Kei Sakao

N-acetylcysteine prevents nitric oxide-induced chondrocyte apoptosis and cartilage degeneration in an experimental model of osteoarthritis.

We investigated whether N‐acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)‐induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0–2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase‐3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO‐induced apoptosis, ROS overproduction, p53 up‐regulation, and caspase‐3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis‐preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO‐induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo.

Osteoblasts derived from osteophytes produce interleukin-6, interleukin-8, and matrix metalloproteinase-13 in osteoarthritis

To clarify the significance of the osteophytes that appear during the progression of osteoarthritis (OA), we investigated the expression of inflammatory cytokines and proteases in osteoblasts from osteophytes. We also examined the influence of mechanical stress loading on osteoblasts on the expression of inflammatory cytokines and proteases. Osteoblasts were isolated from osteophytes in 19 patients diagnosed with knee OA and from subchondral bone in 4 patients diagnosed with femoral neck fracture. Messenger RNA expression and protein production of inflammatory cytokines and proteases were analyzed using real-time RT-PCR and ELISA, respectively. To examine the effects of mechanical loading, continuous hydrostatic pressure was applied to the osteoblasts. We determined the mRNA expression and protein production of IL-6, IL-8, and MMP-13, which are involved in the progression of OA, were increased in the osteophytes. Additionally, when OA pathological conditions were simulated by applying a nonphysiological mechanical stress load, the gene expression of IL-6 and IL-8 increased. Our results suggested that nonphysiological mechanical stress may induce the expression of biological factors in the osteophytes and is involved in OA progression. By controlling the expression of these genes in the osteophytes, the progression of cartilage degeneration in OA may be reduced, suggesting a new treatment strategy for OA.

Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure

OBJECTIVE The aim of this study was to investigate the effect of intermittent hydrostatic pressure (IHP) on chondrogenic differentiation of synovium-derived progenitor cells (SPCs). METHODS SPCs, bone marrow-derived progenitor cells and skin fibroblasts from rabbits were subjected to IHP ranging from 1.0 to 5.0 MPa. The mRNA expression of proteoglycan core protein (PG), collagen type II and SOX-9 was examined using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The production of SOX-9 protein and glycosaminoglycan (GAG) by SPCs was analyzed by Western blot and the dimethylmethylene blue assay. In addition, mitogen-activated protein (MAP) kinase inhibitors for c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and the p38 pathway were used to identify the signal transduction pathways. RESULTS Real-time RT-PCR showed that mRNA expression of PG, collagen type II and SOX-9 was significantly enhanced only in SPCs receiving 5.0 MPa of IHP. The production of SOX-9 protein and GAG by SPCs was also increased by exposure to 5.0 MPa of IHP. These up-regulated expressions were suppressed by pretreatment with an inhibitor of JNK, but not with inhibitors of ERK or p38. CONCLUSION Our results demonstrated that the exposure of SPCs to 5.0 MPa of IHP could facilitate induction of the chondrogenic phenotype by the MAP kinase/JNK pathway. This finding suggests the potential for IHP utilization in regenerative treatments for cartilage injuries or osteoarthritis.

Enhanced expression of interleukin-6, matrix metalloproteinase-13, and receptor activator of NF-κB ligand in cells derived from osteoarthritic subchondral bone

BackgroundThe aim of this study was to clarify the significance of subchondral bone in the pathology of osteoarthritis (OA) by investigating the expression of inflammatory cytokines, proteases, and receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK)/osteoprotegerin (OPG) involved in cartilage degeneration.MethodsSubchondral bone was obtained from 19 patients diagnosed with knee OA and 4 patients diagnosed with femoral neck fracture. Subchondral bone osteoblasts (SBOs) were isolated, and total RNA was extracted. Messenger RNA expression of inflammatory cytokines, proteases, and RANKL/RANK/OPG were analyzed using a real-time reverse transcription-polymerase chain reaction (RT-PCR).ResultsReal-time RT-PCR showed that mRNA expressions of interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13), and RANKL were significantly enhanced in OA SBOs compared to SBOs without OA. The expressions of these genes was greater in patients with severe cartilage damage than in those with mild cartilage damage. A high correlation between mRNA expression of IL-6 and that of MMP-13 was found in OA SBOs.ConclusionThe increases in IL-6, MMP-13, and RANKL expression in OA SBOs suggest that in subchondral bone OA progression involves abnormal osseous tissue remodeling, which induces mechanical property changes. Cartilage degeneration in OA may also be due, at least in part, to IL-6 and MMP-13 produced by SBOs. Comprehensive research on these pathological features may lead to the development of more effective therapies for OA by administration of molecules that affect bone remodeling and metabolism.

Intra-Articular Injection of Hyaluronan Restores the Aberrant Expression of Matrix Metalloproteinase-13 in Osteoarthritic Subchondral Bone

Subchondral bone is a candidate for treatment of osteoarthritis (OA). We investigated the effects of intra‐articular injection of hyaluronan (IAI‐HA) on subchondral bone in rabbit OA model. OA was induced by anterior cruciate ligament transection, with some rabbits receiving IAI‐HA. OA was graded morphologically, and expression of mRNA was assessed by real‐time RT‐PCR. Tissue sections were stained with hyaluronan‐binding protein, and penetration of fluorescent hyaluronan was assessed. The in vitro inhibitory effect of hyaluronan on MMP‐13 was analyzed in human osteoarthritic subchondral bone osteoblasts (OA Ob) by real‐time RT‐PCR and ELISA. Binding of hyaluronan to OA Ob via CD44 was assessed by immunofluorescence cytochemistry. Expression of MMP‐13 and IL‐6 mRNA in cartilage and subchondral bone, and morphological OA grade, increased over time. IAI‐HA ameliorated the OA grade and selectively suppressed MMP‐13 mRNA in subchondral bone. IAI‐HA enhanced the hyaluronan staining of subchondral bone marrow cells and osteocyte lacunae. Fluorescence was observed in the subchondral bone marrow space. In OA Ob, hyaluronan reduced the expression and production of MMP‐13, and anti‐CD44 antibody blocked hyaluronan binding to OA Ob. These findings indicate that regulation of MMP‐13 in subchondral bone may be a critical mechanism during IAI‐HA.

Asporin and transforming growth factor-β gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis

BackgroundTo clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-β1 (TGF-β1), TGF-β2, TGF-β3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism.MethodsOsteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-β1, TGF-β2, TGF-β3, and Runx2 was analyzed using real-time RT-PCR.ResultsExpression of ASPN, TGF-β1, and TGF-β3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-β1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage.ConclusionsThe increased ratio of ASPN mRNA to TGF-β1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-β1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Age-related changes in the hemodynamics of the femoral head as evaluated by early phase of bone scintigraphy

ObjectiveThe femoral head is reported to be in a markedly hypoemic state as compared with other tissues even under normal conditions, and it is therefore necessary to understand its hemodynamics to investigate the pathogenesis of hip disorders. It is known that aspects of intraosseous hemodynamics including blood flow and blood pool can be evaluated soon after radioisotope administration. In this study, hemodynamic changes in the femoral head according to gender and age were examined by investigating accumulation of radioisotope in the tissue during the early phase of bone scintigraphy.MethodsThe subjects of this study consisted of 58 joints of 31 men and 75 joints of 41 women, whose ages ranged from 15 to 87 years (average age: 67.9 years). Images of bone scintigraphy were obtained for 15 to 20 minutes at 5 minutes and at 3 hours after radioisotope administration. The ratio of accumulation in the femoral head to that in the diaphysis (head-to-diaphysis ratio, HD ratio) was calculated.ResultsHD ratios obtained 15-20 minutes later ranged from 0.01 to 7.35 (1.88 ± 0.91, mean ± SD). HD ratios decreased with age, and a significant inverse correlation was observed between age and HD ratio, demonstrating a correlation coefficient of-0.27 (p = 0.001). The HD ratio among men was 0.01-3.57 (1.66 ± 0.71), while that among women was 0.53-7.35 (2.05 ± 1.01), and a significant difference was observed in HD ratio between men and women (p = 0.02). There was a significant difference in HD ratios between men and women in their teens to forties (p = 0.03), while no significant differences was observed in the other age groups. HD ratios obtained 3 hours later ranged from 0.44 to 6.32 (1.95 ± 0.79, mean ± SD), and no significant correlation was observed between age and HD ratio, demonstrating a correlation coefficient of-0.14.ConclusionThe present study demonstrated that blood flow and blood pool of the femoral head decrease with aging particularly in women. This hemodynamic deterioration of the femoral head caused by aging may have an effect on the onset and progression of hip disorders by influencing bone metabolism.

Influence of extracellular matrix on the expression of inflammatory cytokines, proteases, and apoptosis-related genes induced by hydrostatic pressure in three-dimensionally cultured chondrocytes

BackgroundThe purpose of this study was to investigate the influence of hydrostatic pressure (HP) on the gene expression of cartilage matrix, cytokines, and apoptosis-associated factors in chondrocytes in which the cartilage was in extracellular matrix (ECM)-rich or ECM-poor condition.MethodsChondrocytes were isolated from rabbit joints and cultured in alginate beads. Immediately after embedding (0W group) or after 2 weeks culture (2W group), the amounts of glycosaminoglycan (GAG) in the alginate beads were quantified. Both groups were exposed to continuous HP of 10 or 50 MPa for 12 h. The expression of inflammatory cytokines, proteases, and apoptosis-related factors were examined by reverse transcription-polymerase chain reaction (RT-PCR). The expression of proteoglycan core protein (PG) and collagen type II were quantified by real-time RT-PCR.ResultsAll of the GAG components in alginate beads markedly increased in the 2W group. The expression of PG and collagen type II increased after exposure to 10 MPa in both groups. In the 0W group, these levels decreased after exposure to 50 MPa of HP. The expression of interleukins IL-6 and IL-8 increased after exposure to HP in the 0W group. HP at 50 MPa induced mRNA expression of ADAMTS-5 in the 0W group but not in the 2W group. The expression of Fas increased after exposure to HP in the 0W group.ConclusionsThese findings suggested that nonphysiological, excessive HP on chondrocytes with the ECM in poor condition reduced matrix gene expression and increased expression of the genes associated with apoptosis and catabolism of the cartilage matrix. These results might therefore be associated with the pathogenesis of osteoarthritis.

Hydrostatic Pressure Induces Cytokine Production in Human Periodontal Ligament Cells

Abstract Periodontal tissue has a unique structure in that the human periodontal ligament (hPDL) lies between the hard tissues of cementum and alveolar bone. Although the role of cytokines in hPDL function is not clearly understood, we investigated the effect of mechanical stress as hydrostatic pressure (HP) on cytokine expression in hPDL cells. The hPDL cells were obtained from a healthy maxillary third molar. After the 3rd to 4th passage, the cells were exposed to HP ranging from 1 MPa to 6 MPa as previously described. The expression of cytokine mRNA was determined by RT-PCR and cytokines in the culture supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). The exposure to 6 MPa of HP caused no morphological changes of hPDL cells, and did not affect cellular viability. No expression of IL-1β, IL-6, IL-8, TNF-α, RANK, RANKL or OPG mRNA was observed in the control cells under atmospheric pressure, whereas in hPDL cells treated with HP, enhancement of IL-6, IL-8, RANKL and OPG mRNA expression was observed between 10 and 60 minutes after the exposure to HP. After the exposure to HP, the production of IL-6 and TNF-α were induced significantly in hPDL cells, but IL-1β and IL-8 were not produced. These results suggest that hPDL cells may play a role in the production of cytokines in response to mechanical stress in vivo .

164 HYALURONAN AND INTERMITTENT HYDROSTATIC PRESSURE SYNERGISTICALLY SUPPRESSED MMP-13 AND IL-6 EXPRESSIONS IN OSTEOBLASTS FROM OA SUBCHONDRAL BONE

Purpose: Various inflammatory cytokines and proteases are involved in the initiation and progression of osteoarthritis (OA). We previously reported that the expressions of matrix metalloproteinase-13 (MMP-13) and interleukin-6 (IL-6) were augumented in OA subchondral bone. The purpose of this study was to investigate the effects of hyaluronan (HA) and mechanical stress on osteoblasts isolated from OA subchondral bone. Methods: OA subchondral bone from the distal end of the femur was harvested from 9 patients at total knee arthroplasty. The subchondral bone underlying the degenerated articular cartilage was cut into small pieces and incubated in DMEM for 3 weeks, and then osteoblasts were isolated. Subchondral bone osteoblasts (SBOs) were cultured with DMEM conraining 30% fluorescent labeled HA for 48 hours and washed twice with PBS. The monolayer cultured cells were observed with a fluorescence microscope. As control, SBOs cultured without fluorescent labeled HA was used. SBOs were divided into 4 experimental groups. Control group: cultured without stimulation, HA group: incubated with HA (1000 mg/ml, 48 hours), IHP group: applied intermittent hydrostatic pressure (IHP) (1/2 Hz, 5 MPa, 60 minutes), and HA+IHP group: incubated with HA followed by IHP. Total RNA were extracted and mRNA expression was examined by real-time RT-PCR for MMP-13 and IL-6. In control group and HA+IHP group, culture supernatant was harvested 24 hours after the application of HA + IHP, and concentrations of IL-6 and MMP-13 were measured using an enzyme-linked immunosorbent assay (ELISA). Values were analyzed statistically by Tukey-kramer’s test and paired t-test and a p value less than 0.05 was considered significant. Results: In the fluorescent labeled HA group, fluorescence was observed in the area of cytoplasm but not in nuclei 48 hours after the administration. The mRNA expressions of MMP-13 of the each group compared to the control group were 101±18.2%, 89.3±14.1% and 51.2±7.5% respectively, indicating that MMP-13 expression in the HA + IHP group significantly decreased compared to those in the control group and in the HA group. The IL-6 mRNA of the each group were 76.6±11.9 %, 73.2±10.5% and 54.0±18.3%, indicating that IHP treatment and HA + IHP treatment significantly suppressed the IL-6 mRNA.The production of MMP-13 and IL-6 were 51.3±11.6 (pg/ml) and 70.8±25.6 in the control group. In the HA + IHP group, they were significantly reduced to 53.9±25.6 and 45.2±11.1. Conclusions: The role of subchondral bone attracts attention in the onset and/or progression of OA. It was reported that HA influences metabolism in subchondral bone, and that subchondral bone becomes more compliant and thereby reduces cartilage stress. However, the mechanism of the influence of HA on subchondral bone remains unclear. In this study, HA was exposed to osteoblasts, and the enhanced expressions of MMP-13 and IL-6 in OA osteoblasts were significantly suppressed by HA in combination with IHP. In the natural course of OA, a resorption of subchondral bone was documented in the early stage of OA and was considered to take an important role in the progression of the disease. In OA, neovascularization between cartilage and subchondral bone is observed while tidemark disappears. Therefore, intra-articular injected HA could reach the subchondral tissue. The results in this study suggest that intra-articular injection of HA in combination with appropriate exercise could suppress MMP-13 and IL-6 expressions in subchondral bone, which may prevent abnormal metabolism in osseous tissue in OA.