Staining of intracellular granules in fresh epiphyseal cartilage by cationic dyes

Hand-cut sections of fresh epiphyseal cartilage from young rats were stained at pH 4.5 in 0.01% solutions of various cationic dyes of the thiazine, oxazine, azine, triphenylmethane, acridine, and phthallocyanin classes. The intracellular -and -metachromatic granules, previously demonstrated in fresh tissues with toluidine blue, were also demonstrated well with azure A, methylene blue, and brilliant cresyl blue. The granules were also demonstrated, but not as well, by thionin, neutral red, safranin O, toluylene blue, and acridine orange. Under the conditions of staining, the reserve zone matrix and the lower hypertrophic (calcifying) zone matrix stained, whereas the proliferative and upper hypertrophic zone matrix did not stain.Gallocyanin, crystal violet, basic fuchsin, azocarmine B, gallamine blue, and alcian blue either did not stain, or gave a different pattern of staining from that described above.It is suggested that the pK and molecular weight of the dyes are important, but not necessarily the only factors in determining the staining of the granules. The results indicate that there is a change in the metachromatic material (presumably proteinpolysaccharide) in both the matrix and cells of epiphyseal cartilage, which appears to be related to calcification.Supported in part by grant AM 05922 from the National Institute of Arthritis and Metabolic Diseases, Contract AT (30–1) 2960 from the U.S. Atomic Energy Commission, and a grant for general research support given by the USPHS to State University of New York, Downstate Medical Center.     

Ultrastructure of hypertrophic cartilage: Histochemical procedures compared with high pressure freezing and freeze substitution

The effect of cationic dyes on the ultrastructure of hypertrophic cartilage was compared with results obtained with modern cryotechniques in studies on rat epiphyseal growth plate. Addition of alcian blue, acridine orange, cupromeronic blue, ruthenium hexamine trichloride, ruthenium red, or safranin O to conventional glutaraldehyde/ osmium tetroxide fixatives to a large extent resulted in prevention of chondrocyte shrinkage except for alcian blue which showed poor tissue penetration. The fine structure of the matrix in pericellular and territorial compartments appeared very coarse with areas of high contrast in tissue exposed to fixatives containing cationic dyes. This indicates structural collapse and precipitation of electron-dense material, a pattern clearly differing from that observed in specimens prepared by the cryotechniques. The dyes giving a pattern most similar to that seen after high pressure freezing, freeze substitution, and low temperature embedding were acridine orange and safranin O. It is concluded that studies of matrix ultrastructure down to the molecular level necessitate the application of cryotechniques.     

Ultrastructural modifications of proteoglycans coincident with mineralization in local regions of rat growth plate

We carried out histochemical and electron-microscopic studies to examine the relationship between mineral deposition and changes in the organization of proteoglycans during mineralization in the cartilaginous growth plate of the rat. To preserve the distribution and organization of proteoglycans in the extracellular matrix and to stain proteoglycans, acridine orange was included in the solutions that were used for fixation and demineralization. In undecalcified sections, mineral crystals that initially appeared at the level of the penultimate hypertrophic chondrocyte grew into dense, spherical mineral clusters, roughly one micrometer in diameter, in the longitudinal septa of the lowermost hypertrophic zone. Following the removal of mineral with EDTA in the presence of acridine orange, proteoglycan structures with a characteristic rosette-like architecture were revealed within the matrix of the longitudinal septa of the lower hypertrophic zone. The location of these rosette-like proteoglycan structures was identical to that of the dense, spherical mineral clusters in the undemineralized controls, and they were remarkably similar in morphology and size. In the electron micrographs stained with uranyl acetate and lead citrate, the greatly increased density of the rosette-like structures and large diameter of the proteoglycan structures located in mineral clusters contrasted sharply with the slender strands of more faintly stained proteoglycan that was diffusely distributed throughout most of the extracellular matrix. X-ray microprobe analysis for sulphur confirmed the existence of proteoglycans in the rosette-like structures and demonstrated that the concentration of proteoglycans was selectively increased in these regions. The cores of the metaphyseal calcified cartilage also exhibited rosette-like proteoglycan structures, which before demineralization were totally obscured by the diffuse, dense homogeneous deposition of mineral. Without stabilization of the proteoglycans in the extracellular matrix with acridine orange, the rosette-like proteoglycan structures could not be demonstrated. These results clearly indicate that there is a selective increase in the concentration of proteoglycans in exactly the same regions where mineral clusters are formed in the hypertrophic zone of the cartilaginous growth plate.     

Changes in proteoglycan aggregates during cartilage mineralization

Summary The dimensions of proteoglycan aggregates, aggregated monomers, and nonaggregated monomers, and the proportion of aggregated monomers found in the different zones of bovine rib growth plate have been defined by the electron microscopic monolayer technique. Growth plates were divided into the following 1 mm thick transverse slices; the hypertrophic zone, the lower proliferative zone, the upper proliferative zone, a transitional zone, and epiphyseal cartilage. Proteoglycans prepared by associative extraction followed by equilibrium density gradient centrifugation under associative conditions were examined by electron microscopy. Proteoglycan aggregate size decreased sharply in the lower proliferative and hypertrophic zones, as indicated by decreases in hyaluronate filament length and in the number of monomers per aggregate. Aggregated proteoglycan monomers did not show evidence of proteolytic degradation. Nonaggregated monomers were shorter than aggregated monomers, but their mean length did not decrease in the lower proliferative and hypertrophic zones. However, the proportion of nonaggregated monomers increased in these zones. Thus, before the cartilage matrix mineralized in the lower proliferative zone and as the cartilage matrix began to mineralize in the hypertrophic zone, proteoglycan aggregate size decreased and the proportion of aggregated monomers decreased. These changes in matrix proteoglycans may be one of the events that allow cartilage mineralization.     

S-100 protein in human cartilage lesions

S-100 protein is an acidic calcium-binding protein that was originally isolated from the mammalian central nervous system in 1965. Initially, S-100 protein was thought to be specific to neuroectodermal tissues, but its presence in chondrocytes was recently reported. This study is an analysis of the distribution of S-100 protein in lesions of human cartilage and its possible significance. Several cartilaginous tumors, both benign and malignant, as well as normal epiphyseal growth plates, were examined for S-100 protein by the immunoperoxidase technique. Each cartilaginous lesion that was examined showed immunoreactivity for S-100 protein. The staining product was noted only intracellularly. The highest intensity of staining was seen in the hypertrophic chondrocytes of the zone of provisional calcification in the growth plate and in the large chondrocytes located adjacent to areas of matrix mineralization in cartilaginous tumors. In normal epiphyseal growth plates, the intensity of staining increased in chondrocyte cytoplasm as one moved from the proliferating columnar chondrocytes through the zone of hypertrophic chondrocytes to the hypertrophic, degenerating chondrocytes in the zone of provisional calcification. In cartilaginous tumors, the cells of enchondroma and of the cartilaginous cap of osteochondroma were more immunoreactive than those of chondromyxoid fibroma. In benign chondroblastoma, the chondroblasts were less reactive than the chondrocytes in areas of chondroid matrix production. The latter areas of chondroblastomas showed stronger immunoreactivity in the matrix-enclosed cells adjacent to areas of mineral deposition. Among conventional chondrosarcomas, grade-I tumors showed greater immunoreactivity of the chondrocyte cytoplasm than did those of a higher grade, in which chondroid matrix production was less abundant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrolytes of isolated epiphyseal chondrocytes,matrix vesicles,and extracellular fluid

Summary Chondrocyte, matrix vesicle, and membrane fractions, as well as interstitial fluid samples from the proliferating and hypertrophic zones of chicken epiphyseal cartilage were analyzed for electrolyte content. Intracellular Ca levels were 1.4–2.1 mM, over 90% of which was nondiffusible. Isolated hypertrophic chondrocytes had higher intracellular Na and lower K than proliferating cells. Matrix vesicles contained 25 to 50 times higher concentrations of Ca than the adjacent cells. Vesicles from the zone of hypertrophy contained twice as much Ca as did those from the proliferating area. Ca/P₁ molar ratios of matrix vesicles were much higher than those of cells or of later mineral deposits. These findings indicate that Ca is concentrated in matrix vesicles during formation, but acuumulation of Ca and P₁ must continue in the matrix. X-ray diffraction of freeze-dried vesicle and membrane fractions failed to detect crystalline apatite, suggesting that crystals seen in electron micrographs of matrix vesicles may be artifacts. Interstitial fluid expressed from epiphyseal cartilage was higher in K, P_i, Mg and nucleotides, and lower in Na and Cl, than blood plasma. Fluid from the hypertrophic zone was higher in K and nucleotides, but not P_i or Mg, than that from the proliferating layer. These data suggest that selective leakage or extrusion of these constituents, which are normally intracellular, must occur, especially in the hypertrophic zone. More of the Ca and Mg, and less of the P_i, was protein-bound in cartilage fluid than in blood plasma. There was more binding of the divalent cations in fluid from proliferating than from hypertrophic cartilage. The presence of greater amounts of ultrafilterable peptides in fluid from hypertrophic than from proliferating cartilage or blood plasma, suggests that proteolytic activity may release bound divalent cations during mineralization.     

Ultrastructural cytochemistry of complex carbohydrates in osteoblasts, osteoid, and bone matrix

Glycosaminoglycans (GAGs) and glycoproteins are essential components for osteogenesis. We have examined rat osteoblasts, osteoid, transitional zone, and fully calcified bone matrix, utilizing Spicer's high-iron diaminethiocarbohydrazide-silver protein (HID-TCH-SP) method for sulfated glycoconjugates and Thiéry's periodate-TCH-SP (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained cytoplasmic granules of osteoblasts. Stain deposits in the extracellular matrix were observed in decreasing amounts in osteoid, the transitional zone, and fully calcified bone matrix. Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits. PA-TCH-SP staining was observed with increasing intensity in rough endoplasmic reticulum, Golgi saccules, and cytoplasmic granules. Collagen fibrils in osteoid were weakly stained with PA-TCH-SP, and their staining appeared even weaker in fully calcified bone matrix. In contrast, collagen fibrils in calcified cartilage stained intensely with the PA-TCH-SP method. Focal circular profiles (0.1-0.5 mumol in diameter), which lacked collagen fibrils but reacted moderately with PA-TCH-SP, were frequently seen in the transitional zone and fully calcified bone matrix, but were only occasionally present in osteoid. The presence of testicular hyaluronidase-resistant GAG and acid phosphatase in these focal areas suggests that they represent sites of GAG degradation. The eventual loss of HID-TCH-SP staining in the bone matrix suggests that removal of sulfated glycoconjugates may be a requisite for expansion of initial calcification sites and/or complete calcification.     

Legg-Calvé-Perthes disease. Histochemical and ultrastructural observations of the epiphyseal cartilage and physis

Biopsy specimens of the lateral aspect of the femoral head and neck were obtained from five children with Legg-Calvé-Perthes disease and were studied using histochemistry and electron microscopy. Beneath the normal articular cartilage there was a thick zone of hyaline (epiphyseal) cartilage containing sharply demarcated areas of hypercellular and fibrillated cartilage with prominent blood vessels. The fibrillated cartilage was strongly positive to alcian blue, weakly positive to periodic acid-Schiff, and positive to aniline blue. The interterritorial matrix in the hypercellular areas was weakly positive to both alcian blue and periodic acid-Schiff. Ultrastructural examination of these areas revealed many irregularly oriented large collagen fibrils and variable amounts of proteoglycan granules. These results suggest that in the fibrillar areas there are: (1) a high proteoglycan content, (2) a decrease in structural glycoproteins, and (3) a different size of collagen fibrils from that of normal epiphyseal cartilage. The hypercellular areas had a decrease in proteoglycans, glycoproteins, and collagen. The lateral physeal margin was often irregular, with a marked reduction of collagen and proteoglycan granules, and contained numerous large lipid inclusions. CLINICAL RELEVANCE: The abnormal areas in the epiphyseal cartilage of patients with Legg-Calvé-Perthes disease have different histochemical and structural properties from normal cartilage and from fibrocartilage. This suggests that the disease could be a localized expression of a generalized, transient disorder of epiphyseal cartilage that is responsible for delayed skeletal maturation. The cartilage lesions are similar to those seen in the vertebral plates in patients with juvenile kyphosis. Whether the epiphyseal cartilage abnormalities are primary or are secondary to ischemia remains uncertain; however, it appears that the collapse and necrosis of the femoral head could result from the breakdown and disorganization of the matrix of the epiphyseal cartilage, followed by abnormal ossification.     

The effect of enzyme treatment upon the resorption of scapular implants

Pieces of scapula were removed from young rats, decalcified, and treated with pronase, papain, hyaluronidase or trypsin. The epiphyseal cartilages of control scapulae took up the Alcian blue stain, but those treated with pronase, papain or hyaluronidase did not; trypsin did not abolish the staining reaction. When the bones were implanted subcutaneously into the animals from which they had been taken, the epiphyseal cartilages of those treated with the first three enzymes were rapidly invaded by capillaries and small round cells. The cartilages of the control, untreated bones and those from bones treated with trypsin were not invaded in this way. It is postulated that the removal of acid glycosaminoglycans from cartilage may be one of the factors that trigger the capillary invasion that occurs in endochondral calcification.     

Immunohistochemical demonstration of extracellular carbonic anhydrase in epiphyseal growth cartilage

Summary The distribution of carbonic anhydrase isoenzymes C and B in the rat epiphyseal growth cartilage was demonstrated by an immunohistochemical method. The isoenzymes were found in different locations. Isoenzyme C was in the extracellular matrix of the hypertrophic and calcifying cartilage, and no reaction was observed in the chondrocytes. In contrast, the antiserum against isoenzyme B revealed only a weak cellular staining. This supports the hypothesis that carbonic anhydrase isoenzyme C, which is the high-activity form, changes the pH in the extracellular fluid of calcifying cartilage, favoring the deposition of calcium phosphate.     

正常膝关节软骨钙化层形态结构研究

目的探索关节软骨钙化层的结构形态及其与软骨非钙化层和软骨下骨之间的界面连接方式。方法组织库获取自愿捐献的人体正常股骨髁新鲜标本20个,男10个,女10个;年龄17～45岁。常规制备石蜡横、纵切片,番红O／固绿和冯库萨染色观察钙化层形态结构;扫描电镜观察软骨各层之间的界面连接方式;连续切片结合建模技术建立骨软骨三维模型。结果关节骨软骨复合组织番红O／固绿染色结果示软骨红染,软骨下骨蓝染,钙化层位于潮线与黏合线之间;冯库萨染色结果示钙化层黑染,结构及边界清晰,上界面以波浪状潮线结构与非钙化层紧密连接,下界面以凹凸不平的梳齿状结构与软骨下骨相互锚合：关节骨软骨剥离面及断面扫描电镜示钙化层与非钙化层以沟壑镶嵌方式相互嵌合;关节骨软骨复合组织纵切面观察,钙化层与软骨下骨间的黏合线呈凹凸不平的梳齿状结构;骨软骨三维模型观察结果与关节软骨自然剥离横断面扫描电镜观察结果基本一致。结论钙化层是关节软骨的重要结构,它通过特有界面连接方式将软骨牢牢固定在软骨下骨上。     

Morphological and biochemical studies during differentiation and calcification of fracture callus cartilage.

Differentiation and calcification of cartilage of a fracture callus morphologically, ultrastructurally, and histochemically resembles cartilage of growing epiphyseal plate. The fracture callus includes the various cartilage cell types found in the epiphyseal plate. Proliferating and hypertrophic cartilage had higher activities of cytochrome oxidase, alkaline phosphatase and glutamate aspartate transaminase than fibrocartilage. Enzymes controlling glycogen synthesis and glycolysis had higher levels of activity in fibrocartilage than in hypertrophic cartilage. Lysosomal enzymes, catalase, 6-phospho-gluconic acid and glucose 6-phosphate dehydrogenase were uniformly distributed. Alkaline phosphatase was associated with extracellular vesicles found in hypertrophic cartilage. EM dense granules were found in mitochondria in hypertrophic cartilage. There was an increase of total lipids in hypertropic and calcified cartilage as compared to resting cartilage.     

兔Perthes病模型的建立及VEGF表达的实验研究

目的建立兔Perthes病模型并探讨Perthes病程中股骨头局部VEGF表达的变化及意义。方法3月龄新西兰大白兔24只，体重1．6～1．8kg。取16只兔作为实验组，手术切断左侧圆韧带和股骨头支持带血供，建立兔Perthes病模型；剩余8只作为对照组，按照上述程序左侧股骨头进行手术，但不打开关节囊，保持股骨头正常血供。于术后1、2、4、8周分别处死动物，取出股骨头，行大体观察、x线片、组织学、VEGF免疫组织化学染色和VEGF mRNA原位杂交观察。结果实验组动物模型均制备成功，术后5d感染1例，退出实验。大体观察：对照组各时间点股骨头未见坏死改变；实验组股骨头随时问延长逐渐粗糙、失去光泽，变小，可见塌陷。x线片观察：术后1、2周，两组股骨头无明显差异；4、8周，实验组股骨头较对照组密度增高。对照组各时间点HE染色均未见股骨头坏死及修复改变；实验组术后4、8周可见血管及肉芽组织侵入，新骨形成，参与修复。免疫组织化学：对照组股骨头骺软骨中，肥大区表现出较高的VEGF免疫反应性（VEGF immunoreactivity，VEGF—IR），而增殖区VEGF—IR却表现较低水平。术后1周，实验组股骨头增殖区VEGF阳性细胞率开始高于对照组，实验组股骨头肥大区VEGF阳性细胞率明显减少。术后8周，实验组股骨头整个骺软骨区均表现VEGF—IR，增殖区VEGF阳性细胞率较对照组明显增加；坏死股骨头软骨内骨化中心修复重建，肥大区VEGF阳性细胞率较正常接近。术后1、2、4、8周，实验组骺软骨增殖区VEGF阳性细胞率与对照组比较均有统计学意义（P〈0．01）；术后1、2、4周，实验组骺软骨肥大区VEGF阳性细胞率与对照组比较均有统计学意义（P〈0、01）。原位杂交观察结果与免疫组织化学染色观察相似。缺血性坏死后，实验组肥大区VEGFmRNA表达丧失，增殖区VEGF mRNA表达升高。软骨内骨化中心修复重建后，实验组可见新形成的肥大区重新表达VEGF mRNA。结论VEGF在坏死股骨头骺软骨促进血管发生、软骨内骨化中心的修复重建方面起关键的调节作用。     

Calcium localized in juxtanuclear granules of epiphyseal chondrocytes with a dilute glyoxal bis(2-hydroxyanil) solution

The glyoxal bis(2-hydroxyanil) (GBHA) method for staining labile calcium deposits in chondrocytes was modified. Fresh blocks of epiphyseal cartilage stained in this dilute GBHA solution revealed calcium in the juxtanuclear Golgi vesicles, each approximately 1μ in diameter. Prolonged staining revealed previously described 0.5μ granules throughout the cytoplasm which tended to mask the juxtanuclear reaction. The red GBHA reaction product was believed to be a chelate of calcium since it was removable with EGTA, and stable in Na₂CO₃ and KCN solution. When the tissue blocks stained with the dilute GBHA solution were restained with the original concentrated GBHA solution, refractile bodies containing red GBHA-positive granules were revealed at the periphery of the hypertrophied chondrocytes. The presence of calcium in the juxtanuclear Golgi vesicles and in the pericellular refractile bodies may indicate that the Golgi is involved in the transport of minerals to the matrix.     

Parathyroid hormone-related protein regulates proliferation of condylar hypertrophic chondrocytes.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone-related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.     

Analysis of experimental immune synovitis cartilage using monoclonal antibodies reactive to rabbit proteoglycan

This study details the macromolecular changes in cartilage involving proteoglycan molecules in an animal model of rheumatoid arthritis. In experimental chronic immune synovitis, fluorescein-conjugated mouse IgG and three monoclonal antibodies (MAbs 2G2, 2E9, and 6C9) portraying differing fine antigenic specificity for rabbit cartilage proteoglycan monomer were utilized to detail alterations in cartilage proteoglycan. In normal and IgG-immune animals, fluorescein isothiocynate (FITC)-conjugated MAbs 2G2 and 2E9 stained cellular/pericellular (C/PC) region intensely. FITC-MAb 2G2 stained cartilage interterritorial matrix as well. FITC-MAb 6C9 stained only C/PC area lightly but did not stain matrix. A marked decrease in staining intensity with FITC-MAb 2G2 was noted in cartilage sections derived from animals with immune synovitis. A corresponding increase in staining of cartilage was noted with FITC-MAb 6C9. The augmented staining of articular cartilage with FITC-MAb 6C9 was most prominent in femoral condyle tissue sections, which corresponded to the cartilaginous area, with the greatest severity in gross pathology. There was a slight augmentation of staining with FITC-MAb 2E9, especially in the C/PC area of medial/femoral cartilage. In addition, the animals with immune synovitis showed abortive cartilage repair exemplified by the presence of chondrocyte cloning (up to 20 cells) which correlated with increased FITC-MAb 2G2 staining. The differential MAb staining patterns of cartilaginous tissues obtained utilizing FITC-conjugated monoclonal antibodies with known fine antigenic specificity indicates a modulation of proteoglycans involving predominantly core protein epitopes in the articular cartilage of animals with chronic immune synovitis.     

Microchemical studies on acid glycosaminoglycans of the epiphyseal zones during endochondral calcification

The acid glycosaminoglycans of resting, columnar, hypertrophic and calcified zones of calf epiphyseal growth plate and of nasal septum cartilage were extracted with a dissociative solvent, 3M GuCl, according to Sajdera and Hascall (1969), to separate the glycosaminoglycans into an extractable pool and an unextractable pool which remains bound within the tissue. Epiphyseal cartilage required longer extraction times than did nasal septum cartilage to extract comparable amounts of acid glycosaminoglycans suggesting a stronger binding of proteoglycans within the tissue. Towards the calcification front the glycosaminoglycans were extracted more easily while in calcified zone not more than 30% could be extracted. Data obtained by the CPC microfractionation procedure of Antonopouloset al. (1964) indicated similar distribution according to molecular weight and/or charge density for extractable and unextractable chondroitin sulphate in nasal septum cartilage and in resting and columnar zones of epiphyseal growth plate. Unextractable glycosaminoglycans in hypertrophic and calcified zones were of predominantly low molecular weight and/or charge density compared to the extractable pool. Hyaluronic acid was unextractable in nasal septum and in resting, columnar and hypertrophic zones with increasing concentrations towards the calcification front. In calcified zone a shift to mainly extractable hyaluronic acid occurred. The significance of these findings is discussed.     

Resistant proteoglycans in epiphyseal plate cartilage. Variations in their distribution in relationship to age and species

The localizations of resistant proteoglycans (RPGs) in the epiphyseal plates of rats, dogs, and humans are similar. In the epiphyseal plates from young rats, dogs, and humans, the RPGs form a stratum at the junction of the zones of resting and proliferating cells. Non-calcified cartilage RPGs are associated with cells which have the potential for proliferation or column organization. As the individuals age, RPGs are found in intercolumnar regions or at times are even absent. There is also a type of RPGs in calcified cartilage, including the calcified cartilage subjacent to the articular surface, in all species. In human epiphyseal plates, looser fibrillar RPGs change abruptly to a more condensed type in the zone of provisional calcification. Calcified cartilage RPGs stain more intensely with toluidine blue and may represent a different type of RPGs.     

软骨脱细胞基质的研制及其力学性质分析

目的　猪CACM的制作及其力学特征的检测。方法　切取猪耳软骨 ,采取TritonX - 10 0、Tris -HCl液等 4步法制取脱细胞的猪耳CACM。采用光镜、透射电镜、扫描电镜等对结构进行检测 ,同时对CACM进行力学检测。结果　肉眼观察 ,CACM与软骨除颜色略白外无差别。组织学HE染色 ,细胞陷窝已无细胞结构 ,阿尔新兰染色呈淡染 ,阴性 ;透射电镜下CACM软骨陷窝内已无细胞结构 ,细胞膜、核器均已溶解 ;扫描电镜下CACM基质纤维成分与正常软骨基质的胶原纤维在形态上无差异 ;力学试验 ,最大拉伸强度 ,最大拉伸比 ,猪耳软骨与其脱细胞基质比较及其弹性模量无统计学意义。结论　经TritonX - 10 0为主的脱细胞处理方法可以脱去软骨的细胞成分 ,不改变软骨细胞基质的主要成分 ,不改变其原有力学性质