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.     

平衡消肿灌洗液对关节软骨影响的研究与分析

目的观察自制平衡消肿灌洗液对兔活体膝关节灌注后关节软骨的影响，为关节镜手术提供更理想的灌洗液。方法选用健康兔24只（48膝），随机将其分为平衡消肿灌洗液组，透明质酸钠组，林格氏液组。并于灌注后、第1、2、5周末进行关节软骨形态学观察及组织学检查。结果灌注后5周，平衡消肿灌洗液灌注组关节软骨的形态学、组织学完全正常，软骨蛋白多糖含量高于2个对照组。结论平衡消肿灌洗液可维持甚至提高软骨的蛋白多糖含量，对关节软骨有更为显著的保护、营养作用，促进软骨恢复，对关节的影响小，是关节镜手术较为理想的灌洗液。     

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.     

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)     

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.     

骨关节炎的治疗体会

骨关节炎的治疗应是一种综合性治疗,即非药物和药物治疗相结合,非药物治疗十分重要,包括患者教育、超重者减体重、有氧运动、合理使用矫形器和支具以及适量运动,运动即可锻炼肌肉力量又可改善关节功能。重视"年轻人老年膝",积极治疗年轻人由于运动不当或外伤致膝关节半月板和韧带损伤。避免急性滑膜炎反复发作,一旦发生积极治疗,在最短的时间内控制滑膜炎症。建议早期使用保护关节软骨(改善病情)的药物。     

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。     

A constitutive study of the age-dependent mechanical behaviour of deep articular cartilage: Model construction and simulated failure

The tensile properties of deep articular cartilage of the human femoral head have been simulated using a model based on the expected mechanical behaviour of an electrostatically cross-linked network of collagen fibrils. Articular cartilage requires a model incorporating two types of interactions, referred to as type I and type II, which differ in the amount of energy required to bring about their mechanical failure. This modified two-population (MTP) model is shown to accurately simulate the experimental tensile behaviour of 14 specimens of deep articular cartilage. Also, the MTP model simulates a failure behaviour which appears to be comparable to the actual experimental fracture of the articular cartilage specimens. A reduction in the fracture stress of the deep articular cartilage specimens with age can be interpreted through age-related changes which occur in the values of the parameters of the MTP model. This shows that the younger tissues derive their superior tensile properties through an optimum structural arrangement which is associated with a high proportion of binding equivalent to the type I interaction in the tissue model. A decline in the tensile properties with age occurs as the tissue structure falls from its optimal configuration as the proportion of type II interactions increases. Such changes may predispose the articular cartilage to the mechanical damage and deterioration which leads to the osteoarthritic degeneration of a joint.

Relevance

An understanding of the process of osteoarthritic degeneration requires a knowledge of the relationship between the biochemical composition and mechanical behaviour of articular cartilage. An approach is developed to examine this relationship in order to gain insight into the ultrastructural basis of the mechanical weakening of articular cartilage with age.     

玻璃化法保存兔关节软骨的实验研究

目的观察玻璃化法保存兔关节软骨的效果。方法选取10只大白兔,软骨标本取材后,低温冷冻组5只和玻璃化保存组5只,分别通过台盼蓝拒染法检测兔关节关节软骨细胞存活率和MTT法测软骨细胞相对活性。结果玻璃化保存组兔关节关节软骨细胞存活率和软骨细胞相对活性均优于低温冷冻组,差异均有显著性(P<0.05)。结论玻璃化法保存兔软骨,软骨存活率高,软骨修复区的组织更接近正常软骨。     

早期关节软骨损伤及修复后的MRI表现分析

目的:探讨早期关节软骨损伤及修复后的MRI(磁共振成像)表现,为关节软骨损伤早期临床诊治提供依据。方法:择取2018年1月-2019年6月我院23例经关节镜或手术病理证实的关节软骨损伤的患者为研究对象,患者均接受MRI检查,经治疗临床症状消失或显著改善后再行MRI复查,回顾性调研患者两次MRI检查的影像资料,观察关节软骨损伤影像表现与分级。结果:早期关节软骨损伤MRI诊断:关节变薄且凹凸不平1例,局灶性异常低信号4例,信号中断不连续18例。基于Recht标准,I级5例,II级9例,III级6例,IV级3例,影像表现与关节镜检或手术病理所见相符。修复后MRI下观察,21例关节软骨缺损完全填充,融合良好,软骨组织厚度与信号强度同周围软骨信号基本一致,1例缺损未完全填充,1例肥厚增生。结论:MRI是诊断早期关节软骨损伤的有效方法,不仅可以清楚显示病变,较好的反映损伤特点与严重程度,为临床诊治提供依据,亦能用于评价关节软骨损伤修复情况,对预后评估也具有重要参考价值。     

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。     

X型胶原在膝骨关节炎软骨中的表达

目的观察膝骨关节炎患者关节软骨中X型胶原的表达特征,探讨X型胶原在骨关节炎疾病进展中的作用。方法取18例膝骨关节炎患者和13例正常对照者的关节软骨。以Northern杂交、RT-PCR和Western杂交,分别检测各组样品X型胶原mRNA和蛋白水平的表达情况。结果 Northern杂交与RT-PCR的结果均显示膝骨关节炎的X型胶原表达有显著升高。Western杂交发现膝骨关节炎患者软骨基质中X型胶原的含量上升(P<0.01)。但分析mRNA和蛋白水平的变化趋势,未能发现它们之间存在明确的相关性(P>0.05)。结论膝骨关节炎患者的X型胶原表达明显增加,这可能促使基质组分发生改变,从而加速了骨关节炎的进展。     

骨性关节炎模型大鼠关节软骨变化及透骨消痛胶囊干预的作用

背景：透骨消痛胶囊是治疗骨性关节炎的临床验方,作用机制尚未完全阐明。尿激酶型纤溶酶原激活系统参与关节软骨的细胞外基质降解及关节滑膜增生,在骨性关节炎的病理过程中起着重要作用。目的：观察透骨消痛胶囊对膝骨性关节炎模型大鼠软骨中尿激酶型纤溶酶原激活系统的影响。方法：SD大鼠144只,随机取120只采用关节腔注射木瓜蛋白酶复制大鼠膝骨性关节炎模型,并随机分为模型组、壮骨关节丸组[1.2 g/（kg·d）]、透骨消痛胶囊低剂量组[0.092 g/（kg·d）]、透骨消痛胶囊中剂量组[0.184 g/（kg·d）]和透骨消痛胶囊高剂量组[0.368 g/（kg·d）],每组24只,每2周为1个疗程,中间休息2 d,共4个疗程。另取24只正常大鼠为空白组。每2个疗程后,处死一批实验动物,苏木精-伊红染色观察软骨组织病理改变;免疫组织化学反应观察尿激酶型纤溶酶原激活剂、尿激酶型纤溶酶原激活剂受体、纤溶酶原激活物抑制因子阳性表达情况;Western blot检测尿激酶型纤溶酶原激活剂、尿激酶型纤溶酶原激活剂受体、纤溶酶原激活物抑制因子蛋白表达情况。结果与结论：透骨消痛胶囊组和壮骨关节丸组的骨性关节炎大鼠关节软骨 Mankin’s 评分较模型组明显降低（P 〉0.01）,具有时间依赖;免疫组织化学反应显示,透骨消痛胶囊组和壮骨关节丸组尿激酶型纤溶酶原激活剂、尿激酶型纤溶酶原激活剂受体的阳性率明显降低,而纤溶酶原激活物抑制因子明显升高,具有时间依赖。Western blot检测结果与免疫组织化学具有相同的趋势。提示透骨消痛胶囊可能通过调控尿激酶型纤溶酶原激活剂系统对骨性关节炎发挥防治作用。     

Surface fissures in articular cartilage: effect of pathological changes in synovial fluid

Objective. A unified mathematical model of two different modes of inception of fissures at the surface of articular cartilage in healthy and pathological joints.

Design. The superficial tangential zone of articular cartilage is modeled as a three-phase medium consisting of collagen fibers, matrix, and of infiltrated thin constituent of synovial fluid.

Background. The author’s general mesomechanical concept is applied to the analysis of deterioration of articular cartilage.

Methods. Theoretical analysis based on the results of the author’s preceding paper.

Results. The presented analysis shows that superficial fissures in articular cartilage can also be caused by pathological thinning of synovial fluid.

Conclusions. Whereas in healthy joints the probable cause of creation of fissures at the surface of cartilage was shown to be fast impact loading, in joints with inflammatory synovial fluid the fissures can be caused by plain walking.

Relevance  Appearance of surface fissures in articular cartilage is a serious, still not fully clarified problem that deserves attention.     

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)     

Propagation of surface fissures in articular cartilage in response to cyclic loading in vitro

Background. Mechanical overloading of synovial joints can damage the articular cartilage surface and may lead to osteoarthritis. However, causal links between mechanical and biological events in cartilage are poorly understood.

Objectives. To test the hypothesis that surface fissures in cartilage can propagate mechanically if the joint surface is subjected to vigorous cyclic loading.

Methods. Thirty-five cartilage-on-bone specimens, 15-mm square, were removed from mature bovine knee and shoulder joints. Specimens were loaded by means of a 9-mm-diameter flat indenter with a beveled edge, and their compressive strength determined. Failure occurred in the cartilage surface at an average stress of 36 MPa. Cartilage fissures were marked with Indian ink, photographed, and their length and width measured using image analysis software. Each damaged specimen was subjected to cyclic loading at 40% of its compressive strength, at 0.5 Hz, for up to 5 h. Fissure length and width were measured at regular intervals. After testing, fissure depth was measured from histological sections, and compared with measurements from damaged cartilage which was not cyclically loaded.

Results. Cyclic loading caused cartilage fissures to increase in length (mean 353%, P<0.01) and width (360%, P<0.01) but not depth. Propagation was rapid at first, but approached equilibrium after several hundred cycles. Rehydration in saline had no effect on fissure length, but width returned to pre-cyclic loading values.

Conclusion. Cartilage fissures can propagate mechanically when a joint surface is subjected to cyclic compressive loading in vitro. The transient opening-up of fissures to form wide surface “wounds” during cyclic loading could be of biological significance if it occurred in living people.Relevance

In living joints, wide open fissures in the cartilage surface could promote degenerative changes in the tissue.     

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.     

Evaluation of the effect of collagen network degradation on the frictional characteristics of articular cartilage using a simultaneous analysis of the contact condition

Background. Superficial conditions and integrity of collagen network play an important role on the lubrication performance of articular cartilage. In this work, a technique based on the evanescent waves is used for the evaluation of contact condition during friction tests.

Methods. The frictional and superficial characteristics of the normal and degraded articular cartilages with high and low concentration of collagenase were evaluated. The optical apparatus was set in order to decrease the intensity of a light reflected at the interface between a prism and specimens when collagen fibers are found near the interface.

Findings. For all conditions, an increase in the attenuation of reflectance as the friction coefficient increases was observed with reasonable correlation. For the specimens degraded with collagenase, low friction and reduced attenuation of reflectance were observed at the beginning of sliding followed by a gradual increase in both friction and attenuation of reflectance. In comparison to the degraded specimens, normal specimens presented high friction at beginning and low friction at the end of test.

Interpretation. The superficial conditions and the presence of water at the articular surface play an important role in the lubrication of synovial joints. The ability to retain water for degraded specimens is impaired due to the loss of proteoglycan observed in the histological sections and hence, their low friction observed at the beginning of the test is not sustained for a long time. The use of evanescent waves demonstrated to be very useful in the analysis of the contact condition of articular cartilage.     

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。