HBO suppressed nitric oxide and apoptosis in articular cartilage defect via up-regulation of hsp 70 expression —In vitro and in vivo study

Nucleus pulposus cells (NPCs) from degenerating discs produce catabolic and inflammatory factors, including interleukin (IL)-1, and nitric oxide (NO). The enhanced production of NOvia inducible nitric oxide synthase (iNOS) has been implicated in the apoptosis of degenerating disc cells. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs. NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100%O2 at2.5 atmospheres absolute (ATA) inahyperbaric chamber. ERK1/2 and p38MAPK phosphorylation of the NPCswere detected using the phosphor-kinase array kit. RNA was isolated for real-time quantitative polymerase chain reaction (Q-PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL-1β and NOwere quantified by enzymelinked immunosorbent assay (ELISA). In situ analysis of apoptosis was performed using TUNEL staining. Our data showed that HBO treatment decreased expression of IL-1β, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of ERK1/2 and p38 MAPK, decreased NO expression in NPCs as compared with the atmospheric treatment. TUNEL staining showed that HBO treatment suppressed the apoptosis of cultured NPCs. These results support our proposal that HBO treatment suppressed ERK1/2 and p38 MAPK mediate nitric oxide-induced apoptosis on human degenerated intervertebral disc cells. This article of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.299 PP140-M HBO suppressed nitric oxide and apoptosis in articular cartilage defect via up-regulation of hsp 70 expression —In vitro and in vivo study S.-S. Lin⁎, L.-J. Yuan, C.-C. Niu, C.-Y. Yang Department of Orthopaedic Surgery, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan, China Abstract: Heat shock proteins (HSP), inflammatory cytokines, nitric oxide (NO), and localized hypoxia-induced apoptosis are thought to be involved in the occurrence and progression of osteoarthritis (OA). We investigated the effect of hyperbaric oxygen (HBO) on (1) NO-induced apoptosis of rabbit chondrocytes and (2) the healing of rabbit articular cartilage defects. In vitro, interleukin-1β treatment induced NO in chondrocytes, and all hyperoxic cells were exposed to 100% oxygen at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting to detect mRNA and protein expression of HSP 70, inducible NO synthase (iNOS), and caspase 3. In vivo, the HBO group was exposed to 100% oxygen at 2.5 ATA for 2 h, 5 days a week for 10 weeks. The control groupwashoused in the cages containingnormal air. After sacrifice, specimen sectionswere sent for histological and histochemical examinations using a standardized scoring system. In situ analysis of iNOS, HSP 70, and caspase 3 expressionwere performedby immunohistostaining. Our in vitro study demonstrated that HBO treatment up-regulated mRNA and protein expression of HSP70 and suppressed iNOSand caspase3 expression. Invivo, thehistological and histochemical scores showed that HBO treatment significantly enhanced the cartilage repair. Moreover, Heat shock proteins (HSP), inflammatory cytokines, nitric oxide (NO), and localized hypoxia-induced apoptosis are thought to be involved in the occurrence and progression of osteoarthritis (OA). We investigated the effect of hyperbaric oxygen (HBO) on (1) NO-induced apoptosis of rabbit chondrocytes and (2) the healing of rabbit articular cartilage defects. In vitro, interleukin-1β treatment induced NO in chondrocytes, and all hyperoxic cells were exposed to 100% oxygen at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting to detect mRNA and protein expression of HSP 70, inducible NO synthase (iNOS), and caspase 3. In vivo, the HBO group was exposed to 100% oxygen at 2.5 ATA for 2 h, 5 days a week for 10 weeks. The control groupwashoused in the cages containingnormal air. After sacrifice, specimen sectionswere sent for histological and histochemical examinations using a standardized scoring system. In situ analysis of iNOS, HSP 70, and caspase 3 expressionwere performedby immunohistostaining. Our in vitro study demonstrated that HBO treatment up-regulated mRNA and protein expression of HSP70 and suppressed iNOSand caspase3 expression. Invivo, thehistological and histochemical scores showed that HBO treatment significantly enhanced the cartilage repair. Moreover, immunohistostaining showed that HBO treatment enhanced HSP 70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment enhances the expression of HSP 70 and prevents NO-induced apoptosis in articular cartilage injury. This article of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.300 PP141-T The effects of oxygen tension on the terminal differentiation of chondrogenic cells S. Portron ⁎, C. Merceron , M. Masson , M. Perre , P. Weiss , O. Gauthier , J. Guicheux , C. Vinatier a,c a INSERM U791 University of Nantes, France b ONIRIS, Experimental Surgery Department of Nantes, Nantes, France c Graftys SA, Aix en Provence, France Abstract: Mesenchymal stem cells (MCS) have been recently considered as promising autologous cells for the regenerative medicine of articular cartilage. The chondrogenic differentiation of MSC can ultimately lead to the formation of hypertrophic chondrocytes responsible for the calcification of cartilage [1]. To prevent this MSC-dependent production of a calcified matrix in articular site, hypertrophic differentiation has to be carefully controlled. Given that cartilage is an avascular tissue, we questioned whether, in addition to its stimulatory role in the early differentiation of chondrogenic cells [2], hypoxia may also prevent their terminal hypertrophic differentiation. ATDC5 cells differentiate towards the chondrogenic lineage by mimicking the various steps of endochondral ossification including the terminal hypertrophic stage. ATDC5 were cultured in the presence of normoxic (20% O2) for 21 days and for an additional 7 day period in normoxic or hypoxic (5% O2) conditions. The effects of hypoxia on the hypertrophic differentiation of ATDC5 were evaluated by (i) the production of sulfated GAG by alcian blue staining and calcification of the matrix by alizarin red staining, (ii) the expressions of hypertrophic differentiation markers (Mmp13, type X Collagen, Runx2, and AlpL) by realtime PCR and TaqMan Low Density Array, and (iii) the measurement of alkaline phosphatase activity. Cell viability was assessed by cell counting and total protein production. Our data indicate that hypoxia decreases the production of GAG and calcified matrix, down-regulates the expression of hypertrophic markers and reduces alkaline phosphatase activity without affecting cell viability. Our data suggests that a low oxygen tension inhibits the hypertrophic differentiation of chondrogenic cells. These results make hypoxia an instrumental tool to prevent the formation of calcified matrix in MSC-based cartilage tissue engineering. This article of a Special Issue entitled ECTS 2011. Acknowledgement: «Foundation arthritis Courtin», «Societe Francaise de Rhumatologie», ANR Tecsan and «Region Pays de La Loire» (Bioregos). Disclosure of interest: None declared. Mesenchymal stem cells (MCS) have been recently considered as promising autologous cells for the regenerative medicine of articular cartilage. The chondrogenic differentiation of MSC can ultimately lead to the formation of hypertrophic chondrocytes responsible for the calcification of cartilage [1]. To prevent this MSC-dependent production of a calcified matrix in articular site, hypertrophic differentiation has to be carefully controlled. Given that cartilage is an avascular tissue, we questioned whether, in addition to its stimulatory role in the early differentiation of chondrogenic cells [2], hypoxia may also prevent their terminal hypertrophic differentiation. ATDC5 cells differentiate towards the chondrogenic lineage by mimicking the various steps of endochondral ossification including the terminal hypertrophic stage. ATDC5 were cultured in the presence of normoxic (20% O2) for 21 days and for an additional 7 day period in normoxic or hypoxic (5% O2) conditions. The effects of hypoxia on the hypertrophic differentiation of ATDC5 were evaluated by (i) the production of sulfated GAG by alcian blue staining and calcification of the matrix by alizarin red staining, (ii) the expressions of hypertrophic differentiation markers (Mmp13, type X Collagen, Runx2, and AlpL) by realtime PCR and TaqMan Low Density Array, and (iii) the measurement of alkaline phosphatase activity. Cell viability was assessed by cell counting and total protein production. Our data indicate that hypoxia decreases the production of GAG and calcified matrix, down-regulates the expression of hypertrophic markers and reduces alkaline phosphatase activity without affecting cell viability. Our data suggests that a low oxygen tension inhibits the hypertrophic differentiation of chondrogenic cells. These results make hypoxia an instrumental tool to prevent the formation of calcified matrix in MSC-based cartilage tissue engineering. This article of a Special Issue entitled ECTS 2011. Acknowledgement: «Foundation arthritis Courtin», «Societe Francaise de Rhumatologie», ANR Tecsan and «Region Pays de La Loire» (Bioregos). Disclosure of interest: None declared.