Hayley R. Moody

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.

Near Infrared Spectroscopy for Rapid Determination of Mankin Score Components: A Potential Tool for Quantitative Characterization of Articular Cartilage at Surgery

PURPOSE The purpose of this study was to demonstrate the potential of near infrared (NIR) spectroscopy for characterizing the health and degenerative state of articular cartilage based on the components of the Mankin score. METHODS Three models of osteoarthritic degeneration induced in laboratory rats by anterior cruciate ligament (ACL) transection, meniscectomy (MSX), and intra-articular injection of monoiodoacetate (1 mg) (MIA) were used in this study. Degeneration was induced in the right knee joint; each model group consisted of 12 rats (N = 36). After 8 weeks, the animals were euthanized and knee joints were collected. A custom-made diffuse reflectance NIR probe of 5-mm diameter was placed on the tibial and femoral surfaces, and spectral data were acquired from each specimen in the wave number range of 4,000 to 12,500 cm(-1). After spectral data acquisition, the specimens were fixed and safranin O staining (SOS) was performed to assess disease severity based on the Mankin scoring system. Using multivariate statistical analysis, with spectral preprocessing and wavelength selection technique, the spectral data were then correlated to the structural integrity (SI), cellularity (CEL), and matrix staining (SOS) components of the Mankin score for all the samples tested. RESULTS ACL models showed mild cartilage degeneration, MSX models had moderate degeneration, and MIA models showed severe cartilage degenerative changes both morphologically and histologically. Our results reveal significant linear correlations between the NIR absorption spectra and SI (R(2) = 94.78%), CEL (R(2) = 88.03%), and SOS (R(2) = 96.39%) parameters of all samples in the models. In addition, clustering of the samples according to their level of degeneration, with respect to the Mankin components, was also observed. CONCLUSIONS NIR spectroscopic probing of articular cartilage can potentially provide critical information about the health of articular cartilage matrix in early and advanced stages of osteoarthritis (OA). CLINICAL RELEVANCE This rapid nondestructive method can facilitate clinical appraisal of articular cartilage integrity during arthroscopic surgery.

Investigating the potential value of individual parameters of histological grading systems in a sheep model of cartilage damage: the Modified Mankin method

A total histological grade does not necessarily distinguish between different manifestations of cartilage damage or degeneration. An accurate and reliable histological assessment method is required to separate normal and pathological tissue within a joint during treatment of degenerative joint conditions and to sub‐classify the latter in meaningful ways. The Modified Mankin method may be adaptable for this purpose. We investigated how much detail may be lost by assigning one composite score/grade to represent different degenerative components of the osteoarthritic condition. We used four ovine injury models (sham surgery, anterior cruciate ligament/medial collateral ligament instability, simulated anatomic anterior cruciate ligament reconstruction and meniscal removal) to induce different degrees and potentially ‘types’ (mechanisms) of osteoarthritis. Articular cartilage was systematically harvested, prepared for histological examination and graded in a blinded fashion using a Modified Mankin grading method. Results showed that the possible permutations of cartilage damage were significant and far more varied than the current intended use that histological grading systems allow. Of 1352 cartilage specimens graded, 234 different manifestations of potential histological damage were observed across 23 potential individual grades of the Modified Mankin grading method. The results presented here show that current composite histological grading may contain additional information that could potentially discern different stages or mechanisms of cartilage damage and degeneration in a sheep model. This approach may be applicable to other grading systems.

Spatial mapping of proteoglycan content in articular cartilage using near-infrared (NIR) spectroscopy

Diagnosis of articular cartilage pathology in the early disease stages using current clinical diagnostic imaging modalities is challenging, particularly because there is often no visible change in the tissue surface and matrix content, such as proteoglycans (PG). In this study, we propose the use of near infrared (NIR) spectroscopy to spatially map PG content in articular cartilage. The relationship between NIR spectra and reference data (PG content) obtained from histology of normal and artificially induced PG-depleted cartilage samples was investigated using principal component (PC) and partial least squares (PLS) regression analyses. Significant correlation was obtained between both data (R(2) = 91.40%, p<0.0001). The resulting correlation was used to predict PG content from spectra acquired from whole joint sample, this was then employed to spatially map this component of cartilage across the intact sample. We conclude that NIR spectroscopy is a feasible tool for evaluating cartilage contents and mapping their distribution across mammalian joint.

A novel approach to the development of benchmarking parameters for characterizing cartilage health.

This article outlines the motivation and preliminary investigations into a novel method of characterizing cartilage health for potential in vivo application. Current in vivo indentation techniques, which primarily rely on stiffness measurements based on axial data, are unable to adequately distinguish between healthy and degraded tissue. The present in vitro study investigates the effects of controlled artificial degradation on the effective surface stretch, comparing the results with those obtained from the peripheral cartilage surrounding focal osteoarthritis. Results suggest that this technique is highly sensitive, showing a maximum range of 14% effective surface stretch in a normal joint compared with 42% for axial strain measurements. We further demonstrated that the technique can discriminate between degenerative changes and the intrinsic variations in cartilage properties across the normal joint. From these investigations we propose that the relationship between indentation and the in-plane strain field under the indenter can better distinguish degraded tissue than the currently used stiffness techniques.

Characterization of Articular Cartilage Recovery and Its Correlation with Optical Response in the Near-Infrared Spectral Range:

Objectives: In this study, we examine the capacity of a new parameter, based on the recovery response of articular cartilage, to distinguish between healthy and damaged tissues. We also investigate whether or not this new parameter correlates with the near-infrared (NIR) optical response of articular cartilage. Design: Normal and artificially degenerated (proteoglycan-depleted) bovine cartilage samples were nondestructively probed using NIR spectroscopy. Subsequently they were subjected to a load and unloading protocol, and the recovery response was logged during unloading. The recovery parameter, elastic rebound (ER), is based on the strain energy released as the samples underwent instantaneous elastic recovery. Results: Our results reveal positive relationship between the rebound parameter and cartilage proteoglycan content (normal samples: 2.20 ± 0.10 N mm; proteoglycan-depleted samples: 0.50 ± 0.04 N mm for 1 hour of enzymatic treatment and 0.13 ± 0.02 N mm for 4 hours of enzymatic treatment). In addition, multivariate analysis using partial least squares regression was employed to investigate the relationship between ER and NIR spectral data. The results reveal significantly high correlation (R2cal = 98.35% and R2val = 79.87%; P < 0.0001), with relatively low error (14%), between the recovery and optical response of cartilage in the combined NIR regions 5,450 to 6,100 cm−1 and 7,500 to 12,500 cm−1. Conclusion: We conclude that ER can indicate the mechanical condition and state of health of articular cartilage. The correlation of ER with cartilage optical response in the NIR range could facilitate real-time evaluation of the tissue’s integrity during arthroscopic surgery and could also provide an important tool for cartilage assessment in tissue engineering and regeneration research.

A Comparison of the Histochemical and Image-Derived Proteoglycan Content of Articular Cartilage

There are several methods for determining the proteoglycan content of cartilage in biomechanics experiments. Many of these include assay-based methods and the histochemistry or spectrophotometry protocol where quantification is biochemically determined. More recently a method based on extracting data to quantify proteoglycan content has emerged using the image processing algorithms, e.g., in ImageJ, to process histological micrographs, with advantages including time saving and low cost. However, it is unknown whether or not this image analysis method produces results that are comparable to those obtained from the biochemical methodology. This paper compares the results of a well-established chemical method to those obtained using image analysis to determine the proteoglycan content of visually normal (n=33) and their progressively degraded counterparts with the protocols. The results reveal a strong linear relationship with a regression coefficient (R2) = 0.9928, leading to the conclusion that the image analysis methodology is a viable alternative to the spectrophotometry.

A new mechanical indentation framework for functional assessment of articular cartilage

The conventional mechanical properties of articular cartilage, such as compressive stiffness, have been shown to have limited capacity to distinguish visually normal from degraded cartilage samples. In this study, a new mechanical indentation framework for assessing functional properties of articular cartilage during loading/unloading, i.e. deformation and recovery, was established. The capacity of a ring-shaped indenter integrated with an ultrasound transducer to distinguish mechanically intact from proteoglycan-depleted tissue was investigated. To achieve this, normal and enzymatically degraded bovine osteochondral samples were subjected to loading/unloading while the response of the tissue at the middle was captured by ultrasound at the same time. The enzymatic degradation model was characterized by amount of proteoglycan content, glycosaminoglycan release and proteomic analysis. The mechanical response of a wider continuum of articular cartilage in the loaded area and its surrounding region was captured in this framework leading to investigate two parameters, L and TS, related to the surrounding tissue of the loaded area for functional assessment of cartilage. L is the distance between the ultrasound transducer and articular cartilage surface and TS is the transient strain of articular cartilage during loading and unloading. Classification Analysis based on Principal Component Analysis was used to investigate the capacity of the new parameters to assess the functionality of the tissue. Multivariate statistics based on Partial Least Squares regression was employed to identify the correlation between the response of the tissue in the indented area and its surrounding cartilage. The results of this study indicate that L during loading (deformation) can differentiate normal and mildly proteoglycan-depleted samples from severely depleted samples and L during unloading (recovery) can distinguish between normal and proteoglycan-depleted tissue. However, TS during deformation and recovery is unable to discriminate normal cartilage samples from proteoglycan-depleted tissue. The results also demonstrate a strong correlation between mechanical properties of the loaded area with the response of its surrounding cartilage during recovery. It is therefore concluded that L in this newly established framework can discriminate between normal and proteoglycan-depleted cartilage samples. However, more samples will be needed to verify the demarcation between samples degraded for varying amount of time.

Potential enhancement of articular cartilage histological grading with collagen integrity

Background: Histological evaluation of articular cartilage, such as using the Mankin scoring system, is the gold standard for characterization of tissue integrity. This scoring system takes into account several parameters indicative of the tissues health; however, the collagen integrity, which is a primary indicator of cartilage health is not taken into consideration. Thus, there is need to enhance histological grading of articular cartilage by incorporating explicit scoring of collagen degeneration into the Modified Mankin grading system. This paper explores a new histological grading parameter for collagen network degradation and how this information can be used to augment a widely used grading scheme like the Modified Mankin grading system. Methods: Intact and degenerated human cartilage were examined histologically and then subjected to second harmonic generation imaging, leading to qualitative and quantitative description of collagen disruption emanating from the surface to subsurface layers of the tissue. This data was then incorporated into the Modified Mankin grading system. Findings: Second harmonic generation image analysis reveals a relationship between changes in collagen architecture and histologically observed tissue disruption in degenerated articular cartilage. Interpretation: Histological tissue disruption in degenerated human articular cartilage is directly related to the reorganization of collagen fibrils in the form of intense fibril aggregation, either as a result of degeneration or aging. This method of mapping disrupted tissue regions to quantitative collagen fibril damage can be coded into cartilage grading systems and could inform clinical practice and scientific research. Highlights:Damaged cartilage matrix disruption is related to collagen fibril reorganization.Imaging reveals changes in collagen architecture during cartilage degeneration.Collagen integrity information can improve cartilage grading and evaluation.