Development of a microfluidic dispensing device for multivariate data acquisition and application in molecularly imprinting hydrogel preparation.

Molecularly imprinted polymers (MIPs) are superior materials with a molecular recognition ability that apply to various applications. In order to get high specificity recognition for target molecules, selecting polymerization conditions, which provide high interaction with the target template, is crucial. However, it requires time and labor to find the optimal polymerization composition, especially for large biomolecules. The advance in the microfluidic field enables researchers to control the flow rate and divide solutions based on the design of microfluidic devices for acquiring multivariate data by simultaneously preparing samples with different conditions. In this work, we fabricated microfluidic dispensing devices with different flow path widths that can give the solution of different flow rates. The accuracy of the flow rate was compared with the simulation value. As a result, the flow rate data showed almost the same data as the simulation value, and the dispensing volume ratio showed high reproducibility. Besides, the multivariate data from mixing the fluorescent molecule and protein solutions prepared by the dispensing device and a micropipette showed no significant difference with existing laboratory equipment. Finally, the dispensing device was used for preparing MIP hydrogels for lysozyme as a template protein. We successfully acquired multivariate data on the adsorption capacity of proteins, as a result, the hydrogels provided a high imprinting factor and adsorption specificity toward lysozymes.

Radiologic results of additional single screw fixation with lateral locking plate after hybrid closed-wedge high tibial osteotomy.

BACKGROUND: Hybrid closed-wedge high tibial osteotomy (hybrid CWHTO) is an effective surgical treatment for medial compartment osteoarthritis of the knee. Our study investigated whether the combination of a lateral locking plate and a single medial screw promoted bone union after hybrid CWHTO. METHODS: The study cohort consisted of 30 patients (15 men and 15 women) who underwent hybrid CWHTO for medial compartment osteoarthritis or spontaneous osteonecrosis of the knee. Sixteen knees were fixed with a lateral locking plate (LP group), and 17 were fixed with both a lateral locking plate and a cannulated cancellous screw on the medial side of the tibia (LPS group). The times to bone union, radiolucency, and callus formation at the osteotomy site were evaluated radiographically. RESULTS: The mean postoperative time to radiographic confirmation of bone union was 5.5 ± 2.6 months in the LP group and 3.4 ± 1.5 months in the LPS group. Radiolucency at the osteotomy site and excess callus formation on the posterior side of the tibia were lower in the LPS group than in the LP group. CONCLUSIONS: This modified hybrid CWHTO combining a lateral locking plate and a cannulated cancellous screw on the medial side of the tibia improves the stability of the osteotomy site and shortens the period of bone union.

Mild electrical stimulation with heat stimulation increase heat shock protein 70 in articular chondrocyte.

The objective of this study is to investigate the effects of mild electrical stimulation (MES) and heat stress (HS) on heat shock protein 70 (HSP70), that protects chondrocytes and enhances cartilage matrix metabolism, in chondrocyte and articular cartilage. Rabbit articular chondrocytes were treated with MES and/or HS. The safeness was assessed by LDH assay and morphology. HSP70 protein, ubiquitinated proteins and HSP70 mRNA were examined by Western blotting and real-time PCR. Rat knee joints were treated with MES and/or HS. HSP70 protein, ubiquitinated proteins, HSP70 mRNA and proteoglycan core protein (PG) mRNA in articular cartilage were investigated. In vitro, HS increased HSP70 mRNA and HSP70 protein. MES augmented ubiquitinated protein and HSP70 protein, but not HSP70 mRNA. MES + HS raised HSP70 mRNA and ubiquitinated protein, and significantly increased HSP70 protein. In vivo, HS and MES + HS treatment augmented HSP70 mRNA. HS modestly augmented HSP70 protein. MES + HS significantly increased HSP70 protein and ubiquitinated proteins. PG mRNA was markedly raised by MES + HS. This study demonstrated that MES, in combination with HS, increases HSP70 protein in chondrocytes and articular cartilage, and promotes cartilage matrix metabolism in articular cartilage. MES in combination with HS can be a novel physical therapy for osteoarthritis by inducing HSP70 in articular cartilage.

Combined microwave irradiation and intraarticular glutamine administration-induced HSP70 expression therapy prevents cartilage degradation in a rat osteoarthritis model.

The objective of the present study was to investigate the effects of heat stimulation and glutamine (Gln) on the expression of extracellular matrix genes and heat shock protein 70 (HSP70) in rat articular cartilage in vivo and to determine whether HSP70 expression achieved with a combination of microwave (MW) and Gln suppresses osteoarthritis (OA) progression in a rat OA model. Stimulation at 40 W was assumed to be appropriate in the present study, and the effects of heat treatment at this intensity were evaluated. Articular cartilage was collected at 8 h after heat stimulation and/or intraarticular Gln administration, and total RNA was extracted. The expression of HSP70, aggrecan, and type II collagen was quantified using real-time RT-PCR. Cartilage samples from the OA model were subjected to hematoxylin and eosin (HE) and safranin O staining. HSP70 and aggrecan expression was greatest in a group receiving both MW and Gln. In the rat OA model, the severity of OA was significantly milder in a group receiving MW and Gln than in the control group. HSP70, stimulated by the combination of MW heat and Gln, may be involved in the suppression of OA progression.

Lansoprazole inhibits nitric oxide and prostaglandin E(2) production in murine macrophage RAW 264.7 cells.

Aberrantly activated macrophages, which overproduce inflammatory mediators, are involved in the pathogenesis of many inflammatory diseases. We analyzed the anti-inflammatory activity of lansoprazole (LPZ), a typical proton pump (P-ATPase) inhibitor, on RAW264.7 murine macrophages. Treatment of lipopolysaccharide (LPS)-stimulated RAW264.7 cells with LPZ inhibited the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Since P-ATPase expression was not observed in RAW264.7 cells, the anti-inflammatory effect of LPZ was independent of ATPase. In contrast, diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, decreased NO but not PGE(2) levels. LPZ suppressed the LPS-stimulated production by RAW264.7 cells of reactive oxygen species (ROS), which plays an important role in inflammatory responses. ROS elevation in these cells was associated with NO but not PGE(2) production, suggesting that LPZ inhibits NO production by suppressing NADPH oxidase activity. These findings suggest that LPZ may be useful in the treatment of many inflammatory diseases associated with activated macrophages.

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.

MDR1a/1b gene silencing enhances drug sensitivity in rat fibroblast-like synoviocytes.

BACKGROUND: Drug resistance mediated by P-glycoprotein (P-gp) is one of the major reasons for the failure of rheumatoid arthritis (RA) therapy with disease modifying anti-rheumatic drugs and glucocorticoids. In the present study, we aimed to investigate the in vitro effectiveness of small interfering RNA (siRNA) to render rat fibroblast-like synoviocytes (FLS) susceptible to drugs. We also attempted the electroporation-mediated transfer of siRNA against multidrug resistance (MDR) genes into rat knee joints. METHODS: FLS were transfected with siRNAs corresponding to MDR1a and MDR1b genes. FLS were treated with dexamethasone (DEX) and lipopolysaccharide. The mRNA and protein levels of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1beta were measured. Both siRNAs were co-transduced into rat knee joints by an electroporation method and evaluated the target gene expressions in the synovium. RESULTS: Each siRNA could sequence-specifically reduce the target gene expression by over 70% and effectively suppressed P-gp expression and function in the FLS. Both gene expression and protein production of the inflammatory cytokines in the cells transfected with siRNA were reduced by a greater amount compared to in control cells. The in vivo electroporation-mediated transduction of siRNA could significantly inhibit the target gene expressions. CONCLUSIONS: MDR1a/1b gene silencing by siRNA could effectively inhibit P-gp in rat FLS, resulting in a significant enhancement of the anti-inflammatory effects of DEX. The in vivo siRNA transduction could successfully silence MDR gene expression in the rat synovium. These findings indicate that the siRNA targeting MDR gene could be a useful tool for treating refractory arthritis in RA.

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

BACKGROUND: To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. METHODS: Osteoblasts 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-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. RESULTS: Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. CONCLUSIONS: The increased ratio of ASPN mRNA to TGF-beta1 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-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Hyperthermia for the treatment of articular cartilage with osteoarthritis.

Osteoarthritis (OA) is one of the most frequent musculoskeletal disorders in the elderly population. OA is characterised by a gradual loss of extracellular matrix in the articular cartilage of joints. OA can only be managed by artificial joint replacement when joint destruction becomes severe. Therefore, it is preferable to administer conservative therapy that is easy, simple and effective in inhibiting OA progression at the early stage. Heat shock protein 70 (Hsp70) has a protective effect on the cartilage and inhibits the apoptosis of chondrocytes. Heat stimulation by microwave to the joints can increase Hsp70 expression in chondrocytes, and at the same time, Hsp70 expression partially enhances matrix metabolism of the cartilage. These findings suggest that hyperthermia can be positively applied to the treatment of OA. Hyperthermia is therefore expected to be an inexpensive and less-invasive conservative therapy for OA.

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.