Cartilage Matrix Gene Expression Research Articles

Modulation of the long non-coding RNA Mir155hg by high, but not moderate, hydrostatic pressure in cartilage precursor cells.

Osteoarthritis (OA) is the most common joint disease in older adults and is characterized by a gradual degradation of articular cartilage due to decreased cartilage matrix gene expression and increased expression of genes involved in protein degradation, apoptosis and inflammation. Due to the high water content of cartilage, one of the main physical stimuli sensed by chondrocytes is hydrostatic pressure. We previously showed that high pressure above 20 MPa induced gene expression changes in chondrocyte precursor cells similar to what is observed in OA. Micro-RNAs are small non-coding RNAs essential to many physiological and pathological process including OA. As the micro-RNA miR-155 has been found increased in OA chondrocytes, we investigated the effects of high pressure on the expression of the miR-155 host gene Mir155hg. The chondrocyte progenitor cell line ATDC5 was pressurized under hydrostatic pressure up to 25 MPa and the expression of Mir155hg or the resulting micro-RNAs were measured; pharmacological inhibitors were used to identify the signaling pathways involved in the regulation of Mir155hg. We found that Mir155hg is strongly and rapidly up-regulated by high, but not moderate, pressure in chondrocyte progenitor cells. This up-regulation likely involves the membrane channel pannexin-1 and several intracellular signaling molecules including PKC and Src. MiR-155-5p and -3p were also up-regulated by pressure though somewhat later than Mir155hg, and a set of known miR-155-5p target genes, including Ikbke, Smarca4 and Ywhae, was affected by pressure, suggesting that Mir155hg may have important roles in cartilage physiology.

Effects of Mechanical Compression on Chondrogenesis of Human Synovium-Derived Mesenchymal Stem Cells in Agarose Hydrogel.

Mechanical compression is a double-edged sword for cartilage remodeling, and the effect of mechanical compression on chondrogenic differentiation still remains elusive to date. Herein, we investigate the effect of mechanical dynamic compression on the chondrogenic differentiation of human synovium-derived mesenchymal stem cells (SMSCs). To this aim, SMSCs encapsulated in agarose hydrogels were cultured in chondrogenic-induced medium with or without dynamic compression. Dynamic compression was applied at either early time-point (day 1) or late time-point (day 21) during chondrogenic induction period. We found that dynamic compression initiated at early time-point downregulated the expression level of chondrocyte-specific markers as well as hypertrophy-specific markers compared with unloaded control. On the contrary, dynamic compression applied at late time-point not only enhanced the levels of cartilage matrix gene expression, but also suppressed the hypertrophic development of SMSCs compared with unloaded controls. Taken together, our findings suggest that dynamic mechanical compression loading not only promotes chondrogenic differentiation of SMSCs, but also plays a vital role in the maintenance of cartilage phenotype, and our findings also provide an experimental guide for stem cell-based cartilage repair and regeneration.

Polycystin-2 Is Required for Chondrocyte Mechanotransduction and Traffics to the Primary Cilium in Response to Mechanical Stimulation.

Primary cilia and associated intraflagellar transport are essential for skeletal development, joint homeostasis, and the response to mechanical stimuli, although the mechanisms remain unclear. Polycystin-2 (PC2) is a member of the transient receptor potential polycystic (TRPP) family of cation channels, and together with Polycystin-1 (PC1), it has been implicated in cilia-mediated mechanotransduction in epithelial cells. The current study investigates the effect of mechanical stimulation on the localization of ciliary polycystins in chondrocytes and tests the hypothesis that they are required in chondrocyte mechanosignaling. Isolated chondrocytes were subjected to mechanical stimulation in the form of uniaxial cyclic tensile strain (CTS) in order to examine the effects on PC2 ciliary localization and matrix gene expression. In the absence of strain, PC2 localizes to the chondrocyte ciliary membrane and neither PC1 nor PC2 are required for ciliogenesis. Cartilage matrix gene expression (Acan, Col2a) is increased in response to 10% CTS. This response is inhibited by siRNA-mediated loss of PC1 or PC2 expression. PC2 ciliary localization requires PC1 and is increased in response to CTS. Increased PC2 cilia trafficking is dependent on the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4) activation. Together, these findings demonstrate for the first time that polycystins are required for chondrocyte mechanotransduction and highlight the mechanosensitive cilia trafficking of PC2 as an important component of cilia-mediated mechanotransduction.

FOXO1 expression in chondrocytes modulates cartilage production and removal in fracture healing.

Fracture healing is a multistage process characterized by inflammation, cartilage formation, bone deposition, and remodeling. Chondrocytes are important in producing cartilage that forms the initial anlagen for the hard callus needed to stabilize the fracture site. We examined the role of FOXO1 by selective ablation of FOXO1 in chondrocytes mediated by Col2α1 driven Cre recombinase. Experimental mice with lineage-specific FOXO1 deletion (Col2α1Cre+FOXO1L/L) and negative control littermates (Col2α1Cre-FOXO1L/L) were used for in vivo, closed fracture studies. Unexpectedly, we found that in the early phases of fracture healing, FOXO1 deletion significantly increased the amount of cartilage formed, whereas, in later periods, FOXO1 deletion led to a greater loss of cartilage. FOXO1 was functionally important as its deletion in chondrocytes led to diminished bone formation on day 22. Mechanistically, the early effects of FOXO1 deletion were linked to increased proliferation of chondrocytes through enhanced expression of cell cycle genes that promote proliferation and reduced expression of those that inhibit it and increased expression of cartilage matrix genes. At later time points experimental mice with FOXO1 deletion had greater loss of cartilage, enhanced formation of osteoclasts, increased IL-6 and reduced numbers of M2 macrophages. These results identify FOXO1 as a transcription factor that regulates chondrocyte behavior by limiting the early expansion of cartilage and preventing rapid cartilage loss at later phases.

Safflower yellow alleviates osteoarthritis and prevents inflammation by inhibiting PGE2 release and regulating NF-κB/SIRT1/AMPK signaling pathways

BackgroundSafflower yellow (SY) is the main active ingredient of safflower, with various pharmacological effects such as anticoagulating, antioxidant, and anti-arthritis effects. PurposeTo investigate the anti-inflammatory and chondrocyte protecting role of SY, which subsequently leads to the inhibition of cartilage degradation. MethodsRat chondrocytes were stimulated with tumor necrosis factor α (TNF-α) with or without SY treatment. Following this, CCK-8 assay was performed to detect cytotoxicity. RT-qPCR, Western blotting, and immunofluorescence staining were used to detect the gene/protein expression of typical cartilage matrix genes and related inflammatory markers. Subsequently, EdU assay was used to evaluate cell proliferation. RNA sequencing, online target prediction, and molecular docking were performed to determine the possible molecular targets and pathways. ResultsThe results showed that SY restored the TNF-α-induced up-regulation of IL-1β, PTGS2, and MMP-13 and down-regulation of COL2A1 and ACAN. Furthermore, it recovered cell proliferation by suppressing TNF-α. Gene expression profiles identified 717 differentially expressed genes (DEGs) in the cells cultured with or without SY under TNF-α stimulation. After pathway enrichment, PI3K-Akt, TNF, Cytokine-cytokine receptor interaction, NF-κB, NOD-like receptor, and Chemokine signaling pathways were notably selected to highlight NFKBIA, CCL5, CCL2, IL6, and TNF as potential targets in osteoarthritis (OA). SY inhibited TNF-α-induced activation of NF-κB and endoplasmic reticulum (ER) stress by promoting AMPK phosphorylation along with SIRT1 expression. Further, SY reduced MMP-13 expression and targeted COX-2 for decreasing PGE2 release. In addition, anterior cruciate ligament transection-induced OA was ameliorated by local administration of SY. ConclusionThese results demonstrate that SY protects chondrocytes and inhibits inflammation by regulating the NF-κB/SIRT1/AMPK pathways and ER stress, thus preventing cartilage degeneration in OA.

FSTL1 promotes nitric oxide-induced chondrocyte apoptosis via activating the SAPK/JNK/caspase3 signaling pathway

ObjectivePrevious studies have shown that follistatin-like protein 1 (FSTL1) is elevated in the synovial fluid of osteoarthritis and is associated with disease activity. The experiment was performed to stuy the effect and mechanism of FSTL1 on chondrocyte apoptosis in osteoarthritis. DesignAfter the isolation of human normal and osteoarthritis (OA) chondrocytes, the expression of FSTL1 was detected by Q-PCR and western blot analyses. Chondrocytes were pre-transfected with FSTL1 overexpression plasmids then treated with SNP, and chondrocyte viability and apoptosis levels were detected by MTS and flow cytometry, respectively. Cartilage matrix gene expression was measured by Q-PCR and signal pathway-related proteins were assessed by western blot. ResultsThe expression of FSTL1 in OA chondrocytes was markedly up-regulated compared with normal human chondrocytes (P < 0.05). The apoptosis rate of chondrocytes in the FSTL1 overexpression groups was highly elevated in the comparison with the negative control groups (P < 0.05). Additionally, FSTL1 potentiated protein abundances of MMP1, MMP3, MMP-9, and Bax as well as reduced Coll2a1 and Aggrecan and Bcl-2 expression. Furthermore, western blot results showed that the SAPK/JNK/Caspase3 signal pathway was significantly activated and the Ac-DEVD-FMK impaired FSTL1 induced chondrocyte apoptosis. ConclusionFSTL1 promoted SNP-induced chondrocytes apoptosis by activating the SAPK/JNK/Caspase3 signal pathway.

Physioxia Has a Beneficial Effect on Cartilage Matrix Production in Interleukin-1 Beta-Inhibited Mesenchymal Stem Cell Chondrogenesis.

Osteoarthritis (OA) is a degenerative condition that involves the production of inflammatory cytokines (e.g., interleukin-1β (IL-1β), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)) that stimulate degradative enzymes, matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS) resulting in articular cartilage breakdown. The presence of interleukin-1β (IL-1β) is one reason for poor clinical outcomes in current cell-based tissue engineering strategies for treating focal early osteoarthritic defects. Mesenchymal stem cells (MSCs) are a potential cell source for articular cartilage regeneration, although IL-1β has been shown to inhibit in vitro chondrogenesis. In vivo, articular chondrocytes reside under a low oxygen environment between 2–5% oxygen (physioxia) and have been shown to enhance in vitro MSC chondrogenic matrix content with reduced hypertrophic marker expression under these conditions. The present investigation sought to understand the effect of physioxia on IL-1β inhibited MSC chondrogenesis. MSCs expanded under physioxic (2% oxygen) and hyperoxic (20%) conditions, then chondrogenically differentiated as pellets in the presence of TGF-β1 and either 0.1 or 0.5 ng/mL IL-1β. Results showed that there were donor variations in response to physioxic culture based on intrinsic GAG content under hyperoxia. In physioxia responsive donors, MSC chondrogenesis significantly increased GAG and collagen II content, whilst hypertrophic markers were reduced compared with hyperoxia. In the presence of IL-1β, these donors showed a significant increase in cartilage matrix gene expression and GAG content relative to hyperoxic conditions. In contrast, a set of MSC donors were unresponsive to physioxia and showed no significant increase in matrix production independent of IL-1β presence. Thus, physioxia has a beneficial effect on MSC cartilage matrix production in responsive donors with or without IL-1β application. The mechanisms controlling the MSC chondrogenic response in both physioxia responsive and unresponsive donors are to be elucidated in future investigations.

Combinatorial Prg4 and Il-1ra Gene Therapy Protects Against Hyperalgesia and Cartilage Degeneration in Post-Traumatic Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of synovial joints characterized by progressive loss of articular cartilage, subchondral bone remodeling, and intra-articular inflammation with synovitis that results in chronic pain and motor impairment. Despite the economic and health impacts, current medical therapies are targeted at symptomatic relief of OA and fail to alter its progression. Given the complexity of OA pathogenesis, we hypothesized that a combinatorial gene therapy approach, designed to inhibit inflammation with interleukin-1 receptor antagonist (IL-1Ra) while promoting chondroprotection using lubricin (PRG4), would improve preservation of the joint compared to monotherapy alone. Employing two surgical techniques to model mild, moderate and severe posttraumatic OA, we found that combined delivery of helper-dependent adenoviruses (HDVs), expressing IL-1Ra and PRG4, preserved articular cartilage better than either monotherapy in both models as demonstrated by preservation of articular cartilage volume and surface area. This improved protection was associated with increased expression of proanabolic and cartilage matrix genes together with decreased expression of catabolic genes and inflammatory mediators. In addition to improvements in joint tissues, this combinatorial gene therapy prolonged protection against thermal hyperalgesia compared to either monotherapy. Taken together, our results show that a combinatorial strategy is superior to monotherapeutic approaches for treatment of posttraumatic OA.

SFMBT2 positively regulates SOX9 and chondrocyte proliferation.

SRY‑box9(SOX9) is the master regulator of the chondrocyte phenotype, which is essential for differentiating chondrogenic mesenchymal condensations into chondrocytes, and is involved in regulating every stage of chondrocyte differentiation. SOX9 deletion in chondrocytes at the late stages of cartilage development results in decreased chondrocyte proliferation; inhibited expression of cartilage matrix genes, including Indian hedgehog and the downstream parathyroid hormone‑related protein; and premature conversion of proliferating chondrocytes into hypertrophic chondrocytes, which mineralize their matrix prematurely. Therefore, SOX9 is considered vital for the majority of phases of chondrocyte lineage, from early condensations to the differentiation of proliferating chondrocytes, leading to chondrocyte hypertrophy. It has been reported that SOX9 expression is decreased in osteoarthritis(OA) cartilage. Regeneration or repair of cartilage degradation in OA remains a challenge. Previous studies have indicated that overexpression of SOX9 can promote cartilage repair and can be used as a potential therapeutic agentat the early stages of human OA. The present study identified Scm‑like with four malignant brain tumor domains2(SFMBT2) as a novel regulator of SOX9 expression in human chondrocytes. Our previous study revealed that SFMBT2 is negatively regulated in OA cartilage, and decreased levels of SFMBT2 contribute to the catabolic phenotype of chondrocytes. The present study detected increased expression levels of SFMBT2 in early cartilage development and during the early phases of chondrogenesis. Overexpression of SFMBT2 in C28/I2 cells upregulated SOX9 expression in a dose‑dependent manner. Furthermore, SFMBT2 positively regulated C28/I2 cell proliferation and restored the decreased levels of SOX9 in chondrocytes following tumor necrosis factor‑α treatment. Additional studies may reveal novel insights into the molecular mechanism involved and the potential role of SFMBT2 in cartilage repair and OA management.

Delta-like 2 negatively regulates chondrogenic differentiation.

Delta-like 2 (Dlk2), a glycoprotein highly homologous to Dlk1, belongs to the Notch/Delta/Serrata family. Dlk2 has been shown to be an important regulator of adipogenesis; however, its role in other cellular differentiation processes is still unknown. Therefore, in this study, we aimed to determine the role of Dlk2 in chondrogenic differentiation. We found that Dlk2 overexpression promoted the growth of ATDC5 cells but inhibited insulin-induced ATDC5 chondrogenic differentiation, as supported by the reduction in cartilage matrix formation and gene expression of aggrecan (acan), collagentype II (col2a1) and X (col10a1). In contrast, Dlk2 silencing inhibited the proliferation of ATDC5 cells but enhanced their chondrogenic differentiation. We then evaluated the roles of mitogen-activated protein kinases (MAPKs), which are activated by insulin during the chondrogenesis of ATDC5 cells. Overexpression of Dlk2 protein strongly promoted the activation of p38, but not extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK). Moreover, as expected, Dlk2 silencing inhibited the activation of p38, but had no effect on the ERK1/2 and JNK pathways. Finally, we also detected the expression of Dlk2 in mouse epiphyseal cartilage during embryo development. The expression of the Dlk2 protein in the limb bud could be detected at embryonic day 11.5; additionally, it was found to decrease in the superficial zones, but remained unchanged in the deep/hypertrophic zones. In conclusion, our results suggested that Dlk2 acted as an important regulator of chondrogenesis through the p38 pathway. These findings may lead to strategies for the treatment of cartilage-related diseases such as osteoarthritis.

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths—called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture.

Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non-chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P<0.05) increased gene expression of proliferation-associated molecules (cyclin D1 and ki67), as well as significantly (P<0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast-like matrix, type I collagen (COL1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly (P>0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL1 was further decreased. These results reveal that actin polymerization status regulates chondrocyte dedifferentiation. Reorganization of the cytoskeleton by actin depolymerization appears to be an active regulatory mechanism for redifferentiation of passaged chondrocytes.

Human articular chondrocytes express functional leukotriene B4 receptors.

Leukotriene B4 (LTB4) is a potent chemoattractant associated with the development of osteoarthritis (OA), while its receptors BLT1 and BLT2 have been found in synovium and subchondral bone. In this study, we have investigated whether these receptors are also expressed by human cartilage cells and their potential effects on cartilage cells. The expression of LTB4 receptors in native tissue and cultured cells was assessed by immunohistochemistry, immunocytochemistry, polymerase chain reaction (PCR) and electron microscopy. The functional significance of the LTB4 receptor expression was studied by Western blotting, using phospho-specific antibodies in the presence or absence of receptor antagonists. In further studies, the secretion of pro-inflammatory cytokines, growth factors and metalloproteinases by LTB4-stimulated chondrocytes was measured by multiplex protein assays. The effects of LTB4 in cartilage signature gene expression in cultured cells were assessed by quantitative PCR, whereas the LTB4-promoted matrix synthesis was determined using 3D pellet cultures. Both receptors were present in cultured chondrocytes, as was confirmed by immunolabelling and PCR. The relative quantification by PCR demonstrated a higher expression of the receptors in cells from healthy joints compared with OA cases. The stimulation of cultured chondrocytes with LTB4 resulted in a phosphorylation of downstream transcription factor Erk 1/2, which was reduced after blocking BLT1 signalling. No alteration in the secretion of cytokine and metalloproteinases was recorded after challenging cultured cells with LTB4; likewise, cartilage matrix gene expression and 3D tissue synthesis were unaffected. Chondrocytes express BLT1 and BLT2 receptors, and LTB4 activates the downstream Erk 1/2 pathway by engaging the high-affinity receptor BLT1. However, any putative role in cartilage biology could not be revealed, and remains to be clarified.

SAT0568 IL-6-Stat3-Signaling Enhances Chondrogenic Differentiation of Human Mesenchymal Stem Cells

BackgroundMesenchymal stem cells (MSCs) can differentiate into cells of mesenchymal lineages, such as chondrocytes. Given the finding that MSCs exist in articular cartilage, it is conceivable that MSCs contribute to...

Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases

Cartilage repair by mesenchymal stem cells (MSCs) often occurs in diseased joints in which the inflamed microenvironment impairs chondrogenic maturation and causes neocartilage degradation. In this environment, melatonin exerts an antioxidant effect by scavenging free radicals. This study aimed to investigate the anti-inflammatory and chondroprotective effects of melatonin on human MSCs in a proinflammatory cytokine-induced arthritic environment. MSCs were induced toward chondrogenesis in the presence of interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) with or without melatonin. Levels of intracellular reactive oxygen species (ROS), hydrogen peroxide, antioxidant enzymes, and cell viability were then assessed. Deposition of glycosaminoglycans and collagens was also determined by histological analysis. Gene expression of chondrogenic markers and matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. In addition, the involvement of the melatonin receptor and superoxide dismutase (SOD) in chondrogenesis was investigated using pharmacologic inhibitors. The results showed that melatonin significantly reduced ROS accumulation and increased SOD expression. Both IL-1β and TNF-α had an inhibitory effect on the chondrogenesis of MSCs, but melatonin successfully restored the low expression of cartilage matrix and chondrogenic genes. Melatonin prevented cartilage degradation by downregulating MMPs. The addition of luzindole and SOD inhibitors abrogated the protective effect of melatonin associated with increased levels of ROS and MMPs. These results demonstrated that proinflammatory cytokines impair the chondrogenesis of MSCs, which was rescued by melatonin treatment. This chondroprotective effect was potentially correlated to decreased ROS, preserved SOD, and suppressed levels of MMPs. Thus, melatonin provides a new strategy for promoting cell-based cartilage regeneration in diseased or injured joints.

Pronounced Biomaterial Dependency in Cartilage Regeneration Using Nonexpanded Compared with Expanded Chondrocytes

We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. Chondrocytes were both directly and after expansion to passage 2, incorporated into four biomaterials: Polyactive™, Beriplast®, HyStem® and a type II collagen gel. Early cartilage matrix gene expression, cytokine production and glycosaminoglycan (GAG) and DNA content in response to these biomaterials were evaluated. HyStem induced more GAG production, compared with all other biomaterials (p ≤ 0.001). Nonexpanded cells did not always produce more GAGs than expanded chondrocytes, as this was biomaterial-dependent. Cytokine production and early gene expression were not predictive for final regeneration. For chondrocyte-based cartilage treatments, the biomaterial best supporting cartilage matrix production will depend on the chondrocyte differentiation state and cannot be predicted from early gene expression or cytokine profile.

Effects of heme oxygenase‐1 on bacterial antigen‐induced articular chondrocyte catabolism in vitro

This study tested the hypothesis that heme oxygenase-1 (HO-1) expression counteracts bacterial antigen-induced catabolic metabolism in human articular chondrocytes. HO-1 expression was induced in chondrocytes by the iron-containing porphoryin, hemin. Anti-catabolic and anti-apoptotic effects of HO-1 expression were evaluated following bacterial antigen (lipopolysaccharides, LPS) activation of chondrocytes by quantification of cytokine and cartilage matrix protein expression. Effects of HO-1 over-expression on chondrocyte matrix metabolism were evaluated using plasmid-driven protein synthesis. Hemin increased HO-1 expression and LPS increased interleukin-1beta and interleukin-6 gene and protein expression in chondrocytes. Hemin-induced HO-1 decreased LPS-induced interleukin-1beta and interleukin-6 gene and protein expression. Increased HO-1 expression partially reversed LPS-suppression of aggrecan and type II collagen gene expression and suppressed LPS-induced gene expression of IL-6, inducible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs), and IL-1beta. HO-1 induction was inversely correlated with LPS-induced chondrocyte apoptosis. HO-1 over-expression in chondrocytes decreased matrix protein gene expression. With LPS activation, increased HO-1 expression decreased chondrocyte catabolism, partially reversed LPS-dependent inhibition of cartilage matrix protein expression and protected against apoptosis. Without LPS, hemin-induced HO-1 and plasmid-based over-expression of HO-1 inhibited cartilage matrix gene expression. The results suggest that elevated HO-1 expression in chondrocytes is protective of cartilage in inflamed joints but may otherwise suppress matrix turn over.

The pericellular environment regulates cytoskeletal development and the differentiation of mesenchymal stem cells and determines their response to hydrostatic pressure

The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP) in regulating chondrogenesis of mesenchymal stem cells (MSCs) and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker) found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.

Hypoxia promotes redifferentiation and suppresses markers of hypertrophy and degeneration in both healthy and osteoarthritic chondrocytes

IntroductionHypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation.MethodsMonolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured.ResultsIn hypoxia, both healthy and OA chondrocytes had higher human collagen type II, α1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, α1 gene (COL10A1), and human collagen type I, α1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1α and HIF-2α had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time.ConclusionsHypoxic culture of human chondrocytes has long been acknowledged to result in increased matrix accumulation, but still little is known of its effects on catabolism. We show herein that the increased expression of matrix proteins, combined with decreased expression of numerous degradative enzymes by hypoxia, minimizes but does not abolish differences between redifferentiated healthy and OA chondrocytes. Hypoxia-induced HIF expression is associated with hypertrophic marker and degradative enzyme downregulation and increased measures of redifferentiation in both healthy and OA chondrocytes. Therefore, though HIFs may be involved in the pathogenesis of OA, conditions that promote HIF expression in vitro promote matrix accumulation and decrease degradation and hypertrophy, even in cells from OA joints.

A Spectrophotometric Analysis of Human Osteoarthritic Cartilage Explants Subjected to Specific Capacitively Coupled Electric Fields

We have previously shown that capacitively coupled electrical stimulation of either normal bovine articular chondrocytes or osteoarthritic human articular cartilage explants resulted in up-regulation of cartilage matrix gene expression and down-regulation of metalloproteinase gene expression. In addition, collagen and proteoglycan protein levels were also elevated. To determine visually the effect of specific electric fields on modifying cartilage structure, freshly harvested human full-thickness osteoarthritic cartilage explants were stimulated in the absence or presence of interleukin-1β, an inflammatory cytokine, and were examined photographically and spectrophotometrically. Hexosamine and hydroxyproline contents were also determined. Spectrophotometric analysis was used to quantify any changes in the depth of defects in the cartilage ranging from surface level (red-colored) to the deepest affected layer (blue-colored). Interleukin-1β treatment alone caused significant additional cartilage erosion. Electrical stimulation alone resulted in significant decreases in the cartilage defects. Electrical stimulation in the presence of interleukin-1β resulted in a small, but significant, surface improvement. Meta-analysis also confirmed a significant increase in the hexosamine and hydroxyproline contents (indicating matrix deposition). It was concluded that an appropriate electric field could modify osteoarthritic lesions in full-thickness cartilage plugs by increasing matrix production and/or by decreasing matrix destruction. Furthermore, it appears that spectrophotometric analysis is a relatively easy method for quantifying the “filling in” or healing of articular cartilage defects.