Osmolarity-Induced Altered Intracellular Molecular Crowding Drives Osteoarthritis Pathology.

Osteoarthritis (OA) is a multifactorial degenerative joint disease of which the underlying mechanisms are yet to be fully understood. At the molecular level, multiple factors including altered signaling pathways, epigenetics, metabolic imbalance, extracellular matrix degradation, production of matrix metalloproteinases, and inflammatory cytokines, are known to play a detrimental role in OA. However, these factors do not initiate OA, but are mediators or consequences of the disease, while many other factors causing the etiology of OA are still unknown. Here, it is revealed that microenvironmental osmolarity can induce and reverse osteoarthritis-related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism.

Choice of Protein, Not Its Amyloid-Fold, Determines the Success of Amyloid-Based Scaffolds for Cartilage Tissue Regeneration.

The formation of fibrocartilage during articular cartilage regeneration remains a clinical problem affecting adequate restoration of articular cartilage in joints. To stimulate chondrocytes to form articular cartilage, we investigated the use of amyloid fibril-based scaffolds. The proteins α-synuclein, ß-lactoglobulin, and lysozyme were induced to self-assemble into amyloid fibrils and, during dialysis, formed micrometer scale amyloid networks that resemble the cartilage extracellular matrix. Our results show that lysozyme amyloid micronetworks supported chondrocyte viability and extracellular matrix deposition, while α-synuclein and ß-lactoglobulin maintained cell viability. With this study, we not only confirm the possible use of amyloid materials for tissue regeneration but also demonstrate that the choice of protein, rather than its amyloid-fold per se, affects the cellular response and tissue formation.

Implementing Computational Modeling in Tissue Engineering: Where Disciplines Meet.

In recent years, the mathematical and computational sciences have developed novel methodologies and insights that can aid in designing advanced bioreactors, microfluidic setups or organ-on-chip devices, in optimizing culture conditions, or predicting long-term behavior of engineered tissues in vivo. In this review, we introduce the concept of computational models and how they can be integrated in an interdisciplinary workflow for Tissue Engineering and Regenerative Medicine (TERM). We specifically aim this review of general concepts and examples at experimental scientists with little or no computational modeling experience. We also describe the contribution of computational models in understanding TERM processes and in advancing the TERM field by providing novel insights. Impact Statement Although in recent years the use of mathematical and computational sciences has increased in the Tissue Engineering and Regenerative Medicine (TERM) field, we believe that a further integration of experimental and computational approaches has a huge potential for advancing the field due to the ability of models to explain and predict experimental results and efficiently optimize TERM product and process designs. By providing an overview of existing computational models, how they have contributed to the field, as well as a future perspective, this review represents an important step to help realize TERM's ultimate goal: a cure instead of care.

An ECHO of Cartilage: In Silico Prediction of Combinatorial Treatments to Switch Between Transient and Permanent Cartilage Phenotypes With Ex Vivo Validation.

A fundamental question in cartilage biology is: what determines the switch between permanent cartilage found in the articular joints and transient hypertrophic cartilage that functions as a template for bone? This switch is observed both in a subset of OA patients that develop osteophytes, as well as in cell-based tissue engineering strategies for joint repair. A thorough understanding of the mechanisms regulating cell fate provides opportunities for treatment of cartilage disease and tissue engineering strategies. The objective of this study was to understand the mechanisms that regulate the switch between permanent and transient cartilage using a computational model of chondrocytes, ECHO. To investigate large signaling networks that regulate cell fate decisions, we developed the software tool ANIMO, Analysis of Networks with interactive Modeling. In ANIMO, we generated an activity network integrating 7 signal transduction pathways resulting in a network containing over 50 proteins with 200 interactions. We called this model ECHO, for executable chondrocyte. Previously, we showed that ECHO could be used to characterize mechanisms of cell fate decisions. ECHO was first developed based on a Boolean model of growth plate. Here, we show how the growth plate Boolean model was translated to ANIMO and how we adapted the topology and parameters to generate an articular cartilage model. In ANIMO, many combinations of overactivation/knockout were tested that result in a switch between permanent cartilage (SOX9+) and transient, hypertrophic cartilage (RUNX2+). We used model checking to prioritize combination treatments for wet-lab validation. Three combinatorial treatments were chosen and tested on metatarsals from 1-day old rat pups that were treated for 6 days. We found that a combination of IGF1 with inhibition of ERK1/2 had a positive effect on cartilage formation and growth, whereas activation of DLX5 combined with inhibition of PKA had a negative effect on cartilage formation and growth and resulted in increased cartilage hypertrophy. We show that our model describes cartilage formation, and that model checking can aid in choosing and prioritizing combinatorial treatments that interfere with normal cartilage development. Here we show that combinatorial treatments induce changes in the zonal distribution of cartilage, indication possible switches in cell fate. This indicates that simulations in ECHO aid in describing pathologies in which switches between cell fates are observed, such as OA.

Protein Adsorption Enhances Energy Dissipation in Networks of Lysozyme Amyloid Fibrils.

Hydrogels of amyloid fibrils are a versatile biomaterial for tissue engineering and other biomedical applications. Their suitability for these applications has been partly ascribed to their excellent and potentially engineerable rheological properties. However, while in biomedical applications the gels have to function in compositionally complex physiological solutions, their rheological behavior is typically only characterized in simple buffers. Here we show that the viscoelastic response of networks of amyloid fibrils of the protein lysozyme in biologically relevant solutions substantially differs from the response in simple buffers. We observe enhanced energy dissipation in both cell culture medium and synovial fluid. We attribute this energy dissipation to interactions of the amyloid fibrils with other molecules in these solutions and especially to the adsorption of the abundantly present protein serum albumin. This finding provides the basis for a better understanding of the performance of amyloid hydrogels in biomedical applications.

The Expressions of Dickkopf-Related Protein 1 and Frizzled-Related Protein Are Negatively Correlated to Local Inflammation and Osteoarthritis Severity.

OBJECTIVE: To investigate the presence of WNT antagonists Dickkopf-related protein 1 (DKK1), Frizzled-related protein (FRZB) and BMP antagonist Gremlin 1 (GREM1) in synovial fluid (SF) and serum, respectively, from end-stage knee osteoarthritis (OA) patients, and correlate their expression with other markers of OA. DESIGN: In a cross-sectional study, SF and serum were collected from OA patients (n = 132). The concentrations of DKK1, FRZB and GREM1 in SF and serum were determined using immunoassays. Correlation measurements were performed between groups and previously assessed disease markers, such as synovium nitric oxide (NO), inerleukin-1ß (IL1ß), tumor necrosis factor-α (TNFα), and prostaglandin E2 (PGE2). RESULTS: The OA patients with the celecoxib treatment till surgery have higher median SF FRZB values compared with the control (no treatment); the celecoxib 3-days before surgery stopped treatment group has higher median serum FRZB values than the control and the naproxen treatment group. The combinational analysis of SF DKK1 and SF FRZB negatively correlated with macroscopic cartilage scores and histological synovium scores in OA patients. The expression of DKK1 and FRZB in SF showed the same expression trend as their expression in serum. Furthermore, the SF concentration of DKK1 was positively correlated with FRZB in both SF and serum. In contrast, it was negatively correlated with synovium NO and IL1ß. SF FRZB was negatively correlated with synovium NO, IL1ß, cartilage PGE2, and age. CONCLUSIONS: Our findings suggest DKK1 and FRZB were negatively correlated with OA severity and multiple pro-inflammatory cytokines. Our data indicate that DKK1 and FRZB can be joint disease-specific biomarkers.

Engineering Cartilage Tissue by Co-culturing of Chondrocytes and Mesenchymal Stromal Cells.

Co-culture of chondrocytes and mesenchymal stromal cells (MSCs) has been shown to be beneficial in engineering cartilage tissue in vitro. In these co-cultures, MSCs increase the proliferation and matrix deposition of chondrocytes. The MSCs accomplish this beneficial effect by so-called trophic actions. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe different methods for making co-cultures of MSCs and chondrocytes. We also provide detailed protocols for analyzing MSC-chondrocyte co-cultures with cell tracking, proliferation assays, species-specific polymerase chain reactions (PCR), rheological analysis, compression analysis, RNA-sequencing analysis, short tandem repeats analysis, and biochemical examination.

Using FRAP to Quantify Changes in Transcription Factor Dynamics After Cell Stimulation: Cell Culture, FRAP, Data Analysis, and Visualization.

Here we show how to measure the mobility of transcription factors using fluorescence recovery after photobleaching (FRAP). Transcription factors are DNA-binding proteins that, upon binding to specific DNA motifs, regulate transcription of their target genes. FRAP is a simple, fast, and cost-effective method, and is a widely used quantitative method to measure the dynamics of fluorescently labeled molecules in solution, membranes, and inside living cells. Dynamics, specified by the immobile fraction, recovery half-time, diffusion constant, and ratio of molecules contributing to different phases of FRAP recovery, can be quantified by FRAP. This can be useful to understand their function in gene regulation. This tutorial is intended to familiarize the reader with the FRAP procedure to quantify transcription factor dynamics using a standard confocal microscope and analysis using MATLAB (MathWorks®). This article will guide the reader through the preconditions of FRAP, and a detailed and step-by-step procedure of preparing cells, bleaching protocol, data analysis in MATLAB, and visualization of the FRAP data.

Quantitative Molecular Models for Biological Processes: Modeling of Signal Transduction Networks with ANIMO.

Computational modeling of biological networks is increasing in popularity due to the increased demand for understanding biological processes. This understanding requires integration of a variety of experimental data that allows understanding of complex mechanisms regulating cell and tissue function. However, the mathematical complexity of many modeling tools have thusfar prevented broad adaptation and effective use by molecular biologists. In this chapter, we show by example how one can start building a model in ANIMO and how to adapt the model to experimental data. We show how this model can be used for simulating network activities, testing hypotheses, and how to improve the model using wet-lab data.

ECHO, the executable CHOndrocyte: A computational model to study articular chondrocytes in health and disease.

Computational modeling can be used to investigate complex signaling networks in biology. However, most modeling tools are not suitable for molecular cell biologists with little background in mathematics. We have built a visual-based modeling tool for the investigation of dynamic networks. Here, we describe the development of computational models of cartilage development and osteoarthritis, in which a panel of relevant signaling pathways are integrated. In silico experiments give insight in the role of each of the pathway components and reveal which perturbations may deregulate the basal healthy state of cells and tissues. We used a previously developed computational modeling tool Analysis of Networks with Interactive Modeling (ANIMO) to generate an activity network integrating 7 signal transduction pathways resulting in a network containing over 50 nodes and 200 interactions. We performed in silico experiments to characterize molecular mechanisms of cell fate decisions. The model was used to mimic biological scenarios during cell differentiation using RNA-sequencing data of a variety of stem cell sources as input. In a case-study, we wet-lab-tested the model-derived hypothesis that expression of DKK1 (Dickkopf-1) and FRZB (Frizzled related protein, WNT antagonists) and GREM1 (gremlin 1, BMP antagonist) prevents IL1ß (Interleukin 1 beta)-induced MMP (matrix metalloproteinase) expression, thereby preventing cartilage degeneration, at least in the short term. We found that a combination of DKK1, FRZB and GREM1 may play a role in modulating the effects of IL1ß induced inflammation in human primary chondrocytes.

Changes in Fluorescence Recovery After Photobleaching (FRAP) as an indicator of SOX9 transcription factor activity.

Cells respond to their environment via an intricate cellular signaling network, directing cell fate. Changes in cell fate are characterized by changes in gene transcription, dictated by (master) transcription factor activity. SOX9 is the master transcription factor for chondrocyte development. Its impaired function is implicated in osteoarthritis and growth disorders, such as dwarfism. However, the factors regulating SOX9 transcriptional activity are not yet fully mapped. Current methods to study transcription factor activity are indirect and largely limited to quantification of SOX9 target gene and protein expression levels after several hours or days of stimulation, leading to poor temporal resolution. We used Fluorescence Recovery After Photobleaching (FRAP) to study the mobility of SOX9 and correlated the changes in mobility to changes in its transcriptional activity by cross-validating with chromatin immunoprecipitation and qPCR. We show that using FRAP, we can quantify the changes in SOX9 mobility on short time scales as an indication of transcriptional activity, which correlated to changes of SOX9 DNA-binding and long-term target gene expression.

Different response of human chondrocytes from healthy looking areas and damaged regions to IL1ß stimulation under different oxygen tension.

Due to its avascular nature, articular cartilage is relatively hypoxic. The aim of this study was to elucidate the functional changes of macroscopically healthy looking areas chondrocytes (MHC) and macroscopically damaged regions chondrocytes (MDC) at a cellular level in response to the inflammatory cytokine IL1ß under different oxygen tension levels. In this study, two-dimensional (2-D) expanded MHC and MDC were redifferentiated in 3-D pellet cultures in chondrogenic differentiation medium, supplemented with or without IL1ß at conventional culture (normoxia) or 2.5% O2 (hypoxia) for 3 weeks. qPCR, immunohistochemistry and ELISA were used to detect the expression of anabolic and catabolic gene expression. Alcian blue/Safranin O staining and GAG assay were used to measure cartilage matrix production. Cell proliferation and apoptosis were assessed by EdU staining and TUNEL assay, respectively. The results showed that hypoxia enhanced matrix production in both MHC and MDC and this effect was stronger on MDC. Under normoxia, MHC showed higher expression of cartilage markers and lower catabolic genes expression than MDC. Interestingly, hypoxia diminished the difference between MHC and MDC. IL1ß potently induced MMPs expression regardless of cell population and oxygen tension. The fold induction of these MMPs in hypoxia was however much higher than in normoxia. In addition, hypoxia promoted the expression of HIF1α and HIF2α in MHC, while it only enhanced HIF1α expression but decreased the HIF2α expression in MDC. We concluded that hypoxia stimulated the redifferentiation of cultured chondrocytes, particularly in MDC derived from macroscopically diseased cartilage. Oxygen tension may profoundly and differentially influence inflammation-associated cartilage injury and diseases by regulating the expression of HIF1α and HIF2α. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:XX-XX, 2018.

Co-treatment of TGF-ß3 and BMP7 is superior in stimulating chondrocyte redifferentiation in both hypoxia and normoxia compared to single treatments.

Signaling by members of the transforming growth factor-ß (TGF-ß) superfamily, such as TGF-ß3 and BMP7, and oxygen tension play a pivotal role in chondrocyte biology. The objective of this research was to investigate the endogenous BMP7 expression in human osteoarthritis (OA) cartilage and the effect of oxygen tension on the single or combined treatment with TGF-ß3 and BMP7 on OA chondrocyte redifferentiation in three dimensional (3D) pellet cultures. The results showed the expression of BMP7 and its intracellular signaling target SMAD1/5/8 was decreased in early OA, while it was increased in later stages of OA. The combined treatment with TGF-ß3 and BMP7, both in normoxia and hypoxia, was more effective than TGF-ß3 or BMP7 alone in redifferentiating chondrocytes. This was reflected by Alcian blue/Safranin O staining and collagen type II protein expression, as well as by gene expression. Hypoxia elevated TGF-ß3 and BMP7-induced matrix formation of OA chondrocytes and alleviated the catabolic gene expression. Interestingly, cells cultured under normoxia displayed mild signs of an inflammatory stress response, which was effectively counteracted by culturing the cells under low oxygen tension. Our data underscores the important modulatory role of oxygen tension on the chondrocyte's responsiveness to TGF-ß3 and/or BMP7.

The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9, COL2A1, and ACAN, and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5. Inhibition of WNT signaling by PKF increased the expression of SOX9, COL2A1, ACAN, and MMP9, but decreased MMP13 and ADAMTS4/5. In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL, and RUNX2, the dedifferentiation marker COL1A1, and glycolysis genes GULT1 and PGK1. Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were decreased after PKF treatment. All of these chondrogenic effects appeared to be mediated through the canonical WNT signaling pathway, since the target gene Axin2 and other WNT members, such as TCF4 and ß-catenin, were upregulated by BIO and downregulated by PKF, respectively, and BIO induced nuclear translocation of ß-catenin while PKF inhibited ß-catenin translocation into the nucleus. We concluded that addition of BIO to a chondrogenic medium of hMSCs resulted in a loss of cartilage formation, while PKF induced chondrogenic differentiation and cartilage matrix deposition and inhibited hypertrophic differentiation. However, BIO promoted cell survival by inhibiting apoptosis while PKF induced cell apoptosis. This result indicates that either an overexpression or overinhibition of WNT signaling to some extent causes harmful effects on chondrogenic differentiation. Cartilage tissue engineering could benefit from the adjustment of the critical level of WNT signaling during chondrogenesis of hMSC.

Nitric Oxide Mediates Crosstalk between Interleukin 1ß and WNT Signaling in Primary Human Chondrocytes by Reducing DKK1 and FRZB Expression.

Interleukin 1 beta (IL1ß) and Wingless-Type MMTV Integration Site Family (WNT) signaling are major players in Osteoarthritis (OA) pathogenesis. Despite having a large functional overlap in OA onset and development, the mechanism of IL1ß and WNT crosstalk has remained largely unknown. In this study, we have used a combination of computational modeling and molecular biology to reveal direct or indirect crosstalk between these pathways. Specifically, we revealed a mechanism by which IL1ß upregulates WNT signaling via downregulating WNT antagonists, DKK1 and FRZB. In human chondrocytes, IL1ß decreased the expression of Dickkopf-1 (DKK1) and Frizzled related protein (FRZB) through upregulation of nitric oxide synthase (iNOS), thereby activating the transcription of WNT target genes. This effect could be reversed by iNOS inhibitor 1400W, which restored DKK1 and FRZB expression and their inhibitory effect on WNT signaling. In addition, 1400W also inhibited both the matrix metalloproteinase (MMP) expression and cytokine-induced apoptosis. We concluded that iNOS/NO play a pivotal role in the inflammatory response of human OA through indirect upregulation of WNT signaling. Blocking NO production may inhibit the loss of the articular phenotype in OA by preventing downregulation of the expression of DKK1 and FRZB.

Oxygen-Dependent Lipid Profiles of Three-Dimensional Cultured Human Chondrocytes Revealed by MALDI-MSI.

Articular cartilage is exposed to a gradient of oxygen levels ranging from 5% at the surface to 1% in the deepest layers. While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels (≤8%) promotes the chondrogenic phenotype. Exposure of cells to various oxygen levels alters their lipid metabolism, but detailed studies examining how hypoxia affects lipid metabolism in chondrocytes are lacking. To better understand the chondrocyte's behavior in response to oxygen, we cultured 3D pellets of human primary chondrocytes in normoxia (20% oxygen) and hypoxia (2.5% oxygen) and employed matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in order to characterize the lipid profiles and their spatial distribution. In this work we show that chondrocytes cultured in hypoxia and normoxia can be differentiated by their lipid profiles. Among other species, phosphatidylglycerol species were increased in normoxic pellets, whereas phosphatidylinositol species were the most prominent lipids in hypoxic pellets. Moreover, spatial mapping revealed that phospahtidylglyycerol species were less prominent in the center of pellets where the oxygen level is lower. Additional analysis revealed a higher abundance of the mitochondrial-specific lipids, cardiolipins, in normoxic conditions. In conclusion MALDI-MSI described specific lipid profiles that could be used as sensors of oxygen level changes and may especially be relevant for retaining the chondrogenic phenotype, which has important implications for the treatment of bone and cartilage diseases.

Endogenous DKK1 and FRZB Regulate Chondrogenesis and Hypertrophy in Three-Dimensional Cultures of Human Chondrocytes and Human Mesenchymal Stem Cells.

Hypertrophic differentiation occurs during in vitro chondrogenesis of mesenchymal stem cells (MSCs), decreasing the quality of the cartilage construct. Previously we identified WNT pathway antagonists Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in blocking hypertrophic differentiation of human MSCs (hMSCs). In this study, we investigated the role of endogenously expressed DKK1 and FRZB in chondrogenesis of hMSC and chondrocyte redifferentiation and in preventing cell hypertrophy using three relevant human cell based systems, isolated hMSCs, isolated primary human chondrocytes (hChs), and cocultures of hMSCs with hChs for which we specifically designed neutralizing nano-antibodies. We selected and tested variable domain of single chain heavy chain only antibodies (VHH) for their ability to neutralize the function of DKK1 or FRZB. In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan and collagen type II staining were significantly reduced in monocultured hMSCs and monocultured chondrocytes. Furthermore, in cocultures, cells in pellets showed hypertrophic differentiation. In conclusion, endogenous expression of the WNT antagonists DKK1 and FRZB is necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are indispensable for the initial steps of chondrogenic differentiation of hMSCs, second they are necessary for chondrocyte redifferentiation, and finally in preventing hypertrophic differentiation of articular chondrocytes.

A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy.

Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model's results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis.

Modelling with ANIMO: between fuzzy logic and differential equations.

BACKGROUND: Computational support is essential in order to reason on the dynamics of biological systems. We have developed the software tool ANIMO (Analysis of Networks with Interactive MOdeling) to provide such computational support and allow insight into the complex networks of signaling events occurring in living cells. ANIMO makes use of timed automata as an underlying model, thereby enabling analysis techniques from computer science like model checking. Biology experts are able to use ANIMO via a user interface specifically tailored for biological applications. In this paper we compare the use of ANIMO with some established formalisms on two case studies. RESULTS: ANIMO is a powerful and user-friendly tool that can compete with existing continuous and discrete paradigms. We show this by presenting ANIMO models for two case studies: Drosophila melanogaster circadian clock, and signal transduction events downstream of TNF α and EGF in HT-29 human colon carcinoma cells. The models were originally developed with ODEs and fuzzy logic, respectively. CONCLUSIONS: Two biological case studies that have been modeled with respectively ODE and fuzzy logic models can be conveniently modeled using ANIMO. The ANIMO models require less parameters than ODEs and are more precise than fuzzy logic. For this reason we position the modelling paradigm of ANIMO between ODEs and fuzzy logic.

Correlation between Gene Expression and Osteoarthritis Progression in Human.

Osteoarthritis (OA) is a multifactorial disease characterized by gradual degradation of joint cartilage. This study aimed to quantify major pathogenetic factors during OA progression in human cartilage. Cartilage specimens were isolated from OA patients and scored 0-5 according to the Osteoarthritis Research Society International (OARSI) guidelines. Protein and gene expressions were measured by immunohistochemistry and qPCR, respectively. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to detect apoptotic cells. Cartilage degeneration in OA is a gradual progress accompanied with gradual loss of collagen type II and a gradual decrease in mRNA expression of SOX9, ACAN and COL2A1. Expression of WNT antagonists DKK1 and FRZB was lost, while hypertrophic markers (RUNX2, COL10A1 and IHH) increased during OA progression. Moreover, DKK1 and FRZB negatively correlated with OA grading, while RUNX2 and IHH showed a significantly positive correlation with OA grading. The number of apoptotic cells was increased with the severity of OA. Taken together, our results suggested that genetic profiling of the gene expression could be used as markers for staging OA at the molecular level. This helps to understand the molecular pathology of OA and may lead to the development of therapies based on OA stage.