Biomedical subjects

Jarno Rieppo

Publications and source records attributed to Jarno Rieppo.

13 recordsLinked to original sources

PubMed2006

Collagen network of articular cartilage modulates fluid flow and mechanical stresses in chondrocyte.

The extracellular matrix of articular cartilage modulates the mechanical signals sensed by the chondrocytes. In the present study, a finite element model (FEM) of the chondrocyte and its microenvironment was reconstructed using the information from fourier transform infrared imaging spectroscopy. This environment consisted of pericellular, territorial (mainly proteoglycans), and inter-territorial (mainly collagen) matrices. The chondrocyte, pericellular, and territorial matrix were assumed to be mechanically isotropic and poroelastic, whereas the inter-territorial matrix, due to its high collagen content, was assumed to be transversely isotropic and poroelastic. Under instantaneous strain-controlled compression, the FEM indicated that the fluid pressure within the chondrocyte increased nonlinearly as a function of the in-plane Young's modulus of the collagen network. Under instantaneous force-controlled compression, the chondrocyte experienced the highest fluid pressure when the in-plane Young's modulus of the collagen network was approximately 4 MPa. Based on the present results, the mechanical characteristics of the collagen network of articular cartilage can modify fluid flow and stresses in chondrocytes. Therefore, the integrity of the collagen network may be an important determinant in cell stimulation and in the control of the matrix maintenance.

Biophysical Phenomena↗

PubMed2006

Collagen network primarily controls Poisson's ratio of bovine articular cartilage in compression.

The equilibrium Young's modulus of articular cartilage is known to be primarily determined by proteoglycans (PGs). However, the relation between the Poisson's ratio and the composition and structure of articular cartilage is more unclear. In this study, we determined Young's modulus and Poisson's ratio of bovine articular cartilage in unconfined compression. Subsequently, the same samples, taken from bovine knee (femoral, patellar and tibial cartilage) and shoulder (humeral cartilage) joints, were processed for quantitative microscopic analysis of PGs, collagen content, and collagen architecture. The Young's modulus, Poisson's ratio, PG content (estimated with optical density measurements), collagen content, and birefringence showed significant topographical variation (p < 0.05) among the test sites. Experimentally the Young's modulus was strongly determined by the tissue PG content (r = 0.86, p < 0.05). Poisson's ratio revealed a significant negative linear correlation (r = -0.59, p < 0.05) with the collagen content, as assessed by the Fourier transform infrared imaging. Finite element analyses, conducted using a fibril reinforced biphasic model, indicated that the mechanical properties of the collagen network strongly affected the Poisson's ratio. We conclude that Poisson's ratio of articular cartilage is primarily controlled by the content and organization of the collagen network.

Animals↗

PubMed2005

Site-specific ultrasound reflection properties and superficial collagen content of bovine knee articular cartilage.

Previous quantitative 2D-ultrasound imaging studies have demonstrated that the ultrasound reflection measurement of articular cartilage surface sensitively detects degradation of the collagen network, whereas digestion of cartilage proteoglycans has no significant effect on the ultrasound reflection. In this study, the first aim was to characterize the ability of quantitative 2D-ultrasound imaging to detect site-specific differences in ultrasound reflection and backscattering properties of cartilage surface and cartilage-bone interface at visually healthy bovine knee (n = 30). As a second aim, we studied factors controlling ultrasound reflection properties of an intact cartilage surface. The ultrasound reflection coefficient was determined in time (R) and frequency domains (IRC) at medial femoral condyle, lateral patello-femoral groove, medial tibial plateau and patella using a 20 MHz ultrasound imaging instrument. Furthermore, cartilage surface roughness was quantified by calculating the ultrasound roughness index (URI). The superficial collagen content of the cartilage was determined using a FT-IRIS-technique. A significant site-dependent variation was shown in cartilage thickness, ultrasound reflection parameters, URI and superficial collagen content. As compared to R and IRC, URI was a more sensitive parameter in detecting differences between the measurement sites. Ultrasound reflection parameters were not significantly related to superficial collagen content, whereas the correlation between R and URI was high. Ultrasound reflection at the cartilage-bone interface showed insignificant site-dependent variation. The current results suggest that ultrasound reflection from the intact cartilage surface is mainly dependent on the cartilage surface roughness and the collagen content has a less significant role.

Animals↗

PubMed2004

The hyaluronan synthesis inhibitor 4-methylumbelliferone prevents keratinocyte activation and epidermal hyperproliferation induced by epidermal growth factor.

Since excessive epidermal hyaluronan is associated with hyperproliferative states and disturbed terminal differentiation of the keratinocytes, we hypothesized that 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthesis, could counteract these phenotypic features. Cultured epidermal keratinocytes showed a concentration dependent, maximum 83% reduction of hyaluronan in the presence of 0.2-1.0 mM 4-MU, whereas less decline was seen in the synthesis of chondroitin and heparan sulfate. The reduced hyaluronan was associated with no apparent change in its molecular mass. The 4-MU-treated keratinocytes showed an accentuated epithelial morphology with a flat, round cell shape, increased stress fibers and large vinculin-positive adhesion plaques, cytoskeletal changes consistent with the markedly reduced migration rate observed in scratched monolayer cultures. High concentrations of 4-MU also caused a block in keratinocyte proliferation, reversible upon 4-MU withdrawal. In the epidermis of organotypic cultures, 4-MU prevented the hyaluronan accumulation and epidermal hypertrophy induced by epidermal growth factor. The present results concur with earlier data indicating that enhanced cell locomotion and proliferation are associated with hyaluronan synthesis in activated keratinocytes. Cell proliferation, however, was blocked more strongly than expected on the basis of the incomplete hyaluronan synthesis inhibition, and may represent a novel target of 4-MU. At any rate, 4-MU and equivalent hyaluronan synthesis inhibitors might be considered for situations where suppression of epidermal activation and hyperproliferation is warranted.

Animals↗

PubMed2004

Reference sample method reduces the error caused by variable cryosection thickness in Fourier transform infrared imaging.

Fourier transform infrared imaging (FT-IRI) is a novel technique for characterization of the biochemical composition of biological tissues, e.g., articular cartilage. The use of cryosections is preferred in FT-IRI. Unfortunately, significant variation in section thickness often impairs the suitability of cryosections for quantitative FT-IRI analysis. The present study introduces an inexpensive reference sample method for quantitative analysis. In this technique, specimen absorption is normalized with that of nitrocellulose membrane embedded and cryosectioned with the sample. Mean variation of the infrared absorption in cartilage specimens was 11.5%, 12.1%, and 20.6% for 5 microm, 10 microm, and 14 microm thick sections, respectively, without normalization. Normalization reduced the variation to 5.2%, 4.0%, and 4.6% for the same sections, respectively. The normalization method enables usage of cryosections for quantitative work and significantly reduces the cost and time needed for FT-IRI analysis.

Animals↗

PubMed2004

Mechano-acoustic determination of Young's modulus of articular cartilage.

The compressive stiffness of an elastic material is traditionally characterized by its Young's modulus. Young's modulus of articular cartilage can be directly measured using unconfined compression geometry by assuming the cartilage to be homogeneous and isotropic. In isotropic materials, Young's modulus can also be determined acoustically by the measurement of sound speed and density of the material. In the present study, acoustic and mechanical techniques, feasible for in vivo measurements, were investigated to quantify the static and dynamic compressive stiffness of bovine articular cartilage in situ. Ultrasound reflection from the cartilage surface, as well as the dynamic modulus were determined with the recently developed ultrasound indentation instrument and compared with the reference mechanical and ultrasound speed measurements in unconfined compression (n=72). In addition, the applicability of manual creep measurements with the ultrasound indentation instrument was evaluated both experimentally and numerically. Our experimental results indicated that the sound speed could predict 47% and 53% of the variation in the Young's modulus and dynamic modulus of cartilage, respectively. The dynamic modulus, as determined manually with the ultrasound indentation instrument, showed significant linear correlations with the reference Young's modulus (r(2)=0.445, p<0.01, n=70) and dynamic modulus (r(2)=0.779, p<0.01, n=70) of the cartilage. Numerical analyses indicated that the creep measurements, conducted manually with the ultrasound indentation instrument, were sensitive to changes in Young's modulus and permeability of the tissue, and were significantly influenced by the tissue thickness. We conclude that acoustic parameters, i.e. ultrasound speed and reflection, are indicative to the intrinsic mechanical properties of the articular cartilage. Ultrasound indentation instrument, when further developed, provides an applicable tool for the in vivo detection of cartilage mechano-acoustic properties. These techniques could promote the diagnostics of osteoarthrosis.

Animals↗

PubMed2004

Undersulfated chondroitin sulfate does not increase in osteoarthritic cartilage.

OBJECTIVE: To test whether there is undersulfation of chondroitin sulfate in osteoarthritic bovine articular cartilage to support the hypothesis that sulfate deficiency is involved with the development of osteoarthritis. METHODS: Cartilage samples from bovine patellae (n = 32) were divided into 3 groups based on their osteoarthritic progression, as assessed by modified Mankin score. Uronic acid contents of the samples were determined. Fragmentation of the proteoglycans due to proteolytic processing was estimated with agarose gel electrophoresis. The molar ratios of chondroitin sulfate isoforms in the extracted proteoglycans were determined with fluorophore-assisted carbohydrate electrophoresis. RESULTS: Loss of proteoglycans and accumulation of tissue water was evident in groups II and III, and progressive OA increased heterogeneity of aggrecan population in groups II and III. Importantly, the molar ratio of nonsulfated disaccharide was decreased in the osteoarthritic articular cartilage. CONCLUSION: The structure of chondroitin sulfate in degenerated bovine cartilage did not support the hypothesis that sulfate depletion is present in osteoarthritic joint.

Animals↗

PubMed2003

Ultrasound indentation of bovine knee articular cartilage in situ.

We have earlier developed a handheld ultrasound indentation instrument for the diagnosis of articular cartilage degeneration. In ultrasound indentation, cartilage is compressed with the ultrasound transducer. Tissue thickness and deformation are calculated from the A-mode ultrasound signal and the stress applied is registered with the strain gauges. In this study, the applicability of the ultrasound indentation instrument to quantify site-dependent variation in the mechano-acoustic properties of bovine knee cartilage was investigated. Osteochondral blocks (n=6 per site) were prepared from the femoral medial condyle (FMC), the lateral facet of the patello-femoral groove (LPG) and the medial tibial plateau (MTP). Cartilage stiffness (dynamic modulus, E(dyn)), as obtained with the ultrasound indentation instrument in situ, correlated highly linearly (r=0.913, p<0.01) with the values obtained using the reference material-testing device in vitro. Reproducibility (standardized coefficient of variation) of the ultrasound indentation measurements was 5.2%, 1.7% and 3.1% for E(dyn), ultrasound reflection coefficient of articular surface (R) and thickness, respectively. E(dyn) and R were site dependent (p<0.05, Kruskall-Wallis H test). E(dyn) was significantly higher (p<0.05, Kruskall-Wallis Post Hoc test) in LPG (mean+/-SD: 10.1+/-3.1MPa) than in MTP (2.9+/-1.4MPa). In FMC, E(dyn) was 4.6+/-1.3MPa. R was significantly (p<0.05) lower at MTP (2.0+/-0.7%) than at other sites (FMC: 4.2+/-0.9%; LPG: 4.4+/-0.8%). Cartilage glycosaminoglycan concentration, as quantified with the digital densitometry, correlated positively with E(dyn) (r=0.678, p<0.01) and especially with the equilibrium Young's modulus (reference device, r=0.874, p<0.01) but it was not associated with R (r=0.294, p=0.24). We conclude that manual measurements are reproducible and the instrument may be used for detection of cartilage quality in situ. Especially, combined measurement of thickness, E(dyn) and R provides valuable diagnostic information on cartilage status.

Animals↗

PubMed2003

Speed of sound in normal and degenerated bovine articular cartilage.

The unknown and variable speed of sound may impair accuracy of the acoustic measurement of cartilage properties. In this study, relationships between the speed of sound and cartilage composition, mechanical properties and degenerative state were studied in bovine knee and ankle cartilage (n = 62). Further, the effect of speed variation on the determination of cartilage thickness and stiffness with ultrasound (US) indentation was numerically simulated. The speed of sound was significantly (n = 32, p < 0.05) dependent on the cartilage water content (r = -0.800), uronic acid content (per wet weight, r = 0.886) and hydroxyproline content (per wet weight, r = 0.887, n = 28), Young's modulus at equilibrium (r = 0.740), dynamic modulus (r = 0.905), and degenerative state (i.e., Mankin score) (r = -0.727). In addition to cartilage composition, mechanical and acoustic properties varied significantly between different anatomical locations. In US indentation, cartilage is indented with a US transducer. Deformation and thickness of tissue are calculated using a predefined speed of sound and used in determination of dynamic modulus. Based on the simulations, use of the mean speed of sound of 1627 m/s (whole material) induced a maximum error of 7.8% on cartilage thickness and of 6.2% on cartilage dynamic modulus, as determined with the US indentation technique (indenter diameter 3 mm). We believe that these errors are acceptable in clinical US indentation measurements.

Animals↗

PubMed2003

Structure-function relationships in enzymatically modified articular cartilage.

The present study is aimed at revealing structure-function relationships of bovine patellar articular cartilage. Collagenase, chondroitinase ABC and elastase were used for controlled and selective enzymatic modifications of cartilage structure, composition and functional properties. The effects of the enzymatic degradations were quantitatively evaluated using quantitative polarized light microscopy, digital densitometry of safranin O-stained sections as well as with biochemical and biomechanical techniques. The parameters related to tissue composition and structure were correlated with the indentation stiffness of cartilage. In general, tissue alterations after enzymatic digestions were restricted to the superficial cartilage. All enzymatic degradations induced superficial proteoglycan (PG) depletion. Collagenase also induced detectable superficial collagen damage, though without causing cartilage fibrillation or tissue swelling. Quantitative microscopic techniques were more sensitive than biochemical methods in detecting these changes. The Young's modulus of cartilage decreased after enzymatic treatments indicating significant softening of the tissue. The PG concentration of the superficial zone proved to be the major determinant of the Young's modulus (r(2) = 0.767, n = 72, p < 0.001). Results of the present study indicate that specific enzymatic degradations of the tissue PGs and collagen can provide reproducible experimental models to clarify the structure-function relationships of cartilage. Effects of these models mimic the changes observed in early osteoarthrosis. Biomechanical testing and quantitative microscopic techniques proved to be powerful tools for detecting the superficial structural and compositional changes while the biochemical measurements on the whole uncalcified cartilage were less sensitive.

Animals↗

PubMed2002

Spatial assessment of articular cartilage proteoglycans with Gd-DTPA-enhanced T1 imaging.

In Gd-DTPA-enhanced T(1) imaging of articular cartilage, the MRI contrast agent with two negative charges is understood to accumulate in tissue inversely to the negative charge of cartilage glycosaminoglycans (GAGs) of proteoglycans (PGs), and this leads to a decrease in the T(1) relaxation time of tissue relative to the charge in tissue. By assuming a constant relaxivity for Gd-DTPA in cartilage, it has further been hypothesized that the contrast agent concentration in tissue could be estimated from consecutive T(1) measurements in the absence or presence of the contrast agent. The spatial sensitivity of the technique was examined at 9.4 T in normal and PG-depleted bovine patellar cartilage samples. As a reference, spatial PG concentration was assessed with digital densitometry from safranin O-stained cartilage sections. An excellent linear correlation between spatial optical density (OD) of stained GAGs and T(1) with Gd-DTPA was observed in the control and chondroitinase ABC-treated cartilage specimens, and the MR parameter accounted for approximately 80% of the variations in GAG concentration within samples. Further, the MR-resolved Gd-DTPA concentration proved to be an even better estimate for PGs, with an improved correlation. However, the linear relation between MR parameters and PG concentration did not apply in the deep tissue, where MR measurements overestimated the PG content. While the absolute [Gd-DTPA] determination may be prone to error due to uncertainty of relaxivity in cartilage, or to other contributing factors such as variations in tissue permeability, the experimental evidence highlights the sensitivity of this technique to reflect spatial changes in cartilage PG concentration in normal and degenerated tissue.

Animals↗

PubMed2002

Real-time ultrasound analysis of articular cartilage degradation in vitro.

The sensitivity of the reflection coefficient, attenuation and velocity to the enzymatic degradation of bovine patellar cartilage was evaluated in real-time with high-frequency ultrasound (US) (29.4 MHz). These parameters were estimated from the radiofrequency (RF) signal, which was recorded at 5-min intervals during the digestion of the tissue by collagenase or by trypsin. The coefficient of reflection at cartilage surface decreased by 78.5% and 10.5% (p < 0.05) after 6 h of exposure to collagenase and 4 h of exposure to trypsin, respectively. During the trypsin digestion, the attenuation in cartilage increased by 0.274 dB/mm (p < 0.05) and the velocity decreased by 7 m/s (p < 0.05). The coefficient of reflection at the cartilage surface was the most sensitive acoustic parameter to the enzymatic degradation of cartilage and may be the easiest to implement for clinical diagnosis of cartilage quality. US velocity was found to be insensitive to degradation. The small difference in mean velocity between the control and degraded cartilage suggests that a constant predefined US velocity value can be used to obtain diagnostically acceptable measurement of the cartilage thickness.

Animals↗

PubMed2002

Collagenase-induced changes in articular cartilage as detected by electron-microscopic stereology, quantitative polarized light microscopy and biochemical assays.

The purpose of this study was to compare the ability of electron-microscopic (EM) stereology with quantitative polarized light microscopy (PLM) and biochemical collagen (hydroxyproline) and crosslink (pyridinoline) analyses to detect changes in the superficial collagen network of bovine articular cartilage after digestion in vitro with purified bacterial (Clostridium histolyticum) collagenase (30 U/ml) for 24 and 48 h. Collagen volume (V(V)) and surface (S(V)) densities of the uppermost third of the superficial zone were estimated indirectly from zonal isotropic uniform random sections using collagen length density (L(V)) and average collagen fibril diameter, or its average second power. Collagenase digestion caused a significant decrease in fibril diameter (64 to 62%), V(V) (89 to 95%) and S(V) (64 to 86%) after incubation for 24 and 48 h. Collagen L(V) remained unchanged after 24 h incubation but decreased 63% after 48 h. Collagen concentration per dry weight, assayed biochemically from the whole superficial zone, decreased also significantly (29 to 60%) after 24 and 48 h digestions, respectively. The pyridinoline concentration per dry weight of the superficial zone decreased (31 to 57%) whereas the pyridinoline concentration per collagen remained unchanged. PLM revealed that the birefringence of the uppermost third of the superficial zone was decreased by 36% after digestion for 24 h though the total birefringence of the whole zone was not reduced. However, after 48 h, the birefringence of the whole superficial zone was significantly reduced (76%). All of the techniques compared in this study could detect collagen network degradation in bovine articular cartilage but the EM stereological technique was more sensitive at detecting the changes than PLM or biochemical assays.

Animals↗