Cartilage-specific Proteoglycan Research Articles

Calebin A modulates inflammatory and autophagy signals for the prevention and treatment of osteoarthritis.

Osteoarthritis (OA) is associated with excessive cartilage degradation, inflammation, and decreased autophagy. Insufficient efficacy of conventional monotherapies and poor tissue regeneration due to side effects are just some of the unresolved issues. Our previous research has shown that Calebin A (CA), a component of turmeric (Curcuma longa), has pronounced anti-inflammatory and anti-oxidative effects by modulating various cell signaling pathways. Whether CA protects chondrocytes from degradation and apoptosis in the OA environment (EN), particularly via the autophagy signaling pathway, is however completely unclear. To study the anti-degradative and anti-apoptotic effects of CA in an inflamed joint, an in vitro model of OA-EN was created and treated with antisense oligonucleotides targeting NF-κB (ASO-NF-κB), and IκB kinase (IKK) inhibitor (BMS-345541) or the autophagy inhibitor 3-methyladenine (3-MA) and/or CA to affect chondrocyte proliferation, degradation, apoptosis, and autophagy. The mechanisms underlying the CA effects were investigated by MTT assays, immunofluorescence, transmission electron microscopy, and Western blot analysis in a 3D-OA high-density culture model. In contrast to OA-EN or TNF-α-EN, a treatment with CA protects chondrocytes from stress-induced defects by inhibiting apoptosis, matrix degradation, and signaling pathways associated with inflammation (NF-κB, MMP9) or autophagy-repression (mTOR/PI3K/Akt), while promoting the expression of matrix compounds (collagen II, cartilage specific proteoglycans), transcription factor Sox9, and autophagy-associated proteins (Beclin-1, LC3). However, the preventive properties of CA in OA-EN could be partially abrogated by the autophagy inhibitor 3-MA. The present results reveal for the first time that CA is able to ameliorate the progression of OA by modulating autophagy pathway, inhibiting inflammation and apoptosis in chondrocytes, suggesting that CA may be a novel therapeutic compound for OA.

Efficacy of some Herbal Medicines in Osteoarthritis with a Focus on Topical Agents: A Systematic Review.

Osteoarthritis (OA) is a leading cause of musculoskeletal disorders that mainly affects the elderly population. Some herbal medicines have the potential to alleviate the pain associated with OA and improve physical activity mostly through anti-inflammatory and anti-oxidative properties. The aim of this study was to investigate the effects of herbal medicines, especially topical types, on osteoarthritis. In this systematic review, the keywords "osteoarthritis", "herbal compounds", "herbal medicine", "topical drug", "hydrogels", "cream" and "treatment" were used to search publications published from 2010 to 2019 and indexed in databases including PubMed, SCOPUS, Web of Science and Google Scholar. After screening of titles and abstracts and detection of duplicate publications, 38 eligible articles were included in the main review. We also included herbal formulations in vivo. Bioactive fractions of herbal medicines mostly worked on OA through suppression of interleukin-1β (IL-1β), inducing nuclear factor-κB (NF-κB) activation by inhibition of inhibitor of NF-κB (IκBα) phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation, downregulation of NF-κB targets including COX-2 and MMPs, upregulation of collagen type II, cartilage-specific proteoglycans (CSPGs), β1-integrin, and expression of cartilage-specific transcription factor SOX-9 protein. Noticeably, herbal medicines do not produce desirable effects, thereby using their combinations with other therapeutic agents seem to exert substantial clinical outcomes. Herbal gels have demonstrated robustly significant healing effects on knee pain, stiffness and mobility. It is worth considering that because OA is a chronic disease, longer duration of the studies/trials would even lead to obtaining more reliable judgments regarding topical treatment tolerability, safety and efficacy and clarify local or systemic adverse effects. Stability and standardization of a defined amount or concentrations of herbal gels would give promising effects on OA treatment and pain relief.

Chondrotoxicity of Injectable NSAIDs and Narcotics

Objectives:The administration of intra-articular NSAIDs and narcotics has been frequently performed to augment pain control and to decrease inflammation. The chondrotoxicity of these medications have not been evaluated.Methods:Normal human cartilage was arthroscopically harvested from the intercondylar notch in patients undergoing routine ACL reconstruction (N =16 patients). Medication and treatment duration used in this study includes one NSAID, ketorolac at 0.3% and 0.6% for 10 hours, and three narcotics, fentanyl at 0.0005% and 0.001% for 7 hours, meperidine at 0.5%, 1%, and 1.5% for 6 hours, and morphine at 0.01%, 0.02%, and 0.04% for 4 hours. Each medication was delivered in a custom bioreactor over its clinical duration of action. To validate the chondrogenic characteristics of cultured cells, RT-qPCR was applied for the expression of cartilage-specific proteoglycan, aggrecan, the chondrogenic transcription factor, SOX9, the major collagen in cartilage, type II collagen, and the chondrocyte differentiation index, collagen II/I ratio. Undifferentiated human chondrocytes were used as the control. The acute cytotoxicity was determined by fluorescent vital dye staining, followed by flow cytometric analysis, and presented as percentage of dead cells from the total population. The proliferation/AlamarBlue assay was applied, and the growth curves were generated with a two-day interval for up to two weeks.Results:The treatment of a single dose of 0.3% or 0.6% ketorolac demonstrated a significant increase of cell death (0.3% concentration = 16.28% ± 1.26 loss of viability, p < .0001; 0.6% concentration= 41.88 ± 13.42, p < .05) compared to saline control (4.71% ± 1.21). All tested concentrations of meperidine exhibited significant chondrotoxicity (0.5% concentration = 13.61% ± 1.80 loss of viability, p < .01; 1% concentration= 80.53% ± 10.26, p = .001; 1.5% concentration= 89.53% ± 11.00, p < .001). Additionally, ketorolac exhibited a significant dose-response on cell death between 0.3% and 0.6% (p < .05), and higher ranges of meperidine had significant impact on survival (1%, p < .005; 1.5%, p < .005) compared to 0.5% concentration. Treatment with any concentration of fentanyl (0.0005% concentration= 6.13% ± 2.85; 0.001% concentration= 6.02% ± 3.03) or morphine (0.01% concentration= 6.40% ± 1.00; 0.02% concentration= 7.51% ± 2.08; 0.04% concentration= 8.37% ± 2.33) had no significant effect on cell death compared to saline controls. The fentanyl treatments showed no significant difference compared to saline controls up to 8 days with correlation coefficient at 0.1 for concentration = 0.01%, 0.3 for concentration = 0.02%, and -0.5 for concentration = 0.04%. The treatment of morphine exhibited a similar result with no significant difference between the experimental group and saline with correlation coefficient at 0.9 for concentration = 0.0005%, and 0.6 for concentration = 0.001%.Conclusion:A single-dose exposure of 0.3% or 0.6% ketorolac or 0.5-1.5% meperidine resulted in a significant increase in chondrotoxicity and, therefore, should not be used for intra-articular injection. Fentanyl and morphine did not exhibit either acute or delayed chondrotoxicity.

Irx3 and Bmp2 regulate mouse mesenchymal cell chondrogenic differentiation in both a Sox9-dependent and -independent manner.

Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation.

Activin A/BMP2 chimera AB235 drives efficient redifferentiation of long term cultured autologous chondrocytes.

Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.

Chondroprotective effects of taurine in primary cultures of human articular chondrocytes.

Articular cartilage is characterized by the lack of blood vessels and has a poor self-healing potential. Limited cell numbers and dedifferentiation of chondrocytes when expanded in vitro are the major obstacles of autologous chondrocyte implantation. Autologous chondrocyte implantation is a cell-based treatment that can be used as a second-line measure to regenerate chondral or osteochondral defects in younger, active patients. There is an urgent need to find an effective chondrogenic protection agent alleviating or inhibiting chondrocyte dedifferentiation. In this study, we explored the effect of taurine (2-aminoethane sulfonic acid) on proliferation and phenotype maintenance of human articular chondrocytes by analyzing the cell proliferation, morphology, viability, and expression of cartilage specific mRNAs and proteins. Primary chondrocytes were isolated from human articular cartilage tissues. Results showed that taurine effectively promoted chondrocyte growth and enhanced accumulation of glycosaminoglycans and collagens in the conditioned media of chondrocytes. Moreover, taurine exposure caused significant increases in the relative expression levels of mRNAs for cartilage specific markers, including aggrecan, collagen type II and SOX9. Aggrecan is a cartilage-specific proteoglycan, and SOX9 is a chondrogenic transcription factor. In contrast, the mRNA expression of collagen type I, a marker for chondrocyte dedifferentiation, was significantly decreased in cells treated with taurine, indicating that taurine inhibits the chondrocyte dedifferentiation. This study reveals that taurine is effective in proliferation promotion and phenotype maintenance of chondrocytes. Thus, taurine may be a useful pro-chondrogenic agent for autologous chondrocyte implantation in the treatment of cartilage repair.

Extraction of aggrecan-peptide from cartilage by tissue autolysis.

Aggrecan is a cartilage specific proteoglycan containing chondroitin sulfate (CS) and keratan sulfate (KS). CS is an acidic polysaccharide having wide range of applications in pharmaceutical, cosmetic, and food industries. CS is extracted from cartilage by tissue proteolysis with an exogenous proteinase or by activating endogenous proteinases (autolysis) to release aggrecan-peptides from the tissue. This review is focused on the latter technique. Bovine nasal and tracheal cartilages, and broiler chicken sternum cartilage have been used for autolysis studies. To extract aggrecan-peptide, cartilage tissues are cut into small pieces, and incubated in a monovalent or divalent salt solution (e.g., 0.1 M sodium or calcium acetate) at pH 4.5 and 37 &#176;C for 7 - 24 h. Most (~80% or more) of total tissue uronic acid, a constituent sugar of aggrecan, is extracted and released into the salt solution during incubation. Reextraction of the tissue residue results in release of a small amount of uronic acid. Aggrecan-peptides purified using anion exchange chromatography are large compounds containing CS and KS. On gel chromatography, they are excluded from the column of Sephacryl S-300. Chemical composition analysis demonstrated that aggrecan-peptides from either bovine or chicken cartilage contain >90% CS with small amount (< 10%) of either KS or peptide. Patent information included production of aggrecan-peptide substantially free of DNA. The bovine aggrecan-peptide prepared by tissue autolysis has been used as a plate coating antigen in enzyme- linked immunosorbent assay (ELISA) to determine KS.

AB0770 Differential Extracellular Protein Profiles of Osteoarthritic and Normal Mesenchymal Stem Cells Undergoing Chondrogenesis by the Double-Silac Technique

BackgroundChondrogenesis occurs as a result of mesenchymal stem cells (MSCs) condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage. A major...

A lipidomic study of sphingolipid species in human synovial fluid

A lipidomic study of sphingolipid species in human synovial fluid

Quantitative proteomic analysis revealed differentially extracellular protein profiles of osteoarthritic and normal mesenchymal stem cells undergoing chondrogenesis

Purpose: Chondrogenesis occurs as a result of mesenchymal stem cells (MSCs) condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage. A major challenge for the osteoarthitic (OA) cartilage reparation by stem cell-based approaches is the understanding of this multi-step process. In this work, we have analyzed the extracellular protein expression profile of human bone marrow MSCs (hBMSCs) of osteoarthritic patients and control donors undergoing chondrogenesis, in order to compare the mechanisms involved in the cartilage extracellular matrix (ECM) remodeling that occurs during the chondrogenic differentiation process. Methods: hBMSCs isolated from 3 OA patients and 3 healthy controls were grown with different isotope variants of lysine and arginine (Arg6, Lys4 for the control population and Arg10, Lys8 for the OA population) during 4-6 weeks, until achievement of full protein labeling. The labeled populations were then subjected to differentiation in 3D cultures (micromasses) supplemented with chondrogenic inducers for 14 days. Proteins in the conditioned media from the two cell populations were combined, separated by 1D-SDS-PAGE and subjected to in-gel trypsin digestion using an automatic digestor. The resulting peptide mixtures were analyzed by nanoLC coupled on-line to an LTQ-Orbitrap XL mass spectrometer and quantified using the MaxQuant software and the Perseus tool. Results: Real-Time PCR assays showed a relevant difference in the gene expression of collagen type II in the normal donors when compared to the OA patients. Moreover, the chondrocyte phenotype was confirmed in both cases by the proteglycan immunostainings such as aggrecan and chondroitin-6-sulfate after 14 days in chondrogenesis. Using the proteomic approach, we compared the extracellular protein profiles of OA and normal hBMSC at the same time of differentiation. Among the 531 proteins, 56 had significantly altered levels. 35 proteins displayed consistently higher levels in the OA samples compared to normal donors. Many of these proteins are cartilage specific proteoglycans such as hyaluronan and proteoglycan link protein 1, aggrecan core protein or lumican as well as some proteins with a well-known role in the pathogenesis of OA like COMP or MMP3. On the other hand, 21 proteins exhibited a significantly reduced abundance in OA patients when compared to controls. Interestingly, we detected several proteins which belong to the tenascin protein family, like tenascin-X, which accelerates collagen fibril formation. We also found WISP2 decreased at day 14, suggesting a lower activity of the Wnt signaling pathway in OA cells. Conclusions: The identification and quantification of these secreted proteins enhance our knowledge on the extracellular regulation of the chondrogenesis and allow the identification of extracellular markers of this process. Moreover, the lower expression of some of them in OA patients (like tenascin-X or WISP2), suggest their putative usefulness for the molecular monitorization of the chondrogenesis in cell therapy-based approaches for cartilage repair.

Botanical Extracts from Rosehip (Rosa canina), Willow Bark (Salix alba), and Nettle Leaf (Urtica dioica) Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes

The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles.

IGF-1 and PDGF-bb Suppress IL-1β-Induced Cartilage Degradation through Down-Regulation of NF-κB Signaling: Involvement of Src/PI-3K/AKT Pathway

ObjectiveInterleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes.MethodsPrimary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy.ResultsPretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling.ConclusionThe results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects.

Matrix biochemistry and cell biology of dedifferentiated chondrosarcomas

Dedifferentiated chondrosarcoma is an uncommon mesenchymal neoplasm comprised of two different components, low-grade conventional chondrosarcoma and high-grade non-cartilaginous sarcoma. In order to gain better insight into the biology of this tumor, we investigated a large series of dedifferentiated chondrosarcomas by looking at the composition of the extracellular tumor matrix within each of the distinct histological components. Our results showed that the well-differentiated portion of the tumors showed matrix components largely similar to conventional chondrosarcomas or enchondromas. In contrast, the high-grade portions showed a variety of staining patterns related to the matrix being formed. Cartilage-specific proteoglycans and collagens were consistently absent, except in areas showing a chondroblastic osteosarcoma histomorphology. Instead, the most dominant immunostaining was received for type I collagen. Type III and VI collagens were concentrated in the areas showing a fibroblastic phenotype. Our results lend further support to the notion that dedifferentiated chondrosarcoma represents transdifferentiation of a cell towards various blastic mesenchymal cell lineages, most commonly osteoblastic and fibroblastic, but occasionally chondroblastic as well. There was no difference in the clinical outcome of patients with differing high-grade tumor types, emphasizing that grade is a more important predictor of biological behavior than the direction of tumor differentiation.

Curcumin mediated suppression of nuclear factor-κB promotes chondrogenic differentiation of mesenchymal stem cells in a high-density co-culture microenvironment

IntroductionOsteoarthritis (OA) and rheumatoid arthritis (RA) are characterised by joint inflammation and cartilage degradation. Although mesenchymal stem cell (MSC)-like progenitors are resident in the superficial zone of articular cartilage, damaged tissue does not possess the capacity for regeneration. The high levels of pro-inflammatory cytokines present in OA/RA joints may impede the chondrogenic differentiation of these progenitors. Interleukin (IL)-1β activates the transcription factor nuclear factor-κB (NF-κB), which in turn activates proteins involved in matrix degradation, inflammation and apoptosis. Curcumin is a phytochemical capable of inhibiting IL-1β-induced activation of NF-κB and expression of apoptotic and pro-inflammatory genes in chondrocytes. Therefore, the aim of the present study was to evaluate the influence of curcumin on IL-1β-induced NF-κB signalling pathway in MSCs during chondrogenic differentiation.MethodsMSCs were either cultured in a ratio of 1:1 with primary chondrocytes in high-density culture or cultured alone in monolayer with/without curcumin and/or IL-1β.ResultsWe demonstrate that although curcumin alone does not have chondrogenic effects on MSCs, it inhibits IL-1β-induced activation of NF-κB, activation of caspase-3 and cyclooxygenase-2 in MSCs time and concentration dependently, as it does in chondrocytes. In IL-1β stimulated co-cultures, four-hour pre-treatment with curcumin significantly enhanced the production of collagen type II, cartilage specific proteoglycans (CSPGs), β1-integrin, as well as activating MAPKinase signaling and suppressing caspase-3 and cyclooxygenase-2.ConclusionsCurcumin treatment may help establish a microenvironment in which the effects of pro-inflammatory cytokines are antagonized, thus facilitating chondrogenesis of MSC-like progenitor cells in vivo. This strategy may support the regeneration of articular cartilage.

Poly-l-Lactic Acid/Hydroxyapatite Electrospun Nanocomposites Induce Chondrogenic Differentiation of Human MSC

Cartilage and bone tissue engineering has been widely investigated but is still hampered by cell differentiation and transplant integration issues within the constructs. Scaffolds represent the pivotal structure of the engineered tissue and establish an environment for neo-extracellular matrix synthesis. They can be associated to signals to modulate cell activity. In this study, considering the well reported role of hydroxyapatite (HA) in cartilage repair, we focused on the putative chondrogenic differentiation of human mesenchymal stem cells (hMSCs) following culture on membranes of electrospun fibers of poly-L-lactic acid (PLLA) loaded with nanoparticles of HA. hMSCs were seeded on PLLA/HA and bare PLLA membranes and cultured in basal medium, using chondrogenic differentiation medium as a positive control. After 14 days of culture, SOX-9 positive cells could be detected in the PLLA/HA group. Cartilage specific proteoglycan immunostain confirmed the presence of neo-extracellular-matrix production. Co-expression of CD29, a typical surface marker of MSCs and SOX-9, suggested different degrees in the differentiation process. We developed a hydroxyapatite functionalized scaffold with the aim to recapitulate the native histoarchitecture and the molecular signaling of osteochondral tissue to facilitate cell differentiation toward chondrocyte. PLLA/HA nanocomposites induced differentiation of hMSCs in a chondrocyte-like phenotype with generation of a proteoglycan based matrix. This nanocomposite could be an amenable alternative scaffold for cartilage tissue engineering using hMSCs.

Ultrastructural immunolocalization of a cartilage-specific proteoglycan, aggrecan, in salivary pleomorphic adenomas

A pleomorphic adenoma (PA) is the most common epithelial tumor in the salivary glands, but it frequently shows a mesenchyme-like histology, including the presence of myxoid and chondroid areas. Cartilage-specific matrix proteins are deposited in PA. Aggrecan is a major component of cartilage-specific proteoglycans. The present study examined the ultrastructure of the stromal areas in ten salivary PA specimens and investigated the distribution of aggrecan by immunoelectron microscopy. Aggrecan was deposited in the myxoid and chondroid stroma of PA. Ultrastructural observations revealed many proteoglycan cores and fibrils in the myxoid stroma and some spindle-shaped neoplastic myoepithelial cells with vacuoles and actin filaments in the myxoid areas. By immunoelectron microscopy, positivity for aggrecan was observed in the vacuoles of neoplastic myoepithelial cells, which coexisted with the viscous materials, and it was also frequently seen in electron-dense crystals in the myxoid stroma. These findings suggest that neoplastic myoepithelial cells produce aggrecan and release it from vacuoles, and aggrecan is then deposited in the myxoid stroma. Aggrecan deposition is therefore considered to play an important role in the formation of the mesenchyme-like stroma, especially the myxoid stroma.

Altered Synthesis of Cartilage-Specific Proteoglycans by Mutant Human Cartilage Oligomeric Matrix Protein

BackgroundThe mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes.MethodsThe Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the 35S-sulfate incorporation.ResultsNo difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. 35S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells.ConclusionsMutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.

Study of Human Chondrocyte Redifferntiation Capacity in Three-Dimensional Hydrogel Culture

Objective(s) Articular cartilage tissue defects cannot be repaired by the proliferation of resident chondrocytes. Autologous chondrocyte transplantation (ACT) is a relatively new therapeutic approach to cover full thickness articular cartilage defects by in vitro grown chondrocytes from the joint of a patient. Therefore, we investigated the redifferentiation capability of human chondrocytes maintained in alginate culture. Materials and Methods The cartilage specimens obtained from 50 patients who underwent total knee and hip operations at the teaching hospital of Isfahan University of Medical Sciences, Isfahan Iran. Isolated primary chondrocytes were first grown in monolayer cultures for 1 to 6 passages (each passage lasting about 3 days). At each passage, monolayer cells seeded in alginate culture and investigated morphologically and immunocytologically for expression of cartilage-specific markers (collagen type II and cartilage-specific proteoglycans). Results The chondrocytes from monolayer passages P1 to P4 introduced in alginate cultures regained a chondrocyte phenotype. Cells were interconnected by typical gap junctions and after few days, they produced a cartilagespecific extracellular matrix (collagen type II and cartilage-specific proteoglycans). In contrast, cells from monolayer passages P5 and P6 did not redifferentiate to chondrocytes in the alginate cultures. Conclusion Chondrocyte culture was established for the first time in Iran. The alginate culture conditions promote the redifferentiation of dedifferentiated chondrocytes that have still a chondrogenic potential. This procedure opens up a promising approach to produce sufficient numbers of differentiated chondrocytes for ACT. Indeed, in some patients the harvested cells were used immediately and successfully for transplantation.

Fibrin and poly(lactic-co-glycolic acid) hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study

BackgroundSynthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid) (PLGA) are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix.MethodsPLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 106cells/scaffold) and polymerized by dropping thrombin-calcium chloride (CaCl2) solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl)-2-, 5-diphenyltetrazolium-bromide (MTT) assay. Morphological observation, histology, immunohistochemistry (IHC), gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM).ResultsCell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further demonstrated by the expression of genes encoded for cartilage-specific markers, collagen type II and aggrecan core protein. Interestingly, suppression of cartilage dedifferentiation marker; collagen type I was observed after 2 and 3 weeks of in vitro culture. The sulphated-glycosaminoglycan (sGAG) production in fibrin/PLGA was significantly higher than in PLGA.ConclusionFibrin/PLGA promotes early in vitro chondrogenesis of rabbit articular chondrocytes. This study suggests that fibrin/PLGA may serve as a potential cell delivery vehicle and a structural basis for in vitro tissue-engineered articular cartilage.

Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.