Proteoglycan Release Research Articles

Antioxidative therapy in an ex vivo human cartilage trauma-model: attenuation of trauma-induced cell loss and ECM-destructive enzymes by N-acetyl cysteine

Mechanical trauma of articular cartilage results in cell loss and cytokine-driven inflammatory response. Subsequent accumulation of reactive oxygen (ROS) and nitrogen (RNS) species enhances the enzymatic degradation of the extracellular matrix (ECM). This study aims on the therapeutic potential of N-acetyl cysteine (NAC) in a human exvivo cartilage trauma-model, focusing on cell- and chondroprotective features. Human full-thickness cartilage explants were subjected to a defined impact trauma (0.59J) and treated with NAC. Efficiency of NAC administration was evaluated by following outcome parameters: cell viability, apoptosis rate, anabolic/catabolic gene expression, secretion and activity of matrix metalloproteinases (MMPs) and proteoglycan (PG) release. Continuous NAC administration increased cell viability and reduced the apoptosis rate after trauma. It also suppressed trauma-induced gene expression of ECM-destructive enzymes, such as ADAMTS-4, MMP-1, -2, -3 and -13 in a dosage- and time-depending manner. Subsequent suppression of MMP-2 and MMP-13 secretion reflected these findings on protein level. Moreover, NAC inhibited proteolytic activity of MMPs and reduced PG release. In the context of this exvivo study, we showed not only remarkable cell- and chondroprotective features, but also revealed new encouraging findings concerning the therapeutically effective concentration and treatment-time regimen of NAC. Its defense against chondrocyte apoptosis and catabolic enzyme secretion recommends NAC as a multifunctional add-on reagent for pharmaceutical intervention after cartilage injury. Taken together, our data increase the knowledge on the therapeutic potential of NAC after cartilage trauma and presents a basis for future invivo studies.

The activity of aggrecanases and release of proteoglycans from human osteoarthritic cartilage as modulated by biochemical characterized collagen hydrolysates

The activity of aggrecanases and release of proteoglycans from human osteoarthritic cartilage as modulated by biochemical characterized collagen hydrolysates

Value addition of fish waste in the leather industry for dehairing

This study explores the feasibility of utilizing enzymes derived from fish wastes as a low cost depilatory agent. These enzymes are eco-friendly alternative to the conventional lime-sulphide dehairing process. The crude enzyme extract was partially purified with a yield of 60% (Specific protease activity is 4683 U/mg of protein). Optimum pH and temperature for enhanced protease activity of partially purified protease were 8.0 and 30 °C, respectively. The partially purified protease was evaluated for its dehairing efficiency of goat skins. It was observed that the release of proteoglycans and saccharides in the spent liquor after dehairing was 4300 μg/mL and 28 μg/mL, respectively. In the wet blue stage, the enzyme treated leathers exhibited shrinkage temperature of about 110 ± 2 °C, which was found to be on par with that of control leathers. The morphological features were examined through histological and scanning electron microscopic analysis, which reveals a complete removal of hair in the skin matrix. The organoleptic properties and visual assessment of crust leathers were carried out. The physical strength of the crust leathers developed from the enzyme treatment has met the UNIDO norms. Moreover, a significant reduction in the pollution loads was observed in terms of total solids (81%), biological oxygen demand (90%), and chemical oxygen demand (88%) in the dehairing waste stream.

Hsa-miR-15a exerts protective effects against osteoarthritis by targeting aggrecanase-2 (ADAMTS5) in human chondrocytes.

The aim of the present study was to examine the expression levels and role of hsa-miR-15a in osteoarthritis (OA), as well as the associated mechanisms. The expression levels of hsa-miR-15a and A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (ADAMTS5, also known as aggrecanase-2) were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in both OA and normal chondrocytes. hsa-miR‑21 mimics or antisense oligonucleotides (ASO) were co-transfected into the chondrocytes to examine the effects on the putative binding sites compared with the negative control (NC)-mimics or NC-ASO. The relative ADAMTS5 mRNA and protein levels were measured by RT-qPCR and western blot anlaysis, respectively. Moreover, after inhibiting the expression of hsa‑miR‑15a and ADAMTS5 by ASO and small interfering RNA (siRNA), respectively, the amounts of proteoglycan and collagen in the cellular matrix and medium were determined. Additionally, the expression levels of collagen II were measured by western blot analysis. hsa‑miR‑15a expression was downregulated, but ADAMTS5 expression was upregulated in the human OA chondrocytes compared to the normal chondrocytes. Luciferase reporter assay confirmed that the hsa‑miR‑15a binding site was in the ADAMTS5 gene 3'-untranslated region (3'-UTR), and ADAMTS5 was negatively regulated by hsa‑miR‑15a. The downregulation of hsa‑miR‑15a decreased the aggregation of proteoglycan and the collagen content, but increased the release of proteoglycan and collagen; total collagen production was significantly lower, and collagenase activity was markedly higher. The downregulation of ADAMTS5 increased the aggregation of proteoglycan and the collagen content, but decreased the release of proteoglycan and collagen, along with total collagen production. Moreover, collagenase activity was markedly lower. The findings of our study suggest that hsa‑miR‑15a exerts protective effects against OA by targeting ADAMTS5 in human chondrocytes.

Soluble biglycan: a potential mediator of cartilage degradation in osteoarthritis.

BackgroundSoluble biglycan (sBGN) and soluble decorin (sDCN), are two closely related essential components of extracellular matrix which both have been shown to possess proinflammatory properties. We studied whether sBGN or sDCN were present in synovial fluid (SF) of osteoarthritis (OA) or rheumatoid arthritis (RA) patients and studied sBGN or sDCN potential role in the degradation of OA cartilage.MethodsSF obtained from meniscus tear, OA, and RA patients were analysed for sBGN and sDCN using enzyme-linked immunosorbent assays. OA chondrocytes and cartilage explants were stimulated for 48 h with 5 μg/ml sBGN or 1 μg/ml lipopolysaccharide. Messenger RNA (mRNA) levels of Toll-like receptors (TLRs), proteinases and cartilage matrix molecules were determined using quantitative real-time polymerase chain reaction. Protein levels of matrix metalloproteinases (MMPs) and cytokines were measured using Luminex xMap technology. Production of nitric oxide (NO), release of proteoglycans and soluble collagen were measured from conditioned culture media using biochemical assays. OA cartilage explant proteoglycans were stained for Safranin O and quantified using image analysis. TLR4 activation by sBGN and sDCN was studied in engineered HEK-293 cells with TLR4 signalling genes inserted together with a reporter gene.ResultssBGN was found in meniscus tear SF (14 ± 2 ng/ml), OA SF (582 ± 307 ng/ml) and RA SF (1191 ± 482 ng/ml). Low levels of sDCN could also be detected in SF of meniscus tear (51 ± 4) ng/ml, OA (52 ± 3 ng/ml), and RA (49 ± 4 ng/ml). Stimulation of chondrocytes with sBGN increased significantly the mRNA and protein expression of catabolic MMPs, including MMP1, MMP9 and MMP13, and of inflammatory cytokines interleukin (IL)-6 and IL-8, whereas the expression of anabolic markers aggrecan and collagen type II was decreased. sBGN induced release of proteoglycans, collagen and NO from chondrocytes and cartilage explants. The catabolic response in explants was dependent of OA cartilage degradation stage. The mechanism of action of sBGN was mainly mediated through the TLR4-nuclear factor-κB pathway.ConclusionsHigh levels of sBGN was found in advanced OA and RA SF. sBGN activates chondrocytes mainly via TLR4, which results in net loss of cartilage. Thus, sBGN can be a mediator of OA cartilage degradation and also a potential biomarker for arthritis.

Differential Interactive Effects of Cartilage Traumatization and Blood Exposure In Vitro and In Vivo

Background: Sport injuries of the knee often lead to posttraumatic arthritis. In addition to direct damage of the cartilage, trauma-associated intra-articular bleeding may cause hemarthrosis. Both blood exposure and trauma are known to induce cell death and inflammation and to enhance proteoglycan release in cartilage. Hypothesis: Blood exposure increases chondrocyte death as well as inflammatory and degenerative processes in traumatized cartilage. Study Design: Controlled laboratory study. Methods: Human macroscopically intact osteoarthritic (OA) cartilage explants were impacted by a drop-tower system (0.59 J) and cultivated with or without 10% blood. Interactive effects were studied concerning cell survival, gene expression, and the release of mediators over 24 hours and 96 hours. To evaluate the effects of trauma and hemarthrosis in vivo, a newly established blunt cartilage trauma model in the rabbit was used. Treatment of the knee joints of mature New Zealand White rabbits consisted of the following groups: control (C), arthrotomy (A), arthrotomy with cartilage trauma (AT; 1.0 J), and arthrotomy with cartilage trauma and blood injection (ATH). After 1 and 12 weeks, inflammatory mediators in the synovial fluid and histological changes of the cartilage were determined, and immunohistological staining was performed. Results: The in vitro studies revealed a significant additional or synergistic effect of blood exposure on trauma-induced chondrocyte death, interleukin (IL)–1β and prostaglandin-E2 (PGE2) release, and matrix metalloproteinase (MMP)/pro-MMP level. Singular arthrotomy in vivo induced a temporary inflammation. Histologically, cartilage trauma caused significant OA changes that were not aggravated by an additional hemarthrosis. Trauma led to a persistent deposition of terminal complement complex (TCC), being enhanced by hemarthrosis. However, trauma-induced formation of osteophytes and arthrotomy-induced elevation of tumor necrosis factor–α release were reduced by hemarthrosis. Conclusion: While blood exposure clearly aggravated trauma-induced OA processes in the in vitro model, a singular blood injection revealed heterogeneous effects in vivo, enhancing TCC deposition but reducing trauma-induced osteophyte formation while the histological score of traumatized cartilage was not further impaired. Clinical Relevance: The results of this study indicate that a singular, limited bleeding event might not exacerbate early trauma-induced cartilage degeneration in joint injuries. An early removal of intra-articular blood may not prevent the final resulting cartilage damage.

The effect of collagen hydrolysates on the activity of aggrecanases and the release of proteoglycans from human osteoarthritic articular cartilage

The effect of collagen hydrolysates on the activity of aggrecanases and the release of proteoglycans from human osteoarthritic articular cartilage

A4.2 IL4–10 synerkine for the treatment of osteoarthritis

Background and objectives Osteoarthritis (OA) is a degenerative joint disease, clinically characterised by joint pain and disability. Underlying structural changes are degeneration of articular cartilage, intra-articular inflammation with synovitis and changes in peri-articular and subchondral bone. The ideal drug to treat OA should have analgesic, anti-inflammatory and cartilage protective effects. To date, no such drug has been described. In human and animal in and ex-vivo models, the immunoregulatory cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10), especially in combination therapy, can prevent joint degeneration effectively. To use the combined activities of both IL-4 and IL-10, we developed IL4–10 synerkine, preserving the functional activity of each cytokine. In several well-established animal models, we have shown the potential of IL4–10 synerkine to inhibit inflammatory pain in animal models. In this study, we evaluated the potential of the synerkine to reduce inflammation and to provide cartilage protection in an ex vivo model for human OA. Materials and methods Osteoarthritic cartilage (n = 8) and synovium (n = 6) was obtained at joint replacement surgery and was cultured for 72 h in conditioned medium, in the presence or absence of equimolar concentrations of IL-4 (10 ng/ml), IL-10 (10 ng/ml), the combination of IL-4 and IL-10 (10 ng/ml + 10 ng/ml) and IL4-10 synerkine (20 ng/ml). After culture, cartilage was selected to study changes in proteoglycan (PG) synthesis and release. Supernatants of both cartilage and synovium cultures were collected for measurement of a broad spectrum of inflammatory mediators. Results In both synovium and cartilage cultures, elevated levels of pro-inflammatory mediators like IL-6 (resp. 7.5 µg/mg and 531 µg/mg) and IL-8 (resp. 2.6 µg/mg and 142 µg/mg) were measured. These levels were strongly inhibited by the IL4-10 synerkine (about 80%; all p Conclusions IL4-10 synerkine directly affects proteoglycan turnover and cytokine production of OA cartilage, combined with reduced secretion of inflammatory cytokines and proteases by OA synovial tissue. The immunoregulatory and cartilage protective effects in combination with it’s analgesic effects, suggest that the synerkine is an effective disease modifying drug in the treatment of osteoarthritis.

Loss of sclerostin promotes osteoarthritis in mice via β-catenin-dependent and -independent Wnt pathways.

IntroductionSclerostin is a Wnt inhibitor produced by osteocytes that regulates bone formation. Because bone tissue contributes to the development of osteoarthritis (OA), we investigated the role of sclerostin in bone and cartilage in a joint instability model in mice.MethodsTen-week-old SOST-knockout (SOST-KO) and wild-type (WT) mice underwent destabilization of the medial meniscus (DMM). We measured bone volume at the medial femoral condyle and osteophyte volume and determined the OA score and expression of matrix proteins. Primary murine chondrocytes were cultured with Wnt3a and sclerostin to assess the expression of matrix proteins, proteoglycan release and glycosaminoglycan accumulation.ResultsSclerostin was expressed in calcified cartilage of WT mice with OA. In SOST-KO mice, cartilage was preserved despite high bone volume. However, SOST-KO mice with DMM had a high OA score, with increased expression of aggrecanases and type X collagen. Moreover, SOST-KO mice with OA showed disrupted anabolic–catabolic balance and cartilage damage. In primary chondrocytes, sclerostin addition abolished Wnt3a-increased expression of a disintegrin and metalloproteinase with thrombospondin motifs, matrix metalloproteinases and type X collagen by inhibiting the canonical Wnt pathway. Moreover, sclerostin inhibited Wnt-phosphorylated c-Jun N-terminal kinase (JNK) and rescued the expression of anabolic genes. Furthermore, sclerostin treatment inhibited both Wnt canonical and non-canonical JNK pathways in chondrocytes, thus preserving metabolism.ConclusionSclerostin may play an important role in maintaining cartilage integrity in OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0540-6) contains supplementary material, which is available to authorized users.

Interleukin-1β Is Essential for Blood-Induced Cartilage Damage In Vitro

Objective: Joint damage due to recurrent joint bleeds remains the most common complication in hemophilia. The combination of cartilage degeneration and synovial inflammation ultimately lead to hemophilic arthropathy. Cartilage destruction is considered to result from both cartilage matrix-degrading proteases as well as cartilage-destructive pro-inflammatory cytokines such as interleukin (IL)-1b. To unravel the role of IL1b in the pathogenesis of blood-induced cartilage damage, we investigated whether direct blocking of IL1b specifically prevents blood-induced cartilage damage in vitro. Moreover, we investigated whether blocking IL1b after the onset of a bleed can still avert cartilage damage.Methods: Full thickness healthy human articular cartilage explants, obtained post-mortem, were cultured for four days in presence or absence of 50% v/v whole blood. A recombinant human IL1b monoclonal antibody (IL1bmAb) was added at the moment of blood exposure in a concentration of 0, 1, 3, 10, 30, or 100ng/mL (n=8). Furthermore, 100ng/mL IL1bmAb was administered directly or after a delay of several hours up to two days (n=7). Cartilage matrix proteoglycan turnover was determined 12 days later to analyse long-term effects.To investigate the direct effects of IL1bmAb on cartilage, explants were cultured for four days in the presence of 10ng/mL IL1bmAb in the absence of blood (n=7).Results: Exposure to blood decreased the proteoglycan synthesis rate and -content, and increased the proteoglycan release (all p<0.05). Adding IL1bmAb resulted in a dose-dependent increase of the proteoglycan synthesis rate leading to normalisation at higher concentrations (see figure A). Proteoglycan release and -content also normalised after addition of IL1bmAb. Its protective effect was most pronounced when IL1bmAb was administered within 4 hours after the bleed, but significant recovery was still achieved by administration within 8-24 hours (see figure B for proteoglycan synthesis). In the absence of blood, IL1bmAb did not have direct effects on cartilage proteoglycan turnover (for all three parameters p>0.50).Conclusions: This study demonstrates that IL1b is a crucial factor in the development of blood-induced cartilage damage in vitro. Blocking this pro-inflammatory cytokine with a monoclonal antibody protects cartilage from the damaging effects of blood exposure in a dose-dependent way. Early administration after blood-exposure is most beneficial. Further research is warranted to investigate the in vivo capacity of IL1bmAb in prevention and its position as a treatment to limit joint damage upon joint bleeding. [Display omitted] Figure -Effect ofIL1bmAbon cartilage proteoglycan synthesis rate is dose-dependent (A) and time-dependent (B). Median values ± IQR of at least 7 individual cartilage and blood donors are shown. Hash tags indicate a statistically significant difference from control values, whereas asterisks indicate a statistically significant difference from blood-exposed cartilage withoutIL1bmAb addition(p<0.05). DisclosuresSchutgens:CSL Behring: Research Funding.

Parthenolide inhibits pro-inflammatory cytokine production and exhibits protective effects on progression of collagen-induced arthritis in a rat model

Objectives: Progressive destruction of synovial joint cartilage and bone occurs in pathological conditions such as rheumatoid arthritis (RA) because of the overproduction of pro-inflammatory cytokines and activation of nuclear factor kappa B (NF-κB). Through the screening of NF-κB inhibitors by a luciferase reporter gene assay, we identified parthenolide (PAR) as the most potent NF-κB inhibitor, among several PAR analogue compounds. This study was undertaken to determine whether PAR inhibits pro-inflammatory cytokine production, cartilage degradation, and inflammatory arthritis.Method: The mRNA levels of pro-inflammatory cytokines were examined by real-time polymerase chain reaction (PCR). Proteoglycan content and release were determined by measuring glycosaminoglycan (GAG) levels using the dimethylmethylene blue (DMMB) dye-binding assay. The potential role of PAR in treatment of arthritis was studied using a collagen-induced arthritis (CIA) model.Results: We established that PAR, as a prototype compound, suppressed lipopolysaccharide (LPS)- and tumour necrosis factor (TNF)-α-induced increases in matrix metalloproteinase (MMP)-1, MMP-3, inducible nitric oxide synthase (iNOS), and interleukin (IL)-1β mRNA in chondrocytes. In addition, PAR prevented proteoglycan degradation triggered by pro-inflammatory cytokines. PAR treatment at the onset of CIA symptoms significantly reduced synovitis, inflammation, and pannus formation scores. Reduced synovial inflammation after PAR treatment was also reflected in significantly less bone erosion and cartilage damage.Conclusions: These data indicate a protective effect of PAR on the catabolic insults of pro-inflammatory cytokines on chondrocyte metabolism and GAG release in vitro and in CIA. PAR had anti-inflammatory and structure-modifying effects on experimental arthritis, suggesting that PAR may be useful as a potential alternative or adjunct therapy for inflammatory arthritis.

Possible chondroprotective effect of canakinumab: An in vitro study on human osteoarthritic chondrocytes

Canakinumab is a human IgGκ monoclonal antibody that neutralizes the activity of interleukin (IL)-1β blocking interaction with IL-1β receptors. Our study aimed to evaluate the in vitro effect of canakinumab on human osteoarthritic (OA) chondrocytes cultivated in the presence or absence of tumor necrosis factor (TNF)-α. Articular cartilage was obtained from the femoral heads of patients with osteoarthritis (OA). Chondrocytes were incubated with two concentrations (1μg/ml and 10μg/ml) of canakinumab alone or with TNF-α (10ng/ml) for 48h. We evaluated cell viability, release of proteoglycans (PG) and nitric oxide (NO) in culture medium, inducible nitric oxide synthase (iNOS) and metalloproteinanes (MMP)-1,3,13 gene expression, apoptosis, necrosis and morphological feature by transmission electron microscopy (TEM). Canakinumab alone did not have cytotoxic effect. Cell viability was reduced significantly (p<0.001) by TNF-α and restored by canakinumab at both concentrations used. TNF-α determined a significant decrease of PG (p<0.001) and an increase of NO (p<0.001) and MMP-1,3,13 gene expression. Canakinumab significantly increased the PG levels and decreased (1μg/ml, p<0.01; 10μg/ml, p<0.01) NO levels in cells cultured with TNF-α. The NO data were confirmed by the immunocytochemistry assay for iNOS. A significant reduction of MMP-1,3,13 gene expression was induced by canakinumab. Our experiments confirmed the pro-apoptotic effect of TNF-α and demonstrated a protective role of canakinumab. The results concerning biochemical data were further confirmed by the morphological findings obtained by TEM. We showed that canakinumab counteracts the negative effects of TNF-α on OA chondrocyte cultures and may have a potential chondroprotective role in OA.

Fatter through Lipids or Water: Lipohyperplasia Dolorosa versus Lymphedema

Lipohyperplasia dolorosa and lymphedema are completely different disease entities, although both are classified under lymphology. Whereas in lipohyperplasia dolorosa a congenital lipid distribution disorder leads to a high volume insufficiency and corresponding clinical symptoms, lymphedema is characterized by a congenital transport incompetence of the vessels or acquired disorders of transport capacity. According to current knowledge the solid increase in volume with lymphedema is due to a malfunctioning bio-mechanism by which the release of additional proteoglycans in the homeostasis system of the fluid in the interstitial space plays an important role. Therapeutic approaches aim to remove this tissue and the sponge-like proteoglycan substance. Manual lymph drainage and compression can evacuate the sponge but not remove it. Lymphological liposculpture has been successfully used to treat lipohyperplasia dolorosa by removing subcutaneous fatty tissue, present as hyperplasia and not hypertrophy. After lymphological liposculpture, tenderness is no longer present and complex decongestive therapy is no longer necessary because the congenital fatty masses do not recur after surgical removal. The improvement is thus permanent. Lipohyperplasia dolorosa is therefore curable by lymphological liposculpture. The procedure can even achieve a drastic improvement in quality of life for patients with secondary lymphedema by adjusting the symmetry of the extremities and reducing or even avoiding complex decongestive therapy.

Glycogen synthase kinase 3 inhibition stimulates human cartilage destruction and exacerbates murine osteoarthritis.

To assess the role of glycogen synthase kinase 3 (GSK-3) as a regulator of cartilage destruction in human tissue and a murine model of osteoarthritis (OA). Surgical destabilization of the medial meniscus (DMM) was performed to induce experimental murine OA, and joint damage was assessed histologically. Bovine nasal and human OA cartilage samples were incubated with interleukin-1 (IL-1) plus oncostatin M (OSM) and GSK-3 inhibitor. Collagen and proteoglycan release was assessed by hydroxyproline measurement and dye binding assay, collagenase activity was assessed by bioassay, and gene expression was analyzed by real-time polymerase chain reaction. Human articular chondrocytes were isolated by enzymatic digestion and cultured prior to gene silencing and immunoblotting of cell lysates and nuclear fractions. Mice treated with GSK-3 inhibitor exhibited significantly greater cartilage damage compared with sham-operated control mice. GSK-3 inhibition in bovine cartilage dramatically accelerated IL-1 plus OSM-stimulated degradation, concomitant with a profound increase in collagenase activity. GSK-3 inhibitor induced collagen release from human OA cartilage in the presence of IL-1 plus OSM and increased proteoglycan loss. Gene expression profiling of resorbing OA cartilage revealed a marked procatabolic switch in gene expression upon GSK-3 inhibition. This was mirrored in human articular chondrocytes following GSK3 silencing, particularly with the GSK-3β isoform. GSK-3 inhibition or silencing led to enhanced IL-1 plus OSM-stimulated abundance and activity of Jun, and silencing of c-jun ameliorated GSK-3 inhibitor-mediated procatabolic gene expression. GSK-3 is an important regulator of matrix metalloproteinase (MMP)-mediated joint destruction, the inhibition of which by proinflammatory stimuli de-represses catabolic gene expression. Therapeutic strategies that maintain cartilage GSK-3 activity may therefore help curtail aberrant MMP activity during pathologic joint destruction.

SAT0443 In Vitro Effects of Canakinumab on Human Osteoarthritic Chondrocytes

BackgroundCanakinumab is a human IgGκ monoclonal antibody targeting Interleukin (IL)-1β; its action involves the neutralization of the signaling of IL-1β, the most important cytokine in the pathogenesis of Osteoartrhitis (OA)....

Understanding the chemical free enzyme based cleaner unhairing process in leather manufacturing

Abstract Higher cost of enzyme is one of the major factors for the system not being practiced through found environmentally friendly. Through understanding of enzyme based unhairing sans lime and sodium sulfide is essential to develop enzymes of low cost. Understanding of the enzyme–substrate is therefore imminent. The release of PG and carbohydrate was found to be more in the case of EBS compared to Lime–Sulfide System (LSS). The onset of complete removal of hair was found to take place after 4 h in the case of EBS. The trend of release of PG was found to be in congruence to the trend of degree of hair loosening. The micrographs of histological staining also corroborated the findings. Complete hair removal was achieved in 5 h in the case of EBS. The hair loosening in the case of EBS is primarily due to break down of PG present in the hair follicle. If protease that could break down selectively the protein core of the proteoglycans (PG), then with much lesser amount of protease hair loosening could be achieved. And the cost of EBS could therefore be brought down significantly.

Acute Mechanical Injury to the Human Intervertebral Disc Initiates Events Implicated in Disc Degeneration

Introduction Low back pain is a prevalent chronic disorder among individuals worldwide. Intervertebral disc degeneration is a major cause of low back pain in adults. Mechanical overload has been proposed as a potential initiating mechanism of degeneration, and previous studies have demonstrated that complex mechanical loading of lumbar motion segments can disrupt disc cell metabolism.1 Furthermore, mechanical wedge loading of bovine caudal discs induced cell death and causes induction of catabolic genes such as IL-1β and members of the metalloproteinase and ADAMTS family of proteases which leads to loss of aggrecan.2 The aim of this study was to investigate the effect of acute mechanical injury on cellular responses and changes in matrix composition in human intervertebral discs. Materials and Methods Lumbar spine segments were harvested from consenting donors via the Transplant Quebec organ donation program in Montreal, Quebec. This was performed in accordance with the Institutional Review Board guidelines of the Montreal General Hospital. Discs with intact cartilaginous endplates were isolated from the lumbar spine. A single ramp compression of either 5 or 30% resulted in approximately 0.2 MPa (low noninjurious load or LNL) or approximately 1.5 MPa (acute traumatic injury or ATI) peak stress. The rate of compression was 30% strain per second. This compression protocol for acute traumatic injury consistently yields endplate fracture. Discs were, after loading, immediately put in low-serum containing media. The media was changed and collected at days 3 and 7. Proteoglycan release was evaluated in media from all culture days and in guanidium hydrochloride extracts of tissue from the loaded discs. Proteoglycan size distribution was visualized by fractionating conditioned media from low noninjurious load and acute traumatic injury samples by agarose gel electrophoresis. At day 14, disc cultures were terminated and Live/Dead analysis was performed on biopsies from seven different regions of the discs and confocal microscopy was used to quantify live and dead cells. PC12 cells, a neuronal-like cell line, were exposed to conditioned media from the cultured discs to study the potential for neurite sprouting. Nerve growth factor (NGF) levels were measured using a commercially available ELISA assay. Commercially, available cytokine arrays were used to investigate protein levels of cytokines. Disc tissue was fixed in 80% methanol and prepared for cryosectioning and histological analysis with Safranin O, hematoxylin, and Fast Green. Results Considerable cell death was observed in discs subjected to acute traumatic injury 14 days after the load event (NP 40% viable and AF 50% viable). Discs subjected to the low noninjurious load maintained a cell viability of 85% in the NP and the AF (Fig. 1A). Investigation of GAG levels revealed that acute traumatic injury induced a considerable release of GAG from the tissue as compared with low noninjurious load samples (Fig. 1B). This was confirmed by histological analysis where a loss of Safranin O staining was apparent in the tissue of discs subjected to acute traumatic injury. Fractionation of the conditioned media by agarose gel electrophoresis revealed a wider size distribution of the proteoglycans released from the discs after acute traumatic injury as compared with the discs subjected to low noninjurious load. Elevated levels of inflammatory cytokines and pain mediators were detected in the culture media from discs subjected to acute traumatic injury group compared with discs subjected to low noninjurious load. Furthermore, conditioned media from the acute traumatic injury disc cultures caused significantly more neurite outgrowth in PC12 cells compared with disc cultures from discs subjected to low noninjurious load. Conclusion Cell death and cytokine production combined with loss of GAG indicate that acute mechanical injury initiated degradative processes that could lead to disc degeneration. A wider size distribution of proteoglycans in conditioned media from discs subjected to acute traumatic injury further confirmed that this type of load induced expression and/or activation of proteases in the disc. Acute injury also stimulated the remaining viable cells to produce elevated levels of inflammatory cytokines. Once the proteoglycan content in the matrix has been degraded, elevated levels of NGF could lead to neuronal in-growth and directly cause pain. Further investigation may yield insights to novel diagnostics and therapies for degenerative disc disease. Disclosure of Interest None declared References Adams MA, Freeman BJ, Morrison HP, Nelson IW, Dolan P. Mechanical initiation of intervertebral disc degeneration. Spine 2PO.004;25(13):1625–1636 Walter BA, Korecki CL, Purmessur D, Roughley PJ, Michalek AJ, Iatridis JC. Complex loading affects intervertebral disc mechanics and biology. Osteoarthritis Cartilage 2011;19(8):1011–1018

Long non-coding rnas in osteoarthritis and chondrogenesis

Long non-coding rnas in osteoarthritis and chondrogenesis

Osteoclasts reduce the activation of Wnt canonical pathway in chondrocytes

Purpose: Osteoclastogenesis is enhanced in early osteoarthritis (OA). We have previously demonstrated that cartilage degradation is reduced in vivo when osteoclastogenesis is inhibited in murine models with high bone resorption. Wnt activity, known to regulate the bone and chondrocyte cell function, might contribute to the mechanisms that promote cartilage breakdown. Our purpose was to evaluate whether osteoclast-secreting molecules affect directly the metabolism of chondrocyte and the contribution of Wnt pathway. Methods: Osteoclasts were obtained after differentiation of RAW cells cultured with RANKL. Primary murine chondrocyte (Ch) were then cultured in the presence of osteoclast conditioned medium (Oc-CM) for 48h. The gene and protein expressions of catabolism and anabolism were analyzed by RT-qPCR and western blot. To investigate the regulation of canonical Wnt pathway, transactivation assay was performed in primary chondrocytes derived from Topgal mice and cultured with Oc-CM. Western blot and immunocytochemistry were used to confirm the activation of β-catenin canonical Wnt signaling pathway. Finally, in order to confirm the role of Wnt canonical signaling, the β-catenin translocation was activated with the addition of lithium chloride (LiCl) in the Oc-CM. Results: Oc-CM induced a marked decrease in proteoglycan release by chondrocytes. This is the result of both the reduction of the expression of anabolic genes such as collagen type II, Aggrecan and Sox-9 and the increase of the expression of catabolic genes such as metalloproteases (MMP-3 and -13) and aggrecanases (Adamts-4 and-5). We then monitored the nuclear translocation of β-catenin induced by Oc-CM. We observed an abolition of the translocation of β-catenin and subsequently of Topgal activity along with the reduction of Wnt target genes (Axin, Wisp1 C-Myc). In order to rescue the inhibition of canonical pathway induced by Oc-CM, a dose-effect of LiCl was performed (1 - 5 mM). Low dose of LiCl totally reversed the effect of Oc-CM while was aggravated with the highest dose. Conclusion: We here demonstrated that osteoclasts secrete soluble factors able to disrupt the balance of chondrocyte metabolism via the inhibition of the Wnt canonical signaling. Therefore, manipulating bone may affect chondrocyte function.

Galectin fingerprinting in oa cartilage

Galectin fingerprinting in oa cartilage