膝关节原发性骨关节炎软骨和软骨下骨病理改变的定量研究

目的观察膝关节原发性骨关节炎(osteoarthritis,OA)胫骨平台软骨和软骨下骨病理改变特点,对比内、外侧平台软骨和软骨下骨结构参数,探讨钙化层和软骨下骨在OA发病机制中的作用。方法取2009年10月-2011年5月行人工全膝关节置换术治疗的30例30膝原发性OA患者自愿捐赠的新鲜胫骨平台标本进行实验。其中男11例,女19例;年龄55～78岁,平均65.1岁。病程10～25年,平均16.6年;患膝内翻畸形1～23°,平均9.3°。大体观察胫骨平台后在内、外侧中央负重区取材,常规制备脱钙石蜡切片,行HE和番红O/固绿染色,观察关节软骨退变特点,参照Mankin评分标准评分并分期;观察钙化层及软骨下骨病理改变。应用Image Pro Plus 6.0图像分析软件测量软骨和软骨下骨结构参数,包括软骨全层(total articular cartilage,TAC)厚度、钙化层(articular calcified cartilage,ACC)厚度、ACC/TAC比值、软骨下骨板(subchondral bone plate,SCP)厚度以及骨小梁体积分数(trabecular bone volume,BV/TV)。结果大体观察内侧平台软骨退变较外侧严重,内侧平台软骨Mankin评分为(12.4±1.1)分,显著高于外侧平台的(8.3±1.6)分(t=12.173,P=0.000)。根据Mankin评分结果在60个标本中,14个为OA早期,可见软骨浅表层裂隙、潮线复制和软骨下骨增厚;19个为OA中期,可见软骨深层裂隙、多发软骨下骨吸收陷窝和明显增厚的软骨下骨;27个为OA晚期,可见软骨全层缺失、软骨内化骨和"象牙化"软骨下骨。软骨和软骨下骨结构参数测定示:内侧平台TAC厚度显著低于外侧平台,ACC/TAC比值、BV/TV及SCP厚度显著高于外侧平台,差异均有统计学意义(P<0.05)。内、外侧平台ACC厚度比较,差异无统计学意义(P>0.05)。结论钙化层和软骨下骨可能在OA发生与进展中发挥了重要作用。     

In situ measurement of transport between subchondral bone and articular cartilage

Subchondral bone and articular cartilage play complementary roles in load bearing of the joints. Although the biomechanical coupling between subchondral bone and articular cartilage is well established, it remains unclear whether direct biochemical communication exists between them. Previously, the calcified cartilage between these two compartments was generally believed to be impermeable to transport of solutes and gases. However, recent studies found that small molecules could penetrate into the calcified cartilage from the subchondral bone. To quantify the real‐time solute transport across the calcified cartilage, we developed a novel imaging method based on fluorescence loss induced by photobleaching (FLIP). Diffusivity of sodium fluorescein (376 Da) was quantified to be 0.07 ± 0.03 and 0.26 ± 0.22 µm²/s between subchondral bone and calcified cartilage and within the calcified cartilage in the murine distal femur, respectively. Electron microscopy revealed that calcified cartilage matrix contained nonmineralized regions (～22% volume fraction) that are either large patches (53 ± 18 nm) among the mineral deposits or numerous small regions (4.5 ± 0.8 nm) within the mineral deposits, which may serve as transport pathways. These results suggest that there exists a possible direct signaling between subchondral bone and articular cartilage, and they form a functional unit with both mechanical and biochemical interactions, which may play a role in the maintenance and degeneration of the joint. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1347–1352, 2009     

Photoshop-based image analysis of canine articular cartilage after subchondral damage

Introduction The validity of histopathological grading is a major problem in the assessment of articular cartilage. Calculating the cumulative strength of signal intensity of different stains gives information regarding the amount of proteoglycan, glycoproteins, etc. Using this system, we examined the medium-term effect of subchondral lesions on initially healthy articular cartilage.Materials and methods After cadaver studies, an animal model was created to produce pure subchondral damage without affecting the articular cartilage in 12 beagle dogs under MRI control. Quantification of the different stains was provided using a Photoshop-based image analysis (pixel analysis) with the histogram command 6 months after subchondral trauma. Results FLASH 3D sequences revealed intact cartilage after impact in all cases. The best detection of subchondral fractures was achieved with fat-suppressed TIRM sequences. Semiquantitative image analysis showed changes in proteoglycan and glycoprotein quantities in 9 of 12 samples that had not shown any evidence of damage during the initial examination. Correlation analysis showed a loss of the physiological distribution of proteoglycans and glycoproteins in the different zones of articular cartilage.Conclusion Currently available software programs can be applied for comparative analysis of histologic stains of hyaline cartilage. After subchondral fractures, significant changes in the cartilage itself occur after 6 months.     

膝关节原发性骨关节炎软骨-软骨下骨复合单元改变的实验研究

[目的]观察膝关节原发性骨关节炎(osteoarthritis,OA)胫骨平台软骨和软骨下骨病理改变特点,评价不同OA期别的骨形态计量学,探讨软骨和软骨下骨在OA发病机制中的相互作用。[方法]取人工全膝关节置换术后的膝原发性OA患者(n=40)自愿捐赠的新鲜胫骨平台标本。大体观察后在胫骨平台内、外侧中央负重区取软骨-软骨下骨复合体并切割等分为A、B两组。A组行番红O/固绿染色,参照Mankin评分标准评分并分1⁴期,观察软骨及软骨下骨病理改变。B组用Mirco-CT扫描测量骨形态计量学参数。[结果]根据Mankin评分,Ⅰ期OA可见软骨浅表层纤维化、潮线复制,Ⅱ期OA可见软骨磨损腐蚀、微骨折形成、血管侵入钙化软骨层,Ⅲ期OA可见多发性裂隙深达软骨下骨、软骨内囊肿、血管越过潮线生长、明显增厚的软骨下骨,Ⅳ期OA可见软骨全层缺失、软骨内化骨、软骨下骨暴露。随OA发展的期别增高,骨矿化密度(bone mineral density,BMD)、骨体积分数(bone volume fraction,BV/TV)、组织矿化密度(tissue mineralized density,TMD)、骨小梁数目(trabecula number,Tb.N)、骨小梁厚度(trabecular thickness,Tb.Th)呈增长趋势,骨小梁间隔(trabecular spacing,Tb.Sp)及结构模型指数(structure model index,SMI)呈下降趋势,且OA各期之间有显著统计学差异(P<0.05)。评价软骨退变的Makin评分与BMD、BVF、Tb.Th呈显著正相关(P<0.001),与SMI呈显著负相关(P<0.001)。[结论]软骨和软骨下骨构成结构和功能上的复合单元,在OA进展中相互影响。异常力学载荷引发骨重塑,微裂隙和血管等连接通道的存在提示软骨和软骨下骨间存在物质传递和分子交流。软骨下骨的特异性改变和软骨退变密切相关。     

降钙素对大鼠骨关节炎关节软骨和软骨下骨的影响

目的 观察骨吸收抑制剂降钙素对大鼠前交叉韧带切断术后膝关节软骨和软骨下骨的影响,探讨软骨下骨在骨关节炎发病中的作用.方法 建立大鼠右膝前交叉韧带切断的骨关节炎模型,术后第二天隔日皮下注射降钙素,模型组和Sham 组注射等量的生理盐水,持续6 w,并于术后10 w取材.切片后作甲苯胺蓝染色和Ⅱ型胶原免疫组化染色,并测量平均灰度值,用于评估关节软骨中蛋白多糖和Ⅱ型胶原的含量,行HE染色用于软骨下骨组织形态计量分析.结果 甲苯胺蓝、Ⅱ型胶原免疫组化染色平均灰度值和骨组织形态计量学参数测量显示与Sham 组相比模型组关节软骨中蛋白多糖和Ⅱ型胶原丢失,软骨下骨骨形成增加,骨质硬化.早期皮下注射降钙素可抑制大鼠骨关节炎后期软骨下骨骨形成的增加,减少关节软骨中蛋白多糖和Ⅱ型胶原的丢失.结论 软骨下骨在骨关节炎发病中发挥重要作用,骨吸收抑制剂降钙素可作为治疗骨关节炎的有潜力的药物.     

Differential articular calcified cartilage and subchondral bone in postmenopausal women with osteoarthritis and osteoporosis: Two-dimensional analysis

ObjectivesOsteoarthritis (OA) and osteoporosis (OP) are both common problems that affect life quality in aging society. It has been for decades of years to debate an inverse relationship between OA and OP. The objective of this paper was to compare structural properties of the articular calcified cartilage (ACC) and subchondral bone between postmenopausal women with OA and OP.MethodsEight femoral heads were taken from postmenopausal women during total hip replacement surgery due to primary OA. They were compared with nine femoral heads obtained from age-matched women with OP during hemi-hip arthroplasty surgery due to osteoporotic fracture of the proximal femur. Two-dimensional histomorphometric sections were prepared to measure the thickness of ACC and bone histomorphometric parameters. The ratio of ACC to the total articular cartilage (TAC) was also calculated in all bone specimens.ResultsOsteoarthritic donors had higher ACC and the ratio of ACC to TAC (ACC: 117.69 ± 28.16 μm vs. 97.88 ± 16.79 μm, P = 0.041; the ratio of ACC to TAC: 15.74% vs. 10.51%, P = 0.023). Lower bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and the ratio of nodes to termini (Nd/Tm) were demonstrated in donors with OP (BV/TV: 21.55 ± 3.41% vs. 16.64 ± 2.26%, P = 0.003; Tb.Th: 121.33 ± 16.11 μm vs. 98.59 ± 18.56 μm, P = 0.017; Tb.N: 2.41 ± 0.73 mm^?1 vs. 1.37 ± 0.31 mm^?1, P = 0.001; Nd/Tm: 0.78 ± 0.22 vs. 0.50 ± 0.14, P = 0.007). However, trabecular space (Tb.Sp) increased in these osteoporotic patients (Tb.Sp: 345.49 ± 106.18 μm vs. 652.09 ± 159.71 μm, P = 0.0004).ConclusionsThe pathogenesis of OA and OP were influenced by differential properties of ACC and subchondral bone microstructure. Structural properties of the subchondral mineralized tissue supported that an inverse relationship existed between postmenopausal women with OA and OP.     

Viscoelastic properties of bovine articular cartilage attached to subchondral bone at high frequencies

Background  

Articular cartilage is a viscoelastic material, but its exact behaviour under the full range of physiological loading frequencies is unknown. The objective of this study was to measure the viscoelastic properties of bovine articular cartilage at loading frequencies of up to 92 Hz.     

Changes in articular cartilage and subchondral bone histomorphometry in osteoarthritic knee joints in humans

In this study, we have examined the correlation between the histological and histochemical changes of articular cartilage and bone parameters of the underlying subchondral bone. The aim was to elucidate patterns of bone parameter changes within different depths of subchondral bone in the joints with macroscopically normal cartilage and in joints with osteoarthritis (OA). Ten tibial plateaus were taken from patients during total knee replacement surgery due to severe OA. They were compared with 10 sets of tibial condyles obtained from autopsy subjects with no history of bone or joint disease. The cylindrical cartilage-bone samples were taken out from the anterior, posterior, external, and internal areas of the condyles for cartilage assessment (Mankin score) and subchondral bone histomorphometry. Four histomorphometric parameters were measured: bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.S). Our study showed that subchondral bone from the OA group had significantly higher bone volume (54.1 +/- 10.6%) than control group (37.8 +/- 8.1%) (P < 0.01). In addition, trabecular parameters from the OA subchondral bone showed a smaller number of sparsely distributed and thicker trabecules than in control group (P < 0.05). Medial and lateral condyle from the control group did not differ significantly, while medial condyle from OA group showed a high increase of bone volume (62.8 +/- 13.3) and consecutively different trabecular parameters when compared with the lateral condyle from the same group. Also, it was shown that there are regional differences (anterior, posterior, internal, and external) in bone parameters between both condyles within both, control and OA groups. Comparison of bone parameters from three different stage of articular cartilage degeneration (Mankin score) showed that higher degree of cartilage degeneration is followed by significant changes in subchondral bone architecture. Furthermore, we have found that progression of cartilage degeneration leads to changes in bone parameters which affected deeper levels of subchondral bone. According to these results, it can be suggested that changes in histomorphometric parameters of subchondral bone are secondary to cartilage damage and proceed deeper into subchondral bone with increasing cartilage degeneration.     

帕瑞昔布对膝骨关节炎大鼠关节软骨及软骨下骨的影响

[目的]观察帕瑞昔布对膝骨关节炎大鼠关节软骨、软骨下骨及自噬标志物Beclin-1表达的影响。[方法] 30只雄性SD大鼠随机分为假手术组、模型组、治疗组。假手术组仅切开膝关节皮肤,模型组与治疗组应用Hulth法复制OA模型。治疗组自术后首天起腹腔注射帕瑞昔布[10 mg/(kg·d)],每周5次,连续12周。12周后处死大鼠,ELISA法检测血清IL-1、IL-6、CTX-Ⅰ、CTX-Ⅱ、TRACP-5b;关节软骨行HE、番红O染色及Mankin评分;Micro-CT扫描软骨下骨;RT-QPCR技术检测Beclin-1 mRNA表达量。[结果]模型组IL-1、IL-6、CTX-Ⅰ、CTX-Ⅱ、TRACP-5b水平高于假手术组(P0.05);治疗组IL-1、IL-6、CTX-Ⅱ水平低于模型组(P0.05)。治疗组Mankin评分低于模型组(P0.05)。与假手术组比较,模型组骨体积分数(BV/TV)、骨小梁量(Tb.N)显著降低(P0.05),而骨小梁分离度(Tb.Sp)显著增高(P0.05)。与模型组比较,治疗组骨体积分数(BV/TV)、骨小梁量(Tb.N)降低(P0.05),而骨小梁分离度Tb.Sp增高(P0.05)。治疗组Beclin-1 mRNA表达量高于假手术组与模型组(P0.05)。[结论]帕瑞昔布可改善OA大鼠关节软骨退变,其机制可能是通过增强细胞自噬来完成。并能促进大鼠软骨下骨骨量丢失,可能给OA治疗带来负面影响。     

The resurfacing of adult rabbit articular cartilage by multiple perforations through the subchondral bone.

The repair of articular cartilage removed from the femora of adult rabbits at the knee was studied. When multiple perforations were made through the subchondral bone a cartilaginous material, staining heavily with safranin O, first filled the holes. This material resembled hyaline cartilage, and by twelve months there was complete resurfacing of the joint, although the material lost its hyaline appearance after eight months and at one year it resembled dense collagenous tissue.     

大鼠膝关节不稳定骨性关节炎软骨与软骨下骨组织微观结构的变化

[目的]观察大鼠膝关节经前交叉韧带切断术(anterior cruciate ligament transection,ACLT)造成关节不稳定情况下,其关节软骨与软骨下骨骨组织微结构的变化.[方法] 20只3个月龄雄性SD大鼠按体重随机分为对照组与实验组,每组10只动物.实验组切断右膝关节前交叉韧带,对照组只暴露前交叉韧带而不切断,术后12周处死所有动物并取材.取右肢股骨远端膝关节于70％酒精固定后脱钙6周,石蜡包埋切片,经HE染色及Masson染色,普通光学显微镜观察摄片,采用图像分析系统做定量分析;取右肢胫骨膝关节于70％酒精固定后不脱钙做硬组织包埋切片,行Van Kossa染色后,定量分析软骨下骨组织微观结构变化.[结果]术后12周后大体观察发现,实验组关节面较对照组明显失去光泽,呈颗粒感,部分软骨面破溃,关节面周围可见骨赘形成;实验组关节软骨HE及Masson染色着色不均,软骨细胞排列紊乱,且实验组Mankin评分结果显著高于对照组,差异具有统计学意义(P＜0.05);实验组软骨下骨组织微结构较对照组改变明显,差异具有统计学意义(P＜0.05).[结论]ACLT术导致膝关节不稳定,在此情况下对大鼠膝关节软骨形态及软骨下骨组织微结构造成显著破坏,若不能及时采取干预措施,将加速膝关节功能退变进程.     

Quantitative MRI of parallel changes of articular cartilage and underlying trabecular bone in degeneration

OBJECTIVE: To determine the interrelations between degenerative changes in articular cartilage and underlying trabecular bone during development of osteoarthritis and to test the ability of quantitative magnetic resonance imaging (MRI) to detect those changes. METHODS: Human cadaver patellae were investigated with quantitative MRI methods, T(2) and dGEMRIC, at 1.5T. Same measurements for isolated cartilage samples were performed at 9.4T. Bone samples, taken at sites matched with cartilage analyses, were measured with MRI and peripheral quantitative computed tomography (pQCT). Mechanical and quantitative microscopic methods were also utilized for both cartilage and bone samples. RESULTS: Significant differences were found between the samples with different stages of degeneration in mechanical properties, T(2) at 1.5T and proteoglycan (PG) content of articular cartilage. dGEMRIC at 9.4T discerned samples with advanced degeneration from the others. Bone variables measured with pQCT discerned samples with no or minimal and advanced degeneration, and mechanical properties of trabecular bone discerned samples with no or minimal degeneration from the others. Significant linear correlations were found between the bone and cartilage parameters. Characteristically, associations between variables were stronger within the samples with no or minimal degeneration compared to all samples. CONCLUSIONS: Quantitative MRI variables, especially T(2) relaxation time of articular cartilage, may be feasible surrogate markers for early and advanced osteoarthritic changes in joint tissues, including decreased elastic moduli, PG and collagen contents of cartilage and mineral density and volume fraction of trabecular bone. Further work is required to resolve the relaxation mechanisms at clinically applicable field strengths.     

Quantitative assessment of articular cartilage and subchondral bone histology in the meniscectomized guinea pig model of osteoarthritis

OBJECTIVE: A new image analysis system was employed to quantify the main histological parameters reflecting osteoarthritic features, at the cartilage and bone levels, in the meniscectomized guinea pig model of osteoarthritis (OA). METHODS: Meniscectomized (MNX) and sham-operated (SH) guinea pigs were studied 1 and 3 months after partial meniscectomy at the medial side of the left knee (n=10 to 12 animals/group). The left proximal tibias were included in methylmethacrylate. Sections were cut and stained with safranin O or Goldner trichrome. Parameters were quantified using special programs of a Biocom image analyser. The following parameters were evaluated at the medial side of the tibia: cartilage thickness (CT); fibrillation index (FI); proteoglycan content ratio based on safranin O staining intensities (PC); chondrocyte density (CD); bone volume (BV) and subchondral bone plate thickness (SBPT). The degree of user interaction varied from manually tracing objects to almost complete computer automation. RESULTS: Meniscectomy resulted in significant variations of these reproducible histomorphometric parameters both after 1 month (FI: +522%, P<0.01) and 3 months (FI: +162%, P<0.001; PC: -36.7%, P<0.001; CD: -31.8%, P<0.001; SBPT: +8.7%, P<0.05) post-operation (results expressed as percentage variation of MNX vs SH). The linear correlation analysis including data from SH and/or MNX animals at the two grouped time points revealed significant r values, in particular between cartilage (CT) and subchondral bone parameters (SBPT) (r=-0.41, P<0.01). CONCLUSIONS: Contrary to scoring evaluation, this system allowed to show the time-dependent impact of the pathology with an early fibrillation of the medial tibial cartilage appearing as soon as 1 month post-surgery, and the close relationship between bone and cartilage parameters during the progression of OA.     

Magnetic resonance evaluation of the interrelationship between articular cartilage and trabecular bone of the osteoarthritic knee

OBJECTIVE: To use high-resolution magnetic resonance imaging (MRI) to determine the relationship between articular cartilage degeneration and trabecular bone changes of the femur, condyles and tibia in human knees with osteoarthritis (OA).METHODS: Subjects were divided into three groups: without OA (OA0), mild OA (OA1) and severe OA (OA2). Sagittal images of the knee (0.234 x 0.234mm2, 2-mm slice thickness) were obtained at 1.5T and used for calculating the volume and thickness of the femoral and tibial cartilage. Axial images (0.195 x 0.195mm2, 1-mm slice thickness) were used for calculating the trabecular bone structure parameters: apparent bone volume fraction, trabecular number, trabecular separation and trabecular thickness.RESULTS: Cartilage volume and thickness were less in patients with OA compared to normal controls (P<0.1). Articular cartilage thinning is associated with bone structure loss in the opposite femoral condyle (P<0.05). In varus OA, there were extensive correlations between medial tibia and medial femoral cartilage degeneration, and loss of bone structure in the lateral tibia and lateral condyle. Additional correlations existed between the compartmental differences (lateral minus medial) of cartilage thickness and bone structure.CONCLUSION: Degradation of articular cartilage within a compartment correlates with a loss of bone structure in the opposite compartment. The correlation between the (L-M) differences corroborates this relationship. Malalignment of the knee due to cartilage degeneration is associated with bone formation in the diseased condyle and bone resorption in the opposite compartment.