Nanobubbles (<200 nm in diameter) have several unique properties such as long lifetime in liquid owing to its negatively charged surface, and its high gas solubility into the liquid owing to its high internal pressure. They are used in variety of fields including diagnostic aids and drug delivery, while there are no reports assessing their effects on the growth of lives. Nanobubbles of air or oxygen gas were generated using a nanobubble aerator (BUVITAS; Ligaric Company Limited, Osaka, Japan). Brassica campestris were cultured hydroponically for 4 weeks within air-nanobubble water or within normal water. Sweetfish (for 3 weeks) and rainbow trout (for 6 weeks) were kept either within air-nanobubble water or within normal water. Finally, 5 week-old male DBA1/J mice were bred with normal free-chaw and free-drinking either of oxygen-nanobubble water or of normal water for 12 weeks. Oxygen-nanobubble significantly increased the dissolved oxygen concentration of water as well as concentration/size of nanobubbles which were relatively stable for 70 days. Air-nanobubble water significantly promoted the height (19.1 vs. 16.7 cm; P<0.05), length of leaves (24.4 vs. 22.4 cm; P<0.01), and aerial fresh weight (27.3 vs. 20.3 g; P<0.01) of Brassica campestris compared to normal water. Total weight of sweetfish increased from 3.0 to 6.4 kg in normal water, whereas it increased from 3.0 to 10.2 kg in air-nanobubble water. In addition, total weight of rainbow trout increased from 50.0 to 129.5 kg in normal water, whereas it increased from 50.0 to 148.0 kg in air-nanobubble water. Free oral intake of oxygen-nanobubble water significantly promoted the weight (23.5 vs. 21.8 g; P<0.01) and the length (17.0 vs. 16.1 cm; P<0.001) of mice compared to that of normal water. We have demonstrated for the first time that oxygen and air-nanobubble water may be potentially effective tools for the growth of lives.

Cartilage functions at a lower oxygen tension than most other tissues. To determine the role of oxygen tension in chondrocyte differentiation and function, we investigated the influence of oxygen tension in the pluripotent mesenchymal cell line C3H10T1/2 and 14.5E mice embryo forelimb organ culture. 10T1/2 cells and embryo forelimbs were cultured under normoxia (20% O 2 ) or hypoxia (5% O 2 ) in the presence of recombinant human bone morphogenetic protein 2. To elucidate the mechanism by which oxygen tension influences chondrocyte differentiation, the Smad pathway was examined using Smad6 overexpression adenovirus and Smad6 transgenic mice embryo forelimbs. The p38 MAPK pathway was examined using dominant-negative MKK3 and FR167653, a specific p38 MAPK inhibitor. The transcriptional activities of Sox9 and Runx2 were also investigated. Hypoxia promoted bone morphogenetic protein 2-induced glycosaminoglycan production and suppressed alkaline phosphatase activity and mineralization of C3H10T1/2. Thus, hypoxia promoted chondrocytic commitment rather than osteoblastic differentiation. In the mice embryo forelimb organ culture, hypoxia increased cartilaginous matrix synthesis. These effects were primarily mediated by p38 MAPK activation, independent of Sox9. Hypoxia inhibited Col10a1 (type X collagen ␣1) expression via downregulation of Runx2 activity by Smad suppression and histone deacetylase 4 activation. In conclusion, hypoxia promotes chondrocytic differentiation and cartilage matrix synthesis and suppresses terminal chondrocyte differentiation. These hypoxiainduced phenomena may act on chondrocytes to enhance and preserve their phenotype and function during chondrocyte differentiation and endochondral ossification.A number of pathophysiological findings suggest that a correlation exists between hypoxia and chondrogenesis. For example, articular cartilage is an avascular tissue that functions at an oxygen tension that is lower than that of most other tissues. Articular cartilage derives both its nutrition and oxygen supply by diffusion from the synovial fluid and the subchondral bone. It has been estimated that articular chondrocytes in the deepest layers may have access to no more than 1-6% O 2 (1-6). Furthermore, although the majority of mammalian cells derive their energy by using oxygen for mitochondrial oxidative phosphorylation (7), few mitochondria are present in articular chondrocytes (8). Carbohydrate breakdown in articular cartilage is dominated by the conversion of glucose to lactate via the Embden-Meyerhof-Parnas pathway (9 -11) that consumes no O 2 . Similarly, during the endochondral ossification processes that occur in the growth plate, chondromodulin-1, an endogenous inhibitor of neovascularization, is highly expressed by chondrocytes. Of note, most of the growth plate is avascular (12). Recently, in an in vivo experiment, it was found that hypoxia-inducible factor 1, which appears to be one of the major regulators of the hypoxic response, is essential for chondrocyte growth arrest and survival (1...

It has been suggested that interleukin-6 (IL-6)plays a key role in the pathogenesis of rheumatoid arthritis(RA), including osteoporosis not only in inflamed joints but also in the whole body. However, previous in vitro studies regarding the effects of IL-6 on osteoblast differentiation are inconsistent. The aim of this study was to examine the effects and signal transduction of IL-6 on osteoblast differentiation in MC3T3-E1 cells and primary murine calvarial osteoblasts. IL-6 and its soluble receptor significantly reduced alkaline phosphatase (ALP) activity, the expression of osteoblastic genes (Runx2, osterix, and osteocalcin), and mineralization in a dose-dependent manner, which indicates negative effects of IL-6 on osteoblast differentiation. Signal transduction studies demonstrated that IL-6 activated not only two major signaling pathways, SHP2/MEK/ERK and JAK/STAT3, but also the SHP2/PI3K/Akt2 signaling pathway. The negative effect of IL-6 on osteoblast differentiation was restored by inhibition of MEK as well as PI3K, while it was enhanced by inhibition of STAT3. Knockdown of MEK2 and Akt2 transfected with siRNA enhanced ALP activity and gene expression of Runx2. These results indicate that IL-6 negatively regulates osteoblast differentiation through SHP2/MEK2/ERK and SHP2/PI3K/Akt2 pathways, while affecting it positively through JAK/STAT3. Inhibition of MEK2 and Akt2 signaling in osteoblasts might be of potential use in the treatment of osteoporosis in RA.

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Osteocytes are derived from osteoblasts, but reside in the mineralized bone matrix under hypoxic conditions. Osteocyte-like cells show higher expression of ORP150, which is induced by hypoxia, than osteoblast-like cells. Accordingly, we hypothesized that the oxygen tension may regulate the transformation of osteoblasts to osteocytes. MC3T3-E1 cells and calvariae from 4-day-old mice were cultured under normoxic (20% O(2)) or hypoxic (5% O(2)) conditions. To investigate osteoblastic differentiation and tranformation to osteocytes, alizarin red staining was done and the expression of various factors was assessed. Hypoxic culture promoted the increased synthesis of mineralized matrix by MC3T3-E1 cells. Alkaline phosphatase activity was initially increased during hypoxic culture, but decreased during osteogenesis. Osteocalcin production was also increased by hypoxic culture, but decreased after mineralization. Furthermore, expression of Dmp1, Mepe, Fgf23, and Cx43, which are osteocyte-specific or osteocyte-predominant proteins, by MC3T3-E1 cells was greater under hypoxic than under normoxic conditions. In mouse calvarial cultures, the number of cells in the bone matrix and cells expressing Dmp1 and Mepe were increased by hypoxia. In MC3T3-E1 cell cultures, ORP150 expression was only detected in the mineralized nodules under normoxic conditions, while its expression was diffuse under hypoxic conditions, suggesting that the nodules were hypoxic zones even in normoxic cultures. These findings suggest that a low oxygen tension promotes osteoblastic differentiation and subsequent transformation to osteocytes.

The triple composite of rhBMP-2, PLA-PEG, and IP-CHA promotes the repair of full-thickness articular cartilage defects within as short a period as 3 weeks in the rabbit model. Hence, this novel cell-free implant biotechnology could mark a new development in the field of articular cartilage repair.

The purpose of this study was to evaluate the retention and discontinuation reasons of seven biological disease-modifying antirheumatic drugs (bDMARDs) in a real-world setting of patients with rheumatoid arthritis (RA). 1,037 treatment courses with bDMARDs from 2009 to 2016 [female, 81.8%; baseline age, 59.6 y; disease duration 7.8 y; rheumatoid factor positivity 81.5%; Disease Activity Score in 28 joints using erythrocyte sedimentation rate (DAS28-ESR), 4.4; concomitant prednisolone 43.5% and methotrexate 68.6%; Bio-naïve, 57.1%; abatacept (ABT), 21.3%; tocilizumab (TCZ), 20.7%; golimumab (GLM), 16.9%; etanercept (ETN), 13.6%; adalimumab (ADA), 11.1%; infliximab (IFX), 8.5%; certolizumab pegol (CZP), 7.9%] were included in this multi-center, retrospective study. Drug retention and discontinuation reasons at 36 months were estimated using the Kaplan-Meier method and adjusted by potent confounders using Cox proportional hazards modeling. As a result, 455 treatment courses (43.9%) were stopped, with 217 (20.9%) stopping due to inefficacy, 113 (10.9%) due to non-toxic reasons, 86 (8.3%) due to toxic adverse events, and 39 (3.8%) due to remission. Drug retention rates in the adjusted model were as follows: total retention (ABT, 60.7%; ADA, 32.7%; CZP, 43.3%; ETN, 51.9%; GLM, 45.4%; IFX, 31.1%; and TCZ, 59.2%; P < 0.001); inefficacy (ABT, 81.4%; ADA, 65.7%; CZP, 60.7%; ETN, 71.3%; GLM, 68.5%; IFX, 65.0%; and TCZ, 81.4%; P = 0.015), toxic adverse events (ABT, 89.8%; ADA, 80.5%; CZP, 83.9%; ETN, 89.2%; GLM, 85.5%; IFX, 75.6%; and TCZ, 77.2%; P = 0.50), and remission (ABT, 95.5%; ADA, 88.1%; CZP, 91.1%; ETN, 97.5%; GLM, 94.7%; IFX, 86.4%; and TCZ, 98.4%; P < 0.001). In the treatment of RA, ABT and TCZ showed higher overall retention, and TCZ showed lower inefficacy compared to IFX, while IFX showed higher discontinuation due to remission compared to ABT, ETN, GLM, and TCZ in adjusted modeling.