Studies on the composition of the extracellular fluid from calf costal cartilage

Appreciable amounts of free and bound hydroxyproline were found in the tissue fluid obtained by centrifugation from calf costal cartilage. In the tissue fluid the presence of an enzyme capable of splitting synthetic collagenase substrate at pH 7.4 and 37° was also demonstrated. The enzyme has been partially purified by (NH₄₂SO₄ saturation. Peak activity was obtained in the fraction of 50–60% saturation. The enzyme was not capable of degrading insoluble bovine. Achilles tendon collagen. In our opinion the enzyme cannot be considered a collagenase, and we call its activity “collagenase-like peptidase activity”.     

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

Summary Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluidine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue,N, N-Diethylpseudoisocyanine, and a modified PAS-method, and staining method, with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blocking reactions (methylation-saponification, carboxymethylation), were tested to select optimal staining procedures for the semiquantitative histochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 m-thick paraffin and 1 m-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 m apart using a leitz MPV 3 microspectrophotometer, starting at the surface of the cartilage and ending up at the tidemark. The result was compared with the fixed-charge density graph determined from the adjacent articular cartilage.Of the dyes tested, Thionin and Safranin O proved to be excellent cationic dyes for the histochemical quantification of cartilage matrix proteoglycans, since the staining intensity curves showed a linear correlation (r=0.900–0.995) with the fixed charge density curves from the adjacent cartilage. Also, the stain distribution was consistently uniform across the sections. In 1 m-thick glycolmethacrylate sections, the Safranin O staining gradient showed almost perfect identity with the fixed-charge density curve. Cuprolinic Blue and Cupromeronic Blue combined with the critical electrolyte concentration technique were also useful for the microspectrophotometric assays of glycosaminoglycans, but the presence of metachromasia should be checked prior to the measurements. The reliability of blocking procedures for quantitative histochemical work was not convincing.     

Electron microscopic visualization of proteoglycans with Alcian Blue.

The intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage, and chick embryo mesenchymal cells cultured in vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965). Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40-120 A thick filaments which display an orthogonal and longitudinal relationship to collagen fibrils. We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.     

Histochemical evidence of a functional heterogeneity of the chondrocytes of adult canine articular cartilage

Synopsis Twenty humeral heads were collected from 10 adult English Pointer dogs, fixed in 15% formalin containing cetylpyridinium chloride, decalcified, processed for paraffin sections, and cut serially. The articular cartilage was studied by staining principally with Alcian Blue in the presence of 0.4 or 0.9m MgCl₂ with and without a van Gieson counterstain. The results of the differential staining procedures demonstrated the existence of two groups of chondrocytes with distinctly different staining affinities. One group reacted intensely for the presence of protein-polysaccharides within its cytoplasm while the other group completely lacked this property. An approximate proportionality of 31 of the protein polysaccharide-positive and-negative chondrocytes was observed in the tangential layer and upper intermediate zone. In the lower intermediate zone, radiate zone, and zone of calcified cartilage, the chondrocyte types were present in equal proportions. Staining with Alcian Blue in the presence of 0.9m MgCl₂ with and without a van Gieson counterstain indicated a further subdivision of the protein-polysaccharide positive group of chondrocytes. This blocking technique has been reported to distinguish between chondroitin sulphate and high mol. wt. keratosulphate. Thus, based upon a greatly decreased number of the blue-stained chondrocytes after staining with Alcian Blue in the presence of 0.9m MgCl₂ the hypothesis is put forward that some chondrocytes produce primarily chondroitin suphate and others produce both chondroitin sulphate and keratosulphate.     

Staining of demineralized cartilage

Summary Safranin O in the orthochromatic form stains articular cartilage proteoglycan quantitatively in histological sections of demineralized cartilage. This was shown by scanning microdensitometry of stained sections of undemineralized and demineralized articular cartilage and by biochemical analysis of ³⁵S labelled cartilage subjected to demineralization. In constrast, Alcian Blue staining is affected by unknown factors other than simply the amount of proteoglycan present. Alcoholic formalin fixes articular cartilage proteoglycan more successfully than formol Zenker for subsequent rapid demineralization. Alcoholic formalin does not preserve cellular appearance as well as formol Zenker. Staining of articular cartilage with PAS appears unaffected by demineralization.     

Selection of a simple protease procedure for identifying mast cells in routinely processed human tissues

Proteases present in mast cell granules have been harnessed to demonstrate mast cells in human tissues. A number of substrate mixtures were tested. D-Val-Leu-Arg-4-methoxy-2-naphthylamide (MNA) plus Fast Blue B was the best for identifying human mast cells, yielding the most specific and complete staining. The procedure is simple and the results are permanent. Cryostat sections of aldehyde-fixed routine preparations or paraffin sections of Carnoy-fixed tissues give the most satisfactory results. Mast cells are stained a strong red color that stands out distinctly from the surrounding tissues, so that they can be easily identified by simple microscopy. A double-staining technique, first for protease and subsequently using Alcian Blue, showed that as progressive protease staining occurs, the alcianophilia of mast cells is lost. This procedure demonstrated that mast cells in the mucosa of human gut generally required longer incubations to develop protease staining than in other connective tissue sites. In post-mortem tissues, mast cells retain their protease activity well and so can be demonstrated in cryostat sections of aldehyde-fixed material, giving a more complete picture than with Alcian Blue. The synthetic substrate D-Val-Leu-Arg-MNA can be recommended for routine identification of mast cells in human tissues.     

Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.     

Victoria blue B — a nuclear stain for cytology

Summary The aim of this study was to investigate the staining characteristics of Victoria Blue B in alcohol solutions. Cytological specimens (liver and spleen tissue imprints, blood smears) were stained with methanol solutions of commercially available Victoria Blue B-Cl and with pure Victoria Blue B-BF₄. The dye concentration, staining time, and protone concentration of the dye solution were varied. The dye solutions were characterized using spectrophotometry and thin-layer chromatography. Cytophotometry and image analysis were used to quantitate the staining pattern of cell nuclei. Feulgen-stained slides were used as controls. Victoria Blue B-BF₄ gave excellent nuclear staining exhibiting a quantitative dye-substrate relationship, whereas commercial dyes resulted in lower staining intensity and less distinct nuclear texture. Dye concentration and staining time were, over wide ranges, not of critical importance for the quality of the staining. Under certain staining conditions, only cell nuclei were stained, with the background remaining completely unstained. We presume that, in alcohol solutions, Victoria Blue dye binds as a neutral dye molecule in conjunction with its anion. Victoria Blue B-BF₄ staining provides a simple and reproducible staining technique for cytology which is suitable for use in automated cell-pattern recognition.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdayThis study was supported by a grant from the Bundesministerium für Forschung und Technologie, Bonn-Bad Godesberg, Federal Republic of Germany, (no. 01 Z08501/6)     

Distribution of proteoglycans and hyaluronic acid in transverse sections of bovine thoracic aorta

Summary The distribution of hyaluronic acid and proteoglycans in bovine thoracic aorta was studied by Alcian Blue staining of frozen tissue sections under controlled electrolyte conditions with and without prior enzymic digestion. Some sections were digested with chondroitinase ABC, testicular hyaluronidase or bacterial collagenase and subsequent staining permitted conclusions to be drawn about the distribution of specific glycosaminoglycans within the tissue. The total glycosaminoglycan content was maximal in the intima and decreased across the arterial wall to the outermost adventitial layer. The content of proteoglycan containing chondroitin sulphate and/or dermatan sulphate chains paralleled this distribution. However, other glycosaminoglycans also contributed significantly to staining, although there was no evidence for any appreciable concentration of heparin or highly sulphated heparan sulphate.Several experiments indicated that proteoglycan containing chondroitin sulphate and/or dermatan sulphate was associated with elastic laminae which were often seen stained along their periphery. Hyaluronic acid was present at significant concentrations in all locations of the aorta and there was evidence for a similar distribution of heparan sulphate which was possibly also present at a high concentration in the endothelium. Staining of sections after treatment with 4m guanidinium chloride confirmed that this extractant removed most of the proteoglycan from the tissue section.     

An alternative Alcian Blue dye variant for the evaluation of fetal cartilage

BACKGROUND: The most comprehensive evaluation of vertebrate skeletal development involves the use of Alizarin Red S dye to stain ossified bone and various other dyes to stain cartilage. The dye used most widely to stain fetal cartilage in rodents and rabbits is Alcian Blue 8GX. However, the global supply of this specific dye has been exhausted. Several forms of the dye marketed as Alcian Blue 8GX are now available, although they are not synthesized via the original 8GX manufacturing process. METHODS: One new Alcian Blue 8GX form and two Alcian Blue dye variants were evaluated in rats and rabbits using standard staining procedures. The staining quality of these dyes were evaluated relative to the original form of Alcian Blue 8GX based on cartilage uptake of the dye, clarity of the cartilaginous components, staining intensity of the dye, and overall readability of the specimens under stereomicroscopic evaluation. RESULTS: Staining with the newer form of Alcian Blue 8GX resulted in poor staining quality. The Alcian Blue-Pyridine variant performed well, although staining intensity was less than optimal. The Alcian Blue-Tetrakis variant provided staining characteristics that were most similar to the original form of Alcian Blue 8GX. CONCLUSIONS: Alcian Blue-Tetrakis was markedly better in its ability to stain fetal cartilage than the newer form of Alcian Blue 8GX.     

重组人中期因子midkine对大鼠膝关节软骨部分损伤的修复作用

目的:通过外源注射不同剂量的重组人中期因子midkine(rhMK),研究其对大鼠膝关节软骨部分损伤的修复作用。方法:雄性SD大鼠双侧膝关节建立软骨部分损伤的动物模型,术后24小时分别向关节腔内注射生理盐水或rhMK (20μg/kg、60μg/kg、180μg/kg)。于术后8周将大鼠全部处死,取材进行组织学观察,从而确定最佳注射剂量;在药代动力学研究中,按最佳注射剂量向正常大鼠膝关节腔内注射rhMK,分别于注射后1小时、1天、3天、6天、9天、12天和15天处死大鼠,检测膝关节软骨组织中rhMK的含量。结果:不同剂量的重组蛋白对膝关节软骨部分损伤均有不同程度的修复作用,其中180μg/kg的剂量效果最佳;以180μg/kg的剂量向正常大鼠膝关节腔内注射rhMK后,经过Kinetica5.0药代动力学软件拟合后,计算得rhMK在软骨组织中的消除相半衰期为8.69天。结论:rhMK对大鼠膝关节软骨部分损伤有明显的修复作用,最佳注射剂量为180μg/kg,最佳注射时间间隔为8天。     

Conclusions about aminopeptidase in tissue sections from studies of amino acid naphthylamide hydrolysis

Summary Catalytic properties (K_M, V_max) of aminopeptidase in pig kidney sections, in isolated membranes and in a solubilized purified form were investigated using amino acid 2-naphthylamides and 4-methoxy-2-naphthylamides. In the first case these properties were estimated on the basis of the stain intensity resulting from the coupling of product with Fast Blue B, in the latter two cases they were measured fluorometrically. The following observations were made: (1) In all three cases the substrate turnover was shown to be a direct function of time and enzyme concentration. (2) The values obtained for the solubilized and the membrane bound form were practically identical but differed from those found in tissue sections. (3) Each amino acid derivative had defined constants, but these were difficult to obtain in sections, especially if it was necessary, on account of poor solubilities, to use low substrate concentrations. (4) Hydrophilic amino acid derivatives were adsorbed to tissue membranes much less than hydrophobic ones. (5) Fast Blue B caused a non-competitive inhibition of enzymic activity. (6) Binding of antibody against pure aminopeptidase caused inhibition of the enzymic hydrolysis of all the naphthylamides. Thus, histochemical stain intensities per time and area derived from one substrate at a defined concentration are suitable for the determination of enzyme concentrations. However, no conclusions regarding the homogeneity of the enzyme in sections can be drawn by comparing the stain intensities obtained with different substrates in contrast to data from the inhibition of substrate hydrolysis by antibody.     

Kallikrein-like activity in salivary glands using a new tripeptide substrate, including preliminary secretory studies and observations on mast cells

Summary The tripeptide substrated-val-leu-arg-4-methoxy-2-naphthylamine gave a precise localization of reaction product in cryostat sections of aldehyde-fixed salivary glands from a number of species, with Fast Blue B as the capture reagent. In submandibular glands, there was strong staining of the granules in granular tubules of rats and hamsters and somewhat less in mice. Submandibular striated ducts showed variable periluminal staining in a finer granular form; it was abundant in guinea-pigs, strong in cats but somewhat less pronounced in dogs. Parotid glands contained less reactivity with none detectable in hamsters and guinea-pigs. In the rabbit, neither gland showed any reaction. Mast cells were densely stained in glands from cats and dogs; they were less reactive in rats and unstained in the other species.The closely related 7-amino-4-trifluoromethylcoumarin derivative of the tripeptide has been found highly satisfactory for assessing activity in submandibular saliva from cats. Preliminary functional studies indicate that an extensive rapid secretion of enzyme occurs into saliva on sympathetic stimulation, with a corresponding depletion of reactive material from the striated ducts in tissue sections. Far less mobilization of enzyme occurs into saliva on parasympathetic stimulation with no obvious change in the histochemical reaction of striated ducts. The possible significance of these findings in cats is discussed.Extensive qualitative and quantitative studies are required to evaluate enzyme and substrate specificities in each species. Nevertheless, derivatives ofd-val-leu-arg offer great promise for the functional testing of kallikrein-like reactivity both by histochemical means on cells and biochemically in their secretions.     

Occurrence of ornithine decarboxylase and polyamines in cartilage.

The activity of ornithine decarboxylase was investigated in cartilage from chick embryos, rabbits, rats and human foetuses. The enzyme activity in these cartilages was of the same order as the detected in other body tissues. Ornithine decarboxylase activity in chick-embryo cartilage and liver was the same when compared on the basis of total soluble tissue protein. The cartilage enzyme exhibited a pH optimum of 6.5 and a Km for ornithine of 0.16mM. Ornithine decarboxylase activity in chick-embryo pelvic leaflets was maintained at the value in vivo for up to 22h when the isolated tissue was incubated in a modified Waymouth's medium (MB 752/1) at 37 degrees C. After addition of cycloheximide to the incubation medium, ornithine decarboxylase activity declined, with a half-life of 40 min. The concentrations of the polyamines spermidine and spermine in chick-embryo pelvic cartilage and rabbit costal cartilage were of the same order as the concentrations detected in other tissues.     

Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues

Summary To test the value ofStreptomyces hyaluronidase in carbohydrate histochemistry, the effects of digestion with the enzyme on the staining of cartilage and non-cartilaginous tissues by Alcian Blue (AB) pH 1.0, AB pH 2.5, high iron diamine, low iron diamine, aldehyde fuchsin, dialysed iron-ferrocyanide and AB pH 2.5-periodic acid-Schiff were studied by light microscopy. The results obtained lead to the conclusion that theStreptomyces enzyme releases not only hyaluronic acid but also chondroitin sulphates and keratan sulphates in cartilage. Since hyaluronic acid is known to be linked to chondroitin sulphate proteoglycans, the enzyme is of limited value in localizing hyaluronic acid in cartilage. However, it is useful in localizing hyaluronic acid in most non-cartilaginous tissues.     

Cartilage-derived factor (CDF) I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of ³⁵SO₄^?2, [³H]-glucosamine and [³H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

Summary A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H₂O₂ generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-feroprotein in liver and tissue polypeptide antigen in mainmary gland served as models. The immobilized twostep enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

Prostaglandin A2 and S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartlidge, long bone and skull bone than did normal control animals. Administration of prostaglandin A₂ stimulated ³⁵S uptake by lung, kidney and aorta while ³⁵S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

Prostaglandin A2 and 35S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartlidge, long bone and skull bone than did normal control animals. Administration of prostaglandin A₂ stimulated ³⁵S uptake by lung, kidney and aorta while ³⁵S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

Dielectric characterization of costal cartilage chondrocytes

Background

Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

Methods

Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes.

Results

The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance.

Conclusions

We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues.

General significance

The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.