Cartilage mechanobiology and transcriptional effects of combined mechanical compression and IGF-1 stimulation on bovine cartilage explants

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2007.Includes bibliographical references.Background: Investigators have focused on mechano-regulation of upstream signaling and responses at the level of gene transcription, protein translation and post-translational modifications. Intracellular pathways including those involving integrin signaling, mitogen activated protein kinases (MAPKs), and release of intracellular calcium have been confirmed in several laboratories. Studies with IGF-1: Insulin-like growth factor-I (IGF-1) is a potent anabolic factor capable of endocrine and paracrine/autocrine signaling. Previous studies have demonstrated that mechanical compression can regulate the action of IGF-1 on chondrocyte biosynthesis in intact tissue; when applied simultaneously, these stimuli act by distinct cell activation pathways. Our objectives were to elucidate the extent and kinetics of the chondrocyte transcriptional response to combined IGF-1 and static compression in cartilage explants. Discussion: Clustering analysis revealed five distinct groups. TIMP-3 and ADAMTS-5, MMP-l and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed under all conditions tested. In comparing gene expression levels to previously measured aggrecan biosynthesis levels, aggrecan synthesis is shown to be transcriptionally regulated by IGF- 1, whereas inhibition of aggrecan synthesis by compression is not transcriptionally regulated.(cont.) Conclusion: Many genes measured are responsive the effects of IGF-1 under 0% compression and 50% compression. Clustering analysis revealed strong co-expressed gene pairings. IGF-1 stimulates aggrecan biosynthesis in a transcriptionally regulated manner, whereas compression inhibits aggrecan synthesis in a manner not regulated by transcriptional activity.by Cameron A. Wheeler.S.M

Using systems biology to investigate how age-related changes in TGFβ signalling alter pro-inflammatory stimuli

PhD ThesisOsteoarthritis (OA) is a degenerative condition caused by dysregulation of multiple molecular signalling pathways. This dysregulation results in damage to cartilage, a smooth and protective tissue that enables low friction articulation of synovial joints. Matrix metalloproteinases (MMPs), especially MMP13, are key enzymes in the cleavage of type II collagen which is a vital component for cartilage integrity. Various stimuli have been identified as inducers of MMP expression such as excessive load, injury and inflammation. Although previously considered a non-inflammatory arthritis, recent research has shown that inflammation may play an important role in OA development. A novel meta-analysis of microarray data from OA patients was used to create a cytoscape network representative of human OA. This enabled the identification of key processes in OA development, of which inflammation was prominent. Examining various different signalling pathways highlighted a role for transforming growth factor beta (TGFβ) in protecting against pro-inflammatory cytokine-mediated MMP expression. Indeed, TGFβ plays key roles in all facets of cartilage biology including development and maintenance of cartilage integrity. With age there is a change in the ratio of two TGFβ type I receptors (ALK1/ALK5), a shift that results in TGFβ losing its protective role in cartilage homeostasis. Instead, TGFβ promotes cartilage degradation and this correlates with the spontaneous development of OA in murine models. However, the mechanism by which TGFβ protects against pro-inflammatory responses and how this changes with age has not been extensively studied. Mathematical modelling has previously revealed how stochastic changes in TGFβ signalling during ageing led to the upregulation of MMPs. I have expanded the TGFβ section of this model to incorporate the pro-inflammatory stimulus interleukin-1 (IL-1) + oncostatin M (OSM) in order to investigate how TGFβ mediates MMP repression, specifically MMP-13. TGFβ signalling appears to interact with the activator protein 1 (AP-1) complex, which has an important role in MMP upregulation. However, the model indicates this interaction alone is insufficient to mediate the full effect of TGFβ, predicting it may also reduce MMP-13 mRNA stability. Furthermore, the model enabled me to predict how age alters these interactions; it suggested TGFβ would provide limited repression with a prolonged inflammatory response. Combining the modelled genes with the microarray network provided a global overview of how alterations in one pathway can affect others and lead to OA development. This study therefore demonstrates the power of combining computational biology with experimentally-derived data to provide insight into the importance of TGFβ signalling, and how age-related changes can lead to cartilage damage and OA development.Centre of Integrated Research into Musculoskeletal Ageing (CIMA), Arthritis research UK and the Medical Research Counci

Osteoarthritis: From Molecular Pathways to Therapeutic Advances

In this book, we have reported the most recent discoveries and updates regarding molecular pathways in osteoarthritis. In particular, the advances regarding therapeutical options, from a molecular point of view, are largely discussed

Cartilage response to in vitro models of injury in combination with growth factor and antioxidant treatments

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2008.MIT Science Library copy: issued as 1 v.Also issued in 1 v. with pagination as pages.Includes bibliographical references.Approximately one in five Americans is affected by arthritis, making it one of the most prevalent diseases and the leading cause of disability in the United States. Post-traumatic arthritis occurs after joint injury (e.g., ACL rupture or intraarticular fracture) and makes up a substantial proportion of the population with arthritis. In previous clinical studies, patients suffering from a traumatic joint injury have shown an increased risk in osteoarthritis (OA), independent of surgical intervention to stabilize the joint. Thus, the early events post-injury have an important effect on tissue within the joint in the long term. To understand the processes involved in the onset of OA and factors leading to OA post-traumatic injury, in vitro models have been developed to isolate components of the complex processes occurring in vivo. While in vitro models do not mimic true physiologic conditions in vivo, by isolating the effects of mechanical compression, cytokine treatment, and cartilage co-cultured with adjacent tissue, in vitro models can give insight into key biological and mechanical pathways occurring in vivo. This study focuses on changes in cartilage gene and protein expression and associated cartilage matrix degradation in response to static or injurious compression of the tissue in the presence or absence of cytokines including TNF-a and IL-6. In addition, normal or injuriously compressed cartilage explants were co-cultured with injured (excised) joint capsule tissue, another in vitro model of post-traumatic cellular behavior. Both young bovine cartilage and human cartilage from a wide range of ages were used. The growth factors insulin-like growth factor-1 (IGF-1) and Osteogenic protein-i (OP-1), as well as the antioxidant, superoxide dismutase mimetic (SODm), were tested to examine if they had the capability to abrogate the negative effects of these injury models.(cont.) Taking a systems approach, the effects of these stimuli on expression of over 48 genes (in cartilage as well as joint capsule) were quantified, along with measures of chondrocyte viability, biosynthesis, protein expression, and GAG loss. Chondrocyte gene expression was differentially regulated by 50% static compression or IGF- 1 treatment or the combination of compression and IGF- 1. Results showed that IGF- 1 stimulated aggrecan biosynthesis in a transcriptionally regulated manner, whereas compression inhibited aggrecan synthesis in a manner not regulated by transcriptional activity. The injury plus co-culture model was examined in detail, and OP-1 and IGF-1 were unable to rescue changes in transcriptional expressions due to injury. However, these growth factors were able to rescue cells from apoptosis, and slightly increase biosynthesis rates. Human tissue was used to further validate the model of mechanical injury (INJ) combined with co-culture (Co). Immunohistochemical analysis of human cartilage explants after INJ+Co treatment revealed changes in versican and aggrecan protein expression, as well as changes in surface tissue morphology, that mimicked certain changes observed in human osteochondral plugs taken from patients at the time of notchplasty surgery (post ACL reconstruction) at 1, 3, or 57 months post- ACL rupture. The oxidative stress involved in a cytokine plus injury model showed that SODm had no ability to selectively diminish protease transcriptional activity. Cartilage treated with this antioxidant showed significant increases in GAG loss to the medium, but diminished levels of chondrocyte apoptosis. Taken together, this work supports further investigation of the mechanisms of action of OP-1, IGF-1, and SODm in order to elucidate their possible therapeutic value, and demonstrates the usefulness of these complementary in vitro models of cartilage injury.by Cameron A. Wheeler.Ph.D

Molecular basis of Osteoarthritis and aspects of cellular senescence in disease

The aim of this study is to investigate on some molecular mechanisms contributing to the pathogenesis of osteoarthritis (OA) and in particular to the senescence of articular chondrocytes. It is focused on understanding molecular events downstream GSK3β inactivation or dependent on the activity of IKKα, a kinase that does not belong to the phenotype of healthy articular chondrocytes. Moreover, the potential of some nutraceuticals on scavenging ROS thus reducing oxidative stress, DNA damage, and chondrocyte senescence has been evaluated in vitro. The in vitro LiCl-mediated GSK3β inactivation resulted in increased mitochondrial ROS production, that impacted on cellular proliferation, with S-phase transient arrest, increased SA-β gal and PAS staining, cell size and granularity. ROS are also responsible for the of increased expression of two major oxidative lesions, i.e. 1) double strand breaks, tagged by γH2AX, that associates with activation of GADD45β and p21, and 2) 8-oxo-dG adducts, that associate with increased IKKα and MMP-10 expression. The pattern observed in vitro was confirmed on cartilage from OA patients. IKKa dramatically affects the intensity of the DNA damage response induced by oxidative stress (H2O2 exposure) in chondrocytes, as evidenced by silencing strategies. At early time point an higher percentage of γH2AX positive cells and more foci in IKKa-KD cells are observed, but IKKa KD cells proved to almost completely recover after 24 hours respect to their controls. Telomere attrition is also reduced in IKKaKD. Finally MSH6 and MLH1 genes are up-regulated in IKKαKD cells but not in control cells. Hydroxytyrosol and Spermidine have a great ROS scavenging capacity in vitro. Both treatments revert the H2O2 dependent increase of cell death and γH2AX-foci formation and senescence, suggesting the ability of increasing cell homeostasis. These data indicate that nutraceuticals represent a great challenge in OA management, for both therapeutical and preventive purposes

USE OF 3D PRINTED POLY(PROPYLENE FUMARATE) SCAFFOLDS FOR THE DELIVERY OF DYNAMICALLY CULTURED HUMAN MESENCHYMAL STEM CELLS AS A MODEL METHOD TO TREAT BONE DEFECTS

This project investigates the use of a tissue engineering approach of an absorbable polymer, poly(propylene fumarate) (PPF) to provide long term mechanical stability while delivering a bioactive material, precultured human mesenchymal stem cells (hMSC) encapsulated in hydrogel, to repair bone defects. Annually over 2.2 million bone grafting procedures are performed worldwide; however, current treatment options are limited for critically sized and load bearing bone defects. Much progress has been made in development of bone tissue replacements within the field of bone tissue engineering. The combination of a polymer scaffold seeded with cells for the eventual replacement by host tissue has shown significant promise. One such polymer is PPF, a synthetic linear polyester. PPF has been shown to be biocompatible, biodegradable and provide sufficient mechanical strength for bone tissue engineering applications. Additionally PPF is able to be photocrosslinked and therefore can be fabricated into specific geometries using advanced three-dimensional (3-D) rapid prototyping. Current technology to culture and differentiate hMSCs into osteoblasts has been enhanced with the development of the tubular perfusion system (TPS). The TPS bioreactor has been shown to enhance osteoblastic differentiation in hMSCs when encapsulated in alginate beads. Although this system is effective in differentiating hMSCs it lacks the sufficient mechanical strength for the treatment of bone defects. Therefore this work suggests a combination strategy of harnessing the ability of the TPS bioreactor to enhance osteoblastic differentiation with the mechanical properties of poly(propylene fumarate) to develop a porous PPF sleeve scaffold for the treatment of bone defects. This is accomplished through four steps. The first step investigates the cytotoxicity of the polymer PPF. Concurrently the second step focuses on designing, fabricating and characterizing PPF scaffolds. The third step investigates the degradation properties of 3D printed porous PPF scaffolds. The fourth step characterizes alginate bead size and composition for use within the PPF sleeve scaffolds. The successful completion of these aims will develop a functional biodegradable bone tissue engineering strategy that utilizes PPF fabricated scaffolds for use with the TPS bioreactor

Liver X Receptor and Retinoid X Receptor in Cartilage Development and Homeostasis

Osteoarthritis (OA) is a heterogeneous and multifactorial degenerative disease characterized by cartilage degradation in the joint. Available treatment options target symptoms but do not address the underlying issue of joint tissue degeneration. A better understanding of the molecular mechanisms maintaining cartilage health is essential for developing novel therapeutic strategies. Previous studies have shown the nuclear receptor Liver X Receptor (LXR) to possess protective roles against cartilage breakdown in OA, however the underlying mechanisms behind this process remain unknown. Since LXR regulates transcription by forming obligate heterodimers with another nuclear receptor, the Retinoid X Receptor (RXR), I hypothesized that LXR and RXR regulate cartilage development, maturation and lipid homeostasis. The first study of this thesis investigated the effect of LXR activation on chondrocyte differentiation in order to elucidate the molecular mechanisms behind LXR’s protection against OA. Three different chondrogenic culture systems were treated with the specific LXR agonist, GW3965, and it was found that LXR activation suppressed chondrocyte hypertrophy, in part through the delay of cell-cycle exit and consequent retention of chondrocyte proliferation. To further identify the intracellular changes that occur to elicit this suppression in hypertrophy, I conducted microarray analysis on growth plate chondrocytes treated with GW3965 in my second study. LXR activation caused differential regulation of various genes involved in lipid metabolism, including central players mediating cellular cholesterol efflux. These findings demonstrate a potential link between LXR’s role in lipid metabolism and the differentiation of developing chondrocytes. Lastly, to gain insight into LXR and RXR’s transcriptional effects in articular chondrocytes, I conducted microarray analysis on immature murine articular chondrocytes (IMACs) treated with either a LXR or RXR agonist. Both LXR and RXR activation differentially regulated cellular lipid metabolism. However, they appear to exert opposing effects on cellular oxidative stress response and maintenance of extracellular matrix (ECM) homeostasis, suggesting contrasting roles in OA progression. Collectively, these data demonstrate that LXR and RXR regulate various cellular mechanisms involved in metabolic homeostasis of both growth plate and articular chondrocytes. Deeper understanding of nuclear receptor function in chondrocytes will contribute to better understanding of the molecular pathways controlling cartilage health and disease

Novel Exogenous Agents for Improving Articular Cartilage Tissue Engineering

This thesis demonstrated the effects of exogenous stimuli on engineered articular cartilage constructs and elucidated mechanisms underlying the responses to these agents. In particular, a series of studies detailed the effects of chondroitinase-ABC (C-ABC), hyaluronic acid (HA), and TGF-β1 on the biochemical and biomechanical properties of self-assembled articular cartilage. Work with C-ABC showed that this catabolic agent can be employed to improve the tensile properties of constructs. When constructs were cultured for 6 weeks, treating with C-ABC at 2 weeks enhanced the tensile stiffness. Furthermore, treating at 2 and 4 weeks synergistically increased tensile properties and allowed compressive stiffness to recover to control levels. Another study showed that combining C-ABC and TGF-β1 synergistically enhanced the biochemical and biomechanical properties of neotissue. Microarray analysis demonstrated that TGF-β1 increased MAPK signaling in self-assembled neocartilage whereas C-ABC had minimal effects on gene expression. SEM analysis showed that C-ABC increased collagen fibril diameter and fibril density, indicating that C-ABC potentially acts via a biophysical mechanism. Constructs treated with C-ABC and TGF-β1 also showed stability and maturation in vivo , exhibiting a tensile stiffness of 3.15±0.47 MPa compared to a pre-implantation stiffness of 1.95±0.62 MPa. To assess the response to HA application, studies were conducted to optimize HA administration and examine its effects in conjunction with TGF-β1. Applying HA increased the compressive stiffness 1-fold and increased GAG content by 35%, with these improvements depending on HA molecular weight, application commencement time, and concentration. Microarray and PCR analyses showed that HA also influenced genetic signaling, up-regulating multiple genes associated with the TGF-β1 pathway. In addition to genetic effects, the enhanced GAG retention due to HA treatment could increase the fixed charge density of the matrix and thereby increase resistance to compressive loading. Additive effects were observed when HA was applied in conjunction with TGF-β1, with the combined treatment increasing compressive stiffness and GAG content by 150% and 65%, respectively. In general, results demonstrated mechanisms underlying C-ABC, HA, and TGF-β1 treatments and showed how these agents can be applied to improve cartilage regeneration efforts

A systems biology approach to musculoskeletal tissue engineering: transcriptomic and proteomic analysis of cartilage and tendon cells

Disorders of cartilage and tendon account for a high incidence of disability and are highly prevalent co-morbidities within the ageing population; therefore, musculoskeletal disorders represent a major public health policy issue. Despite considerable efforts to characterise biochemical and biomechanical cues that promote a stable differentiated cartilage or tendon phenotype in vitro the benchmarks by which progress is measured are limited. Common regenerative interventions, such as autologous cartilage implantation, have a required period of monolayer expansion that induces a loss of the functional phenotype, termed dedifferentiation. Dedifferentiation has no definitive mechanism yet is widely described in both regenerative and degenerative contexts; in addition to stem cell transplantation and cell-seeding in three-dimensional scaffolds, dedifferentiation represents the third approach to the development of regenerative mechanisms for mammalian tissue repair. Cartilage and tendon show a number of common features in structure, develop, disease, and repair. The extracellular matrix is a dynamic and complex structure that confers the functional mechanical properties of cartilage and tendon. Dysregulation of production and degradation are critical to the pathophysiology of musculoskeletal disorders, therefore, reparative interventions require a stable, functional phenotype from the outset. Cartilage and tendon demonstrate a commonality in terms of function defining structure both being sparsely cellular with a preponderance of collagenous matrix. Parity of functionality with the pre- injury state after healing is rarely achieved for cartilage and tendon. Cartilage and  tendon also share common embryological origins. Common mesenchymal progenitor cells differentiate into many musculoskeletal tissues with diverse functions. Specialist sub-populations of tendon and cartilage progenitors enable formation of transitional zones between these developing tissues. The development of musculoskeletal structures does not occur in isolation, however, cartilage and tendon have not previously been considered together in a systems context. An integrated understanding of the differentiation of these tissues should inform regenerative therapies and tissue engineering strategies. Systems biology is paradigm shift in scientific thinking where traditional reductionist strategies to complex biological problems have been superseded by a holistic philosophy seeking to understand the emergent behavior of a system by the integrative and predictive modeling of all elements of that system. Whole transcriptome and proteome profiling studies are used to collect quantitative data about a system, which may then be exploited by systems biology methodologies including the analysis of gene and protein networks. Gene-gene co-expression relationships, which are core regulatory mechanisms in biology, are often not part of a comprehensive gene expression analysis. Many biological networks are sparse and have a scale-free topology, which generally indicates that the majority of genes have very few connections, whilst certain key regulators, or ‘hubs’, are highly interconnected. Co-expression networks may be used to define regulatory sub- networks and ‘hubs’ that have phenotypic associations. This approach allows all quantitative data to be used and makes no a priori assumptions about relationships in the system and, therefore, can facilitate the exploration of emergent behavior in the system and the generation of novel hypotheses. The ultimate goal of tissue engineering is the replacement of lost or damaged cells, and in vitro, to develop biomimetic (organotypic) structures to serve as experimental models. Tissues, and the strategies to functionally replicate them ex vivo, are complex and require an integrated, multi-disciplinary approach. Systems biology approaches, using data arising from multiple-levels of the biological hierarchy, can facilitate the development of predictive models for bioengineered tissue. The iterative refinement, quantification, and perturbation of these models may expedite the translation of well-validated organotypic systems, through legal regulatory frameworks, into regenerative strategies for musculoskeletal disorders in humans. In this thesis the systems under consideration are the major cell populations of cartilage and tendon (chondrocytes and tenocytes, respectively). They are described in three environmental conditions: native tissue, monolayer (two- dimensional), or three-dimensional models. There has been no systematic investigate of the global gene and protein profiles of cartilage and tendon in their native state relative to monolayer or three-dimensional cultures. There is no clear mechanistic description of the impact of in vitro environmental perturbations on the system or indeed the adequacy of these models as proxies for cartilage and tendon. A discovery approach using transcriptomic and proteomic profiling is undertaken to define a robust and consistent gene and protein profile for each condition. Differentially expressed elements are functionally annotated and pathway topology approaches employed to predict major signalling pathways associated with the observed phenotype. This study defines dedifferentiated chondrocytes and tenocytes in monolayer culture as expressing markers of musculoskeletal development, including scleraxis (Scx) and Mohawk (Mkx). Furthermore, there is reproducible synthetic profile convergence in monolayer culture between cartilage and tendon cells. Standard three-dimensional culture systems for chondrocyte and tenocytes fail to replicate the gene expression profile of cartilage and tendon. The PI-3K/Akt signaling pathway is predicted to be the predominant canonical pathway associated with de- and re-differentiation in vitro. Using novel, and publically available, transcriptomic data sets a meta-analysis of microarray gene expression profiles is performed using weighted gene co- expression network analysis. This is employed for transcriptome network decomposition to isolate highly correlated and interconnected gene-sets (modules) from gene expression profiles of cartilage and tendon cells in different environmental conditions. Sub-networks strongly associated with de- and re- differentiation phenotypes are defined. Comparison of global transcriptome network architecture was performed to define the conservation of network modules between a model species (rat) and human data. In addition to the annotation of an osteoarthritis-associated module in the rat a class-prediction analysis defined a minimal gene signature for the prediction of three-dimensional cultures from standard monolayer culture. Finally, proteomic and transcriptomic data sets are integrated by defining common upstream regulators (TGFB and PDGF BB) and unified mechanistic networks are generated for de- and re- differentiation. The studies collected in this thesis contribute to a wider understanding of cartilage and tendon tissue engineering and organotypic culture development. A clear mechanistic understanding of the regulatory networks controlling differentiation of cartilage and tendon progenitor cells is required in order to develop improved in vitro models and bio-engineered tissue that are physiologically relevant. The findings presented here provide practical outputs and testable hypotheses to drive future evidence-based research in organotypic culture development for musculoskeletal tissues