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Invossa™ (TISSUEGENE-C) induces an anti-inflammatory environment in the arthritic knee joints via macrophage polarization
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
The 35Sulphate incorporation rate was normalized to the specific activity of the pulse medium, labeling time, and weight of cartilage. In vivo: in seven F8 KO rats a unilateral joint bleed was induced by needle puncture and in the following four days until euthanasia, the animals received analgesia. At UMC Utrecht, the tibial cartilage was removed and proteoglycan synthesis activity determined according to the previously described method. All animal experiments were performed under anesthesia and analgesia in accordance with and approved by the Danish Animal Experiments Council, Ministry of Food, Agriculture and Fisheries, Denmark. Results: On average, a total of 1.6mg (0.8e3.1mg) cartilage per tibia could be obtained. The proteoglycan synthesis rate of healthy cartilage determined after four days of culturing in vitro was on average 49.5 nmol/h.g. This could be modulated by in vitro exposure to blood, resulting in a diminished synthesis: 7.7 nmol/h.g (p ¼ 0.0191), corresponding to an 84% decrease comparable to previously published human experiments. Moreover, when a joint bleed was induced in vivo a diminished proteoglycan synthesis rate could also be demonstrated by this assay (13.5 vs 7.5 nmol/h.g, p ¼ 0.0151), although more variation was seen.
S157
Conclusions: These findings suggest that HMGB2 may play important roles in chondrocytes following exposure to mechanical loading, which may involve in the pathogenesis of osteoarthritis.
Cartilage Repair 237 INVOSSA™ (TISSUEGENE-C) INDUCES AN ANTI-INFLAMMATORY ENVIRONMENT IN THE ARTHRITIC KNEE JOINTS VIA MACROPHAGE POLARIZATION K. Choi, H. Lee, D. Kim, H. Lee, M. Kim, C.-L. Lim, Y.-J. Lee, B. Lee, S. Kim, M. Lee, H. Choi. Kolon Life Sci., Seoul, Republic of Korea
Conclusions: This study demonstrates for the first time that proteoglycan synthesis activity, by use of the 35Sulphate incorporation assay, can be determined in tibial cartilage of the rat. Moreover, this synthetic activity is affected by blood exposure in vitro and in vivo. As such, this assay can be a valuable tool to detect cartilage changes, both in pathophysiological and interventional experiments, using joint degenerative rat models. 236 SPATIAL AND TEMPORAL EXPRESSION PATTERN OF HMGB2 IN RABBIT CONDYLAR CARTILAGE FOLLOWING ANTERIOR DISC DISPLACEMENT M. Wu, Y. Zhou. Hosp. of Stomatology, Zhejiang Univ., Hangzhou, China Purpose: The chromatin protein HMGB2 plays important roles in development, remodeling and disruption of the articular cartilage. The objective of present study was to investigate whether the expression of HMGB2 in the condylar cartilage was correlated to the continuing forward mandibular positioning (FMP), and explore its potential relationship with b-catenin pathway. Methods: Fourty-four healthy adult Japanese white rabbits were used in this experiment. The right tempromandibular joints (TMJ) of rabbits were subjected to surgical and sham surgical operation of anterior disc displacement (ADD) (N ¼ 8). The TMJ condyles of rabbits were collected and fixed at 1-, 2-, 4-, and 8- weeks after the surgery. Histological micrographs of the condyle cartilage were mearsured by HE staining. The distribution and expression of HMGB2, Col-II, MMP1, MMP13 and b-catenin were evaluated through immunohistochemical staining. Results: Histological results by HE staining showed that the condyle cartilage turned thinner as well as disordered cell arrangement in cartilage with ADD. The expression of HMGB2 chiefly distributed in fibrous layer and proliferative layer of normal condylar cartilage. After ADD treatment, expression of HMGB2 and b-catenin enhanced at the 1 week ADD, and subsequently decreased at 2 and 4 weeks. COL-II and MMP13 expression also increased at the 1e2 weeks after ADD, suggesting osteoarthritis may occur in condylar cartilage. Most of the cartilages were repaired and chondrocytes were arranged in order at 8 weeks, suggesting spontaneous remodeling of condylar cartilage after external force loading.
Purpose: INVOSSA™ (TissueGene-C) is a novel cell and gene therapy for osteoarthritis (OA). The phase III clinical study has been completed and its outcome showed that INVOSSA™ improved pain, daily activities, sports functions and cartilage structure in patients with OA. Here we hypothesized that treatment of INVOSSA™ may induce an antiinflammatory environment, especially M2 macrophage differentiation, which contributes to the reduction of the pain and cartilage regeneration. In this study, we evaluated the effect of INVOSSA™ on pain and structural improvements with its anti-inflammatory modulations in the arthritic knee joints of a rodent OA model. Methods: The in vivo efficacy of INVOSSA™ or each component of INVOSSA™ and a vehicle control (CS-10) were analyzed in a rat MIA model. Pain behavior was assessed by von Frey filament test, and cartilage regeneration was analyzed by various histological stainings. To evaluate the anti-inflammatory effect of INVOSSA™, various cytokines were analyzed by a multiplex assay using synovial fluid. The synovial macrophage differentiation profiles were investigated by immunohistochemistry with CD86 as M1 marker and Arginase 1 as M2 marker. Gene expression profiles were analyzed by quantitative RT-PCR. Results: Pain relief was shown initially at day 15 and maintained up to 56 days post INVOSSA™ treatment. The regenerated cartilage showed hyaline cartilage characteristics post INVOSSA™ treatment. Cytokine expression profiles in synovial fluid showed that INVOSSA™ induced IL10, which was consistent to the observation of CD68-positive monocyte infiltration to the synovial membrane. Furthermore, the INVOSSA™ attracted more arginase 1-positive cells to the synovial membrane. However, the number of CD86-positive cells post INVOSSA™ treatment was comparable to the control treatment. Quantitative RT-PCR analysis also showed that M2 macrophage related markers were highly upregulated in the synovial membranes in INVOSSA™ treated group. Conclusions: This study supports that INVOSSA™ holds a great potential for a disease modifying osteoarthritis drug. 238 ENDOGENOUS PRRX1þ PROGENITORS ARE PRESENT AFTER FULL THICKNESS CARTILAGE INJURY BUT DO NOT DIRECTLY CONTRIBUTE TO FIBROCARTILAGE OR SUBCHONDRAL BONE TISSUES DURING THE REPAIR PROCESS C.L. Jablonski, C. Leonard, R.J. Krawetz. Univ. of Calgary, Calgary, AB, Canada Purpose: While it is known that full thickness cartilage defects (FTCD) in mice and humans repair with a fibrocartilage patch rather than native articular cartilage (AC), it remains unknown what cell type(s) are responsible for producing the fibrocartilage patch. It has long been assumed clinically that mesenchymal stem cells (MSCs) present in the bone marrow are required for this fibrocartilage repair (e.g. rationale behind micro-fracture procedures), but to our knowledge this has never been demonstrated in vivo with endogenous cells. MSCs are unique in their ability to self-renew or differentiate into chondrocytes (cartilage cells), however, we have recently shown that exogenously delivered MSCs (GFPþ) from C57BL/6 mice (C57) do not migrate to AC defects
The 35Sulphate incorporation rate was normalized to the specific activity of the pulse medium, labeling time, and weight of cartilage. In vivo: in seven F8 KO rats a unilateral joint bleed was induced by needle puncture and in the following four days until euthanasia, the animals received analgesia. At UMC Utrecht, the tibial cartilage was removed and proteoglycan synthesis activity determined according to the previously described method. All animal experiments were performed under anesthesia and analgesia in accordance with and approved by the Danish Animal Experiments Council, Ministry of Food, Agriculture and Fisheries, Denmark. Results: On average, a total of 1.6mg (0.8e3.1mg) cartilage per tibia could be obtained. The proteoglycan synthesis rate of healthy cartilage determined after four days of culturing in vitro was on average 49.5 nmol/h.g. This could be modulated by in vitro exposure to blood, resulting in a diminished synthesis: 7.7 nmol/h.g (p ¼ 0.0191), corresponding to an 84% decrease comparable to previously published human experiments. Moreover, when a joint bleed was induced in vivo a diminished proteoglycan synthesis rate could also be demonstrated by this assay (13.5 vs 7.5 nmol/h.g, p ¼ 0.0151), although more variation was seen.
S157
Conclusions: These findings suggest that HMGB2 may play important roles in chondrocytes following exposure to mechanical loading, which may involve in the pathogenesis of osteoarthritis.
Cartilage Repair 237 INVOSSA™ (TISSUEGENE-C) INDUCES AN ANTI-INFLAMMATORY ENVIRONMENT IN THE ARTHRITIC KNEE JOINTS VIA MACROPHAGE POLARIZATION K. Choi, H. Lee, D. Kim, H. Lee, M. Kim, C.-L. Lim, Y.-J. Lee, B. Lee, S. Kim, M. Lee, H. Choi. Kolon Life Sci., Seoul, Republic of Korea
Conclusions: This study demonstrates for the first time that proteoglycan synthesis activity, by use of the 35Sulphate incorporation assay, can be determined in tibial cartilage of the rat. Moreover, this synthetic activity is affected by blood exposure in vitro and in vivo. As such, this assay can be a valuable tool to detect cartilage changes, both in pathophysiological and interventional experiments, using joint degenerative rat models. 236 SPATIAL AND TEMPORAL EXPRESSION PATTERN OF HMGB2 IN RABBIT CONDYLAR CARTILAGE FOLLOWING ANTERIOR DISC DISPLACEMENT M. Wu, Y. Zhou. Hosp. of Stomatology, Zhejiang Univ., Hangzhou, China Purpose: The chromatin protein HMGB2 plays important roles in development, remodeling and disruption of the articular cartilage. The objective of present study was to investigate whether the expression of HMGB2 in the condylar cartilage was correlated to the continuing forward mandibular positioning (FMP), and explore its potential relationship with b-catenin pathway. Methods: Fourty-four healthy adult Japanese white rabbits were used in this experiment. The right tempromandibular joints (TMJ) of rabbits were subjected to surgical and sham surgical operation of anterior disc displacement (ADD) (N ¼ 8). The TMJ condyles of rabbits were collected and fixed at 1-, 2-, 4-, and 8- weeks after the surgery. Histological micrographs of the condyle cartilage were mearsured by HE staining. The distribution and expression of HMGB2, Col-II, MMP1, MMP13 and b-catenin were evaluated through immunohistochemical staining. Results: Histological results by HE staining showed that the condyle cartilage turned thinner as well as disordered cell arrangement in cartilage with ADD. The expression of HMGB2 chiefly distributed in fibrous layer and proliferative layer of normal condylar cartilage. After ADD treatment, expression of HMGB2 and b-catenin enhanced at the 1 week ADD, and subsequently decreased at 2 and 4 weeks. COL-II and MMP13 expression also increased at the 1e2 weeks after ADD, suggesting osteoarthritis may occur in condylar cartilage. Most of the cartilages were repaired and chondrocytes were arranged in order at 8 weeks, suggesting spontaneous remodeling of condylar cartilage after external force loading.
Purpose: INVOSSA™ (TissueGene-C) is a novel cell and gene therapy for osteoarthritis (OA). The phase III clinical study has been completed and its outcome showed that INVOSSA™ improved pain, daily activities, sports functions and cartilage structure in patients with OA. Here we hypothesized that treatment of INVOSSA™ may induce an antiinflammatory environment, especially M2 macrophage differentiation, which contributes to the reduction of the pain and cartilage regeneration. In this study, we evaluated the effect of INVOSSA™ on pain and structural improvements with its anti-inflammatory modulations in the arthritic knee joints of a rodent OA model. Methods: The in vivo efficacy of INVOSSA™ or each component of INVOSSA™ and a vehicle control (CS-10) were analyzed in a rat MIA model. Pain behavior was assessed by von Frey filament test, and cartilage regeneration was analyzed by various histological stainings. To evaluate the anti-inflammatory effect of INVOSSA™, various cytokines were analyzed by a multiplex assay using synovial fluid. The synovial macrophage differentiation profiles were investigated by immunohistochemistry with CD86 as M1 marker and Arginase 1 as M2 marker. Gene expression profiles were analyzed by quantitative RT-PCR. Results: Pain relief was shown initially at day 15 and maintained up to 56 days post INVOSSA™ treatment. The regenerated cartilage showed hyaline cartilage characteristics post INVOSSA™ treatment. Cytokine expression profiles in synovial fluid showed that INVOSSA™ induced IL10, which was consistent to the observation of CD68-positive monocyte infiltration to the synovial membrane. Furthermore, the INVOSSA™ attracted more arginase 1-positive cells to the synovial membrane. However, the number of CD86-positive cells post INVOSSA™ treatment was comparable to the control treatment. Quantitative RT-PCR analysis also showed that M2 macrophage related markers were highly upregulated in the synovial membranes in INVOSSA™ treated group. Conclusions: This study supports that INVOSSA™ holds a great potential for a disease modifying osteoarthritis drug. 238 ENDOGENOUS PRRX1þ PROGENITORS ARE PRESENT AFTER FULL THICKNESS CARTILAGE INJURY BUT DO NOT DIRECTLY CONTRIBUTE TO FIBROCARTILAGE OR SUBCHONDRAL BONE TISSUES DURING THE REPAIR PROCESS C.L. Jablonski, C. Leonard, R.J. Krawetz. Univ. of Calgary, Calgary, AB, Canada Purpose: While it is known that full thickness cartilage defects (FTCD) in mice and humans repair with a fibrocartilage patch rather than native articular cartilage (AC), it remains unknown what cell type(s) are responsible for producing the fibrocartilage patch. It has long been assumed clinically that mesenchymal stem cells (MSCs) present in the bone marrow are required for this fibrocartilage repair (e.g. rationale behind micro-fracture procedures), but to our knowledge this has never been demonstrated in vivo with endogenous cells. MSCs are unique in their ability to self-renew or differentiate into chondrocytes (cartilage cells), however, we have recently shown that exogenously delivered MSCs (GFPþ) from C57BL/6 mice (C57) do not migrate to AC defects