Cartilage Matrix Synthesis Research Articles

Understanding CpG Methylation in the Context of Osteoarthritis

Worldwide, osteoarthritis (OA) affects approxi‐ mately 40% of people older than 70 years and is therefore the most common form of arthritis. OA is a complex degenerative disease of the joint but is epitomized by focal degradation of articular cartilage [1]. Cartilage itself is a highly special‐ ized tissue composed of an extracellular matrix (ECM) rich in type‐II collagen and the proteo‐ glycan aggrecan. The tissue is sparsely populated by the sole resident cell, the chondrocyte, which maintains tissue integrity by balancing the syn‐ thesis and degradation of ECM components [2]. In OA, this balance is perturbed in favor of ECM degradation, mediated by proteolytic enzymes including MMP13 and ADAMTS5, which prob‐ ably degrade the type‐II collagen and aggrecan, respectively [3]. Inhibition of metalloproteinase activity should protect against cartilage destruc‐ tion, but has failed in clinical trials in part owing to nonselectivity and resulting adverse side effects [4]. Amongst other things, this has led to the sug‐ gestion that targeting the regulation of expression of specific proteases, or regulating factors, could be an important therapeutic option to slow or halt OA progression. There is an urgent need for such disease‐modifying, noninvasive treatments for OA since the only current therapy is joint replacement surgery. Epigenetic regulation involves three basic mechanisms: DNA methylation, histone modi‐ fications and ncRNAs. DNA methylation rep‐ resents the most stable epigenetic modification and involves the addition of a methyl group to the cytosine at CpG dinucleotides, converting cytosine to 5‐methylcytosine. This functions to disrupt the binding of transcription factors or enhance the binding of repressors, leading to an altered transcriptional state of the cell [5]. In terms of OA research, a number of studies have inves‐ tigated the role that epigenetics, especially DNA methylation, could play in OA, particularly in terms of regulating the balance between matrix synthesis and degradation in cartilage. These studies have focused on correlating DNA methyla‐ tion status with gene expression at a few candidate genes (loci), including those coding for aggrecan, collagens, MMPs, LEPTIN, SOX9 and RUNX2 [6]. Broadly, these works have shown a correlation between promoter methylation and the expression of the associated gene. Recently, we took this work one step further and identified a specific differen‐ tially methylated CpG locus within the MMP13 promoter whose methylation status regulates the expression of the gene by controlling the binding of the transcriptional regulator CREB [7]. These and ongoing ‘candidate’ gene studies will continue to identify differentially methylated regions that may be important in onset of the disease.

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis. A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2-inducible gene expression to prevent articular cartilage degeneration. Col2-Cre;Smad3(fl/fl) mice allowed study of the chondrocyte-intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3(fl/fl) mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP-13) messenger RNA (mRNA) and protein expression. Chondrocyte-specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2-Cre;Smad3(fl/fl) mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP-13-mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2-inducible MMP-13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP-13 expression. Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2-inducible MMP-13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

In Vivo Effects of a Single Intra‐Articular Injection of 2% Lidocaine or 0.5% Bupivacaine on Articular Cartilage of Normal Horses

To assess the effects of an intra-articular (IA) lidocaine or bupivacaine injection on synovial fluid (SF) biomarkers of cartilage metabolism. Experimental. Horses (n = 6). The study had 2 components, each with an identical design: the first arm assessed the effects of bupivacaine (0.5%) IA in the intercarpal joints and, after a 2-week washout period, the second was conducted to evaluate the effects of IA lidocaine (2%) in the tarsocrural joints. The mares were randomly assigned to receive the test local anesthetic in the target joint or the placebo (0.9% NaCl) in the contralateral joint. After a 2-week washout period, treatments were reversed, yielding 6 joints for each treatment. SF samples were collected for measurement of biomarkers of cartilage metabolism. IA bupivacaine or lidocaine induced a significant increase in both markers of cartilage matrix synthesis (CS846-aggrecan and CPII-type II collagen) suggesting an anabolic effect. Bupivacaine also resulted in an unanticipated decrease in both collagen degradation markers (C2C and C1,2C). These results reveal an anabolic effect of single IA injections of local anesthetics on cartilage metabolism. Although collagen degradation biomarker results were not increased, it is possible that the anabolic response is because of an undetected cartilage insult, and requires further investigation.

Metabolic syndrome meets osteoarthritis

Metabolic osteoarthritis (OA) has now been characterized as a subtype of OA, and links have been discovered between this phenotype and metabolic syndrome (MetS)--both with individual MetS components and with MetS as a whole. Hypertension associates with OA through subchondral ischaemia, which can compromise nutrient exchange into articular cartilage and trigger bone remodelling. Ectopic lipid deposition in chondrocytes induced by dyslipidemia might initiate OA development, exacerbated by deregulated cellular lipid metabolism in joint tissues. Hyperglycaemia and OA interact at both local and systemic levels; local effects of oxidative stress and advanced glycation end-products are implicated in cartilage damage, whereas low-grade systemic inflammation results from glucose accumulation and contributes to a toxic internal environment that can exacerbate OA. Obesity-related metabolic factors, particularly altered levels of adipokines, contribute to OA development by inducing the expression of proinflammatory factors as well as degradative enzymes, leading to the inhibition of cartilage matrix synthesis and stimulation of subchondral bone remodelling. In this Review, we summarize the shared mechanisms of inflammation, oxidative stress, common metabolites and endothelial dysfunction that characterize the aetiologies of OA and MetS, and nominate metabolic OA as the fifth component of MetS. We also describe therapeutic opportunities that might arise from uniting these concepts.

Icariin: a potential promoting compound for cartilage tissue engineering

To investigate whether icariin, which is a widely used pharmacological constituent in traditional Chinese herbal medicine, can be a potential promoting compound for cartilage tissue engineering. Icariin was added into cell-hydrogel constructs derived from neonatal rabbit chondrocytes and collagen type I. The chondrogenic gene expressions and the synthesis of cartilage matrix of the seeded cells were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Biochemical assay. The effects of icariin-added cell-hydrogel constructs on the restoration of supercritical-sized osteochondral defects of adult rabbit were investigated by histological observation. The cell-hydrogel constructs without Icariin were set for controls. Icariin obviously up-regulate the expressions included aggrecan, sox9, and collagen type II of seeded chondrocytes from 99.7% to 248%. It increases the synthesis of glycosaminoglycan and collagen type II about fourfold to fivefolds from week 1 to week 4, and accelerates the formation of chondroid tissue in the cell-hydrogel constructs. Even, it improves the restoration efficiency of supercritical-sized osteochondral defects in adult rabbit model, and enhances the integration of new-formed cartilage with subchondral bone. Icariin can be a potential promoting compound for cartilage tissue engineering, and it can be a substitute for the use of some growth factors. The long history and extensive cases of safe use in China, Japan and Korea make it more attractive.

Injectable perlecan domain 1-hyaluronan microgels potentiate the cartilage repair effect of BMP2 in a murine model of early osteoarthritis

The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2-free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2-treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

Purpose: Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties, and effectiveness of aggregated MSCs for cartilage regeneration. Methods: Synovial MSCs. Human synovium was harvested from donors during ACL reconstruction surgery. Rabbit synovium was harvested from the knee joint. After collagenase digestion, nucleated cells were expanded, and colony forming cells were harvested. Preparation of aggregates of MSCs. 2.5×105 synovial MSCs suspended in 35μl of culture medium were aggregated using the hanging drop technique. Microarray analysis. Total RNA was extracted from human synovial MSCs in a monolayer culture, aggregates of human synovial MSCs cultured for 3 days. In vitro chondrogenic differentiation assay. 2.5 × 105 human synovial MSCs cultured as monolayer were pelleted by trypsinization and centrifugation. The pellets or aggregate of human synovial MSCs cultured for 3 days in hanging drop were cultured in 400μl chondrogenic medium. The medium was changed every 3-4 days for 21 days. Transplantation of aggregates of synovial MSCs in rabbits. Osteochondral defects (5×5 mm wide, 1.5 mm deep) were created in the femoral groove, and 5, 10, 20, 40, 80 aggregates of autologous synovial MSCs were transplanted to the defect. MSCs labeled with DiI and MSCs derived from GFP rabbit were also used. The cartilage was evaluated at 1 day, 2, 4, 12 weeks after the operation (n = 5). Statistical analysis. To assess differences, the Kruskal-Wallis test and the Steel test were used. A P value of < 0.05 was considered statistically significant. Results: Aggregates of MSCs cultured for three days became visible, approximately 1mm in diameter, and solid and durable by manipulation, and most of the cells were viable. Microarray analysis revealed upregulation of chondrogenesis-related genes (SOX5, -6, -9, and BMP2) and anti-inflammatory (TSG-6, and STC-1) genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to apoptosis and nutrient deprivation of aggregates of MSCs. Conclusions: Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential, and efficient attachment.

Inhibition of asporin signaling is critical in the prevention of cartilage damage by physiotherapies

Purpose. Physiotherapies are the most frequently recommended non-invasive treatment options recommended for arthritic diseases. Here, we examined the signaling pathways responsible for the beneficial effects of exercise on various stages of monoiodoacetate-induced arthritis (MIA). We demonstrate that both success and failure of the physiotherapy is mediated via discrete signaling pathways involving Asporin, a susceptibility gene in osteoarthritis (OA). Asporin-mediated regulation of matrix synthesis in turn may regulate Transforming Growth Factor-β (TGF-β) networks essential for cartilage matrix synthesis. Methods. Treadmill walking (TW), used as a means of exposing MIA afflicted knees to physiotherapy, was initiated in rats at different stages of MIA induced cartilage damage and compared to unexercised MIA afflicted knees. TW started at 1 day post-MIA induction, or after cartilage damage had progressed to Grade I or Grade II. The cartilage specimens were analyzed by macroscopic, microscopic, μCT imaging, transcriptome-wide gene expression analysis and activation/suppression of signaling networks. Data was further analyzed by Ingenuity Pathways Analysis to construct molecular functional networks and signaling pathways to dissect pathways regulated by exercise. One-way ANOVA and a post hoc Tukey test were used for statistical analysis. Results. Treadmill walking immediately following initiation of MIA (1 day post-MIA induction) demonstrated a significant prevention of MIA progression. However, the efficacy of this intervention was significantly reduced when implemented on knees showing close to Grade I or greater cartilage damage. On the contrary, TW accelerated damage in the knees with close to Grade II cartilage pathologies. Transcriptome-wide gene expression analysis revealed that exercise intervention started 1-day post-MIA inception significantly suppressed signaling networks that inhibit matrix synthesis. In parallel, TW upregulated gene networks associated with matrix synthesis. However, TW intervention following Grade I damage was less effective in preventing cartilage damage and regulating matrix synthesis. Interestingly, Asporin and networks associated with TGF-β expression and signaling were the major gene products that were regulated by TW to control matrix synthesis (Aggrecan, Collagen type II, Matrilin, FRZB, Col 9A2 and Col9A3) and prevent cartilage damage as evident by microscopic grading of cartilage. Conclusions. The findings demonstrate that Asporin might be one of the critical genes controlled by TW, that in turn controls expression of TGF-β family of molecules, essential for cartilage matrix synthesis. The findings underscore the importance of physiotherapies by showing suppression of Asporin, an OA susceptibility gene by TW. Nevertheless, the extent of cartilage damage at the initiation of physiotherapy is an important determinant for the effectiveness of TW.

Regulation of Human Chondrocyte Function through Direct Inhibition of Cartilage Master Regulator SOX9 by MicroRNA-145 (miRNA-145)

Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator transcription factor SOX9. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how SOX9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of SOX9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses SOX9 expression in human cells through a unique binding site in its 3'-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

IntroductionTransplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration.MethodsHuman and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined.ResultsAggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs.ConclusionsAggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment.

The prostaglandin E2 system: A toolbox for skeletal repair?

Prostaglandins, a group of lipid mediators, govern a multitude of biologic processes, including inflammation, platelet aggregation, and cell growth. Because of the key role of prostaglandins in inflammation and pain, their metabolism represents the final target of nonsteroidal antiinflammatory drugs (NSAIDs). In the US, sales volumes for NSAIDs of far more than $1 billion per year highlight the enormous economic impact of these agents and the important role of prostaglandin inhibition in clinical practice (1). Prostaglandin E2 (PGE2), the most widely produced member of the prostaglandin group, originates from arachidonic acid that is released from the cell membrane by phospholipase A2, followed by enzymatic conversion by cyclooxygenase (COX) and PGE synthase 1 (PGES-1). PGE2 not only triggers pain and inflammation but also promotes matrix degradation and tissue damage by stimulating the expression of matrixdegrading enzymes (2). Inflammatory cytokines such as interleukin-1 or tumor necrosis factor strongly increase PGE2 levels, but PGE2 secretion may also increase in response to other stimuli including growth factors, hypoxia, and a number of hormones. Apart from the well-defined role of PGE2 in tissue inflammation, there is increasing evidence that it is involved in the maintenance and repair of the skeletal system. At first glance, the effects of PGE2 on tissue homeostasis of bone and cartilage seem confusing, because they conflict with the long-known destructive potential of the prostaglandins. Nevertheless, both catabolic and anabolic effects have already been described for PGE2 in these tissues. In terms of catabolism, PGE2 can promote osteoclastic bone resorption (3,4) and enhance breakdown of the extracellular matrix by increasing the expression of matrix metalloproteinase 3 (MMP-3) or MMP-13 (2,5). Regarding its anabolic aspects, PGE2 may stimulate chondrogenesis, chondrocyte proliferation (6), cartilage matrix synthesis (7), as well as osteoblast activity (8). To shed light on the apparent ambivalent role of PGE2, a multidimensional view is necessary that addresses the developmental stage, type of tissue, disease status, concentration of PGE2, and specific receptor status of the different cell types. So far, 4 different receptors of PGE2 have been identified, designated EP1, EP2, EP3, and EP4 (9). Specific signal transduction properties characterize each receptor, as follows: EP1 stimulates intracellular Ca2 mobilization, whereas EP2 and EP4 bind GS proteins to enhance cAMP levels and activate protein kinase A. In contrast to EP2 and EP4, binding to EP3 results in decreased cAMP concentrations through Gi, Gq, or GS, depending on the EP3 isoform. The 4 receptor subtypes are expressed differentially within various types of tissues. Within articular cartilage and growth plate cartilage, all 4 receptor subtypes are detectable. Their respective expression patterns, however, vary between the different types of cartilage tissues and depend on the stage of development. Early in the process of cartilage development, PGE2 promotes proliferation and DNA synthesis of chondrocytes via binding to EP1 within the growth plate (6), while PGE2 stabilizes the phenotype of chondrocytes within articular cartilage via binding to EP2 and EP4 in later stages (10). Concentration-dependent effects of PGE2 may also contribute to its pleiotropic and sometimes even contradictory effects, as particularly shown in cartilage metabolism. In experimental settings, administration of PGE2 at low doses (slightly above the physiologic level) can promote chondrocyte proliferation (6) and suppress the expression of the degradative enzymes MMP-3 and MMP-13 (11). PGE2 may also stimulate the expression of cartilage-specific genes, including type II collagen, SOX9, and aggrecan, and inhibit the expression of Supported by the DFG (GE 1975/2-1). Kolja Gelse, MD, Christian Beyer, MD: University of Erlangen–Nuremberg and University Hospital Erlangen, Erlangen, Germany. Address correspondence to Kolja Gelse, MD, Department of Orthopaedic Trauma Surgery, University Hospital Erlangen, Krankenhausstrasse 12, 91054 Erlangen, Germany. E-mail: kolja. gelse@web.de. Submitted for publication November 1, 2010; accepted in revised form November 9, 2010.

Post-transcriptional gene regulation in chondrocytes

The control of gene expression in articular chondrocytes is an essential factor in maintaining the homoeostasis of extracellular matrix synthesis and turnover necessary in healthy articular cartilage. Although much is known of how steady-state levels of gene expression and rates of transcription are altered, there has been a poorer understanding of gene control at the post-transcriptional level and its relevance to cartilage health and disease. Now, an emerging picture is developing of the importance of this tier of gene regulation, driven by in vitro studies and mouse genetic models. This level of cellular regulation represents an as yet unexplored area of potential intervention for the treatment of degenerative cartilage disorders such as osteoarthritis.

Heterotopic Autologous Chondrocyte Transplantation—A Realistic Approach to Support Articular Cartilage Repair?

Injured articular cartilage is limited in its capacity to heal. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but it requires articular cartilage biopsies for sufficient autologous chondrocyte expansion in vitro. Hence, ACT is restricted by donor-site morbidity and autologous articular chondrocytes availability. The use of nonarticular heterotopic chondrocytes such as auricular, nasoseptal, or costal chondrocytes for ACT might overcome these limitations: heterotopic sources show lesser donor-site morbidity and a comparable extracellular cartilage matrix synthesis profile to articular cartilage. However, heterotopic (h)ACT poses a challenge. Particular tissue characteristics of heterotopic cartilage, divergent culturing peculiarities of heterotopic chondrocytes, and the advantages and drawbacks related to these diverse cartilage sources were critically discussed. Finally, available in vitro and in vivo experimental (h)ACT approaches were summarized. The quality of the cartilage engineered using heterotopic chondrocytes remains partly controversy due to the divergent methodologies and culture conditions used. While some encouraging in vivo results using (h)ACT have been demonstrated, standardized culturing protocols are strongly required. However, whether heterotopic chondrocytes implanted into joint cartilage defects maintain their particular tissue properties or can be adapted via tissue engineering strategies to fulfill regular articular cartilage functions requires further studies.

Three-dimensional poly(1,8-octanediol–co-citrate) scaffold pore shape and permeability effects on sub-cutaneous in vivo chondrogenesis using primary chondrocytes

The objective of this study was to evaluate the coupled effects of three-dimensional poly(1,8-octanediol–co-citrate) (POC) scaffold pore shape and permeability on chondrogenesis using primary chondrocytes in vivo. Chondrogenesis was characterized as cartilage matrix formation by sulfated glycosaminoglycan (sGAG) quantification, relative mRNA expression of the cartilage-related proteins collagen types I, II and X, aggrecan and matrix metalloproteinases 13 and 3 and the compressive mechanical properties of the tissue/scaffold construct. A low permeability design with a spherical pore shape showed a significantly greater increase in cartilage matrix formation over 6 weeks in vivo than a high permeability design with a cubical pore shape. This increase in cartilage matrix synthesis corresponded with increases in mechanical compressive nonlinear elastic properties and histological data demonstrating darker red Safranin-O staining. There was higher mRNA expression for both cartilage-specific proteins and matrix degradation proteins in the high permeability design, resulting in overall less sGAG retained in the high permeability scaffold compared with the low permeability scaffold. Controlled POC scaffolds with a spherical pore shape and low permeability correlated with significantly increased cartilage matrix production using primary seeded chondrocytes. These results indicate that the low permeability design with a spherical pore shape provided a better microenvironment for chondrogenesis than the high permeability design with a cubical pore shape. Thus, scaffold architecture and material design may have a significant impact on the success of matrix-based clinical cartilage repair strategies.

Magnesium enhances adherence and cartilage formation of synovial mesenchymal stem cells through integrins

We previously reported that more than 60% of synovial mesenchymal stem cells (MSCs) placed on osteochondral defects adhered to the defect within 10 min and promoted cartilage regeneration. The efficiency of adherence is considered to depend on the interaction between cells and extracellular matrix (ECM), in which integrins may play some important roles. Divalent cations such as calcium, magnesium, and manganese may affect functions of integrins, and the integrins may be involved in differentiation of MSCs. Among divalent cations, magnesium is used in clinical practice as a therapeutic agent and increases the affinity of integrin to ECM. In this study, we investigated whether magnesium enhanced adherence and chondrogenesis of synovial MSC through integrins. We performed assays for adherence of human synovial MSCs to collagen-coated slides, in vitro chondrogenesis, ex vivo assays for adherence of human synovial MSCs to osteochondral defect, and in vivo assays for adherence and cartilage formation of synovial MSCs in a rabbit osteochondral defect model. Magnesium increased adhesion of human synovial MSCs to collagen, and this effect was inhibited by neutralizing antibodies for integrin α3 and β1. Magnesium also promoted synthesis of cartilage matrix during in vitro chondrogenesis of synovial MSCs, which was diminished by neutralizing antibodies for integrin β1 but not for integrin α3. Ex vivo analyses demonstrated that magnesium enhanced adherence of human synovial MSCs to osteochondral defects. In vivo studies in rabbits showed that magnesium promoted adherence at 1 day and cartilage formation of synovial MSCs at 2 weeks. Magnesium enhanced adherence of synovial MSCs through integrins, which promoted synthesis of cartilage matrix at an early phase.

Inhibition of Hypoxia-inducible Factor-targeting Prolyl Hydroxylase Domain-containing Protein 2 (PHD2) Enhances Matrix Synthesis by Human Chondrocytes

Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-alpha isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2alpha stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2alpha levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

The ability of insulin-like growth factor I (IGF-I) to stimulate cartilage matrix synthesis is reduced in aged and osteoarthritic cartilage. Aging and osteoarthritis are associated with an increase in reactive oxygen species, which we hypothesized would interfere with normal IGF-I signaling. We compared IGF-I signaling in normal and osteoarthritic human articular chondrocytes and investigated the effects of oxidative stress induced by tert-butylhydroperoxide (tBHP). In normal human chondrocytes, IGF-I initiated a strong and sustained phosphorylation of IRS-1 (Tyr-612) and Akt (Ser-473) and transient ERK phosphorylation. In contrast, in osteoarthritic chondrocytes, which possessed elevated basal IRS-1 (Ser-312) and ERK phosphorylation, IGF-I failed to stimulate IRS-1 (Tyr-612) or Akt phosphorylation. In normal human chondrocytes, tBHP triggered strong IRS-1 (Ser-312 and Ser-616) and ERK phosphorylation and inhibited IGF-I-induced IRS-1 (Tyr-612) and Akt phosphorylation. Lentivirus-mediated overexpression of constitutively active (CA) Akt significantly enhanced proteoglycan synthesis, whereas both dominant negative Akt and CA MEK inhibited proteoglycan synthesis. CA Akt also promoted type II collagen and Sox9 expression, whereas tBHP treatment and CA MEK inhibited aggrecan, collagen II, and Sox9 mRNA expression. In osteoarthritic chondrocytes, the antioxidants Mn(III) tetrakis(4-benzoic acid)porphyrin and N-acetylcysteine increased the ratio of Akt to ERK phosphorylation and promoted IGF-I-mediated proteoglycan synthesis. Chemical inhibition of ERK significantly enhanced IGF-I phosphorylation of Akt and alleviated tBHP inhibition of Akt phosphorylation. These results demonstrate opposing roles for phosphatidylinositol 3-kinase-Akt and MEK-ERK in cartilage matrix synthesis and suggest that elevated levels of reactive oxygen species cause chondrocyte IGF-I resistance by altering the balance of Akt to ERK activity.

Improved Cartilage Regeneration Utilizing Mesenchymal Stem Cells in TGF-β1 Gene–Activated Scaffolds

Recently, bone marrow-derived mesenchymal stem cells (MSCs) have been paid more attention for cartilage regeneration. This study evaluated the potential of using MSCs seeded in plasmid transforming growth factor beta1 (pTGF-beta1)-activated three-dimensional chitosan/gelatin scaffolds for improving cartilage repair in vivo. Significant cell proliferation and transforming growth factor beta1 protein expression were observed in vitro in pTGFbeta1-activated scaffolds. Transforming growth factor beta1-activated scaffolds showed high collagen type II and aggrecan expression and low collagen type I expression during in vitro cultivation. MSC-based pTGF-beta1-activated scaffolds also exhibited cartilage histology with high secretion of collagen type II in vitro under the stimulation of pTGF-beta1. In rabbits with full-thickness cartilage defects, the implantation of MSC-based pTGF-beta1-activated scaffolds not only significantly promoted chondrogenic differentiation of MSCs and hyalin-like cartilage matrix synthesis, but also remarkably improved the overall repair of rabbit cartilage defects and exhibited favorable tissue integrity at 10 weeks postsurgery. These results suggest that MSC-based localized pTGF-beta1-activated scaffolds have potential applications for in vivo cartilage repair.

Integrated Model of IGF-I Mediated Biosynthesis in a Deformed Articular Cartilage

Maintenance of articular cartilage’s functional mechanical properties ultimately depends on the balance between the extracellular matrix component biosynthesis, degradation, and loss. A variety of factors are known to modulate the rate of cartilage matrix synthesis (e.g., growth factors and stress/strain environment). In the present study, we develop an integrated mathematical model that quantifies biological processes within cartilage tissue modulated by insulin-like growth factors (IGFs). Specifically, the model includes IGF transport through a deforming porous media, competitive binding to binding proteins and cell receptors, and matrix macromolecule biosynthesis—particularly glycosaminoglycans (GAGs). These newly synthesized matrix molecules are then able to modify the material properties of cartilage. The model is used to investigate the effect of synovial fluid IGF-I concentration on cartilage homeostasis. The results presented here suggest that GAG production can be rapidly “switched on” when the concentration of IGF-I reaches a certain threshold, while it is predicted that high receptor concentration leads to heterogeneous matrix production. As for the combined effect of IGF-I and mechanical loading on biosynthesis, the current model predicts that a loading regime with high strain magnitude (e.g., 10%) can achieve a synergistic effect on matrix protein production. Furthermore, dynamic loading is seen to promote spatial homogeneous GAG production.

A stable isotope method for the simultaneous measurement of matrix synthesis and cell proliferation in articular cartilage in vivo

Measurements of cell proliferation and matrix synthesis in cartilage explants have identified regulatory factors [e.g., interleukin-1 (IL-1)] that contribute to osteoarthritis and anabolic mediators [e.g., bone morphogenic protein-7 (BMP-7)] that may have therapeutic potential. The objective of this study was to develop a robust method for measuring cell proliferation and glycosaminoglycan synthesis in articular cartilage that could be applied in vivo. A stable isotope-mass spectrometry approach was validated by measuring the metabolic effects of IL-1 and BMP-7 in cultures of mature and immature bovine cartilage explants. The method was also applied in vivo to quantify physiologic turnover rates of matrix and cells in the articular cartilage of normal rats. Heavy water was administered to explants in the culture medium and to rats via drinking water, and cartilage was analyzed for labeling of chondroitin sulfate (CS), hyaluronic acid (HA) and DNA. As expected, IL-1 inhibited the synthesis of DNA and CS in cartilage explants. However, IL-1 inhibited HA synthesis only in immature cartilage. Furthermore, BMP-7 was generally stimulatory, but immature cartilage was significantly more responsive than mature cartilage, particularly in terms of HA and DNA synthesis. In vivo, labeling of CS and DNA in normal rats for up to a year indicated half-lives of 22 and 862 days, respectively, in the joint. We describe a method by which deuterium from heavy water is traced into multiple metabolites from a single cartilage specimen to profile its metabolic activity. This method was demonstrated in tissue culture and rodents but may have significant clinical applications.