Objective. To test a fluid flow system for the investigation of the influence of shear stress on expression of plasminogen activator inhibitor 1 (PAI-1) in human osteoarthritic (OA) articular chondrocytes (from lesional and nonlesional sites) and human SW-1353 chondrocytes.Methods. Human SW-1353 chondrocytes and OA and normal human articular chondrocytes were cultured on type II collagen-coated glass plates under static conditions or placed in a flow chamber to form a closed fluid-circulation system for exposure to different levels of shear stress (2-20 dyn/cm 2 ). Real-time polymerase chain reaction was used to analyze PAI-1 gene expression, and protein kinase C (PKC) inhibitors and small interfering RNA were used to investigate the mechanism of shear stress-induced signal transduction in SW-1353 and OA (lesional and nonlesional) articular chondrocytes.Results. There was a significant reduction in PAI-1 expression in OA chondrocytes obtained from lesional sites compared with those obtained from nonlesional sites. In SW-1353 chondrocytes subjected to 2 hours of shear flow, moderate shear stresses (5 and 10 dyn/cm 2 ) generated significant PAI-1 expression, which was regulated through PKC␣ phosphorylation and Sp-1 activation. These levels of shear stress also increased PAI-1 expression in articular chondrocytes from nonlesional sites and from normal healthy cartilage through the activation of PKC␣ and Sp-1 signal transduction, but no effect of these levels of fluid shear stress was observed on OA chondrocytes from lesional sites.Conclusion. OA chondrocytes from lesional sites and those from nonlesional sites of human cartilage have differential responses to shear stress with regard to PAI-1 gene expression, and therefore diverse functional consequences can be observed.Osteoarthritis (OA) is the most common joint disease among older persons. The most common sites of clinical manifestations of degenerative cartilage in OA are the hips, knees, and spine. The symptoms of OA include pain, stiffness, and deformation of the knee joints (1). OA occurs more frequently in women and elderly individuals, but being overweight and engaging in a regimen of intense exercise are also factors that convey a high risk of OA. Excessive and repetitive mechanical stresses on the articular joint generate cartilage wear and may induce OA (2). Despite the widespread incidence of OA in the human population, its etiology is still largely unknown.

Curcumin (Cur) and bisdemethoxycurcumin (BDMC), extracted from Curcuma longa, are poorly water-soluble polyphenol compounds that have shown anti-inflammatory potential for the treatment of osteoarthritis. To increase cellular uptake of Cur and BDMC in bone tissue, soybean phosphatidylcholines were used for liposome formulation. In this study, curcuminoid (Cur and BDMC)-loaded liposomes were characterized in terms of particle size, encapsulation efficiency, liposome stability, and cellular uptake. The results show that there is about 70% entrapment efficiency of Cur and BDMC in liposomes and that particle sizes are stable after liposome formation. Both types of liposome can inhibit macrophage inflammation and osteoclast differential activities. In comparison with free drugs (Cur and BDMC), curcuminoid-loaded liposomes were less cytotoxic and expressed high cellular uptake of the drugs. Of note is that Cur-loaded liposomes can prevent liposome-dependent inhibition of osteoblast differentiation and mineralization, but BDMC-loaded liposomes could not. With interleukin (IL)-1β stimulation, curcuminoid-loaded liposomes can successfully downregulate the expression of inflammatory markers on osteoblasts, and show a high osteoprotegerin (OPG)/receptor activator of nuclear factor κB ligand (RANKL) ratio to prevent osteoclastogenesis. In the present study, we demonstrated that Cur and BDMC can be successfully encapsulated in liposomes and can reduce osteoclast activity and maintain osteoblast functions. Therefore, curcuminoid-loaded liposomes may slow osteoarthritis progression.

IntroductionSynovial macrophages, which can release proinflammatory factors, are responsible for the upregulation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). In addition, shear stress exerts multifunctional effects on chondrocytes by inducing the synthesis of catabolic or anabolic genes. However, the interplay of macrophages, chondrocytes, and shear stress during the regulation of cartilage function remains poorly understood. We investigated the mechanisms underlying the modulation of human chondrocyte urokinase plasminogen activator (uPA) expression by macrophages and shear stress.MethodsHuman chondrocytes were stimulated by peripheral blood-macrophage- conditioned medium (PB-MCM), or exposure of chondrocytes cultured in PB-MCM to different levels of shear stress (2 to 20 dyn/cm2). Real-time polymerase chain reaction was used to analyze uPA gene expression. Inhibitors and small interfering RNA were used to investigate the mechanism for the effects of PB-MCM and shear stress in chondrocytes.ResultsStimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that activation of the JNK and Akt pathways and NF-κB are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression in chondrocytes. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK and Akt phosphorylation, NF-κB activation, and uPA expression. The PB-MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression.ConclusionsThese data support the hypothesis that uPA upregulation stimulated by macrophages may play an active role in the onset of OA and in the shear-stress protection against this induction.

Biodegradable polymers, such as polycaprolactone (PCL), have been well studied in the application of tissue engineering and drug delivery systems due to their favourable biocompatibility profiles. However, little research has discussed the influence of the molecular weight of these polymers on cell behaviours. Here, PCL films were synthesized using polymers with various molecular weight and the physical characteristics of these films and their effect on cell growth were evaluated. Results showed that the tensile strength and elongation rate of PCL films increased from 1.82 MPa and 95.3% to 2.01 MPa and 741.6% when the molecular weight (Mn) of polymers increased from 40,000 to 80,000. A significant difference in polymer crystallinity was also observed between these formulations. Moreover, we found that prolonged exposure to UV radiation of these films could lead to the breakdown of polymer chains and consequently increased the crystallinity of PCL films with a corresponding reduction in the tensile strength. Using Hs68 human foreskin fibroblasts, PCL films prepared using high molecular weight polymers (CAPA 6800) displayed a higher cell proliferation rate compared to ones prepared using low molecular weight polymers (CAPA 6400 and 6500). In contrast, a higher cell proliferation rate was observed for PCL films made of low molecular weight polymers, especially CAPA 6500, when H9c2 rat heart myoblasts were used. In conclusion, polymer molecular weight can alter the