Effects of lower leg muscle quality on gait variability in community-dwelling older and healthy young adults.

[Purpose] We aimed to determine whether lower leg muscle echo intensity, an indicator of muscle quality, is a useful predictor of gait variability after examining the relationship between physical activity and gait variability in community-dwelling older and healthy young adults. [Participants and Methods] This study comprised two tasks. In the first task, 18 older and 25 young adults were included as participants. We examined the relationship between the amount of physical activity and gait variability in both groups. In the second task, muscle echo intensity related to gait variability in each group was measured using ultrasound echoes after identifying common factors related to gait variability in 19 older and 19 younger adults, and trends were compared. [Results] In the first task, gait variability was significantly higher in the younger group than in the older group. A significant negative correlation was found between the amount of physical activity and gait variability in both groups. In the second task, multiple regression analysis was performed for gait variability, and lower leg muscle echo intensity was identified as a significant factor. There was no difference in the correlation coefficient between gait variability and lower leg muscle echo intensity between the two groups. [Conclusion] Lower leg muscle quality was one of the causes of gait variability, suggesting that it is a useful predictor of gait sway status.

Lower-body positive pressure diminishes surface blood flow reactivity during treadmill walking.

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 < .05 for each), whereas an increasing trend in blood flow was observed during standing still (p < .05). Furthermore, in the forehead and thigh, the extent of blood flow increase at the onset of walking was found to decrease in accordance with the applied pressure (p < .01 for each). These findings suggest that during walking, lower-body positive pressure modulates the blood flow, which implies safeness of this novel apparatus for use during orthopedic rehabilitation treatment.

Three-dimensional motion analysis for comprehensive understanding of gait characteristics after sciatic nerve lesion in rodents.

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.

Ultrasound Parameters for Human Osteoarthritic Subchondral Bone ex Vivo: Comparison with Micro-Computed Tomography Parameters.

The aim of this study was to identify ultrasound parameters reflecting subchondral porosity (Po), subchondral plate thickness (Tpl) and bone volume fraction at the trabecular bone region (BV/TVTb). Sixteen osteoarthritic human lateral femoral condyles were evaluated ex vivo using a 15-MHz pulsed-echo ultrasound 3-D scanning system. The cartilage-subchondral bone (C-B) surface region (layer 1) and inner subchondral bone region (layer 2) were analyzed; we newly introduced entropy (ENT) and correlation (COR) of ultrasound texture parameters of the parallel (x) or perpendicular (z) direction to the C-B interface for this analysis. Po, Tpl and BV/TVTb were evaluated as reference measurements using micro-computed tomography. ENTL1x (ENT of layer 1, x-direction) and ENTL1z were significantly correlated with Po (both r values = 0.58), CORL2x with Tpl (r = -0.73) and CORL2z with BV/TVTb (r = -0.66). These are efficient indicators of the characteristics of osteoarthritis-related subchondral bone; the other texture parameters were not significant.

Pathohistological investigation of osteochondral tissue obtained during total knee arthroplasty after osteochondral autologous transfer: a case report.

BACKGROUND: Osteochondral autologous transfer is one of the repair techniques for cartilage defects of knee with promising knee function recovery. There are no reports including histopathological images concerning human osteochondral tissue after osteochondral autologous transfer. This is the first report to present pathohistological findings of transplanted plugs and host tissues extracted from the human body 3 years after osteochondral autologous transfer. This study aimed to explore the cause factor of chronic pain using histological techniques. CASE PRESENTATION: A 67-year-old Japanese man presented with adjusted total knee arthroplasty 3 years after osteochondral autologous transfer. Although in pain, arthroscopic assessment was not severe. The specimens which was gained during total knee arthroplasty were investigated in gross and microscopically using immunohistochemical staining technic. Histological examination revealed that the gap between grafted plugs and host osteochondral tissues was filled with fibrous tissue that stained positive for type I collagen. A degenerative change and some neovascularity were observed in the regenerated tissue and host trabecular bone. Furthermore, cysts and bone marrow edema were observed. CONCLUSION: Our data suggests that the host osteochondral morbidity around grafted plugs might be related to chronical pain and revision surgery.

Effect of Low-Intensity Pulsed Ultrasound after Mesenchymal Stromal Cell Injection to Treat Osteochondral Defects: An In Vivo Study.

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.

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.

Remobilization causes site-specific cyst formation in immobilization-induced knee cartilage degeneration in an immobilized rat model.

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.

Effect of microfabricated microgroove-surface devices on the morphology of mesenchymal stem cells.

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 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 24h 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 p16(INK4A) (cell senescence marker) mRNA expression was retained under hypoxic conditions up to 72h, but it was up-regulated under normoxic conditions. Interestingly, the p16(INK4A) 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.

Alteration of cartilage surface collagen fibers differs locally after immobilization of knee joints in rats.

The purpose of this study was to examine the ultrastructural changes of surface cartilage collagen fibers, which differ by region and the length of the experimental period in an immobilization model of rat. Male Wistar rats were randomly divided into histological or macroscopic and ultrastructural assessment groups. The left knees of all the animals were surgically immobilized by external fixation for 1, 2, 4, 8 or 16 weeks (n = 5/time point). Sagittal histological sections of the medial mid-condylar region of the knee were obtained and assessed in four specific regions (contact and peripheral regions of the femur and tibia) and two zones (superficial and deep). To semi-quantify the staining intensity of the collagen fibers in the cartilage, picrosirius red staining was used. The cartilage surface changes of all the assessed regions were investigated by scanning electron microscopy (SEM). From histological and SEM observations, the fibrillation and irregular changes of the cartilage surface were more severe in the peripheral region than in the contact region. Interestingly, at 16 weeks post-immobilization, we observed non-fibrous structures at both the contact and peripheral regions. The collagen fiber staining intensity decreased in the contact region compared with the peripheral region. In conclusion, the alteration of surface collagen fiber ultrastructure and collagen staining intensity differed by the specific cartilage regions after immobilization. These results demonstrate that the progressive degeneration of cartilage is region specific, and depends on the length of the immobilization period.

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.

Intermittent application of hypergravity by centrifugation attenuates disruption of rat gait induced by 2 weeks of simulated microgravity.

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.

Discordance in recovery between altered locomotion and muscle atrophy induced by simulated microgravity in rats.

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.

Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes.

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.

Regional comparisons of porcine menisci.

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.

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.

Evaluation of reference genes for human chondrocytes cultured in several different thermal environments.

PURPOSE: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.

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.

Optimum temperature for extracellular matrix production by articular chondrocytes.

PURPOSE: The purpose of this study was to investigate the influence of temperature on the ability of the chondrocytes to produce extracellular matrix (ECM). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.