Three-dimensional Ultrasound Imaging For Quantifying Knee Cartilage Volume

Arthritis is the most common chronic health condition in Canada, with the most common form being osteoarthritis (OA). There is a great clinical need for an objective imaging-based point-of-care tool to assess OA status, progression, and response to treatment. This thesis aims to validate a handheld mechanical three-dimensional (3D) ultrasound (US) device against the current clinical standard of magnetic resonance imaging (MRI) for quantifying femoral articular cartilage (FAC) volume. Knee images of 25 healthy volunteers were acquired using 3D US and 3.0 Tesla MRI scans. Two raters manually segmented the trochlear FAC during separate sessions to assess intra- and inter-rater reliabilities. The results demonstrated that 3D US has excellent reliability and strong concurrent validity with MRI for measuring healthy FAC volume. 3D US is a promising, inexpensive, and widely accessible imaging modality that will enable clinicians and researchers to obtain additional information without added complexity or discomfort to patients

Advanced Magnetic Resonance Imaging of Osteoarthritis

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.Funding: Addenbrooke's Charitable Trust, Experimental Medicine Initiative, non-investigator sponsored study grant from GlaxoSmithKlin

The role of subchondral bone in osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis. Affected individuals commonly suffer with chronic pain, joint dysfunction, and reduced quality of life. OA also confers an immense burden on health services and economies. Current OA therapies are symptomatic and there are no therapies that modify structural progression. The lack of validated, responsive and reliable biomarkers represents a major barrier to the development of structure-modifying therapies. MRI provides tremendous insight into OA structural disease and has highlighted the importance of subchondral bone in OA. The hypothesis underlying this thesis is that novel quantitative imaging biomarkers of subchondral bone will provide valid measures for OA clinical trials. The Osteoarthritis Initiative (OAI) provided a large natural history database of knee OA to enable testing of the validity of these novel biomarkers. A systematic literature review identified independent associations between subchondral bone features with structural progression, pain and total knee replacement in peripheral joint OA. However very few papers examined the association of 3D bone shape with these patient-centred outcomes. A cross-sectional analysis of the OAI established a significant association between 3D bone area and conventional radiographic OA severity scores, establishing construct validity of 3D bone shape. A nested case-control analysis within the OAI determined that 3D bone shape was associated with the outcome of future total knee replacement, establishing predictive validity for 3D bone shape. A regression analysis within the OAI identified that 3D bone shape was associated with current knee symptoms but not incident symptoms, establishing evidence of concurrent but not predictive validity for new symptoms. In summary, 3D bone shape is an important biomarker of OA which has construct and predictive validity in knee OA. This thesis, along with parallel work on reliability and responsiveness provides evidence supporting its suitability for use in clinical trials

Self-Assembling Peptides for Cartilage Regeneration

Loss of glycosaminoglycans (GAGs) in osteoarthritic (OA) cartilage contributes to a decrease in mechanical properties and function in vitro, and is considered to be a major contributor to disease progression. The aims of this investigation were to test the hypothesis that a combination of self-assembling peptides (SAPs) and chondroitin sulfate (glycosaminoglycan; GAG) would restore the biomechanical properties of GAG depleted porcine condylar cartilage, ideally to a level intrinsic to native porcine condylar cartilage. The SAPs investigated were members of the P11 series which have been designed to spontaneously self-assemble into three-dimensional fibrilar hydrogels, in response to physiological conditions. Initial studies were carried out to determine which of three peptides (P11-4, P11-8 and P11-12) demonstrated high β-sheet percentage, long-woven fibrilar networks and high stiffness; when mixed with chondroitin sulfate at two different GAG molar ratios (1:16 and 1:64) in physiological conditions, using FTIR analysis, transmission electron microscopy and rheology. The β-sheet percentage, dimensions of fibrils and stiffness were dependent upon the peptide, GAG molar ratio and Na2+ salt concentration. P11-4 and P11-8: GAG mixtures had high β-sheet percentage ranging from 50.6-91 % and 81.7-92 %, respectively. Fibril lengths of the P11-4 and P11-8: GAG mixtures were in the range 498- 3518 nm and the elastic shear modulus (G’) ranged from 4,479-10,720 Pa and 7,722-26,854 Pa, respectively. P11-4 and P11-8: GAG mixtures were selected for further investigation. In order to produce a GAG depleted cartilage model, porcine femoral condylar cartilage was subjected to three different methods of GAG depletion (1) coating the surface with chondroitinase ABC (2) injecting chondroitinase ABC into the cartilage (3) washing the condyles in sodium dodecyl sulfate (SDS). GAG depletion was successfully achieved following two 24 hour washes in 0.1 % (w/v) SDS and buffer washes. Histological analysis of safranin O stained sections revealed an absence of GAGs. Quantification of GAGs using the dimethylemethylene blue assay revealed that 75 % of GAGs had been removed. In order to assess the effects of peptide: GAG mixtures on the biomechanical properties of the GAG depleted porcine condylar cartilage a biomechanical test method was developed. A series of indentation tests using different loads, followed by finite element analysis of the data were performed on native and GAG depleted porcine condylar cartilage; to identify a suitable load for detection of a significant difference in the deformation, equilibrium elastic modulus and permeability between the native and GAG depleted porcine condylar cartilages. A load of 0.31 N was identified as the most appropriate. GAG depleted porcine condylar cartilage was injected with P11-4 and P11-8 alone, P11-4 and P11-8 : GAG mixtures at a molar ratio of 1:64 and chondroitin sulfate alone. The average percentage deformation of the medial condylar cartilage samples injected with P11-4 alone and P11-4: GAG mixture was 15.5 % and 8.7 % and for P11-8 alone and P11-8: GAG mixture was 11.4 % and 9.1 % respectively; compared to 6.3 % for the native cartilage and 12.6 % for the GAG depleted cartilage. The average equilibrium elastic modulus of the medial cartilage samples injected with P11-4 alone and P11-4: GAG mixture was 0.16 MPa and 0.43 MPa and for P11-8 alone and P11-8: GAG, 0.23 MPa and 0.35 MPa, respectively; compared to 0.49 MPa for the native cartilage and 0.21 MPa for the GAG depleted cartilage. Statistical analysis (ANOVA) showed that a mixture of P11-4: GAG, but not P11-8: GAG restored both the percentage deformation and equilibrium elastic modulus of the GAG depleted cartilage to levels that were not significantly different to the native cartilage. This study has shown that the use of P11-4 in combination with chondroitin sulfate has future potential for development as a minimally invasive treatment for early stage osteoarthritis

In-vitro Modelling of Stromal and Immune Cell Interactions Following Surgical Bone Marrow Stimulation

Osteoarthritis (OA) results in degeneration of cartilage and bone within a joint, leading to pain and loss-of-function. While effective treatments exist for end-stage OA, earlier disease stages lack treatment options. Micro-fracture and micro-drilling, often termed bone marrow stimulation techniques, offer an early-stage repair and regenerative strategy. These techniques result in the formation of a haematoma containing Bone Marrow Stromal Cells (BMSC) and immune cells at the joint surface, leading to repair. However, the quality of repair after marrow stimulation is, at present, sub-optimal. BMSC are known to interact with immune subpopulations during repair, although the consequences of these interactions for repair outcomes are not fully understood. This thesis aims to further the current understanding of how interactions between immune subpopulations and BMSCs in the environment of a haematoma may influence repair outcomes following surgical bone marrow stimulation techniques. In order to understand the influence of hypoxia on BMSC, strains of human BMSCs were isolated and cultured under normoxic (18.9% oxygen) and hypoxic (3.0% oxygen) conditions. Characterisation demonstrated that hypoxic culture had functional consequences on BMSC phenotype and behaviour. To explore the effect of BMSC-immune cell interactions on BMSC migration, immuno-regulation, and cell phenotypes, parallel experiments were performed under normoxic and hypoxic conditions. A unique approach was taken to identify an immune subpopulation resulting in BMSC migration, which was used as an indicator of potential co-localisation and interaction. BMSC were exposed to paired enriched and depleted populations of peripheral blood mononuclear cells, which had been sequentially fractionated using specific markers. The migration stimulated in the BMSC by the enriched and depleted fractions was compared. A Natural Killer (NK) cell population was found to have induced the greatest BMSC migration relative to its paired fraction. Co-cultures indicated that the influence of NK cells on BMSC, particularly NK cell-mediated cytotoxicity, were heavily dependent on culture conditions, including NK cell number and oxygen levels. In a further study, co-cultures of BMSC and monocytes, which interact with BMSC and have a potential role in fibrosis, were co-cultured under normoxic and hypoxic conditions. A pro-angiogenic, immunomodulatory phenotype developed in normoxic co-cultures, which was significantly reduced in hypoxic co-cultures. In conclusion, these data suggest that BMSC and immune subpopulations have diverse interactions which are strongly influenced by the local oxygen tension and cellular environment. These findings demonstrate the importance of the microenvironment formed by bone marrow stimulation techniques on cellular interactions, with potential consequences for the outcome of the repair. Furthermore, this work indicates that manipulation of the haematoma and surrounding environment following bone marrow stimulation could improve repair and regenerative outcomes