H.J. Nieminen

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.

MINIMALLY INVASIVE ULTRASOUND METHOD FOR INTRA-ARTICULAR DIAGNOSTICS OF CARTILAGE DEGENERATION

Quantitative ultrasound imaging (QUI) can be used to evaluate the integrity of articular cartilage and for diagnosing the early signs of osteoarthritis (OA). In this study, we applied a minimally invasive ultrasound imaging technique and investigated its ability to detect superficial degeneration of bovine knee articular cartilage. Intact (n=13), collagenase-digested (n=6) and mechanically degraded (n=7) osteochondral samples (dia.=25 mm) and custom-made phantoms with different degrees of surface roughness (n=8) were imaged using a high-frequency (40 MHz) QUI system. For each sample and phantom, the ultrasound reflection coefficient (R), integrated reflection coefficient (IRC) and ultrasound roughness index (URI) were determined. Furthermore, to evaluate the clinical applicability of intra-articular ultrasound (IAUS) in diagnostics, one intact bovine knee joint was investigated ex vivo using a simulated arthroscopic approach. Differences in the surface characteristics of the phantoms were detected by monitoring changes in the reflection and surface roughness parameters. Both mechanically- and enzymatically-induced degradation were sensitively diagnosed by decreased (p<0.05) reflection (R and IRC) at the cartilage surface. Furthermore, mechanical degradation was detected in the increased (p<0.05) surface roughness (URI). The intra-articular investigation of a bovine knee joint suggested that the IAUS technique may enable minimally invasive, straightforward diagnostics of the degenerative status of the articular surfaces. We conclude that quantitative IAUS imaging can be used for detecting collagen disruption and increased roughness of the articular surface. This quantitative in vivo ultrasound technique could have great clinical value in the diagnostics of joint diseases.

Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography

Summary Objective Collagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC. Method We labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland–Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC. Results Both, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4). Conclusions PTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA.

Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos.

Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) at 2–14 hours post fertilization (hpf) for 1000 (n = 47) or 2000 seconds (n = 47). We compared the size and number of trunk neuromasts and otoliths in sonicated samples to controls (n = 94), and found no statistically significant differences (p > 0.05). While mortality rate was lower in the control group (22.3%) compared to that in the 1000 s (34.0%) and 2000 s (42.6%) levitation groups, the differences were statistically insignificant (p > 0.05). The results suggest that acoustic levitation for less than 2000 sec does not interfere with the development of zebrafish embryos, but may affect mortality rate. Acoustic levitation could potentially be used as a non-contacting wall-less platform for characterizing and manipulating vertebrae embryos without causing major adverse effects to their development.

The potential utility of high-intensity ultrasound to treat osteoarthritis.

Osteoarthritis (OA) is a widespread musculoskeletal disease that reduces quality of life and for which there is no cure. The treatment of OA is challenging since cartilage impedes the local and systemic delivery of therapeutic compounds (TCs). This review identifies high-intensity ultrasound (HIU) as a non-contact technique to modify articular cartilage and subchondral bone. HIU enables new approaches to overcome challenges associated with drug delivery to cartilage and new non-invasive approaches for the treatment of joint disease. Specifically, HIU has the potential to facilitate targeted drug delivery and release deep within cartilage, to repair soft tissue damage, and to physically alter tissue structures including cartilage and bone. The localized, non-invasive ultrasonic delivery of TCs to articular cartilage and subchondral bone appears to be a promising technique in the immediate future.

Strain-dependent modulation of ultrasound speed in articular cartilage under dynamic compression.

Mechanical properties of articular cartilage may be determined by means of mechano-acoustic indentation, a clinically feasible technique for cartilage diagnostics. Unfortunately, ultrasound speed varies in articular cartilage during mechanical compression. This can cause significant errors to the measured mechanical parameters. In this study, the strain-dependent variation in ultrasound speed was investigated during dynamic compression. In addition, we estimated errors that were induced by the variation in ultrasound speed on the mechano-acoustically measured elastic properties of the tissue. Further, we validated a computational method to correct these errors. Bovine patellar cartilage samples (n = 7) were tested under unconfined compression. Strain-dependence of ultrasound speed was determined under different compressive strains using an identical strain-rate. In addition, the modulation of ultrasound speed was simulated using the transient compositional and structural changes derived from fibril-reinforced poroviscoelastic (FRPVE) model. Experimentally, instantaneous compressive strain modulated the ultrasound speed (p < 0.05) significantly. The decrease of ultrasound speed was found to change nonlinearly as a function of strain. Immediately after the ramp loading ultrasound speed was found to be changed -0.94%, -1.49%, -1.84%, -1.87%, -1.89% and -2.15% at the strains of 2.4%, 4.9%, 7.3%, 9.7%, 12.1% and 14.4%, respectively. The numerical simulation revealed that the compression-related decrease in ultrasound speed induces significant errors in the mechano-acoustically determined strain (39.7%) and dynamic modulus (72.1%) at small strains, e.g., at 2.4%. However, at higher strains, e.g., at 14.4%, the errors were smaller, i.e., 12.6% for strain and 14.5% for modulus. After the proposed computational correction, errors related to ultrasound speed were decreased. By using the correction, with e.g., 2.4% strain, errors in strain and modulus were decreased from 39.7% to 7.2% and from 72.1% to 35.3%, respectively. The FRPVE model, addressing the changes in fibril orientation and void ratio during compression, showed discrepancy of less than 1% between the predicted and measured ultrasound speed during the ramp compression.

Delivering Agents Locally into Articular Cartilage by Intense MHz Ultrasound

There is no cure for osteoarthritis. Current drug delivery relies on systemic delivery or injections into the joint. Because articular cartilage (AC) degeneration can be local and drug exposure outside the lesion can cause adverse effects, localized drug delivery could permit new drug treatment strategies. We investigated whether intense megahertz ultrasound (frequency: 1.138 MHz, peak positive pressure: 2.7 MPa, Ispta: 5 W/cm2, beam width: 5.7 mm at −6 dB, duty cycle: 5%, pulse repetition frequency: 285 Hz, mechanical index: 1.1) can deliver agents into AC without damaging it. Using ultrasound, we delivered a drug surrogate down to a depth corresponding to 53% depth of the AC thickness without causing histologically detectable damage to the AC. This may be important because early osteoarthritis typically exhibits histopathologic changes in the superficial AC. In conclusion, we identify intense megahertz ultrasound as a technique that potentially enables localized non-destructive delivery of osteoarthritis drugs or drug carriers into articular cartilage.

Ultrasonic transport of particles into articular cartilage and subchondral bone

Osteoarthritis (OA) is a debilitating musculoskeletal disease without a cure. Delivery of therapeutic compounds is a problem and localized drug therapy could enable new treatment strategies. In this study high-intensity ultrasound was used to deliver micro- and nano-particles (MNPS) into three bovine osteochondral samples: (1) control (C) that was exposed to the particles without sonication, (2) UST-1 that was sonicated prior to immersion in a MNPS and (3) UST-2 that was sonicated in the presence of MNPS. Following treatment samples were cut into 2.9 ± 0.3 mm sections and digital images were obtained by light microscopy. In the sonicated samples (UST-1 and UST-2) MNPS penetrated into articular cartilage and subchondral bone. No MNPS penetration was observed in C. The proposed technique could potentially be used for local drug treatment of OA.

Differences in Acoustic Properties of Intact and Degenerated Human Patellar Cartilage During Compression

Ultrasound indentation measurements have been shown to provide means to assess cartilage integrity and mechanical properties. To determine cartilage stiffness in the ultrasound indentation geometry, cartilage is compressed with an ultrasound transducer to determine the induced strain from the ultrasound signal using the time-of-flight principle. As the ultrasound speed in cartilage has been shown to vary during compression, the assumption of constant speed generates significant errors in the values of mechanical parameters. This variation in ultrasound speed has been investigated in intact cartilage, however, its existence and significance in degenerated tissue is unknown. In the present study, we investigate this issue with both intact and spontaneously degenerated human tissue. To accomplish this aim, we determined ultrasound speed and attenuation in human patellar cartilage (n=68) during mechanical loading. For reference, cartilage mechanical properties and proteoglycan, collagen and water contents were determined. The acoustic properties were related to the composition and mechanical properties of the samples. Ultrasound speed showed significant, site-dependent variation and it was significantly associated (r=0.79-0.81, p<0.01) with the mechanical properties of cartilage. The compression related decrease in ultrasound speed showed statistically significant variation between different stages of degeneration. Error simulations revealed that changes in ultrasound speed during 2% compression could generate errors up to 15% in the values of elastic moduli of samples with early degeneration, if determined with the ultrasound indentation technique. In samples with advanced degeneration, the error was significantly (p<0.05) smaller being 2% on average. As the compression related variation in ultrasound speed was lower in more degenerated samples, the mechanical parameters could be diagnosed more reliably in tissue showing advanced degeneration. The present results address the need to consider possible uncertainties in mechano-acoustic measurements of articular cartilage and call for methods to correct the effect of variable sound speed during compression.

Effects of articular cartilage constituents on phosphotungstic acid enhanced micro-computed tomography

Contrast-enhanced micro-computed tomography (CEμCT) with phosphotungstic acid (PTA) has shown potential for detecting collagen distribution of articular cartilage. However, the selectivity of the PTA staining to articular cartilage constituents remains to be elucidated. The aim of this study was to investigate the dependence of PTA for the collagen content in bovine articular cartilage. Adjacent bovine articular cartilage samples were treated with chondroitinase ABC and collagenase to degrade the proteoglycan and the collagen constituents in articular cartilage, respectively. Enzymatically degraded samples were compared to the untreated samples using CEμCT and reference methods, such as Fourier-transform infrared imaging. Decrease in the X-ray attenuation of PTA in articular cartilage and collagen content was observed in cartilage depth of 0–13% and deeper in tissue after collagen degradation. Increase in the X-ray attenuation of PTA was observed in the cartilage depth of 13–39% after proteoglycan degradation. The X-ray attenuation of PTA-labelled articular cartilage in CEμCT is associated mainly with collagen content but the proteoglycans have a minor effect on the X-ray attenuation of the PTA-labelled articular cartilage. In conclusion, the PTA labeling provides a feasible CEμCT method for 3D characterization of articular cartilage.