Search

Roll tilt vestibular perceptual thresholds, an assay of vestibular noise, were recently found to correlate with postural sway. We therefore hypothesized that roll tilt perceptual training would yield improvements in both perceptual precision and balance. Our data show that roll tilt perceptual thresholds and quiet stance postural sway can be significantly improved after less than 5 h of roll tilt perceptual training, supporting the hypothesis that vestibular noise contributes to increased postural sway.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

Objective This study aimed to clarify the effects of periodic mild heat stimuli on extracellular matrix (ECM) synthesis of adult human chondrocytes in 3-dimensional pellet culture. Results Human articular chondrocytes were subjected to pellet culture at 37 °C for 3 days. Thereafter, the pellets were divided into three groups: 32 °C group which was cultured at 32 °C without heat stimuli, 32 °C + Heat group which was cultured at 32 °C and applied periodic heat stimuli, 37 °C group which was cultured at 37 °C. Heat stimuli were given by transferring the pellets into a CO2 incubator set at 41 °C for 20 min/day, 6 times/week. ECM synthesis ability was evaluated by analyzing the mRNA expressions. Additionally, the collagen and proteoglycan content in the pellet was quantified. DNA content was also measured for estimating the cell amount. We found that there were no significant differences in the mRNA expression of COL2A1, COL1A1, and ACAN between the 32 °C group and 32 °C + Heat group. However, the collagen content per cell and DNA content were significantly lower in the 32 °C + Heat group compared to other groups. Our results indicate that periodic mild heat stimuli may diminish ECM synthesis due to inhibition of collagen production and loss of cells. Electronic supplementary material The online version of this article (10.1186/s13104-019-4058-x) contains supplementary material, which is available to authorized users.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

The growing field of regenerative rehabilitation has great potential to improve clinical outcomes for individuals with disabilities. However, the science to elucidate the specific biological underpinnings of regenerative rehabilitation-based approaches is still in its infancy and critical questions regarding clinical translation and implementation still exist. In a recent roundtable discussion from International Consortium for Regenerative Rehabilitation stakeholders, key challenges to progress in the field were identified. The goal of this article is to summarize those discussions and to initiate a broader discussion among clinicians and scientists across the fields of regenerative medicine and rehabilitation science to ultimately progress regenerative rehabilitation from an emerging field to an established interdisciplinary one. Strategies and case studies from consortium institutions-including interdisciplinary research centers, formalized courses, degree programs, international symposia, and collaborative grants-are presented. We propose that these strategic directions have the potential to engage and train clinical practitioners and basic scientists, transform clinical practice, and, ultimately, optimize patient outcomes.

Objective The purpose of this study was to determine the effects of the lower-body positive pressure on surface blood flow during standing still and treadmill walking to explore cardiovascular safety for application to rehabilitation treatment. Thirteen healthy volunteers participated in the experiment and surface blood flows were measured in the forehead, thigh, calf, and the top of the foot during standing still and walking under various pressure conditions (0 kPa, 5 kPa, and 6.7 kPa). Results Lower-body positive pressure decreased the blood flow in the forehead and the thigh during walking (p p p

We investigated the effect of low-intensity pulsed ultrasound (LIPUS) treatment combined with mesenchymal stromal cell (MSC) injection for cartilage repair and subchondral bone reconstitution for treatment of osteochondral defects. An osteochondral defect was created on both femur grooves of Wistar rats. Four weeks later, bone marrow MSCs were injected into the right knee joint. The rats were divided into two intervention groups: without or with LIPUS irradiation. Cartilage repair was evaluated histologically based on the Wakitani cartilage repair score. Subchondral bone reconstitution was evaluated as bone volume (BV)/tissue volume (TV) by micro-computed tomography analysis. MSC injection improved the cartilage repair score, and LIPUS irradiation improved BV/TV. Combination treatment promoted both cartilage repair and BV/TV improvement. Thus, MSC injection combined with LIPUS irradiation is more effective than either treatment alone in promoting concurrent cartilage repair and subchondral reconstitution.

Summary Objective This study aimed to determine whether treadmill walking (TW) prevents the progression of post-traumatic osteoarthritic changes in cartilage-subchondral bone unit, and whether the exercise timing changes the exercise efficacy in destabilized medial meniscus (DMM) rat knees. Design Twelve-week-old male Wistar rats underwent DMM surgery on their right knees and sham surgery on their left knees and were assigned to either the sedentary ( n = 10) or walking ( n = 24) groups. The rats in the walking group were subjected to TW from day 2 through 4 weeks, from 4 through 8 weeks, or from day 2 through 8 weeks ( n = 8 per group). Osteoarthritic changes of cartilage and subchondral bone were assessed with micro-computed tomography, histology, and immunohistochemistry 8 weeks after surgery. Results TW prevented the progression of cartilage and subchondral bone lesions induced by the DMM, and increased bone morphogenetic protein (BMP)-2 and -6 expressions in superficial zone chondrocytes and bone-lining cells including osteoblasts. Furthermore, the TW-induced increase in BMPs varied with the exercise timing. Beginning TW 4 weeks after DMM surgery was the best option for increasing BMPs, coinciding with the most robust prevention of osteoarthritic changes. Conclusions TW increased the expression of BMPs and prevented the progression of cartilage-subchondral bone lesions in rat knees with a DMM. Selective exercise timing may be a key factor in the development of an exercise regimen for preventing the progression of post-traumatic osteoarthritis (PTOA). Furthermore, exercise may have favorable effects even after the PTOA has been developed.

An understanding of the articular cartilage degenerative process is necessary for the prevention and treatment of joint disease. The present study aimed to examine how long‐term immobilization‐induced cartilage degeneration is aggravated by remobilization. Sixty 8‐week‐old male Wistar rats were used in this study. The unilateral knee joint was immobilized using an external fixator for 8 weeks. The rats were killed at 0 and 3 days, and at 1, 2, 4 and 8 weeks after removing the fixator. After the rats were killed, the maximum knee extension angles were measured. Histological sections at the medial mid‐condylar region (non‐contact, transitional and contact regions of the femur and tibia) were prepared and scored. The cartilage thickness and number of chondrocytes were measured, and CD44 and Col2‐3/4c expression levels were assessed immunohistochemically. The histological assessment revealed progressive aggravation of cartilage degeneration in the transitional region, with a decreased number of chondrocytes and CD44‐positive chondrocytes as well as poor scoring over time, particularly in the tibia. Cyst formation was confirmed in the transitional region of the tibia at 8 weeks post‐remobilization. The cartilage thickness in the transitional region was thicker than that in the contact region, particularly in the tibia. Col2‐3/4c expression was observed in the non‐contact and transitional regions, and the knee extension angle was recovered. In conclusion, immobilization‐induced cartilage degeneration was aggravated by remobilization over time in the transitional region, followed by observations of a decreased number of chondrocytes and morphological disparity between different cartilage regions.

Summary Objective This study investigated the association between spatiotemporal cartilage-subchondral bone plate alterations and mechanical load during ambulation in an experimental rat model of destabilized medial meniscus (DMM). Design Twelve-week-old Wistar rats ( n = 38) underwent DMM surgery on the right knee and sham surgery on the left knee. At 2 and 4 weeks after surgery, subchondral bone changes were evaluated via micro-computed tomography with various knee flexion angles to simulate weight-bearing during rat ambulation under a 3-dimensional motion capture apparatus. Additionally, the biomechanical properties, histology, and ultrastructure of the medial tibia and femoral condyle were evaluated. Results Focal subchondral bone plate perforations were confirmed in the medial tibia within 2 weeks after surgery and were aggravated rapidly 2 weeks later. This subchondral plate porosity colocalized with articular cartilage lesions as confirmed by histology and scanning electron microscopy, and coincided with the likely point of contact between the posterior femoral condyle and tibial plateau during ambulation. Biomechanical properties were confirmed at the medial tibia, at which stiffness was reduced to approximately half that of the sham-operated knee at 4 weeks after surgery. Conclusions Cartilage-subchondral bone plate alterations localized in the region of the point of mechanical load during ambulation in DMM-operated knees, at which the mechanical integrity of cartilage was impaired. These results indicate that DMM-induced increases in mechanical load play an important role in the pathogenesis of early post-traumatic osteoarthritis (OA), and it might accelerate the development of the disease via cartilage-subchondral bone plate crosstalk through increased subchondral plate perforations.

Rodent models of sciatic nerve lesion are regularly used to assess functional deficits in nerves. Impaired locomotor functions induced by sciatic nerve lesion are currently evaluated with scoring systems despite their limitations. To overcome these shortcomings, which includes low sensitivity, little significance, and the representation of only marginal components of motion profiles, some additional metrics have been introduced. However, a quantitative determination of motion deficits is yet to be established. We used a three-dimensional motion analysis to investigate gait deficits after sciatic nerve lesion in rats. This enabled us to depict the distorted gait motion using both traditional parameters and novel readouts that are specific for the three-dimensional analysis. Our results suggest that three-dimensional motion analysis facilitates a comprehensive understanding of the gait impairment specifically, but not limited to, a sciatic lesion rat model. A broad application of these methods will improve understanding and standardized motor assessment.

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

Exposure to a microgravity environment leads to adverse effects in motion and musculoskeletal properties. However, few studies have investigated the recovery of altered locomotion and muscle atrophy simultaneously. The authors investigated altered locomotion in rats submitted to simulated microgravity by hindlimb unloading for 2 weeks. Motion deficits were characterized by hyperextension of the knees and ankle joints and forward-shifted limb motion. Furthermore, these locomotor deficits did not revert to their original form after a 2-week recovery period, although muscle atrophy in the hindlimbs had recovered, implying discordance in recovery between altered locomotion and muscle atrophy, and that other factors such as neural drives might control behavioral adaptations to microgravity.

To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes.

Similarities in the development of locomotion between young children and other mammals are explored by reanalysis of data accrued over ∼18 years. Supported stepping in children was tested on a treadmill. Although the time course of development is more protracted in humans compared to other mammals, the same trends are seen. For example, the duration of the stepping cycle shortens rapidly in the first 5 months of life. Hypermetric flexion of the hip and knee during stepping is seen in children

Background: Although autologous nerve grafting is the gold standard treatment of peripheral nerve injuries, several alternative methods have been developed, including nerve conduits that use supportive cells. However, the seeding efficacy and viability of supportive cells injected in nerve grafts remain unclear. Here, we focused on a novel completely biological, tissue-engineered, scaffold-free conduit. Methods: We developed six scaffold-free conduits from human normal dermal fibroblasts using a Bio 3D Printer. Twelve adult male rats with immune deficiency underwent mid-thigh-level transection of the right sciatic nerve. The resulting 5-mm nerve gap was bridged using 8-mm Bio 3D conduits (Bio 3D group, n = 6) and silicone tube (silicone group, n = 6). Several assessments were conducted to examine nerve regeneration eight weeks post-surgery. Results: Kinematic analysis revealed that the toe angle to the metatarsal bone at the final segment of the swing phase was significantly higher in the Bio 3D group than the silicone group (-35.78 ± 10.68 versus -62.48 ± 6.15, respectively; p < 0.01). Electrophysiological studies revealed significantly higher compound muscle action potential in the Bio 3D group than the silicone group (53.60 ± 26.36% versus 2.93 ± 1.84%; p < 0.01). Histological and morphological studies revealed neural cell expression in all regions of the regenerated nerves and the presence of many well-myelinated axons in the Bio 3D group. The wet muscle weight of the tibialis anterior muscle was significantly higher in the Bio 3D group than the silicone group (0.544 ± 0.063 versus 0.396 ± 0.031, respectively; p < 0.01). Conclusions: We confirmed that scaffold-free Bio 3D conduits composed entirely of fibroblast cells promote nerve regeneration in a rat sciatic nerve model., バイオ3Dプリンターを用いた末梢神経損傷に対する神経再生技術の開発. 京都大学プレスリリース. 2017-02-27., バイオ3Dプリンタを用いた神経再生技術の開発に成功 --末梢神経損傷に対する三次元神経導管の医師主導治験を開始--. 京都大学プレスリリース. 2021-01-15.

The purpose of this study was to analyze histologic, biochemical, and biomechanical differences between zonal, regional, and anatomic locations of porcine menisci. We evaluated six menisci removed from pigs. Medial and lateral menisci were divided into three regions: anterior, middle, and posterior. In each portion, the central zone (CZ) and peripheral zone (PZ) were examined histologically (hematoxylin & eosin, safranin O/Fast green, and picrosiriusred staining), using scanning electron microscopy, biochemically (hydroxyproline assay for collagen content and dimethylmethylene blue assay for glycosaminoglycan [GAG] content), and biomechanically (compression testing). Collagen content in the CZ was lower than that in the PZ. GAG content in the CZ was higher than that in the PZ. GAG content in the PZ of the posterior portion was significantly higher than that in the anterior and middle portions. Compression strength in the CZ was higher than that in the PZ. The differences in cellular phenotype, vascular penetration, and ECM not only between CZ and PZ but also among the anterior, middle, and posterior portions were clarified in the immature porcine meniscus. This result helps further our understanding of the biological characteristic of the meniscus. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1602-1611, 2014.

The purpose of this study was to investigate the influence of temperature on the ability of the chondrocytes to produce extracellular matrix (ECM).Articular chondrocytes were isolated from porcine knee joints. The chondrocytes were cultured at three different temperatures: 32 °C, 37 °C, and 41 °C. The ability to produce ECM was assessed by gene expression analysis, histological, and biochemical evaluation in a pellet culture system.Wet weight of the pellets generated after 21 days, was significantly heavier when cultured at lower temperatures. Picrosirius red staining, employed to evaluate collagen production, was higher at lower temperatures, and safranin-O staining, used to evaluate sulphated glycosaminoglycan (GAG), was lower at 32 °C than at 37 °C and 41 °C. Collagen type IIA1 mRNA expression was markedly up-regulated at 41 °C. However, picrosirius red staining was inhibited at 41 °C. GAG and DNA content were measured by 1,9-dimethylmethylene blue (DMMB) assay and PicoGreen® assay, respectively. The GAG content per pellet was significantly low at 41 °C compared to that at 32 °C and 37 °C. The DNA content per pellet was larger at lower temperatures. The GAG content normalised with the DNA content per pellet was significantly lower at 32 °C compared to that at 37 °C and 41 °C.Our results suggest that a culture temperature of approximately 41 °C inhibits ECM production by decreasing DNA content and perhaps by collagen misfolding. Taken together, the optimum temperature for ECM production in articular chondrocytes may be between 32 °C and 37 °C.

Aim: To facilitate establishment of an effective thermotherapy for osteoarthritis (OA), we investigated the effects of the thermal environment on articular chondrocyte metabolism in vitro. Methods: Chondrocytes were isolated from porcine knee joints, and cultured at 32°C, 37°C and 41°C. Cell proliferation and viability were assessed at Days 2, 4 and 8. In addition, TdT-mediated dUTP nick end labeling (TUNEL) assay was performed at Day 3 to determine the proportion of apoptotic chondrocytes. Analysis of genes specific for factors related to the cartilage extracellular matrix (ECM), cartilage destruction, and cartilage protection was performed at Day 2. Furthermore, evaluation of heat stress tolerance, and heat shock protein 70 (HSP70) mRNA expression and protein synthesis was performed at Day 2 and 3, respectively. Results: Cell proliferation was more at 37°C than at 32°C and 41°C. Cell viability and the number of TUNEL-positive cells were not affected until Day 8 and 3, respectively. The expression of the ECM-related genes was up-regulated at higher temperature. The expression of MMP13, a type II collagen destructive enzyme, and that of TIMP1 and TIMP2, which are MMP inhibitors, were up-regulated at higher temperatures. Finally, the chondrocytes cultured at 41°C may acquire heat stress tolerance, in part, due to the up-regulation of HSP70, and may inhibit apoptosis induced by various stresses, which is observed in OA. Conclusions: The thermal environment affects articular chondrocyte metabolism, and a heat stimulus of approximately 41°C could enhance chondrocyte anabolism and induce heat stress tolerance.

The aim of this study was to elucidate whether the levels of serum biomarkers reflect the progression of osteoarthritis (OA) induced by different levels of exercise. Thirty-five Wistar rats subjected to anterior cruciate ligament transaction (ACLT) were divided into three groups: Control, moderate running, and intense running. Twelve rats (moderate running without ACLT) were allocated as a naive group. Running was performed on a motorized treadmill, at a speed of 18 m/min for 30 min/day (moderate and naive) or 60 min/day (intense) for 3 days per week. After 2 or 4 weeks, OA histopathology in the knees was evaluated using the Osteoarthritis Research Society International (OARSI) score, and the serum levels of cleaved collagen type II (C2C) and procollagen II C-propeptide (CPII) were analyzed. The OARSI score deteriorated in the intense running group after 2 weeks and the serum C2C/CPII ratio suggested the development of OA. At 4 weeks, the C2C/CPII ratio suggested there would be deterioration in the OARSI score but the score did not differ significantly between the moderate and intense running groups. C2C/CPII ratio had 13-25% correlation with the OARSI histological score. Thus, in rat experimental OA, the OARSI score could be partially predicted by the C2C/CPII ratio as a serum biomarker of OA.

Summary Objective To evaluate the dose–response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. Design Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. Results Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. Conclusions Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted.

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.

The surface of a material that is in contact with cells is known to affect cell morphology and function. To develop an appropriate surface for tendon engineering, we used zigzag microgroove surfaces, which are similar to the tenocyte microenvironment. The purpose of this study was to investigate the effect of microgroove surfaces with different ridge angles (RAs), ridge lengths (RLs), ridge widths (RWs), and groove widths (GWs) on human bone marrow-derived mesenchymal stem cell (MSC) shape. Dishes with microgroove surfaces were fabricated using cyclic olefin polymer by injection-compression molding. The other parameters were fixed, and effects of different RAs (180 - 30 °), RLs (5 - 500 μm), RWs (5 - 500 μm), and GWs (5 - 500 μm) were examined. Changes in the zigzag shape of the cell due to different RAs, RLs, RWs, and GWs were observed by optical microscopy and scanning electron microscopy. Cytoskeletal changes were investigated using Phalloidin immunofluorescence staining. As observed by optical microscopy, MSCs changed to a zigzag shape in response to microgroove surfaces with different ridge and groove properties. . As observed by scanning electron microscopy, the cell shape changed at turns in the microgroove surface. Phalloidin immunofluorescence staining indicated that F-actin, not only in cell filopodia but also inside the cell body, changed orientation to conform to the microgrooves. In conclusion, the use of zigzag microgroove surfaces microfabricated by injection-compression molding demonstrated the property of MSCs to alter their shapes to fit the surface.

The effects of intermittent hypergravity on gait alterations and hindlimb muscle atrophy in rats induced by 2 weeks of simulated microgravity were investigated. Rats were submitted to hindlimb unloading for 2 weeks (unloading period), followed by 2 weeks of reloading (recovery period). During the unloading period, animals were subjected to the following treatments: (1) free in cages (Control); (2) continuous unloading (UL); (3) released from unloading for 1 hour per day (UL+1G); (4) hypergravity for 1h per day using a centrifuge for small animals (UL+2G). The relative weights of muscles to the whole body weight and kinematics properties of hindlimbs during gait were evaluated. UL rats walked with their hindlimbs overextended, and the oscillation of their limb motion had become narrowed and forward-shifted after the unloading period, and this persisted for at least 2 weeks after the termination of unloading. However, these locomotor alterations were attenuated in rats subjected to UL+2G centrifugation despite minor systematic changes in muscle recovery. These findings indicate hypergravity application could counteract the adverse effects of simulated or actual microgravity environments.

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.

SummaryObjectiveSubchondral bone cyst (SBC) growth, caused by osteoclast activity during early knee osteoarthritis (OA) pathogenesis, should be treated to prevent further progressions of OA. In the present study, we evaluated the effects of gentle treadmill walking on subchondral bone and cartilage changes in an experimental rat model of destabilized medial meniscus (DMM).MethodTwelve-week-old Wistar rats underwent DMM surgery in their right knee and sham surgery in their left knee and were assigned to either the sedentary group or walking group (n = 42/group). Animals in the walking group were subjected to treadmill exercise 2 days after surgery, which included walking for 12 m/min, 30 min/day, 5 days/week for 1, 2, and 4 week(s). Subchondral bone and cartilage changes were evaluated by micro-CT analysis, histological analysis, and biomechanical analysis.ResultsTreadmill walking had a tendency to suppress SBC growth, which was confirmed by micro-CT (P = 0.06) and positive staining for tartrate-resistant acid phosphatase (TRAP) activity for the osteoclast number per bone surface (P = 0.09) 4 weeks after surgery. These changes coincide with the prevention of cartilage degeneration as evaluated by the Osteoarthritis Research Society International (OARSI) score (P

To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes.

Similarities in the development of locomotion between young children and other mammals are explored by reanalysis of data accrued over ~18 years. Supported stepping in children was tested on a treadmill. Although the time course of development is more protracted in humans compared to other mammals, the same trends are seen. For example, the duration of the stepping cycle shortens rapidly in the first 5 months of life. Hypermetric flexion of the hip and knee during stepping is seen in children3 mo old. Stability of the locomotor rhythm both with respect to cycle duration within a limb and coupling between limbs improves slowly. Finally, coordination between the left and right legs can be manipulated with training, indicating experience-dependent learning at a young age. The possible reasons for these remarkably similar trends in development are explored as a function of maturational time tables for neural structures.

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

The purpose of this study was to identify reference genes showing stable expression in chondrocytes cultured under several different thermal environments and in different culture systems.Human articular chondrocytes were cultured by monolayer or pellet culture system at 32 °C, 37 °C, and 41 °C for 3 days. Thereafter, the total RNA was extracted, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed. The qRT-PCR data was analysed using three different algorithms (geNorm, NormFinder, and BestKeeper) to identify reference genes exhibiting stable expression from among the seven candidate reference genes (B2M, ACTB, GAPDH, HSPCB, RPL13a, YWHAZ, and 18S).The candidate reference genes, except for HSPCB and YWHAZ, showed systematic variations in expression. In the monolayer culture, RPL13a was the most stable gene identified using NormFinder and BestKeeper; on using geNorm, ACTB and GAPDH showed the highest expression stability. In the pellet culture, ACTB was the most stable gene identified using NormFinder and BestKeeper, whereas GAPDH and RPL13a were the most stable reference genes as determined using geNorm. In the combined group, B2M and GAPDH were the most stable genes identified using geNorm, whereas RPL13a and YWHAZ were the most stable as per NormFinder and BestKeeper, respectively. The best combination of two candidate reference genes among all the groups determined using NormFinder was RPL13a and YWHAZ.The combination of RPL13a and YWHAZ might be suitable as reference genes for human chondrocytes cultured at 32 °C, 37 °C, and 41 °C in monolayer, pellet, or combined cultures.

Summary Objective This study aimed to investigate subchondral bone changes using micro-computed tomography (micro-CT) and regional differences in articular cartilage degeneration, focusing on changes of cartilage covered by menisci, in the early phase using a destabilization of the medial meniscus (DMM) model. Method The DMM model was created as an experimental rat osteoarthritis (OA) model (12 weeks old; n = 24). At 1, 2, and 4 weeks after surgery, the rats were sacrificed, and knee joints were scanned using a Micro-CT system. Histological sections of the medial tibial plateau, which was divided into inner, middle, and outer regions, were prepared and scored using the modified OARSI scoring system. The cartilage thickness was also calculated, and matrix metalloproteinase 13 (MMP13), Col2-3/4c, and vascular endothelial growth factor (VEGF) expression was assessed immunohistochemically. Results Subchondral bone defects were observed in the middle region, in which the cartilage thickness decreased over time after surgery, and these defects were filled with MMP13- and VEGF-expressing fibrous tissue. The OARSI score increased over time in the middle region, and the score was significantly higher in the middle region than in the inner and outer regions at 1, 2, and 4 weeks after surgery. Col2-3/4c and MMP13 expression was observed primarily in the meniscus-covered outer region, in which the cartilage thickness increased over time. Conclusion Loss of meniscal function caused cartilage degeneration and subchondral bone defects in the early phase site-specifically in the middle region. Furthermore, our results might indicate cartilage covered by menisci is easily degraded resulting in osmotic swelling of the cartilage in early OA.

The purpose of this study was to analyze histologic, biochemical, and biomechanical differences between zonal, regional, and anatomic locations of porcine menisci. We evaluated six menisci removed from pigs. Medial and lateral menisci were divided into three regions: anterior, middle, and posterior. In each portion, the central zone (CZ) and peripheral zone (PZ) were examined histologically (hematoxylineosin, safranin O/Fast green, and picrosiriusred staining), using scanning electron microscopy, biochemically (hydroxyproline assay for collagen content and dimethylmethylene blue assay for glycosaminoglycan [GAG] content), and biomechanically (compression testing). Collagen content in the CZ was lower than that in the PZ. GAG content in the CZ was higher than that in the PZ. GAG content in the PZ of the posterior portion was significantly higher than that in the anterior and middle portions. Compression strength in the CZ was higher than that in the PZ. The differences in cellular phenotype, vascular penetration, and ECM not only between CZ and PZ but also among the anterior, middle, and posterior portions were clarified in the immature porcine meniscus. This result helps further our understanding of the biological characteristic of the meniscus. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1602-1611, 2014.

s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S208 Figure 3. Aggrecanchanges in articularcartilage exposed toTIMP-3. Co-culturewith TIMP3-transduced cells significantly decreased the amount of thedegraded -aggrecan fragment, ARGSVIL released into the media of IL-1b-cultured cartilage. Error bars represent SE. 393 CHANGES ON THE EXPRESSION OF CD44 IN IMMOBILIZED KNEE IN RATS M. Nagai y, A. Ito y, X. Zhang y, S. Yamaguchi y, H. Iijima y, J. Tajino y, T. Aoyama z, H. Kuroki y. yDept. of Motor Function Analysis, Graduate Sch. of Med.,Kyoto Univ., Kyoto, Japan; zDept of Dev. and Rehab. of Motor Function, Graduate Sch. of Med., Kyoto Univ., Kyoto, Japan Purpose: Both acute and chronic high-intensity loads on the knee joint caused cartilage degeneration, which may eventually lead to osteoarthritis. Likewise, joint immobilization following spinal cord injuries or secondary to treatments for acute musculoskeletal injury could also result in catabolic responses on articular cartilage. Previous studies have demonstrated that immobilization of the knee joint caused cartilage thinning, tissue softening and reduced proteoglycan content in vivo. Turnover of matrix hyaluronan and the hyaluronan binding region of aggrecan is mediated by CD44. The capacity for cell signaling by CD44 exhibits cell-type specificity, and those cascades in chondrocyte have not yet been defined clearly. In this study, we examined the expression of CD 44, which could be the indicative of the chondrocytemetabolism and the histological changes of the cartilage in the immobilized knee in rats. Methods: The unilateral knee joint of Wistar rats aged 8-week old were immobilized at 140 5 degrees of knee flexion with metal screws, wire and resin for 1,2,4,8 or 16 weeks. Sham operated had holes drilled in the femur and tibia with screws, but the joint motion was not restricted. The immobilized operation and sham-operation made up the immobilized group. A control group had no surgery on their knees. Forty rats were prepared for histological analysis (immobilized group: n1⁄45/each period, control group: n1⁄43/each group). Also we identified the loss of extension of the range of motion (ROM) at the immobilized knee joint. Rats were sacrificed at 1, 2, 4, 8 and 16 weeks after the operation. The knees were removed, fixed with 4% PFA, decalcified in 10% EDTA and embedded in paraffin. Six-mm sections were obtained at the medial mid-condylar region of the knee in the sagittal plane. The sections were stained with hematoxylin-eosin. In the two areas (non-contact and contact) from the articular surface of femur and tibia, the number of chondrocytes was counted and thickness of total articular cartilage of each area was measured. The sections were also stained with rabbit anti-rat CD44 antibody (Abcam, ab65829) and the number of CD44(+) cells per region was counted. Results: There was a progressive loss of extension ROM in the immobilized knee joint with time in immobilized groups. In histology, in the non-contact area, no significant change in the cartilage thickness was observed in control and immobilized group. The cartilage thickness in the contact area was gradually decreased throughout the experimental period in the immobilized group especially at tibia region. The number of chondrocytes in the contact area changed significantly throughout the experimental periods after immobilization especially at 16 weeks in the tibia region. There was not a significant change in the non-contact area. In control group, CD44 expression has been observed in whole area of cartilage. In immobilized group, Depth-dependent less expression pattern of CD44 was observed with time after immobilization, especially in 8 and 16 weeks. This pattern observed both of areas, noncontact and contact in immobilized groups. The less expression pattern was observed near the calcified cartilage and subchondral bone. The ratio of CD44 positive chondrocytes was gradually decreased in immobilized group, especially contact area. Conclusions: CD44 participates in the internalization and turnover of HA in articular chondrocytes. Current results indicated that disruption of the HA-CD44 interaction occurred and may disturb cartilage homeostasis at the cartilage of immobilized joint. Furthermore, these change occur deeper layer than superficial layer at the cartilage. 394 PLATELET RICH PLASMA FROM DIFFERENT PREPARATIONS: EFFECT ON CHONDROCYTES AND SYNOVIOCYTES OF OSTEOARTHRITIS PATIENTS E. Assirelli y, C. Cavallo y,z, L. Pulsatelli y,z, B. Grigolo y,z, V. Canella y, E. Mariani y,z, G. Filardo x, E. Kon x, A. Facchini y,z. y Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Istituto Ortopedico Rizzoli, Bologna, Italy; z Laboratorio RAMSES, Istituto Ortopedico Rizzoli, Bologna, Italy; x Laboratorio di Biomeccanica, III Clinica, Istituto Ortopedico Rizzoli, Bologna, Italy Purpose: Platelet Rich Plasma (PRP) is an autologous blood derived product used as promising intra-articular treatment for cartilage degenerative lesions in osteoarthritis (OA). However, the results on the potentiality of this biological treatment are still controversial and confounding, due to the lack of well-designed studies and to the different preparation procedures. Aim of this study was to increase knowledge concerning the biological effect of PRP treatment on clinically relevant joint target cells. For this purpose, we compared two different PRP preparations on the expression of a panel of biomolecules by OA cultured synoviocytes and chondrocytes. Methods: Blood collected from 7 volunteers was used to obtain the two PRP preparations, according to Anitua E. et al, (J Periodontal 2008)(PRP1) and according to Filardo G. et al, (Knee Surg Sports Traumatol Arthrosc 2012) (PRP-2). Cartilage and synovial tissues were collected from OA patients (Kellgren-Lawrence grade I-III) undergoing knee replacement surgery. Isolated chondrocytes and synoviocytes were cultured in appropriate medium, supplemented with 5%, 10% and 20% PRP-1, PRP-2 and Poor Platelet Plasma (PPP). Alamar Blue test was performed at day 0, 3 and 6 to test cell viability and proliferation. Gene expression analysis was performed at day 7 by semi-quantitative RT-PCR. IL-1b, IL-6, IL-8, TNF, MMP-13, TIMP-1, VEGF, TGFb1, IL-10, FGF2, HGF, Hyaluronic acid synthases (HAS)-1, -2, -3 were evaluated on both cell types; TIMP-3, -4, IL-4,-13 on synoviocytes only and Coll-II, SOX-9, aggrecan on chondrocytes only. Hyaluronic acid (HA) production was analyzed in culture supernatants by ELISA. PRP treated samples were compared to PPP ones at the same concentration and all results were expressed as relative increments. Statistical analysis was carried out using the GraphPad Prism for Windows. Results: a) PRP-2 white blood and platelets concentrations were significantly higher than PRP-1 concentrations (p

The aim of this study was to elucidate whether the levels of serum biomarkers reflect the progression of osteoarthritis (OA) induced by different levels of exercise. Thirty-five Wistar rats subjected to anterior cruciate ligament transaction (ACLT) were divided into three groups: Control, moderate running, and intense running. Twelve rats (moderate running without ACLT) were allocated as a naive group. Running was performed on a motorized treadmill, at a speed of 18 m/min for 30 min/day (moderate and naive) or 60 min/day (intense) for 3 days per week. After 2 or 4 weeks, OA histopathology in the knees was evaluated using the Osteoarthritis Research Society International (OARSI) score, and the serum levels of cleaved collagen type II (C2C) and procollagen II C-propeptide (CPII) were analyzed. The OARSI score deteriorated in the intense running group after 2 weeks and the serum C2C/CPII ratio suggested the development of OA. At 4 weeks, the C2C/CPII ratio suggested there would be deterioration in the OARSI score but the score did not differ significantly between the moderate and intense running groups. C2C/CPII ratio had 13-25% correlation with the OARSI histological score. Thus, in rat experimental OA, the OARSI score could be partially predicted by the C2C/CPII ratio as a serum biomarker of OA.

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A308 484 EFFECT OF INITIATION TIMING OF GENTLE TREADMILL EXERCISE ON CARTILAGE AND SUBCHONDRAL BONE IN A MODEL OF DESTABILIZATION OF MEDIAL MENISCUS OF RATS H. Iijima, T. Aoyama, A. Ito, S. Yamaguchi, M. Nagai, J. Tajino, X. Zhang, W. Kiyan, H. Kuroki. Human Hlth.Sci., Graduate Sch. of Med., Kyoto Univ.