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

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

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

Crystallographic Characterization of Extraterrestrial Materials by Energy-Scanning X-ray Diffraction

We have continued our long-term project using X-ray diffraction to characterize a wide range of extraterrestrial samples. The stationary sample method with polychromatic X-rays is advantageous because the irradiated area of the sample is always same and fixed, meaning that all diffraction spots occur from the same area of the sample, however, unit cell parameters cannot be directly obtained by this method though they are very important for identification of mineral and for determination of crystal structures. In order to obtain the cell parameters even in the case of the sample stationary method, we apply energy scanning of a micro-beam of monochromatic SR at SPring-8

Measuring the Shock Stage of Asteroid Regolith Grains by Electron Back-Scattered Diffraction

We have been analyzing Itokawa samples in order to definitively establish the degree of shock experienced by the regolith of asteroid Itokawa, and to devise a bridge between shock determinations by standard light optical petrography, crystal structures as determined by electron and X-ray diffraction. These techniques would then be available for samples returned from other asteroid regoliths

Measuring Shock Stage of ltokawa Regolith Grains by Electron Back-Scattered Diffraction and Synchrotron X-Ray Diffraction

We have been analyzing Itokawa samples in order to definitively establish the degree of shock experienced by the regolith of asteroid Itokawa, and to devise a bridge between shock determinations by standard light optical petrography, crystal structures as determined by electron and X-ray diffraction techniques. We are making measurements of olivine crystal structures and using these to elucidate critical regolith impact processes. We use electron back-scattered diffraction (EBSD) and synchrotron X-ray diffraction (SXRD). We are comparing the Itokawa samples to L and LL chondrite meteorites chosen to span the shock scale experienced by Itokawa, specifically Chainpur (LL3.4, Shock Stage 1), Semarkona (LL3.00, S2), Kilabo (LL6, S3), NWA100 (L6, S4) and Chelyabinsk (LL5, S4). In SXRD we measure the line broadening of olivine reflections as a measure of shock stage. In this presentation we concentrate on the EBSD work. We employed JSC's Supra 55 variable pressure FEG-SEM and Bruker EBSD system. We are not seeking actual strain values, but rather indirect strain-related measurements such as extent of intra-grain lattice rotation, and determining whether shock state "standards" (meteorite samples of accepted shock state, and appropriate small grain size) show strain measurements that may be statistically differentiated, using a sampling of particles (number and size range) typical of asteroid regoliths. Using our system we determined that a column pressure of 9 Pa and no C-coating on the sample was optimal. We varied camera exposure time and gain to optimize mapping performance, concluding that 320x240 pattern pixilation, frame averaging of 3, 15 kV, and low extractor voltage yielded an acceptable balance of hit rate (>90%), speed (11 fps) and map quality using an exposure time of 30 ms (gain 650). We found that there was no strong effect of step size on Grain Orientation Spread (GOS) and Grain Reference Orientation Deviation angle (GROD-a) distribution; there was some effect on grain average Kernel Average Misorientation (KAM) (reduced with smaller step size for the same grain), as expected. We monitored GOS, Maximum Orientation Spread (MOS) and GROD-a differences between whole olivine grains and sub-sampled areas, and found that there were significant differences between the whole grain dataset and subsets, as well as between subsets, likely due to sampling-related "noise". Also, in general (and logically) whole grains exhibit greater degrees of cumulative lattice rotation. Sampling size affects the apparent strain character of the grain, at least as measured by GOS, MOS and GROD-a. There were differences in the distribution frequencies of GOS and MOS between shock stages, and in plots of MOS and GOS vs. grain diameter. These results are generally consistent with those reported this year. However, it is unknown whether the differences between samples of different shock states exceeds the clustering of these values to the extent that shock stage determinations can still be made with confidence. We are investigating this by examination of meteorites with higher shock stage 4 to 5. Our research will improve our understanding of how small, primitive solar system bodies formed and evolved, and improve understanding of the processes that determine the history and future of habitability of environments on other solar system bodies. The results will directly enrich the ongoing asteroid and comet exploration missions by NASA and JAXA, and broaden our understanding of the origin and evolution of small bodies in the early solar system, and elucidate the nature of asteroid and comet regolith

ラット膝関節骨軟骨欠損モデルに対する間葉系間質細胞移植後の低出力超音波刺激が骨軟骨の再生へおよぼす影響について

京都大学0048新制・課程博士博士(人間健康科学)甲第20572号人健博第48号新制||人健||4(附属図書館)京都大学大学院医学研究科人間健康科学系専攻(主査)教授 坪山 直生, 教授 市橋 則明, 教授 妻木 範行学位規則第4条第1項該当Doctor of Human Health SciencesKyoto UniversityDA