In the last decade, skeletal muscle has been recognized as an endocrine organ able to release molecules that may act as paracrine or endocrine factors, namely myokines. Among these, irisin is secreted upon muscle contraction after physical exercise (PE) and has been demonstrated to yield anabolic effects on different cell types. Recently, irisin has been shown to improve cortical bone mass, geometry and strength, hence resembling the effect of PE. It has also been reported that irisin levels in the serum and synovial fluid of patients with knee osteoarthritis (OA) were negatively correlated with OA severity. Therefore, we hypothesized that irisin may improve cartilage metabolism and blunt the osteoarthritic process.

Human osteoarthritic chondrocytes (hOAC) were isolated from osteochondral specimens of patients undergoing total knee joint replacement. After in vitro expansion, hOAC were put in a three-dimensional culture system (alginate beads) and treated with either phosphate-buffered saline (control) or irisin (25 ng/mL). After 1 week, the amount of glycosaminoglycans (GAG) was evaluated using dimethylmethylene blue (DMMB) and PicoGreen assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect interleukin (IL)-1 and -6, matrix metalloproteinase (MMP)-1 and -13, inducible nitric oxide synthase (iNOS) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -3 gene expression levels.

hOAC treated with irisin showed a significant higher GAG content compared to the control group (p < 0.01). Moreover, irisin was able to reduce the expression of catabolic (MMP-1, -13, iNOS) and pro-inflammatory (IL-1, IL-6) markers, while incrementing the expression of TIMP-1 and -3 (p < 0.001).

Our results showed that irisin was able to stimulate GAG synthesis and diminish extracellular matrix catabolism in hOAC, demonstrating the existence of a cross-talk between cartilage and muscle possibly supporting the beneficial role of PE on cartilage homeostasis.

Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects. Articular cartilage was isolated from donors undergoing total hip or knee replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase, and expanded in DMEM-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1, COL2A1, COL10A1, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13, on an ABI 7500 fast qPCR system. Although IL-1β did not significantly decrease the expression of matrix proteins, it did increase the expression of ADAMTS-4, −5, and MMP3 and −13 when compared to controls (Kruskal-Wallis, p < 0 .05, n=3). TUDCA treatment alone did not significantly increase the expression of catabolic enzymes but it did increase the expression of collagen type II. When IL-1β was coincubated with TUDCA, the expression of ADAMTS-4, ADAMTS-5, and MMP-13 significantly decreased by ∼40-fold, ∼10-fold, and ∼3-fold, respectfully. We provide evidence that the catabolic activities of IL-1β on human cartilage can be abrogated through modulation of the ER stress response

Mechanical loading regulates the metabolism of chondrocytes in cartilage1. Nowadays, studies exploring the in vitro response of cartilage towards loading often rely on bioreactor experiments applying only compressive loading. This is likely not sufficiently representative for the complex multi-directional loading profile in vivo (i.e. where typical compressive and shear loading are both present). The impact of multi-axial loading is specifically relevant in the context of the onset of osteoarthritis (OA) due to joint destabilization. Here, alterations in the 3D loading profile, and in particular increased shear forces, are suggested to initiate catabolic molecular responses leading to cartilage degeneration3. However, in vitro/ex vivo data confirming this hypothesis are currently lacking. Therefore, we aim to investigate how increased shear loading affects the metabolism and ECM deposition of a healthy chondrogenic cell line and if this response is different in osteoarthritic primary chondrocytes.

A murine chondrogenic precursor cell line (ATDC5) and primary human osteoarthritic articular chondrocytes (hOACs) were encapsulated in 2.2% alginate disks and cultured in DMEM medium for three days. Hydrogels seeded with the different cell groups were loaded in the TA ElectroForce BioDynamic Bioreactor and subjected to following loading conditions: (a) 10% compression at 1Hz for 1h, (b) 10% compression and 10° shear loading at 1Hz for 1h. Unloaded constructs were used as control. After loading, hydrogel constructs were stabilized in culture medium for 2 hours, to facilitate adequate gene expression responses, before being dissolved and snap frozen. RNA was isolated and gene expression levels specific for anabolic pathways, characterized by extracellular matrix (ECM) genes (Col2a1, Aggrecan and Perlecan), catabolic processes (MMP-3 and MMP-13) and chondrogenic transcription factor (Sox9) were evaluated using RT-qPCR. The TA ElectroForce BioDynamic Bioreactor was successfully set-up to mimic cartilage loading.

In ATDC5 cells, compression elicits an increase in all measured ECM genes (Col2a1, Aggrecan and Perlecan) compared to unloaded controls, suggesting an anabolic response. This upregulation is decreased when adding additional shear strain. In contrast to ATDC5 cells, the anabolic response of proteoglycans Aggrecan and Perlecan to compressive loading was lower in osteoarthritic chondrocytes, and Col2a1 expression appeared decreased. Adding shear strain reversed this effect on Col2a1 expression. Multi-directional loading increased transcription factor Sox9 expression compared to compression in both ATDC5 and OA chondrocytes. In OA chondrocytes, both loading regimens increased MMP-3 and MMP-13 expression. Shear loading reduces the anabolic effect of compressive loading in both cell types. OA cells presented more catabolic response to mechanical loading compared to precursors, given the increase in catabolic enzymes MMP-3 and MMP-13.

Summary Statement. IL-1β stimulation of human OA chondrocytes induces NFκB, ERK1/2, c-JUN, IκB and P38 signalling pathways. Pre-treatment with cannabinoid WIN-55 for 48 hours inhibits certain pathways, providing mechanisms for cannabinoids inhibitory actions on IL-1β induced cartilage degradation. Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) breakdown in osteoarthritis (OA) and their expression is regulated by nuclear factor kappa B (NFκB). In addition signalling pathways ERK1/2, c-JUN, IκB and P38 are activated in OA and are induced by inflammatory cytokine interleukin 1 (IL-1). Cannabinoids have been shown to reduce joint damage in animal models of arthritis. Synthetic cannabinoid WIN-55, 212-2 mesylate (WIN-55) significantly reduces IL-1β induced expression of MMP-3 and -13 in human OA chondrocytes, indicating a possible mechanism via which cannabinoids may act to prevent ECM breakdown. Here the effects of WIN-55 on IL-1β induced NFκB, ERK1/2, c-JUN, IκB and P38 phosphorylation in human OA chondrocytes has been investigated. Primary human chondrocytes were obtained from articular cartilage removed from patients with symptomatic OA during total knee replacement (Ethic approval:SMB002). Cartilage tissue was graded macroscopically 0–4 using the Outerbridge Classification method. Chondrocytes isolated from grade 2 cartilage and cultured in monolayer were pre-treated with 10 μM WIN-55 for 1 hour prior to stimulation with 10 ng/ml IL-1β for 30 minutes for investigation of NFκB, c-JUN, IκB and P38 phosphorylation. In addition chondrocytes were pre-treated with 10 μM WIN-55 for 30 minutes, 1, 3, 6, 24 and 48 hours prior to 10 ng/ml IL-1β stimulation for 30 minutes to investigate ERK1/2 phosphorylation. Dimethyl sulfoxide (DMSO) was used as a vehicle control at 0.1%. Immunocytochemistry was used to investigate the phosphorylation and translocation of NFκB. ERK1/2, c-JUN, IκB, and P38 activation was investigated using cell based ELISA. Immunocytochemical analysis showed chondrocytes stimulated with IL-1β induced NFκB phosphorylation and translocation to the nucleus. Chondrocytes treated with IL-1β with WIN-55 for 1 hour pre-treatment showed no inhibition of the IL-1β induced NFκB phosphorylation and translocation to the nucleus. WIN-55 treatment alone for 1 hour stimulated NFκB phosphorylation in the cytoplasm but not the nucleus. ELISA showed that phosphorylation of ERK1/2, c-JUN, IκB, and P38 was significantly induced by IL-1β following 30 minutes stimulation (p<0.05). Pre-treatment with WIN-55 for 1 hour had no significant effect on this IL-1β induced phosphorylation. However WIN-55 pre-treatment for 48 hours prior to IL-1β stimulation for 30 minutes, resulted in a significant decrease in ERK1/2 phosphorylation compared to IL-1β stimulation alone (p<0.05). WIN-55 treatment alone for 1 hour significantly induced c-JUN phosphorylation (p<0.05), but had no effect on IκB and P38 phosphorylation compared to DMSO control. IL-1β stimulation of ERK1/2 phosphorylation was not significantly affected by WIN-55 pre-treatment of 30 minutes, 1, 3, 6 and 24 hours. WIN-55 treatment alone for 48 hours significantly reduced ERK1/2 phosphorylation compared to DMSO control (p<0.05). WIN-55 treatment alone for 30 min, 1, 3, 6 and 24 hours had no significant effect on ERK1/2 phosphorylation compared to DMSO control. The results show that following 48 hours pre-treatment WIN-55 inhibits IL-1β induced ERK1/2 phosphorylation in human OA chondrocytes. Thus inhibitory effects of cannabinoids on IL-1β induced cartilage degradation may be mediated via modulation of ERK1/2 signalling

Introduction

In vitro expansion of human articular chondrocytes (HACs) is required for cell-based strategies to treat cartilage defects. We have earlier shown that culturing HACs at increased osmolarity (i.e., 380 mOsm), as compared to plasma osmolarity (i.e., 280 mOsm), increases collagen type II (COL2A1) expression in vitro. Our earlier results showed that knockdown of TGF-β2, a prototypic member of the TGF-β superfamily and an accepted key regulator of chondrocyte differentiation, resulted in increased COL2A1 production. BMPs are members of the TGF-β superfamily which are known to be involved in the regulation of COL2A1 expression. In this study, we aimed to elucidate the role of BMP signaling, in the upregulation of COL2 production upon TGF-β2 knockdown (KD) under hyperosmotic culture conditions.

Introduction: Autologous chondrocyte implantation (ACI) has been developed in order to repair cartilage successfully. Experimental models are based on osteochondral defects with potentially triphasic chondrogenic system: periosteal flaps, bone marrow cells and transplanted chondrogenic cells. All these three have chondrogenic activity so it is difficult to determinate the role of the implanted cells unless appropriate control is set up. The purpose of this study is to determinate if the inoculation of chondrocytes under periosteal flaps does improve the chondrogenic potential of periosteal flaps. MATERIALS AND Methods: 10 New Zealand rabbits, 8 months old were used. Right knees served as study group (ACI Group; N5: Chondrocytes + Periosteal Flap) – (Fibroblast Group: N5 Fibroblast + Periosteal Flap) and left knees as control group (N: 10: osteochondral defect alone). During the first procedure dermal fibroblast cells were isolated from skin biopsy and chondrocytes were isolated from the medial femoral condyle as a full thickness of the right and left knee were done. Chondrocytes and dermal fibroblasts cells were incubated for 4 weeks. Then they were implanted under periostel flap according to study group. Chondrocyte and Fibroblast Implantation:. A parapatellar incision was performed on both knees. Defect was cleaned and on study group the periosteum taken from the tibia was sutured leaving one edge free to inoculate the chondrocytes or fibroblast according to group using a needle Then the defect was closed using fibrin glue. The animals were euthanatized 8 months postoperative. Analysis: Specimens were evaluated using Hematoxylin and Eosin. Safranine and inmunohistochemistry for Collagen Type 2 using the ICRS score system. Statistical Analysis: T student, Fisher and confidence interval were used. A p value < 0,05 was considered significant. Results: Control non treated group presented a histological score grade mean IV (95% CI: 44–97). The ACI group showed a tissue type means II (ICRS) (95% CI: 28–99%) Collagen type 2 was evident only in the deep layers. The fibroblast group did show a reparative tissue, tissue type mean II (95% CI: 28–99%) Collagen type 2 was evident in deep layers. DISCUSSION: According to this study the inoculation of chondrocytes under periosteal flaps does not improve significally the chondrogenic potential of periosteal flaps.(p: 0,77). Comparing the same procedure with chondrogenic and non chondrogenic cell lines could determinate the role of different chondrogenic components (periosteum and chondrocytes). Probably the chondrogenic capacity of the periosteum is sufficient to stimulate a reparative tissue. However none of these procedures could establish an adult normal cartilage hyaline tissue

Purpose. Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold. Method. Chondrocytes were isolated from Outerbridge grade 0 and 1 AC from four patients undergoing total knee arthroplasty and embedded within 216 bovine collagen I scaffolds. Scaffolds were incubated in hypoxic (3% O2) or normoxic (21% O2) conditions for 1hr, 21hr and 14 days. Gene expression was determined using Q-rt-PCR for col I/II/X, COMP, SOX9, aggrecan and B actin. Matrix production was determined using glycosaminoglycan (GAG) content relative to cell count determined by DNA quantification. Cell viability and location within the matrix was determined by Live/Dead assay and confocal microscopy. Statistical analysis was performed using a two-tailed T-test. Results. Chondrocytes cultured under hypoxic conditions showed an upregulation of all matrix related genes compared to normoxic conditions noted most markedly in col II, COMP and SOX9 expression. There were similar numbers of chondrocytes between hypoxic and normoxic groups (P=0.68) but the chondrocytes in the hypoxic group produced more GAG per cell (P= 0.052). Viable cells were seen throughout the matrix in both groups. Conclusion. Important matrix related genes (col II, COMP, SOX9) were most significantly upregulated in hypoxic conditions compared to normoxic conditions. This was supported by an increase in GAG production per cell in hypoxic conditions. The results indicate that hypoxia induces an upregulation in the production of extracellular matrix components typical of AC with only modest increases in col I (possibly related to the col I based scaffold used in this experiment). These results indicate that hypoxic conditions are important for the maintenance of chondrocyte phenotype even when the cells are cultured in a 3D environment. In conclusion, hypoxic culture conditions should be used to help maintain chondrocyte phenotype even when culturing these cells in a 3D scaffold

The use of intra-articular corticosteroid injections for their anti-inflammatory effects is widespread amongst clinicians. Despite their use in both rheumatoid arthritis and osteoarthritis, the effect of these agents on articular chondrocytes is not fully established. Previous reports suggest a detrimental effect on cartilage explants resulting from inhibition of matrix synthesis. 1. However it has also been suggested that the beneficial effects in vivo may be due to prevention of inflamed synovium causing cartilage degradation. 2. Our aim was to assess the effect of a commercially available preparation of methylprednisolone (MP), at clinical doses, on articular chondrocytes cultured in vitro. Bovine articular chondrocytes were isolated by sequential digestion with pronase and collagenase and seeded in 2% alginate at 1x10. 7. cells/ml. The constructs were cultured for up to 15 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of MP, including doses equivalent to those found in vivo. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA and glycosaminoglycans. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate. Chondrocytes cultured in MP containing medium showed a significant abnormality in cell morphology compared to controls at the day 15 time point. Histologically there was evidence of cell necrosis, reduced amounts of extracellular matrix and loss of collagen type II staining. The effects were dose dependant, with significant damage occurring even at clinical doses. Biochemical analysis revealed a reduction in DNA content and an inhibition of glycosaminoglycan and collagen type II synthesis. In contrast, in the controls, there was cell proliferation with a cell doubling time of 14 days, collagen type II containing extracellular matrix synthesis occurred and the chondrocytes maintained their phenotype throughout the culture period. Methylprednisolone has a significant detrimental effect on cultured articular chondrocytes in vitro. There was significant cell necrosis associated with inhibition of extracellular matrix synthesis. Based on these results, intra-articular corticosteroid injections should be used with extreme caution

Aim: To determine the pattern of gene expression induced in cultured human chondrocytes in response to compressive mechanical loads. Methods: Chondrocytes were obtained from tissue discarded at the time of a number of total knee replacements and where established in primary cell culture. The cultured chondrocytes were then subjected to compressive and tensile loads using a Flexcell machine. The RNA was subsequently extracted from these chondrocytes and the alterations in gene expression determined using the Affymetrix Gene Array machine. Results: Intended as an in vitro model for Osteoarthritis, it was found that mechanical stimulation of human chondrocytes caused a significant alteration in the expression of a number of classes of compounds. These included enzymes, inflammatory mediators and structural proteins. Conclusions: This study identified several interesting candidate genes whose expression was significantly altered after being exposed to a laboratory model for osteoarthrosis. Further study of these genes and their expression may lead to important clinical applications

The use of intra-articular hyaluronic acid injections for the treatment of early osteoarthritis is in widespread clinical use. Hyaluronate (HA) is a major component of connective tissue. 1. and is available commercially for the intra-articular injective treatment of osteoarthritis of the knee and periarthritis of the shoulder. Although it is known to improve intra-articular lubrication it is also thought to promote articular cartilage structure and prevent catabolism of matrix proteoglycans in osteoarthritis. Clinical studies have shown beneficial effects lasting for many months after cessation of therapy unlike anti-inflammatory drugs that have relatively short term relieving effects. 2,. 3. . Documentation of the true chondroprotective effects of hyaluronic acid (HA) at the cellular level is lacking and therefore this study aimed to identify the effects of HA on chondrocytes cultured in vitro. Bovine articular chondrocytes were isolated by sequential digestion with pronase and collagenase and seeded in 2% alginate at 1x10. 7. cells/ml. The constructs were cultured for up to 14 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of HA (Sigma), including doses equivalent to those found in vivo. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA, glycosaminoglycans and Collagen. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate. Chondrocytes cultured in the HA system proliferated (increase in DNA) at a faster rate than the controls. There was a 2.2 fold increase in cell concentration at 14 days compared to a 1.2 fold increase in the controls. Total GAG levels at each time point were significantly greater for cells cultured in HA than in controls. Histologically, constructs were characterised by extensive cell cluster formation and intense Safranin-O staining. The newly synthesised matrix also stained positive for type II collagen. By contrast, control constructs exhibited minimal cluster formation, Safranin-O and type II collagen staining. Cells maintained with HA exhibited a significantly greater rate of proliferation and matrix production. The presence of matrix rich in type II collagen indicates maintenance of chondrocytic phenotype. By contrast, cells cultured without HA did not show these features. These results support the use of intra-articular injections for the treatment of osteoarthritis. The benefits of HA injections may be due to cellular mechanisms as well as mechanical

Introduction: In the growth plate, chondrocyte swelling (hypertrophy) is a crucial event during endochondral ossification and bone lengthening, accounting for ~80% of the increase in bone length (. 1. ,. 3. ). The swelling is dramatic (~10x) and closely regulated. Failure of chondrocyte hypertrophy may underlie the chondrodysplasias of the vertebrate skeleton (. 1. ). However, the mechanisms which control cell swelling are poorly understood although there must be a key role for chondrocyte osmolyte transporters which are sensitive to an increase in cell volume. We have used confocal scanning laser microscopy (CLSM) to study volume regulation by living in situ growth plate chondrocytes at varying degrees of hypertrophy. Methods: Bovine growth plates were taken from the ends of young (~12d) bovine ribs. In situ growth plate chondrocytes at the proliferative through to hypertrophic stages were fluorescently-labelled (calcein-AM; 5μM), imaged (Zeiss CLSM510) and volumes determined quantitatively as described (. 2. ). An acute osmotic challenge (280-140mOsm) was delivered by perfusion to determine volume-regulatory capacity by cells in the various zones. Results: The resting volumes of proliferative and hypertrophic cells were 550±63μm. 3. and 5227±1974μm. 3. respectively. Reducing osmolarity resulted in a rapid (within ~1min) cell swelling, proliferative and hypertrophic chondrocytes increasing in volume by 126±2% and 146±5% (n=5) respectively. Chondrocytes within the proliferative zone then recovered in volume by ~60% over the following 20mins (p=0.04), whereas no volume recovery was detected in hypertrophic cells (p=0.94). Conclusions: For the increase in growth plate chondrocyte volume to produce hypertrophy it is essential that the membrane transporters which normally prevent cell swelling are suppressed, otherwise the increase in volume will be compromised. These results suggest that chondrocyte hypertrophy is associated with reduced activity of the swelling-stimulated osmolyte transporter whereas the pathway is active in proliferating chondrocytes. Changes in the activity of this pathway are likely to be an important component in the control of chondrocyte hypertrophy. It is clear that the contributions of other membrane transporters in mediating chondrocyte swelling must be identified in order to understand the overall hypertrophic process

Programmed cell death (PCD) contributes to the pathogenesis of many diseases including osteoarthritis. The principal method is apoptosis that has a well-defined and very characteristic morphology and biochemistry. The aim of the present study was examine whether the mechanism of cell death in OA chondrocytes was classical apoptosis. Rat thymocytes were used as controls since these cells are known to undergo classical apoptosis. Human OA cartilage was obtained from femoral head of patients (50 – 80 years) who were undergoing joint replacement surgery. Pieces of OA samples were processed into paraffin and sections incubated with the following antibodies: M3O, an antibody that recognizes the cleavage of cytokeratin 18 by caspases; annexin V, which recognizes phosphatidylserine “flip-flop” that occurs early in the apoptotic process; bcl-2, a protein whose presence protects apoptosis and c-myc, a transcription factor thought to be associated with apoptosis. To induce apoptosis, some samples were incubated with etoposide and staurosporine. In sections of thymus we noticed the presence of numerous apoptotic bodies. The number increased when the tissue was treated with etoposide and staurosporine. Some thymocytes were immunopositive for M3O and annexin V, and the number of positive cells increased when treated for 2h with etoposide. Chondrocytes of the articular cartilage showed chromatin condensation and many vacuoles but no fragmentation into apoptotic bodies, even when treated with etoposide or staurosporine. The OA chondrocytes were immunonegative for M3O and annexin-V, even after incubation with etoposide and staurosporine. With respect to c-myc and bcl-2, both non-weight bearing and weight-bearing areas in OA sample showed more positive cells then the thymus. More chondrocytes stained for c-myc in the superficial zone of the articular cartilage in the non-weight bearing, while in the weight-bearing areas it was more in the intermediate zone. On the other hand, there were no differences in the distribution of the cells stained for bcl-2 in the articular cartilage. It is known that some events like the phosphatidylserine flip, caspase activation and apoptotic bodies fragmentation occur quickly during apoptosis, so may be difficult to detect. The results suggest that some features of classical apoptosis, such as phosphatidylserine flip,caspase activation and apoptotic bodies formation did not take place in OA cartilage. It is known that the molecular machinery for apoptosis is not always present in tissues that are undergoing programmed cell death, which seems to be case for OA chondrocytes

It was aimed to investigate the isolated effect of hydrostatic pressure on chondrocyte metabolism. Chondrocytes obtained from bovine metatarso-phalangeal joints were cultured in cylindrical 2% agarose gels. A special apparatus which was designed and constructed, allowed the application of hydrostatic pressure of either 2 MPa or 5 MPa on the chondrocytes for 4 hours either in a pulsatile (1Hz) or a static manner. Changes in the syntheses of glycosaminoglycan (GAG) and DNA during and after the application of the hydrostatic pressure were analysed with . 35. S-sulphate and . 3. H-thymidine incorporation, respectively. Radiolabelling was carried out for the following conditions: (a) 4 hours during the application of hydrostatic pressure; (b) 4 hours and (c) 20 hours immediately after the application of load. In addition, the experiments were carried out at 2 days, 7 days and 14 days after embedding the chondrocytes in agarose gels. Static hydrostatic pressure of 5 MPa caused a significant increase by 13% on average in the GAG synthesis during the load application on Day 2 7 and 14 (p < 0.05). On the contrary, pulsatile pressure of 2 MPa caused a significant decrease by 17% in the GAG synthesis measured at 20 hours after the loading on Day 14 (p < 0.01). In addition, there was a significant decrease by 29% in the DNA synthesis measured at 4 hours after the pulsatile loading of 5 MPa on Day 7 (p < 0.01). The results suggest that hydrostatic pressure alone, which causes no cell deformation, can affect the GAG synthesis and proliferation of chondrocytes. In addition, the effect of hydrostatic pressure on the chondrocyte metabolism varies according to the regimes of loading and with the period of cell culture

Purpose and Background: There is increasing evidence that events within the diseased intervertebral disc (IVD) are mediated by locally synthesised cytokines. A prominent histological, imaging and surgical feature of IVD disease is degradation of the cartilaginous discal matrix. Whilst the mechanism by which this is mediated is unknown, in other situations where connective tissues are degraded degradation is the result of production of matrix-degrading enzymes by local connective tissue cells stimulated by cytokines, particularly the beta isoform of interleukin-1 (IL-1β). Included amongst these disorders is osteoarthritis (OA) of diarthrodial joints. OA has many similarities to the discal “degeneration” seen in mechanical back pain syndromes. In the current study, we have used a combination of in-situ techniques to establish if IL-1β is responsible for stimulating matrix degradation in the IVD. Methods: Using a combination of radioactive in-situ hybridisation (ISH) and competitive in situ zymography (ISZ) we have studied expression of IL-1β and IL-1R – its type 1 receptor (ISH) and matrix degradation (ISZ) in five diseased lumbar IVD taken at spinal fusion surgery and 10 cadaveric IVD (five normal and five diseased). The nucleus pulposus (NP) was separated from the annulus fibrosus and diced into 0.5cm cubes. Half the cubes (typically three) were fixed in formalin and processed into paraffin wax for ISH, and half were used for ISZ. For ISH, 5 μm sections of paraffin-embedded tissue were reacted with cDNA probes radiolabelled with 35S to 580 and 530 base segments of the IL-1β and IL-1R molecules. Hybridisation was disclosed using autoradiography. For ISZ, 50 μm vibratome sections were placed into wells on microscope slides precoated with gelatin. Sections were incubated for 10 days, half in culture medium and half in medium supplemented with human recombinant IL-1 receptor antagonist (IL-1Ra – an inhibitor of IL-1). Sections were photographed at daily intervals to detect evidence of gel degradation. Results: Chondrocytes within patient and cadaveric diseased but not normal discs expressed mRNA for both IL-1β and IL-1R. By ISZ, the same cells degraded gelatin. Degradation was inhibited by recombinant IL-1Ra. Conclusion: This study shows that chondrocytes of diseased discs express IL-1 and its receptor. The same cells produced matrix-degrading enzymes by a mechanism that can be inhibited by the IL-1 inhibitor IL-1Ra. IL-1 is a potential therapeutic target for the management of IV disc disease

Osteoarthritis (OA) is characterised by the progressive loss of the articular cartilage. This is accompanied by change in phenotype from cells expressing chondrocytic genes to cells, termed ‘degradative’ chondrocytes, that express aggrecanases and collagenases. To understand the cellular events involved, human articular cartilage was obtained from femoral heads after arthroplasty due to OA, fracture of the neck of femur (#NOF) due to osteoporosis, or from a 14 year old male (CDH). Samples were graded according to the new OARSI system (. Osteoarthritis and Cartilage. , . 2006. , . 14. :. 13. –29. ) and par-affin sections were immunostained for c-Myc (marker of cellular activation), S100 (typically expressed in chon-drocytes), Sox-9 (expressed in early-stage chondrocytes) and nucleostemin (a stem-cell marker). In addition, some specimen were incubated with fluorescent probes to identify metabolically activated cells (CellTracker green). All chondrocytes, irrespective of OA grade, were immunopositive for S100, but there were differences in the other parameters. Cartilage from the 14-year old (OARSI grade =0) was characterized by no loss of proteoglycans (safranin-O) in the superficial zone and absence of c-Myc, Sox-9 and nucleostemin in all articular chondrocytes. In #NOF cartilage, proteoglycan loss was evident in the very superficial zone. Many chondro-cytes in that zone showed bright green fluorescence with CellTracker-green and were c-Myc positive, consistent with cellular activation. Sox-9 and nucleostemin were absent. Mid-zone and deep zone chondrocytes showed no change. In low-grade OA samples (OARSI = 1-2), the zone of proteoglycan loss had increased, the Cell-Tracker-green/c-Myc positive chondrocytes in that zone had divided to form clusters of 4-8 cells. Occasional cells were positive for nucleostemin. Mid-zone and deep zone chondrocytes still showed no change. In high-grade fib-rillated OA cartilage (OARSI = 3-4) the superficial and mid zones had been eroded, leaving the deep zone at the surface. Chondrocytes were typically found in large clones, which were all immunopositive for c-Myc as well as for nucleostemin and Sox-9. The results show that cellular activation starts near the surface and progresses to the deep zone. The presence of nucleostemin and Sox-9 suggests that de-differentiation may be involved in the phenotypic change from the chondrocytic to the degradative phenotype

The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia.

Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3).

Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and COL2A1 expression analysis showed that MSC chondrogenesis was not compromised by any treatment. Importantly, hypertrophy markers (COL10A1, ALPL, SPP1, IBSP) were significantly reduced by PGE2 treatment, but increased by all inhibitors. Additionally, PGE2 significantly decreased ALP activity (2.9-fold), whereas the inhibitors caused a significant increase (1.3-fold, 1.7-fold, 1.8-fold). This identified PGE2 as an important inhibitor of chondrocyte hypertrophy.

Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment.

Regenerative medicine techniques are currently being investigated to replace damaged cartilage. Critical to the success of these techniques is the ability to expand the initial population of cells while minimising de-differentiation to allow for hyaline cartilage to form. Three-dimensional culture systems have been shown to enhance the differentiation of chondrocytes in comparison to two-dimensional culture systems. Additionally, bioreactor expansion on microcarriers can provide mechanical stimulation and reduce the amount of cellular manipulation during expansion. The aim of this study was to characterise the expansion of human chondrocytes on microcarriers and to determine their potential to form cartilaginous tissue in vitro. High-grade human articular cartilage was obtained from leg amputations with ethics approval. Chondrocytes were isolated by collagenase digestion and expanded in either monolayers (104 cells/cm2) or on CultiSpher-G microcarriers (104 cells/mg) for three weeks. Following expansion, monolayer cells were passaged and cells on microcarriers were either left intact or the cells were released with trypsin/EDTA. Pellets from these three groups were formed and cultured for three weeks to establish the chondrogenic differentiation potential of monolayer-expanded and microcarrier-expanded chondrocytes. Cell viability, proliferation, glycosaminoglycan (GAG) accumulation, and collagen synthesis were assessed. Histology and immunohistochemistry were also performed. Human chondrocytes remained viable and expanded on microcarriers 10.2±2.6 fold in three weeks. GAG content significantly increased with time, with the majority of GAG found in the medium. Collagen production per nanogram DNA increased marginally during expansion. Histology revealed that chondrocytes were randomly distributed on microcarrier surfaces yet most pores remained cell free. Critically, human chondrocytes expanded on microcarriers maintained their ability to redifferentiate in pellet culture, as demonstrated by Safranin-O and collagen II staining. These data confirm the feasibility of microcarriers for passage-free cultivation of human articular chondrocytes. However, cell expansion needs to be improved, perhaps through growth factor supplementation, for clinical utility. Recent data indicate that cell-laden microcarriers can be used to seed fresh microcarriers, thereby increasing the expansion factor while minimising enzymatic passage

INTRODUCTION. Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis. 1). Recently it has been suggested that LIPUS may be transmitted via Integrin: a protein which mediates cellular attachment between cells and extracellular matrix. 2). In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chndrocyte response to LIPUS was evaluated. EXPERIMENTAL METHODS. Three kinds of culture dishes coated with three diffrent fibroin aqueous solutions were prepared: 1 wild-type, 2 transgenic and 3 mixed. The wild-type aqueous solution was prepared from Bombyx mori silkworm cocoons. The transgenic aqueous solution was prepared from Bombyx mori silkworm cocoons in which RGD was interfused in the fibroin light chain. 3). The mixed aqueous solution was prepared simply by blending RGD peptides with the wild-type fibroin aqueous solution. Chondrocytes were asepically harvested from the joints of 4-week-old Japanese white rabbits and then subcultured on T-flasks and seeded at 2.0 × 10. 5. cells/dish. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm. 2. and a frequency of 1.71 MHz with a 200 ms tone burst repeated at 1.0 kHz, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. GAG production and the number of chondrocytes were measured by the Dimethylmethylene blue (DMMB) method. 4). and the LDH method. 5). , respectively. Extracted mRNA from the chondrocytes was analyzed by using the Syber Green method, where the primers were designed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the house-keeping gene, aggrecan and Sox 9. This data was analyzed using the two-sided Student's t-test. RESULTS and DISCUSSION. In the transgenic group, the number of chondrocytes and GAG production were increased by the LIPUS stimulation in 1 day of culture (Fig. 1,2), and the mRNA expression levels of agrrecan (Fig. 3) and Sox 9 were increased in 2 days of culture. However the mRNA expression level of aggrecan was decreased after 3 days of culture. These LIPUS-derived changes were not found in the wild-type and mixed groups. We previously reported that the adhesive force between chondrocytes and RGD transgenic fibroin surfaces was higher than that for mixed fibroin, suggesting that adhesive force is translated via RGD which bonds covalently to the fibroin proteins for the transgenic group. The present results suggest that the early biological adhesion via RGD on the transgenic fibroin is sensitive to LIPUS

Summary Statement. Differential expression of canonical and noncanonical Wnt signalling along cartilage canals and osteochondral junctions is dependent on age. Increased gene expression of PTHrP along cartilage canals and Ihh along osteochondral junctions suggests paracrine feedback in articular-epiphyseal cartilage. Introduction. Wnt signaling has been shown to regulate chondrocyte differentiation during pre-/post-natal cartilage development. In addition, parathyroid-related peptide(PTHrP) and Indian hedgehog(Ihh) create a negative feedback loop in growth cartilage, but less is known in articular cartilage. The objective of this study was to elucidate expression of regulatory molecules in chondrocytes surrounding cartilage canals and osteochondral junctions during neonatal and pre-adolescent development. We hypothesised there would be increased expression of canonical Wnt signalling molecules and Ihh in osteochondral junction chondrocytes compared to cartilage canal chondrocytes. In addition, we hypothesised that Wnt signaling and PTHrP expression would be greater in neonates than pre-adolescents. Patients & Methods. Osteochondral samples were obtained(IACUC-approved) from normal femoropatellar joints of 14 euthanised immature horses(6 neonates, 8 pre-adolescents). Samples were frozen in OCT for laser capture microdissection(LCM) or fixed in 4% paraformaldehyde and paraffin-embedded for immunohistochemistry. Chondrocytes surrounding cartilage canals and osteochondral junctions were captured using LCM. Following RNA isolation, equine-specific β-catenin, Wnt-4, Wnt-5b, Wnt-11, Dickkopf-1(Dkk-1), low-density lipoprotein receptor-related protein-4,-6(Lrp4, Lrp6), Axin1, Wnt inhibitory factor-1(WIF)-1, secreted Frizzled-related protein-1,-3,-5(sFRP), retinoic acid receptor gamma(RARG), RAR-inducible serine carboxypeptidase(SC-PEP), Ihh, PTHrP, VEGF, PDGF, MMP-13, and 18S mRNA expression levels were evaluated by two-step real-time qPCR. Following immunohistochemistry using rabbit polyclonal or mouse monoclonal primary antibodies (confirmed by Western blot), spatial tissue protein expression was scored (0–3). Statistical analysis included Wilcoxon signed rank test(paired samples) or rank sum test(unpaired samples)(P<0.05). Results. Gene expression in chondrocytes along cartilage canals was significantly higher for PTHrP, β-catenin, Lrp6, Axin1, sFRP5, RARgamma, and SC-PEP than osteochondral junctions. Conversely, gene expression of Ihh, Wnt4, Wnt11, sFRP3, and VEGF were higher in osteochondral junction chondrocytes than cartilage canals. There was higher protein expression of β-catenin, PDGF, VEGF, and MMP-13 along osteochondral junctions than cartilage canals of pre-adolescents. Neonates had higher gene expression of PTHrP, Wnt-5b, sFRP3, Lrp6, and RARG in cartilage canal chondrocytes than pre-adolescents, while Ihh, Wnt-11, Lrp4, and Dkk1 were significantly higher in pre-adolescents. Immunostaining was higher for β-catenin and Wnt-11 in pre-adolescent osteochondral junction cartilage than neonates. No differences were found between age groups for Wnt-4 immunostaining. Dkk1 protein expression was significantly higher in the middle cartilage layer of pre-adolescents than neonates. Immunostaining was greater for Ihh and PTHrP in the deep cartilage layer of pre-adolescents than neonates. PDGF, VEGF, and MMP13 protein expression was higher in the superficial cartilage layer of pre-adolescents than neonates. Discussion. Wnt/β-catenin and Ihh/PTHrP signaling regulate cartilage differentiation during development and are important in endochondral ossification. This study revealed cell-specific, age-related differences in gene/protein expression of both regulatory pathways. Cells surrounding cartilage canals typically appeared small/rounded compared to larger chondrocytes along osteochondral junctions, likely due to different developmental stages. Higher PTHrP gene expression along cartilage canals and Ihh expression along osteochondral junctions may reflect these stages, suggesting paracrine feedback in articular-epiphyseal cartilage. β-catenin signaling may induce chondrocyte hypertrophy, potentially by enhancing Ihh and MMP-13 expression. Differential expression of canonical(β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signalling(Wnt-5b, Wnt-11) and Wnt inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding cartilage canals and osteochondral junctions provides evidence of age-related interactions during postnatal development

Monolayer expansion of human articular chondrocytes (HAC) is known to result in progressive dedifferentiation and loss of stable cartilage formation capacity in vivo. For optimal outcome of chondrocyte based repair strategies, HAC capable of ectopic cartilage formation may be required. Thus, the aim of this study was to establish appropriate quality control measures capable to predict the ectopic cartilage formation capacity of HAC from culture supernatants. This strategy would avoid the waste of cells for quality control purposes, in order to improve cell therapy and tissue-engineering approaches for the repair of joint surface lesions. Standardized medium supernatants (n=5) of freshly isolated HAC and chondrocytes expanded for 2 (PD2) or 6 population doublings (PD6) were screened for 15 distinct interleukins, 8 MMPs and 11 miscellaneous soluble factors by a multiplexed immunoassay. Cartilage differentiation markers like COMP and YKL-40 were determined by ELISA. Corresponding HAC were subcutaneously transplanted into SCID-mice and their capacity to form stable ectopic cartilage was examined histologically 4 weeks later. While freshly isolated chondrocytes generated stable ectopic cartilage positive for collagen type II, none of the PD6 transplants formed cartilaginous matrix. Loss of ectopic stable cartilage formation capacity between PD0 and PD6 correlated with a drop of MMP3 secretion to < 10% of initial levels, while changes for other investigated molecules were not predictive. Chondrocytes from donors with low MMP3 levels (< 10%) at PD2 failed to regenerate ectopic cartilage at PD2, indicating that MMP3 levels of cultured chondrocytes, independent of the number of cell doublings and the time in culture, predicted ectopic cartilage formation. In conclusion, loss of stable ectopic cartilage formation capacity in the course of HAC dedifferentiation can be predicted by determination of relative MMP3 levels demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches