Cartilage Gene Research Articles

Intra-articular injection of urinary bladder matrix reduces the development of post traumatic osteoarthritis

Purpose: Urinary bladder matrix (UBM) is a naturally derived biomaterial derived from a cellular epithelial basement membrane and lamina propria of the porcine urinary bladder. The objective of this study was to assess the disease modifying ability of UBM in a mouse model of post traumatic osteoarthritis (OA). Methods: C57BL6 male mice underwent anterior cruciate ligament transection (ACLT) to induce OA. Two weeks after ACLT, 10 μl of UBM (ACell, Inc.) or saline was injected intra-articularly into the mouse joint. Cartilage integrity was assessed using OARSI scoring of histology at 4 and 8 weeks post ACLT and correlated with functional pain testing. Additionally, joints were harvested for quantitative PCR (qPCR) of cartilage and inflammatory markers at 4 and 8 weeks post ACLT. Results: In UBM treated animals, the average OARSI score was reduced from 3.28 (saline control) to 1.43 at 4 weeks, and to 1.92 at 8 weeks (p < 0.05), with more intense proteoglycan staining and intact articular cartilage. qPCR analysis was performed and fold changes calculated with respect to un-operated, age matched controls. This revealed that, compared to saline controls, UBM injected animals expressed significantly higher levels of the structural cartilage gene collagen 2α1 (on average 13.27 fold compared to 4.14 fold), aggrecan (9.24 fold compared to 3.99 fold), and the anti-inflammatory genes IL-4 (2.64 fold compared to 1.19 fold) and IL-10 (5.00 fold compared to 1.22 fold). These results correlated with the functional tests, in which the mice displayed a significant reduction of pain on the hot plate and incapacitance tester. Conclusions: UBM lessens cartilage degeneration in the ACLT model of OA. This therapeutic effect maybe due to the reduced inflammation in the joint and maintenance of high expression levels of proteoglycans, which may be helping to retain normal cartilage.

Exercise-driven metabolic pathways in healthy cartilage

Exercise is vital for maintaining cartilage integrity in healthy joints. Here we examined the exercise-driven transcriptional regulation of genes in healthy rat articular cartilage to dissect the metabolic pathways responsible for the potential benefits of exercise. Transcriptome-wide gene expression in the articular cartilage of healthy Sprague-Dawley female rats exercised daily (low intensity treadmill walking) for 2, 5, or 15 days was compared to that of non-exercised rats, using Affymetrix GeneChip arrays. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for Gene Ontology (GO)-term enrichment and Functional Annotation analysis of differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway mapper was used to identify the metabolic pathways regulated by exercise. Microarray analysis revealed that exercise-induced 644 DEGs in healthy articular cartilage. The DAVID bioinformatics tool demonstrated high prevalence of functional annotation clusters with greater enrichment scores and GO-terms associated with extracellular matrix (ECM) biosynthesis/remodeling and inflammation/immune response. The KEGG database revealed that exercise regulates 147 metabolic pathways representing molecular interaction networks for Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, and Diseases. These pathways collectively supported the complex regulation of the beneficial effects of exercise on the cartilage. Overall, the findings highlight that exercise is a robust transcriptional regulator of a wide array of metabolic pathways in healthy cartilage. The major actions of exercise involve ECM biosynthesis/cartilage strengthening and attenuation of inflammatory pathways to provide prophylaxis against onset of arthritic diseases in healthy cartilage.

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes. However, its role in articular cartilage remains obscure. Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes, the distribution of Twist1 proteins in normal versus OA human articular cartilage, and its role in modulating OA development in mice. High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features. The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro. In normal human articular cartilage, Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones. However, our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage. Moreover, Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples. To assess the role of Twist1 in OA pathophysiology, we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus. At 12 weeks post-surgery, micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice. Collectively, the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration.

The next generation of stem cell therapy: “From cartilage explants to arthritic patients”

The next generation of stem cell therapy: “From cartilage explants to arthritic patients”

Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage.

This study reports the effects of the iron chelator deferoxamine (DFO) on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA) articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1–50 μM). Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK) concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10–50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA), AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

A developmental transcriptomic analysis ofPax1andPax9in embryonic intervertebral disc development

ABSTRACTPax1 and Pax9 play redundant, synergistic functions in the patterning and differentiation of the sclerotomal cells that give rise to the vertebral bodies and intervertebral discs (IVD) of the axial skeleton. They are conserved in mice and humans, whereby mutation/deficiency of human PAX1/PAX9 has been associated with kyphoscoliosis. By combining cell-type-specific transcriptome and ChIP-sequencing data, we identified the roles of Pax1/Pax9 in cell proliferation, cartilage development and collagen fibrillogenesis, which are vital in early IVD morphogenesis. Pax1 is up-regulated in the absence of Pax9, while Pax9 is unaffected by the loss of Pax1/Pax9. We identified the targets compensated by a single- or double-copy of Pax9. They positively regulate many of the cartilage genes known to be regulated by Sox5/Sox6/Sox9 and are connected to Sox5/Sox6 by a negative feedback loop. Pax1/Pax9 are intertwined with BMP and TGF-B pathways and we propose they initiate expression of chondrogenic genes during early IVD differentiation and subsequently become restricted to the outer annulus by the negative feedback mechanism. Our findings highlight how early IVD development is regulated spatio-temporally and have implications for understanding kyphoscoliosis.

The infrapatellar fat pad from diseased joints inhibits chondrogenesis of mesenchymal stem cells.

Cartilage repair by bone marrow derived mesenchymal stem cells (MSCs) can be influenced by inflammation in the knee. Next to synovium, the infrapatellar fat pad (IPFP) has been described as a source for inflammatory factors. Here, we investigated whether factors secreted by the IPFP affect chondrogenesis of MSCs and whether this is influenced by different joint pathologies or obesity. Furthermore, we examined the role of IPFP resident macrophages. First, we made conditioned medium from IPFP obtained from osteoarthritic joints, IPFP from traumatically injured joints during anterior cruciate ligament reconstruction, and subcutaneous adipose tissue. Additionally, we made conditioned medium of macrophages isolated from osteoarthritic IPFP and of polarised monocytes from peripheral blood. We evaluated the effect of different types of conditioned medium on MSC chondrogenesis. Conditioned medium from IPFP decreased collagen 2 and aggrecan gene expression as well as thionin and collagen type 2 staining. This anti-chondrogenic effect was the same for conditioned medium from IPFP of osteoarthritic and traumatically injured joints. Furthermore, IPFP from obese (Body Mass Index >30) donors did not inhibit chondrogenesis more than that of lean (Body Mass Index <25) donors. Finally, conditioned medium from macrophages isolated from IPFP decreased the expression of hyaline cartilage genes, as did peripheral blood monocytes stimulated with pro-inflammatory cytokines. The IPFP and the resident pro-inflammatory macrophages could therefore be targets for therapies to improve MSC-based cartilage repair.

Catabolic cytokines disrupt the circadian clock and the expression of clock-controlled genes in cartilage via an NFкB-dependent pathway

SummaryObjectiveTo define how the catabolic cytokines (Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFα)) affect the circadian clock mechanism and the expression of clock-controlled catabolic genes within cartilage, and to identify the downstream pathways linking the cytokines to the molecular clock within chondrocytes.MethodsEx vivo cartilage explants were isolated from the Cry1-luc or PER2::LUC clock reporter mice. Clock gene dynamics were monitored in real-time by bioluminescence photon counting. Gene expression changes were studied by qRT-PCR. Functional luc assays were used to study the function of the core Clock/BMAL1 complex in SW-1353 cells. NFкB pathway inhibitor and fluorescence live-imaging of cartilage were performed to study the underlying mechanisms.ResultsExposure to IL-1β severely disrupted circadian gene expression rhythms in cartilage. This effect was reversed by an anti-inflammatory drug dexamethasone, but not by other clock synchronizing agents. Circadian disruption mediated by IL-1β was accompanied by disregulated expression of endogenous clock genes and clock-controlled catabolic pathways. Mechanistically, NFкB signalling was involved in the effect of IL-1β on the cartilage clock in part through functional interference with the core Clock/BMAL1 complex. In contrast, TNFα had little impact on the circadian rhythm and clock gene expression in cartilage.ConclusionIn our experimental system (young healthy mouse cartilage), we demonstrate that IL-1β (but not TNFα) abolishes circadian rhythms in Cry1-luc and PER2::LUC gene expression. These data implicate disruption of the chondrocyte clock as a novel aspect of the catabolic responses of cartilage to pro-inflammatory cytokines, and provide an additional mechanism for how chronic joint inflammation may contribute to osteoarthritis (OA).

Collagen duplicate genes of bone and cartilage participate during regeneration of zebrafish fin skeleton

The zebrafish fin is widely used as a model for skeleton regeneration. For years, the nature of the fin skeleton has been controversial as its extracellular matrix shows hybrid characteristics of both bone and cartilage. The presence of co-orthologs genes also increases the complexity of these tissues. In this article, we have identified and described the expression of fibrillar collagens in zebrafish fin skeleton. We found that genes coding for types I, II, V, XI and XXVII collagens are duplicated, showing in several cases, different expression domains. We also identified specific genomic features, such as the presence of type XXIV collagen and the absence of type III collagen in the zebrafish genome. Our study showed that actinotrichia-forming cells and osteoblasts synthesize a wide variety of these fibrillar collagens during fin regeneration. An intertrichial domain expressing most of the collagens was located in the transition between the mesenchyme condensations of actinotrichia and lepidotrichia and may determine an important niche associated with fin skeleton morphogenesis. We also confirmed the hybrid nature of the fin exoskeleton and provided a complete description of those fibrillar collagens expressed during the formation of the fin skeleton.

Comparing effects of perfusion and hydrostatic pressure on gene profiles of human chondrocyte.

Hydrostatic pressure and perfusion have been shown to regulate the chondrogenic potential of articular chondrocytes. In order to compare the effects of hydrostatic pressure plus perfusion (HPP) and perfusion (P) we investigated the complete gene expression profiles of human chondrocytes under HPP and P. A simplified bioreactor was constructed to apply loading (0.1MPa for 2h) and perfusion (2ml) through the same piping by pressurizing the medium directly. High-density monolayer cultures of human chondrocytes were exposed to HPP or P for 4 days. Controls (C) were maintained in static cultures. Gene expression was evaluated by sequencing (RNAseq) and quantitative real-time PCR analysis. Both treatments changed gene expression levels of human chondrocytes significantly. Specifically, HPP and P increased COL2A1 expression and decreased COL1A1 and MMP-13 expression. Despite of these similarities, RNAseq revealed a list of cartilage genes including ACAN, ITGA10 and TNC, which were differentially expressed by HPP and P. Of these candidates, adhesion related molecules were found to be upregulated in HPP. Both HPP and P treatment had beneficial effects on chondrocyte differentiation and decreased catabolic enzyme expression. The study provides new insight into how hydrostatic pressure and perfusion enhance cartilage differentiation and inhibit catabolic effects.

Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the BCL2 gene family. Further investigation may reveal novel prognostic biomarkers and potential targets for therapeutic interventions in the early stages of OA.

Interplay between Genetic Risk Factors and Protective Mechanisms for Intervertebral Disc Degeneration in Mice

Introduction Intervertebral disc degeneration is a major cause of back pain. It is clear that there is a significant genetic contribution to the susceptibility of an individual-to-disc degeneration. Study in Hong Kong population cohort for disc degeneration cohort showed that while approximately 90% of the people will develop some form of disc degeneration by the age of 50 years, there are individuals who showed no indication of disc degeneration by MRI, suggesting the possibility of “protective factors,” either genetic and/or environmental. We have previously shown that Aspirin, an extracellular matrix protein, is a genetic risk factor of disc degeneration. Furthermore, our unpublished data have revealed that transgenic mice (ASPN-tg) overexpressing human ASPN gene in cartilage and disc tissues results in an accelerated or abnormal differentiation of cells in the mouse nucleus pulposus that could enhance initiation of disc degeneration. It is possible to study protective genes in humans using case–control association studies, but this would require the collection of a very large cohort of “protected” individuals. Another approach is to study in animal models and map the identified loci to the human genome. In mice, there are “super healer” strains (e.g., LG/J) with superior healing potentials of damaged tissues including ear elastic cartilage and hyaline cartilage of synovial joints, and potential genetic loci for improved joint cartilage repair have been identified in these mice. Materials and Methods Using the LG/J mice as a source of “protective” genes, we aim to perform sequential backcross of the ASPN-tg mice in C57 background to an LG/J background and compare the degenerative outcome in each generation. Our hypothesis is that if there is a protective or modulating effect, this will be reflected in histological analysis of the tail discs, coupled with the use of specific molecular analysis of differentiation markers for the different population of nucleus pulposus (NP) cells. Results Our data showed that ASPN-tg in successive backcross generations to LG/J background showed progressive improvement in the repair potential to ear punch wound compared with the parental strain, ASPN-tg/C57, for the repair of a 2 mm through-and-through ear punch wound. This is our positive indicator for the presence of “healing” genes in corresponding F1, F2, and F3 generations for the repair of damages to ear cartilage. In the NP for the backcrossed mice at F3, histological and immunostaining of cells in the NP showed fewer or absence of clusters of small round-shaped cells and Brachyury/Sox-9 positive chondrocyte-like cells were observed in ASPN-tg/C57 mice, suggesting there are genes in the LG/J background that reduces the risk or susceptibility to disc degeneration. Conclusion Our study reveals that genetic components in mice with a genetic background for better wound healing can provide protection against the morphological changes to chondrocyte-like cells in the NP that we use as an indicator of disc degeneration in intervertebral discs, induced by enhanced expression of ASPN in NP cells. Further investigation is needed to map the protective genes and study their functional impact on genetic risk factors to provide mechanistic insight for this protection or modulation of disc degeneration.

Transcriptional associations of osteoarthritis-mediated loss of epigenetic control in articular cartilage.

To identify osteoarthritis (OA) progression-modulating pathways in articular cartilage and their respective regulatory epigenetic and genetic determinants in end-stage disease. Transcriptional activity of CpG was assessed using gene expression data and DNA methylation data for preserved and lesional articular cartilage samples. Disease-responsive transcriptionally active CpG were identified by means of differential methylation between preserved and lesional cartilage. Transcriptionally relevant genetic determinants were addressed by means of single-nucleotide polymorphisms (SNPs) proximal to the OA-responsive transcriptionally active CpG. Statistical analyses were corrected for age, sex, joint, and technical covariates. A random effect was included to correct for possible correlations between paired samples. Of 9,838 transcribed genes in articular cartilage, 2,324 correlated with the methylation status of 3,748 transcriptionally active CpG; both negative (n = 1,741) and positive (n = 2,007) correlations were observed. Hypomethylation and hypermethylation (false discovery rate of <0.05, |Δβ| > 0.05) were observed for 62 and 25 transcriptionally active CpG, respectively, covering 70 unique genes. Enrichment for developmental and extracellular matrix maintenance pathways indicated possible reactivation of endochondral ossification. Finally, we observed 31 and 26 genes for which methylation and expression, respectively, were additionally affected by genetic variation. We identified tissue-specific genes involved in OA disease progression, reflected by genetic and pathologic epigenetic regulation of transcription, primarily at genes involved in development. Therefore, transcriptionally active SNPs near these genes may serve as putative susceptibility alleles. Our results constitute an important step toward understanding the reported widespread epigenetic changes occurring in OA articular cartilage and toward subsequent development of treatments targeting disease-driving pathways.

Transcriptional profiling of PRKG2-null growth plate identifies putative down-stream targets of PRKG2.

BackgroundKinase activity of cGMP-dependent, type II, protein kinase (PRKG2) is required for the proliferative to hypertrophic transition of growth plate chondrocytes during endochondral ossification. Loss of PRKG2 function in rodent and bovine models results in dwarfism. The objective of this study was to identify pathways regulated or impacted by PRKG2 loss of function that may be responsible for disproportionate dwarfism at the molecular level.MethodsMicroarray technology was used to compare growth plate cartilage gene expression in dwarf versus unaffected Angus cattle to identify putative downstream targets of PRGK2.ResultsPathway enrichment of 1284 transcripts (nominal p < 0.05) was used to identify candidate pathways consistent with the molecular phenotype of disproportionate dwarfism. Analysis with the DAVID pathway suite identified differentially expressed genes that clustered in the MHC, cytochrome B, WNT, and Muc1 pathways. A second analysis with pathway studio software identified differentially expressed genes in a host of pathways (e.g. CREB1, P21, CTNNB1, EGFR, EP300, JUN, P53, RHOA, and SRC). As a proof of concept, we validated the differential expression of five genes regulated by P53, including CEBPA, BRCA1, BUB1, CD58, and VDR by real-time PCR (p < 0.05).ConclusionsKnown and novel targets of PRKG2 were identified as enriched pathways in this study. This study indicates that loss of PRKG2 function results in differential expression of P53 regulated genes as well as additional pathways consistent with increased proliferation and apoptosis in the growth plate due to achondroplastic dwarfism.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1136-6) contains supplementary material, which is available to authorized users.

DIO2-knockout modulates circadian clock genes in articular cartilage through thyroid hormone signaling

DIO2-knockout modulates circadian clock genes in articular cartilage through thyroid hormone signaling

The tissue specific role of SirT1 in joint inflammation and anabolic cartilage gene expression

The tissue specific role of SirT1 in joint inflammation and anabolic cartilage gene expression

Expression of hedgehog signal pathway in articular cartilage is associated with the severity of cartilage damage in rats with adjuvant-induced arthritis.

BackgroundCartilage damage is a crucial step in rheumatoid arthritis (RA) disease progress while its molecular mechanisms are not fully understood. Here we investigated the expression of hedgehog (Hh) signal pathway in articular cartilage of adjuvant-induced arthritis (AIA) rats and its possible pathological role in cartilage damage.Methods30 rats were divided into sham and AIA group (n = 15). Complete Freund’s adjuvant was used to induce AIA. Secondary paw swelling was measured on day 10, 14, 18, 22 and 26 after induction. Rats were sacrificed on day 26 and knee joints and cartilage tissues were collected. Paw swelling, cartilage histopathologic changes and OARSI scores were used to evaluate AIA in rats. The protein expression of Hh signal related genes (Shh, Ptch1, Smo and Gli1) in cartilage were assayed by immunohistochemistry. The mRNA levels of Shh, Ptch1, Smo, Gli1, type-II collagen (COII) and aggrecan in cartilage were assayed by real-time PCR. In vitro study, cultured AIA chondrocytes were treated with cyclopamine (a specific inhibitor of Hh signal) and the mRNA levels of Hh signal and ECM components (COII and aggrecan) were measured by real-time PCR.ResultsImmunohistochemical results revealed that Shh, Ptch1, Smo and Gli1 proteins showed higher expression in the articular cartilage of AIA rats than those of sham rats. Real-time PCR results confirmed that Shh, Ptch1, Smo and Gli1 mRNA levels in cartilage tissues of AIA rats were significantly increased compared with those of sham rats (1.6, 1.4, 1.6, 2.0 fold, respectively). The mRNA levels of Shh, Ptch1, Smo, and Gli1 were associated with the severity of cartilage damage (indicated by OARSI scores, COII and aggrecan mRNA levels in cartilage). In vitro, cyclopamine effectively decreased the mRNA levels of Shh, Ptch1, Smo and Gli1, and increased the mRNA levels of COII and aggrecan in AIA chondrocytes, suggesting Hh signal inhibition might directly promote ECM production.ConclusionsOur findings present certain experimental evidence that Hh signal pathway is involved in the pathogenesis of cartilage damage in RA.

Cartilage regeneration for treatment of osteoarthritis: a paradigm for nonsurgical intervention.

Osteoarthritis (OA) is associated with articular cartilage abnormalities and affects people of older age: preventative or therapeutic treatment measures for OA and related articular cartilage disorders remain challenging. In this perspective review, we have integrated multiple biological, morphological, developmental, stem cell and homeostasis concepts of articular cartilage to develop a paradigm for cartilage regeneration. OA is conceptually defined as an injury of cartilage that initiates chondrocyte activation, expression of proteases and growth factor release from the matrix. This regenerative process results in the local activation of inflammatory response genes in cartilage without migration of inflammatory cells or angiogenesis. The end results are catabolic and anabolic responses, and it is the balance between these two outcomes that controls remodelling of the matrix and regeneration. A tantalizing clinical clue for cartilage regrowth in OA joints has been observed in surgically created joint distraction. We hypothesize that cartilage growth in these distracted joints may have a biological connection with the size of organs and regeneration. Therefore we propose a novel, practical and nonsurgical intervention to validate the role of distraction in cartilage regeneration in OA. The approach permits normal wake-up activity while during sleep; the index knee is subjected to distraction with a pull traction device. Comparison of follow-up magnetic resonance imaging (MRI) at 3 and 6 months of therapy to those taken before therapy will provide much-needed objective evidence for the use of this mode of therapy for OA. We suggest that the paradigm presented here merits investigation for treatment of OA in knee joints.

Differentiation of Human Induced Pluripotent Stem Cells to Chondrocytes.

Human induced pluripotent stem (iPS) cells are relevant tools for modeling human skeletal development and disease, and represent a promising source of patient-specific cells for the regeneration of skeletal tissue, such as articular cartilage. Devising efficient and reproducible strategies, which closely mimic the physiological chondrogenic differentiation process, will be necessary to generate functional chondrocytes from human iPS cells. Our previous study demonstrated the generation of chondrogenically committed human iPS cells via the enrichment of a mesenchymal-like progenitor population, application of appropriate high-density culture conditions, and stimulation with bone morphogenetic protein-2 (Bmp-2). The differentiated iPS cells showed temporal expression of cartilage genes and the accumulation of a cartilaginous extracellular matrix in vitro. In this chapter, we provide detailed methodologies for the differentiation of human iPS cells to the chondrogenic lineage and describe protocols for the analysis of chondrogenic differentiation.

Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration.

IntroductionTo investigate if decreased histone deacetylase 4 (HDAC4) is associated with human osteoarthritis (OA) cartilage degeneration by releasing HDAC4 inhibition of runt-related transcription factor-2 (Runx2) resulting in increase of OA cartilage degeneration-related genes.MethodsThe mRNA and protein levels of HDAC4, Runx2, matrix metalloproteinase (MMP)-13, Indian hedgehog (Ihh) and type X collagen were detected by performing real-time PCR (RT-PCR), western blotting and immunohistochemistry on specimens from human OA and normal cartilage. To further explore the mechanism of regulation of Runx2 and OA-related genes by HDAC4, changes in these OA-related genes were further quantified by RT-PCR after overexpression of HDAC4 and knockdown of HDAC4 by siRNA. Runx2 and MMP-13 promoter activities were measured by dual luciferase assays.ResultsThe levels of HDAC4 in the cartilage from OA patients and healthy 40- to 60-year-old donors were decreased to 31% and 65% compared with specimens from 20- to 40-year-old healthy donors, respectively (P <0.05). Decreased HDAC4 was associated with increased Runx2 and other OA-related genes in human OA cartilage, specifically: MMP-13, Ihh and type X collagen. Exogenous HDAC4 decreased the mRNA levels of Runx2, MMP1, MMP3, MMP-13, type X collagen, Ihh, ADAMTS-4 and -5, and increased the mRNA of type II collagen. In addition, the data also shows that overexpression of HDAC4 not only decreased the expression of interleukin (IL)-1β, Cox2 and iNos and increased the expression of aggrecan, but also partially blocked the effect of IL-1β on expression of catabolic events in human OA chondrocytes. HDAC4 also inhibited Runx2 promoter activity and MMP13 promotor activity in a dose-dependent manner. In contrast, inhibition of HDAC4 by TSA drug had an opposite effect.ConclusionsOur study is the first to demonstrate that decreased HDAC4 contributes, at least in part, to the pathogenesis of OA cartilage degeneration. Thus, HDAC4 may have chondroprotective properties by inhibiting Runx2 and OA-related genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0491-3) contains supplementary material, which is available to authorized users.