Affect Chondrocyte Proliferation Research Articles

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

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

Molecular mechanisms of long bone growth and chondrocyte regulation: A narrative review

Long bone growth is a fundamental determinant of final height. Growth, metabolism, and differentiation of chondrocytes, which are the key cellular players in this process, are regulated by systemic hormones, local factors, and cellular signaling pathways. This review provides an overview of the structural aspects of the growth plate, factors influencing chondrocyte function, and their impact on longitudinal bone growth. Systemic factors, including growth hormone, sex hormones, thyroid hormone, glucocorticoids, leptin, and insulin significantly affect chondrocyte proliferation and hypertrophy. Local factors, including transcription factors such as SRY-box 9 protein (SOX9), Runt-related transcription factor 2 (RUNX2), and bone morphogenetic proteins (BMPs), along with signaling pathways such as the Wnt pathway, play critical roles in chondrocyte proliferation and differentiation. These factors regulate gene expression, cell differentiation, and extracellular matrix synthesis. Additionally, Indian hedgehog (Ihh) and C-type natriuretic peptide (CNP) are involved in the complex signaling network governing chondrocyte function. Understanding molecular mechanisms underlying normal growth plate function has provided valuable insights into the genetic defects that impact growth and foundation for the development of effective therapeutic strategies for individuals with growth disorders.

MiR-1 is a critical regulator of chondrocyte proliferation and hypertrophy by inhibiting Indian hedgehog pathway during postnatal endochondral ossification in miR-1 overexpression transgenic mice.

Endochondral bone formation from the growth plate plays a critical role in vertebrate limb development and skeletal homeostasis. Although miR-1 is mainly expressed in the hypertrophic region of the growth plate during this process, its role in the endochondral bone formation is unknown. To elucidate the role of miR-1 in cartilage development, chondrocyte-specific transgenic mice with high expression of miR-1 were generated (Col2a1-Cre-ERT2-GFPfl/fl-RFP-miR-1). Transgenic mice showed short limbs and delayed formation of secondary ossification centers. In the tibia growth plate of miR-1-overexpressing transgenic mice, the chondrocytes in the proliferative zone were disorganized and their proliferation decreased, and the ColX, MMP-13 and Indian Hedgehog (IHH) in chondrocytes showed a downward trend, resulting in decreased terminal differentiation in the hypertrophic zone. In addition, the apoptosis index caspase-3 also showed a downward trend in the tibia growth plate. It was concluded that miR-1 overexpression affects chondrocyte proliferation, hypertrophic differentiation, and apoptosis, thereby delaying the formation of secondary ossification centers and leading to short limbs. It was also verified that miR-1 affects endochondral ossification through the IHH pathway. The above results suggest that miR-1 overexpression can affect endochondral osteogenesis by inhibiting chondrocyte proliferation, hypertrophic differentiation, and apoptosis, thus causing limb hypoplasia in mice. This work gives potential for new therapeutic directions and insights for the treatment of dwarf-related diseases.

MIR-140-5p affects chondrocyte proliferation, apoptosis, and inflammation by targeting HMGB1 in osteoarthritis.

This study aimed to test the expression and biological function of miR-140-5p in osteoarthritis (OA), and identify its target gene and explore its mechanism in OA. Differential genes were screened and analyzed by gene microarray and WGCNA analysis. The normal human chondrocytes C28/I2 were induced by IL-1β to construct the OA cell model. The expression of miR-140-5p and high mobility group box 1 (HMGB1) was quantified by quantitative real-time PCR (qRT-PCR) in OA tissues and IL-1β-induced chondrocytes. Western blotting was performed to evaluate the expression of HMGB1 and PI3K/AKT pathway activation. The concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, MMP-1 and MMP-3 were determined by ELISA. CCK-8 and flow cytometry were conducted to determine the cellular capabilities of proliferation and cell apoptosis. Bioinformatics analysis demonstrated that HMGB1 was highly expressed in OA and activated PI3K/AKT pathway. Also, HMGB1 was predicted as a target of miR-140-5p. The levels of miR-140-5p were negatively correlated with HMGB1 in OA tissues and IL-1β-induced chondrocytes. The overexpression of miR-140-5p reduced the expression of HMGB1 protein, p-AKT (Ser473) and p-PI3K in IL-1β-induced chondrocytes. Besides, the expression of p-AKT (Ser473) and p-PI3K was significantly upregulated by employing miR-140-5p inhibitor, but retrieved after treating with LY294002. Furthermore, miR-140-5p inhibited inflammation, matrix metalloprotease expression and apoptosis in IL-1β-induced chondrocytes through regulating HMGB1. MiR-140-5p was down-regulated while HMGB1 was upregulated in OA. MiR-140-5p could inhibit the PI3K/AKT signaling pathway and suppress the progression of OA through targeting HMGB1.

MiR-140-5p/miR-149 Affects Chondrocyte Proliferation, Apoptosis, and Autophagy by Targeting FUT1 in Osteoarthritis.

Osteoarthritis (OA), the most prevalent chronic and degenerative joint disease, is characterized by articular cartilage degradation and chondrocyte injury. Increased cell apoptosis and defective cell autophagy in chondrocytes are a feature of degenerative cartilage. MicroRNAs (miRNAs) have been identified as potential regulators of OA. This study aimed to determine the potential role of miR-140-5p and miR-149 in apoptosis, autophagy, and proliferation in human primary chondrocytes and investigate the underlying mechanism. We revealed the differential expressional profiles of miR-140-5p/149 and fucosyltransferase 1 (FUT1) in the articular cartilage tissues of OA patients and normal people and validated FUT1 was a direct target of miR-140-5p/149. The overexpression of miR-140-5p/149 inhibited apoptosis and promoted proliferation and autophagy of human primary chondrocytes via downregulating FUT1. On the contrary, the downregulation of miR-140-5p/149 inhibited chondrocyte proliferation and autophagy, whereas the effect was reversed by FUT1 knockdown. Taken together, our data suggested that miR-140-5p and miR-149 could mediate the development of OA, which was regulated by FUT1. miR-140-5p/miR-149/FUT1 axis might serve as a predictive biomarker and a potential therapeutic target in OA treatment.

MiR-195 inhibits the proliferation and migration of chondrocytes by targeting GIT1

Previous studies have demonstrated that G-protein coupled receptor kinase interacting protein-1 (GIT1) and microRNAs (miRNAs) serve an important role in chondrocyte proliferation and migration. However, a limited number of studies conducted thus far have investigated the association between GIT1 and miRNAs. In the present study, putative miR‑195 binding sites in the GIT1 3'‑untranslated region were identified using common bioinformatic algorithms (miRanda, TargetScan, miRBase and miRWalk), and it was demonstrated that they may be involved in regulating GIT1 expression. Following transfection of miR‑195 mimics in chondrocytes, the expression of GIT1 was significantly reduced, whereas the expression was significantly increased following transfection with miR‑195 inhibitors. In addition, the results of the current study demonstrated that increased miR‑195 expression may downregulate chondrocyte proliferation and reduce cell migration. However, chondrocyte proliferation and migration was enhanced following suppression of miR‑195 expression. Furthermore, upon co‑transfection of miR‑195 and GIT1 expression vectors, the inhibitory effect of miR‑195 on chondrocyte proliferation and migration was attenuated. Therefore, miR‑195 may affect chondrocyte proliferation and migration via targeted regulation of GIT1 expression. The results of the current study provide novel evidence for the regulatory mechanisms of miRNAs in bone and cartilage tissues, which may facilitate further research and provide a greater understanding of different osteoarticular diseases.

Longitudinal bone growth is impaired by direct involvement of caffeine with chondrocyte differentiation in the growth plate.

We showed previously that caffeine adversely affects longitudinal bone growth and disrupts the histomorphometry of the growth plate during the pubertal growth spurt. However, little attention has been paid to the direct effects of caffeine on chondrocytes. Here, we investigated the direct effects of caffeine on chondrocytes of the growth plate invivo and invitro using a rapidly growing young rat model, and determined whether they were related to the adenosine receptor signaling pathway. A total of 15 male rats (21days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180mgkg-1 day-1 for 4weeks. After sacrifice, the tibia processed for the analysis of the long bone growth and proliferation of chondrocytes using tetracycline and BrdU incorporation, respectively. Caffeine-fed animals showed decreases in matrix mineralization and proliferation rate of growth plate chondrocytes compared with the control. To evaluate whether caffeine directly affects chondrocyte proliferation and chondrogenic differentiation, primary rat chondrocytes were isolated from the growth plates and cultured in either the presence or absence of caffeine at concentrations of 0.1-1mm, followed by determination of the cellular proliferation or expression profiles of cellular differentiation markers. Caffeine caused significant decreases in extracellular matrix production, mineralization, and alkaline phosphatase activity, accompanied with decreases in gene expression of the cartilage-specific matrix proteins such as aggrecan, type II collagen and type X. Our results clearly demonstrate that caffeine is capable of interfering with cartilage induction by directly inhibiting the synthetic activity and orderly expression of marker genes relevant to chondrocyte maturation. In addition, we found that the adenosine type 1 receptor signaling pathway may be partly involved in the detrimental effects of caffeine on chondrogenic differentiation, specifically matrix production and mineralization, as evidenced by attenuation of the inhibitory effects of caffeine by blockade of this receptor. Thus, our study provides novel information on the integration of caffeine and adenosine receptor signaling during chondrocyte maturation, extending our understanding of the effect of caffeine at a cellular level on chondrocytes of the growth plate.

Synergistic Effect of TGF-β1 And BMP–7 on Chondrogenesis and Extracellular Matrix Synthesis: An In Vitro Study

Introduction:The purpose of the present study seeks to determine the signal timing of BMP–7 and TGF-β1 from a novel chitosan based hydrogel system that may affect chondrocyte proliferation resulting in the presence of a synergism seen conspicuously in consecutive controlled delivery.Methods:Four groups of cultured chondrocytes were seeded on a novel designed chitosan based hydrogel. The hydrogel was left empty (control) in one group and loaded with BMP–7, TGF-β1 and their combination in the other groups, respectively. Hydrogel structure was analyzed with scanning electron microscope. The release kinetics of Growth Factors (GFs) was determined with ELISA. Chondrocyte viability and toxicity after being tested with MTS and collagen type II synthesis, were quantified with western blotting. Canonical regression analysis was used for measuring statistical evaluation.Results:Chitosan based hydrogel allowed controlled release of GFs in different time intervals for BMP–7 and TGF-β1. Double peak concentration gradient was found to be present in the group loaded with both GFs. In this group, substantially higher chondrocyte growth and collagen synthesis were also detected. Conclusions:We concluded that, chitosan based hydrogel systems may be adjusted to release GFs consecutively during biodegradation at the layers of surface, which may increase the cell number and enhance collagen type II synthesis.

Evaluation of nanoarchitectured collagen type II molecules on cartilage engineering

Scaffold architecture, including the geometry and dimension of scaffolds, is an important parameter in cell adhesion, migration, proliferation, and differentiation. Following the characterization of collagen type II nanoarchitectured molecules, collagen fibrils (CNFs) and collagen spheres (CNPs) prepared using a high-voltage electric field in our laboratory, we proposed to use these nanoarchitectured molecules to assess their influence on the culturing of chondrocytes in stirred bioreactors. The results demonstrate that chondrocytes rapidly formed more and larger chondrocyte pellets (spheroids) after the addition of nanoarchitectured molecules into the culture medium. The maintenance of chondrocytes with round morphology and increased glycosaminoglycan secretion indicated that these spheroids contained viable and un-dedifferentiated chondrocytes. No significant increases in DNA content were detected. These results show that the introduction of these molecules did not affect chondrocyte proliferation during a 3-day culture period. After the addition of CNPs and CNFs into the culture medium, the expression levels of collagen type II and aggrecan genes in chondrocytes increased significantly as demonstrated by real-time PCR analysis. Interestingly, chondrocytes exhibited distinct collagen type II and aggrecan gene expression profiles in culture with CNPs and CNFs. The aggrecan gene expression level of the chondrocytes was 2.5-fold greater following CFN addition than following the addition of CNPs. In contrast, the collagen type II expression level of the chondrocytes was 2.2-fold greater following the addition of CNPs than following the addition of CNFs. The chondrocyte pellets rapidly restored defects in articular cartilage during a 1-month implantation period in a rabbit model.

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro

IntroductionChondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.MethodsHuman articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.ResultsModerately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.ConclusionsPhysiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.

Influence of Traumeel on cultured chondrocytes and recombinant human matrix metalloproteinases: Implications for chronic joint diseases

Background Chronic joint diseases, such as osteoarthritis, are associated with insults to articular cartilage. Imbalance between extracellular matrix production and degradation as well as defects in chondrocyte proliferation and differentiation lead to progressive degeneration of cartilage. In this study we have investigated whether Traumeel, an anti-inflammatory and wound-healing agent, affects chondrocyte proliferation and differentiation as well as activity of matrix metalloproteinases (MMPs) that are implicated in matrix degradation. Methods Chondrocytes from porcine knee joints were cultured in agarose layers in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Foetal Calf Serum (FCS). To maintain the differentiation state of the chondrocytes they were treated with collagen type II in absence of FCS. Viability and proliferation of chondrocytes upon Traumeel application in combination with transforming growth factor beta (TGF-s) treatment were determined by MTT assay and 3 H-thymidine incorporation, respectively. Levels of sulfated glycosaminoglycans (sGAG), which are characteristic of chondrocyte functional activity, were measured with the dimethylmethylene blue assay. Activities of recombinant catalytic domains of human MMPs were determined with a chromogenic assay. Results Addition of Traumeel significantly enhanced TGF-β-induced proliferation, but did not affect basal proliferation of chondrocytes in the presence of FCS. In chondrocytes in the differentiated state, Traumeel enhanced viability of the cells and stimulated biosynthesis of sGAG. Several MMPs were screened for inhibition by Traumeel; MMP-13 was found to be most inhibited (by 30%), while MMPs-2, -3, and -9 were not affected. Discussion While influence of Traumeel on proliferation of chondrocytes appears to be context-dependent, differentiation was supported under all tested conditions. Since Traumeel is also known to inhibit the production of cytokines which may reduce the functional capacity of chondrocytes, survival and function of these cells is likely to be improved by Traumeel on various levels. Remarkably, Traumeel inhibited MMP-13 which is closely associated with the pathology of joint destruction. Thus, Traumeel might also indirectly slow down the progression of cartilage degeneration. Conclusion Our data suggest that Traumeel offers a potential therapeutic option for chronic joint diseases which needs to be further investigated.

Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo

To investigate the direct effect of prolyl-hydroxyproline (Pro-Hyp) on chondrocytes under in vivo and in vitro conditions in an attempt to identify Pro-Hyp as the bioactive peptide in collagen hydrolysate (CH). The in vivo effects of CH and Pro-Hyp intake on articular cartilage were studied by microscopic examination of sections of dissected articular cartilage from treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp, amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and mineralization of chondrocytes were determined by MTT activity and staining with alkaline phosphatase, alcian blue and alizarin red. Expression of chondrogenesis-specific genes in ATDC5 cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the articular cartilage layer caused by phosphorus-induced degradation. In the in vitro study, CH and Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-hydroxyprolyl-glycine did not affect chondrocyte proliferation or differentiation. Moreover, CH and Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosaminoglycan in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by two-thirds and one-tenth, respectively. Pro-Hyp is the first bioactive edible peptide derived from CH to be shown to affect chondrocyte differentiation under pathological conditions.

The effectiveness of pulsed electrical stimulation (E-PES) in the management of osteoarthritis of the knee: a protocol for a randomised controlled trial

BackgroundOsteoarthritis (OA) of the knee is one of the main causes of musculoskeletal disability in the western world. Current available management options provide symptomatic relief (exercise and self-management, medication and surgery) but do not, in general, address the disease process itself. Moreover, adverse effects and complications with some of these interventions (medication and surgery) and the presence of co-morbidities commonly restrict their use. There is clearly a need to investigate treatments that are more widely applicable for symptom management and which may also directly address the disease process itself.In two randomised controlled trials of four and 12 weeks duration, pulsed electrical stimulation was shown to be effective in managing the symptoms of OA of the knee. Laboratory and animal studies demonstrate the capacity of externally applied electric and electromagnetic fields to positively affect chondrocyte proliferation and extracellular matrix protein production. This latter evidence provides strong theoretical support for the use of electrical stimulation to maintain and repair cartilage in the clinical setting and highlights its potential as a disease-modifying modality.Methods/DesignA double-blind, randomised, placebo-controlled, repeated measures trial to examine the effectiveness of pulsed electrical stimulation in providing symptomatic relief for people with OA of the knee over 26 weeks.Seventy people will be recruited and information regarding age, gender, body mass index and medication use will be recorded. The population will be stratified for age, gender and baseline pain levels.Outcome measures will include pain (100 mm VAS and WOMAC 3.1), function (WOMAC 3.1), stiffness (WOMAC 3.1), patient global assessment (100 mm VAS) and quality of life (SF-36). These outcomes will be measured at baseline, four, 16 and 26 weeks. Activity levels will be measured at baseline and 16 weeks using accelerometers and the Human Activity Profile questionnaire. A patient global perceived effect scale (11-point Likert) will be completed at 16 and 26 weeks.DiscussionThis paper describes the protocol for a randomised, double-blind, placebo-controlled trial that will contribute to the evidence regarding the use of sub-sensory pulsed electrical stimulation in the management of OA of the knee.Trial registrationAustralian Clinical Trials Registry ACTRN12607000492459.

A-Raf and B-Raf Are Dispensable for Normal Endochondral Bone Development, and Parathyroid Hormone-Related Peptide Suppresses Extracellular Signal-Regulated Kinase Activation in Hypertrophic Chondrocytes

Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.

Leptin Stimulates Parathyroid Hormone Related Peptide Expression in the Endochondral Growth Plate

We have previously shown that growth plate chondrocytes expressed the long form of leptin receptor, and that within the growth plate, leptin stimulated cell proliferation and differentiation and epiphyseal growth in a balanced manner. These three cell processes are known to be regulated by the interactions of parathyroid hormone-related peptide (PTHrP) and Indian hedgehog (Ihh) protein. The aim of the present study was to examine the effect of leptin on the PTHrP/Ihh feedback loop. The effect of leptin was investigated in vivo in pair-fed experiments in ICR mice, and ex vivo in mandibular condyle explants incubated with leptin. Immunohistochemistry and in situ hybridization showed that in the pair-fed in vivo system as well as in the organ culture, leptin increased the level of PTHrP and reduced that of Ihh. Leptin may affect chondrocyte proliferation and differentiation by activating the PTHrP/Ihh growth-restraining feedback loop in the postnatal growth plate.

Downregulation of Akt activity contributes to the growth arrest induced by FGF in chondrocytes

Unregulated FGF signaling produced by activating FGFR3 mutations causes several forms of dwarfism-associated chondrodysplasias in humans and mice. FGF signaling inhibits chondrocyte proliferation by activating multiple signal transduction pathways that all contribute to chondrocyte growth arrest and induction of some aspects of differentiation. Previous studies had identified the Stat1 pathway, dephosphorylation of the Rb family proteins p107 and p130, induction of p21 expression and sustained activation of MAP kinases as playing a role in the FGF response of chondrocytes. We have examined the role of Akt (PKB) in the response of chondrocytes to FGF signaling. Differently from what is observed in many other cell types, FGF does not activate Akt in chondrocytes, and Akt phosphorylation is actually downregulated after FGF treatment. By expressing a constitutively activated, myristylated form of Akt (myr-Akt) in the RCS chondrosarcoma cell line, we show that Akt activation partially counteracts the inhibitory effect of FGF signaling. The response of myr-Akt expressing cells to FGF is identical to parental RCS in the first few hours after treatment, but then diverges as myr-Akt cells show decreased p130 phosphorylation, increased cyclin E/cdk2 activity and continue to proliferate at a slow rate. Constitutive Akt activation does not affect p21 expression but appears to influence directly cdk/cyclin activity. On the other hand, the induction of differentiation-related genes is unchanged in myr-Akt cells. These results identify Akt downregulation as an important aspect of the response of chondrocytes to FGF that, however, only affects chondrocyte proliferation and not the ability of FGF to induce differentiation genes.

The in vitro effects of dehydroepiandrosterone on chondrocyte metabolism

To investigate the in vitro effects of dehydroepiandrosterone (DHEA) on neonatal rat chondrocytes. Chondrocytes isolated from neonatal rat cartilage were cultured in three-dimensionally agarose beads and were treated with DHEA. Primary culture of chondrocytes was harvested from newborn Wistar rats. The DHEA effects on chondrocyte activities were evaluated by analyzing chondrocyte proliferation, matrix protein synthesis, gene expressions of collagen, matrix metalloproteinase-1, -3 and -13 (MMP-1, -3 and -13), and cyclooxygenase-2 (COX-II), and protein synthesis of interleukin-6 (IL-6), prostaglandin E2 (PGE2) and tissue inhibitor of metalloproteinase-1 (TIMP-1). The DHEA treatment did affect chondrocyte proliferation and glycosaminoglycan (GAG) synthesis. DHEA suppressed the expression of MMP-1, -3 and -13 genes and PGE2 protein synthesis enhanced by lipopolysaccharide (LPS) while the COX-II and inducible nitric oxide synthase (iNOS) gene expressions were down-regulated by DHEA. Our study demonstrates that DHEA has an ability to modulate the imbalance between MMPs and PGE2 in the neonatal chondrocytes which suggest that it has a potential protective role against articular cartilage damage.

Growth hormone therapy in calcium-loaded rats with renal failure.

GH increases linear growth in children with chronic renal failure, but the response remains suboptimal in some patients. Some of the factors that may explain the poor response to GH include high doses of calcitriol and exogenous calcium loading to prevent hyperphosphatemia. High doses of exogenous calcium adversely affect chondrocyte proliferation and delay mineralization in the growth plate of rats with renal failure; bone histomorphometric changes in these animals are comparable to adynamic bone. To evaluate GH effects on adynamic bone in renal failure, 48 weanling rats underwent sham nephrectomy (Intact-Control) or 5/6 nephrectomy (Nx). Nx animals were fed a high-calcium diet (Nx-Ca(2+)) to induce adynamic bone. After 4 wk, the Nx-Ca(2+) animals were treated with GH (Nx-Ca(2+) + GH), calcitriol (Nx-Ca(2+) + D), or a combination of GH and calcitriol (Nx-Ca(2+)GH + D) for 2 wk. Serum intact PTH and IGF-I levels did not differ among all nephrectomized groups given high calcium. GH did not increase body length or tibial length at the end of study period. In the proximal tibia, the width of the growth plate and the growth plate architecture did not improve with GH. There was a decline in histone-4 expression, IGF-I protein, IGF binding protein-3, and bone morphogenetic protein-7 staining and a mild increase in IGF-I receptor, GH receptor, and gelatinase B expression in the Nx-Ca(2+) + GH group when compared with the Intact-Control group. Calcitriol blunted some of the mitogenic effects of GH in the growth plate. Thus, there was a poor response to GH therapy in calcium-loaded animals with renal failure.

The in vitro effects of dehydroepiandrosterone on human osteoarthritic chondrocytes

Objective: To investigate the in vitro effects of dehydroepiandrosterone (DHEA) on human osteoarthritic chondrocytes. Design: Chondrocytes isolated from human osteoarthritic knee cartilage were three-dimensionally cultured in alginate beads, except for cell proliferation experiment. Cells were treated with DHEA in the presence or absence of IL-1β. The effects on chondrocytes were analyzed using a 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (for chondrocyte proliferation), a dimethylmethylene blue (DMB) assay (for glycosaminoglycan (GAG) synthesis), and an indole assay (for DNA amount). Gene expressions of type I and II collagen, metalloproteinase-1 and -3 (MMP-1 and -3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as the IL-1β-induced gene expressions of MMP-1 and -3 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein synthesis of MMP-1 and -3 and TIMP-1 was determined by Western blotting. Results: The treatment of chondrocytes with DHEA did not affect chondrocyte proliferation or GAG synthesis up to 100 μM of concentration. The gene expression of type II collagen increased in a dose-dependent manner, while that of type I decreased. DHEA suppressed the expression of MMP-1 significantly at concentrations exceeding 50 μM. The gene expression of MMP-3 was also suppressed, but this was without statistical significance. The expression of TIMP-1 was significantly increased by DHEA at concentrations exceeding 10 μM. The effects of DHEA on the gene expressions of MMP-1 and -3 were more prominent in the presence of IL-1β, in which DHEA suppressed not only MMP-1, but also MMP-3 at the lower concentrations, 10 and 50 μM, respectively. Western blotting results were in agreement with RT-PCR, which indicates that DHEA acts at the gene transcription level. Conclusions: Our study demonstrates that DHEA has no toxic effect on chondrocytes up to 100 μM of concentration and has an ability to modulate the imbalance between MMPs and TIMP-1 during OA at the transcription level, which suggest that it has a protective role against articular cartilage loss.

Effect of Surface Roughness and Composition on Costochondral Chondrocytes is Dependent on Cell Maturation State

During endochondral bone formatin, as occurs in fracture healing, chondrocytes are one of the first cells to see an implant surface. We tested the hypothesis that chemical composition and surface roughness affect chondrocyte differentiation matrix synthesis, and local factor production and that the nature of the response is dependent on the state of maturation of the cells. To do this, we harvested rat growth zone and resting zone chondrocytes and examined their tesponce to smooth and rough disk surfaces manufactured from either commercially pure titanium or titanium alloy. profilometry, scanning electron microscopy, Auger spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the surfaces. Average roughness values were 0.22 μm for smooth titanium surfaces, 0.23 μm for smooth titanium alloy surfaces, 4.24 μm for rough titanium surfaces. and 3.20 μm for rough titanium alloy surface. Cells were grown on the different disk surfaces until the cultures had reached confluence on plastic. The effect of the surfaces was determined by assaying cell number and [3H]thymidine incorporation as measurs of cell proliferatin, cell layer and cell alkaline phosphatase specific activity as markers of differentiation, and collagen production and [35S]sulfate incorporation as indicators of extracellular matrix production. In addition, the synthesis of prostaglandin E2 and transforming growth factor-β were examined to measure changes in local factor synthesis. In growth Zone and resting Zone cultures, cell number and [3H]thymidine incorporation were decreased on rough surfaces: however, this effect was greater on commercially pure titanium surfaces. Cell layer and cell alkaline phosphatase specific activity were decreased in resting zone cells grown on rough surfaces. Cell alkaline phosphatase specific activity in growth zone cells was decreased on rough surfaces, whereas cell layer alkaline phosphatase specific activity was increased only in growth zone cells growth on rough commercially pure titanium surfaces, Resting Zone cell collagen production was decreased only on rough commercially pure titanium, whereas in growth zone cells, collagen production was increased. Increased prostaglandin E2 release into the media was found for growth zone and resting zone cell cultures on the disks with rough surfaces. The observed effect was greater on rough commercially pure titanium. Production of transforming growth factor-β by resting zones was similarly affected. whereas an increase in its production by growth zone cells was measured only on rough commercially pure titanium. These results indicate that surface roughness affects chondrocyte proliferation, differentiation, matrix synthesis. and local factor production and that these parameters are also affected by chemical composition. Furthermore. the nature and extent of the cell response is dependent on cell maturation. The overriding variable in response to an implant material, however, appears to be roughness of the surface.