Quantitative Evaluation of Spontaneously and Surgically Repaired Rabbit Articular Cartilage Using Intra-Articular Ultrasound Method in situ

During the last decade, a major effort has been devoted to developing surgical methods for repairing localized articular cartilage lesions. Despite some promising results no ultimate breakthrough in surgical cartilage repair has been achieved. Improvements in repair techniques would benefit from more sensitive and quantitative methods for long-term follow-up of cartilage healing. In this study, the potential of a new ultrasound technique for detecting the compositional and structural changes in articular cartilage after surgery, using recombinant human type II collagen gel and spontaneous repair was, investigated. Rabbit knee joints containing intact (n = 13) and surgically (n = 8) or spontaneously (n = 5) repaired tissue were imaged in situ at 6 months after the operation using a clinical intravascular high-frequency (40 MHz) ultrasound device. Based on the ultrasound raw data, ultrasound reflection coefficient (R), integrated ultrasound reflection coefficient (IRC), apparent integrated backscattering coefficient (AIB) and ultrasound roughness index (URI) were determined for each sample. URI was significantly higher in both repair groups than in intact cartilage (p < 0.05). The reflection parameters (R and IRC) were significantly lower in surgically repaired cartilage (p < 0.05) than in intact cartilage. Furthermore, AIB was significantly higher in surgically repaired cartilage than in intact tissue (p < 0.05). To conclude, the integrity of the rabbit articular cartilage repair could be quantitatively evaluated with the nondestructive ultrasound approach. In addition, clinically valuable qualitative information on the changes in cartilage integration, structure and composition could be extracted from the ultrasound images. In the present study, the structure and properties of repaired tissue were inferior to native tissue at 6 months after the operation. The applied ultrasound device and probes are FDA approved and, thus, applicable for the quantitative in vivo evaluation of human articular cartilage. (E-mail: tviren@hytti.uku.fi)     

Arthroscopic Ultrasound Technique for Simultaneous Quantitative Assessment of Articular Cartilage and Subchondral Bone: An In Vitro and In Vivo Feasibility Study

Traditional arthroscopic examination is subjective and poorly reproducible. Recently, we introduced an arthroscopic ultrasound method for quantitative diagnostics of cartilage lesions. Here we describe our investigation of the feasibility of ultrasound arthroscopy for simultaneous measurements of articular cartilage and subchondral bone. Human osteochondral samples (n = 13) were imaged using a clinical 9-MHz ultrasound system. Ultrasound reflection coefficients (R, IRC), the ultrasound roughness index (URI) and the apparent integrated backscattering coefficient (AIB) were determined for both tissues. Mechanical testing, histologic analyses and micro-scale computed tomography imaging were the reference methods. Ultrasound arthroscopies were conducted on two patients. The ultrasound reflection coefficient correlated with the Mankin score and Young's modulus of cartilage (|r| > 0.56, p < 0.05). Ultrasound parameters (R, IRC, AIB) for subchondral bone correlated with the bone surface/volume ratio (|r| > 0.70, p < 0.05) and trabecular thickness (|r| > 0.59, p < 0.05). Furthermore, R and subchondral bone mineral density were significantly correlated (|r| > 0.65, p < 0.05). Arthroscopic ultrasound examination provided diagnostically valuable information on cartilage and subchondral bone in vivo.     

Ultrasound Evaluation of Site-Specific Effect of Simulated Microgravity on Articular Cartilage

Space flight induces acute changes in normal physiology in response to the microgravity environment. Articular cartilage is subjected to high loads under a ground reaction force on Earth. The objectives of this study were to investigate the site dependence of morphological and ultrasonic parameters of articular cartilage and to examine the site-specific responses of articular cartilage to simulated microgravity using ultrasound biomicroscopy (UBM). Six rats underwent tail suspension (simulated microgravity) for four weeks and six other rats were kept under normal Earth gravity as controls. Cartilage thickness, ultrasound roughness index (URI), integrated reflection coefficient (IRC) and integrated backscatter coefficient (IBC) of cartilage tissues, as well as histological degeneration were measured at the femoral head (FH), medial femoral condyle (MFC), lateral femoral condyle (LFC), patello-femoral groove (PFG) and patella (PAT). The results showed site dependence not significant in all UBM parameters except cartilage thickness (p < 0.01) in the control specimens. Only minor changes in articular cartilage were induced by 4-week tail suspension, although there were significant decreases in cartilage thickness at the MFC and PAT (p < 0.05) and a significant increase in URI at the PAT (p < 0.01). This study suggested that the 4-week simulated microgravity had only mild effects on femoral articular cartilage in the rat model. This information is useful for human spaceflight and clinical medicine in improving understanding of the effect of microgravity on articular cartilage. However, the effects of longer duration microgravity experience on articular cartilage need further investigation. (E-mail: rsguoxia@inet.polyu.edu.hk)     

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.     

Characterization of center frequency and bandwidth of broadband ultrasound reflected by the articular cartilage to subchondral bone interface

Osteoarthritis (OA) produces degenerative changes both in articular cartilage and subchondral bone. During OA, reflection of high frequency ultrasound from the cartilage-bone interface is affected by both changes in attenuation of the cartilage layer and acoustic properties of the interface. The objective of this study was to experimentally investigate the spectral content of ultrasound reflection from the cartilage-bone interface. Specifically, we analyzed the center frequency and -6 dB bandwidth of the broadband high-frequency (40 MHz) ultrasound signal. Intact bovine articular cartilage samples with and without the underlying subchondral bone (n = 6) were measured in vitro using a commercial high-frequency ultrasound scanner. Furthermore, the diagnostic potential of the measurement of center frequency and bandwidth for OA was studied with another series of bovine articular cartilage samples (n = 40) after enzymatic degradations of tissue proteoglycans and collagen. Compared with the reference spectrum at the same depth from a perfect reflector, a major downshift (>51%) of the center frequency and a reduction (>42%) of the bandwidth were observed in both sample groups when analyzing the ultrasound reflection from the cartilage-bone interface. The results suggest that attenuation in the cartilage layer primarily controls the observed downshift of the center frequency and acoustic properties of the subchondral bone play only a minor role in affecting the spectrum of the cartilage-bone interface. Changes in the ultrasound bandwidth of the cartilage-bone interface signals, compared with reference signals, were found to vary more than those in the center frequency in both cartilage sample groups. Compared with pretreatment values, a significant downshift in center frequency (p < 0.01) and a minor reduction in bandwidth of spectra from the cartilage-bone interface were recorded after chemical degradation of proteoglycans with trypsin. In contrast, center frequency and bandwidth of the echoes from the cartilage-bone interface did not change after the chemical degradation of cartilage collagen fibrils. The results suggest that proteoglycan loss, typical to OA, may be detected via the changes in the center frequency of the ultrasound reflected from the cartilage-bone interface. (E-mail: ypzheng@ieee.org, simo.saarakkala@oulu.fi)     

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. (E-mail: panu.kiviranta@uku.fi)     

High-Frequency Ultrasound Imaging of Tidemark In Vitro in Advanced Knee Osteoarthritis

High-frequency ultrasound imaging has been widely adopted for assessment of the degenerative changes of articular cartilage in osteoarthritis (OA). Yet, there are few reports on investigating its capability to evaluate subchondral bone. Here, we employed high-frequency ultrasound imaging (25?MHz) to examine in vitro the tidemark in cylindrical osteochondral disks (n?=?33) harvested from advanced OA knees of humans. We found good correspondence in morphology observed by ultrasound imaging and micro-computed tomography. Ultrasound roughness index (URI) of tidemark was derived from the raw radiofrequency signals to compare with bone quality factors, including bone volume fraction (BV/TV) and bone mineral density (BMD) measured by micro-computed tomography, using the Spearman correlation (ρ). URI of the tidemark was negatively associated with the subchondral plate BV/TV (ρ?=??0.73, p?<0.001), BMD (ρ?=??0.40, p?=?0.020), as well as the underneath trabecular bone BV/TV (ρ?=??0.39, p?=?0.025) and BMD (ρ?=??0.43, p?=?0.012). In conclusion, this preliminary study demonstrated that morphology measured by high-frequency ultrasound imaging could reflect the quality of the subchondral bone. High-frequency ultrasound is a promising imaging tool to evaluate the changes of the subchondral bone in addition to those of the overlying cartilage in OA.     

Effects of Ultrasound Beam Angle and Surface Roughness on the Quantitative Ultrasound Parameters of Articular Cartilage

High-resolution arthroscopic ultrasound imaging provides a potential quantitative technique for the diagnostics of early osteoarthritis. However, an uncontrolled, nonperpendicular angle of an ultrasound beam or the natural curvature of the cartilage surface may jeopardize the reliability of the ultrasound measurements. We evaluated systematically the effect of inclining an articular surface on the quantitative ultrasound parameters. Visually intact (n = 8) and mechanically degraded (n = 6) osteochondral bovine patella samples and spontaneously fibrillated (n = 1) and spontaneously proteoglycan depleted (n = 1) osteochondral human tibial samples were imaged using a 50-MHz scanning acoustic system. The surface of each sample was adjusted to predetermined inclination angles (0, 2, 5 and 7°) and five ultrasound scan lines along the direction of the inclination were analyzed. For each scan line, reflection coefficient (R), integrated reflection coefficient (IRC) and ultrasound roughness index (URI) were calculated. Nonperpendicularity of the cartilage surface was found to affect R, IRC and URI significantly (p < 0.05). Importantly, all ultrasound parameters were able to distinguish (p < 0.05) the mechanically degraded samples from the intact ones even though the angle of incidence of the ultrasound beam varied between 0 and 5° among the samples. Diagnostically, the present findings are important because the natural curvature of the articular surface varies, and a perfect perpendicularity between the ultrasound beam and the surface of the cartilage may be challenging to achieve in a clinical measurement. (Email: erna.kaleva@uku.fi)     

Quantitative Evaluation of Enzyme-Induced Porcine Articular Cartilage Degeneration Based on Observation of Entire Cartilage Layer Using Ultrasound

Enzyme-induced articular cartilage degeneration resembling osteoarthritis was evaluated using a newly defined acoustic parameter, the “averaged magnitude ratio” (AMR), which has been suggested as an indicator of articular cartilage degeneration. In vitro experiments were conducted on porcine cartilage samples digested with trypsin for 2?h (n?=?10) and 4?h (n?=?13) and healthy control samples (n?=?13). AMR was determined with 15- and 25-MHz ultrasound, and the integrated reflection coefficient (IRC) and apparent integrated backscattering coefficient (AIB) were also calculated for comparison. The Young's modulus of superficial cartilage was measured using atomic force microscopy. Performance of the AMR differs between 15 and 25?MHz, possibly because of frequency-related attenuation and resolution of ultrasound. At the proper settings, AMR exhibited a competence similar to that of IRC and AIB in detecting cartilage degeneration and could also detect differences in deeper positions. Furthermore, AMR has the advantages of being easy to measure and requiring no reference material.     

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.     

Novel mechano-acoustic technique and instrument for diagnosis of cartilage degeneration

Fibrillation of articular surface and depletion of proteoglycans are the structural changes related to early osteoarthrosis. These changes make cartilage softer and prone to further degeneration. The aim of the present study was to combine mechanical and acoustic measurements towards quantitative arthroscopic evaluation of cartilage quality. The performance of the novel ultrasound indentation instrument was tested with elastomers and bovine articular cartilage in vitro. The instrument was capable of measuring elastomer thickness (r = 1.000, p < 0.01, n = 8) and dynamic modulus (r = 0.994, p < 0.01, n = 13) reliably. Osteochondral plugs were tested before and after enzymatic degradation of cartilage proteoglycans by trypsin or chondroitinase ABC, and of cartilage collagens by collagenase. Trypsin and collagenase induced a mean decrease of -31.2 +/- 12.3% (+/- SD, p < 0.05) and -22.9 +/- 20.8% (p = 0.08) in dynamic modulus, respectively. Rate of cartilage deformation, i.e. creep rate, increased by +117.8 +/- 71.4% (p < 0.05) and +24.7 +/- 35.1% (p = 0.17) in trypsin and chondroitinase ABC treatments, respectively. Collagenase induced a greater decrease in the ultrasound reflection from the cartilage surface (-54.2 +/- 29.6%, p < 0.05) than trypsin (-17.1 +/- 13.5%, p = 0.08). In conclusion, combined quantitation of tissue modulus, viscoelasticity and ultrasound reflection from the cartilage surface provides a sensitive method to distinguish between normal and degenerated cartilage, and even to discern proteoglycan loss and collagen degradation from each other.     

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. (E-mail: tviren@hytti.uku.fi)     

Ultrasound Arthroscopy of Human Knee Cartilage and Subchondral Bone in Vivo

Arthroscopic ultrasound imaging enables quantitative evaluation of articular cartilage. However, the potential of this technique for evaluation of subchondral bone has not been investigated in vivo. In this study, we address this issue in clinical arthroscopy of the human knee (n = 11) by determining quantitative ultrasound (9 MHz) reflection and backscattering parameters for cartilage and subchondral bone. Furthermore, in each knee, seven anatomical sites were graded using the International Cartilage Repair Society (ICRS) system based on (i) conventional arthroscopy and (ii) ultrasound images acquired in arthroscopy with a miniature transducer. Ultrasound enabled visualization of articular cartilage and subchondral bone. ICRS grades based on ultrasound images were higher (p < 0.05) than those based on conventional arthroscopy. The higher ultrasound-based ICRS grades were expected as ultrasound reveals additional information on, for example, the relative depth of the lesion. In line with previous literature, ultrasound reflection and scattering in cartilage varied significantly (p < 0.05) along the ICRS scale. However, no significant correlation between ultrasound parameters and structure or density of subchondral bone could be demonstrated. To conclude, arthroscopic ultrasound imaging had a significant effect on clinical grading of cartilage, and it was found to provide quantitative information on cartilage. The lack of correlation between the ultrasound parameters and bone properties may be related to lesser bone change or excessive attenuation in overlying cartilage and insufficient power of the applied miniature transducer.     

Dendritic Cells Loaded with Ultrasound-Ablated Tumour Induce in vivo Specific Antitumour Immune Responses

Previous studies have shown that high-intensity focused ultrasound (HIFU) ablation can induce a local inflammation with marked infiltration of dendritic cells (DCs). The purpose of this study was to investigate whether DCs could capture and present activating signals delivered by necrotic tumour cells that remain in situ after HIFU, thus initiating specific antitumour immunity. Tumour debris was derived from a mouse H22 tumour model after HIFU ablation. Bone marrow-derived DCs were loaded with HIFU-treated tumour, tumour lysate and mouse serum. Syngeneic naïve C57BL/6J mice were immunised with three loaded DCs followed by a subsequent H22 tumour challenge. Tumour size and survival were then recorded in each vaccinated mouse. The results showed that both HIFU-ablated tumour and tumour lysate could significantly increase the number of mature DCs and the secretion of IL-12 and IFN-γ (p < 0.001). The proliferation of splenic lymphocytes co-incubated with the loaded-DCs was significantly higher in both HIFU-ablated tumour and tumour lysate groups (p < 0.01). Cytotoxocity and TNF-α and IFN-γ secretion of cytotoxic T lymphocytes against H22 cells were significantly higher in HIFU-ablated tumour group than that in tumour lysate group (p < 0.01). After the H22 tumour challenge, a significant decrease of tumour volume was observed in HIFU-ablated tumour group (p < 0.01). However, there was no statistical difference of long-term survival rates among three groups (p > 0.05). It is concluded that DCs can be activated by HIFU-ablated tumour debris and, thus, initiate host specific antitumour immune response after HIFU therapy. (E-mail: mfengwu@yahoo.com)     

Numerical Analysis of Uncertainties in Dual Frequency Bone Ultrasound Technique

Quantitative ultrasound (QUS) measurements are used in the diagnostics of osteoporosis. However, the variation in the thickness and composition of the overlying soft tissue causes significant errors to the bone QUS parameters and diminishes the reliability of the technique in vivo. Recently, the dual frequency ultrasound (DFUS) technique was introduced to minimize the errors related to soft tissue effects. In this study, the significance of soft tissue induced errors and their elimination with the DFUS technique were simulated using the finite difference time domain technique. Furthermore, we investigated the potential of the DFUS corrected integrated reflection coefficient (IRC) of bone to detect changes in the cortical bone density. The effects of alterations in the thickness of fat and lean tissue layers and the inclination between the soft-tissues and between the soft tissue-bone layers were simulated. When the angle of the soft tissue interface was zero, i.e., perpendicular to the incident ultrasound beam, the DFUS-calculated soft tissue composition correlated highly linearly with the true soft tissue composition. The inclination between the soft tissue-bone layers was found to be critical. Even a 2-degree inclination between the soft tissue and the bone surface induced an almost 18% relative error in the corrected IRC. Increasing the inclination between the soft tissue layers increased the error in the DFUS-calculated lean and fat tissue thickness. This error was especially significant at inclination angles greater than 20 degrees. The significant soft tissue induced errors in IRC values (>300 %) could be effectively minimized (<10 %) by means of the DFUS correction. Importantly, after the DFUS correction, physiologically relevant variation in the cortical bone density could be detected (p < 0.05). (E-mail: Markus.Malo@uef.fi)     

Ultrasound Biomicroscopy Imaging for Monitoring Progressive Trypsin Digestion and Inhibition in Articular Cartilage

This study reports an ultrasound biomicroscopy (UBM) imaging approach to monitor the progressive trypsin-induced depletion of proteoglycan (PG) and its inhibition in articular cartilage. Three fresh, normal bovine patellae were obtained and four full-thickness cartilage-bone specimens were prepared from the lower medial side of each patella. One sample was used as a control and the other three were divided into three groups: Groups A, B and C (n = 3 for each group). After a 40 min 0.25% trypsin digestion, samples from group A were continuously digested in trypsin solution, while those in groups B and C were immersed in physiologic saline and fetal bovine serum (FBS), respectively, for another 280 min. The trypsin penetration front was observed by UBM and M-mode images were acquired using 50 MHz focused ultrasound and custom-developed software. The results show that the 40 min trypsin digestion degraded nearly the whole surface layer of the cartilage tissue. Further digestion in trypsin or residual digestion in saline for 280 min depleted most of the PG content, as observed in groups A and B. The replacement of trypsin with a physiologic saline solution only slightly slowed the digestion process (group B), while trypsin inhibitors in FBS stopped the digestion in approximately 1.5 h (group C). The normalized digestion fractions of the digested tissues were calculated from ultrasound data and histology sections, and then compared between the groups. Without the use of FBS, 80% to 100% of the full thickness was digested, while this number was only approximately 50% when using FBS. Our findings indicate that the UBM imaging system could provide two-dimensional (2-D) visual information for monitoring progressive trypsin-induced PG depletion in articular cartilage. The system also potentially offers a useful tool for preparing cartilage degeneration models with precisely controlled PG depletion. (E-mail: ypzheng@ieee.org)     

Real-time electro-mechano-acoustic imaging for monitoring interactions between trypsin and different inhibitors in articular cartilage

The purpose of this study was to observe the real-time interactions between trypsin and various inhibitors in articular cartilage in vitro using a novel electro-mechano-acoustic imaging method. Monitored in real-time, articular cartilage specimens from bovine patellae were first treated with trypsin to reach half proteoglycan depletion (Phase I), then the trypsin solution was replaced with (i) physiological saline buffer (PS), (ii) fetal bovine serum (FBS), (iii) protease inhibitor cocktail (PI) and (iv) 10% formalin (F), respectively, to observe their effects on residual digestion (Phase II). Ultrasound radio frequency signals from the articular cartilage were used to form a M-mode image, where the interface between trypsin digested and intact cartilage tissues could be observed with an additional echo generated. The inhibition time, the digestion depth and digestion fraction were measured for each specimen. The results showed that the dilution of trypsin using saline solution was not sufficient to stop the enzyme action instantly. Although groups FBS and PI had a similar inhibition time of approximately 1.5 h, their digestion depth was obviously different (0.25 ± 0.03 and 0.06 ± 0.06 mm, respectively). In contrast, formalin only took <30 min to stop the trypsin digestion with almost no further digestion. The results demonstrated that the current system was capable of monitoring the trypsin digestion and inhibition process in real time. Also, different chemicals affected the residual trypsin digestion to different degrees. (E-mail: ypzheng@ieee.org)     

Expression of synovial fluid and articular cartilage VIP in human osteoarthritic knee: A new indicator of disease severity?

Objectives

Vasoactive intestinal peptide (VIP) is a molecule shared by the neuroendocrine immune network and is considered to be a potential candidate for treatment of inflammatory and autoimmune diseases. Although some recent studies demonstrate that VIP has a protective role in animal RA models, its variant in different disease grade of OA remains uncertain.

Design and methods

Fifty patients with primary knee OA and ten controls with severe trauma were enrolled. Synovial fluid and articular cartilage samples were collected from specimens of total knee arthroplasty (TKA) or knee above amputation. VIP levels in these samples were assessed by ELISA and immunohistochemistry. Kellgren–Lawrence criteria and Mankin score were taken to determine the disease severity.

Results

Compared to the controls, OA patients have lower VIP concentration in synovial fluid (659.70 ± 112.79, 95%CI 579.01–740.38 vs 470.83 ± 156.40, 95%CI 426.38–515.28 pg/mL, P < 0.001) and articular cartilage (0.26 ± 0.02, 95%CI 0.24–0.28 vs 0.20 ± 0.04, 95%CI 0.18–0.21, P < 0.001). Subsequent analysis show that the VIP expression in synovial fluid is markedly correlated with its OD in articular cartilage (Pearson's r = 0.580, P < 0.001). Furthermore, the synovial fluid and articular cartilage levels of VIP both demonstrated to be negatively correlated with severity of disease (Spearman's ρ = 0.838, P < 0.001; Spearman's ρ = 0.814, P < 0.001).

Conclusions

VIP in synovial fluid and articular cartilage is negatively associated with progressive joint damage in OA and is a potential indictor of disease severity.     

Ultrasound Backscatter Imaging Provides Frequency-Dependent Information on Structure,Composition and Mechanical Properties of Human Trabecular Bone

The strength as well as the acoustic properties of trabecular bone are determined by its structure and composition. Consequently, tissue structure and compositional properties also affect the ultrasound propagation in bone. The diagnostic potential of ultrasound has not been fully exploited in clinical quantitative ultrasound devices. The aim of this study was to investigate the ability of quantitative ultrasound pulse-echo imaging, conducted over a broad range of frequencies (1 to 5 MHz), to predict the mechanics, composition and microstructure of trabecular bone. Ultrasound reflection and backscatter parameters correlated significantly with the ultimate strength of the trabecular bone and the bone volume fraction (r = 0.76–0.90, n = 20, p < 0.01). Ultrasound backscatter associated significantly (independently of bone structure or mineral content) with the collagen content of the bone matrix (r = 0.75, r_adjusted = 0.66, p < 0.01). Interestingly, the applied ultrasound frequency seemed to relate the sensitivity of ultrasound backscatter to different properties of trabecular bone. At frequencies ranging from 1 to 3.5 MHz, the ultrasound backscatter associated significantly with the tissue mechanical and structural parameters. At 5 MHz, the composition of the bone matrix was a more significant determinant of the measured backscatter. This study provides useful information for optimizing the use of pulse-echo measurements, and thereby further emphasizes the diagnostic potential of the ultrasound backscatter measurements of trabecular bone.     

In-vitro comparison of time-domain, frequency-domain and wavelet ultrasound parameters in diagnostics of cartilage degeneration

Quantitative ultrasound imaging (QUI) is a promising preclinical method for detecting early osteoarthrotic (OA) changes in articular cartilage. The aim of this study was to compare time-domain, frequency-domain and wavelet transform (WT) QUI parameters in terms of their performance in revealing degenerative changes in cartilage in vitro. Mankin score and Cartilage Quality Index (CQI) were used as a reference for quantifying cartilage degeneration. Intact (n = 11, Mankin score = 0) and spontaneously degenerated (n = 21, Mankin score = 1-10, mean = 4) osteochondral samples (diameter 19 mm) from bovine patellae, prepared and scanned with an ultrasound instrument in our earlier study, were further analyzed. Ultrasound reflection coefficient (R), integrated reflection coefficient (IRC) and ultrasound roughness index (URI) for cartilage surfaces were obtained from our earlier study. In the present study, maximum magnitude (MM) and echo duration (ED) for the cartilage surface were determined from the WT analysis. All ultrasound (US) parameters were capable of distinguishing intact and degenerated cartilage groups (p < 0.01, Mann-Whitney U test). Significant correlations were established between all QUI parameters and CQI or Mankin score (p < 0.01, Spearman's correlation test). The receiver operating characteristic (ROC) analysis indicated that the simple time-domain parameters (R and URI) were diagnostically as sensitive and specific as the more complex frequency-domain (IRC) or WT (MM, ED) parameters. Although QUI shows significant potential for OA diagnostics, complex signal processing techniques may provide only limited additional benefits for diagnostic performance compared with simple time-domain methods. However, certain technical challenges must be met before any of these methods can be used clinically.