FOXO1 transcription factor regulates chondrogenic differentiation through transforming growth factor ß1 signaling.

The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor ß1 (TGFß1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFß1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFß1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9 To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFß1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFß1 signaling.

Alignment factors affecting the medial meniscus extrusion increases the risk of osteoarthritis development.

PURPOSE: Varus alignment is known as one of the major causes of medial compartment osteoarthritis (OA). Medial meniscus extrusion also plays a critical role in the in the development of OA. However, studies on the exact relationship between alignment parameters and medial meniscus extrusion are limited. Therefore, this study aimed to investigate this relationship in patients with knee OA. METHODS: Based on a retrospective analysis of the outpatient magnetic resonance imaging (MRI) database, 190 knees were identified to be examined using weight-bearing, whole-leg radiographs and MRIs within 3 months from the first consultation. Subsequently, various parameters of lower leg alignment were measured, which affected the knee varus in radiographs. Finally, a statistical analysis was performed to assess the relationships between the OA grade, distance of medial meniscus extrusion (MME), and alignment parameters; hip-knee-ankle angle (HKAA), percentage of mechanical axis (% MA), medial proximal tibial angle (MPTA), and joint line convergence angle (JLCA). The subjects were divided according to the presence or absence of MME (Group A: MME distance below 3 mm, Group B: MME distance 3 mm and above) to assess the differences in each alignment parameter correlated with MME distance between the groups. RESULTS: MME distance significantly increased with OA grade progression. HKAA, % MA, MPTA, and JLCA significantly correlated with medial meniscus extrusion distance (r = - 0.21, - 0.23, - 0.16, 0.3, respectively). Multiple regression analysis of each significant alignment combined with age, sex, and body mass index revealed that HKAA, % MA, MPTA, and JLCA were significant independent factors of MME distance (P = 0.008, 0.0026, 0.011, 0.0001, respectively). These significant findings were reinforced in group B. In contrast, the correlation between alignment parameters and medial meniscus extrusion distance was not significant in group A. CONCLUSION: Varus alignment factors are related to MME distance especially in extruded meniscus knees, as the OA grade progressed. Therefore, the coexistence of varus alignment and MME can be the risk factors for OA progression. As the low MPTA was an independent alignment factor for generating varus alignment, patients with osteoarthritis of the knee with both, low MPTA and MME could be the appropriate candidates for early intervention by high tibial osteotomy. LEVEL OF EVIDENCE: III.

Deposits from Creams Containing 20% (w/w) Urea and Suppression of Crystallization (Part 1): Rate of Crystallization from Cream Containing 20% (w/w) Urea and Evaluation of the Properties of the Deposit.

Two creams containing 20% (w/w) urea and various emulsifiers, a nonionic surfactant (NS) and lecithin (LEC), were prepared, and the rate of crystallization following application of the cream and differences in the properties of the deposits were investigated. Post-application crystallization was slower with the LEC formulation. Differences in the crystals obtained from the two formulations and from a 20% aqueous solution of urea were evaluated by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), powder X-ray-DSC (PXRD-DSC) and Fourier transform infrared spectrophotometry (FT-IR). PXRD and PXRD-DSC measurements showed that the diffraction patterns of both formulations differed from that of urea. The NS formulation provided diffraction peaks for urea and a urea composite, whereas only the urea composite was evident in the LEC formulation. DSC scans of urea showed an endotherm at around 134°C, whereas the deposits from both formulations provided an endotherm 23-25°C below that of urea; the NS formulation also showed a peak at around 140°C. These results indicate a tendency for urea crystallization in the NS formulation. FT-IR measurements showed that both deposits have a urea-based structure. The effects of the LEC formulation components on the physical properties of urea were investigated by PXRD and showed that all diffraction peaks were evenly weakened, suggesting that urea tends to be amorphous and that the formulation impacts post-application urea crystallization. Consequently, the amorphous state of urea can be maintained post-application by optimizing the formulation, thereby increasing the clinical efficacy of the cream.

Deposits from Creams Containing 20% (w/w) Urea and Suppression of Crystallization (Part 2): Novel Analytical Methods of Urea Accumulated in the Stratum Corneum by Tape stripping and Colorimetry.

The transfer of urea from a urea formulation to the stratum corneum varies with the formulation base and form, and impacts the formulation's therapeutic effect. Consequently, determining the amount of urea transferred is essential for developing efficient formulations. This study assessed a simple method for measuring the amount of urea accumulated in the stratum corneum. Conventional methods rely on labeling urea used in the formulation with radiocarbon ((14)C) or other radioactive isotopes (RIs), retrieving the transferred urea from the stratum corneum by tape stripping, then quantitating the urea. The handling and use of RIs, however, is subject to legal regulation and can only be performed in sanctioned facilities, so methods employing RIs are neither simple nor convenient. We therefore developed a non-radiolabel method "tape stripping-colorimetry (T-C)" that combines tape stripping with colorimetry (urease-glutamate dehydrogenase (GLDH)) for the quantitative measurement of urea. Urea in the stratum corneum is collected by tape stripping and measured using urease-GLDH, which is commonly used to measure urea nitrogen in blood tests. The results indicate that accurate urea measurement by the T-C method requires the application of 1400 mg (on hairless rats) of a 20% urea solution on a 50 cm(2) (5×10 cm) area. Further, we determined the amount of urea accumulated in the stratum corneum using formulations with different urea concentrations, and the time course of urea accumulation from formulations differing in the rate of urea crystallization. We demonstrate that the T-C method is simple and convenient, with no need for (14)C or other RIs.

Deposits from Creams Containing 20% (w/w) Urea and Suppression of Crystallization (Part 3): Novel Analytical Methods Based on Raman Spectroscopy for the Characterization of Deposits and Deposition Phenomena of Creams Containing 20% (w/w) Urea.

In drug formulations for external application, variations in the state of pharmaceutical agents within the base formulation may affect the transfer of agents to the skin. Here, we use Raman spectroscopic methods to acquire more detailed information on the state of the active pharmaceutical ingredients within an externally applied formulation. The combination of wide-field Raman spectroscopy with an experimental method in which drug formulations are applied to glass surfaces provided a new method for characterizing the state of pharmaceutical agents within drug formulations. Here, we demonstrate the usefulness of this new method, called application to glass-wide-field Raman spectroscopy (AG-WRS). In addition to allowing rapid and easy wide-field observations, the use of WRS allows Raman imaging in a manner that is insensitive to variations in the thickness of the formulations applied to sample slides. We consider two types of urea-compound creams with different crystal deposition rates, using AG-WRS to characterize the post-application time-evolving state of deposited crystals. Differences in the base pharmaceutical produce different spectra for the deposits, indicating that the deposits differ in composition and structure. In addition, we use microscopic laser Raman measurements to demonstrate that the process of crystal formulation differs significantly for formulations with different compositions. Our results demonstrate that the combination of AG-WRS with existing analytical techniques such as powder X-ray diffraction or thermal analysis yields more detailed and timely post-application information on the state of pharmaceuticals in external application. We believe this will be a valuable analytical tool for future studies related to the development of external application.

Fluvastatin promotes chondrogenic differentiation of adipose-derived mesenchymal stem cells by inducing bone morphogenetic protein 2.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADMSCs) are a promising source of material source for medical regeneration of cartilage. Growth factors, including transforming growth factor-ß (TGFß) subfamily members and bone morphogenetic proteins (BMPs), play important roles in inducing and promoting chondrogenic differentiation of MSCs. However, these exogenous growth factors have some drawbacks related to their cost, biological half-life, and safety for clinical application. Several studies have reported that statins, the competitive inhibitors of 3-hydroxy-2-methylglutaryl coenzyme A (HMG-CoA) reductase, induce the expression of BMP2 in multiple cell types as the pleotropic effects. The objective of this study was to investigate the effects of fluvastatin during chondrogenic differentiation of human ADMSCs (hADMSCs). METHODS: The effects of fluvastatin were analyzed during chondrogenic differentiation of hADMSCs in the pellet culture without exogenous growth factors by qRT-PCR and histology. For functional studies, Noggin, an antagonist of BMPs, mevalonic acid (MVA) and geranylgeranyl pyrophosphate (GGPP), metabolites of the mevalonate pathway, ROCK inhibitor (Y27632), or RAC1 inhibitor (NSC23766) were applied to cells during chondrogenic differentiation. Furthermore, RhoA activity was measured by RhoA pulldown assay during chondrogenic differentiation with or without fluvastatin. Statistically significant differences between groups were determined by Student's t-test or the Tukey-Kramer test. RESULTS: Fluvastatin-treated cells expressed higher levels of BMP2, SOX9, ACAN, and COL2A1 than control cells, and accumulated higher levels of glycosaminoglycans (GAGs). Noggin significantly inhibited the fluvastatin-mediated upregulation of ACAN and COL2A1. Both MVA and GGPP suppressed the effects of fluvastatin on the expressions of BMP2, SOX9, ACAN, and COL2A1. Furthermore, fluvastatin suppressed the RhoA activity, and inhibition of RhoA-ROCK signaling by Y27632 increased the expressions of BMP2, SOX9, ACAN, and COL2A1, as well as fluvastatin. CONCLUSIONS: Our results suggest that fluvastatin promotes chondrogenic differentiation of hADMSCs by inducing endogenous BMP2, and that one of the mechanisms underlying the effects is inhibition of RhoA-ROCK signaling via suppression of GGPP. Fluvastatin is a safe and low-cost compound that holds promise for use in transplantation of hADMSCs for cartilage regeneration.

The influence of post-operative knee coronal alignment parameters on long-term patient-reported outcomes after closed-wedge high tibial osteotomy.

BACKGROUND: Both intra-articular and extra-articular knee alignment pathologies can affect clinical outcomes after high tibial osteotomy. The purpose of this study was to investigate post-operative knee coronal alignment parameters that affect long-term patient-reported outcomes after closed-wedge high tibial osteotomy (CW-HTO). METHODS: This study included 105 osteoarthritic knees that underwent CW-HTO. Long-term patient-reported outcomes were defined by the 2011 Knee Society Score (KSS) and were collected at an average follow-up of 10.2 years. Post-operative knee coronal alignment parameters, consisting of the femoral tibial angle (FTA), lateral distal femoral angle (LDFA), medial proximal tibial angle (MPTA), tibial plateau inclination (TPI), and joint line convergence angle (JLCA), were measured using standing radiographs of the knee at an average follow-up of 11.0 months. The correlations between these parameters and KSS were then assessed by Spearman's correlation analysis. Comparisons of groups classified by MPTA and TPI cutoff values were performed by the Wilcoxon rank-sum test. RESULTS: Post-operative JLCA showed a significant negative correlation with two KSS sub-scores: satisfaction (R = -0.2232, P = 0.022) and total function (R = -0.2059, P = 0.035). There was no significant difference in any KSS sub-score between the "low" groups (MPTA and TPI less than 95 and 5°, respectively) and the "high" groups (MPTA and TPI greater than 98 and 7°, respectively). CONCLUSIONS: Among knee coronal alignment parameters, a large post-operative residual JLCA, which is an intra-articular varus deformity, was independently associated with worsened long-term clinical outcomes after CW-HTO. LEVEL OF EVIDENCE: Level Ⅳ, Retrospective cohort study.

GRK5 Inhibition Attenuates Cartilage Degradation via Decreased NF-κB Signaling.

OBJECTIVE: NF-κB-dependent signaling is an important modulator in osteoarthritis (OA), and G protein-coupled receptor kinase 5 (GRK5) regulates the NF-κB pathway. This study was undertaken to investigate the functional involvement of GRK5 in OA pathogenesis. METHODS: GRK5 expression in normal and OA human knee joints was analyzed immunohistochemically. Gain- or loss-of-function experiments were performed using human and mouse chondrocytes. OA was induced in GRK5-knockout mice by destabilization of the medial meniscus, and histologic examination was performed. OA was also induced in wild-type mice, which were then treated with an intraarticular injection of amlexanox, a selective GRK5 inhibitor, every 5 days for 8 weeks. RESULTS: GRK5 protein expression was increased in human OA cartilage. In vitro, expression levels of OA-related factors and NF-κB transcriptional activation were down-regulated by suppression of the GRK5 gene in human OA chondrocytes (3.49-fold decrease in IL6 [P < 0.01], 2.43-fold decrease in MMP13 [P < 0.01], and 2.66-fold decrease in ADAMTS4 [P < 0.01]). Conversely, GRK5 overexpression significantly increased the expression of OA-related catabolic mediators and NF-κB transcriptional activation. On Western blot analysis, GRK5 deletion reduced IκBα phosphorylation (up to 4.4-fold decrease [P < 0.05]) and decreased p65 nuclear translocation (up to 6.4-fold decrease [P < 0.01]) in mouse chondrocytes. In vivo, both GRK5 deletion and intraarticular amlexanox protected mouse cartilage against OA. CONCLUSION: Our results suggest that GRK5 regulates cartilage degradation through a catabolic response mediated by NF-κB signaling, and is a potential target for OA treatment. Furthermore, amlexanox may be a major compound in relevant drugs.

Single intra-articular injection of fluvastatin-PLGA microspheres reduces cartilage degradation in rabbits with experimental osteoarthritis.

Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme of the mevalonate pathway. The anti-inflammatory effect of statins has been reported in recent years. The present study investigated therapeutic effects of the local administration of statin in osteoarthritis (OA). We assessed clinically used statins and selected fluvastatin for further experimentation, as it showed potent anabolic and anti-catabolic effects on human OA chondrocytes. To achieve controlled intra-articular administration of statin, we developed an intra-articular injectable statin using poly(lactic-co-glycolic acid) (PLGA) as a drug delivery system (DDS). The release profile of the statin was evaluated in vitro. Finally, therapeutic effects of fluvastatin-loaded PLGA microspheres (FLU-PLGA) were tested in a rabbit OA model. Rabbit knees were divided into four subgroups: group 1-A, PLGA-treated group; group 1-B, PLGA contralateral saline control group; group 2-A, FLU-PLGA-treated group; and group 2-B, FLU-PLGA contralateral saline control group. Histological analysis 5 weeks after intra-articular injection revealed that OARSI scores were lower in group 2-A. No significant differences in OARSI scores were observed between groups 1-A, 1-B, and 2-B. This study indicates that a single intra-articular injection of fluvastatin-loaded PLGA microspheres could be a novel therapeutic approach for treating patients with OA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2465-2475, 2017.