Med. and Dental Univ., Tokyo, Japan; x Ctr. for Stem Cell and Regenerative Med., Tokyo Med. and Dental Univ., Tokyo, Japan Purpose: Synovial fluid is an interstitial fluid secreted by fibroblastic cells in the synovial membrane. The physiological functions of synovial fluid may include reduction of friction, shock absorption, nutrient, and waste transportation in the joint. In addition, previous studies showed various types of cells reside in synovial fluid. Those are inflammatory cells such as macrophages and T cells, and mesenchymal stem cells (MSCs), those are considered to contribute the tissue homeostasis and regeneration. We have reported that pathological conditions such as joint injury or joint inflammation increased both cellular components and inflammatory cytokine levels in synovial fluid. We predict that both of them may play important roles during the recovery process after joint injury or surgery. In this study, we aimed to analyze the dynamic changes of cellular components in synovial fluid after anterior cruciate ligament reconstruction surgery (ACL-R) and compared them with the biochemical parameters such as inflammatory cytokine levels in synovial fluid from each patient. Here we report that the population of CD66b-positive Granulocytes surged in the early stage after ACL-R and positively correlated with IL1 beta and IL8 levels in synovial fluid. Methods: This study was approved by the Ethics Committee of this institute. All patients included in this study gave their full, written, informed consent for participation prior to the operative procedure. Synovial fluid was obtained from patients who underwent ACL-R from February 2013 till March 2014 in our University’s Hospital (just before surgery, day 3e4, day 19e21, and day 35 post surgery: n¼58). Cellular components in synovial fluid were analyzed by flow cytometry (BD FACS Verse). In this study, we quantitated the population of T-cells (CD3, CD4, CD8), Granulocytes (CD66b), Macrophages (CD11b, CD14), and MSCs (CD73, CD90, CD105, CD44) in each time point. Inflammatory cytokine levels in synovial fluid (IL1 beta, IL2, IL6, IL8, IL10, TNF alpha, and IFN gamma) were quantitated by ELISA. Kruskal-Wallis test and Peasons correlation analysis were employed for statistical analysis and probability values less than 0.05 were considered as significant. Results: Flow cytometric analyses detected T cells, Granulocytes, Macrophages, and MSCs in synovial fluid. Dynamic analysis indicated that T cell and Macrophage populations in synovial fluid surged at day 19e21 post surgery (1.3-fold and 7.8-fold respectively if compared with that before surgery). Population of MSC gradually increased with time post surgery (1.2-fold at day 35; compared to the population before surgery). Granulocyte population significantly increased at day 3e4 post surgery. We observed the highest individual variation in Granulocyte population between the patients (minimum 0.1% and maximum 96.5% at day 3e4). To examine if the individual variation of Granulocyte population correlates clinical conditions of each patient, we compared it with cytokine levels in synovial fluid at day 3e4 post ACL-R. Among the cytokines tested, we found that Granulocyte population in synovial fluid significantly correlated with the levels of an inflammatory cytokine, IL1 beta (r¼0.55, p<0.01) and chemokine, IL8 (r¼0.52, p<0.01). Conclusions: Dynamic analyses of cellar components in synovial fluid depicted the time sequence of inflammatory cells migrated post ACL-R. And severity of joint inflammation post ACL-R was significantly correlated with the Granulocyte population in synovial fluid. 558 SITE SPECIFIC INFLUENCE OF WEIGHT OR NON-WEIGHT BEARING CONDITION ON THE STIFFNESS OF THE CARTILAGE DURING SHORTTERM IMMOBILIZATION INTERVENTION ON RAT KNEE JOINT M. Nagai y, H. Iijima z, A. Ito x, J. Tajino z, S. Yamaguchi z, X. Zhang z, W. Kiyan z, T. Aoyama z, H. Kuroki z. y Congenital Anomaly Res. Cent., Graduate Sch. of Med., Kyoto Univ., Kyoto, Japan; z Physical Therapy, Human Hlth. Sci.s, Graduate Sch. of Med., Kyoto Univ., Kyoto, Japan; x Orthop Surg, Graduate Sch. of Med., Kyoto Univ., Kyoto, Japan Purpose: Joint immobilization has various influences on joint structure. Previous reports using rat immobilization model which maintained
weight-bearing condition indicated that joint immobilization causes various degeneration of articular cartilage such as thinning, softening and reducing proteoglycan content. These degeneration were different according to the assessment regions because of the different patterns of mechanical stress. Mechanical loading affects synthesis of cartilage extracellular matrix and is important for maintaining of cartilage structure. We reported that degenerated cartilage after immobilization was aggravated by re-mobilization only at around the contact region (Nagai et al., 2014 World Congress on Osteoarthritis). According to the reports, it was thought that focal degradation concerned the characteristic of cartilage region due to weight-bearing condition. However, it is still unknown how does the load condition influence and how cartilage stiffness is different on assessment regions during immobilization period. The purpose of this study was to examine the alteration of cartilage stiffness at each regions respectively, when the knee joint was immobilized with weight or non-weight bearing condition. Understanding of the differences in the cartilage stiffness is necessary to develop strategies to prevent and treat joint disease and cartilage degeneration after immobilization. Methods: The knee joint of Wistar rats aged 12-week old was immobilized at 140 ± 5 degrees of knee flexion with K-wire and resin for 1-, 2, 4-weeks. The experimental rats were separated two groups; weightbearing group (WG: n¼4 legs/ period) and non-weight-bearing group (NWG: n¼4 legs/ period). The rats in the WG were maintained in normal gages. The rats in the NWG were applied hind limb suspension intervention. Control group did not apply intervention and maintained in normal gage for same period as that of the experimental rats (n¼4 legs/ period). The rats were sacrificed at the end of experimental period. The knees were extracted and observed macroscopically. Thereafter, the extracted knee were stored in PBS and trimmed for micro indentation test. The two regions (contact region and transitional region) of cartilage at the medial mid-tibia plateau segment were analyzed. A cartilage elasticity (N/mm2) and displacement (um) were obtained from the micro-indentation test. Results: In macroscopic observation, there was no apparent degeneration of the cartilage in the control group throughout experimental periods. However, in the both of the WG and the NWG, the surface irregularity was observed. The surface irregularity of whole region in the NWG was more severe than that of the WG. Besides, about the color of cartilage, a pale red got reduced slightly in the NWG. The surface of the contact region was concave and the transitional regions clearly encircled at the contact region in the WG compared with that of the NWG. The results of micro indentation test, increased cartilage elasticity and decreased displacement at contact region were confirmed in WG over time (P < 0.05). On the other hand, decreased cartilage elasticity and increased displacement at transitional region were confirmed in the WG and the NWG compare with control group. However, at transitional region, there were not significant different between WG and NWG. Conclusions: Current results showed that the cartilage stiffness increased at contact region with weight bearing condition throughout the experimental period in rat immobilization model. However, the cartilage stiffness decreased at transitional region regardless of weightbearing condition. It means that the cartilage stiffness was different due to weight-bearing condition and assessment region after short-term immobilization period. It might suggest that the intervention such as joint remobilization after immobilization would cause of different alteration on cartilage due to weight-bearing condition or the difference of cartilage region. 559 A SYNOVIOCYTES AND SYNOVIAL FLUID INTERACTION MODEL FOR OSTEOARTHRITIS STUDY: MODULATION OF ANABOLIC AND CATABOLIC MARKERS S. Koppikar y, P. Kulkarni y, S. Deshpande z, D. Shinde x, D. Ingale y, A. Harsulkar y. y Interactive Res. Sch. for Hlth.Affairs (IRSHA), Pune, India; z Dept. of Orthopedics, Bharati Hosp., Pune, India; x Dept. of Kayachikisa, Bharati Ayurvedic Hosp., Pune, India Purpose: Osteoarthritis (OA) is the most common degenerative disease characterized by cartilage loss. A pre-requisite to maintain cartilage integrity is the balance of anabolic and catabolic processes, which are largely compromised in OA to the extent that tissue degradation predominates over the capacity of tissue repair. Where most of research targets cartilage to study anabolism and catabolism, studies on synovial