Cyclic compressive loading induces a mature meniscal cell phenotype in mesenchymal stem cells with an atelocollagen-based scaffold.

Introduction: Biomechanical stimulation is reportedly pivotal in meniscal regeneration, although its effect on mesenchymal stem cell (MSC) meniscal differentiation remains elusive. In this study, we investigated how cyclic compressive loading (CCL) could impact MSCs using three-dimensional cultures in atelocollagen-based meniscal substitute (ACMS). Methods: We extracted MSCs from the meniscus, synovium, and articular cartilage, cultured them in three-dimensional cultures, and exposed them to CCL for 7 days. We then compared the transcriptomes of MSCs treated with and without CCL. Results: Our RNA-seq analysis revealed that CCL induced significant transcriptome changes, significantly affecting chondrocyte-related genes, including SOX9, TGFB1, and PRG4 upregulation. CCL induced transcriptional differentiation of meniscus progenitors toward mature meniscal cells. Conclusion: This study unveils the potential of mechanical stress in promoting MSC meniscal differentiation within ACMS. Our investigations provide new insights for mechanisms underlying meniscal regeneration with ACMS.

Basic fibroblast growth factor promotes meniscus regeneration through the cultivation of synovial mesenchymal stem cells via the CXCL6-CXCR2 pathway.

OBJECTIVE: To investigate the efficacy of basic fibroblast growth factor (bFGF) in promoting meniscus regeneration by cultivating synovial mesenchymal stem cells (SMSCs) and to validate the underlying mechanisms. METHODS: Human SMSCs were collected from patients with osteoarthritis. Eight-week-old nude rats underwent hemi-meniscectomy, and SMSCs in pellet form, either with or without bFGF (1.0 × 106 cells per pellet), were implanted at the site of meniscus defects. Rats were divided into the control (no transplantation), FGF (-) (pellet without bFGF), and FGF (+) (pellet with bFGF) groups. Different examinations, including assessment of the regenerated meniscus area, histological scoring of the regenerated meniscus and cartilage, meniscus indentation test, and immunohistochemistry analysis, were performed at 4 and 8 weeks after surgery. RESULTS: Transplanted SMSCs adhered to the regenerative meniscus. Compared with the control group, the FGF (+) group had larger regenerated meniscus areas, superior histological scores of the meniscus and cartilage, and better meniscus mechanical properties. RNA sequencing of SMSCs revealed that the gene expression of chemokines that bind to CXCR2 was upregulated by bFGF. Furthermore, conditioned medium derived from SMSCs cultivated with bFGF exhibited enhanced cell migration, proliferation, and chondrogenic differentiation, which were specifically inhibited by CXCR2 or CXCL6 inhibitors. CONCLUSION: SMSCs cultured with bFGF promoted the expression of CXCL6. This mechanism may enhance cell migration, proliferation, and chondrogenic differentiation, thereby resulting in superior meniscus regeneration and cartilage preservation.

A novel anti-TNF-α drug ozoralizumab rapidly distributes to inflamed joint tissues in a mouse model of collagen induced arthritis.

In clinical studies, the next-generation anti-tumor necrosis factor-alpha (TNF-α) single domain antibody ozoralizumab showed high clinical efficacy shortly after the subcutaneous injection. To elucidate the mechanism underlying the rapid onset of the effects of ozoralizumab, we compared the biodistribution kinetics of ozoralizumab and adalimumab after subcutaneous injection in an animal model of arthritis. Alexa Fluor 680-labeled ozoralizumab and adalimumab were administered by subcutaneous injection once (2 mg/kg) at five weeks after induction of collagen-induced arthritis (CIA) in an animal arthritis model. The time-course of changes in the fluorescence intensities of the two compounds in the paws and serum were evaluated. The paws of the CIA mice were harvested at four and eight hours after the injection for fluorescence microscopy. Biofluorescence imaging revealed better distribution of ozoralizumab to the joint tissues than of adalimumab, as early as at four hours after the injection. Fluorescence microscopy revealed a greater fluorescence intensity of ozoralizumab in the joint tissues than that of adalimumab at eight hours after the injection. Ozoralizumab showed a significantly higher absorption rate constant as compared with adalimumab. These results indicate that ozoralizumab enters the systemic circulation more rapidly and is distributed to the target tissues earlier and at higher levels than conventional IgG antibodies. Our investigation provides new insight into the mechanism underlying the rapid onset of the effects of ozoralizumab in clinical practice.

Meniscus surface texture is associated with degenerative changes in biological and biomechanical properties.

Meniscal degeneration is defined by semi-quantitative assessment of multiple histological findings and has been implicated in biomechanical dysfunction, yet little is known about its relationship with biological properties. This paper aimed to quantitatively evaluate degenerative findings in human meniscus to examine their relationship with gene expression and biomechanical properties, and to extract histological findings that reflect biological properties like gene expression and cytokine secretion. This study included lateral menisci of 29 patients who underwent total knee arthroplasty. The menisci were divided into six samples. For each sample, Pauli's histological evaluation and corresponding quantitative assessment (surface roughness, DNA content, collagen orientation, and GAG content) were performed, with surface roughness showing the highest correlation with the histological evaluation in a single correlation analysis (r = 0.66, p < 0.0001) and multivariate analysis (p < 0.0001). Furthermore, surface roughness was associated with gene expression related to meniscal degeneration and with tangent modulus which decreases with increasing degeneration (r = - 0.49, p = 0.0002). When meniscal tissue was classified by surface integrity, inflammatory cytokine secretion tended to be higher in severe degenerated menisci. These results suggest that the evaluation of meniscal surface texture could predict the degree of degeneration and inflammatory cytokine secretion.

Temporary Renal Enlargement in Children with a First Episode of Febrile Urinary Tract Infection is a Significant Risk of Recurrent Infection.

PURPOSE: Although morphological renal abnormalities in children with febrile urinary tract infection (fUTI) have been showed a predictive factor for recurrent infection, there are no available data on recurrence regarding sonographic renal enlargement at first fUTI episode, especially focusing on whether renal enlargement is temporary or not. MATERIALS AND METHODS: This cohort study reviewed the medical records of children who underwent renal ultrasound during their first fUTI during 2005-2013 and who were aged <15 years at diagnosis. We defined a kidney as temporary enlarged when the kidney length was ≥2 standard deviation above normal renal length for that age on sonography or a difference of ≥1 cm in sonographic length between the right and left kidneys, following normal renal length after antibiotic treatment. RESULTS: A total of 132 children were enrolled, of whom 11 had sonographic temporary temporal renal enlargement during their first fUTI. After completing antibiotic therapy for a first fUTI episode, 20 (15%) children had fUTI recurrence. The clinical characteristics at first episode of fUTI were not significantly different between renal enlargement and nonrenal enlargement groups. Children with temporary renal enlargement at a first fUTI episode had significantly lower fUTI recurrence-free survival proportion than those with nonrenal enlargement according to the Kaplan-Meier method (p = 0.003) Conclusion: Identification of temporary temporal renal enlargement as a predictor of recurrent fUTI may help identify children with a first episode of fUTI who will be warned of close monitoring.

Effects of iguratimod on glucocorticoid-induced disorder of bone metabolism in vitro.

INTRODUCTION: Glucocorticoids are widely used to treat various diseases including rheumatoid arthritis (RA); however, one of the most frequent and severe adverse effects is glucocorticoid-induced osteoporosis (GIOP). Iguratimod (IGU) is a novel conventional synthetic disease-modifying anti-rheumatic drug developed in Japan. The aim of this study is to investigate the effects of IGU on glucocorticoid-induced disorder of bone metabolism in vitro. MATERIALS AND METHODS: In osteoclastogenesis of mouse bone marrow-derived cells, tartrate-resistant acid phosphatase staining, resorption pit assay, western blotting, real-time polymerase chain reaction (PCR), and mRNA sequencing were performed. In osteoblastogenesis of MC3T3-E1 cells, alkaline phosphatase (ALP) staining and activity, alizarin red staining, and mRNA sequencing were performed, and real-time PCR and western blotting were conducted in MC3T3-E1 cells and murine osteocyte-like cell line MLO-Y4 cells. RESULTS: IGU significantly suppressed a dexamethasone-induced increase in osteoclasts, differentiation, and bone resorption activity by inhibition of the receptor activator of the nuclear factor kappa-B (RANK)/tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)/nuclear factor kappa-B (NFκB)-p52 pathway. In MC3T3-E1 cells, IGU significantly upregulated dexamethasone-induced downregulation of ALP activity, bone mineralization, and osteoblast-related gene and protein expression. In MLO-Y4 cells, IGU significantly upregulated dexamethasone-induced downregulation of the gene expression of ALP and osteocalcin, and also downregulated receptor activator of NFκB ligand (RANKL)/osteoprotegerin gene expression ratio without dexamethasone. CONCLUSION: These results suggest that IGU may improve glucocorticoid-induced disorder of bone metabolism and may exhibit positive effects against GIOP associated with RA.

Integrin α2ß1 plays an important role in the interaction between human articular cartilage-derived chondrocytes and atelocollagen gel.

Although atelocollagen gel is used as a scaffold for culturing human articular cartilage-derived chondrocytes, little is known about cell-gel interactions. In this study, we investigated the mechanism via which atelocollagen gel affects human articular cartilage-derived chondrocytes. Two types of three-dimensional cultures of human articular cartilage-derived chondrocytes (i.e., with and without atelocollagen gel) were compared. While the amount of atelocollagen gel in culture gradually decreased with time, it promoted the expression of matrix metalloproteinases (MMPs) during the early stages of culture. Genome-wide differential gene expression analysis revealed that cell membrane- and extracellular matrix-related genes were highly ranked among up- and down-regulated groups in cells cultured in the presence of atelocollagen gel. Among the integrin family of genes, the expression of integrin subunit alpha 2 and integrin subunit alpha 10 was significantly increased in the presence of atelocollagen gel. Blocking α2ß1 integrin with the specific inhibitor BTT 3033 had a significant effect on cell proliferation, MMP expression, and cell shape, as well as on the response to mechanical stimulation. Taken together, our findings indicate that the α2ß1 integrin pathway plays an important role in the interaction of atelocollagen gel with human articular cartilage-derived chondrocytes and may be a potential therapeutic target for articular cartilage disorders.

Pathophysiological Role of Transient Receptor Potential Ankyrin 1 in a Mouse Long-Lasting Cystitis Model Induced by an Intravesical Injection of Hydrogen Peroxide.

Chronic inflammatory bladder disorders, such as interstitial cystitis/bladder pain syndrome, are associated with poor quality of life. The exact pathological processes remain unclear, but accumulating evidence suggests that reactive oxidative species (ROS) are involved in urinary bladder disorders. Transient receptor potential ankyrin 1 (TRPA1), the most sensitive TRP channel to ROS, was shown to be responsible for urinary bladder abnormalities and hyperalgesia in an acute cystitis model. However, the roles of TRPA1 in chronic inflammatory bladder are not fully understood. We previously established a novel mouse cystitis model induced by intravesical injection of hydrogen peroxide (H2O2), resulting in long-lasting frequent urination, bladder inflammation, pain-related behavior, and histopathological changes. In the present study, we investigated the pathophysiological role of TRPA1 in the H2O2-induced long-lasting cystitis mouse model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female wild-type (WT) and TRPA1-knockout mice and maintained for 30 min. This increased the number of voids in WT mice at 1 and 7 days after injection, but reduced the number in TRPA1-knockout mice at 1 day but not 7 days after injection. Spontaneous locomotor activities (increase in freezing time and decrease in distance moved) were reduced at 3 h after injection in WT mice, whereas the spontaneous visceral pain-related behaviors were attenuated in TRPA1-knockout mice. Furthermore, upregulation of c-fos mRNA in the spinal cord at 1 day after injection was observed in WT but not TRPA1-knockout mice. However, there was no difference in histopathological changes in the urinary bladder, such as edematous thickening in the submucosa, between WT and TRPA1-knockout mice at 1 or 7 days after injection. Finally, Trpa1 mRNA levels in the L5-S1 dorsal root ganglion were not altered, but levels in the urinary bladder were drastically increased at 1 and 7 days after injection. Taken together, these results suggest that TRPA1 contributes to acute bladder hyperactivity such as frequent urination and bladder pain, but does not appear to play a major role in the pathological processes of long-lasting cystitis.

A rat long-lasting cystitis model induced by intravesical injection of hydrogen peroxide.

Novel longer lasting inflammatory bladder animal models are needed to better understand the pathophysiology of chronic cystitis. We previously developed a relatively long-lasting mouse cystitis model by intravesical injection of hydrogen peroxide (H2O2). To further evaluate its pathophysiology, in this study, we established and analyzed a rat cystitis model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female rats, and kept for 30 min. The H2O2 injection significantly increased the number of micturition events up to day 14 and decreased urine volume per micturition, with the smallest volumes on day 3, compared with the vehicle-treated group. Cystometric analysis on day 7 revealed that intercontraction intervals were significantly shortened without affecting the baseline, threshold, or maximum pressures. Intravesical resiniferatoxin-evoked nociceptive behaviors, such as freezing, were significantly enhanced on days 7 and 14. Furthermore, histopathology revealed hemorrhage, edema, infiltration of neutrophils into the lamina propria, and urothelial denudation in the early phase (day 1). These damages were gradually repaired, while hyperplasia of the urothelium, vascularization, increases in fibroblast counts, and infiltration of mast cells and eosinophils were observed through the later phase (days 7 and 14). These results suggest that intravesical H2O2 injection induces relatively long-lasting cystitis with enhanced bladder activity and pain sensation in rats. This approach thus provides a novel rat long-lasting cystitis model that allows us to analyze detailed symptoms and pathophysiology of H2O2-induced cystitis model than the mouse model and may be used to investigate the pathophysiology and treatment of chronic bladder hypersensitive disorders, such as bladder pain syndrome/interstitial cystitis.

Long-lasting pain-related behaviors in mouse chronic cystitis model induced by a single intravesical injection of hydrogen peroxide.

We previously established a long-lasting cystitis model by an intravesical injection of hydrogen peroxide (H2O2) into mice. In this study, we assessed the pain-related behaviors in the cystitis model. An intravesical injection of 1.5% H2O2 transiently decreased spontaneous locomotor activity at 3 h after injection, indicative of acute spontaneous pain. In contrast, licking response to a bladder distention was slowly observed as licks to the lower abdomen at 7 and 14 days after injection, which was attenuated by amitriptyline and morphine, but not by oxybutynin. These results suggest that H2O2-induced chronic cystitis model shows delayed and long-lasting painful pathological condition.

[Vagal Nerve Schwannoma Complicated with Bloody Pleural Effusion；Report of a Case].

A 51-year-old male who had received hemodialysis twice a week was referred to our hospital for a further examination of bloody pleural effusion in the right chest. He has been suffering from a fever and cough for 2 months. Chest computed tomography and magnetic resonance imaging revealed a pleural effusion in the right pleural cavity and posterior mediastinal tumor in paravertebral lesion. Chest drainage was performed, and cytological diagnosis did not show malignant findings. To make a definite diagnosis and treatment, surgical resection was carried out. During surgery, posterior mediastinal tumor originated from vagal nerve, and a schwannoma was diagnosed by frozen section. After resection, postoperative course was uneventful, and bloody pleural effusion disappeared.