Maintenance Of Articular Cartilage Research Articles

Inflammatory conditions partly impair the mechanically mediated activation of Smad2/3 signaling in articular cartilage.

BackgroundJoint trauma, which is frequently related with mechanical overloading of articular cartilage, is a well-established risk for osteoarthritis (OA) development. Additionally, reports show that trauma leads to synovial joint inflammation. In consequence, after joint trauma, cartilage is influenced by deleterious excessive loading combined with the catabolic activity of proinflammatory mediators. Since the activation of TGF-β signaling by loading is considered to be a key regulatory pathway for maintaining cartilage homeostasis, we tested the effect of proinflammatory conditions on mechanically mediated activation of TGF-β/Smad2/3P signaling in cartilage.MethodsCartilage explants were subjected to dynamic mechanical compression in the presence of interleukin-1 beta (IL-1β) or osteoarthritic synovium-conditioned medium (OAS-CM). Subsequently, the activation of the Smad2/3P pathway was monitored with QPCR analysis of reporter genes and additionally the expression of receptors activating the Smad2/3P pathway was analyzed. Finally, the ability for mechanically mediated activation of Smad2/3P was tested in human OA cartilage.ResultsIL-1β presence during compression did not impair the upregulation of Smad2/3P reporter genes, however the results were affected by IL-1β-mediated upregulations in unloaded controls. OAS-CM significantly impaired the compression-mediated upregulation of bSmad7 and Tgbfb1. IL-1β suppressed the compression-mediated bAlk5 upregulation where 12 MPa compression applied in the presence of OAS-CM downregulated the bTgfbr2. Mechanically driven upregulation of Smad2/3P reporter genes was present in OA cartilage.ConclusionsProinflammatory conditions partly impair the mechanically mediated activation of the protective TGF-β/Smad2/3P pathway. Additionally, the excessive mechanical compression, applied in the presence of proinflammatory conditions diminishes the expression of the type II TGF-β receptor, a receptor critical for maintenance of articular cartilage.

Diseases of Subchondral Bone 1.

The subchondral zone plays an important role in both the structural and biochemical maintenance of articular cartilage. Knowledge of the structure, function, and pathophysiology of the normal subchondral bone/articular surface interface is essential for an understanding of the pathogenesis of many of the disease entities that we will review in this chapter.

The Effect of Mechanical Loading on Articular Cartilage

The effect of mechanical loading on articular cartilage is the topic chosen for the second editorial of this newly launched journal. The aim of this interesting editorial is to illustrate the cell signaling correlated to the mechanical loading, some aspects of the mechanobiology and the positive and negative effects of the mechanical loading on articular cartilage. The benefits of the mechanical loading on articular cartilage have been shown to have a short- and long-term effectiveness. In this article, the role of mechanical signaling in the maintenance of articular cartilage and how the alterations in normal signaling can lead to joint pathology have been discussed.

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.

All-Arthroscopic Reconstruction of the Acetabular Labrum by Capsular Augmentation

The acetabular labrum plays an important role in hip joint stability and articular cartilage maintenance. As such, reconstitution of the labral complex is ideal. In cases in which the labrum is too degenerative to allow adequate reconstruction with current repair techniques, a capsular augmentation is a novel technique that can be used to restore the labral structure. Use of capsular augmentation enables preservation of the donor-tissue blood supply with local tissue transfer, without adding significant complexity to the procedure or significant donor-site morbidity.

Targeted mutation of NOV/CCN3 in mice disrupts joint homeostasis and causes osteoarthritis-like disease

SummaryObjectiveThe matricellular protein NOV/CCN3, is implicated in osteoarthritis (OA) and targeted mutation of NOV in mice (Novdel3) leads to joint abnormalities. This investigation tested whether NOV is required for joint homeostasis and if its disruption causes joint degeneration.MethodNOV expression in the adult mouse joint was characterized by immunohistochemistry. A detailed comparison of the joints of Novdel3−/− and Novdel3+/+ (wild-type) males and females at 2, 6 and 12 months of age was determined by X-ray, histology and immunohistochemistry.ResultsNOV protein was found in specific cells in articular cartilage, meniscus, synovium and ligament attachment sites in adult knees. Novdel3−/− males exhibited severe OA-like pathology at 12 months (OARSI score 5.0 ± 0.5, P < 0.001), affecting all tissues of the joint: erosion of the articular cartilage, meniscal enlargement, osteophytic outgrowths, ligament degeneration and expansion of fibrocartilage. Subchondral sclerosis and changes in extracellular matrix composition consistent with OA, were also seen. The density of articular cartilage cells in Novdel3+/+ knee joints is maintained at a constant level from 2 to 12 months of age whereas this is not the case in Novdel3−/− mice. Compared with age and sex-matched Novdel3+/+ mice, a significant increase in articular cartilage density was seen in Novdel3−/− males at 2 months, whereas a significant decrease was seen at 6 and 12 months in both Novdel3−/− males and females.ConclusionNOV is required for the maintenance of articular cartilage and for joint homeostasis, with disruption of NOV in ageing Novdel3−/− male mice causing OA-like disease.

Recent progress in osteoarthritis research.

Osteoarthritis (OA) is a degenerative joint disease and is characterized by articular cartilage degeneration, subchondral bone sclerosis, and osteophyte formation with major clinical symptoms, including chronic pain, joint instability, stiffness, and radiographic joint space narrowing.1 OA is the most common form of arthritis and is a leading cause of impaired mobility in the elderly. It has been forecast that 25% of the adult population, or >50 million people in the United States, will be affected by OA by the year 2020, and it will be a major cause of morbidity and physical limitation among individuals older than 40 years. In addition to the well-documented impact of OA on physical function and quality of life, depression, and anxiety, there is a significant financial burden, with aggregate annual medical expenditures exceeding $185 billion in 2008.2 A variety of risk factors has been identified in the initiation and/or progression of OA, including age, gender, traumatic injury, obesity, metabolic dysfunction, and environmental and genetic factors.1 Despite extensive research over the past 20 years to delineate the pathogenic mechanism or mechanisms of OA, a full understanding of the initiators of the disease and the factors that accelerate OA progression is yet to be achieved. Thus, there is no clinical diagnosis for early OA and no effective disease-modifying treatment of late OA other than pain-relieving medication or the replacement of damaged joints.1 Normal articular cartilage that emerges during the postnatal stage as a permanent tissue distinct from the growth plate cartilage is a smooth, hard, white tissue that lines the surface of all diarthrodial joints. Collagens and proteoglycans are the principle extracellular matrix (ECM) molecules of articular cartilage. Mutations of ECM-related factors, including types II, IX, and XI collagen, have been reported in OA patients.1 Articular chondrocytes are the cells responsible for the maintenance of articular cartilage. As such, the dysregulation of this cell is directly connected to the process of cartilage degeneration in OA. Thus, understanding the phenotypic behavior of articular chondrocytes in homeostasis and disease has made us aware of several key environmental and genetic factors that affect OA initiation and progression. In earlier decades, the surgically induced destabilization of medial meniscus model, as well as genetic mouse models, were developed and demonstrated potential roles of affected genes in OA pathogenesis. Transforming growth factor (TGF)-β/Smad, Wnt/β-catenin, Notch, and Indian hedgehog pathways have demonstrated the critical and unique roles of chondrocytes during OA development and progression by stimulating chondrocytes toward hypertrophy.1 Recent genetic findings further suggest that Runx2, Mmp13, and Adamts5 are common target genes involved in the above-mentioned signaling networks to disrupt the metabolic and catabolic balance in chondrocytes; they eventually degrade cartilage matrix by upregulation of matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) activity and by downregulation of type II collagen and aggrecan synthesis.1 Recent studies of genome-wide association screens have been performed in large numbers of OA and control populations throughout the world and have confirmed several critical signaling molecules previously implicated by mouse genetic and injury-induced animal models, including the Wnt (Sfrp3), bone morphogenetic protein (Gdf5), and TGF-β (Smad3) signaling pathways.1 In addition to the single-nucleotide polymorphisms analysis, growing evidence points to the fact that the gene expression profile can be largely regulated by epigenetic machinery, modulating the local transcriptional activity and manipulating mRNA expression through a microRNA-mediated regulatory mechanism.3 Recent genome-wide methylation screening in patients with OA revealed different DNA methylation signatures in both synoviocytes and chondrocytes, indicating that epigenetic changes can influence OA susceptibility and severity.4 Epigenetic modification of Mmp13 and Adamts5 was also observed during OA development and progression, indicating that epigenetic factors may also play a role in the pathophysiology of OA.3 In addition to articular chondrocytes, other cell types, such as the mesenchymal stem cell in subchondral bone and synovial fibroblasts, contribute to OA progression. TGF-βs, in response to abnormal mechanical loading, were found to be released, activated, and accumulated in the subchondral bone in patients with OA, leading to aberrant bone formation and angiogenesis through recruitment of osteoprogenitor cells.5 Both injury and obesity-induced low-grade inflammation have been widely recognized as contributing factors to synovial tissue expansion and to hyperplasia in the early onset of OA. The inflammatory factors and ECM-degrading enzymes facilitate OA progression.6 As these underlying mechanisms are further delineated, manipulation of several critical molecules could serve as potential key targets for therapeutic intervention for the treatment of OA disease.

HIF-1α regulates configuration and maintenance of articular cartilage through induction of anabolic factors and suppression of catabolic factors

Purpose: Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in hypoxic conditions for cell survival in various tissues. In chondrocytes, previous studies showed that HIF-1α was necessary for chondrocyte survival in endochondral ossification and for interzone formation in joint development; however, the underlying molecular mechanisms remain unclear. Moreover, physiological and pathological functions of HIF-1α in articular cartilage are yet to be revealed. Here we have investigated HIF-1α functions and the underlying mechanisms of the configuration and maintenance of articular cartilage by HIF-1α. Methods: We generated tissue-specific knockout mice of HIF-1α by mating Sox9-Cre knock-in mice (Sox9-Cre) or tamoxifen-inducible Col2a1-Cre transgenic mice (Col2a1-CreERT2), with mice homozygous for a floxed Hif1a allele (Hif1afl/fl). For analyses of cartilage configuration, we examined Sox9-Cre;Hif1afl/fl mice at E15.5, E18.5 and P1. To analyze articular cartilage maintenance and osteoarthritis (OA) progression after maturation, we injected tamoxifen into 7-week-old Col2a1-CreERT2;Hif1afl/fl mice, and created a surgical OA model by inducing instability in the knee joints one week later. OA severity was quantified by the OARSI histopathology grade 8 weeks after surgery. For histological analyses of these knockout mice, we performed Safranin O staining, TUNEL staining, and immunofluorescence. Quantitative gene expression in limb cartilages of Sox9-Cre;Hif1afl/fl and the control Hif1afl/fl neonates was determined based on real-time RT-PCR analyses using samples obtained directly from their limb cartilages. To assess the catabolic ability of HIF-1α, we measured the concentration of aggrecan released from the 3-week-old mouse femoral heads cultured with or without CoCl2, a potent enhancer of HIF-1α protein stability, utilizing dimethylmethylene blue dye-binding assay. Mouse primary articular chondrocytes were used for further in vitro functional analyses. Results: Sox9-Cre;Hif1afl/fl embryos and neonates showed dwarfism with severe limb shortening throughout the embryonic stages and died in the perinatal period. This phenotype was consistent with previous reports using Prx1-Cre and Col2a1-Cre mice. Notably, the knee joints of Sox9-Cre;Hif1afl/fl embryos showed a widespread epiphysis with a massive cavity continuing from the joint surface forming a trumpet shape, in which the columnar architecture was completely abolished and the cell numbers were vastly reduced(Fig.1). Safranin O staining showed a decrease in proteoglycan content in comparison with the control Hif1afl/fl embryo. TUNEL staining accentuated ectopic apoptosis broadly across the limb cartilage, and immunofluorescence showed enhanced expression of Mmp13 surrounding the defective area. When we created the experimental OA model one week after the tamoxifen injection into Col2a1-CreERT2;Hif1afl/fl mice, the OA development in the knee joints was markedly accelerated as compared to the control Hif1afl/fl joints. Apoptosis and Mmp13 expression were upregulated by the conditional knockout of HIF-1α after maturation in articular cartilage, as well as in the limb cartilage. To reveal altered gene expression by HIF-1α deficiency, we obtained RNA samples directly from the limb cartilages of Sox9-Cre;Hif1afl/fl and the control Hif1afl/f embryo. Real-time RT-PCR using these samples revealed increases of catabolic factors including Mmp13 and Mmp9, and decreases of anabolic factors including Col2a1 and Sox9 by the conditional knockout of HIF-1α. When we deleted HIF-1α in primary articular chondrocytes from Hif1afl/fl mice by adenoviral vector expressing Cre recombinase, expressions of the catabolic and the anabolic genes were changed in ways similar to those in the in vivo analyses, under both normoxic and hypoxic (1% O2 concentration) conditions. Similar results were also obtained under both O2 conditions by HIF-1α silencing in primary articular chondrocytes from WT mice using siRNA. Furthermore, in the organ culture of mouse femoral heads, stabilizing HIF-1α protein by CoCl2 treatment markedly decreased aggrecan release into the medium. Conclusions: HIF-1α regulates the configuration and maintenance of articular cartilage through induction of anabolic factors and suppression of catabolic factors. Elucidation of the molecular network related to HIF-1α may lead to cartilage regeneration and OA treatment.

Determination of differential gene expression profiles in superficial and deeper zones of mature rat articular cartilage using RNA sequencing of laser microdissected tissue specimens.

Superficial zone (SFZ) cells, which are morphologically and functionally distinct from chondrocytes in deeper zones, play important roles in the maintenance of articular cartilage. Here, we established an easy and reliable method for performance of laser microdissection (LMD) on cryosections of mature rat articular cartilage using an adhesive membrane. We further examined gene expression profiles in the SFZ and the deeper zones of articular cartilage by performing RNA sequencing (RNA-seq). We validated sample collection methods, RNA amplification and the RNA-seq data using real-time RT-PCR. The combined data provide comprehensive information regarding genes specifically expressed in the SFZ or deeper zones, as well as a useful protocol for expression analysis of microsamples of hard tissues.

Design and validation of an in vitro loading system for the combined application of cyclic compression and shear to 3D chondrocytes-seeded agarose constructs

Physiological loading is essential for the maintenance of articular cartilage by regulating tissue remodelling, in the form of both catabolic and anabolic processes. To promote the development of tissue engineered cartilage which closely matches the long term functionality of native tissue, bioreactors have been developed to provide a combination of loading modalities, which reflect the nature of normal physiological loads. This study describes the design and validation of an in vitro mechanical system for the controlled application of bi-axial loading regimes to chondrocyte-seeded agarose constructs.The computer-controlled system incorporates a robust gripping system, which ensures the delivery of precise values of cyclic compressive and shear strain to 3D cell-seeded constructs. Sample prototypes were designed, optimised using finite element analysis and validated performing compressive and shear fatigue mechanical tests. The horizontal and vertical displacements within the bioreactor are precisely controlled by a dedicated programme that can be easily implemented. The synchronisation of the orthogonal displacements was shown to be accurate and reproducible.Constructs were successfully loaded with a combined compressive and shear loading regimen at 1Hz for up to 48h with no appreciable loss of cell viability or mechanical integrity. These features along with the demonstrated high consistency make the system ideally suitable for a systematic investigation of the response of chondrocytes to a complex physiologically relevant deformation profile.

Inflammation Is More Distinct in Temporomandibular Joint Osteoarthritis Compared to the Knee Joint

Most of the current understanding of articular cartilage maintenance and degradation is derived from large load-bearing synovial joints, in particular the knee joint. The aim of this study was to identify valuable degradation markers for cartilage degradation in the temporomandibular joint (TMJ) by comparing the relative concentrations of carboxyterminal telopeptides of collagen types I and II (CTX-I and CTX-II), cartilage oligomeric matrix protein (COMP), and prostaglandin E2 (PGE2) in synovial fluid (SF) of TMJ and knee joints with cartilage degradation. In this cross-sectional comparative study, participants were recruited from the University Medical Center Groningen, The Netherlands. Patients with TMJ osteoarthritis were compared with patients with knee osteoarthritis. The outcome variables were the relative SF concentrations of CTX-I, CTX-II, COMP, and PGE2. An independent samples Mann-Whitney U test was used to compare the relative concentrations. Thirty consecutive patients (9 male, 21 female; mean age, 40.1 yr; standard deviation, 15.3 yr) with TMJ osteoarthritis and 31 consecutive patients (20 male, 11 female; mean age, 37.4 yr; standard deviation, 13.7 yr) who were scheduled for arthroscopy of the knee joint participated in this study. Significant differences were found between relative concentrations of COMP (P = .000) and PGE2 (P = .005), and no significant differences were found between relative concentrations of CTX-I (P = .720) and CTX-II (P = .242). Relative SF concentrations of COMP and PGE2 showed significant differences between the TMJ and the knee joint, suggesting that there are differences in pathophysiology and that the inflammatory component may be more distinct in the TMJ.

Mesenchyme-specific Knockout of ESET Histone Methyltransferase Causes Ectopic Hypertrophy and Terminal Differentiation of Articular Chondrocytes

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

Prolonged local delivery of IL-1Ra prevents post-traumatic arthritis in mice

Prolonged local delivery of IL-1Ra prevents post-traumatic arthritis in mice

Identification of the patients who respond safely and optimally to intervention with biologics; lesson learned from rheumatoid arthritis

Identification of the patients who respond safely and optimally to intervention with biologics; lesson learned from rheumatoid arthritis

Identification of fibroblast growth factor-18 as a molecule to protect and regenerate articular cartilage

Purpose: Aiming at the disease-modifying treatment of osteoarthritis (OA), we sought to identify genes that maintain the homeostasis of adult articular cartilage and regenerate its lesions by gene expression profile analyses. The mechanisms of the identified molecule underlying the protection and regeneration were further investigated. Methods: To identify genes that have both protective and regenerative effects on adult articular cartilage, we performed two sets of microarray analyses. First, to select genes that work for maintenance of articular cartilage, we compared gene expression profiles between adult articular (AA) and adult growth plate (AG) cartilages in 10-week-old rats. Second, to find genes that function for regeneration of articular cartilage, we compared the profiles between infant superficial (IS) and infant deep (ID) layers of epiphyseal cartilage in 6-day-old rats. For genes which were up-regulated ≥10-fold both in AA than in AG and in IS than in ID, we performed real-time RT-PCR for the confirmation. In vivo expression was examined by immunohistochemistry of articular and growth plate cartilage of 14-week-old rats. The therapeutic effect of intra-articular injection of sprifermin (recombinant human fibroblast growth factor 18 (FGF18)) was examined in the experimental OA model by surgical induction of instability in the knee joints of adult rats. To further learn the underlying mechanism, the protective ability of articular cartilage was assessed by measuring the amount of sulfated glycosaminoglycan (sGAG) released into the medium in the ex vivo culture of bilateral femoral heads of 3-week-old mice using dimethylmethylene blue dye-binding assay. Proliferation and migration were analyzed in the cultures of mouse articular chondrocytes using Cell Counting Kit-8 and Oris Cell Migration assay systems, respectively. Expression levels of catabolism-related factors (Mmp9, Mmp13, Adamts4, Adamts5, Timp1, Timp2, and Timp3) and anabolism-related factors (Col2a1 and aggrecan) in the cultures of mouse femoral heads and mouse articular chondrocytes were analyzed by real-time RT-PCR. Results: Microarray analyses revealed that 40 and 186 genes had ≥10-fold higher expression ratios of AA/AG and IS/ID, respectively, and 16 genes showed ≥10-fold of both AA/AG and IS/ID ratios. The ratios of the 16 genes were confirmed to be ≥10 fold by real-time RT-PCR analysis. Among them three genes were expressed more strongly in AA than in IS. In these three genes, FGF18 was the extracellular and secreted factor of which the AA/AG ratio was the highest in the microarray analysis. Immunohistochemistry showed that FGF18 was strongly expressed in the articular cartilage chondrocytes of adult rats but was hardly detected in the growth plate cartilage. In the rat surgical OA model, a once-weekly injection of sprifermin given 3 weeks post-surgery prevented cartilage degeneration in a dose-dependent manner at 6 and 9 weeks after surgery, with a significant effect at 10 μg/week of sprifermin. As an underlying mechanism, sprifermin suppressed the sGAG release into the culture medium in the ex vivo culture of mouse femoral heads. Furthermore, sprifermin accelerated proliferation and migration of cultured mouse articular chondrocytes. Among catabolic and anabolic factors, sprifermin decreased Adamts4 and increased Timp1 expressions in the cultures of mouse femoral heads and murine articular chondrocytes; however it decreased Col2a1 and aggrecan expressions in both cultures. Conclusions: The present gene expression profiling analysis identified sprifermin as a molecule to protect and regenerate adult articular cartilage, causing prevention of cartilage degeneration by the once-weekly intra-articular injection in a rat model. This effect may be due to inhibition of cartilage catabolism, and acceleration of proliferation and migration of articular chondrocytes, providing a disease-modifying OA treatment.

Ets-transcription factors in formation and maintenance of permanent articular cartilage

Ets-transcription factors in formation and maintenance of permanent articular cartilage

Articular cartilage degradation induced by extensive treadmill exercise is greatly exacerbated by estrogen depletion in mice

Purpose: Osteoarthritis (OA) is considered to be a multifactorial disease with factors such as chronic inflammation, aging, menopause, obesity, and joint instability. For example, more than 39% of the patients develop OA in the knee joint after anterior cruciate ligament reconstruction within 8 years. Too much mechanical stress by treadmill exercise induces severe articular cartilage degradation in rats. Estrogen depletion by ovariectomy (OVX) accelerates cartilage degradation in mice. These data clearly show that each factor is involved in the pathogenesis of OA, however, orchestrated effects of all these factors are still unclear. To analyze the crosstalk between estrogen signal and mechanotransduction in articular cartilage homeostasis, we examined articular cartilage damage after treadmill exercise in OVX mice. Here we report that degree of articular cartilage degradation induced by extensive treadmill exercise is greatly exacerbated by estrogen depletion. Methods: Twenty-four Balb/c mice (8 wks) were randomly divided into 4 groups, OVX+CAGE, OVX+RUN, SHAM+CAGE and SHAM+RUN. Two weeks after ovariectomy or sham operation, RUN group was subjected to a forced running for 6 weeks (5 days a week) at 20m/min for 100 minutes by treadmill while CAGE group were left in cage ad libitum. Integrity of articular cartilage was assessed by Hematoxylin and Eosin staining and type II collagen immunostaining of sagittal sections. To assess articular cartilage damage, three sections (apart from 150μm respectively) were stained by Safranin O and 400μm in width of articular cartilage between anterior and posterior edge of medial meniscus was contoured into 3 areas according to the dyeability: Grade I; intact cartilage, Grade II; mildly denatured cartilage with reduced Safranin O staining, and Grade III; severely denatured cartilage with no Safranin O staining. Each area was measured using Zeiss Axio Vision Image Analysis system. To analyze the structural alteration of knee joint after treadmill exercise and OVX, right knee joints were fixed in 70% ethanol and subjected for μCT analyses. Animal care and experimental procedures were in accordance with the guidelines of the ethics committee of our university. Kruskal-Wallis test followed by Tukey-Kramer methods was used for statistical analysis. Results: μCT analyses did not reveal the apparent alteration in bone structure and osteophyte formation between OVX and SHAM group regardless of treadmill exercise. However we observe significant loss in trabecular bone volume in the OVX group. Image analyses of the articular cartilage indicated that % area of Grade III was significantly higher in OVX+RUN group (72.1%) if compared to that of SHAM+CAGE (10.6%, p<0.05). Loss of proteoglycan in articular cartilage was also observed in both OVX+CAGE and SHAM+RUN groups (Grade III; 24.2% and 20.1% respectively) although the difference was not statistically significant. Immunohistochemical staining for type II collagen further support these data, since we observe marked decrease in type II collagen amount in OVX+RUN group. Hematoxylin and Eosin staining showed that OVX enhanced cellularity of synovial membrane after forced running suggesting that depletion of estrogen might enhance syovitis in the damaged joint. Conclusions: In this study, we showed that the combination of OVX and forced running greatly accelerated articular cartilage denaturation in mice. In contrast, the effects of OVX or forced running respectively were not so apparent on articular cartilage maintenance. These data strongly support the evidence that OA is a multifactorial disease and our data strongly indicate the great contribution of hormonal regulation on articular cartilage homeostasis in adults. Crosstalk between hormonal regulation and mechanotransduction on articular cartilage homeostasis is still unclear, our data suggest that estrogen depletion enhances inflammatory response after articular cartilage damage. Since prevalence of OA greatly increased after menopause, our experimental system will be of great use for analyzing OA progression in aged women.

Mesenchymal stem cell-based treatment for cartilage defects in osteoarthritis

Osteoarthritis (OA) is a common disorder and the restoration of the diseased articular cartilage in patients with OA is still a challenge for researchers and clinicians. Currently, a variety of experimental strategies have investigated whether mesenchymal stem cells (MSCs) instead of chondrocytes can be used for the regeneration and maintenance of articular cartilage in OA. MSCs can modulate the immune response of individuals and positively influence the microenvironment of the stem cells already present in the diseased tissue. Through direct cell-cell interaction or the secretion of various factors, MSCs can initiate endogenous regenerative activities in the OA joint. Targeted gene-modified MSC-based therapy might further enhance the cartilage regeneration in OA. Conventionally, delivery of MSCs was attained by graft of engineered constructs derived from cell-seeded scaffolds. However, intra-articular MSCs transplantation without scaffolds is a more attractive option for OA treatment. This article briefly summarizes the current knowledge about MSC-based therapy for prevention or treatment of OA, discussing the direct intra-articular injection of MSCs for the treatment of OA in animal models and in clinical applications, as well as potential future strategies for OA treatment.

Depletion of primary cilia in articular chondrocytes results in reduced Gli3 repressor to activator ratio, increased Hedgehog signaling, and symptoms of early osteoarthritis

Primary cilia are present in almost every cell type including chondrocytes. Studies have shown that defects in primary cilia result in skeletal dysplasia. The purpose of this study was to understand how loss of primary cilia affects articular cartilage. Ift88 encodes a protein that is required for intraflagellar transport and formation of primary cilia. In this study, we used Col2aCre;Ift88(fl/fl) transgenic mice in which primary cilia were deleted in chondrocytes. Col2aCre;Ift88(fl/fl) articular cartilage was characterized by histological staining, real time RT-PCR, and microindentation. Hedgehog (Hh) signaling was measured by expression of Ptch1 and Gli1 mRNA. The levels of Gli3 proteins were determined by western blot. Col2aCre;Ift88(fl/fl) articular cartilage was thicker and had increased cell density, likely due to decreased apoptosis during cartilage remodeling. Mutant articular cartilage also showed increased expression of osteoarthritis (OA) markers including Mmp13, Adamts5, ColX, and Runx2. OA was also evident by reduced stiffness in mutant cartilage as measured by microindentation. Up-regulation of Hh signaling, which has been associated with OA, was present in mutant articular cartilage as measured by expression of Ptch1 and Gli1. Col2aCre;Ift88(fl/fl) cartilage also demonstrated reduced Gli3 repressor to activator ratio. Our results indicate that primary cilia are required for normal development and maintenance of articular cartilage. It was shown that primary cilia are required for processing full length Gli3 to the truncated repressor form. We propose that OA symptoms in Col2aCre;Ift88(fl/fl) cartilage are due to reduced Hh signal repression by Gli3.

Physical Stimulation of Chondrogenic Cells In Vitro: A Review

Mechanical stimuli are of crucial importance for the development and maintenance of articular cartilage. For conditioning of cartilaginous tissues, various bioreactor systems have been developed that have mainly aimed to produce cartilaginous grafts for tissue engineering applications. Emphasis has been on in vitro preconditioning, whereas the same devices could be used to attempt to predict the response of the cells in vivo or as a prescreening method before animal studies. As a result of the complexity of the load and motion patterns within an articulating joint, no bioreactor can completely recreate the in vivo situation. This article aims to classify the various loading bioreactors into logical categories, highlight the response of mesenchymal stem cells and chondrocytes to the various stimuli applied, and determine which data could be used within a clinical setting. We performed a Medline search using specific search terms, then selectively reviewed relevant research relating to physical stimulation of chondrogenic cells in vitro, focusing on cellular responses to the specific load applied. There is much data pertaining to increases in chondrogenic gene expression as a result of controlled loading protocols. Uniaxial loading leads to selective upregulation of genes normally associated with a chondrogenic phenotype, whereas multiaxial loading results in a broader pattern of chondrogenic gene upregulation. The potential for the body to be used as an in vivo bioreactor is being increasingly explored. Bioreactors are important tools for understanding the potential response of chondrogenic cells within the joint environment. However, to replicate the natural in vivo situation, more complex motion patterns are required to induce more physiological chondrogenic gene upregulation.