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Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor composed of an alpha and a beta subunit. HIF-1α is a master regulator of cellular response to hypoxia by activating the transcription of genes that facilitate metabolic adaptation to hypoxia. Since chondrocytes in mature articular cartilage reside in a hypoxic environment, HIF-1α plays an important role in chondrogenesis and in the physiological lifecycle of articular cartilage. Accumulating evidence suggests interactions between the HIF pathways and the circadian clock. The circadian clock is an emerging regulator in both developing and mature chondrocytes. However, how circadian rhythm is established during the early steps of cartilage formation and through what signaling pathways it promotes the healthy chondrocyte phenotype is still not entirely known. This narrative review aims to deliver a concise analysis of the existing understanding of the dynamic interplay between HIF-1α and the molecular clock in chondrocytes, in states of both health and disease, while also incorporating creative interpretations. We explore diverse hypotheses regarding the intricate interactions among these pathways and propose relevant therapeutic strategies for cartilage disorders such as osteoarthritis. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a currently incurable, chronic, progressive, and debilitating musculoskeletal (MSK) condition. One of its hall-mark symptoms is chronic nociceptive and neuropathic pain, which significantly reduces the quality of life of patients with OA. Although research into the pathomechanisms of OA pain is ongoing and several pain pathways are well understood, the true source of OA pain remains unclear. Ion channels and transporters are key mediators of nociceptive pain. In this narrative review article, we summarize the state-of-the-art in relation to the distribution and function of ion channels in all major synovial joint tissues in the context of pain generation. We provide an update on the ion channels likely involved in mediating peripheral and central nociceptive pathways in the nervous system in OA pain, including voltage-gated sodium and potassium channels, members of the transient receptor potential (TRP) channel family, and purinergic receptor complexes. We focus on ion channels and transporters that have the potential to be candidate drug targets for pain management in patients with OA. We propose that ion channels expressed by the cells of constituent tissues of OA-afflicted synovial joints including cartilage, bone, synovium, ligament, and muscle, should be more thoroughly investigated and targeted in the context of OA pain. Based on key findings from recent basic research articles as well as clinical trials, we propose novel directions for the development of future analgesic therapies to improve the quality of life of patients with OA. [ABSTRACT FROM AUTHOR]

Musculoskeletal disorders represent one of the main causes of disability worldwide, and their prevalence is predicted to increase in the coming decades. Stem cell therapy may be a promising option for the treatment of some of the musculoskeletal diseases. Although significant progress has been made in musculoskeletal stem cell research, osteoarthritis, the most-common musculoskeletal disorder, still lacks curative treatment. To fine-tune stem-cell-based therapy, it is necessary to focus on the underlying biological mechanisms. Ion channels and the bioelectric signals they generate control the proliferation, differentiation, and migration of musculoskeletal progenitor cells. Calcium- and voltage-activated potassium (KCa) channels are key players in cell physiology in cells of the musculoskeletal system. This review article focused on the big conductance (BK) KCa channels. The regulatory function of BK channels requires interactions with diverse sets of proteins that have different functions in tissue-resident stem cells. In this narrative review article, we discuss the main ion channels of musculoskeletal stem cells, with a focus on calcium-dependent potassium channels, especially on the large conductance BK channel. We review their expression and function in progenitor cell proliferation, differentiation, and migration and highlight gaps in current knowledge on their involvement in musculoskeletal diseases. [ABSTRACT FROM AUTHOR]

The biomechanical environment plays a key role in regulating cartilage formation, but the current understanding of mechanotransduction pathways in chondrogenic cells is incomplete. Among the combination of external factors that control chondrogenesis are temporal cues that are governed by the cell‐autonomous circadian clock. However, mechanical stimulation has not yet directly been proven to modulate chondrogenesis via entraining the circadian clock in chondroprogenitor cells. The purpose of this study was to establish whether mechanical stimuli entrain the core clock in chondrogenic cells, and whether augmented chondrogenesis caused by mechanical loading was at least partially mediated by the synchronised, rhythmic expression of the core circadian clock genes, chondrogenic transcription factors, and cartilage matrix constituents at both transcript and protein levels. We report here, for the first time, that cyclic uniaxial mechanical load applied for 1 h for a period of 6 days entrains the molecular clockwork in chondroprogenitor cells during chondrogenesis in limb bud‐derived micromass cultures. In addition to the several core clock genes and proteins, the chondrogenic markers SOX9 and ACAN also followed a robust sinusoidal rhythmic expression pattern. These rhythmic conditions significantly enhanced cartilage matrix production and upregulated marker gene expression. The observed chondrogenesis‐promoting effect of the mechanical environment was at least partially attributable to its entraining effect on the molecular clockwork, as co‐application of the small molecule clock modulator longdaysin attenuated the stimulatory effects of mechanical load. This study suggests that an optimal biomechanical environment enhances tissue homoeostasis and histogenesis during chondrogenesis at least partially through entraining the molecular clockwork. [ABSTRACT FROM AUTHOR]

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription‐polymerase chain reaction, we examined the role of calcium‐dependent potassium channels in CPCs and observed functional large‐conductance calcium‐activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in‐depth characterisation of CPCs and their phenotype in the context of cartilage regeneration. [ABSTRACT FROM AUTHOR]

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro‐death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine‐stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real‐time polymerase chain reaction. MMP‐3, MMP‐13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down‐regulated 7‐days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker. [ABSTRACT FROM AUTHOR]

Background: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the 'surfaceome'. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting. Results: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1–2 and annexin A1 were considered to be of special interest and were analysed further by western blotting. Conclusions: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli. [ABSTRACT FROM AUTHOR]

Background: In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. Methods: We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. Results: Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. Conclusions: We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts. [ABSTRACT FROM AUTHOR]

Basic, translational, and clinical research in the area of stem cell biology is currently aimed at investigating the ability of adipose stem cells (ASCs) to enhance tissue and organ regeneration and suggest that ASCs in 3D scaffolds may have potential for application in wound healing, orthopaedic tissue repair, and tissue reconstruction after surgery [[1]]. The development of regenerative medicine strategies requires an appropriate phenotypically stable and well-characterized cell source and a 3-dimensional scaffold or "smart" biomaterial (novel "intelligent" biomaterial with appropriate physical properties that can support I in vivo i the commitment of ASCs), together with a suitable microenvironment to provide the necessary cues and signals for cell growth and tissue formation. However, 3D tissue engineering scaffolds are better able to mimic the in vivo cellular microenvironment and more effectively recapitulate the physiological setting, resulting in improved localization, attachment, proliferation, and differentiation of ASCs. [Extracted from the article]

Abstract: Novel synthetic biomaterials able to support direct tissue growth and retain cellular phenotypical properties are promising building blocks for the development of tissue engineering platforms for accurate and fast therapy screening for cancer. The aim of this study is to validate an aligned, pristine multi‐walled carbon nanotube (CNT) platform for in vitro studies of pancreatic cancer as a systematic understanding of interactions between cells and these CNT substrates is lacking. Our results demonstrate that our CNT scaffolds—which are easily tuneable to form sheets/fibers—support growth, proliferation, and spatial organization of pancreatic cancer cells, indicating their great potential in cancer tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1637–1644, 2018. [ABSTRACT FROM AUTHOR]

The effective growth of chondrocytes and the formation of cartilage is demonstrated on scaffolds of aligned carbon nanotubes; as two dimensional sheets and on three dimensional textiles. Raman spectroscopy is used to confirm the presence of chondroitin sulfate, which is critical in light of the unreliability of traditional dye based assays for carbon nanomaterial substrates. The textile exhibits a very high affinity for chondrocyte growth and could present a route to implantable, flexible cartilage scaffolds with tuneable mechanical properties. [ABSTRACT FROM AUTHOR]

Current cell-based repair strategies have proven unsuccessful for treating cartilage defects and osteoarthritic lesions, consequently advances in innovative therapeutics are required and mesenchymal stem cell-based (MSC) therapies are an expanding area of investigation. MSCs are capable of differentiating into multiple cell lineages and exerting paracrine effects. Due to their easy isolation, expansion, and low immunogenicity, MSCs are an attractive option for regenerative medicine for joint repair. Recent studies have identified several MSC tissue reservoirs including in adipose tissue, bone marrow, cartilage, periosteum, and muscle. MSCs isolated from these discrete tissue niches exhibit distinct biological activities, and have enhanced regenerative potentials for different tissue types. Each MSC type has advantages and disadvantages for cartilage repair and their use in a clinical setting is a balance between expediency and effectiveness. In this review we explore the challenges associated with cartilage repair and regeneration using MSC-based cell therapies and provide an overview of phenotype, biological activities, and functional properties for each MSC population. This paper also specifically explores the therapeutic potential of each type of MSC, particularly focusing on which cells are capable of producing stratified hyaline-like articular cartilage regeneration. Finally we highlight areas for future investigation. Given that patients present with a variety of problems it is unlikely that cartilage regeneration will be a simple "one size fits all," but more likely an array of solutions that need to be applied systematically to achieve regeneration of a biomechanically competent repair tissue. [ABSTRACT FROM AUTHOR]

Musculoskeletal conditions are a major burden on individuals, healthcare systems, and social care systems throughout the world, with indirect costs having a predominant economic impact. Aging is a major contributing factor to the development and progression of arthritic and musculoskeletal diseases. Indeed, aging and inflammation (often referred to as 'inflammaging') are critical risk factors for the development of osteoarthritis (OA), which is one of the most common forms of joint disease. The term 'chondrosenescence' has recently been introduced to define the age-dependent deterioration of chondrocyte function and how it undermines cartilage function in OA. An important component of chondrosenescence is the age-related deregulation of subcellular signaling pathways in chondrocytes. This mini-review discusses the role of age-related alterations in chondrocyte signaling pathways. We focus our attention on two major areas: age-dependent alterations in transforming growth factor-? signaling and changes in protein kinase and phosphoprotein phosphatase activities in aging chondrocytes. A better understanding of the basic signaling mechanisms underlying aging in chondrocytes is likely to facilitate the development of new therapeutic and preventive strategies for OA and a range of other age-related osteoarticular disorders. [ABSTRACT FROM AUTHOR]

Context: There is insufficient knowledge about the chondrocyte membranome and its molecular composition. Objective: To develop a Triton X-114 based separation technique using nanoLC-MS/MS combined with shotgun proteomics to identify chondrocyte membrane proteins. Materials and methods: Articular chondrocytes from equine metacarpophalangeal joints were separated into hydrophobic and hydrophilic fractions; trypsin-digested proteins were analysed by nanoLC-MS/MS. Results: A total of 315 proteins were identified. The phase extraction method yielded a high proportion of membrane proteins (56%) including CD276, S100-A6 and three VDAC isoforms. Discussion: Defining the chondrocyte membranome is likely to reveal new biomarker targets for conventional and biological drug discovery. [ABSTRACT FROM AUTHOR]

Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells. [ABSTRACT FROM AUTHOR]

Chondrocytes, the single cell type in adult articular cartilage, have conventionally been considered to be non-excitable cells. However, recent evidence suggests that their resting membrane potential (RMP) is less negative than that of excitable cells, and they are fully equipped with channels that control ion, water and osmolyte movement across the chondrocyte membrane. Amongst calcium-specific ion channels, members of the voltage-dependent calcium channel (VDCC) family are expressed in chondrocytes where they are functionally active. L-type VDCC inhibitors such as nifedipine and verapamil have contributed to our understanding of the roles of these ion channels in chondrogenesis, chondrocyte signalling and mechanotransduction. In this narrative review, we discuss published data indicating that VDCC function is vital for chondrocyte health, especially in regulating proliferation and maturation. We also highlight the fact that activation of VDCC function appears to accompany various inflammatory aspects of osteoarthritis (OA) and, based on in vitro data, the application of nifedipine and/or verapamil may be a promising approach for ameliorating OA severity. However, very few studies on clinical outcomes are available regarding the influence of calcium antagonists, which are used primarily for treating cardiovascular conditions in OA patients. This review is intended to stimulate further research on the chondrocyte 'channelome', contribute to the development of novel therapeutic strategies and facilitate the retargeting and repositioning of existing pharmacological agents currently used for other comorbidities for the treatment of OA. [ABSTRACT FROM AUTHOR]

Articular cartilage is a unique load-bearing connective tissue with a low intrinsic capacity for repair and regeneration. Its avascularity makes it relatively hypoxic and its unique extracellular matrix is enriched with cations, which increases the interstitial fluid osmolarity. Several physicochemical and biomechanical stimuli are reported to influence chondrocyte metabolism and may be utilized for regenerative medical approaches. In this review article, we summarize the most relevant stimuli and describe how ion channels may contribute to cartilage homeostasis, with special emphasis on intracellular signaling pathways. We specifically focus on the role of calcium signaling as an essential mechanotransduction component and highlight the role of phosphatase signaling in this context. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is the most common form of chronic musculoskeletal disorders. A migratory stem cell population termed chondrogenic progenitor cells (CPC) with in vitro chondrogenic potential was previously isolated from OA cartilage. Since intracellular Ca signalling is an important regulator of chondrogenesis, we aimed to provide a detailed understanding of the Ca homeostasis of CPCs. In this work, CPCs immortalised by lentiviral administration of the human telomerase reverse transcriptase (hTERT) and grown in monolayer cultures were studied. Expressions of all three IPRs were confirmed, but no RyR subtypes were detected. Ca oscillations observed in CPCs were predominantly dependent on Ca release and store replenishment via store-operated Ca entry; CPCs express both STIM1 and Orai1 proteins. Expressions of adenosine receptor mRNAs were verified, and adenosine elicited Ca transients. Various P2 receptor subtypes were identified; P2Y can bind ADP; P2Y is targeted by UTP; and ATP may evoke Ca transients via detected P2X subtypes, as well as P2Y and P2Y. Enzymatic breakdown of extracellular nucleotides by apyrase completely abrogated Ca oscillations, suggesting that an autocrine/paracrine purinergic mechanism may drive Ca oscillations in these cells. As CPCs possess a broad spectrum of functional molecular elements of Ca signalling, Ca-dependent regulatory mechanisms can be supposed to influence their differentiation potential. [ABSTRACT FROM AUTHOR]

Presence of the pituitary adenylate cyclase-activating polypeptide (PACAP) signalling has been proved in various peripheral tissues. PACAP can activate protein kinase A (PKA) signalling via binding to pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1), vasoactive intestinal polypeptide receptor (VPAC) 1 or VPAC2 receptor. Since little is known about the role of this regulatory mechanism in bone formation, we aimed to investigate the effect of PACAP on osteogenesis of UMR-106 cells. PACAP 1-38 as an agonist and PACAP 6-38 as an antagonist of PAC1 were added to the culture medium. Surprisingly, both substances enhanced protein expressions of collagen type I, osterix and alkaline phosphatase, along with higher cell proliferation rate and an augmented mineralisation. Although expression of PKA was elevated, no alterations were detected in the expression, phosphorylation and nuclear presence of CREB, but increased nuclear appearance of Runx2, the key transcription factor of osteoblast differentiation, was shown. Both PACAPs increased the expressions of bone morphogenetic proteins (BMPs) 2, 4, 6, 7 and Smad1 proteins, as well as that of Sonic hedgehog, PATCH1 and Gli1. Data of our experiments indicate that activation of PACAP pathway enhances bone formation of UMR-106 cells and PKA, BMP and Hedgehog signalling pathways became activated. We also found that PACAP 6-38 did not act as an antagonist of PACAP signalling in UMR-106 cells. [ABSTRACT FROM AUTHOR]

Pituitary adenylate cyclase activating polypeptide (PACAP) is an important neurotrophic factor influencing differentiation of neuronal elements and exerting protecting role during traumatic injuries or inflammatory processes of the central nervous system. Although increasing evidence is available on its presence and protecting function in various peripheral tissues, little is known about the role of PACAP in formation of skeletal components. To this end, we aimed to map elements of PACAP signalling in developing cartilage under physiological conditions and during oxidative stress. mRNAs of PACAP and its receptors (PAC1,VPAC1, VPAC2) were detectable during differentiation of chicken limb bud-derived chondrogenic cells in micromass cell cultures. Expression of PAC1 protein showed a peak on days of final commitment of chondrogenic cells. Administration of either the PAC1 receptor agonist PACAP 1-38, or PACAP 6-38 that is generally used as a PAC1 antagonist, augmented cartilage formation, stimulated cell proliferation and enhanced PAC1 and Sox9 protein expression. Both variants of PACAP elevated the protein expression and activity of the Ca-calmodulin dependent Ser/Thr protein phosphatase calcineurin. Application of PACAPs failed to rescue cartilage formation when the activity of calcineurin was pharmacologically inhibited with cyclosporine A. Moreover, exogenous PACAPs prevented diminishing of cartilage formation and decrease of calcineurin activity during oxidative stress. As an unexpected phenomenon, PACAP 6-38 elicited similar effects to those of PACAP 1-38, although to a different extent. On the basis of the above results, we propose calcineurin as a downstream target of PACAP signalling in differentiating chondrocytes either in normal or pathophysiological conditions. Our observations imply the therapeutical perspective that PACAP can be applied as a natural agent that may have protecting effect during joint inflammation and/or may promote cartilage regeneration during degenerative diseases of articular cartilage. [ABSTRACT FROM AUTHOR]

Murine micromass models have been extensively applied to study chondrogenesis and osteogenesis to elucidate pathways of endochondral bone formation. Here we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1 at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2) were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models. [ABSTRACT FROM AUTHOR]

Background: Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca2+- signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation. Methodology/Principal Findings: Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated NaV1.4, KV1.1, KV1.3 and KV4.1 channels, although KV1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of NaV1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K+ channel blocker tetraethylammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced KV currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca2+-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive KV1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive KV4.1 did not affect cartilage formation. Conclusions/Significance: These data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca2+-oscillations, which are modulated by KV-driven membrane potential changes. KV1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use. [ABSTRACT FROM AUTHOR]

We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells. [ABSTRACT FROM AUTHOR]

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation. [ABSTRACT FROM AUTHOR]

Diseases such as osteoarthritis (OA) are commonly characterized at the molecular scale by gene expression and subsequent protein production; likewise, the effects of pharmaceutical interventions are typically characterized by the effects of molecular interactions. However, these phenomena are usually preceded by numerous precursor steps, many of which involve significant ion influx or efflux. As a consequence, rapid assessment of cell electrophysiology could play a significant role in unravelling the mechanisms underlying drug interactions and progression of diseases, such as OA. In this study, we used dielectrophoresis (DEP), a technique that allows rapid, label-free determination of the dielectric parameters to assess the role of potassium ions on the dielectric characteristics of chondrocytes, and to investigate the electrophysiological differences between healthy chondrocytes and those from an in vitro arthritic disease model. Our results showed that DEP was able to detect a significant decrease in membrane conductance (6191 ± 738 vs. 8571 ± 1010 S/m2), membrane capacitance (10.3 ± 1.47 vs. 14.5 ± 0.01 mF/m2), and whole cell capacitance (5.4 ± 0.7 vs. 7.5 ± 0.3 pF) following inhibition of potassium channels using 10 mM tetraethyl ammonium, compared to untreated healthy chondrocytes. Moreover, cells from the OA model had a different response to DEP force in comparison to healthy cells; this was seen in terms of both a decreased membrane conductivity (782 S/m2 vs. 1139 S/m2) and a higher whole cell capacitance (9.58 ± 3.4 vs. 3.7 ± 1.3 pF). The results show that DEP offers a high throughput method, capable of detecting changes in membrane electrophysiological properties and differences between disease states. [ABSTRACT FROM AUTHOR]

The complement of cell surface proteins, collectively referred to as the surfaceome, is a useful indicator of normal differentiation processes, and the development of pathologies such as osteoarthritis (OA). We employed biochemical and proteomic tools to explore the surfaceome and to define biomarkers in chondrogenic progenitor cells (CPC) derived from human OA knee articular cartilage. These cells have great therapeutic potential, but their unexplored biology limits their clinical application. We performed biotinylation combined with glycocapture and high throughput shotgun proteomics to define the surface proteome of human bone marrow mesenchymal stem cells (MSCs) and human CPCs. We prepared cell surface protein-enriched fractions from MSCs and CPCs, and then a proteomic approach was used to compare and evaluate protein changes between undifferentiated MSCs and CPCs. 1256 proteins were identified in the study, of which 791 (63%) were plasma membrane, cell surface or extracellular matrix proteins. Proteins constituting the surfaceome were annotated and categorized. Our results provide, for the first time, a repository of quantitative proteomic data on the surfaceome of two closely related cell types relevant to cartilage biology and OA. These results may provide novel insights into the transformation of the surfaceome during chondrogenic differentiation and phenotypic changes during OA development. [ABSTRACT FROM AUTHOR]