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Affecting over 250 million people worldwide, Osteoarthritis (OA) is the most prevalent musculoskeletal disease and the leading cause of disability among elderly people. An accelerated form of OA, termed post-traumatic OA (PTOA), is triggered by one or more injurious, high impact loading events which result in chondrocyte cell death and a 'phenotypic shift' in residual cells toward a more catabolic state. Conversely, cyclic loading within the physiological range has been shown to have beneficial effects on joint health, with longitudinal patient studies demonstrating a lack of rhythmic mechanical stimulation results in cartilage atrophy. Here, the differences in cartilage tissue response to physiological cyclic and pathological impact loads were examined, to elucidate the mechanosensitive pathways responsible for destabilising chondrocyte homeostasis, driving the initiation of PTOA. Assembly and optimisation of a novel ex vivo porcine loading model, incorporating an ElectroForce 5110 mechanical testing instrument, delivered compressive strains comparable to that seen in the joint, providing physiological cyclic (10% stain) and pathological impact (50% strain) loading regimes for further investigation. Biological readouts including cell death, sGAG release and structural damage demonstrated the catabolic and anti-catabolic effects of these two loading programmes. Subsequent development of a protein extraction protocol and employment of a data-driven discovery proteomics study permitted unbiased examination of the two aforementioned loading regimes and their short-term (< 72 h) global effects on cartilage health. Similarities were observed through a sub-population of indiscriminately mechanosensitive proteins associated with cytoskeletal remodelling, whilst divergence was detected in IL-1, integrin and mTOR signalling pathways, among others. Most notably, this work elucidated the induction of a number of small ubiquitin related modifier (SUMO)-associated proteins via mechanical stress ex vivo, chiefly following impact loading. Further investigation of this stress response pathway was conducted in a human chondrocyte cell line in vitro via co-immunoprecipitation mass spectrometry. A global shift towards deSUMOylation was observed following PIEZO1-mediated calcium influx, with the greatest alteration in protein SUMOylation state associated with RNA processing and the cytoskeleton. Overall, this study demonstrated the effective use of a novel ex vivo mechanical loading model and protein extraction methodology for the proteomic examination of the chondrocyte response to variable mechanical load. These techniques and the identification of a unique mechanosensitive pathway (SUMOylation) aim to further the field of orthopaedic research by progressing our understanding of PTOA disease progression in the hope of identifying new therapeutic targets for the future.

Osteoarthritis (OA) and osteoporosis (OP) are highly prevalent and debilitating conditions that share some common risk factors. Exercise and/or physical activity can be beneficial to both conditions, however, there still remain many unanswered questions regarding how different types, durations and frequencies of exercise/physical activity affect bone and cartilage metabolism and joint health. The aim of this thesis was to examine the role of physical activity and exercise in bone and cartilage physical properties, metabolism, and joint symptoms. The first study of the thesis examined the acute response to a bout of high impact exercise (IE) and a resistance exercise (RE) in healthy young men. Participants completed a control (CON) and IE or RE trial. In the IE trial 120 maximum effort jumps were performed, in the RE trial 120 lifts were performed. Both included rest pauses. Venous blood samples were taken before and immediately and 24 hours after exercise and at corresponding times in the CON trial. The concentration of serum sclerostin ([sSOST]), cartilage oligomeric matrix protein ([sCOMP]) Procollagen I N-Terminal Propeptide ([PINP]) and C-Terminal Telopeptide of Type I Collagen ([CTx-I]) was measured. Repeated measures ANOVAs were used to assess whether there was trial*time interaction. Participants were 26 men (mean (SD) age: 23.4 (2.9) years: body mass index 23.1 (1.7) kg/m²). High forces were recorded during IE; countermovement jump peak force at take-off and vertical ground reaction force were 1.6 (0.3) and 6.2 (2.1) times body weight respectively and one repetition maximums (1RM) for the RE participants were all greater than one times body weight. In the impact exercise trial, changes in [sSclerostin] differed significantly from those in control (p for trial*time interaction = < 0.0001); with the post exercise value being significantly higher than baseline and 24 hours later (+35.6% and +35.6% respectively; both P < 0.05); there was no significant time*trial interaction in the resistance exercise trial (p > 0.05). [sPINP] also differed significantly between exercise and control in the impact exercise trial (p for trial*time interaction = 0.006) with post values being significantly higher than baseline (+11.5% post exercise, p = 0.001). After adjustment for haemoconcentration [sPINP] also changed significantly in the resistance exercise (p for trial*time interaction = < 0.0001) but decreased after exercise (-13.2% p < 0.001). sPIICP and sCOMP also showed different responses between impact exercise and control trials (p for time*trial interaction = 0.030 and 0.049 respectively), with increases of 4.2 and +20.2% immediately after the exercise. Impact exercise was associated with an acute increase in [sSclerostin] which was accompanied by an increase in [sPINP] after impact exercise and a decrease after resistance exercise once adjusted for haemoconcentration. This was also accompanied by changes in [sCOMP] and [sPIICP] however, no change in [sCTx-I]. However, it is uncertain whether an acute increase in [sSclerostin] relates to an increase in synthesis or an increased distribution of pre-existing sclerostin from bone to systemic circulation; future research should examine the underlying mechanism. Study two investigated changes in hip shape after unilateral high impact exercise interventions in older adults and applied a previously developed model associated with OA risk. Dual X-ray absorptiometry scans were analysed using statistical shape modelling, an image analysis technique employing principal component analysis, and statistically derived modes of variation were generated, describing how much each image varies from the mean shape of all images. Femoral neck BMD increased in exercise compared to control leg (p < 0.05). In men there was a non-significant decrease in LHM2 mean score (p = 0.034 before adjustment, p = 0.68 after) possibly indicating a general widening of the femur. MrOSM modes 2, 3 and 4 were negatively associated with OA and 9 positively associated. When applied to our model there were no significant changes in any MrOSM after adjustments were made for multiple comparisons. However, before adjustment there was a decrease in MrOSM 2 in the exercise leg of men (p = 0.007 before adjustment, p = 0.056 after) suggesting a potential detrimental effect of exercise on hip shape, increasing OA risk. Both interventions increased femoral neck BMD and however, had no effect on hip shape after adjustments for multiple comparisons. Unilateral high impact exercise seemed to have limited effect on proximal femur shape despite increasing density. The COVID-19 restrictions in the UK have influenced physical activity engagement and sedentary behaviour due to stay at home orders and the closure of leisure facilities which may have impact upon joint symptoms. Study three was an online survey examining physical activity (Community Healthy Activities Model Program for Seniors (CHAMPS)), sedentary behaviour (Longitudinal Aging Study Amsterdam (LASA)), and joint symptoms (Knee Injury and Osteoarthritis Outcome Score (KOOS)) before and during the UK 2020 COVID-19 lockdown and one year later in adults ≥60 years old. One hundred and eighty-eight participants were included in analysis, 95 completing the follow up survey one year later. There was no significant change in physical activity over lockdown (p>0.05) but a significant decrease in sedentary behaviour during week days and increase during the weekend (p<0.05). These changes in physical activity and sedentary behaviour were accompanied by some small inconsistent changes in joint symptoms (KOOS pain over lockdown and KOOS quality of life (QOL) one year later) indicative of a moderate worsening. In conclusion, participants maintained their physical activity levels over the first COVID-19 lockdown in the UK although sedentary time decreased on weekdays and increased at weekends with some modest worsening of joint symptoms. This research has examined the role of physical activity and exercise on several aspects of bone, cartilage, and joint health. Impact exercise in the form of jumping increased bone signalling molecules sclerostin which was accompanied by increases in bone formation and cartilage metabolism. RE had no effect on sclerostin but decreased bone formation. The first COVID-19 lockdown in the UK did not significantly decrease individuals PA levels as we had hypothesised but did have a small significant effect on sedentary behaviour and some self-reported joint symptoms. Finally, six months of unilateral high impact exercise seemed to have limited effect on proximal femur shape despite increasing density. Overall, this research indicated that physical activity and exercise had a small but mostly positive effect on bone and cartilage physical properties, metabolism, and joint symptoms each being an independent contributor to bone and joint health. Cumulatively these small effects could amount to substantial benefits to bone and cartilage health and future research should incorporate all elements; physical properties, metabolism, and joint symptoms, to investigate the impact of exercise and other lifestyle factors on the joint as a whole.

Osteoarthritis (OA) is a highly prevalent condition that affects more than 40% of individuals over the course of their lifetime. There are currently no disease-modifying OA drug (DMOAD) licensed for the treatment of this disease. An Icelandic genome wide association study (GWAS) study identified common polymorphic variants in ALDH1A2, the gene which encodes the enzyme involved in the synthesis of all-trans retinoic acid (atRA), with severe hand OA. Subsequent data from our lab showed that atRA-responsive genes are downregulated on cartilage injury. Mechanical injury is one of the principal risk factors in the development of OA. Cartilage injury is known to activate downstream inflammatory signalling that is driven in a transforming growth factor activated-beta 1 (TAK1) dependent manner and thereby increase inflammatory gene regulation, in a process known as mechanoflammation. Talarozole (TLZ), a CYP26 inhibitor, which prevents the breakdown of atRA, suppresses the upregulation of inflammatory genes on cartilage injury. My project sought to identify the mechanism by which atRA-responsive genes were downregulated and determine how atRA suppressed mechanoflammation on cartilage injury. The downregulation of atRA-responsive genes on cartilage injury was mediated in both cytosolic phospholipase A2 (cPLA2) - and reactive oxygen species (ROS)-sensitive manner. TAK1 phosphorylation on cartilage injury was partially mediated in a nicotinamide adenine dinucleotide phosphate oxidase (NOX)-sensitive manner, whereas TAK1 was responsible for the partial phosphorylation of cPLA2 on cartilage injury. Apoptosis signal regulating kinase 1 (ASK1) was the key driver of mitogen activated protein kinases (MAPKs) (Jun N terminal kinase (JNK) and extracellular signal regulated kinase (ERK)) on cartilage injury and downstream inflammatory gene regulation. atRA suppressed mechanoflammation in a peroxisome proliferator activated receptor gamma (PPARG)-sensitive manner. These findings suggest that both boosting atRA level and ASK1 inhibition can suppress mechanoflammation in articular cartilage and identifies each pathway as targets for potential disease modifying drugs in the treatment of OA.

MRI plays an important role in the continuing search for a sensitive osteoarthritis (OA) imaging biomarker able to detect early, pre-morphological alterations in cartilage composition. Determining the compositional recovery pattern of cartilage following acute joint loading could potentially present a more sensitive biomarker for defining cartilage health [1]. However, only a limited amount of studies have assessed both the immediate effect of joint loading on cartilage, as well as its post-loading recovery. In addition, when assessing the compositional responses of cartilage to joint loading, previous studies usually did not incorporate the measurement error of the used quantitative MRI technique into their analysis. Therefore, an uncertainty persists whether or not compositional MRI techniques are sensitive enough to measure changes in water and macromolecular content of cartilage, or if previous studies were merely measuring noise. Consequently, an objective of this thesis is to increase our understanding of and reliability in quantitative T2 and T1ρ relaxation time mapping to detect compositional responses of cartilage following a joint loading activity. Furthermore, to obtain the quantitative morphological and compositional measures of cartilage, detailed region-specific delineation of cartilage is required. This delineation (or segmentation) of cartilage is laborious and time-consuming as it is usually performed manually by an expert observer. Many new advances in image analysis, particularly those in convolutional neural networks (CNNs) and deep learning, have enabled a time-efficient semi- or fully-automated alternative to this process [2, 3]. This thesis explores the utility of deep CNNs generated segmentations for accurate surface-based analysis of cartilage morphology and composition from knee MRIs as well as of cortical bone thickness from knee CTs. Chapter 1 will provide an introduction into the structure and biomechanics of articular cartilage and the role of MRI in imaging the degenerative joint disorder, osteoarthritis as well as the effects of different joint loading activities on cartilage morphology and composition. Chapter 2 explains the principle of MRI and the pulse sequences used in the following chapter for the morphometric and compositional assessment of articular cartilage. Chapter 3 describes the use of 3D Cartilage Surface Mapping (3D-CaSM) [3] to assess variations in cartilage T1ρ and T2 relaxation times of young, healthy participants following a mild, unilateral stepping activity. By evaluating and incorporating the intrasessional repeatability of the T1ρ and T2 mapping techniques, I aim to highlight those cartilage areas experiencing exercise-induced compositional changes greater than measurement error. A significant amount of time is needed to manually segment the regions-of-interest required to perform the 3D-CaSM used in Chapter 3. Therefore, in Chapter 4, I assessed the use of deep convolutional neural networks for automating the segmentation process for multiple knee joint tissues simultaneous and increase the time-efficiency for evaluating knee MR datasets. I evaluated the use of a conditional Generative Adversarial Network (cGAN) as a potentially improved method for automated segmentation compared to the widely used convolutional neural network, U-Net. In Chapter 5 I combined the 3D-CaSM and automated segmentation methods presented in Chapters 3 and 4, respectively to assess the use of fully automatic segmentations of femoral and tibial bone-cartilage structures for accurate surface-based analysis of cartilage morphology and composition on knee MR images. This was performed on publicly available data from the Osteoarthritis Initiative, a multicentre observational study with expert manual segmentations provided by the Zuse Institute in Berlin. Chapter 6 describes an automated pipeline for subchondral cortical bone thickness mapping from knee CT data. I developed a method of using automated segmentations of articular cartilage and bone from knee MRI data to determine the periarticular bone surface which is covered by cartilage. This surface was then used to perform cortical bone thickness measurements on corresponding CT data. I validated this pipeline using data from the EU-funded, multi-centre observational study called Applied Private-Public partneRship enabling OsteoArthritis Clinical Headway (APPROACH). Chapter 7 summarises the main conclusions and contributions of the works presented in this thesis as well as providing directions for future work.

Cartilage is a viscoelastic tissue that absorbs shocks and facilitates low friction of the joint. Adult articular cartilage is limited in its ability to self-repair. Lesions or gradual wear-and-tear affect cartilage integrity and lead to damage that, when untreated, can ultimately develop to osteoarthritis. Osteoarthritis is a considerable health burden and is a leading cause of disability worldwide. In order to address this burdensome condition, several treatments have been developed. In particular, therapies that allow delivery of bone marrow and its constituent cell populations to the site of cartilage damage to form a regenerative clot have proved promising in repairing joint cartilage defects. In order to improve such regenerative technique, further research is required to provide a deeper understanding of the mechanisms underlying cartilage regeneration. One of the critical players are the cells that originally reside in the cartilage surrounding the damage. How these resident cells contribute to the activity of cells within the repair tissue at the site of cartilage damage is largely undescribed. In this thesis chondrocytes from three different cartilage areas were compared: chondrocytes from 1) the superficial (SZ) and 2) the middle-deep (MDZ) zone of non-weight bearing femoral condyles, and from 3) the osteoarthritic zone (OAZ) of patients undergoing knee replacement. More inflammatory factors and cytokines are present in MSCs co-culture with OA cartilage chips, and with MDZ cartilage chips. To assess how chondrocyte-MSC crosstalk would affect MSCs chondrogenesis, cartilage chips from the three different zones were co-cultured with BMSC pellets. Results indicated that the SZ induce chondrogenic differentiation of BMSCs, whereas MDZ and OAZ have a negative effect, compared to control conditions. The findings suggest that the presence of SZ, which has been reported to reduce with age, is important to direct BMSCs differentiation towards the chondrogenic fate. In order to better understand the molecular mechanism that differentiate SZ from MDZ and OA chondrocytes, DACTs (Dapper antagonist of catenin) were studied. DACT1 and DACT2 are known to be Wnt and TGFβ pathways regulators, but their role in chondrocytes and MSCs has not been described before. Both proteins are present in chondrocytes throughout the osteoarthritic human tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 expression is localised to the articular surface. In mouse embryos (E.15.5), DACT2 is expressed at the interzone, site of developing synovial joint, indicating that DACT2 is expressed in cells that give rise to the articular joint. Subsequently the expression of DACT1 and DACT2 was analysed in MSCs: both are expressed in synovial and bone marrow-derived MSCs. Following this observation an RNAi knockdown experiment showed that DACT1 knockdown in both chondrocyte and MSCs causes the cells to undergo apoptosis within 24 hours. To understand the pathway regulated by DACT1, next generation sequencing gene expression analysis was performed on BMSCs where DACT1 had been reduced by RNAi. The study showed that loss of DACT1 influences the expression (p<0.05) of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways (Ingenuity® Pathway Analysis). SMURF2 and other genes involved in protein phosphorylation and degradation are downregulated following DACT1 knockdown. This suggests that DACT1 is an upstream regulator of SMAD protein phosphorylation affecting proliferation and survival of MSCs. The data presented in this thesis has indicated that different types of chondrocytes are present within human articular cartilage and that they cross talk with MSCs differently according to their regional origin. This information offers a new level of complexity to consider to improve regenerative techniques. In addition, this work describes for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. DACT1 is involved in MSCs survival and is downregulated in OA which suggest that this is an important regulatory protein. Further studies of DACT1 could help elucidate mechanisms involved in OA, but also uncover the relevance of cartilage progenitors loss in the development of cartilage degeneration.

This thesis examines the potential utility of magnetic resonance (MR) quantitative imaging biomarkers (QIBs) of knee osteoarthritis (OA) for rapid assessment of treatment efficacy in experimental medicine studies. The development of treatments able to modify disease in OA is hampered by an inability to evaluate treatment response over a timeframe relevant to clinical trials. There are particular challenges in the experimental medicine setting due to the small numbers of participants and short follow-up duration relative to the expected time course of OA development and progression. Multiple MR QIBs of OA exist which may help address the problem of early evaluation of treatment response. However, their use in early phase studies has remained limited. Possible reasons for this include incomplete characterisation of the performance of QIBs in this setting and lack of head-to-head comparison of candidate QIBs to determine which would be optimal. This thesis aims to address these shortcomings and provide new information on the likely utility of MR QIBs in the setting of experimental medicine studies, as well as their potential for improving our general understanding of OA pathophysiology. I start by examining the reliability and ability to discriminate between OA and healthy knees of cartilage compositional MR imaging in a systematic review and meta-analysis. I then describe the development and validation of a novel semi-automatic surface-based method for analysing articular cartilage composition and morphology at the knee which may offer improved responsiveness and spatial localisation of change. Moving to QIBs of subchondral bone, I evaluate the association between measures of subchondral bone architecture derived from MR texture analysis and OA progression in the Osteoarthritis Initiative. The remainder of the thesis describes a prospective observational study where the utility of MR QIBs of synovium, subchondral bone and cartilage in experimental medicine studies is assessed. In summary, this thesis will inform decisions regarding the use of MR-based QIBs in future longitudinal and interventional studies. Their inclusion in experimental medicine studies may allow early assessment of treatment efficacy at a structural level and improve efficiency of treatment development pipelines.

Osteoarthritis (OA) is one of the leading causes of morbidity worldwide. Single injurious mechanical impact to joints as seen in sports injury is known to be associated with a high risk of later development of OA, known as post-traumatic OA (PTOA). PTOA affects a younger population of patients and significantly increases the socio-economic burden of the disease on society. Breakdown of articular cartilage in the joint is one of the characterising features of OA. Cartilage is populated with chondrocytes responsible for production and turnover of the matrix. Chondrocyte death is known to occur following impact, suggesting a chondroprotective agent might provide a promising candidate as a disease modifying drug. Urocortin (Ucn), a small peptide member of the Corticotrophin- Releasing Factor (CRF) family, has previously been seen to be essential for chondrocyte survival, where depletion of Ucn led to chondrocyte cell death. This thesis sought to elucidate the mechanism of this chondroprotection by Ucn. Initial chondrocyte cell line and monolayer primary cell studies showed a significant increase in calcium influx when cells were deprived of Ucn. A siRNA screen of candidate ion channels highlighted Piezo1 as being involved in this excessive influx. An ex-vivo model of single impact then was developed to study the effect of overloading on cartilage explants over a short timeframe. Both pre-impact and crucially post-impact addition of Ucn to porcine explants significantly increased chondrocyte survival 72 hrs after challenge. This corresponded to a reduction in excessive calcium influx. Further study with specific mechanosensitive ion channel blockers and activators confirmed Piezo1 as the ion channel modulated by Ucn in order to prevent this calcium overload. Observation of these cells on a subcellular level highlighted a distinct programmed cell death response to impact, although molecular biology techniques indicated this was in the absence of caspase-3 activation. This work has detailed a previously unseen chondroprotective role of Ucn in response to single impact. In particular, the protection observed even when Ucn was added postimpact suggests Ucn as an interesting candidate for further study in a post-impact injury context, thereby limiting chondrocyte cell death early after damage and potentially leading to a reduced incidence of PTOA.

Osteoarthritis is a debilitating condition with an increasing global burden. Regenerative strategies to treat damaged articular cartilage aim to relieve pain and maintain joint function. Procedures for osteochondral repair, such as drilling or microfracture of the subchondral bone, are thought to restore cartilage by delivering bone marrow cells to the defect site. However, the specific identity or contribution of these cells to the repair process is not well understood. Mesenchymal stem/stromal cells are thought to contribute to tissue repair by either progenitor or trophic action. In the bone marrow niche mesenchymal stem/stromal cells (BMSCs) contribute to haematopoietic stem and progenitor cell (HSPC) maintenance and the dynamics of this are disrupted by osteochondral repair procedures. In addition, the bone marrow niche can be further manipulated by the HSPC mobilising agent granulocyte-colony stimulating factor (G-CSF). This cytokine has previously been shown to improve the repair of osteochondral tissues, however, the cellular mechanisms underlying this response are poorly described. This thesis investigates BMSC and HSPC interactions in bone and cartilage repair, and the mechanism by which G-CSF may act to improve repair outcomes. To look at BMSC-HSPC interactions a co-culture system was designed and then used to demonstrate that HSPCs inhibit the differentiation of BMSCs towards osteogenic and chondrogenic lineages. To explore the interaction and role of these cells in vivo during osteochondral repair, a mouse defect model was established. Defects were 729 (± 42.6) µm wide at the articular surface, occupied 41.6 (± 2.5) % of the width of the patella groove and penetrated 1205 (± 58.6) µm into the distal femur. The coefficient of variation (CV) for each parameter was below 6%. The area of injury demonstrated regenerated articular cartilage, an observation not previously described in the typically poor healing C57Bl/6 strain of mice. Further, cells known to respond to G-CSF were seen to have specific spatial profiles in the osteochondral repair tissue over time. For example at 24 hours post-surgery, defect sites were filled with a blood clot within which neutrophils (anti NIMP) were embedded. After 1 week, these cells were no longer present within the injury whilst macrophages (anti-CD68) had infiltrated the wound site having previously been rarely observed at 24hrs. To begin to understand the role of BMSCs in this repair model, Nestin-GFP reporter mice were studied. GFP signal was detected at the defect site at 24 hours after injury. Mice treated with G-CSF showed an increase in Nestin-driven GFP in their spleens. In addition, GFP was detected in the defect of G-CSF treated mice at 4 days post-surgery. To explore the role of G-CSF on BMSCs directly, in vitro techniques were used. These studies demonstrated the presence of a functional G-CSF receptor in the non-haematopoietic bone marrow-derived BMSCs. The impact on MSC cell function was explored, including proliferation and differentiation, showing minimal impact and suggesting a more likely role for G-CSF in the regulation of the trophic activity of BMSCs. The data presented in this thesis further illustrates that the cross talk of multiple bone marrow cell types influences their cell fate. The osteochondral model developed during these studies can be exploited further to investigate the role of individual cell types during repair. In addition, the studies presented here illustrate that the actions of G-CSF are likely exerted on a diverse range of cell types during the repair process and a better understanding of the mechanism of action will allow greater precision, translation and clinical utility.

As the natural turnover level of cartilage and its ability to repair itself are both relatively slow, minor injuries or lesions may lead to progressive damage of cartilage. Unlike other types of tissues that can heal intrinsically, cartilage, being avascular, has a confined supply of nutrients. For the past 25 years, researchers have turned to tissue engineering (TE) in treating cartilage injury. Cells, matrix and signalling molecules are three essential ingredients in TE. An ideal matrix such as biomaterial scaffold would support the cells at the primitive stage of tissue development, in terms of promoting desired phenotype and regeneration of neo-tissues. Physiologically, ~80% of the extracellular matrix of healthy cartilage comprises of water. This makes hydrogels, which are composed of >90% water, the best scaffold candidate to support the cells during cartilage regeneration. Self-assembling peptide hydrogels (SAPH) are designed by embracing the nature of self-assembly in protein folding. SAPHs are seen to have great potential in TE applications; as the properties of peptide fibrils can be controlled by changing peptide sequences, concentration and pH. Other advantages of using SAPH in TE are their biocompatibility, biodegradability, tunability for desired mechanical properties, injectability and they can be functionalised. The primary objective of this thesis was to develop fully chemically and physically defined peptide-based hydrogels that can be used as three dimensional (3D) scaffolds for TE applications. In the present study, we have shown that these peptide hydrogels have different storage moduli depending on the concentration and the pH of the hydrogels. This thesis also discusses the effects of charge in peptide hydrogels on cell viability, proliferation and production of GAGs. It was seen that bovine and human chondrocytes were viable for the duration of the study (21 days). In addition, these charged peptide hydrogels, when cultured without chondrogenic media, were observed to inhibit the production of GAG and type II collagen. In addition, cells were also found to respond differentially to the dynamic mechanical properties of the peptide hydrogels. Cell - material interaction has been shown to play a vital role in cartilage engineering. Different biomaterial properties significantly influence the initial cell attachment to the substrate, which is crucial for chondrogenesis. The interaction of chondrocytes with the SAPH has been explored in this project. We show the effects of using different peptide hydrogels on the attachment, proliferation and spreading of immortalised human chondrocytes. Cell adhesion rate on SAPH was comparable to those on collagen hydrogels. Arginine was seen to induce cells' elongated morphology. This thesis shows that modifying the peptide sequence changes the gelation and degradation behaviour of the SAPH, which affects the cells bio-response due to cells' mechanosensitivity. The work also contributes to the knowledge of substituting phenylalanine with arginine to the pioneering peptide sequence, FEFKFEFK. The study led an inaugural investigation on the interaction between the cells and SAPH.

Articular cartilage plays a vital role in load-bearing joints, providing an almost frictionless surface to articulating bones. However, the avascular nature and low cell density of the tissue means that following injury, there is limited potential for regeneration and repair. With the ageing population, the prevalence and economic burden associated with osteoarthritis (OA) is increasing rapidly, but as of yet there are no fully effective ways to treat the condition. Research into novel therapies has therefore become a popular avenue of investigation, and human mesenchymal stem/stromal cells (hMSCs) have been highlighted as particularly promising targets. However, current, methods for inducing the chondrogenic differentiation of hMSCs, which typically employ the use of transforming growth factor beta 1 or 3 (TGFβ1/3), result in the production of hypertrophic rather than hyaline tissue, hampering translational progress. Growth differentiation factor 5 (GDF5) belongs to the TGFβ superfamily of proteins and is vital for skeletal formation, however its use in cartilage tissue engineering (TE) strategies has been somewhat neglected. Here we demonstrate that GDF5 significantly increases aggrecan gene expression (a marker of articular cartilage), without affecting collagen type X expression (a marker of chondrocyte hypertrophy), in chondrocyte pellet cultures derived from hMSCs, making it a promising target for the formation of permanent articular cartilage. The therapeutic application of growth factors is, at present, limited due to their expense, susceptibility to proteolytic degradation, and rapid clearance, leading to large quantities being required to get anywhere near the desired outcome. The highly sulfated glycosaminoglycan (GAG), heparin, is already extensively used in the clinic as an anticoagulant, and is also able to bind and potentiate the activity of a wide range of growth factors. As such, researchers are now using it to enhance stem cell expansion/ differentiation protocols, as well as to improve the delivery/ activity of growth factors in TE strategies. Here, we identify GDF5 as a novel heparin/heparan sulfate (HS)-binding protein, and show that endogenous HS proteoglycans (HSPGs) are vital for localizing GDF5 to the cell surface, but are not required for its signalling activity. Importantly, we report that clinically relevant doses of heparin (≥ 10 nM), but not equivalent concentrations of HS, inhibit GDF5’s biological activity, in both hMSC-derived chondrocyte pellet cultures, and in the skeletal cell line ATDC5. We demonstrate that these inhibitory effects are due to heparin (but not HS) inhibiting both GDF5 binding to endogenous HSPGs and GDF5-induced induction of Smad 1/5/8 signalling. This study may therefore explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE, and the adverse skeletal effects, such as osteoporosis, that have been reported in the clinic following long-term heparin treatment. Together, our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g. HS derivatives or synthetics), with better-defined structures and fewer off-target effects, if optimal therapy is to be achieved. In the case of GDF5, we have used a variety of developed techniques to begin uncovering important structural and functional information regarding the HS-GDF5 interaction, which are hoped to ultimately pave the way towards achieving this aim. Although further analysis is necessary, our data indicate that relatively long HS sequences are required for binding, and that both ionic and non-ionic interactions play a role in the interaction. In addition we suggest that low- rather than high-affinity HS variants may be key to potentiating the activity of this growth factor.

There is a huge unmet clinical need to treat damaged articular cartilage such as that caused by osteoarthritis (OA) with an estimated 8.75 million people in the UK having sought treatment for OA (ARUK 2013). Embryonic stem cells (ESCs) offer a promising alternative therapeutic approach, potentially providing an unlimited source of chondrocytes capable of regenerating the damaged cartilage however this is limited by the efficiency of the chondrogenic differentiation protocol. An improved understanding of the posttranscriptional regulation of chondrogenesis by microRNAs (miRNAs) may enable us to improve hESC chondrogenesis. Also the recent discovery that miRNAs are selectively packaged into exosomes which can then be transferred to and be functionally active within neighbouring cells suggests they may have a role in cell-cell communication. This project investigated the regulation of miRNA expression in relation to the transcriptome during hESCs-directed chondrogenesis and the possible role for exosomes during differentiation and in stem cell maintenance of hESCs. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis using the Directed Differentiation Protocol (DDP) developed in our lab. Also small RNA-seq was performed on exosomes isolated from hESCs and chondroprogenitors along with the donor cells that the exosomes originated from. This revealed significant changes in the expression of several miRNAs during hESC-directed chondrogenesis including: upregulation of miRNAs transcribed from the four Hox complexes, known cartilage associated miRNAs and the downregulation of pluripotency associated miRNAs. Overall miRome and transcriptome analysis revealed the two hESC lines exhibited slightly different miRome and transcriptome profiles during chondrogenesis, with Man7 displaying larger changes in miRNA and mRNA expression as it progressed through the DDP suggesting it may be more predisposed to undergo chondrogenesis. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified four key functionally related clusters of co-expressed miRNAs and protein coding genes: pluripotency associated cluster, primitive streak cluster, limb development cluster and an extracellular matrix cluster. Further investigation of these gene/miRNA clusters allowed the identification of several potential novel regulators of hESC-directed chondrogenesis. In accordance with the reported literature the exosomal miRNAs from hESCs and hESC-chondroprogenitors were enriched with a guanine rich motif. Notably, several of these were enriched with targets associated with embryonic skeletal system development suggesting they may play a role in regulating differentiation. Preliminary functional experiments examining pluripotency-associated exosomes suggests they may have a role in regulating hESC stem cell maintenance. However the molecular mechanism by which this is achieved has not been investigated. This research identified main miRome and transcriptome changes during hESC-directed chondrogenesis leading to the identification of several potential novel regulators of chondrogenesis and pluripotency which can be further investigated. This project has also highlighted the potential of exosomal miRNAs to regulate hESC stem cell maintenance and differentiation.

A hallmark of the joint disease osteoarthritis (OA) is the degradation of the articular cartilage in the affected joint, debilitating pain and decreased mobility. At present there are no disease modifying drugs for treatment of osteoarthritis. This represents a significant, unmet medical need as there is a large and increasing prevalence of OA. Using a systems biology approach, we aimed to better understand the pathogenic mechanisms of OA and ultimately aid development of therapeutics. This thesis focuses on the analysis of gene expression data from human OA cartilage obtained at total knee replacement (TKR). This transcriptomics approach gives a genome-wide overview of changes, but can be challenging to interpret. Network-based algorithms provide a framework for the fusion of knowledge so allowing effective interpretation. The PhenomeExpress algorithm was developed as part of this thesis to aid the interpretation of gene expression data. PhenomeExpress uses known disease gene associations to identify relevant dysregulated pathways in the data. PhenomeExpress was further developed into an 'app' for Cytoscape, the widely used network analysis and visualisation platform. To investigate the processes that occur during the degradation of cartilage we examined the gene expression of damaged and intact OA cartilage using RNA-Seq and identified key altered pathways with PhenomeExpress. A regulatory network driven by four transcription factors accounts for a significant proportion of the observed differential expression of damage-associated genes in the PhenomeExpress identified pathways. We further explored the role of the cytokines IL-1 and TNF that have been reported to β drive the progression of OA. Comparison of the expression response of in vitro cytokine-treated explants with the in vivo damage response revealed major differences, providing little evidence for any significant role of IL-1 and TNF as drivers of OA β damage in vivo. Finally, we examined the heterogeneity of OA through analysis of cartilage expression profiles at TKR. Through a network-based clustering method, we found two subgroups of patients on the basis of their gene expression profiles. These subgroups were found to have distinct OA expression perturbations and we identified TGF and S100A8/9 β signalling as potentially explaining the observed differential expression. We developeda RT-qPCR based classifier that allowed classification of new samples into these subgroups so allowing future assessment of the clinical significance of these subgroups. The work presented in this thesis includes a novel, widely-accessible tool for the analysis of disease gene expression data, which we used to give new insights into the pathogenesis of osteoarthritis. We have produced a rich dataset for future research and our analysis of this data has increased our understanding of cartilage damage processes and the heterogeneity of OA.

Rib and costal cartilage fractures are the most common injuries resulting from blunt thoracic loading scenarios, including motor vehicle collisions. The costal cartilage is a cylindrical hyaline cartilage composed of two layers: a core interstitial matrix enveloped by the perichondrium. The perichondrium itself has an inner chondrogenic layer and an outer fibrous layer. The objective of this study was to evaluate the tensile material properties of human costal cartilage perichondrium at two loading rates for a range of subject demographics. Fifty-six (n=56) samples containing the fibrous layer and chondrogenic layer (i.e., two-layered samples) were fabricated from thirty-three (n=33) donors aged from 11 to 69 years of age (19 M, 14 F). Thirteen (n=13) samples without the fibrous layer (i.e., one-layered samples) were fabricated from eight (n=8) donors aged from 11 to 54 years of age (5 M, 3 F). The perichondrium was isolated from the interstitial matrix for all samples and the fibrous layer was removed for one-layered samples to assess the effect of the absence of the fibrous layer. The tissue was then stamped into a dog bone-shaped coupon and sanded down to a uniform thickness of ~1.3 mm for two-layered samples and ~1 mm for one-layered samples. The gage length of the completed coupons was marked with a black ink dot pattern to facilitate strain calculations via video tracking. The coupons were loaded axially in tension to failure at either a slow (0.005 s⁻¹) or fast (0.5 s⁻¹) target loading rate using a material testing system. The elastic modulus, ultimate stress, ultimate strain, failure stress, failure strain, and strain energy density (SED) were then calculated for each test. Material property data were compared by sample type and loading rate. Since there was no significant influence of sex on any material properties, the data were grouped together for the analysis. Modulus, ultimate stress, failure stress, and SED were found to significantly decrease with donor age at both loading rates and ultimate and failure strain also significantly decreased with donor age at the 0.5 s⁻¹ target loading rate. Failure stress in the two-layered samples was found to be greater than that of the one-layered samples at both loading rates. One-layered samples had a greater failure strain than two-layered samples at both loading rates. Perichondrium data were compared to interstitial matrix data from a previous study to further investigate the role of cartilage layer on material properties. The modulus, ultimate stress, and failure stress of costal cartilage decreased moving radially inward (greatest in two-layered perichondrium samples, least in interstitial matrix samples). The opposite was true for ultimate and failure strain, with the greatest failure strain values occurring in the interstitial matrix and the least in the two-layered perichondrium samples. The sample size of one-layered samples was too small to draw any substantial conclusions regarding age trends. This was the first study to analyze the material property trends in costal cartilage perichondrium. The results of this study can be incorporated into virtual human body models to improve the accuracy of thoracic injury prediction in the context of motor vehicle safety.

During open orthopaedic surgical procedures, the articular cartilage covering the exposed joint surfaces can be exposed to air for prolonged periods. This exposure facilitates cartilage drying, characterised by changes to the extracellular matrix and chondrocyte death. Due to cartilage’s limited capacity for regeneration, it has been proposed that this may lead to post-operative joint degeneration. It has been proposed that chondrocyte death occurs as a result of both drying and nutritional deficiency. It is known that death during drying correlates with the drying interval and is initiated in the superficial chondrocytes, progressing to deeper layers at higher intervals. Additionally, it is well established that periodic rewetting (e.g. using 0.9% saline solution) can reduce chondrocyte death. The work presented in this thesis aimed to characterise chondrocyte death during drying, with particular reference to the chondrotoxic/protective effect of environmental variables (airflow) and surgical interventions (irrigation solutions), mechanism of injury and cell death, and the effect of in vivo drying on joint health. An ex vivo model of cartilage drying was developed and carried out on bovine and human intact cartilage and osteochondral explants, while varying environmental factors (drying interval, airflow velocity, oxygen concentration) and interventions (irrigation solutions and protective coverings. Throughout the study, cartilage drying was assessed in terms of 1) cartilage macroscopic appearance, 2) percent chondrocyte death (PCD), 3) cartilage water content, and 4) chondrocyte morphology. Histologically and fluorescently labelled samples were imaged using light and confocal laser scanning microscopy respectively, which formed the basis of the qualitative and quantitative assessments. Experimental drying at high airflow velocities had a more severe effect on cartilage appearance, PCD, and water content than in static air. This relationship was apparent in dried intact joints and osteochondral explants and in bovine and human samples. This suggests that the effects of surgical drying (where ventilation systems and airflow are routine) may be more pronounced than previously suggested and demonstrates a correlation between PCD and water-loss. Irrigation solutions supplemented with glucose (25-100 mM) had no significant effect on the PCD or water content in dried samples. Additionally, PCD was minimal in osteochondral explants cultured in the absence of glucose, even after 24 hr. This suggests that nutritional deficiency is unlikely to contribute to PCD during drying. However, chondrocyte death (in intact bovine cartilage) was reduced when drying was carried out at an oxygen concentration more reflective of the in vivo environment (5 %), which suggests that cell death during drying may be facilitated by a hyperoxic shock. Finally, in vivo cartilage drying was carried out on murine cartilage. Compared to sham operated controls, dried cartilage demonstrated a loss of surface integrity (4 weeks post-surgery) and fibrillations (8 weeks) and an increased modified Mankin score (at 4 and 8 weeks). Microscopically, an altered cartilage thickness, and chondrocyte density and arrangement were visible. These changes are comparable with changes in osteoarthritis. The results of this study demonstrate the importance of maintained cartilage hydration in order to avoid unnecessary chondrocyte death articular cartilage degeneration.

Osteoarthritis (OA) is a common disease with a strong genetic component. Despite this, previous attempts to identify genetic variants that predispose to OA have met with limited success. Recently, the results of a large genome wide association study in OA has identified a novel susceptibility locus on chromosome 3 tagged by two SNPs, rs11177 (p=1.25x10-10) which lies within the coding region of GNL3 and rs6976 (p=7.24x10-11) situated in the 3’UTR of GLT8D1. The GNL3 gene encodes the protein nucleostemin which is found within the nucleolus of stem cells and tumour cells. It functions to regulate cell cycle progression, embryogenesis, tumorigenesis, tissue regeneration and ribosome biogenesis but its role in the joint is unknown. In an attempt to identify the causal variant(s) at locus 3p21.1 I conducted a mutation screen of GNL3 which identified a common non-synonymous coding variant, rs2289247, which was in strong LD (r2=0.92) with rs11177, as well as several other variants. Localisation studies showed that GNL3 was expressed at the mRNA and protein level in several joint tissues. While levels of mRNA expression were found to be significantly higher in human articular chondrocytes from OA patients as compared with controls, levels of GNL3 protein were significantly lower in OA chondrocytes than controls. Further studies showed that cytokines which have been implicated in the pathogenesis of OA such as IL1β, IL13, TNFα and FGF2 had no effect on GNL3 mRNA in cartilage. Knockdown of GNL3 using siRNA in articular chondrocytes and the chondrosarcoma cell line, JJ012, did not alter the mRNA expression of chondrogenic markers; COL2A, ACAN, MMP3, MMP13, RUNX2 and SOX9. Cultures of mesenchymal stem cells and articular chondrocytes from patients of different rs11177 genotypes, showed no difference in chondrogenic potential. Furthermore, genotypes at rs11177 and rs2289247 did not influence the expression of p53, MDM2 or GNL3 in response to stressful stimuli, including cisplatin and hypoxia, when cloned into a melanoma cell line. Studies of zebrafish carrying a loss of function mutation in gnl3 revealed a significant reduction in cartilage volume and an alteration in cartilage structure, as evident by a reduced number of chondrocytes, disorganised stacking and an increase in cartilage extracellular matrix in the mutant fish. This research has shown that gnl3 plays a vital role in chondrogenesis in zebrafish and has shown evidence of dysregulation of GNL3 expression in OA human articular chondrocytes. The in vitro studies failed to identify any specific effects of the variants rs11177 and rs2289247 on GNL3 expression, chondrogenesis or p53 stress response although, it remains possible that the variants may have modest effects that were not detected by the assays used. The zebrafish studies illustrate that gnl3 plays a critical role in normal cartilage development however further studies on GNL3 in OA would be of interest.

Septic arthritis has the potential to be a highly destructive joint disease. Although numerous bacterial species are capable of inducing septic arthritis, Staphylococcus aureus is most commonly implicated, accounting for up to 65% of cases. Whilst this organism is known to produce a diverse array of potential virulence factors, studies investigating a variety of S. aureus-related infections have implicated alpha(Hla)-, beta(Hlb)- and gamma(Hlg)-haemolysins as key damaging toxins, with the ‘pore-forming’ Hla considered to be the most potent. The work presented in this study focused on gaining further insight into the interaction between S. aureus toxins and in situ chondrocytes during an episode of septic arthritis. An in vitro bovine osteochondral explant model of S. aureus-induced septic arthritis was developed in this study. Utilising fluorescence-mode confocal laser scanning microscopy (CLSM), the model, which avoided the complexities of a host immune response, permitted an assessment of the following: (1) the spatial and temporal quantification of in situ chondrocyte viability following exposure to both a laboratory ‘wild-type’ (S. aureus 8325-4) and clinical strains of S. aureus; (2) the influence of Hla, Hlb and Hlg on in situ chondrocyte viability through the use of specific ‘haemolysin-knockout’ mutant strains; (3) the influence of altered culture medium osmolarity and extracellular Ca2+ on Hla-induced in situ chondrocyte death; and (4) dynamic changes in intracellular Ca2+ within in situ chondrocytes following Hla exposure. S. aureus 8325-4 and S. aureus clinical strains rapidly reduced in situ chondrocyte viability ( > 45% chondrocyte death at 40hrs). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) of cartilage and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins (achieved with the use of subchondral bone-free explants) demonstrated that SZ chondrocytes were more susceptible to the toxins than DZ chondrocytes. When explants were cultured in the presence of a selection of isogenic S. aureus mutants, with varying Hla, Hlb and Hlg production capabilities (all originating from S. aureus 8325-4), Hla-producing mutants induced significant in situ chondrocyte death compared to toxin deficient controls (Hla-Hlb-Hlg-). In contrast, mutants producing Hlb and Hlg in the absence of Hla were unable to induce significant chondrocyte death. Hla alone was therefore identified as the key damaging toxin to in situ chondrocyte viability. Raised culture medium osmolarity had no influence on Hla-induced in situ chondrocyte death. In the absence of Hla, a high extracellular Ca2+ concentration (20mM) had no influence on chondrocyte viability during the experimental period. Hla-induced chondrocyte death increased in the presence of raised extracellular Ca2+ concentrations thereby confirming a role of Ca2+ in the chondrocyte death pathway. There was no significant difference between S. aureus growth in high and low Ca2+ culture media. Finally, when live osteochondral explants stained with the Ca2+-sensitive fluorophore Fluo-4 were cultured with an Hla-containing S. aureus supernatant (S. aureus 8325-4 (Hla+Hlb+Hlg+)) there was a significant rise in intracellular Ca2+ in comparison to those explants exposed to a non-Hla-containing supernatant (S. aureus DU5938 (Hla- Hlb-Hlg-)). The Hla-induced Ca2+ transients were always followed by chondrocyte death. Thus, it is likely that Hla-induced chondrocyte death was associated with a rise in intracellular Ca2+. These findings are of translational relevance. Firstly, toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes, thereby advocating the prompt and thorough removal of bacteria and their toxins during the treatment of septic arthritis. Secondly, the identification of Hla alone as the key damaging toxin to in situ chondrocyte viability, with its destructive action being associated with a rise in intracellular Ca2+, may enable the development of future targeted therapeutic strategies in order to reduce the extent of cartilage destruction during and after an episode of septic arthritis.

Post mortem interval (PMI – the time elapsed since death and discovery) is important to medicolegal investigations. It helps to construct crucial time lines and assists with the identification of unknown persons by inclusion or exclusion of a suspect’s known movements. Accurate methodologies for establishing PMI are limited to about 48-hours. Such methods involve use of increasing levels of potassium in vitreous humour, and algor mortis. This study is two-fold. Firstly, it explores the biomolecular changes in degrading porcine cartilage buried in soil environments and its potential to determine PMI in the crucial two days to two months period. Trotters were interred in a number of graves at two distinct locations exhibiting dissimilar soil environments. Weekly disinterments (for 6 weeks) resulted in dissection for cartilage samples which were processed for protein immunoblot analyses and cell vitality assays. Results demonstrate that aggrecan, a major structural proteoglycan, produces high (230kDa) and low (38kDa) molecular weight cross-reactive polypeptides (CRPs) within cartilage extracellular matrix. The 230kDa CRP degrades in a reproducible manner irrespective of the different soil environments utilised. As PMI increases, aggrecan diminishes and degrades forming heterogeneous subpopulations with time. Immunodetection of aggrecan ceases when joint exposure to the soil environment occurs. At this time, aggrecan is metabolised by soil microbes. The molecular breakdown of cartilage proteoglycans has potential for use as a reliable indicator of PMI, irrespective of differing soil environments, beyond the 48-hours period. Likewise, vitality assays also demonstrated viable chondrocytes for as long as 35 PM days. The second component of this study examined the fungal activity associated with trotters buried below ground. Results indicate that fungal growth was considerably influenced by soil chemistry and changes in the environment. Fungal colonisation did not demonstrate temporal patterns of succession. The results of this study indicate that cartilage has the potential to prolong PMI determination well beyond the current 48- and 100-hour limitations posed by various other soft tissue methods. Moreover, the long-term post mortem viability of chondrocytes presents an opportunity to explore DNA extraction from these cells for the purpose of establishing a positive identification for unidentified remains. On the contrary, the growth and colonisation patterns of post putrefactive fungi in relation to decomposing porcine trotters proved to be futile for estimating PMI. Therefore, fungi may not be a suitable candidate for evaluating PMI during the early phase fungal activity.

The application of prolonged mechanical loading to cartilage alters the osmolality of the extracellular environment, with osmotic challenge known to alter the gene expression and the metabolic activity of chondrocytes. However, the mechanisms by which osmolality controls chondrocyte activity remain unclear. Previous study on various cell types, including chondrocytes, showed that hyper-osmotic challenge induces the condensation of chromatin, with highly condensed chromatin often associated with gene poor regions of DNA and gene silencing. The present study investigated the effect of osmotic challenge on chromatin organisation, genome wide gene-expression and the cellular and nuclear deformability of chondrocytes. In order to observe a broad effect of osmotic challenge on the nuclei, the chondrocytes were subjected to a range of hypo- and hyper-osmotic challenge and imaged by confocal microscopy. Chromatin condensation was quantified by the Sobel edge algorithm in MATLAB. Hyper-osmotic challenge on chondrocytes induced an increase in chromatin condensation. Interestingly, the most marked condensation occurred within the osmolality range of articular cartilage in vivo. The effect of osmotic challenge varied between the monolayer cultured and agarose seeded chondrocytes, which may be due to the differences in cytoskeleton organisation between the two culture conditions. Additionally, chromatin condensation induced by hyper-osmotic challenge was shown to be reversible. Marked differences were observed in the deformability of the cell and nucleus in chondrocytes post osmotic challenge, compared to the 300 mOsm/kg conditions typically used for in vitro isolated chondrocyte studies. From the microarray study, the application of 500 mOsm/kg for both 1 and 5 hours altered the gene expression, including the expression of histone related genes, with a higher number of genes affected by the 5 hours hyper-osmotic challenge. The findings of this study suggest that osmotically-induced alterations in nuclei morphology and chromatin structure may provide a direct biophysical mechanism that controls chondrocytes activity.

Chondroplasia, the development of cartilage-like characteristics in tendinous tissue, is a form of degeneration found in tendons including those of the rotator cuff. The molecular mechanism of its development is currently unknown. An examination of the features of the torn rotator cuff and the cartilage literature led to the identification of several potential drivers of chondroplasia including cell shape change/actin cytoskeleton and hypoxia. Lovastatin caused actin cytoskeleton disruption and promoted cartilage matrix deposition in the ATDC5 model. It was the most effective member of a panel of cytoskeletal inhibitors, increasing expression of the chondrocytic markers Sox5 and Sox9 and decreasing expression of COL1A1 and COL3A1 in primary human tenocytes. Its effects were dose dependent, reversible by mevalonate addition and long term treatment induced de novo expression of collagen II. Short term hypoxia upregulated VEGF-A and chondrocytic marker gene DEC1 expression but not other chondrocyte markers. Combination treatment with hypoxia did not enhance the effects of lovastatin. These data suggest that modulation of pathways that regulate the actin cytoskeleton and cell shape may alter tenocyte phenotype.

Determining the postmortem interval (PMI) remains one of the most important but challenging factors to establish in a suspicious death investigation. Unfortunately, as time passes current methods lose accuracy and only allow investigators to approximate how long ago death occurred. Bodies interred in clandestine graves prove particularly challenging due to an abundance of variables that need to be taken into consideration. Due to the problems associated with determining the PMI of buried remains this study will utilise macroscopic, microscopic, molecular, chemical and microbiological analyses to systematically document the decompositional changes to human hair and porcine cartilage and bone in a burial environment. The aim was to correlate decompositional changes with time and develop new methods for estimating the PMI of remains found in this context. Whole trotters (from which the cartilage was harvested) exhibited decompositional changes including darkening of the dermis, skin slippage, liquefaction of soft tissues and complete skeletonisation. The decompositional changes to cartilage included a loss of cartilage covering articular facets, changes in colour and texture, formation of orthorhombic crystals, a change in surface pH and colonisation by bacteria. The bacteria found on the cartilage surface were in close proximity to the crystals and when cultured on a B-41 medium were found to precipitate crystals of the same morphology and chemical composition to those found on the cartilage surface. Three species of bacteria (Acinetobacter calcoaceticus, Acinetobacter iwoffii and Grimontia hollisae) were identified based on gas chromatography–mass spectrometry (GC-MS) of their fatty acids and one species (Comamonas sp.) was identified by DNA analysis. Formation of crystals on goat and cow cartilage proved that this was not a porcine specific phenomenon. Human hair exhibited a gradual degradation over time but this was dependent on the characteristics of the burial environment. Decompositional changes included colonisation by fungi, erosions to the cortical surface and formation of tunnels and breaks to the hair shaft. Two fungal species (Aspergillus fumigatus and Penicillium sp.) were identified based on DNA analysis of fungal ribosomal (rDNA) internally transcribed spacer (ITS) regions. The Penicillium sp. was linked with fungal tunnelling of hair. Bone exhibited little modification over time but changes were observed. These included a change in colour of the cortical surface, a change in colour and gradual loss of bone marrow and erosions, cracking and flaking of the cortical bone. Fungi were found to colonise both the bone marrow and bone surface. Whole piglets were buried to document the time period taken to reach skeletonisation. This data was used as a correction factor and combined with the bone results to give an overall time period for the decomposition changes observed. The results of this study suggest that the decompositional changes to cartilage could be used to determine the postmortem interval of buried remains. However, the degradation of hair and bone was too variable to be of use in this context.

The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

Tissue regeneration, once a dream or a fantasy, is now becoming a reality. Science has advanced so much that cells can be cryopreserved and expanded into millions under laboratory conditions. Cells can be manipulated to become something they were not before and can be coerced into forming actual tissues. The discovery of powerful growth factors and the ability to synthesize them, helps to achieve these goals. Our understanding of pathologic processes and aging also plays an essential role in our ability to regenerate tissues. Laboratory methods have advanced immensely, allowing more efficient and rapid progress in regenerative medicine. This thesis addresses the regenerative efforts of two types of tissues: cartilage and skin. These tissues have an essential function in our bodies, but their structure and biology are dramatically different. Yet the common denominator is that people can benefit greatly from learning more about the regeneration of these tissues.Cartilage is a shock-absorbing tissue lining the ends of diarthrodial joints that aims to provide a frictionless surface for joint articulation. In disease, damage of articular cartilage is often accompanied by tissue thinning and an irreversible yet progressive degeneration. Cartilage’s innately poor regenerative capacity and progressively increasing demand for its regeneration have resulted in astonishing developments and achievements in the field of tissue engineering (TE) and regenerative therapies. However, despite advancements in cartilage tissue engineering and current clinical transplantation practices, the ability to replace deficient or damaged cartilage remains limited. In response, researchers are urgently trying to develop novel and clinically applicable solutions for the treatment of cartilage degenerative disorders.The translation of cartilage tissue engineering practices is hindered by the lack of suitable cell sources and implant integration with surrounding native tissue. The lack of implant integration is further heightened within the joint environment, where constant motion and loading generate tensile, compressive, and shear forces. The lack of native tissue-graft integration and long-term efficacious cartilage implantation suggest that further exploration is needed to see if immunology is playing a role in the lack of tissue integration. In addition, the quality of cartilage tissue generated varies greatly with different cell sources. Cartilage replacement therapies primarily utilize chondrocytes (cartilage cells) as their main cell source for generating cartilage tissue. Moreover, the chondrocytes can be sourced from multiple anatomical locations. The current harvesting procedure to extract chondrocytes for cartilage tissue engineering can be quite limited and invasive. Therefore, the exploration of different cell sources or chondrocyte harvesting locations would be beneficial for improving cartilage tissue engineering techniques. Another indicator of cartilage tissue quality is the mechanical durability of the tissue to withstand daily forces. A current limitation of cartilage tissue engineering practices is the inability to generate tissues of sufficient biomimicry to that of native tissue. Previous attempts in our laboratory have shown that tissue engineered cartilage is inferior to native tissue in terms biochemical composition and mechanical durability. Our laboratory has explored several applications and interventions to improve the quality of tissue engineered cartilage. This discrepancy between native tissue and tissue engineered cartilage is also due to our lack of understanding of cartilage biomechanics and limiting testing modalities. Tissue regeneration is not limited to cartilage tissue. The demand for skin regeneration primarily aims to repair wounds caused from burns, infections, or other reconstructive surgeries. However, skin regenerative therapies are limited in the availability of sufficient autograft tissue. In addition, facilitating internal regeneration is difficult due to deficient vascularity within the wound. With this approach, wound closure may be delayed or improper healing. Improper healing could lead to scarring or deformity which could lead to mental adversities. Researchers may have found a speedy and miraculous therapy to facilitate wound closure and wound vascularization: fish skin bandages. The science of this therapy has yet to be explored in a comprehensive fashion until now.Chapter 1 of this dissertation provides a concise review of cartilage immunology at the tissue and cellular level as it relates to immune privilege. It covers the current cartilage replacement therapies and how the lack of efficacy of each one could be explained by chondrocyte expression of the major histocompatibility complex. The chapter summarizes the current arguments and evidence supporting and refuting the immune privilege status of cartilage. The chapter also outlines the immunomodulatory mechanisms seen in other immune-privileged tissues and cancer. It concludes with a provision of how current methodologies to manipulate immune recognition could be beneficial to cartilage replacement therapies. Chapter 2 presents a comprehensive study that accomplishes the following: 1) It explores the immunogenicity of chondrocytes. 2) It explores a novel strategy for evading immune detection of allogeneic chondrocytes through genetic editing. 3) It constructs genetically engineered immune-universal tissue-engineered cartilage constructs. 4) It evaluates the safety and efficacy of genetically engineered chondrocytes as a cell source for tissue-engineered cartilage implants.Chapter 3 explores auricular chondrocytes as a cell source for cartilage tissue engineering. This chapter first outlines the current sources of cartilage tissue engineering and the disadvantages. It next uses previously published methods to construct cartilage tissue constructs from auricular chondrocytes. Upon completion, the tissue was evaluated mechanically and biochemically to determine its credibility as a sufficient as a cell source to construct durable cartilage tissue. Chapter 4 explores the biomechanical properties of canine articular cartilage across age using an in-situ testing model. It provides extensive characterization of biochemical composition-mechanical function relationships of canine articular surfaces and presents a comparison of these values across different joints. Specifically, it reports the instantaneous modulus at 15% strain, thickness, collagen content, glycosaminoglycan content, cellularity, and histological changes across age. Also, this chapter provides an innovative, quantitative, and visual assessment of regional distribution of mechanical properties of cartilage using 3D mapping of each surface. This chapter suggests that additional unknown factors may play a role in influencing cartilage’s mechanical properties.Chapter 5 explored the feasibility of using fish skin bandages as a therapeutic option for third-degree burns. The study induces third-degree burns on mice and treats the burns with either tilapia-skin or hydrocolloid adhesive bandages. The study explored the therapeutic efficacy of fish-skin bandages vs hydrocolloid adhesive bandages at the histologic, hematologic, molecular, and gross level. This study may seem out of place with the other chapters. However, this chapter reflects on how to induce internal tissue regeneration using external therapeutics. By studying the healing mechanisms behind tilapia skin, perhaps these factors can be applied to cartilage and induce tissue regeneration in vivo. In summary, this dissertation delineates the cumulative progression of studies providing robust techniques methodologies for tissue regeneration and durability following transplantation. This work provides a solid foundation for future assessment directed at full tissue replacement conditions. Furthermore, this platform can be optimized for regeneration of other tissue structures, such as skin.

Arthritis is a pathological condition whereby a persistent inflammatory response leads to breakdown of articular cartilage in synovial joints. Cartilage is a specialised avascular tissue containing chondrocytes embedded in an extracellular matrix. The cartilage matrix is composed of collagen to provide strength with aggregated proteoglycan to facilitate hydration. Cartilage has been reported to loose proteoglycans with concordant loss of integrity observed in arthritic disease pathology. Proteoglycans loss from cartilage has also been reported in in vitro models. Application of interleukin-1 (lL-1β) to cartilage in vitro has been demonstrated to increase loss of proteoglycans and modulate production of inflammatory mediators such as Nitric Oxide (NO) and Prostaglandin-E2 (PGE2). NO and PGE2 have also been associated with cartilage breakdown. Other cytokines such as colony stimulating factors (CSFs) may regulate cartilage function. The aim of this study was to select a cartilage explant system and compare the effects of interleukin-1 (lL-1) with those of colony stimulating factors (CSFs) by measuring the production of NO and PGE2 and release of proteoglycans. It was found that IL-1β increased PGE2 and NO production, but not loss of proteoglycans from rat cartilage explants. Granulocyte-CSF (G-CSF) and lL-3 increased production of NO and PGE2, respectively. When combined, IL-1β / Granulocyte-Macrophage (GM-CSF) increased production of PGE2 and G-CSF / IL-1β produced increased proteoglycan loss from explants. The model was then modified by integrating Swiss 3T3 Fibroblasts monolayers with explants. Fibroblasts were initially screened to determine their separate response to these cytokines. Fibroblasts did not release proteoglycans into the culture media, but produced elevated concentrations of NO and PGE2 in response to IL-lβ.·Fibroblast-cartilage co-cultures treated with IL-lβ produced increased NO, PGE2 and proteoglycan release. G-CSF, GM-CSF and IL-3 caused increased levels of PGE2 in co-cultures, however, IL-1β was required to generate significant proteoglycan loss from cartilage explants. Finally, extra-cellular signal related protein kinases I and 2 (ERK 1&2) and p38 intracellular signalling pathways were shown to be involved in IL-1β mediated production of NO fibroblasts and explants. These studies show that IL-1β has increased potential to mediate cartilage breakdown when interacting with other cytokines, such as G-CSF, and other cell types, such as Swiss 3T3 fibroblasts. IL-1β has defined intracellular signalling pathways that may produce a range of responses in cartilage explants and fibroblasts. These studies may relate to production of inflammatory processes and loss of cartilage integrity and function in pathological conditions.

Tissues are often complex heterogeneous systems, where individual cells coordinate responses to internal and external stimuli. To fully understand cell function in such environments, it is critical to connect the behavior of individual cells with the full tissue-scale response. Articular cartilage is one such system where the complex extracellular matrix and heterogeneous cell responses make it difficult to understand how chondrocytes respond to injury-inducing strain. In this thesis I will explore the methods I developed to map the spatiotemporal behaviors of chondrocytes in articular cartilage after impact. These methods combine microscopy techniques with large scale data analysis, making use of a custom-programmed decision tree algorithm, supervised time series classifiers, and unsupervised clustering via a variational autoencoder to identify and categorize cell phenotypes. Time series data collected from thousands of chondrocytes \textit{in situ} during and after impact allow me to probe responses through the lenses of calcium signaling, mitochondrial polarity, and nuclear membrane permeability. This thesis serves to outline the experimental and analytical methods developed to probe cellular response to external stimuli. While the focus is on method development, I will touch on some of the biological implications of impact on articular cartilage, and identify questions and hypotheses generated through the experiments conducted for this work.

A study on a solution to repair osteochondral defects was investigated. This workcontained the use of a novel collagen-based biomaterial that was structured to mimic thecomposition and structure of osteochondral tissue. Collagen extraction from the bovineachilles was optimized in terms of atelocollagen yield and stability. It was found thatcollagen enzymatically digested at a 1.25:10 pepsin to tendon weight ratio in the superiortendon region, gave optimal results in terms of atelocollagen quantity and hydrogelformation. Mineralized collagen scaffolds were fabricated to reflect the composition ofsubchondral bone. Controlled freezing was applied, which successfully oriented collagenfibers mimicking those in each native zonal tissue. Multiple approaches were attemptedto replicate the collagen orientations of osteochondral tissue, ultimately a T-shaped moldwas designed to guide directional freezing, resulting in an anisotropic scaffold structure.Native composition of bone hydroxyapatite and cartilage hyaluronic acid were also takeninto consideration when fabricating such multizonal scaffolds.

Articular cartilage has limited healing capability, resulting in cartilage injuries to often progress into post traumatic osteoarthritis (PTOA), a painful debilitating disease. Adult human mesenchymal stem cell (hMSC)-based tissue engineering strategies have been widely explored to achieve functional cartilage repair due to their ability to differentiate into chondrocytes and secrete cartilage matrix macromolecules collagen II and aggrecan. However, clinical application of MSC-based cartilage repair strategies faces two significant challenges. First, upon chondrogenic differentiation, MSC-derived chondrocytes (hMdChs) undergo hypertrophic maturation driven by master transcription factor RUNX2. Second, the inflammatory environment of the injured joint, established predominantly by pro-inflammatory macrophages, accelerates chondrocyte hypertrophy and matrix degradation, a process also mediated by RUNX2 activity. Inflammatory cytokines induce hMdChs to secrete further inflammatory and catabolic factors, establishing a positive feed forward loop that sustains the joint inflammation. This results in matrix loss which compromises the functionality of engineered cartilage. The central objective of this thesis is to increase matrix accumulation and retention by hMdChs under hypertrophic and inflammatory conditions to improve cartilage regeneration. We hypothesize RUNX2 suppression can increase matrix accumulation by 1) maintaining the production of cartilage structural proteins, 2) protecting the cells from hypertrophy and inflammation induced matrix degradation by inhibiting matrix metalloproteinase (MMP) expression and, 3) inhibiting the pro-inflammatory crosstalk between hMdChs and M1 macrophages. To evaluate these hypotheses, we used a synthetic gene circuit that induces autonomous and tunable RUNX2 suppression in hMdChs based on intracellular RUNX2 concentrations. In Aim 1, I show that suppressing RUNX2 activity using our gene circuit increases matrix accumulation and inhibits matrix mineralization under hypertrophic conditions. Moreover, RUNX2 suppression stabilizes the expression of chondrogenic markers collagen II and aggrecan and inhibits the expression of hypertrophic markers MMP13 and collagen X in hMdChs under hypertrophic stimulus. These results indicate that the gene circuit increases the stability of hMdCh chondrogenic phenotype. In Aim 2, I show that RUNX2 suppression protects hMdChs from inflammatory cytokine IL-1β induced cartilage matrix catabolism. Moreover, I show that under inflammatory stimulus, RUNX2 suppression in hMdChs increases matrix retention by preserving the expression of cartilage matrix macromolecules and inhibiting the expression of hypertrophic and catabolic markers. In Aim 3, using a conditioned media-based co-culture model to recapitulate the injured joint environment, I show that RUNX2 suppression protects hMdChs from M1 conditioned media (M1CM) induced matrix catabolism partly by inhibiting MMP13 expression. Moreover, RUNX2 suppression abates the pro-inflammatory response of hMdChs to M1CM. Finally, pro-inflammatory M1 macrophages treated with the secretome of RUNX2 suppressing hMdChs exhibited downregulation of inflammatory markers and upregulation of anti-inflammatory markers indicating a shift towards anti-inflammatory phenotype. This thesis reveals that RUNX2 mediates many pathological features of PTOA progression. Although the role of RUNX2 in mediating hypertrophy and cartilage matrix catabolism is well known, I demonstrate the application of autoregulated gene circuits to study the RUNX2 mediated pro-inflammatory crosstalk between hMdChs and pro-inflammatory macrophages that reinforces macrophage inflammation, resulting PTOA progression. As demonstrated in this dissertation, the ability to concomitantly improve cartilage matrix accumulation and resolve joint inflammation utilizing autoregulated gene circuits, is a significant step towards clinical application of MSC-based tissue engineering strategies for cartilage regeneration.

Copper chloride (CuCl2) is an essential trace element found in humans, which helps to aid in brain and nerve function, glucose metabolism, calcium absorption, and bone formation. Bone formation utilizes a variety of cell types, including, osteoblasts, osteoclasts, and chondrocytes. Chondrocytes deposit cartilage components like collagen and proteoglycans that provide the necessary scaffold for osteoblasts to synthesize and mineralize bone. Copper chloride is known to aid in osteogenesis, maintain bone metabolism, and regulate bone mineral density, but its role in chondrogenesis is still unclear (15). In this study, it is hypothesized that CuCl2 treatments would enhance chondrogenic differentiation in the murine chondrogenic ATDC5 cell line. To test this hypothesis, chondrocytes were treated with a no treatment control (DMEM/F12) (Corning, Corning NY), a positive control of insulin (2.8mM), and increasing doses CuCl2 (2.6mM, 26mM, 260mM) over 14 days. Cell media was collected 18-20 hours post-treatment on days 0, 1, 3, 7, 10, and 14. Additional experiments were run with spent media to observe the effect of copper chloride on functionality. The amount of alkaline phosphatase (ALP) activity, an important biomarker of chondrocyte activity was measured overtime throughout treatments. At 14 days post-treatment, alizarin red staining was performed to quantify calcium deposited in the presence of CuCl2 treated cells. To observe gene expression changes, quantitative polymerase chain reaction (qPCR) was run for each sample. Specific genes to chondrocytes were run including, VEGF, aggrecan, COL2A1, COL2B1, KISS1, and RHEB. Results found that CuCl2 decreased cell proliferation and ALP activity at medium and high doses. Calcium deposition as well as proteoglycan activity both increased as doses of CuCl2 increased. Overall, by understanding these effects on chondrogenesis advances our knowledge on the environmental impact they have on organismal health.

Articular cartilage is the soft tissue found at the end of long bones within the joint capsule and plays an important role in load transmission and facilitating joint motion. However, traumatic injury delivered to joints generates extreme supraphysiologic shear and compressive forces which result in joint inflammation and significant damage to both cartilage tissue and chondrocytes. Chronic persistence of post-injury changes often results in the development of post-traumatic osteoarthritis (PTOA). Treatment options for cartilaginous injuries are limited due to the lack of vasculature or lymphatics, which limit the transport of anabolic cytokines to injured areas. Furthermore, development of disease modifying treatments is hindered by the lack of suitable PTOA models to conduct experimental trials upon as current models are unable to accurately model the post-injury joint environment, thereby limiting their translational relevance.Thus, the overall goal of this thesis was to generate an ex vivo model of PTOA that could more closely capture the characteristics of an injured synovial joint. This was achieved by demonstrating that simultaneous compressive and shear forces delivered to cartilage generated a profile of chondrocyte damage that is more physiologically relevant to what is expected in vivo (Chapter 1). Secondly, by assessing changes in cartilage mechanical properties pre- and post-injury and relating these changes to resulting chondrocyte damage, to evaluate chondrocyte sensitivity to mechanical loading (Chapter 2). Finally, the effect of negatively altering the lubricating qualities of synovial fluid on the relationship between local shear strains and cellular damage was also assessed (Chapter 3). Collectively, this work offers crucial insight into the mechanisms behind the early stages of PTOA pathogenesis and provides a physiologically relevant model to be utilized for future testing of potential PTOA therapeutics.

Articular cartilage is a seemingly simple biological tissue with mechanical properties that far exceed the simplicity of its structure. In this thesis I will explore how we are able to model the shear mechanical properties with a simple rigidity percolation framework, and are able to show how the two constituents of the extracellular matrix interplay to create a network greater than the sum of its two parts. I will then investigate how we are able to adapt these models with spatial correlation to dramatically change their rigidity percolation properties. Finally, I adapt the degradation protocol we developed in the first chapter to investigate the effect of the extracellular matrix constituent concentrations on the lubrication properties of articular cartilage. Overall, this thesis champions the utility of the interplay of the experimental and the computational, by using computational models to map experimental results and provide predictive power outside the realms of testable limits, as well as by using experimental knowledge and understanding to inform our model making and create introducing theories to explore the natural world.

The objective of this study was to evaluate the effects of sex, loading rate, and age on the compressive and tensile material properties of human costal cartilage over a wide range of subject ages and sexes. Cylindrical compression samples and dog-bone shaped tension samples were tested to failure on a material testing system using target strain rates of 0.005 strain/s and 0.5 strain/s. Compression data were obtained from forty (n = 40) subjects (M = 26, F = 14) ranging in age from 11 – 69 years (Avg. = 39.1 ± 18.2 yrs.), and matched loading rate data were obtained for thirty-four (n = 34) samples. Tension data were obtained from forty-one (n = 41) subjects (M = 30, F = 11) ranging in age from 10 – 59 years (Avg. = 32.9 ± 14.9 yrs.), and matched loading rate data were obtained for seventeen (n = 17) samples. For both compression and tension, load and sample deflection data were collected and used to calculate stress and strain. For the compression data, the toe region was fit using a second-order polynomial, and the toe transition stress, toe transition strain, second-order polynomial coefficient A, and second-order polynomial coefficient B were calculated. In addition, the elastic modulus, ultimate stress, ultimate strain, and strain energy density (SED) were also calculated for each test. For the tension data, only the elastic modulus, ultimate stress, ultimate strain, and SED were calculated for each test. There were no effects of sex on the material properties for either method of loading or strain rate. Therefore, male and female data were combined for the age and loading rate analyses. For compression, toe transition stress, toe transition strain, A, elastic modulus, ultimate stress, and SED were all found to be significantly higher at 0.5 strain/s compared to 0.005 strain/s. For tension, no material properties were found to differ with respect to loading rate. Regarding the effects of age, toe transition stress, toe transition strain, A, B, ultimate stress, ultimate strain and SED were found to significantly decrease with advancing age for the 0.005 strain/s compression data. At 0.5 strain/s, toe transition stress, toe transition strain, elastic modulus, ultimate stress, ultimate strain, and SED all significantly decreased with advancing age. For tension, ultimate stress, ultimate strain, and SED were found to significantly decrease with advancing age at 0.005 strain/s and 0.5 strain/s. Comparing the two loading modes, the ultimate stress, elastic modulus, and SED were significantly higher in compression than in tension. For the compression samples, sample density and percent calcification were also obtained for each sample using physical measurements and micro-CT scans, respectively. However, since there were only a few samples with large calcifications, no meaningful trends were found. This is the first study of its kind to analyze the effects of sex, loading rate, and age on both the compressive and tensile material properties on human costal cartilage from such a wide range of subject ages. The results from this study can be used to develop more accurate finite element models of the human body, which will allow researchers to better evaluate human occupant response and injury risk in motor vehicle collisions for both young and old individuals.

The projects presented here revolve around the utilization of three different animal models of developmental and degenerative orthopaedic diseases, with the work described in order of typical clinical onset in the relevant patient populations. These projects share an integral focus on characterization and validation of the chosen models by histological assessment. The objective of the first phase of this work was to histologically characterize the effects of surgically induced epiphyseal ischemia in a piglet model of Legg-Calvé-Perthes disease on the proximal femoral growth plate, with histomorphometry and immunohistochemical characterization. The objective of the second phase of this work was to determine the sensitivity and specificity of noninvasive quantitative MRI methods in detection of early naturally occurring osteochondrosis lesions ex vivo in the pig, with co-registration and characterization of lesions identified with advanced imaging and identified histologically. The objective of the third phase of this work was to determine the optimal approach for histological assessment of osteoarthritis in murine models, including comparison of two widely recognized murine-specific histological grading schemes and additional microscopic means of evaluation relevant to determination of osteoarthritis severity. Collectively, the results of this work highlight the need for careful evaluation and validation of animal models of orthopaedic diseases, with a focus on repeatability of the model, complete evaluation with reflection on how accurately the model mirrors the human disease, and statistical support for the approach to analysis.

Proteoglycans are a class of biomolecules that play an important and ubiquitous role in the body, with their roles ranging from maintaining tissue mechanical properties to regulating cell signaling. Many of these functions are carried out by the glycosaminoglycan (GAG) chains that decorate the proteoglycan core protein, with glycans chondroitin sulfate (CS), dermatan sulfate, and heparan sulfate regulating growth factors and water content through their negatively charged polysaccharide chains. Additionally, the free-floating GAGs heparin and hyaluronic acid (HA) play important roles in tissue function, such as modulation of enzymes and supporting cell mobility. Because of their important biological roles, as well as their relatively simple structure, multiple easily accessible reactive moieties, and commercial availability, many groups have sought to GAGs for biomedical applications. In this dissertation, we explore the engineering of GAGs to suppress inflammation, starting with a study regarding the balance of the chemical modification of GAGs and the perseveration of their bioactivity. Here, we discuss the fabrication of hydrogels from thiolated CS (CS-SH) and thiolated HA (HA-SH), demonstrating how increased thiolation of these GAGs impairs their recognition by hyaluronidase and GAG-targeting peptides, suggesting reduced biorecognition. Subsequent studies build off these findings, with these DPN hydrogels employed in the context of osteoarthritic joint inflammation. Articular chondrocytes encapsulated in GAG DPN hydrogels demonstrated a suppressed inflammatory response, as shown through reduced secretion of pro-inflammatory cytokines and reduced proliferation. Finally, the GAGs CS and heparin were conjugated with collagen targeting peptides to prevent platelet activation following endothelial denudation. The spacer sequence and C-terminal modification of the peptides were optimized to improve conjugation efficiency and collagen binding ability, improving the effectiveness of the peptide-glycan molecule. Overall, these studies serve as groundwork studies for the engineering of GAGs in the contexts of the prevention of inflammation through balancing the modification of GAGs for increased functionality with the preservation of the potent bioactive signals from the GAG themselves.

Articular cartilage, the near-frictionless tissue allowing smooth movement of joints, has a poor intrinsic healing capacity and can degenerate to osteoarthritis due to disease, trauma, or age. Clinical osteoarthritis is a late-stage condition for which disease-modifying opportunities are limited. Therefore, early detection of osteoarthritis is critical to support a paradigm shift from palliation of late disease towards prevention. Unfortunately, early diagnosis of OA is still a challenging, unmet clinical need that must be addressed. On the other hand, cartilage tissue engineering is an emerging strategy at the threshold of clinical translation, holding immense potential to deliver effective curative therapies for osteoarthritis. However, tissue engineers currently depend predominantly on time-consuming, expensive, and destructive techniques to monitor the maturation of engineered tissue, which can be inappropriate or impractical for large-scale biomanufacturing. Therefore, there is also an immediate need to develop nondestructive tools to monitor tissue-engineered cartilage products for both research and clinical applications.Optical techniques such as fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) have great potential to address these unmet needs due to their capacity to probe tissue composition and structure spatially and temporally in a nondestructive and noninvasive manner. In this dissertation, the feasibility of using FLIm and OCT for early detection of osteoarthritis and nondestructive monitoring of engineered cartilage is demonstrated, a novel solid-state multispectral FLIm system with improved performance is developed, addressing the limitations of current FLIm instrumentation for clinical and tissue engineering applications. Chapter two describes the basics principle and instrumentation of FLIm and OCT as well as the rationale for their application to cartilage research. In chapter three, the fluorescence properties of native cartilage are studied and the feasibility of using FLIm for the early detection of osteoarthritis is demonstrated. Chapter four demonstrates the potential of bimodal FLIm-OCT as a nondestructive quality control technique for monitoring the growth of tissue-engineered cartilage in a pre-clinical animal model. Chapter five reports the design, development, and characterization of a novel multispectral FLIm system with four-times faster imaging speed, five-times lower measurement variability, and automated gain control for each spectral channel utilizing state-of-art solid-state detectors, addressing the limitation in imaging speed and signal-to-noise ratio of the current FLIm instrument for clinical and tissue engineering applications.

Recent evidence suggests that muscle quality may play an important role in the pathogenesis of hip osteoarthritis (OA). Muscle quality describes the extent of adipose tissue accumulation, or fatty infiltration, within skeletal muscle, as adipose tissue stored within the muscle has the capacity to impair muscle function. The hip abductors are important pelvic stabilizers during gait; therefore, dysfunction of these muscles may impact joint loading and lead to degeneration of cartilage and bone. Despite evidence of increased fatty infiltration in individuals with hip OA, minimal research has evaluated muscle quality in a population with mild-to-moderate OA, and relationships of muscle quality with physical function, joint loading, and cartilage health have been sparsely characterized. In this study, chemical shift-based water-fat separated magnetic resonance (MR) imaging was used to quantitatively evaluate hip abductor muscle quality in a cohort of individuals with and without mild-to-moderate hip OA. Measures of hip abductor muscle quality were related to various disease outcomes including radiographic OA severity, patient-reported disability, functional performance, isometric strength, gait kinematics and kinetics, semi-quantitatively evaluated morphological cartilage degeneration, and biochemical cartilage composition evaluated using quantitative MR imaging. Hip abductor fatty infiltration was found to be significantly associated with worse patient-reported outcomes, functional impairment, cartilage lesions, muscle weakness, and increased frontal plane joint loading. These findings suggest that hip abductor fatty infiltration may contribute to an abnormal mechanical loading environment at the joint, potentially leading to greater cartilage degeneration and disability. These findings are clinically relevant, as muscle quality may be a modifiable risk factor for OA which can be targeted through exercise intervention to improve disease outcomes.

Cartilage, the tissue that provides a lubricious surface in diarthrodial joints, does not regenerate when injured or diseased. No treatments for cartilage degeneration succeed at regenerating native cartilage, and most fail in the long term. Tissue-engineered cartilage is a promising therapeutic for cartilage repair and regeneration; this is particularly pertinent for people who have ailments of the temporomandibular joint (TMJ) cartilages — up to 8-16% of the population. However, a deep understanding of the extracellular matrix (ECM) of tissue-engineered cartilage is limited by a dearth of analytic techniques for collagens, the proteins that play essential functional roles in cartilage ECM. Through novel liquid chromatography–mass spectrometry (LC-MS) methods, the global objectives of this work were to: 1) develop methods for the quantification of collagen subtypes and crosslinks, 2) interrogate the temporal changes in collagen subtype and crosslink profiles of native cartilage and tissue-engineered neocartilage during tissue development, and 3) investigate the collagen subtypes and crosslinks of cartilage regeneration through the use of tissue-engineered neocartilage implants in a large animal model.Toward addressing the dearth of techniques for collagen subtype quantification, novel LC-MS methods were developed to quantify individual collagen subtypes and crosslinks in biological samples, and bottom-up proteomics techniques were used to quantify all proteins in tissue ECM, including collagen subtypes, using the cartilages of the Yucatan minipig as a model. The high-throughput LC-MS methods for collagen subtype and crosslink quantification are low in cost and operator time and can be translated to any facility that has access to a triple quadrupole mass spectrometer. These novel collagenomic techniques are widely applicable to researchers studying tissue characterization, disease states, and tissue engineering of all collagenous tissues, including cartilage, skin, bone, ligament, tendon, and many others. The novel collagenomic methods were applied to study the development of native and engineered cartilages. In addition to well-known developmental changes in native cartilage such as total collagen and DNA contents, trends in the collagen subtype and crosslink profiles throughout maturation of knee cartilage were elucidated. The self-assembling process of tissue engineered cartilage was also examined with collagenomic techniques, and many similarities in development between the self-assembled cartilage and native cartilage, such as an increase in collagen type II, were determined. These trends showed evidence that self-assembled engineered cartilage recapitulates aspects of the development of native cartilages. Toward developing a deeper understanding of cartilage regeneration with self-assembled neocartilage implants, Yucatan minipig TMJ disc perforation models were evaluated. Small perforation (3-mm) and large perforation (6-mm) defects were regenerated with self-assembled neocartilage implants and evaluated at 24 weeks and 8 weeks, respectively. In both in vivo studies, treatment with self-assembled neocartilage implants resulted in regenerated fibrocartilaginous repair tissue, an important step toward translating self-assembled cartilage implants to clinical usage. Quantitative collagenomics was used to show that the repair tissue of empty defects in the small perforation group had a collagen subtype profile similar to that of scar tissue (more collagen type III and less collagen type I) than repair tissue of implant-treated TMJ discs, which had a collagen subtype profile of native TMJ disc tissue. Overall, this work is highly significant in that it introduces novel methods for collagen subtype and crosslink quantification, which are applicable not only to cartilage, but to a wide breadth of tissue fields. Because of the critical roles of major and minor collagens in providing structure to tissues, collagenomics techniques are particularly relevant to researchers studying tissue characterization, disease states, structure-function relationships, and tissue engineering of cartilage, bone, heart valve, ligament, tendon, blood vessels, and skin. Applying collagenomics techniques to the development of native and engineered cartilages allows for a deeper understanding of the self-assembling process of cartilage tissue engineering beyond traditional benchtop biochemical assays. Finally, showing the fibrocartilaginous collagenomic profile of regenerated cartilage tissue in a large animal model is a major milestone toward translating tissue-engineered cartilage to the clinic for human usage, which can drastically improve the quality of life in millions of patients who suffer from TMJ disorders.

Osteoarthritis (OA) is a degenerative disease of articular cartilage that causes cartilage degradation, osteophyte formation, synovial inflammation, angiogenesis, and subchondral bone alteration. OA causes chronic disability in older people. Various factors are associated with its pathogenesis, including aging, obesity, joint instability, and joint inflammation. In healthy conditions, cartilage remodeling involves balanced interactions of synthesis and degradation to achieve homeostasis of the extracellular matrix (ECM) However, in OA this process becomes unbalanced, leading to pathologic changes in the affected joint. OA is also a highly prevalent rheumatic musculoskeletal disorder, that affected 303 million people globally in 2017. In the U.S. only, OA affects more than 32.5 million adults and is estimated to affect approximately 70 million more (i.e., 25% of the U.S. population) by 2030. Multiple OA factors might lead to stimulate the chondrocytes in the articular cartilage to produce the proteolytic enzymes which include matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) which work together to degrade joint articular cartilage leading to osteophyte formation and stiffening of joints. So far, there are no medications that can treat OA and all medicines such as analgesics, corticosteroids, and non-steroid anti-inflammatory drugs (NSAIDs) are used to reduce pain and inflammation.Trafficking protein particle complex subunit 9 (TRAPPC9) is a protein subunit part of the Transport Protein Particle II (TRAPPII), a highly conserved trafficking pathway from yeast to human. Not only TRAPPC9 is implicated in protein trafficking, but it has been also reported that TRAPPC9 regulates/potentiates multiple cellular activities such as prefiltration, differentiation, and function for several cell types through NF-kB mediation. To emphasize the relationship between TRAPPC9 and NF-kB, a study showed that TRAPPC9 physically interacts with NF-kB-inducing kinase (NIK) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) but not IKKα or IKKγ. While the role of TRAPPC9 in cartilage is unknown, our study aims for the first time to discover the role of TRAPPC9 in cartilage homeostasis in OA. On the other hand, we also studied L-Plastin (LPL). LPL belongs to a family of plastins that exist in three isoforms: L-, I-, and T-plastin and they are actin-bundling proteins essential for actin regulation in eukaryotes. LPL, also called lymphocyte cytosolic protein 1 (LCP1), has been described as one of the 15 most abundant proteins in human monocytes and T cells. LPL phosphorylation is essential to its function and it occurs on serine 5 and serine 7, however, other plastins isoforms not phosphorylated. In recent studies, LPL was shown to have a role in inflammation, a study by Ozturk et al, showed that patients with chronic periodontitis and generalized aggressive periodontitis exhibited significantly higher tissue LPL gene expression and gingival crevicular fluid levels compared to the control group. This indicates that LPL might be involved in OA inflammation.The current study is composed of three hypothesizes. First, TRAPPC9 has a pro-inflammatory role in osteoarthritis by inducing the catabolic genes such as MMP-3, MMP- 9, MMP-13. Second, TRAPPC9 binds physically to LPL in chondrocytes. Third, LPL has also a pro-inflammatory role in osteoarthritis by inducing the catabolic genes such as MMP-3, MMP- 9, MMP-13. Three mouse models were used in this study to define the function, mechanism, and regulation of LPL in cartilage which involved C57BL/6, Life Act/W.T, and Life act/LPL-/-.Our data showed that TRAPPC9 and LPL were expressed in normal articular chondrocytes, and their expression level was higher in post-IL-1β treatment. IL-1β- induced inflammatory factors involved interleukin-6 (IL-6) and cartilage degradation enzymes such as MMP-3, MMP-9, and MMP-13, were upregulated following TRAPPC9 overexpression. Conversely, MMP-3, MMP-9, and MMP-13 were downregulated following TRAPPC9 knockdown or LPL phosphorylation inhibition respectively. Furthermore, immunoprecipitation data showed that TRAPPC9 binds to LPL in chondrocytes. We also found that LPL inhibitor inhibits P-P100 a subunit of NF-kB. We also showed that LPL knockout has chondroprotective effects in post-traumatic OA in mice.In conclusion, TRAPPC9 and LPL are highly expressed in post-IL-1β treatment compared to the control. TRAPPC9 and LPL both have an anti-inflammatory effect by inhibiting the expression of MMP-3, MMP-9, and MMP-13, which are induced by IL-1β. Also, the post-traumatic OA model (DMM) showed that LPL knockout is essential to reduce inflammation. Our results determined that LPL knockout mice have less cartilage damaged compared to the WT mice. Overall, the TRAPPC9 and LPL inhibition may be used as a therapeutic approach in osteoarthritis and may replace the anti-inflammatory medicine that causes several side effects which include elevated blood pressure, gastrointestinal bleeding, and kidney disorders.