A combined experimental atomic force microscopy-based nanoindentation and computational modeling approach to unravel the key contributors to the time-dependent mechanical behavior of single cells.

Cellular responses to mechanical stimuli are influenced by the mechanical properties of cells and the surrounding tissue matrix. Cells exhibit viscoelastic behavior in response to an applied stress. This has been attributed to fluid flow-dependent and flow-independent mechanisms. However, the particular mechanism that controls the local time-dependent behavior of cells is unknown. Here, a combined approach of experimental AFM nanoindentation with computational modeling is proposed, taking into account complex material behavior. Three constitutive models (porohyperelastic, viscohyperelastic, poroviscohyperelastic) in tandem with optimization algorithms were employed to capture the experimental stress relaxation data of chondrocytes at 5 % strain. The poroviscohyperelastic models with and without fluid flow allowed through the cell membrane provided excellent description of the experimental time-dependent cell responses (normalized mean squared error (NMSE) of 0.003 between the model and experiments). The viscohyperelastic model without fluid could not follow the entire experimental data that well (NMSE = 0.005), while the porohyperelastic model could not capture it at all (NMSE = 0.383). We also show by parametric analysis that the fluid flow has a small, but essential effect on the loading phase and short-term cell relaxation response, while the solid viscoelasticity controls the longer-term responses. We suggest that the local time-dependent cell mechanical response is determined by the combined effects of intrinsic viscoelasticity of the cytoskeleton and fluid flow redistribution in the cells, although the contribution of fluid flow is smaller when using a nanosized probe and moderate indentation rate. The present approach provides new insights into viscoelastic responses of chondrocytes, important for further understanding cell mechanobiological mechanisms in health and disease.

Ability of ultrasound imaging to detect erosions in a bone phantom model.

OBJECTIVES: The authors examined the validity, interobserver reliability and interscanner variation in detecting bone erosions with ultrasonography using a custom-made phantom. METHODS: 21 bovine bones were used. Artificial erosions were made into 15 bones and six bones were left as controls. In the processed bones the numbers of erosions, their depths and widths varied between 1-7, 1-4 and 1.5-5 mm, respectively. Each bone was coated with polyvinyl alcohol cryogel to mimic overlying soft tissue and to hide the erosions. Four musculoskeletal sonography experts scanned the 21 blind-coded phantoms using one of the three sets of ultrasound equipment. Finally, quality assurance measurements of the ultrasound equipment was carried out using two additional bone samples. RESULTS: The sonographers detected the erosions successfully with ultrasound. The mean correlation coefficient for a correct result in terms of the number of erosions detected was 0.88 (range 0.75-0.975). The overall Cohen's kappa coefficient for interobserver agreement was 0.683 in terms of discrimination between healthy bones and bones with erosions. The different sets of equipment showed that their overall performance was equal. CONCLUSIONS: The sonographers had good correlations with the number of erosions and they were successful in separating healthy bones from bones with erosions. It seems that neither depth nor width is crucial but that in experimental conditions a 1.5 mm erosion width was the limit for the resolution with current ultrasound equipment. Ultrasound is a valid and reliable method of detecting cortical bone erosions in vitro, when the round erosion is at least 1 mm deep and 1.5 mm wide.

Quantitative ultrasound imaging detects degenerative changes in articular cartilage surface and subchondral bone.

Previous studies have suggested that quantitative ultrasound imaging could sensitively diagnose degeneration of the articular surface and changes in the subchondral bone during the development of osteoarthrosis (OA). We have recently introduced a new parameter, ultrasound roughness index (URI), for the quantification of cartilage surface roughness, and successfully tested it with normal and experimentally degraded articular surfaces. In this in vitro study, the applicability of URI was tested in bovine cartilage samples with spontaneously developed tissue degeneration. Simultaneously, we studied the sensitivity of quantitative ultrasound imaging to detect degenerative changes in the cartilage-bone interface. For reference, histological degenerative grade of the cartilage samples was determined. Mechanical reference measurements were also conducted. Cartilage surface roughness (URI) was significantly (p<0.05) higher in histologically degenerated samples with inferior mechanical properties. Ultrasound reflection at the cartilage-bone interface was also significantly (p<0.05) increased in degenerated samples. Furthermore, it was quantitatively confirmed that ultrasound attenuation in the overlying cartilage significantly affects the measured ultrasound reflection values from the cartilage-bone interface. To conclude, the combined ultrasound measurement of the cartilage surface roughness and ultrasound reflection at the cartilage-bone interface complement each other, and may together enable more sensitive and quantitative diagnosis of early OA or follow up after surgical cartilage repair.

Acoustic properties of articular cartilage under mechanical stress.

Mechano-acoustic and elastographic techniques may provide quantitative means for the in vivo diagnostics of articular cartilage. These techniques assume that sound speed does not change during tissue loading. As articular cartilage shows volumetric changes during compression, acoustic properties of cartilage may change affecting the validity of mechano-acoustic measurements. In this study, we examined the ultrasound propagation through human, bovine and porcine articular cartilage during stress-relaxation in unconfined compression. The time of flight (TOF) technique with known cartilage thickness (true sound speed) as well as in situ calibration method [Suh, Youn, Fu, J. Biomech. 34 (2001), 1347-1353] were used for the determination of sound speed. Ultrasound speed and attenuation decreased in articular cartilage during ramp compression, but returned towards the level of original values during relaxation. Variations in ultrasound speed induced an error in strain and compressive moduli provided that constant ultrasound speed and time-of-flight data was used to determine the tissue thickness. Highest errors in strain (-11.8 +/- 12.0%) and dynamic modulus (15.4 +/- 17.9%) were recorded in bovine cartilage. TOF and in situ calibration methods yielded different results for changes in sound speed during compression. We speculate that the variations in acoustic properties in loaded cartilage are related to rearrangement of the interstitial matrix, especially to that of collagen fibers. In human cartilage the changes, are, however relatively small and, according to the numerical simulations, mechano-acoustic techniques that assume constant acoustic properties for the cartilage will not be significantly impaired by this phenomenon.

Cellulose sponge as a scaffold for cartilage tissue engineering.

One goal of functional tissue engineering is to manufacture scaffolds infiltrated with chondrocytes which are suitable for transplantation into the lesion areas of articular cartilage. Various research strategies are used to fabricate cartilage transplants which would have the correct phenotype, contain enough extracellular matrix components, and have structural and biomechanical properties equivalent to normal articular cartilage. We have investigated the suitability of viscose cellulose sponges as a scaffold for cartilage tissue engineering. The sponges were tested alone, or with recombinant human type II collagen cross-linked inside the material. Scanning electron microscopy and confocal microscopy were used to study the structure of the scaffold during four weeks of cultivation. Cellulose and cellulose/recombinant type II collagen sponges were biocompatible for at least four weeks in cultivation, and gradual filling of the scaffold was observed. However, the constructs remained soft during the observation period, and were devoid of extracellular matrix composition typical for normal articular cartilage.

In situ and ex vivo evaluation of an arthroscopic indentation instrument to estimate the health status of articular cartilage in the equine metacarpophalangeal joint.

OBJECTIVE: To evaluate an arthroscopic indentation instrument (Artscan 200) for assessment of the health status of equine articular cartilage. STUDY DESIGN: In vitro experiment using equine isolated proximal phalanx (P1) specimens. SAMPLE POPULATION: P1 specimens from 39 horses (aged 1.5-22 years). METHODS: Reproducibility was tested by determination of the coefficient of variation (CV). Dynamic modulus and cartilage degeneration index (CDI) values were measured at 2 predefined sites (site 1, joint margin; site 2, joint center) to assess the accuracy and to evaluate the relation with surface integrity. RESULTS: CV was 9.0%. A significant decrease in indenter force was identified when dynamic modulus values decreased to <2.5 MPa (range of tested samples 0.9-8.1 MPa) and when CDI values at site 1 increased to >50% (range 5.4-72.8%). CONCLUSIONS: Technique reproducibility was adequate but accuracy was limited. The device identified degeneration-associated decreases in cartilage stiffness only when the mechanical properties of the cartilage were considerably changed. CLINICAL RELEVANCE: Usefulness of this indentation instrument during arthroscopic surgery would be limited in the initial phase of OA-like cartilage degeneration, but may yield important information in more advanced OA.

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.

Influence of age, site, and degenerative state on the speed of sound in equine articular cartilage.

OBJECTIVE: To determine the speed of sound (SOS) in equine articular cartilage and investigate the influence of age, site in the joint, and cartilage degeneration on the SOS. SAMPLE POPULATION: Cartilage samples from 38 metacarpophalangeal joints of 38 horses (age range, 5 months to 22 years). PROCEDURE: Osteochondral plugs were collected from 2 articular sites of the proximal phalanx after the degenerative state was characterized by use of the cartilage degeneration index (CDI) technique. The SOS was calculated (ratio of needle-probe cartilage thickness to time of flight of the ultrasound pulse), and relationships between SOS value and age, site, and cartilage degeneration were evaluated. An analytical model of cartilage indentation was used to evaluate the effect of variation in true SOS on the determination of cartilage thickness and dynamic modulus with the ultrasound indentation technique. RESULTS: The mean SOS for all samples was 1,696 +/- 126 m/s. Age, site, and cartilage degeneration had no significant influence on the SOS in cartilage. The analytical model revealed that use of the mean SOS of 1,696 m/s was associated with maximum errors of 17.5% on cartilage thickness and 70% on dynamic modulus in an SOS range that covered 95% of the individual measurements. CONCLUSIONS AND CLINICAL RELEVANCE: In equine articular cartilage, use of mean SOS of 1,696 m/s in ultrasound indentation measurements introduces some inaccuracy on cartilage thickness determinations, but the dynamic modulus of cartilage can be estimated with acceptable accuracy in horses regardless of age, site in the joint, or stage of cartilage degeneration.

Localization of sentinel nodes in breast cancer: novel method and device to help pen marking of active nodes during gamma camera imaging.

Gamma camera imaging with Tc-99m marking is a widely used method to locate sentinel lymph nodes (SNs) in breast cancer patients. Prior to SN biopsy, the anterior and lateral location of the SN is marked on the patient's skin using an ink pen. The pen marks guide the surgeon during an operation. However, in many cases the marking is difficult due to limited space under the detectors of a gamma camera. The aim of this study was to improve the pen marking method. Eleven female patients were imaged 3-4 h after injection of Tc-99m labelled Nanocol. Injection was performed to parenchyma surrounding the breast tumour. To facilitate pen marking, two polycarbonate (PC) plates with 40 x 32 holes (spacing=10 mm) were engineered for anterior and lateral side imaging and then installed on the bed of a dual-head gamma camera. Two drops of Tc-99m were placed into the top corners of both the PC plates, in order to trace the corresponding x-y coordinates first from the acquired images and then from the plates. After imaging, the x-y coordinates of the SN(s) were determined from the anterior and lateral side images. Subsequently, the location of each SN was marked with an ink pen on the skin through the small holes in the PC plates. According to the surgeon's evaluation, the distance between the marks and the true location of the SNs was 4.5+/-6.9 mm. Measurements with a custom made phantom revealed that the accuracy of the novel method was significantly (P=0.06) higher as compared with the traditional method (2.7+/-3.0 mm versus 9.2+/-3.0 mm). In addition, we were not able to mark the weakest activity (0.02 MBq) with the traditional method. Taken together, the marking process was considerably easier with the novel method, it had better accuracy and sensitivity than the traditional method and the device is simple enough to be adapted for most gamma cameras.

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.

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.

Ultrasonic quantitation of superficial degradation of articular cartilage.

Ultrasound (US) has been suggested as a means for the quantitative detection of early osteoarthrotic changes in articular cartilage. In this study, the ability of quantitative US 2-D imaging (20 MHz) to reveal superficial changes in bovine articular cartilage after mechanical or enzymatic degradation was investigated in vitro. Mechanical degradation was induced by grinding samples against an emery paper with the grain size of 250 microm, 106 microm, 45 microm or 23 microm. For enzymatic degradation, samples were digested with collagenase, trypsin or chondroitinase ABC. Variations of the US reflection coefficient induced by the degradation were investigated. Furthermore, two novel parameters, the US roughness index (URI) and the spatial variation of the US reflection coefficient (SVR), were established to quantitate the integrity of the cartilage surface. Statistically significant decreases (p < 0.05) in US reflection coefficient were observed after mechanical degradations or enzymatic digestion with collagenase. Increases (p < 0.05) in URI were also revealed after these treatments. We conclude that quantitative US imaging may be used to detect collagen disruption and increased roughness in the articular surface. These structural damages are typical of early osteoarthrosis.

Ultrasound attenuation in normal and spontaneously degenerated articular cartilage.

High-frequency ultrasound (US) measurements may provide means for the quantification of articular cartilage quality. Bovine patellar cartilage samples (n = 32) at various degenerative stages were studied using US attenuation measurements in the 5- to 9-MHz frequency range. The results were compared with the histologic, biochemical and mechanical parameters obtained for the same samples, to identify which structural or functional factors could be related to the attenuation and its variations. Attenuation, as calculated in the frequency or time domain, correlated significantly with the histologic tissue integrity (i.e., Mankin score, Spearman r = -0.576 or -0.571, p < 0.01), but the slope of attenuation vs. frequency was not related to Mankin score. Ultrasound speed was, however, the most sensitive indicator of Mankin score (r = -0.755, p < 0.01). Cartilage quality index (CQI), a combination of structural and functional parameters, correlated significantly with the attenuation or speed (r = -0.655 or -0.872, p < 0.01). Our results suggest that US attenuation and speed may be suited for the diagnostics of cartilage degeneration. (E-mail: )

Prediction of biomechanical properties of articular cartilage with quantitative magnetic resonance imaging.

Quantitative magnetic resonance imaging (MRI) is the most potential non-invasive means for revealing the structure, composition and pathology of articular cartilage. Here we hypothesize that cartilage mechanical properties as determined by the macromolecular framework and their interactions can be accessed by quantitative MRI. To test this, adjacent cartilage disk pairs (n=32) were prepared from bovine proximal humerus and patellofemoral surfaces. For one sample, the tissue Young's modulus, aggregate modulus, dynamic modulus and Poisson's ratio were determined in unconfined compression. The adjacent disk was studied at 9.4T to determine the tissue T(2) relaxation time, sensitive to the integrity of the collagen network, and T(1) relaxation time in the presence of Gd-DTPA, a technique developed for the estimation of cartilage proteoglycan (PG) content. Quantitative MRI parameters were able to explain up to 87% of the variations in certain biomechanical parameters. Correlations were further improved when data from the proximal humerus was assessed separately. MRI parameters revealed a topographical variation similar to that of mechanical parameters. Linear regression analysis revealed that Young's modulus of cartilage may be characterized more completely by combining both collagen- and PG-sensitive MRI parameters. The present results suggest that quantitative MRI can provide important information on the mechanical properties of articular cartilage. The results are encouraging with respect to functional imaging of cartilage, although in vivo applicability may be limited by the inferior resolution of clinical MRI instruments.

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.

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.

Experimental and numerical validation for the novel configuration of an arthroscopic indentation instrument.

Softening of articular cartilage, mainly attributable to deterioration of superficial collagen network and depletion of proteoglycans, is a sign of incipient osteoarthrosis. Early diagnosis of osteoarthrosis is essential to prevent the further destruction of the tissue. During the past decade, a few arthroscopic instruments have been introduced for the measurement of cartilage stiffness; these can be used to provide a sensitive measure of cartilage status. Ease of use, accuracy and reproducibility of the measurements as well as a low risk of damaging cartilage are the main qualities needed in any clinically applicable instrument. In this study, we have modified a commercially available arthroscopic indentation instrument to better fulfil these requirements when measuring cartilage stiffness in joints with thin cartilage. Our novel configuration was validated by experimental testing as well as by finite element (FE) modelling. Experimental and numerical tests indicated that it would be better to use a smaller reference plate and a lower pressing force (3 N) than those used in the original instrument (7-10 N). The reproducibility (CV = 5.0%) of the in situ indentation measurements was improved over that of the original instrument (CV = 7.6%), and the effect of material thickness on the indentation response was smaller than that obtained with the original instrument. The novel configuration showed a significant linear correlation between the indenter force and the reference dynamic modulus of cartilage in uncontined compression, especially in soft tissue (r = 0.893, p < 0.001, n = 16). FE analyses with a transversely isotropic poroelastic model indicated that the instrument was suitable for detecting the degeneration of superficial cartilage. In summary, the instrument presented in this study allows easy and reproducible measurement of cartilage stiffness, also in thin cartilage, and therefore represents a technical improvement for the early diagnosis of osteoarthrosis during arthroscopy.

Mechano-acoustic diagnosis of cartilage degeneration and repair.

BACKGROUND: The combined use of high-frequency ultrasound and mechanical indentation has been suggested for the evaluation of cartilage integrity. In this study, we investigated the usefulness of high-resolution B-mode ultrasound imaging and quantitative mechanical measurements for the diagnosis of cartilage degeneration and for monitoring tissue-healing after autologous chondrocyte transplantation. METHODS: In the first study, osteochondral samples (n = 32) were obtained from the lateral facet of a bovine patella, and the samples were visually classified as intact (n = 13) or degenerated (n = 19) and were graded with use of the Mankin scoring system. Samples were imaged with use of a 20-MHz ultrasound instrument, and the dynamic modulus (Edyn) of cartilage was determined in unconfined compression with use of a high-resolution materials tester. In the second study, cartilage chondrocytes were harvested from the low-weight-bearing area of six-month-old porcine knee joints and cultured. A month later, a cartilage lesion was created on the facet of the femoral trochlea and was repaired with use of the autologous chondrocyte transplantation technique (n = 10). Three months later, to estimate cartilage Edyn, the repair tissue, the adjacent cartilage, and the sham-operated contralateral joint cartilage (control) were analyzed in situ with an arthroscopic indentation instrument. Subsequently, the same sites were imaged with ultrasound. RESULTS: All visually degenerated bovine samples (mean Mankin score = 4) and five visually normal samples (Mankin score = 1) showed reduced Edyn (<2.1 MPa) as compared with histologically normal cartilage (Edyn = 13.8 +/- 3.2 MPa, Mankin score = 0). Cartilage stiffness, as shown by the indenter force, was lower (0.6 +/- 0.3 N, p < 0.05, Wilcoxon's signed-rank test) in the porcine tissue repaired with autologous chondrocyte transplantation than it was in the adjacent (1.6 +/- 0.1 N) or the control (1.9 +/- 0.4 N) tissue. The superficial and internal structure of the degenerated and repaired tissue, including the subchondral erosion at the repair site, was sensitively demonstrated by the ultrasound imaging. CONCLUSIONS: Measurement of cartilage Edyn is an objective method with which to follow changes in the mechanical integrity of cartilage. B-mode ultrasound imaging offers detailed information on the structural properties of cartilage and subchondral bone.

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.