Low-field one-dimensional and direction-dependent relaxation imaging of bovine articular cartilage

The structure of articular cartilage is separated into three layers of differently oriented collagen fibers, which is accompanied by a gradient of increasing glycosaminoglycan (GAG) and decreasing water concentration from the top layer towards the bone interface. The combined effect of these structural variations results in a change of the longitudinal and transverse relaxation times as a function of the distance from the cartilage surface. In this paper, this dependence is investigated at a magnetic field strength of 0.27 T with a one-dimensional depth resolution of 50 μm on bovine hip and stifle joint articular cartilage. By employing this method, advantage is taken of the increasing contrast of the longitudinal relaxation rate found at lower magnetic field strengths. Furthermore, evidence for an orientational dependence of relaxation times with respect to an axis normal to the surface plane is given, an observation that has recently been reported using high-field MRI and that was explained by preferential orientations of collagen bundles in each of the three cartilage zones. In order to quantify the extent of a further contrast mechanism and to estimate spatially dependent glycosaminoglycan concentrations, the data are supplemented by proton relaxation times that were acquired in bovine articular cartilage that was soaked in a 0.8 mM aqueous Gd++ solution.     

Hydroxyapatite/polyamide66 porous scaffold with an ethylene vinyl acetate surface layer used for simultaneous substitute and repair of articular cartilage and underlying bone

In this paper, tissue scaffold made from polyamide66 (PA66) and hydroxyapatite (HA) was prepared by co-precipitation and thermal-induced phase inversion method, in which biomimetic HA crystals were uniformly distributed in PA66 matrix. The porosity of the scaffold is about 81% and the macropore size is from 50 to 500 μm. The ethylene vinyl acetate (EVA) layer was thermally molded on one surface of HA-PA66 scaffold to develop EVA/HA-PA66 composite for articular cartilage/bone substitute, i.e., upper EVA layer for cartilage substitute and underlying HA-PA66 scaffold for bone bonding and fixation. The physicochemical and mechanical properties of EVA were also investigated. The results indicate that the tensile and compressive strength of EVA is about 4.65 MPa and 9.44 MPa respectively, while its mean friction coefficient is very small, only about 0.23. The cell culture of EVA and HA-PA66 scaffold shows that these materials possess good cytocompatibility. The proposed preparation method is novel and effective, and the EVA/HA-PA66 composite has good potential for simultaneous substitute of articular cartilage and underlying bone.     

Bilaminar pattern of tibial condyle cartilage layer on the fat-suppressed 3D gradient echo images: artifact or structural and biochemical difference in composition of cartilage?

The purpose of this study was to examine if an unusual bilaminar pattern of lateral tibial condyle cartilage layer on the fat-suppressed three-dimensional (3D) spoiled gradient echo sequence is artifactual or correlates with structural and/or biochemical composition of cartilage. The laminar appearance of the lateral tibial condyle cartilage layer was studied on fat-suppressed 3D spoiled gradient echo MR images of the knee joint in 67 patients (mean age: 28y) performed at 1.0 Tesla. After i.v. administration of gadopentetate dimeglumine, diffusion of the contrast media into cartilage layer was qualitatively analysed over time on inversion recovery spin echo images of knee joints of five asymptomatic volunteers (mean age: 25y). In a patient with osteosarcoma and total knee replacement, MR examination of cartilage layer of lateral tibial plateau was compared with histologic specimens stained with Safranin-O, demonstrating proteoglycan distribution in cartilage. The retrospective analysis of 67 knee joints revealed a bilaminar appearance of lateral tibial condyle cartilage layer in the gradient echo images in the majority of cases (81%) with a statistically significant tendency to a trilaminar pattern in patients older than 20 years. With i.v. contrast administration, the contrast enhancement was only observed in the superficial zone of tibial cartilage layer. Histologic specimens in one patient demonstrated a good correlation between thickness of proteoglycan-free and proteoglycan-rich laminae of lateral tibial condyle on Safranin-O staining with hyperintense and hypointense zones, respectively, on corresponding fat-suppressed 3D spoiled gradient echo images (correlation coefficient of 0.87). Bilaminar pattern of tibial condyle cartilage layer on fat-suppressed 3D spoiled gradient echo images in younger subjects is not an artifact or an intrachondral lesion, but it may represent a regional difference in composition of extracellular cartilage matrix possibly produced by a highly-oriented collagen fiber structure associated with a high concentration of proteoglycans in the middle and deep portion of the cartilage layer.     

In vivo structural analysis of articular cartilage using diffusion tensor magnetic resonance imaging

Purpose

The articular cartilage is a small tissue with a matrix structure of three layers between which the orientation of collagen fiber differs. A diffusion-weighted twice-refocused spin-echo echo-planar imaging (SE-EPI) sequence was optimized for the articular cartilage, and the structure of the three layers of human articular cartilage was imaged in vivo from diffusion tensor images.

Materials and Methods

The subjects imaged were five specimens of swine femur head after removal of the flesh around the knee joint, five specimens of swine articular cartilage with flesh present and the knee cartilage of five adult male volunteers. Based on diffusion-weighted images in six directions, the mean diffusivity (MD) and the fractional anisotropy (FA) values were calculated.

Results

Diffusion tensor images of the articular cartilage were obtained by sequence optimization. The MD and FA value of the specimens (each of five examples) under different conditions were estimated. Although the articular cartilage is a small tissue, the matrix structure of each layer in the articular cartilage was obtained by SE-EPI sequence with GRAPPA. The MD and FA values of swine articular cartilage are different between the synovial fluid and saline. In human articular cartilage, the load of the body weight on the knee had an effect on the FA value of the surface layer of the articular cartilage.

Conclusion

This method can be used to create images of the articular cartilage structure, not only in vitro but also in vivo. Therefore, it is suggested that this method should support the elucidation of the in vivo structure and function of the knee joint and might be applied to clinical practice.     

Differences in Cartilage Repair between Loading and Unloading Environments in the Rat Knee

We investigated the histopathological and immunohistochemical effects of loading on cartilage repair in rat full-thickness articular cartilage defects. A total of 40 male 9-week-old Wistar rats were studied. Full-thickness articular cartilage defects were created over the capsule at the loading portion in the medial condyle of the femur. Twenty rats were randomly allocated into each of the 2 groups: a loading group and a unloading group. Twenty rats from these 2 groups were later randomly allocated to each of the 2 groups for evaluation at 1 and 2 weeks after surgery. At the end of each period, knee joints were examined histopathologically and immunohistochemically. In both groups at 1 and 2 weeks, the defects were filled with a mixture of granulation tissue and some remnants of hyaline cartilage. The repair tissue was not stained with toluidine blue in both groups. Strong staining of type I collagen was observed in the repair tissue of both groups. The area stained with type I collagen was smaller in the unloading group than in the loading groups, and the stained area was smaller at 2 weeks than at 1 week. In the staining for type II collagen, apparent staining of type II collagen was observed in the repair tissue of both groups at 1 week. At 2 weeks, there was a tendency toward a higher degree of apparent staining in the loading group than in the unloading group. Accordingly, these results indicated that loading and unloading in the early phase of cartilage repair have both merits and demerits.     

The zonal architecture of human articular cartilage described by T2 relaxation time in the presence of Gd-DTPA2-

The depth-wise variation of T(2) relaxation time is known to reflect the collagen network architecture in cartilage, while the delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC) technique is sensitive to tissue proteoglycan (PG) concentration. As the cartilage PG content varies along the tissue depth, the depth-dependent accumulation of the contrast agent may affect the inherent T(2) of cartilage in a nonconstant manner. Therefore, T(2) and dGEMRIC are typically measured in separate MRI sessions. In the present in vitro MRI study at 9.4 T, depth-wise T(2) profiles and collagenous zone thicknesses as determined from T(2) maps in the absence and presence of Gd-DTPA(2-) (T(2) and T(2Gd), respectively) were compared in samples of intact human articular cartilage (n=65). These T(2) measures were further correlated with birefringence (BF) of polarized light microscopy (PLM) to quantify the ability of MRI to predict the properties of the collagen fibril network. The reproducibility of the T(2) measurement in the current setup was also studied. Typical tri-laminar collagen network architecture was observed both with and without Gd-DTPA(2-). The inverse of BF (1/BF) correlated significantly with both T(2) and T(2Gd) (r=0.91, slope=0.56 and r=0.90, slope=0.63), respectively. The statistically significant linear correlations between zone thicknesses as determined from T(2) and T(2Gd) were r=0.55 (slope=0.49), r=0.74 (slope=0.71) and r=0.95 (slope=0.94) for superficial, middle and deep tissue zones, respectively. Reproducibility of the T(2) measurement was worst for superficial cartilage. Consistent with PLM, T(2) and T(2Gd) measurements reveal highly similar depth-dependent information on collagen network in intact human cartilage. Thus, dGEMRIC and T(2) measurements in one MRI session are feasible for intact articular cartilage in vitro.     

使用超声研究关节软骨的深度依赖瞬态膨胀行为

关节软骨是覆盖关节表面的一层承载生物重量的组织，关节软骨在正常状态下，软骨蛋白多糖的弹性和胶原纤维的张力保持平衡。这种平衡的微小变化会引起关节软骨的退化。量化关节软骨，特别是在表层区域的膨胀影响，可以表征骨关节炎的退化变化。本文的主要目的是使用一种新的实时超声膨胀测量系统来研究在改变浸泡关节软骨的盐水浓度，以及改用胰岛素溶液时的关节软骨的深度依赖瞬态膨胀行为。     

Evaluation of water content by spatially resolved transverse relaxation times of human articular cartilage

Non-invasive assessment of cartilage properties, specifically water content, could prove helpful in the diagnosis of early degenerative joint diseases. Transverse relaxation times T(2) of human articular cartilage (34 cartilage slices of three donors) were measured on a pixel-by-pixel basis in a clinical whole body MR system in vitro. In vivo feasibility to measure quantitative T(2) maps was shown for human patellar cartilage. The relaxation times of cartilage with collagen in the radial zone oriented perpendicular to the magnetic field increased from approximately 10 ms near the bone to approximately 60 ms near the articular surface. Cartilage water content of the tibial plateau and femoral condyles could be determined from the correlation with T(2) (R(2) = 0.71) with an error of approximately 2 wt.%. In vivo, directional variation would need to be considered. If confirmed in vivo, T(2) measurements could potentially serve as a non-invasive tool for the evaluation of the status and distribution of water content in articular cartilage.     

Raman analysis of proteoglycans simultaneously in bone and cartilage

Bone and cartilage are connective tissues with distinct organic matrix (collagen and non‐collagenous proteins) composition facilitating their biological function. Proteoglycans (PGs), a member of the non‐collagenous proteins fulfill functions that are determined by both their core protein and their glycosaminoglycan chains. The purpose of the present study was to identify Raman bands that are representative of PG concentration and may be used in both bone and cartilage tissues. To achieve this goal, we analyzed a series of reference PGs and collagens, as well as turkey leg tendon to verify the laser polarization independency of the identified bands. Additionally, the applicability of these bands in both cartilage and bone tissue simultaneously was tested in a healthy femoral head by Raman imaging and hierarchical cluster analysis to describe the distribution of PGs at the micron level from articular cartilage to subchondral bone. The results of the study show that the Raman band ~1375 cm⁻¹ can be used as a PGs marker band in both cartilage and bone. Moreover, articular cartilage has a lower content of organic matrix (mostly type II collagen), while the middle and deep transitional zone haves a higher concentration of PGs. The calcified cartilage is characterized by a lower content of PGs and total organic matrix (estimated from the integrated area of the amide III band). Copyright © 2014 John Wiley & Sons, Ltd.     

骨关节炎软骨的近红外光谱学研究及分期诊断

骨关节炎是一种威胁中老年人群公共健康和生活质量的重大医学疾病。骨关节炎的早期病变主要表现在细胞外基质成分含量的变化,患者自身很难发现,现有的临床方法和实验方法也不能较准确地识别骨关节炎的早期病变。近年来,傅里叶变换近红外（FTNIR）光谱技术因为其分析速度快、成本低、易于穿透组织获得样本的光谱信息等特点已被用于手术导航、无损检测和疾病诊断等各个领域。基于以上优势,采用FTNIR技术对不同深度分区（表层区、过渡区、深层区）的健康和骨关节炎的关节软骨进行NIR光谱采集和预处理,结合主成分分析（PCA）和Fisher判别（FDA）分别研究不同的预处理方式对判别结果的影响、不同深度下基质成分含量的变化以及骨关节炎分期识别。比较其他2种（基线校正、二阶导数3次多项式25点Savitzky-Golay平滑）预处理方式,同分区中一阶导数2次多项式21点Savitzky-Golay平滑预处理的判别结果最优,其中表层区的识别率高达95%（初始案例）和90%（交互验证案例）;表层区的判别结果优于过渡区,更优于深层区,恰可证明骨关节炎的早期病变主要发生在表层区。在骨关节炎分期识别中,经数据优化后模型的初始案例识别率为100%,交互验证识别率为93.3%,预测集的识别率为87.5%。结果表明：NIR光谱的一阶导数预处理结合PCA-FDA方法能有效地鉴别关节软骨病变与否并进行骨关节炎的分期诊断,对骨关节炎监测和早期诊断研究具有重要意义,并有潜力应用于骨关节炎的原位分期和早期临床诊断。     

Ultrastructural analysis of Wuchereria bancrofti (Nematoda: Filarioidea) body wall

Bancroftian filariasis constitutes the principal mosquito-borne nematode infection of humans and the surface of adult of Wuchereria bancrofti seems to be especially important in the intricate interplay between host and parasite. The study of the parasite's surface structure might help to understand the localization and function of various organelles. W. bancrofti adult worms were recovered from untreated patients during hydrocele repair surgery and studied by transmission electron microscopy. The body wall of adult parasite is composed of cuticle, hypodermis and muscular layer. Cuticle is the external layer and shows transverse cuticular striation. It is composed by an epicuticle, cortical layers, median layer, fibrous layers and basal layer. The epicuticle is the most external cuticular layer and appears as a single laminar electron-dense layer. The cortical external region is more electron-dense and granular in appearance than the inner cortical layer. Electron-dense structures, called bosses are randomly distributed filling the cuticular striation. The median layer is formed by an electron-dense and continuous thick line. The fibrous layer is subdivided in inner and external layers connected by projections. The basal layer includes a large quantity of membranous projections directed toward the hypodermis. The hypodermis is a syncytium where some cellular organelles are observed. The somatic musculature is meromyarian. The muscle fibers consist of contractile and non-contractile regions and the contractile region is composed of myofilaments separated by dense body. This is the first study of W. bancrofti adult worms obtained from untreated patients and studied by transmission electron microscopy.     

Inter-rater and intra-rater reliability of subchondral bone and cartilage thickness measurement from MRI

MRI is often used to visualize and quantify the articular cartilage layer of load bearing joints affected by degenerative diseases, such as osteoarthritis (OA). Although the role played by the subchondral bone in the etiology and/or progression of OA may be important, the ability to visualize and quantify subchondral bone with MRI has received little attention. In this report we examined the inter-rater and intra-rater reliability of subchondral bone and cartilage thickness measurements from MR images of cadaver femoral head specimens. A 3D-SPGR pulse sequence tuned to eliminate chemical shift artifact through phase cancellation was used to image the specimens. Three raters manually segmented four specimens on two different occasions. Subchondral bone and cartilage thickness measurements were calculated from the segmented images. Inter-rater and intra-rater reliabilities were very high (>.98) for both cartilage and subchondral bone thickness measurements. We conclude that subchondral bone thickness can be measured as reliably as cartilage thickness from MR images.     

Vibrational spectroscopy of articular cartilage

Articular cartilage is a connective tissue that is located at the ends of long bones. Type II collagen, proteoglycans, water, and chondrocytes are the main constituents of articular cartilage. Osteoarthritis, the most common joint disease in the world, causes degenerative changes in articular cartilage tissue. Fourier transform infrared, Raman, and near infrared spectroscopic techniques offer versatile tools to assess biochemical composition and quality of articular cartilage. These vibrational spectroscopic techniques can be used to broaden our understanding about the compositional changes during osteoarthritis, and they also hold promise in disease diagnostics. In this article, the current literature of articular cartilage spectroscopic studies is reviewed.     

Chondrocyte recovery in cryopreserved porcine articular cartilage after bone carrier alteration

In order to investigate the consequences on the distribution of cell recovery through a cross-section of articular cartilage, the pathway for ice nucleation and diffusion of water and solutes in porcine osteochondral tissue was altered by drilling a 2mm diameter hole through the subchondral bone to the base of the cartilage. Samples equilibrated with 1M dimethyl sulfoxide were cooled at 1 C/min to -30 degrees C then stored in liquid nitrogen. A significant increase in chondrocyte recovery was documented when compared to samples cryopreserved without holes (48.3 percent vs 28.6 percent, P=0.003). The most significant change due to bone base modification was an increase in recovery in the middle section of the cartilage. These results provide insight into mechanisms of cryoinjury in tissue systems.     

Differential distribution of some extracellular matrix fibers in an experimentally denervated rat megaileum

Absence of enteric neurons is associated with thickening of the intestinal muscularis externa in Chagas' disease. The thickening is due to hyperplasia and hypertrophy of the smooth muscle cells and increased extracellular matrix components. The influence of the nervous system on the structure of the smooth muscle cells and its associated matrix has been poorly investigated. An experimental model of denervation of the ileum in rats was performed by application of the surfactant agent benzalkonium chloride that selectively destroys the myenteric plexus. Three months later, ileal tissue samples were obtained and studied by histochemistry and transmission electron microsocopy. Sham operated rats were used as controls. The diameter of collagen fibrils was evaluated in electron micrographs. The histopathological analysis showed thickening of the muscular layer. The thin and weakly arranged collagen and reticulin fibers surrounding the smooth muscle cells, observed in control cases by Picrosirius polarization (PSP) stain method, corresponded to a population of loosely packed thin collagen fibrils of uniform diameters (mean=29.16 nm) at the ultrastructural level. In contrast, the thick and strongly birefringent fibers around the muscle cells, observed in the treated group, stained by PSP, corresponded to densely packed thicker fibrils with large variation in diameter (mean=39.41 nm). Comparison of the data demonstrated statistically significant difference between the groups suggesting that the replacement of loosely arranged reticulin fibers by fibrous tissue (with typical collagen fiber), may alter the biomechanical function resulting in impairment of muscular contraction.     

关节软骨内胶原纤维各向异性的红外光谱学显微成像研究

福尔马林溶液对于固化关节软骨组织、防止在长时间测量过程中组织的分解退化起到很好的作用,但对福尔马林溶液浸泡后软骨组织的结构变化(固化)过程及其胶原纤维各向异性的改变却鲜有研究。采用傅里叶变换红外光谱显微成像技术与偏振技术相结合的方法,通过关节软骨内胶原纤维(蛋白)的红外光谱特征吸收峰(Amide Ⅰ,Amide Ⅱ带)的吸光度随福尔马林溶液浸泡时间及偏光角度的变化来研究福尔马林溶液对软骨组织结构即胶原纤维各向异性的影响,并利用与各向异性方程拟合得到的决定系数(R2)对胶原蛋白纤维各向异性程度进行表征。研究发现,关节软骨Amide Ⅰ和Amide Ⅱ带的各向异性随着福尔马林浸泡时间的增长而愈加明显(Amide Ⅰ带变化尤为明显),说明福尔马林溶液中甲醛分子诱发了胶原蛋白分子新的交联,最终获得较好的固化效果,有利于关节软骨的各向异性分析。本研究将为今后关节软骨样本的制备、储存及各向异性研究提供参考。     

Magnetic resonance imaging of the knee with ultrashort TE pulse sequences

BACKGROUND: We wished to assess the feasibility of imaging the knee with ultrashort TE (UTE) pulse sequences. SUBJECTS AND METHODS: Five volunteers and 16 patients were studied with UTE (TE=0.08 ms) sequences including later echoes. Conventional fat-suppressed images and difference images were also produced by subtracting a later echo from the first. Gadodiamide enhancement was used. RESULTS: High signal was obtained in tendons, ligaments, menisci and periosteum. Normal contrast enhancement was seen in these structures. Deep and superficial layers were seen in the articular cartilage. Cartilage defects were identified. The red zone could be differentiated from the white zone of the meniscus. Meniscal tears and degeneration were observed with low signal on subtraction images. Enhancement was seen within the anterior and posterior cruciate ligaments and associated scar tissue. CONCLUSION: Ultrashort TE imaging provides new options to visualize anatomy, manipulate conspicuity, observe contrast enhancement and demonstrate disease of the knee.     

关节软骨光透明的拉曼光谱研究

为探讨原位条件下关节软骨及下骨组织成分含量变化等深层次微观信息,采用拉曼光谱技术结合组织光透明技术来研究软骨组织的光透明效果。选用甘油、碘海醇作为光透明剂,对在不同光透明剂浓度和浸渍时间下的犬膝关节股骨端软骨样本进行拉曼光谱采集。通过计算磷酸基团(920~960 cm-1 ) 和酰胺Ⅰ带(1 595~1 700 cm-1)的积分强度,获得拉曼强度比(磷酸基团/酰胺Ⅰ),探索在不同浸渍时间(10~60 min)、不同浓度下(甘油40%,60%,80%和100%,碘海醇50,150,250和350 mg·mL-1)组织光透明的规律,即寻找合适的透明浓度和透明时间。结果显示：相比于无透明剂情形,两种透明剂均使该强度比信号增强。在同一时间范围内,甘油和碘海醇分别在60%和150 mg·mL-1 浓度下能获得较好的透明效果;而在不同浓度下,甘油的透明效果均在20 min最强,而碘海醇的透明效果一般是在50 min后开始增强。在光透明剂的作用下激发光透过软骨可以直接探测到软骨下骨的拉曼光谱信息,这为研究骨关节炎深层次病因提供了方法和思路。     

Structural aspects of an unusual choroidal polarized hyaline cartilage in the eyeball of Bufo ictericus and Rana catesbeiana (Amphibia, Anura): morphological and physiological significance

Amphibian eyes play an important role in vision and in several physiological processes, such as food capture and breathing. To maintain the integrity of the eyeball there is a unique cuplike hyaline cartilage as a supporting tissue. In Bufo ictericus and Rana catesbeiana the cartilage layer is located between the retina and the choroids, being designated as choroidal cartilage, important to visual performance. On the retinal surface, there is no perichondrium, and the pigmented epithelium exhibits an intimate relationship to the cartilage layer.     

太赫兹技术用于关节软骨检测的研究进展

骨关节炎是主要由软骨组织损伤与退化引起的常见关节疾病,是影响人类健康的重大疾病之一,对于关节软骨组织早期病变的检测可以大大提高疾病的治愈率,然而相关的临床诊断技术尚未发现。近年来,太赫兹技术在医学领域日益受到关注。与传统方法相比,太赫兹辐射能量低,不会产生电离辐射,可高灵敏、无损伤地对生物组织进行成像检测,因此在关节软骨诊断方面具有较大的应用潜力。本文简要介绍了关节软骨的生理与病理情况以及目前关节软骨检测的主要方法,重点总结了太赫兹技术应用于关节软骨检测方面的相关研究工作,分别包括对动物与人类关节软骨的检测,探讨了太赫兹技术在关节软骨检测中所面临的挑战与未来研究展望。