Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems

Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32° C, 37° C, and 41° C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32° C and 37° C in pellet cultures, but the levels were significantly lower at 41° C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37° C than at 32° C and 41° C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32° C was maintained or enhanced compared to that at 37° C. However, chondrogenesis-related genes were further induced at 37° C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis

The effects of short-term hypoxia on human mesenchymal stem cell proliferation, viability and p16(INK4A) mRNA expression: Investigation using a simple hypoxic culture system with a deoxidizing agent

A hypoxic environment is thought to be important for the maintenance of stemness and suppressing cell senescence, in stem cells. Therefore, a hypoxic condition is induced during cell expansion and/or induction of intended differentiation. However, the induction of these conditions requires a specially equipped hypoxia chamber and expensive gas mixtures, which are expensive and space-consuming. Owing to these restrictions, appropriate hypoxic conditions cannot be provided during cell transportation, which is increasingly required for regenerative medicine. Hence, a simple and economical culture system is required. The purpose of this study was to investigate the effects of short-term hypoxic conditions on human mesenchymal stem cell (MSC) proliferation, viability, and senescence, utilizing the CulturePal system (CulturePal-Zero and CulturePal-Five), a novel and simple hypoxic culture system with a built-in deoxidizing agent. The O₂ concentration in the CulturePal-Zero was observed to reduce to <0.1% within 1 h, and to 5% within 24 h in the CulturePal-Five system. Cell proliferation under these hypoxic conditions showed a sharp increase at 5% O₂ concentration, and no noticeable cell death was observed even at severe hypoxic conditions (<0.1% O₂) up to 72h. The p16INK4A (cell senescence marker) mRNA expression was retained under hypoxic conditions up to 72h, but it was up-regulated under normoxic conditions. Interestingly, the p16INK4A expression altered proportionately to the O₂ concentration. These results indicated that the short-term hypoxic condition, at an approximate O₂ concentration of 5%, would be suitable for promoting cell proliferation and repressing cell senescence, without aggravating the MSC viability. Therefore, the CulturePal systems may be suitable for providing an appropriate hypoxic condition in stem cell research and transportation

Immature articular cartilage and subchondral bone covered by menisci are potentially susceptive to mechanical load

BACKGROUND: The differences of mechanical and histological properties between cartilage covered by menisci and uncovered by menisci may contribute to the osteoarthritis after meniscectomy and these differences are not fully understood. The purpose of this study is to investigate potential differences in the mechanical and histological properties, and in particular the collagen architecture, of the superficial cartilage layer and subchondral bone between regions covered and uncovered by menisci using immature knee. METHODS: Osteochondral plugs were obtained from porcine tibial cartilage that was either covered or uncovered by menisci. Investigation of the thickness, mechanical properties, histology, and water content of the cartilage as well as micro-computed tomography analysis of the subchondral bone was performed to compare these regions. Collagen architecture was also assessed by using scanning electron microscopy. RESULTS: Compared to the cartilage uncovered by menisci, that covered by menisci was thinner and showed a higher deformity to compression loading and higher water content. In the superficial layer of cartilage in the uncovered regions, collagen fibers showed high density, whereas they showed low density in covered regions. Furthermore, subchondral bone architecture varied between the 2 regions, and showed low bone density in covered regions. CONCLUSIONS: Cartilage covered by menisci differed from that uncovered in both its mechanical and histological properties, especially with regards to the density of the superficial collagen layer. These regional differences may be related to local mechanical environment in normal condition and indicate that cartilage covered by menisci is tightly guarded by menisci from extreme mechanical loading. Our results indicate that immature cartilage degeneration and subchondral microfracture may occur easily to extreme direct mechanical loading in covered region after meniscectomy

Contributions of biarticular myogenic components to the limitation of the range of motion after immobilization of rat knee joint

BACKGROUND: Muscle atrophy caused by immobilization in the shortened position is characterized by a decrease in the size or cross-sectional area (CSA) of myofibers and decreased muscle length. Few studies have addressed the relationship between limitation of the range of motion (ROM) and the changes in CSA specifically in biarticular muscles after atrophy because of immobilization. We aimed to determine the contribution of 2 distinct muscle groups, the biarticular muscles of the post thigh (PT) and those of the post leg (PL), to the limitation of ROM as well as changes in the myofiber CSAs after joint immobilization surgery. METHODS: Male Wistar rats (n = 40) were randomly divided into experimental and control groups. In the experimental group, the left knee was surgically immobilized by external fixation for 1, 2, 4, 8, or 16 weeks (n = 5 each) and sham surgery was performed on the right knee. The rats in the control groups (n = 3 per time point) did not undergo surgery. After the indicated immobilization periods, myotomy of the PT or PL biarticular muscles was performed and the ROM was measured. The hamstrings and gastrocnemius muscles from the animals operated for 1 or 16 weeks were subjected to morphological analysis. RESULTS: In immobilized knees, the relative contribution of the PT biarticular myogenic components to the total restriction reached 80% throughout the first 4 weeks and decreased thereafter. The relative contribution of the PL biarticular myogenic components remained <20% throughout the immobilization period. The ratio of the myofiber CSA of the immobilized to that of the sham-operated knees was significantly lower at 16 weeks after surgery than at 1 week after surgery only in the hamstrings. CONCLUSIONS: The relative contribution of the PT and PL components to myogenic contracture did not significantly change during the experimental period. However, the ratio of hamstrings CSAs to the sham side was larger than the ratio of medial gastrocnemius CSAs to the sham side after complete atrophy because of immobilization

The Effect of Exercise on the Early Stages of Mesenchymal Stromal Cell-Induced Cartilage Repair in a Rat Osteochondral Defect Model

The repair of articular cartilage is challenging owing to the restriction in the ability of articular cartilage to repair itself. Therefore, cell supplementation therapy is possible cartilage repair method. However, few studies have verified the efficacy and safety of cell supplementation therapy. The current study assessed the effect of exercise on early the phase of cartilage repair following cell supplementation utilizing mesenchymal stromal cell (MSC) intra-articular injection. An osteochondral defect was created on the femoral grooves bilaterally of Wistar rats. Mesenchymal stromal cells that were obtained from male Wistar rats were cultured in monolayer. After 4 weeks, MSCs were injected into the right knee joint and the rats were randomized into an exercise or no-exercise intervention group. The femurs were divided as follows: C group (no exercise without MSC injection); E group (exercise without MSC injection); M group (no exercise with MSC injection); and ME group (exercise with MSC injection). At 2, 4, and 8 weeks after the injection, the femurs were sectioned and histologically graded using the Wakitani cartilage repair scoring system. At 2 weeks after the injection, the total histological scores of the M and ME groups improved significantly compared with those of the C group. Four weeks after the injection, the scores of both the M and ME groups improved significantly. Additionally, the scores in the ME group showed a significant improvement compared to those in the M group. The improvement in the scores of the E, M, and ME groups at 8 weeks were not significantly different. The findings indicate that exercise may enhance cartilage repair after an MSC intra-articular injection. This study highlights the importance of exercise following cell transplantation therapy

Association of Pain History and Current Pain With Sagittal Spinal Alignment and Muscle Stiffness and Muscle Mass of the Back Muscles in Middle-aged and Elderly Women

[Study Design] A cross-sectional study. [Objective] To investigate the association of low back pain history (LBPH) and LBP with sagittal spinal alignment, stiffness assessed using ultrasonic shear wave elastography, and mass of the back muscle in community-dwelling middle-aged and elderly women. [Summary of Background Data] The association of LBPH and LBP with sagittal spinal alignment, stiffness, and mass of the back muscles remains unclear in middle-aged and elderly women. [Participants and Methods] The study comprised 19 asymptomatic middle-aged and elderly women [control (CTR) group], 16 middle-aged and elderly women with LBPH (LBPH group), and 23 middle-aged and elderly women with LBP (LBP group). Sagittal spinal alignment in the standing and prone positions (kyphosis angle in the thoracic spine, lordosis angle in the lumbar spine, and anterior inclination angle in the sacrum) was measured using a Spinal Mouse. The stiffness of the back muscles (lumbar erector spinae and multifidus) in the prone position was measured using ultrasonic shear wave elastography. The mass of the back muscles (thoracic and lumbar erector spinae, lumbar multifidus, and quadratus lumborum) was also measured. [Results] Multiple logistic regression analysis with a forward selection method showed that the stiffness of the lumbar multifidus muscle was a significant and independent factor of LBPH. The stiffness of the lumbar multifidus muscle was significantly higher in the LBPH group than in the CTR group. Multiple logistic regression analysis also indicated that lumbar lordosis angle in the standing position was a significant and independent factor of LBP. The lumbar lordosis angle was significantly smaller in the LBP group than in the CTR group. [Conclusions] Our results suggest that LBPH is associated with increased stiffness of the lumbar multifidus muscle in the prone position, and that LBP is associated with the decreased lumbar lordosis in the standing position in community-dwelling middle-aged and elderly women

Evaluation of Setup Errors at the Skin Surface Position for Whole Breast Radiotherapy of Breast Cancer Patients

We used image-processing software to analyze the setup errors at the skin surface position of breast cancer patients (n=66) who underwent post-operative whole breast irradiation at our hospital in 2014-2015. The sixty-six digital reconstructed radiographs (DRR) were created at the treatment planning for each patient. The lineacgraphies (n=377) were taken after the patients’ setup during radiotherapy. The lineacgraphies and DRR were superimposed at the skin surface position for each patient with the image-processing software. We measured the deviations of the isocenters for the nipple-lung (X) direction and craniocaudal (Y) direction and the deviation of the rotation angle of the XY axes between the lineacgraphy and DRR on the superimposed images. The systematic error (μ, Σ) and random error (σ) were calculated from the X and Y deviations and rotation angle deviation. The μ of X, Y, and rotation angle were 0.01 mm, −1.2 mm, and 0.05°, respectively. The Σ of X, Y, and rotation angle were 1.8 mm, 1.5 mm, and 0.9°, respectively. The σ of X, Y, and rotation angle were 2.0 mm, 1.5 mm, and 1.0°, respectively. Our analyses thus revealed that evaluations using image-processing software at the skin surface position in routine breast radiotherapy result in sufficiently small setup errors

Folliculotropic mycosis fungoides treated with electron beam therapy that evolved into fatal, tumor-stage mycosis fungoides and erythroderma with multiple ulcerations

　A 71-year-old woman diagnosed with mycosis fungoides with multiple erythematous plaques and follicular papules on the scalp, trunk, and thigh was referred to our institution. Folliculotropic mycosis fungoides was histologically diagnosed, and the erythematous papules and plaques regressed temporarily after total-skin electron beam therapy. The patient then developed tumors and erythroderma. The area of painful erosion spread, and her condition rapidly worsened. The patient died 3 years and 4 months after the first examination due to multiple organ failure caused by sepsis. The cause of rapid evolution into erythroderma remains elusive and requires further investigation in similar cases

Field measurements of absolute gravity in East Antarctica

This paper reports the results of field-based absolute gravity measurements aimed at detecting gravity change and crustal displacement caused by glacial isostatic adjustment. The project was initiated within the framework of the 53rd Japanese Antarctic Research Expedition (JARE53). Absolute gravity measurements, together with GPS measurements, were planned at several outcrops along the Prince Olav Coast and Sôya Coast of East Antarctica, including at Syowa Station. Since the icebreaker Shirase (AGB 5003) was unable to moor alongside Syowa Station, operations were somewhat restricted during JARE53. However, despite this setback, we were able to complete measurements at two sites: Syowa Station and Langhovde. The absolute gravity value at the Syowa Station IAGBN (A) site, observed using an FG-5 absolute gravimeter (serial number 210; FG-5 #210), was 982 524 322.7±0.1 μGal, and the gravity change rate at the beginning of 2012 was –0.26 μGal∙a-1. An absolute gravity value of 982 535 584.2±0.7 μGal was obtained using a portable A-10 absolute gravimeter (serial number 017; A-10 #017) at the newly located site AGS01 in Langhovde