Development of a finite element model of the knee using patient specific magnetic resonance imaging data and biomechanical testing of soft tissues

This thesis presents the findings of investigations carried out relating to the creation of full joint contact patient specific finite element models for correlation with biological studies in the study of Osteoarthritis (OA) development. To understand the relationship between altered loading and biological changes in articular cartilage (AC), a method for predicting stresses and strains experienced inside the tissues is required. An in-vitro study was conducted to explore the possibility of correlating finite element (FE) and gene expression study results. FE models were used to predict the stresses and strains inside the AC for explants subjected to different loading conditions. The study demonstrated that the accurate representation of AC surface geometry is crucial and current flat surface axisymmetric cylinder representations used in AC explant modelling introduces significant error in the prediction of tissue mechanical behaviour. Cutting of the AC explant to achieve a flat surface can affect the biological, mechanical and tribology behaviour of the tissue. Thus, a method for creating explant specific finite element models with the use of digital image correlation (DIC) was developed and is presented, allowing for surface layer preservation in AC explants for correlated gene expression and inverse FE. Reconstruction of tissue geometries from magnetic resonance (MR) imaging scan data of the knee was explored. It was possible to segment both hard and soft tissues from the same set of MR imaging scan data. Meshing of the geometries using a fundamentally voxel based algorithm proved to cause significant error in the segmented volume. An alternative contour based algorithm needs to be explored. Uncertainties concerning the presence and modelling of meniscotibial ligaments (MTLs) in full joint contact FE models found in literature were addressed. An anatomy study revealed that the MTLs are found in both the medial and lateral side of the joint around the periphery of the anterior, middle and posterior portion of the menisci. With the use of cross polarised light microscopy, it was established Page | VII that MTLs consist of Type I collagen orientated in the circumferential direction around the menisci. As a result, the MTLs were modelled as an anisotropic membrane. Using the full joint contact finite element model, the influence of MTLs on knee joint kinematics was investigated. It was found that the MTLs reinforce the function of the meniscal horns and circumferential fibres in the meniscus and help constrain the meniscus. Therefore, it was concluded that the MTLs are mechanically significant in the stabilisation of knee joints and should be included in knee models for accurate prediction of knee joint behaviour

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field