The effect of collagen degradation on chondrocyte volume and morphology in bovine articular cartilage following a hypotonic challenge

Collagen degradation is one of the early signs of osteoarthritis. It is not known how collagen degradation affects chondrocyte volume and morphology. Thus, the aim of this study was to investigate the effect of enzymatically induced collagen degradation on cell volume and shape changes in articular cartilage after a hypotonic challenge. Confocal laser scanning microscopy was used for imaging superficial zone chondrocytes in intact and degraded cartilage exposed to a hypotonic challenge. Fourier transform infrared microspectroscopy, polarized light microscopy, and mechanical testing were used to quantify differences in proteoglycan and collagen content, collagen orientation, and biomechanical properties, respectively, between the intact and degraded cartilage. Collagen content decreased and collagen orientation angle increased significantly (p < 0.05) in the superficial zone cartilage after collagenase treatment, and the instantaneous modulus of the samples was reduced significantly (p < 0.05). Normalized cell volume and height 20 min after the osmotic challenge (with respect to the original volume and height) were significantly (p < 0.001 and p < 0.01, respectively) larger in the intact compared to the degraded cartilage. These findings suggest that the mechanical environment of chondrocytes, specifically collagen content and orientation, affects cell volume and shape changes in the superficial zone articular cartilage when exposed to osmotic loading. This emphasizes the role of collagen in modulating cartilage mechanobiology in diseased tissue.     

Peculiarities of cytometrical methods of DNA content determination in the nucleus

This work has proposed a new theoretical approach to analysis of histograms of DNA content, which are obtained by the method of flow cytometry, in cells of Drosophila melanogaster imaginal discs. The precision of measurements of the DNA amount in G₁ and G₂(M) phases has been shown to be limited by precision of instrument tuning of zero of the flow cytometer. Use of the calculative zero of the flow cytometer and of dividing cells as standards of the DNA content is able to increase severalfold the precision of the DNA measurements in nuclei of the species. Comparative analysis of errors of various methods of measurement of the DNA content in cell nuclei is also performed. For methods of flow fluorescent cytometry, confocal scanning, and cytophotometry of the Feulgen-stained nuclei, it has been shown that, at present, the mean square errors of the DNA content measurements are within the interval of values considered acceptable for biological studies (0.02 < CV < 0.06).     

Measurement of local diffusion and composition in degraded articular cartilage reveals the unique role of surface structure in controlling macromolecular transport

Developing effective therapeutics for osteoarthritis (OA) necessitates that such molecules can reach and target chondrocytes within articular cartilage. However, predicting how well very large therapeutic molecules diffuse through cartilage is often difficult, and the relationship between local transport mechanics for these molecules and tissue heterogeneities in the tissue is still unclear. In this study, a 150 kDa antibody diffused through the articular surface of healthy and enzymatically degraded cartilage, which enabled the calculation of local diffusion mechanics in tissue with large compositional variations. Local cartilage composition and structure was quantified with Fourier transform infrared (FTIR) spectroscopy and second harmonic generation (SHG) imaging techniques. Overall, both local concentrations of aggrecan and collagen were correlated to local diffusivities for both healthy and surface-degraded samples (0.3 > R² < 0.9). However, samples that underwent surface degradation by collagenase exhibited stronger correlations (R² > 0.75) compared to healthy samples (R² < 0.46), suggesting that the highly aligned collagen at the surface of cartilage acts as a barrier to macromolecular transport.     

Detection of cartilage matrix degradation by autofluorescence lifetime

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.     

Effects of inserting a pressensor film into articular joints on the actual contact mechanics.

Fuji film has been widely used in studies aimed at obtaining the contact mechanics of articular joints. Once sealed for practical use in biological joints, Fuji Pressensor film has a total effective thickness of 0.30 mm, which is comparable to the cartilage thickness in the joints of many small animals. The average effective elastic modulus of Fuji film is approximately 100 MPa in compression, which is larger by a factor of 100-300 compared to that of normal articular cartilage. Therefore, inserting a Pressensor film into an articular joint will change the contact mechanics of the joint. The measurement precision of the Pressensor film has been determined systematically; however, the changes in contact mechanics associated with inserting the film into joints have not been investigated. This study was aimed at quantifying the changes in the contact mechanics associated with inserting sealed Fuji Pressensor film into joints. Spherical and cylindrical articular joint contact mechanics with and without Pressensor film and for varying degrees of surface congruency were analyzed and compared by using finite element models. The Pressensor film was taken as linearly elastic and the cartilage was assumed to be biphasic, composed of a linear elastic solid phase and an inviscid fluid phase. The present analyses showed that measurements of the joint contact pressures with Fuji Pressensor film will change the maximum true contact pressures by 10-26 percent depending on the loading, geometry of the joints, and the mechanical properties of cartilage. Considering this effect plus the measurement precision of the film (approximately 10 percent), the measured joint contact pressures in a joint may contain errors as large as 14-28 percent.     

Fibril reinforced poroelastic model predicts specifically mechanical behavior of normal,proteoglycan depleted and collagen degraded articular cartilage

Degradation of collagen network and proteoglycan (PG) macromolecules are signs of articular cartilage degeneration. These changes impair cartilage mechanical function. Effects of collagen degradation and PG depletion on the time-dependent mechanical behavior of cartilage are different. In this study, numerical analyses, which take the compression-tension nonlinearity of the tissue into account, were carried out using a fibril reinforced poroelastic finite element model. The study aimed at improving our understanding of the stress-relaxation behavior of normal and degenerated cartilage in unconfined compression. PG and collagen degradations were simulated by decreasing the Young's modulus of the drained porous (nonfibrillar) matrix and the fibril network, respectively. Numerical analyses were compared to results from experimental tests with chondroitinase ABC (PG depletion) or collagenase (collagen degradation) digested samples. Fibril reinforced poroelastic model predicted the experimental behavior of cartilage after chondroitinase ABC digestion by a major decrease of the drained porous matrix modulus (-64+/-28%) and a minor decrease of the fibril network modulus (-11+/-9%). After collagenase digestion, in contrast, the numerical analyses predicted the experimental behavior of cartilage by a major decrease of the fibril network modulus (-69+/-5%) and a decrease of the drained porous matrix modulus (-44+/-18%). The reduction of the drained porous matrix modulus after collagenase digestion was consistent with the microscopically observed secondary PG loss from the tissue. The present results indicate that the fibril reinforced poroelastic model is able to predict specifically characteristic alterations in the stress-relaxation behavior of cartilage after enzymatic modifications of the tissue. We conclude that the compression-tension nonlinearity of the tissue is needed to capture realistically the mechanical behavior of normal and degenerated articular cartilage.     

Vertiquant: A Device for Automatically Recording Vertical Migration of Plankton

Vertical migration is a key subject in understanding zooplankton ecology and its influence on aquatic ecosystems. This paper introduces a device for automatically recording vertical plankton migrations to study proximate factors regulating the stimulus, timing and amplitude of these movements under controlled laboratory conditions. The instrument records the light scattered by organisms at their respective depths and processes the signals in real time to a graphic representation of the organisms vertical distribution. Organisms of different taxa from a size of <40 μ, to > 10 000 μm were used for these experiments. Daphnia migrations in response to UV light are used to demonstrate the basic functions of the instrument.     

Systemic lupus erythematosus and C1q: A quantitative ELISA for determining C1q levels in serum

C1q is of interest in systemic lupus erythematosus (SLE) research due to deficiencies in its activity being associated with the disease. Current published protocols for measuring C1q vary greatly in their results and ease of reproducibility. Due to this, average C1q concentrations have been reported between 56 and 276 μg/mL in non-SLE serum. We present an improved method for quantifying C1q concentrations, which employs a sandwich ELISA. This method has improved precision, cost efficiency, up-scaling, reproducibility, and uses significantly lesser volumes of serum sample when compared to RID and other methods for quantifying C1q. We report an average concentration of 113 ± 40 μg/mL for C1q in non-SLE serum. The assay designed here will be useful in the high-throughput measurement of serum C1q in SLE cases.     

Multiplex single molecule counting technology used to generate interleukin 4, interleukin 6, and interleukin 10 reference limits

Detecting biomarkers at pg/ml concentrations or below is, in many situations, critical for quantifying levels in healthy individuals as well as the changes that can occur in the progression of disease states. The ability to detect multiple biomarkers from the same sample allows for better diagnoses, more efficient testing, and lower volumes of sample required. Based on single molecule counting technology, a multiplex instrument was designed and built that is capable of detecting cytokines and other low-abundance proteins at sub-pg/ml quantities in human plasma samples. The multiplex single molecule counting instrument was used to generate 95% reference limits for interleukin 4 (IL-4, <0.61 pg/ml), interleukin 6 (IL-6, <6.53 pg/ml), and interleukin 10 (IL-10, <1.08 pg/ml) from 100 healthy human donor plasma samples, with more than 90% of IL-4 concentrations and 100% of IL-6 and IL-10 concentrations above the limit of detection.     

Utility of NucleoCounter for the chondrocyte count in the collagenase digest of human native cartilage

In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R² = 0.9999) and reproducibility (%CV: 2.00–8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R² = 0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20–1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions.     

Death and proliferation of chondrocytes in the degraded mandibular condylar cartilage of rats induced by experimentally created disordered occlusion

Objective  To investigate the effect of experimentally created disordered occlusion (ECDO) on cell death and proliferation in rat mandibular condylar cartilage. Methods  Sprague–Dawley rats were randomly assigned to experimental and control groups. In the experimental groups, ECDO was created by the dental orthodontic method. By means of histological evaluation, immunohistochemistry and TUNEL staining, we studied the histomorphological changes, the death and proliferation of chondrocytes. Results  Time- and sex-related progressive histologic degradation was observed in the condylar cartilage of ECDO rats, accompanied with diminished chondrocyte proliferation in the female 12-week ECDO subgroup (P < 0.05). An increase in the number of apoptotic chondrocytes was seen in both the female 8- and 12-week ECDO subgroups and in the male ECDO 12-week subgroup (all P < 0.05), but not in the male ECDO 8-week subgroup (P > 0.05). Conclusion  ECDO induces degradation in the rat condylar cartilage accompanied by an increase in chondrocyte death.     

Rapid determination of the anti-cancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C₁₈ sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4–800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 μl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran™ following single doses of oral tablet formulations.     

Observations on preadipocytes and their distribution patterns in rat adipose tissue

Microscopic examination of adipocytes isolated from adult rat epididymal adipose tissue revealed numerous small cells (< 10 μm) morphologically similar to larger adipocytes. These small adipocytes appear identical to a new classification of adipose cells termed preadipocytes. Electron micrographs of these preadipocytes revealed examples of cells < 10 μm in diameter in various stages of maturation and lipid accumulation. The percent distribution pattern of these small adipocytes was not significantly altered by exercise although exercise shifted the distribution patterns of the larger cells (> 30 μm) toward a smaller mean cell size. The quantitative significance of preadipocytes is not established but these preliminary observations indicate that adipocytes < 10 μm in diameter may account for a numerically greater proportion of the total adipocytes observed in collagenase isolated preparations than heretofore recognized, although their contribution to total adipose mass is probably negligible.     

An in vitro model for the pathological degradation of articular cartilage in osteoarthritis

The objective of this study was to develop an in vitro cartilage degradation model that emulates the damage seen in early-stage osteoarthritis. To this end, cartilage explants were collagenase-treated to induce enzymatic degradation of collagen fibers and proteoglycans at the articular surface. To assess changes in mechanical properties, intact and degraded cartilage explants were subjected to a series of confined compression creep tests. Changes in extracellular matrix structure and composition were determined using biochemical and histological approaches. Our results show that collagenase-induced degradation increased the amount of deformation experienced by the cartilage explants under compression. An increase in apparent permeability as well as a decrease in instantaneous and aggregate moduli was measured following collagenase treatment. Histological analysis of degraded explants revealed the presence of surface fibrillation, proteoglycan depletion in the superficial and intermediate zones and loss of the lamina splendens. Collagen cleavage was confirmed by the Col II–3/4C_short antibody. Degraded specimens experienced a significant decrease in proteoglycan content but maintained total collagen content. Repetitive testing of degraded samples resulted in the gradual collapse of the articular surface and the compaction of the superficial zone. Taken together, our data demonstrates that enzymatic degradation with collagenase can be used to emulate changes seen in early-stage osteoarthritis. Further, our in vitro model provides information on cartilage mechanics and insights on how matrix changes can affect cartilage's functional properties. More importantly, our model can be applied to develop and test treatment options for tissue repair.     

Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the in vivo cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish the value of intra-articular injections of polyphenol in stabilization of cartilage collagen against degradation and indicate the unique beneficial role of injectable polyphenols in protecting the cartilage in arthritic conditions.     

Effect of collagenase–gelatinase ratio on the mechanical properties of a collagen fibril: a combined Monte Carlo–molecular dynamics study

Loading in cartilage is supported primarily by fibrillar collagen, and damage will impair the function of the tissue, leading to pathologies such as osteoarthritis. Damage is initiated by two types of matrix metalloproteinases, collagenase and gelatinase, that cleave and denature the collagen fibrils in the tissue. Experimental and modeling studies have revealed insights into the individual contributions of these two types of MMPs, as well as the mechanical response of intact fibrils and fibrils that have experienced random surface degradation. However, no research has comprehensively examined the combined influences of collagenases and gelatinases on collagen degradation nor studied the mechanical consequences of biological degradation of collagen fibrils. Such preclinical examinations are required to gain insights into understanding, treating, and preventing degradation-related cartilage pathology. To develop these insights, we use sequential Monte Carlo and molecular dynamics simulations to probe the effect of enzymatic degradation on the structure and mechanics of a single collagen fibril. We find that the mechanical response depends on the ratio of collagenase to gelatinase—not just the amount of lost fibril mass—and we provide a possible mechanism underlying this phenomenon. Overall, by characterizing the combined influences of collagenases and gelatinases on fibril degradation and mechanics at the preclinical research stage, we gain insights that may facilitate the development of targeted interventions to prevent the damage and loss of mechanical integrity that can lead to cartilage pathology.

     

The Intra‐ and Inter‐instrument Reliability of DXA Based on Ex Vivo Soft Tissue Measurements

Objective: Comparison of ex‐vivo soft tissue measurements using the GE/Lunar pencil (DPX‐L; GE/Lunar Co., Madison, WI) and fan beam (Prodigy dual‐energy X‐ray absorptiometers (DXA) GE/Lunar Co.). Research Methods and Procedures: Intra‐instrument reliability was assessed by repeatedly scanning soft tissue phantoms for lean tissue (water) and fat tissue (methanol) using one DPX‐L and two identical Prodigy DXAs at fast, medium, and slow scan modes. For each machine, 10 scans of each phantom were performed at each scan speed. The number of scans per instrument totaled 60. Data were analyzed using ANOVA to ascertain whether scan speed affected the intra‐instrument reliability and to test whether soft tissue measurements differed among instruments. Percentage fat (phantom density) was the outcome variable. Results: Intra‐instrument reliability, expressed as coefficient of variation, ranged between 0.7% and 5.2% for the DPX‐L and 0.4% and 4.5% for the Prodigy, with the lowest coefficients of variation observed when scanning the fat tissue phantom. Scan speed also affected the intra‐instrument reliability (p < 0.01). Furthermore, differences in the measurement of percentage body fat for both the lean and fat tissue phantoms were observed among all three absorptiometers (all p < 0.01). After adjusting for scan speed, differences persisted for all three instruments. Discussion: Intra‐ and inter‐instrument reliability of DXA machines, even those from the same manufacturer, remains unpredictable. Thus, when measuring body composition using DXA, it is important to consider that even in the absence of measurement bias, the use of different DXA machines, particularly when using a variety of speed settings, will increase the residual error around the true value.     

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.     

An experimental comparison of respiration measuring techniques in fermenters and shake flasks: exhaust gas analyzer vs. RAMOS device vs. respirometer

Respiration measurement is applied as a universal tool to determine the activity of biological systems. The measurement techniques are difficult to compare, due to the vast variety of devices and analytical procedures commonly in use. They are used in fields as different as microbiology, gene engineering, toxicology, and industrial process monitoring to observe the physiological activity of living systems in environments as diverse as fermenters, shake flasks, lakes and sewage plants. A method is introduced to determine accuracy, quantitation limit, range and precision of different respiration measurement devices. Corynebacterium glutamicum cultures were used to compare an exhaust gas analyzer (EGA), a RAMOS device (respiration measurement in shake flasks) and a respirometer. With all measuring devices it was possible to determine the general culture characteristics. The EGA and the RAMOS device produced almost identical results. The scatter of the respirometer was noticeably higher. The EGA is the technique of choice, if the reaction volume is high or a short reaction time is required. The possibility to monitor cultures simultaneously makes the RAMOS device an indispensable tool for media and strain development. If online monitoring is not compulsive, the respiration of the investigated microbial system extremely low, or the sample size small, a respirometer is recommended.     

Estimation of the commercial height of trees with laser meter: a viable alternative for forest management in the Brazilian Amazon

Commercial height of the tree is a key variable for estimating the wood stock in tropical forests managed for timber production purposes. Most available measurement devices suffer limitations in this type of forest, promoting low precision measurements with high variation errors. The laser meter device appears as a viable alternative, as in addition to using trigonometric principles, it is not necessary that the device is close to the eyes of the meter to carry out the measurement. The device can be used to measure commercial height of trees on flat or sloping terrain, at different distances from the tree. However, there are no studies evaluating the precision of this device. The objective of this study was to determine the precision of the laser meter method for estimating the commercial height of trees, as compared to the actual measurement in a tropical forest in the Brazilian Amazon. Measurements were made on 300 trees with commercial height between 7 and 14 m. Actual commercial heights were measured with graduated ruler. Applied tests were paired t test, graphical analysis of residuals and calculations of bias statistics, mean absolute deviation, standard deviation of differences, and coefficient of determination (R²). Paired t test indicated that the mean of the heights measured by the laser meter is statistically equal to that of the graduated ruler. Measurements with laser meter did not show bias and had mean error of 0.0745. The standard deviation of the differences indicated dispersion of errors of 0.97, equal to that shown in the graduated rule. Laser meter presents an alternative method for estimating the commercial height of trees in tropical forest in the Brazilian Amazon. There was no tendency to underestimate or overestimate the commercial heights of trees. Use of the laser meter is potentially of use for measuring the commercial height of trees in tropical forests.