Cameron P. Brown

Non-invasive imaging of cartilage in early osteoarthritis.

Treatment for osteoarthritis (OA) has traditionally focused on joint replacement for end-stage disease. An increasing number of surgical and pharmaceutical strategies for disease prevention have now been proposed. However, these require the ability to identify OA at a stage when it is potentially reversible, and detect small changes in cartilage structure and function to enable treatment efficacy to be evaluated within an acceptable timeframe. This has not been possible using conventional imaging techniques but recent advances in musculoskeletal imaging have been significant. In this review we discuss the role of different imaging modalities in the diagnosis of the earliest changes of OA. The increasing number of MRI sequences that are able to non-invasively detect biochemical changes in cartilage that precede structural damage may offer a great advance in the diagnosis and treatment of this debilitating condition.

Imaging and modeling collagen architecture from the nano to micro scale

The collagen meshwork plays a central role in the functioning of a range of tissues including cartilage, tendon, arteries, skin, bone and ligament. Because of its importance in function, it is of considerable interest for studying development, disease and regeneration processes. Here, we have used second harmonic generation (SHG) to image human tissues on the hundreds of micron scale, and developed a numerical model to quantitatively interpret the images in terms of the underlying collagen structure on the tens to hundreds of nanometer scale. Focusing on osteoarthritic changes in cartilage, we have demonstrated that this combination of polarized SHG imaging and numerical modeling can estimate fibril diameter, filling fraction, orientation and bundling. This extends SHG microscopy from a qualitative to quantitative imaging technique, providing a label-free and non-destructive platform for characterizing the extracellular matrix that can expand our understanding of the structural mechanisms in disease.

Rough fibrils provide a toughening mechanism in biological fibers.

Spider silk is a fascinating natural composite material. Its combination of strength and toughness is unrivalled in nature, and as a result, it has gained considerable interest from the medical, physics, and materials communities. Most of this attention has focused on the one to tens of nanometer scale: predominantly the primary (peptide sequences) and secondary (β sheets, helices, and amorphous domains) structure, with some insights into tertiary structure (the arrangement of these secondary structures) to describe the origins of the mechanical and biological performance. Starting with spider silk, and relating our findings to collagen fibrils, we describe toughening mechanisms at the hundreds of nanometer scale, namely, the fibril morphology and its consequences for mechanical behavior and the dissipation of energy. Under normal conditions, this morphology creates a nonslip fibril kinematics, restricting shearing between fibrils, yet allowing controlled local slipping under high shear stress, dissipating energy without bulk fracturing. This mechanism provides a relatively simple target for biomimicry and, thus, can potentially be used to increase fracture resistance in synthetic materials.

In vitro degradation of articular cartilage: does trypsin treatment produce consistent results?

It is common practice in laboratories to create models of degraded articular cartilage in vitro and use these to study the effects of degeneration on cartilage responses to external stimuli such as mechanical loading. However, there are inconsistencies in the reported action of trypsin, and there is no guide on the concentration of trypsin or the time to which a given sample can be treated so that a specific level of proteoglycan depletion is achieved. This paper argues that before any level of confidence can be established in comparative analysis it is necessary to first obtain samples with similar properties. Consequently, we examine the consistency of the outcome of the artificial modification of cartilage relative to the effects of the common enzyme, trypsin, used in the process of in vitro proteoglycan depletion. The results demonstrate that for a given time and enzyme concentration, the action of trypsin on proteoglycans is highly variable and is dependent on the initial distribution and concentration of proteoglycans at different depths, the intrinsic sample depth, the location in the joint space and the medium type, thereby sounding a note of caution to researchers attempting to model a proteoglycan‐based degeneration of articular cartilage in their experimental studies.

Diffuse reflectance near infrared spectroscopy can distinguish normal from enzymatically digested cartilage.

A non-destructive, diffuse reflectance near infrared spectroscopy (DR-NIRS) approach is considered as a potential tool for determining the component-level structural properties of articular cartilage. To this end, DR-NIRS was applied in vitro to detect structural changes, using principal component analysis as the statistical basis for characterization. The results show that this technique, particularly with first-derivative pretreatment, can distinguish normal, intact cartilage from enzymatically digested cartilage. Further, this paper establishes that the use of DR-NIRS enables the probing of the full depth of the uncalcified cartilage matrix, potentially allowing the assessment of degenerative changes in joint tissue, independent of the site of initiation of the osteoarthritic process.

Damage initiation and progression in the cartilage surface probed by nonlinear optical microscopy

With increasing interest in treating osteoarthritis at its earliest stages, it has become important to understand the mechanisms by which the disease progresses across a joint. Here, second harmonic generation (SHG) microscopy, coupled with a two-dimensional spring-mass network model, was used to image and investigate the collagen meshwork architecture at the cartilage surface surrounding osteoarthritic lesions. We found that minor weakening of the collagen meshwork leads to the bundling of fibrils at the surface under normal loading. This bundling appears to be an irreversible step in the degradation process, as the stress concentrations drive the progression of damage, forming larger bundles and cracks that eventually form lesions.

Analysis of forward and backward Second Harmonic Generation images to probe the nanoscale structure of collagen within bone and cartilage

Collagen ultrastructure plays a central role in the function of a wide range of connective tissues. Studying collagen structure at the microscopic scale is therefore of considerable interest to understand the mechanisms of tissue pathologies. Here, we use second harmonic generation microscopy to characterize collagen structure within bone and articular cartilage in human knees. We analyze the intensity dependence on polarization and discuss the differences between Forward and Backward images in both tissues. Focusing on articular cartilage, we observe an increase in Forward/Backward ratio from the cartilage surface to the bone. Coupling these results to numerical simulations reveals the evolution of collagen fibril diameter and spatial organization as a function of depth within cartilage.

Characterization of early stage cartilage degradation using diffuse reflectance near infrared spectroscopy.

Interest in localized and early stage treatment technologies for joint conditions such as osteoarthritis is growing rapidly. It has therefore become important to develop objective measures capable of characterizing the earliest (non-visible) changes associated with degeneration to aid treatment procedures. In addition to assessing tissue before treatment, it is further important to develop an effective, non-destructive means of monitoring post-treatment tissue healing, and of providing the high-quality data needed for trials of developing treatment methods. To investigate its ability to detect the early stages of degeneration in cartilage-on-bone, diffuse reflectance near infrared spectroscopy was applied to normal and osteoarthritic joints. A discriminating function was developed to relate absorbance peaks of interest and track degradation around focal osteoarthritic defects. The function could distinguish between normal and degraded tissue (100% separation of normal tissue from that within 25 mm of a defect) and between different stages of osteoarthritic progression (p < 0.05). This technique allows simple, practical and non-destructive assessment of component-level properties over the full depth of the tissue. It has the potential to increase our understanding of the underlying etiologic and pathogenic processes in early stage degeneration, to assist classification and the development of new treatment methods.

An alternative mechanical parameter for assessing the viability of articular cartilage.

Abstract This paper is a sequel to previously published findings showing that mechanical indentation alone cannot clearly discriminate between normal and degraded articular cartilage. Consequently, the structural elasticity potential ℜc = ɛr/σi, which combines indentation stress σi with near-instantaneous rebound ɛr following unloading, is hypothesized as a potential cartilage assessment parameter, which arguably measures the integrity of the collagen fibre—proteoglycan entrapment system. To establish the validity of our hypothesis, samples of normal intact, artificially degraded, and osteoarthritic bovine cartilage were subjected to quasi-static compression at 0.1 s−1 and 0.025 s−1 to 30 per cent strain and then unloaded. A significant reduction in recovery was observed for artificially and naturally degraded samples in the first 5 s following unloading (p<0.01). The structural elasticity potential provided a considerable improvement over the results obtained using the individual indentation and rebound parameters to distinguish between paired normal and artificially degraded samples and indicated a high statistical significance of p<0.005 when applied to the differentiation of normal and osteoarthritic samples.

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy.

We image the relative orientation of organized groups of noncentrosymmetric molecules (like collagen or myosin) at the micron scale in biological tissues by combining interferometry and Second Harmonic Generation (SHG) microscopy.