Real-time ultrasonic assessment of progressive proteoglycan depletion in articular cartilage

The loss of proteoglycan (PG) is regarded as one of the early signs of osteoarthritis (OA), thus observing the progress of PG loss would be useful for the early detection of OA. In this study, high-frequency ultrasound was used to monitor and analyze the trypsin-induced progressive degeneration in articular cartilage. Full thickness cartilage-bone specimens (n = 10) prepared from normal bovine patellae were digested using 0.25% trypsin solution for different periods of time to evaluate the dynamics of the digestion process. The trypsin penetration front was observed in M-mode image, which was acquired using a nominal 50 MHz focused transducer. The transient speed of the digestion process was estimated from the image. The digestion fraction, which represents the ratio of the digestion depth to the total cartilage thickness, was estimated from ultrasound data and histology sections. With ultrasound, the digestion fraction observed in the 10 specimens ranged from 64% to 99% and was correlated to that measured by histology (R(2) > or = 0.63, p < 0.05). It was found that the digestion speed decreased nonlinearly with depth from 0.61 +/- 0.16 microm/s (mean +/- SD) in the superficial zone to 0.04 +/- 0.02 microm/s in a region located at 70% of the cartilage thickness in depth. The relationship between the digestion depth and the exposure duration in trypsin could be described using a third order polynomial function. The full thickness of digested and undigested tissues was also measured using caliper, estimated from ultrasound data and histology sections, and compared. These findings indicate that ultrasound could provide useful information about the trypsin-induced progressive PG depletion in articular cartilage. Therefore, ultrasound represents a useful tool to evaluate the dynamics of models of OA in vitro in cartilage specimens in a research environment and this would ultimately help the in vitro examination of articular cartilage for research related to model of OA from the early stages of tissue degradation.     

磁共振关节软骨成像新技术——MapIt

关节疾病早期的软骨改变,长期以来缺乏敏感的检查手段,磁共振量化参数图成像技术--MapIt可反映关节软骨的生化和微观结构的改变,显示出在软骨微小损伤诊断和疗效检测中的强劲潜力.本文对这一技术做一综述.     

A review on the mechanical quality of articular cartilage - implications for the diagnosis of osteoarthritis

The functional behaviour of articular cartilage in diarthrodial joints is determined by its morphological and biomechanical properties. Whereas morphological changes are mainly detectable in the progressed stages of osteoarthritis, biomechanical properties seem to be more sensitive to early degenerative variations since they are determined by the biochemical composition and structural arrangement of the extracellular matrix. The objective of this paper is to review studies focussing on variations in the mechanical compressive properties during the early pre-osteoarthritic stage. The aim is to quantify the requirements to detect the early cartilage degeneration in pre-osteoarthritis based on the mechanical parameters and to create an updated basis for a better understanding of inherent relationships between characteristic parameters in articular cartilage.

Correlations between mechanical and biochemical parameters as well as magnetic resonance, ultrasonic, histological and structural parameters were observed. In early osteoarthritis, static moduli decrease below 80% of healthy controls and dynamic moduli below 30% of controls. To identify osteoarthritic changes of articular cartilage based on static or dynamic mechanical parameters in an early stage of the disease progression the accuracy of a mechanical testing method has to be adequate to detect changes of 10% in cartilage stiffness.     

Ultrasound speed in articular cartilage under mechanical compression

Ultrasound elastography is a method that can be used to determine the elastic properties of soft tissues and it has been recently applied to study of articular cartilage. While ultrasound elastography techniques assume a constant ultrasound speed in tissue under mechanical compression, ultrasound speed in articular cartilage has been found to vary depending on the loading conditions. This may limit the quantitative use of the technique for determination of the elastic properties of articular cartilage along the axis of ultrasound propagation. The aim of the present study was to investigate the origin of the load-related variation in ultrasound speed. Samples of human and bovine articular cartilage (n = 82) were mechanically and acoustically tested during unconfined compression. A statistically significant (p < 0.05) variation of ultrasound speed was found in cartilage during a stress-relaxation test. A finite element model was constructed by exploiting microscopically determined collagen and proteoglycan contents, collagen orientation and biochemical analyses of water content. From the finite element simulations, collagen orientation and the void ratio (fluid-to-solid ratio) as a function of time were assessed and, together with the experimentally determined ultrasound speed, a linear model predicting variation of the ultrasound speed in human articular cartilage under mechanical compression was established. The model predicted compression-related ultrasound speed with an error of <0.3% at each time point. The effect of strain rate on variation of ultrasound speed was tested in bovine cartilage samples. The decrease in ultrasound speed was found to be proportional to the strain rate. The results suggest that ultrasound speed in articular cartilage is controlled mainly by collagen orientation and the void ratio and depends on the imposed strain rate. A numerical simulation revealed that the compression-related decrease in ultrasound speed induces notable errors in mechano-acoustically determined strain. A method to eliminate the compression-related errors in measured strain and elastic properties may be needed in mechano-acoustic measurements of articular cartilage.     

磁共振关节软骨成像的不同序列信号比较

目的:通过比较软骨信号噪声比(SNR)和对比度噪声比(CNR),分析临床常用的磁共振(MR)序列显示关节软骨的能力与限度。方法:选取12例成人离体膝关节标本行软骨磁共振成像(MRI)。应用的MR序列包括:①自旋回波T1加权序列(SE T1W);②快速自旋回波T2加权序列(FSE T2W);③短T1翻转恢复序列(STIR);④脂肪抑制质子密度序列(FS-PD);⑤三维脂肪抑制扰相梯度回波序列(3D-FS-SPGR);⑥重度T2加权梯度回波序列(T2*W GRE)。在股骨髁间凹中央层面上分别测量股骨髁软骨、股骨干骺端骨髓、小腿腓肠肌和背景噪声信号强度(SI),计算软骨SNR和软骨-关节液、软骨-骨髓、软骨-肌肉CNR。结果:3D-FS-SPGR和FS-PD序列软骨SNR最高(P<0.001)。结论:在目前临床应用的序列中,3D-FS-SPGR和FS-PD序列是进行软骨MRI的理想序列组合。     

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)     

Strain dependence of ultrasound speed in bovine articular cartilage under compression in vitro

The change of the ultrasound (US) speed in articular cartilage (artC) under applied strain conditions may induce significant measurement errors of the mechanical properties of the artC during both indentation and compression tests using US. In this paper, the strain dependence of the US speed in bovine artC (n = 20) under compression in vitro was investigated by virtue of using a custom-made US compression testing system. The US speed of the artC at the instant after the compression and that after a period of stress-relaxation were estimated under the applied strain ranging from 0% to 20%. Moreover, the instantaneous modulus and the modulus after the stress-relaxation of the artC were measured and correlated with the US speed. There was no significant difference (p > 0.05) between the US speed at the instant after the compression and that after the stress-relaxation, although there was a discrepancy between the instantaneous modulus and the modulus after stress-relaxation. The US speed was found to be highly correlated to the applied strain (r(2) = 0.98, p < 0.001) in a quadratic relation and changed by 7.8% (from 1581 +/- 36 m/s to 1671 +/- 56 m/s) when the applied strain reached 20%. The results suggest that the strain-dependent effect on the US speed in artC should be considered when the US is deployed for the assessment of artC using the compression or indentation test.     

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)     

磁共振膝关节软骨分层表现的组织学对照研究

目的对照研究关节软骨磁共振分层表现与组织学层次的异同点。方法分别对12例新鲜离体人膝关节标本采用以下序列行软骨磁共振成像:①SET1加权序列;②FSET2加权序列;③STIR序列;④3DFSSPGR序列;⑤FSPD序列;⑥T2*加权GRE序列。选取各序列图像髁间凹中央层面髌软骨最厚区域为感兴趣区,观察软骨分层表现并测定每一层次平均厚度。然后在标本相应部位取材,获得深及骨质的软骨组织并行甲苯胺兰染色。光镜下测定软骨厚度各组织层次厚度。对照分析MR和组织学分层表现。结果FSPD、FSSPGR和T2*GRE所测软骨整体厚度与组织学测量值之间无显著差异(P=1.432,0.097,0.064),以FSPD序列显示的髌软骨厚度值的组织学相关性最高(r=0.787);MR所显示的软骨分层与组织层次具有显著差异,各层厚度之间存在显著性差异(χ2=13.12,P=0.012);MR与组织学层次之间无相关性(P<0.05)。结论MR软骨分层表现受多种因素共同影响,与组织学层次之间无相关性。     

正常成年人股骨远端关节面软骨厚度的超声测量

目的通过超声测量正常成年人股骨远端关节面软骨的厚度,建立正常参考值范围。方法随机选择20～25岁的健康者92名,超声测量股骨远端关节面软骨厚度。结果男性负重关节面内、外侧软骨厚度测量值分别为（1．65±0．29）mm、（1．81±0．27）mm,女性分别为（1．51±0．26）mm、（1．62±0．25）mm。男性关节面软骨厚度大于女性,膝关节外髁面的关节软骨厚度大于内髁面,滑车关节面软骨厚度大于负重面（均P〈0．05）。男性负重关节面软骨厚度内、外侧分别大于1．12mm、1．37mm,女性分别大于1．09mm、1．21mm。结论超声测量股骨远端关节面软骨的厚度,可为建立中国人股骨远端关节面软骨厚度的正常标准奠定基础。     

T2^*mapping技术评估早期膝关节软骨退变

目的:探讨T2^*mapping技术诊断早期膝关节软骨退变的应用价值。方法:常规MR成像显示关节软骨正常而关节镜诊断软骨退变的65个膝关节,先记录常规MR成像显示关节软骨正常的具体关节面和数量,再记录T2^*mapping诊断为软骨退变的具体关节面和数量并与关节镜结果对照。测量软骨内退变区域T2^*值以及正常区域T2^*值,统计分析两者是否存在差异。结果:65个膝关节,关节镜诊断软骨退变113处,T2^*mapping诊断软骨退变72处,T2^*mapping诊断阳性率63.7%。软骨退变区域的T2^*均值为30±8.1ms,正常区域的T2^*均值为23±5.1ms,差异有统计学意义(t=5.585,P<0.05)。结论:T2^*mapping技术可明显提高MR术前诊断早期软骨退变的阳性率。     

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.     

DTI在正常成人髌骨软骨的初步应用及临床意义

目的探讨扩散张量成像技术(diffusion tensor imaging,DTI)在正常成人髌骨软骨的初步应用并探讨其临床意义。材料与方法采用Philips Achieva 3.0 T超导型MR扫描仪对114例健康成年志愿者的右膝关节髌骨软骨进行三维双回波稳态序列(3D dual-echo steady state,3D-DESS)及DTI扫描,获得不同图像后测量表观弥散系数(apparent diffusion coefficient,ADC)、各向异性分数(fractional anisotropy,FA)值。结果随着年龄的增长,髌骨软骨的FA值逐渐下降,而ADC值逐渐增加(P0.05)。相关性分析表明,年龄与FA值呈负相关(r=-0.8321,P=0.0270.05),年龄与ADC值呈正相关性(r=0.698,P=0.0150.05)。结论DTI可探查髌骨软骨成分及胶原纤维结构的细微变化,且重复性及准确性良好,可为髌骨软骨病变早期诊断、监测病情进展及评价治疗效果提供一种重要的方法。     

An Ultrasound Biomicroscopic and Water Jet Ultrasound Indentation Method for Detecting the Degenerative Changes of Articular Cartilage in a Rabbit Model of Progressive Osteoarthritis

It is important to assess the early degeneration of articular cartilage associated with osteoarthritis (OA) for early intervention and treatment planning. Previously, we have developed a high frequency ultrasound and water jet indentation method for the morphologic, acoustic and mechanical assessment of articular cartilage, using the enzymatic digestion as a model of osteoarthritic degeneration. No naturally degenerated articular cartilage has been tested with the developed method. In this study, we aimed to determine the usefulness of the developed method for detecting the natural degeneration of articular cartilage in a standard surgical model of OA in rabbits. Forty adult New Zealand white female rabbits were used in this study, which included 30 experimental rabbits undergoing the right anterior cruciate ligament transection surgery and 10 control rabbits. At the 3^rd, 6^th, and 9^th week post-surgery, 10 experimental rabbits were sacrificed, respectively, for assessment of the knee cartilage quality. The cartilage at the medial and lateral femoral condyles and tibial plateaus (four points) was measured by the high frequency ultrasound biomicroscopy, the water jet ultrasound indentation and a contact mechanical indentation test before a histopathologic analysis for grading of degeneration severity. Measured parameters were compared among different groups classified either by post-surgery time or by histopathologic grade. The results showed a general trend of increase for ultrasound roughness index and a general trend of decrease for integrated reflection coefficient, stiffness coefficient from water-jet indentation and Young's modulus (E) from the mechanical indentation with the increase of post-surgery time. Comparisons among groups with different histopathologic grades showed similar trend with the increase of degeneration severity. The water jet ultrasound indentation method was demonstrated to be an effective method to measure the mechanical properties of the articular cartilage and with further development of arthroscopic ultrasound probe; it has the ability to assess the early degeneration of articular cartilage with measurement of morphologic, acoustic and mechanical properties of the cartilage in vivo.     

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.     

持续被动运动对兔膝全层关节软骨缺损修复的影响

目的 研究持续被动运动(CPM)对全层关节软骨缺损修复的影响。方法 将60个兔膝关节建立全层关节软骨缺损模型，随机分为3组，术后A组每天进行CPM 8h；B组每天进行CPM 2h，2组均连续进行CPM 4周，然后笼内自由活动至12周。C组仅笼内自由活动。分别于术后4周和12周处死各组半数兔，并取材，行HE、番红O以及Ⅰ、Ⅱ胶原免疫组化染色，再进行评分。结果 A组全层关节软骨缺损修复的效果较B、C两组明显。结论 兔膝全层关节软骨缺损后给予CPM干预可显著促进组织的修复，同时可促进Ⅱ型胶原和蛋白多糖的恢复。     

膝关节软骨退变的MRI与病理对照研究

目的:通过不同MR序列对膝关节软骨病变的检出与病理进行对照比较,总结适于软骨退变的MR检查方法。材料与方法:选择尸体膝关节9只,采用7种不同的序列进行扫描,即:FS 3D FAST、FS 3D RF-spoiled FAST、3D FAST、3D RF-spoiled FAST、SE T1WI、SE T2WI、SE PDWI。将各序列发现的病变数目及分级与病理结果进行对照,所有结果采用SPSS软件包进行统计学处理。结果:FS 3D FAST及FS 3D RF-spoiled FAST发现病变的敏感度为92%和95%,均优于其它MR序列,统计学结果显示与其它各序列比较有统计学显著性差异(P<0.05),而这两个序列的扫描结果之间无统计学显著差异(P>0.05)。结论:FS 3D FAST及FS 3D RF-spoiled FAST序列是发现软骨病变的敏感序列。     

Characterization of costal cartilage and its suitability as a cell source for articular cartilage tissue engineering

Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articular cartilage tissue engineering. Limited knowledge exists, however, on costal cartilage characteristics. This study describes the characterization of costal cartilage and articular cartilage properties and compares neocartilage engineered with costal chondrocytes to native articular cartilage, all within a sheep model. Specifically, we (a) quantitatively characterized the properties of costal cartilage in comparison to patellofemoral articular cartilage, and (b) evaluated the quality of neocartilage derived from costal chondrocytes for potential use in articular cartilage regeneration. Ovine costal and articular cartilages from various topographical locations were characterized mechanically, biochemically, and histologically. Costal cartilage was stiffer in compression but softer and weaker in tension than articular cartilage. These differences were attributed to high amounts of glycosaminoglycans and mineralization and a low amount of collagen in costal cartilage. Compared to articular cartilage, costal cartilage was more densely populated with chondrocytes, rendering it an excellent chondrocyte source. In terms of tissue engineering, using the self‐assembling process, costal chondrocytes formed articular cartilage‐like neocartilage. Quantitatively compared via a functionality index, neocartilage achieved 55% of the medial condyle cartilage mechanical and biochemical properties. This characterization study highlighted the differences between costal and articular cartilages in native forms and demonstrated that costal cartilage is a valuable source of chondrocytes suitable for articular cartilage regeneration strategies.     

Pluronic F-127负载骨髓间充质干细胞和碱性成纤维细胞生长因子修复兔膝关节软骨缺损

目的探讨应用Pluronic F-127负载骨髓间充质干细胞（MSCs）和碱性成纤维细胞生长因子（bFGF）修复兔膝关节软骨缺损的可行性。方法抽取青紫蓝兔骨髓,行MSCs原代及传代培养,将3代MSCs作为种子细胞。将32只青紫蓝兔造成双膝关节4 mm×4 mm关节软骨缺损模型,随机分为四组,实验组缺损内植入负载了MSCs和bFGF的Plu-ronic F-127;细胞组植入负载了MSCs的Pluronic F-127;生长因子组植入负载了bFGF的Pluronic F-127;对照组缺损不做处理。于术后4周和16周,处死动物取材,进行大体观察、HE染色、甲苯胺蓝染色,且对各组修复组织行组织学评分及比较。结果术后4周时实验组与其他三组比较修复效果不明显;术后16周,大体观察及HE染色示实验组缺损已不易辨认,以透明样软骨修复为主,Pluronic F-127基本降解仅残余少量颗粒,甲苯胺蓝染色示细胞周围基质有较多异染质,而细胞组和生长因子组缺损表面不平整,界限清晰,修复组织以纤维组织为主,仅周缘见少许纤维软骨出现,对照组仅缺损底部有纤维组织覆盖。根据Wakitani评分标准术后16周时实验组的修复效果明显好于其他三组。结论 Plu-ronic F-127结合MSCs和bFGF的复合物最终形成类关节软骨样组织,基本修复关节软骨缺损,此方法简便易行,有实用价值,有望成为临床治疗软骨缺损的一种新方法。