Morphology of the synovium during its differentiation and development in the mouse knee joint

Summary The prenatal and postnatal development of the mouse knee joint was investigated by transmission and scanning electron microscopy. In the prenatal stage, following the appearance of a narrow intercellular cleft between two skeletal elements on the 16th fetal day, clefting extended into the lateral synovial mesenchyme. In some regions, the extension of the cleft was very rapid, but in a certain region (future fat pad region), it was somewhat slower. Macrophage-like cells appeared in the synovial mesenchyme on the 16th fetal day, and then increased in number, and were distributed as if they were clustering around the presumptive clefting zone in the future fat pad region on the 17th–18th fetal day. This suggests that macrophage-like cells may participate in joint development, as they phagocytize and remove some kinds of solid extracellular matrix, and facilitate the cleft extension. In the early postnatal stage, scanning electron microscopic observations showed that there were two different types of cell in the synovial lining. One of them exhibited a surface morphology corresponding to that of macrophages: a spherical cell body and numerous pseudopodia. The other type of cell exhibited various cell shapes with many cytoplasmic processes extending along the synovial surface.     

Ultracytochemistry of glycosaminoglycans in the canine knee synovium.

The accurate localization and nature of glycosaminoglycans (GAGs) in the canine knee synovium were studied by ultracytochemical methods that involved high or low iron diamine-thiocarbohydrazide-silver proteinate (physical development) staining in combination with enzyme digestion control procedures. The results obtained indicated that heparan sulfates and hyaluronan were present mainly in the plasma membrane of the B (fibroblast-like) cells. In contrast, the plasma membrane of the A (macrophage-like) cells showed negative reactions after the histochemical examination. Dermatan sulfates, chondroitin sulfates (A and/or C) and hyaluronan were localized in the extracellular matrix of the synovial intima, whereby dermatan sulfates were confined to the fibrous component, whereas chondroitin sulfates and hyaluronan were found in the interfibrous matrix. Heparan sulfate was the only notable GAG molecular species localized in the basement membrane of the capillary wall. It is obvious that differences in the quality and localization of glycosaminoglycans in the canine synovial tissue are of specific interest in understanding normal functions as well as pathological alterations of the knee synovium in mammals.     

长距离跑步大鼠膝关节滑膜基质金属蛋白酶的表达

背景：很多研究表明基质金属蛋白酶 1,3,9和13 对关节软骨的退变存在影响,但是针对关节滑膜进行的专项研究相对较少。 目的：观察长距离跑步运动对基质金属蛋白酶1,3, 9以及基质金属蛋白酶13在滑膜上的表达的影响。 方法：Wistar雄性大鼠15只随机分为3组：对照组、平板组和上坡组。对照组普通笼养;平板组每天在平板0°的水平面跑步机上以1 km/h,运动1 h,持续45 d;上坡组先在平板0°跑步机上以1 km/h,运动1 h,持续15 d,然后在上坡+20°的跑步机上每天以1 km/h,运动1 h,持续30 d。造成不同程度的膝关节滑膜损伤模型,造模成功后取双后肢膝关节,进行石蜡包埋,矢状面整体切片,而后进行苏木精-伊红和免疫组织化学染色,观察并分析实验结果。 结果与结论：长距离跑步运动后,平板组和上坡组的滑膜组织中基质金属蛋白酶1的表达都比对照组增高    (P < 0.05),但平板组和上坡组之间差异无显著性意义(P > 0.05);而3组基质金属蛋白酶3的表达并无明显变化(P > 0.05);基质金属蛋白酶9和基质金属蛋白酶13在滑膜组织中的表达呈梯度递增状态(P < 0.05),对照组表达最低,平板组有所升高,上坡组的表达最高。说明长距离跑步运动可通过改变基质金属蛋白酶的表达而影响大鼠膝关节滑膜组织的正常生理结构。     

The effect of intra-articular capsaicin on nerve fibres within the synovium of the rat knee joint

The aim of this study was to establish the effects of intra-articular capsaicin (pelargonic acid vallinylamide) on synovial innervation of the rat knee. Rats were sacrificed 1, 2, 4 and 7 days after intra-articular injection of capsaicin and joint tissues stained with either conventional haematoxylin and eosin (H and E) or with specific antibodies to the calcitonin gene-related peptide (CGRP), substance P (both of which are markers for primary afferent fibres), the C-flanking peptide of neuropeptide Y (CPON) (localised in postganglionic sympathetic fibres), or protein gene product 9.5 (a pan-neuronal marker). At lower concentrations (0.1% and 0.25%), capsaicin produced no change in peptide staining pattern or histological appearance. At 0.5% capsaicin, there was complete loss of nerve fibres showing positive staining for CGRP and substance P at all time points. Staining for CPON and protein gene product 9.5 was still present, but decreased, 1 and 2 days after treatment and virtually absent at 4 and 7 days. These findings provide evidence for partially selective denervation induced by 0.5% capsaicin, in contrast to 1% capsaicin which abolished staining for all peptide markers, indicating a total ablation of nerve fibres. A consistent but unexpected finding was the presence of a severe inflammatory response in joints treated with 0.5% and 1% capsaicin. An influx of polymorphonuclear leucocytes was found to occur within 4 h of injection, with progressive appearance of mononuclear cells after this time. We conclude that it is difficult to specifically deplete sensory nerve fibres from the synovium by means of local capsaicin injection. Although selective loss of staining for sensory nerve fibres could be achieved by injection of 0.5% capsaicin, there was progressive non-specific loss of post-ganglionic autonomic fibres which may be related to the severe inflammatory response provoked by the higher doses of capsaicin.     

Ultrastructural localization of acid phosphatase in denervated and diabetic striated muscles.

Catabolism in denervated and diabetic rat skeletal muscle undergoing accelerated protein degradation has been investigated with methods for demonstrating acid phosphatase ultrastructurally. Control muscles displayed strong acid phosphatase activity in lateral sacs and in sparse secondary hysosomes distributed mainly near nuclear poles. Muscles from diabetic rats and, to a lesser extent, 2-day denervated rats, revealed increased secondary lysosomes apparently derived from fusion of mitochondria with acid-phosphatase-reactive vesicles and cisternae. The latter were interpreted as possibly originating from T tubules. Reaction product was also noted in the junctional folds of the motor end plate of a denervated muscle. At the longer post denervation intervals studied, deposits indicative of acid phosphatase were dispersed throughout the sarcoplasm with greater concentration in the I band and appeared more abundant in denervated than in contralateral control muscles. The enzymatic basis for the sarcoplasmic deposits and other deposits was confirmed by their absence from cytochemical controls, which included incubation in substrate-free medium, heat or NaF inactivation of enzyme, and exposure sequentially to PbNO3 and NaH2PO or PbNO3 and beta-glycerophosphate.     

Influence of additive hyaluronic acid on the lubricating ability in the temporomandibular joint

In synovial fluid, hyaluronic acid (HA) is an essential component for the lubrication of joints, thus preventing friction. The relationship between HA and joint friction is not unambiguously established yet. In the present study, the effect of the application of HA on the frictional coefficient in the temporomandibular joint was evaluated. After measuring the frictional coefficient in intact porcine joints (n = 10), the subsequent effect of phosphate-buffered saline (PBS) washing and gauze scouring and the application of HA was examined. Compared with the intact joint, the frictional coefficient was significantly larger after PBS washing and gauze scouring. Subsequent application of HA resulted in a significant decrease (50-75%) of the frictional coefficient. However, it did not recover to the same value as in the intact joints. Observations by scanning electron microscopy showed that after PBS washing, the amorphous layer of the articular cartilage was still intact, whereas it was partially collapsed after gauze scouring. In conclusion, the addition of HA did reduce the coefficient of friction under the experimental conditions in this study; the relevance to the clinical condition and the duration of the treatment effect in vivo require further investigation.     

The innervation of the synovium of the knee joint in the guinea pig: an immunohistochemical and ultrastructural study

 The innervation of the knee joint synovial membrane of the guinea pig, i.e., the synoviocyte layer, the subjacent connective tissue and the connective tissue region beneath, was analyzed with immunohistofluorescence and electron microscopy. A screening of the innervation with antibodies against the general axon marker – protein gene product (PGP) 9,5 – revealed the presence of nerve fibers distributed in various regions of the knee joint synovial membrane. Confirmating previous studies, some of these nerve fibers stained with antibodies to tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP). In addition, dynorphin (DYN)-containing fibers were detected, which have not been reported previously in normal joints. In general, the immunoreactive fibers were observed close to the synoviocytes and at blood vessels. Fibers with colocalization of NPY- and TH-like immunoreactivities (LIs), as well as of DYN- and TH-LIs were demonstrated. In the electron microscope, bundles of unmyelinated fibers as well as single fibers were found in the connective tissue region below the synoviocytes. Varicose parts of the nerve fibers contained mainly small, clear vesicles. Small and large dense-cored vesicles were also seen, but less frequently. Denser portions of the plasma membranes of some axons were observed in these regions, facing the extracellular space. Myelinated fibers were also observed in some nerve bundles. These findings emphasize the complex innervation of the synovial membrane, with nerve fibers containing a host of neuroactive substances. Altogether, these fibers are probably involved in many functions such as vasoregulation and control of synovial secretion in addition to being a source of mediators in joint inflammation. Accepted: 22 November 1997     

关节腔内注射透明质酸钠与富血小板血浆治疗膝关节骨性关节炎的比较

文题释义： 富血小板血浆：1993年HOOD等首先提出富血小板血浆的概念,人全血经过离心后获得富含血小板的血浆,在其中加入凝血酶后可变为胶状物,因此也被称为富血小板凝胶。富血小板血浆中含有大量的生长因子如血小板源性生长因子、转化生长因子β、胰岛素样生长因子1等,可促进骨再生。富血小板血浆的制备方法尚未形成统一的标准,主要有血浆分离置换法和密度梯度离心法2种方法。 透明质酸钠：是一种葡聚糖醛酸,广泛存在于胎盘、羊水、晶状体、关节软骨、皮肤真皮层等组织。它是关节腔滑液的主要成分,对关节起润滑作用,可以减少组织间的磨擦,关节腔内注入透明质酸钠的作用：明显改善滑液组织的炎症反应,增强关节液的黏稠性和润滑功能,保护关节软骨,促进关节软骨的愈合与再生,缓解疼痛,增加关节的活动度。  背景：富血小板血浆因其富含生长因子,在组织修复及再生中起促进作用,其已逐渐应用于临床治疗骨性关节炎。透明质酸钠可改善滑液组织的炎症反应,保护关节软骨,促进关节软骨的愈合与再生,缓解疼痛。 目的：对比观察富血小板血浆、透明质酸钠治疗膝关节骨性关节炎的疼痛和功能改善程度。 方法：符合标准的膝关节骨性关节炎患者被随机分配到富血小板血浆组和透明质酸钠组。富血小板血浆组患者在21 d内接受3次关节穿刺注射富血小板血浆治疗,透明质酸钠组患者在35 d内接受5次关节穿刺注射透明质酸钠治疗。注射前以及注射后2,4,6个月随访评估疼痛及功能的改善程度,所使用的评估量表包括可视量化分级量表、膝关节和骨性关节炎症状系统分级表、HSS评分量表。 结果与结论：富血小板血浆组最终有35例患者,透明质酸钠组有36例患者进入结果分析。在6个月的随访期末,两组患者的疼痛、功能症状都有所减轻。膝关节损伤和骨性关节炎症状系统分级评分表明,相对于透明质酸钠组,富血小板血浆治疗关节炎分级Ⅱ级患者更有效。最后1次治疗后,富血小板血浆治疗组患者可视量化分级量表分值相对于初始值减少50%的患者数明显多于透明质酸钠组。两组患者治疗后2,4,6个月HSS评分差异无显著性意义(P > 0.05)。结果表明,富血小板血浆治疗方法适用于骨性关节炎分级较低的患者。 ORCID: 0000-0003-4453-6221(孙仁义) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

骨桥蛋白对人膝骨关节炎软骨细胞透明质酸表达的影响

BACKGROUND: Both osteopontin and hyaluronic acid involve in the pathological process of osteoarthritis, resulting in the abnormal expression levels of various cytokines and enzymes. However, the relationship between the high expression of osteopontin and hyaluronic acid in chondrocytes remains unclear. OBJECTIVE: To investigate the effect of osteopontin on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes in vitro by modulating the level of osteopontin. METHODS: Chondrocytes from human knee osteoarthritic cartilage were cultured in vitro, and were then divided into three groups: blank control group without any treatment; osteopontin group and and pontin siRNA group were treated with 1 mg/L recombinant human osteopontin and osteopontin siRNA, respectively. Expression levels of osteopontin, hyaluronic acid synthase 1, 2 and 3 mRNA were detected by real-time PCR, and the levels of hyaluronic acid were measured using ELISA. RESULTS AND CONCLUSION: Compared with the blank control group, the mRNA expressions of hyaluronic acid synthase 1, 2 and 3 were remarkably increased in the osteopontin group, while siRNA made the significantly inhibitory effects on the hyaluronic acid synthase 1, 2 and 3 mRNA expressions (P < 0.05). The level of hyaluronic acid in chondrocytes in the osteopontin group was significantly higher than that in the other two groups (P < 0.05). Our results suggest that osteopontin induces the synthesis of hyaluronic acid in osteoarthritic chondrocytes through upregulating the hyaluronic acid synthases expression levels. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Ultracytochemical demonstration of glycoproteins in the canine knee synovium

By various ultracytochemical methods, glycoconjugates of the synoviocytes, the intercellular matrix and the wall of the small capillaries were studied in the synovial intimal tissues of the canine knee joint. Glycoconjugates with vicinal diol groups could be visualized in certain elements of the Golgi complex, lysosomes, vacuoles, the majority of intracellular cytomembranes, the surface coat of the plasma membrane and glycogen particles in type A cells. In type B cells, less-developed Golgi complexes, and fewer lysosomes and vacuoles were present in the cytoplasm than in that of type A cells. In contrast, a large number of cytoplasmic glycogen particles and abundant vicinal diol-containing groups in the surface coat of the plasma membrane became especially obvious in the B cells. Abundant neutral and acidic glycoproteins were observed in fibrous components in the intercellular matrix. In the small capillaries, strongly positive staining intensities for neutral and acidic glycoconjugates were observed in the basement membrane and perivascular connective tissue, as well as in the surface coat of the luminal plasma membrane of the endothelial cells, although to a somewhat weaker degree. Sialic acid, particularly, was notable in the surface coat of the latter cells. In addition, glycoproteins in the type A cells were shown by lectin ultracytochemistry to contain a variety of saccharide residues such as -D-mannose, -D-glucose, -L-fucose, N-acetyl-β-D-glucosamine, and N-acetyl-neuraminic acid, which were also found in the plasma membrane of the B cells.

The properties of the glycoconjugates found are discussed in relation to the basic functions assigned to the synovial membrane of the canine knee joint.     

Ultrastructural localization of viral nucleic acid by in situ hybridization

In situ hybridization has become a standard technique in the localization of viral nucleic acids in tissue sections and cytologic preparations at the light microscopic level. We have extended this technique to the electron microscopic level using human cytomegalovirus (CMV) infection in cultured human foreskin fibroblasts, and have shown for the first time that colloidal gold can be used to study intranuclear localization of viral replication. CMV-infected fibroblasts exhibiting early (4-day) and late (18-day) cytopathic effect were fixed in formalin, gently permeabilized with detergent and protease, and hybridized with a biotinylated CMV DNA probe. Hybridized sequences were localized by a pre-embedding technique using streptavidin-conjugated 15 to 20 nm colloidal gold particles. Ultrastructural nuclear and cytoplasmic architecture were well preserved through permeabilization and hybridization steps. Viral DNA was clearly detected in fibroblast nuclei containing nascent and well-formed electron-dense viral inclusions. Gold particles were localized to the periphery of electron-dense nuclear inclusions, occasionally in association with 70 nm nuclear dense bodies, but not with complete viral nucleocapsids. DNA hybridization was abolished by pretreatment of infected cells with DNase. Cross-hybridization of CMV DNA sequences with human DNA or with herpes simplex virus genome was not observed. The ultrastructural findings suggest that CMV DNA replication may occur at the margins of electron-dense regions in maturing viral inclusions, and that viral DNA associated with core dense bodies is available for hybridization with complementary nucleic acid sequences. This technique can be useful in studies of viral pathogenesis.     

骨关节炎患者膝关节滑膜CD1c+髓源树突细胞的鉴定

CD1c~+髓样树突细胞(myeloid dendritic cells, mDC)是一类重要的DC亚群,参与T细胞的免疫应答。为探讨膝骨关节炎患者关节滑膜是否存在CD1c~+mDC的分布,收集20例骨关节炎(osteoarthritis, OA)患者的外周血和膝关节滑膜,采用FACS检测外周血和关节滑膜内的CD1c~+mDC、单核/巨噬细胞和T细胞,免疫组化方法检测关节滑膜中CD1c、CD3、CD33分子的表达情况。结果显示,OA患者膝关节滑膜中的CD1c~+mDC和巨噬细胞的比例显著高于外周血(P<0.000 1),而T细胞比例显著低于外周血(P<0.000 1);关节滑膜中存在CD1c和CD3的弥漫性浸润且两者定位邻近。该研究提示,OA关节滑膜中存在一群比例显著高于外周血的CD1c~+mDC,CD1c~+mDC在骨关节炎发生、发展过程中具有重要意义。     

Ultrastructural localization of histones in epiphyseal chondrocytes.

Epiphyseal growth plate chondrocyte nuclei of growing rats were studied for histone content using the ammoniacal silver technique. This stain, which is specific for the demonstration of the arginine-rich fractions of histones, revealed that growth plate nuclei vary in their histone content. Nuclei of cells of the proliferating zone revealed a significantly greater amount of postformalin ammoniacal silver deposit consistent with the presence of arginine-rich histones.     

人膝骨性关节炎滑膜中表达的骨桥蛋白

背景：目前研究显示骨桥蛋白与骨性关节炎关节软骨退变密切相关,但骨桥蛋白与骨性关节炎滑膜病变是否相关,仍少见报道。 目的：研究骨桥蛋白在原发性骨性关节炎发生与发展过程中的作用。 方法：选取膝骨性关节炎患者关节滑膜标本与下肢外伤患者作对照,根据综合评分法进行分组,采用免疫组化方法检测关节滑膜中骨桥蛋白水平,比较不同程度骨性关节炎组膝关节滑膜中骨桥蛋白的差异,同时比较标本滑膜衬里层和衬里下层骨桥蛋白的水平。 结果与结论：骨性关节炎组织中骨桥蛋白阳性呈黄色、棕黄色、棕褐色表达,病变程度越重,颜色表达越深。骨性关节炎组滑膜中骨桥蛋白明显高于对照组,差异有显著性意义(P < 0.05),且随着骨性关节炎病情加重,骨桥蛋白的表达逐渐增多,呈正相关(ρ=0.663,P < 0.01)。但滑膜衬里层和衬里下层骨桥蛋白的水平差异无显著性意义(P > 0.05)。说明骨桥蛋白可能与骨性关节炎的发病有关。     

Ultrastructural localization of acid phosphatase in thyrocytes of rats with thyroid hyperfunction

Acid phosphatase was detected electron-cytochemically in lysosomes which appeared in large numbers in the follicular cells of the hyperplastic rat thyroid gland. The remaining types of granules (mature secretory granules, lipid granules), the number of which also increased appreciably during functional stress on the thyrocytes, did not contain the reaction product. By the character of distribution of acid phosphatase the lysosomes could be subdivided into three main groups: those with a dense homogeneous residue and those with residue in the form of densely or infrequently distributed circular grains. The heterogeneity of the lysosomes may be attributed to differences in their functional state. Besides in the lysosomes, the reaction product was found in pale cells as a sprinkling of widely scattered dark grains. These drops evidently appeared as the result of fusion of droplets of colloid with lysosomes. The acid phosphatase of the lysosomes in this case participates in hydrolysis of the secretion product of the cells with the formation of the active substances.Electron Microscopy Room, N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 622–625, May, 1977.     

Ultrastructural localization of acid phosphatase activity in the jejunal absorptive cells of fasted rats

Ultrastructural localization of acid phosphatase activity was investigated in the jejunal absorptive cells of 21 d fasted rats (about 500 g). The enzyme activity was localized on the membrane of microvilli, the lateral cell membrane, the Golgi complex, the lysosomes and the GERL of Novikoff (a part of the smooth surfaced endoplasmic reticulum located in close proximity to the Golgi saccules) of jejunal absorptive cells. Moreover, the lysosomes of various sizes and shapes with acid phosphatase activity was characteristically encountered in the infranuclear cytoplasm. The lysosomes appeared to be autolysosomes .     

Ultrastructural localization of acid phosphatase in rosetted T and B lymphocytes of normal human blood.

Using electron microscopy and cytochemical techniques, the authors determined the distribution of acid phosphatase (AcPase) within the organelles of lymphocytes from blood rosetted with either neuraminidase-treated sheep erythrocytes (En) or sheep erythrocytes coated with antibody and complement (EACs). Subsequently, the various reactive organelles of the rosetted lymphocytes were counted, affording a comparison of T and B cells. It was found that AcPase was present in approximately 80% of T cells and 45% of B cells and was most frequently observed in secondary lysosomes of varying size and content. Although more T cells than B cells were reactive for AcPase, the extent of reaction in some B cells clearly precludes the use of AcPase for differentiating the two cell lines. It should be recognized that while the En rosetting procedure detects T cells in a nonselective manner, the EAC rosette is a marker of a major subpopulation of B lymphocytes, ie, those bearing complement receptors. We believe that the distribution of lysosomal enzymes in B and T lymphocytes probably reflects the functional state of individual cells rather than being a reliable indicator of cell lineage. A surprising finding (which could be established only by a fine-structural study) was the fact that 20% of circulating "resting" T cells contained reaction product for AcPase within endoplasmic reticulum and the perinuclear cisterna indicating that these cells are actively synthesizing AcPase, probably due to a foregoing inductive event. Such stimulus could be the result of recent endocytosis of surface receptors in combination with antigen, antibody, or immune complexes and/or recent mitosis, or possibly some unrelated autophagic incident.