Evidence that TD-198946 enhances the chondrogenic potential of human synovium-derived stem cells through the NOTCH3 signaling pathway.

Human synovium-derived stem cells (hSSCs) are an attractive source of cells for cartilage repair. At present, the quality of tissue and techniques used for cartilage regeneration have scope for improvement. A small compound, TD-198946, was reported to enhance chondrogenic induction from hSSCs; however, other applications of TD-198946, such as priming the cell potential of hSSCs, remain unknown. Our study aimed to examine the effect of TD-198946 pretreatment on hSSCs. HSSCs were cultured with or without TD-198946 for 7 days during expansion culture and then converted into a three-dimensional pellet culture supplemented with bone morphogenetic protein-2 (BMP2) and/or transforming growth factor beta-3 (TGFß3). Chondrogenesis in cultures was assessed based on the GAG content, histology, and expression levels of chondrogenic marker genes. Cell pellets derived from TD-198946-pretreated hSSCs showed enhanced chondrogenic potential when chondrogenesis was induced by both BMP2 and TGFß3. Moreover, cartilaginous tissue was efficiently generated from TD-198946-pretreated hSSCs using a combination of BMP2 and TGFß3. Microarray analysis revealed that NOTCH pathway-related genes and their target genes were significantly upregulated in TD-198946-treated hSSCs, although TD-198946 alone did not upregulate chondrogenesis related markers. The administration of the NOTCH signal inhibitor diminished the effect of TD-198946. Thus, TD-198946 enhances the chondrogenic potential of hSSCs via the NOTCH3 signaling pathway. This study is the first to demonstrate the gradual activation of NOTCH3 signaling during chondrogenesis in hSSCs. The priming of NOTCH3 using TD-198946 provides a novel insight regarding the regulation of the differentiation of hSSCs into chondrocytes.

Enhancement of chondrogenic differentiation supplemented by a novel small compound for chondrocyte-based tissue engineering.

PURPOSE: Chondrocyte -based tissue engineering has been a promising option for the treatment of cartilage lesions. In previous literature, TD198946 has been shown to promote chondrogenic differentiation which could prove useful in cartilage regeneration therapies. Our study aimed to investigate the effects of TD198946 in generating engineered cartilage using dedifferentiated chondrocyte-seeded collagen scaffolds treated with TD198946. METHODS: Articular chondrocytes were isolated from mini pig knees and expanded in 2-dimensional cell culture and subsequently used in the experiments. 3-D pellets were then cultured for two weeks. Cells were also cultured in a type I collagen scaffolds for four weeks. Specimens were cultured with TD198946, BMP-2, or both in combination. Outcomes were determined by gene expression levels of RUNX1, SOX9, ACAN, COL1A1, COL2A1 and COL10A1, the glycosaminoglycan content, and characteristics of histology and immunohistochemistry. Furthermore, the maturity of the engineered cartilage cultured for two weeks was evaluated through subcutaneous implantation in nude mice for four weeks. RESULTS: Addition of TD198946 demonstrated the upregulation of gene expression level except for ACAN, type II collagen and glycosaminoglycan synthesis in both pellet and 3D scaffold cultures. TD198946 and BMP-2 combination cultures showed higher chondrogenic differentiation than TD198946 or BMP-2 alone. The engineered cartilage maintained its extracellular matrices for four weeks post implantation. In contrast, engineered cartilage treated with either TD198946 or BMP-2 alone was mostly absorbed. CONCLUSIONS: Our results indicate that TD198946 could improve quality of engineered cartilage by redifferentiation of dedifferentiated chondrocytes pre-implantation and promoting collagen and glycosaminoglycan synthesis.

Effect of the small compound TD-198946 on glycosaminoglycan synthesis and transforming growth factor ß3-associated chondrogenesis of human synovium-derived stem cells in vitro.

As an alternative to chondrocytes-based cartilage repair, stem cell-based therapies have been investigated. Specifically, human synovium-derived stem cells (hSSCs) are a promising cell source based on their highly capacities for chondrogenesis, but some methodological improvements are still required towards optimal cartilage regeneration. Recently, a small compound, TD-198946, was reported to promote chondrogenesis of several stem cells, but the effect on hSSCs is still unknown. This study aimed to examine the effects of TD-198946 on chondrocyte differentiation and cartilaginous tissue formation with hSSCs. A range of concentrations of TD-198946 were examined in chondrogenic cultures of hSSC-derived cell pellets. The effect of TD-198946 on glycosaminoglycan (GAG) production, chondrocyte marker expression, and cartilaginous tissue formation was assessed. At concentrations >1 nM, TD-198946 dose-dependently enhanced GAG production, particularly hyaluronan, whereas chondrocyte differentiation was not impacted. When combined with transforming growth factor ß3 (TGFß3), TD-198946 promoted chondrocyte differentiation and production of cartilaginous matrices at doses <1 nM as judged by SOX9, S100, and type 2 collagen upregulation. Conversely, doses >1 nM TD-198946 attenuated TGFß3-associated chondrocyte differentiation, but aggrecan was efficiently produced at 1 to 10 nM TD-198946 as judged by safranin O staining. Thus, TD-198946 exhibited different dose ranges for either GAG synthesis or chondrocyte differentiation. Regarding use of TD-198946 for in vitro engineering of cartilage, cartilaginous particles rich in type 2 collagen and GAG were predominately created with TGFß3 + 0.25 nM TD-198946. These studies have demonstrated that TD-198946 synergistically enhances chondrogenesis of hSSCs in a unique dose range, and such findings may provide a novel strategy for stem cell-based cartilage therapy.

Enhanced repair of meniscal hoop structure injuries using an aligned electrospun nanofibrous scaffold combined with a mesenchymal stem cell-derived tissue engineered construct.

Damage to the meniscal hoop structure results in loss of biomechanical function, which potentially leads to the extrusion of the meniscus from the weight bearing area. However, there have been no established, effective treatments for such injuries. The purpose of this study was to investigate the applicability of cell-seeded nanofibrous scaffolds to repair the damaged meniscal hoop structure along with the prevention of subsequent cartilage degeneration using a rabbit model. Meniscal radial defects (5 mm width) in the medial meniscus were treated by wrapping and suturing with either an aligned electrospun nanofibrous scaffold alone or a scaffold combined with a tissue engineered construct (TEC) derived from synovial mesenchymal stem cells (MSCs), with the scaffold fiber direction matching that of the meniscal circumferential fibers. The MSC-based TEC-combined nanofibrous scaffolds contributed significantly to the prevention of meniscal extrusion and exerted a chondroprotective effect, compared with either scaffold alone or the untreated control groups. Also, meniscal defects treated with such TEC-combined nanofibrous scaffolds were consistently repaired with a fibrocartilaginous tissue. In this study, we have demonstrated the feasibility of a combined TEC-nanofibrous scaffold to repair the meniscal hoop structure, and prevent the progression to cartilage degeneration, as a potential tissue engineering method.

Meniscal repair and regeneration: Current strategies and future perspectives.

The management of meniscal injuries remains difficult and challenging. Although several clinical options exist for the treatment of such injuries, complete regeneration of the damaged meniscus has proved difficult due to the limited healing capacity of the tissue. With the advancements in tissue engineering and cell-based technologies, new therapeutic options for patients with currently incurable meniscal lesions now potentially exist. This review will discuss basic anatomy, current repair techniques and treatment options for loss of meniscal integrity. Specifically, we focus on the possibility and feasibility of the latest tissue engineering approaches, including 3D printing technologies. Therefore, this discussion will facilitate a better understanding of the latest trends in meniscal repair and regeneration, and contribute to the future application of such clinical therapies for patients with meniscal injuries.

Evaluation of the relationship between the minimum inhibitory concentration of ceftiofur and third-generation cephalosporins in Escherichia coli isolates from food-producing animals.

To enable future comparison of the antimicrobial susceptibility data between bacteria obtained from animals and humans, it is necessary to compare the relationships between minimum inhibitory concentrations (MICs) of veterinary and human medicine. We evaluated the relationship between the MIC of ceftiofur (CTF) and the MICs of other third-generation cephalosporins (TGCs): cefotaxime (CTX), cefpodoxime (CPDX), and ceftazidime (CAZ), determined by the broth microdilution method using 118 cefazolin-resistant Escherichia coli isolates from food-producing animals. Using the Clinical and Laboratory Standards Institute criteria, very major classification errors were observed only in CAZ (17.8%, 21 of 118); major and minor errors were observed in all TGCs (CTX: 0.8% [1 of 118] and 9.3% [11 of 118]; CPDX: 9.3% [11 of 118] and 6.8% [8 of 118]; CAZ: 2.5% [3 of 118] and 9.3% [11 of 118], respectively). The Spearman correlation coefficients between the MICs of CTF and CTX, CPDX, and CAZ were 0.765, 0.731, and 0.306, respectively. The sensitivity and specificity values were 100.0% and 81.8% for CTX, 99.0% and 27.3% for CPDX, and 76.0% and 86.4% for CAZ compared with CTF. The C-statistic was 0.978 for CTF and CTX, 0.953 for CPDX, and 0.798 for CAZ. For the TGCs evaluated in our study, testing for CTX susceptibility results showed the highest correlation with the results given when testing for CTF susceptibility.

Scaffold-free, stem cell-based cartilage repair.

Various approaches to treat articular cartilage have been widely investigated due to its poor intrinsic healing capacity. Stem cell-based therapy could be a promising approach as an alternative to chondrocyte-based therapy and some of these therapies have been already applied in clinical condition. This review discusses the current development of stem cell-based therapies in cartilage repair, specifically focusing on scaffold-free approaches.

Molecular Typing of Fluoroquinolone-Resistant Campylobacter jejuni Isolated from Broilers in Japan Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis.

Fluoroquinolone-resistant Campylobacter jejuni isolates from broilers in Japan were characterized using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) in order to elucidate the genetic relationship between these strains. Forty-three of the isolates were classified into 20 sequence types and were clustered into 21 PFGE types with 70% similarity. The most dominant clonal complex (CC) was CC-21 (41.9%). Diverse PFGE patterns were observed within the same CC, but the combined analysis of PFGE type and CC revealed that the strains with the same combination were isolated from the same district or neighboring districts. On the other hand, strains with the same combination pattern were also isolated from geographically distant districts. Our results elucidate two possible reasons for the prevalence of fluoroquinolone-resistant C. jejuni among broiler farms: (1) the resistant C. jejuni is clonally disseminated within the limited area, and (2) susceptible C. jejuni acquired fluoroquinolone resistance during the use of fluoroquinolone on the farms.

Decreased Resistance to Broad-Spectrum Cephalosporin in Escherichia coli from Healthy Broilers at Farms in Japan After Voluntary Withdrawal of Ceftiofur.

The high prevalence of broad-spectrum cephalosporin (BSC) resistance in Escherichia coli isolates from healthy broilers at farms is a source of grave concern in Japan. In an effort to solve this problem, the off-label use of ceftiofur (CTF) at hatcheries was voluntarily withdrawn around March 2012. The objective of this study was to assess the effect of the voluntary withdrawal on the prevalence of BSC resistance in E. coli from healthy broilers at farms. A total of 693 E. coli isolates collected from 362 fecal samples of healthy broilers at farms between 2010 and 2013 were examined to determine their antimicrobial resistance profiles and ß-lactamase genes. ß-Lactamase genes were characterized by polymerase chain reaction and sequencing. BSC resistance was detected in 84 of the 693 E. coli isolates (12.1%) from healthy broilers between 2010 and 2013. The percentage of BSC-resistant E. coli isolates was significantly decreased: from 16.4% (32/195) in 2010 and 16.8% (27/161) in 2011 to 9.2% (19/206) in 2012 and 4.6% (6/131) in 2013 (2010 versus 2012: p=0.024, 2010 versus 2013: p=0.001, 2011 versus 2012: p=0.038, and 2011 versus 2013: p=0.001). Regarding ß-lactamase genes, 58 of the 84 BSC-resistant E. coli isolates (69.0%) harbored blaCMY-2. The prevalence of BSC resistance in E. coli isolated from healthy broilers at farms was markedly decreased within a year after the voluntary withdrawal from CTF use at hatcheries. This indicates that BSC resistance in E. coli isolates from broilers could be controlled by restricting the use of CTF at the hatchery level.

Primary osteosarcoma of the heart with long-term survival: A case report of laparoscopic resection of a metastatic sarcoma in the intestine.

Primary cardiac tumors are uncommon and cardiac osteosarcoma is a rare disease. While complete surgical resection is considered to be the best treatment option for cardiac osteosarcomas, local and metastatic recurrences present challenges and indicate a poor prognosis. A combination of surgical resection with radio- and/or chemotherapy is a more effective course of treatment for osteosarcoma. In the present case, the patient underwent a complete resection of a primary cardiac osteosarcoma, and received chemotherapy and radiotherapy following local recurrence and metastasis to the bone post-operatively. Following these treatments, a rectal metastatic tumor was detected as causative of anemia. There is currently a lack of guidelines on the treatment of metastatic osteosarcomas in the intestine and there are few reports on rectal metastases. The present study described a laparoscopic resection of the osteosarcoma. The patient recovered without any complications and radiotherapy and chemotherapy were administered post-surgery to treat the bone metastases. The patient remained healthy at a follow-up examination, 61 months post surgery.

Control of the development and prevalence of antimicrobial resistance in bacteria of food animal origin in Japan: a new approach for risk management of antimicrobial veterinary medicinal products in Japan.

Antimicrobial agents are essential for controlling bacterial disease in food-producing animals and contribute to the stable production of safe animal products. The use of antimicrobial agents in these animals affects the emergence and prevalence of antimicrobial resistance in bacteria isolated from animals and animal products. As disease-causing bacteria are often transferred from food-producing animals to humans, the food chain is considered a route of transmission for the resistant bacteria and/or resistance genes. The Food Safety Commission of Japan (FSC) has been assessing the risk posed to human health by the transmission of antimicrobial-resistant bacteria from livestock products via the food chain. In addition to the FSC's risk assessments, the Japanese Ministry of Agriculture, Forestry and Fisheries has developed risk-management guidelines to determine feasible risk-management options for the use of antimicrobial veterinary medicinal products during farming practices. This report includes information on risk assessment and novel approaches for risk management of antimicrobial veterinary medicinal products for mitigating the risk of development and prevalence of antimicrobial resistance in bacteria originating from food-producing animals in Japan.