Staining of demineralized cartilage. I. Alcoholic versus aqueous demineralization at neutral and acidic pH

Demineralization of cartilage with alcoholic EDTA provides cartilage staining that is no better, as measured by scanning microdensitometry, than that of adequately fixed specimens demineralized with aqueous EDTA. Aqueous EDTA is a faster demineralizing agent than alcoholic EDTA. Certain fixatives can preserve maximal proteoglycan staining in articular cartilage even with subsequent rapid demineralization in formate buffer at pH 3.3. Although alcoholic formalin fixation provided optimum quantitative cartilage staining, cetylpyridinium chloride (CPC) in aqueous buffered formalin improved cellular detail, but CPC partially suppressed matrix staining.     

Chemical and Spectrometric Evaluation of Glycogen After Routine Histological Fixatives

A detailed comparison of fixatives used for the demonstration of glycogen has been based on chemical assay and microspectrophotometry. Rat liver containing known amounts of glycogen was fixed in formol alcohol, Rossman's fluid, 10% neutral formalin, Bouin, Helly, SUSA, and Zenker's fluid at 4 C and 18 C. Chemical assay was carried out before and after fixation and paraffin sections were prepared from the fixed material. Sections were stained with PAS and the silver methenamine method. Visual examination was carried out with a comparison microscope and quantitative estimations on PAS-stained sections were performed by scanning microspectrophotometry. The histochemical methods were compared with the chemical results obtained from the same tissue and a reasonable degree of correlation between the sets of results was observed. Cold formol alcohol and cold Rossman's fluid preserved the most glycogen and Zenker and SUSA fixation preserved the least. Cold formol alcohol was the only fixative that preserved threshold values of glycogen i.e. 0.3% and the silver methenamine method is recommended for the demonstration of these small amounts.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [³⁵S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

Persistence of cartilage proteoglycan and link protein during matrix-induced endochondral bone development: An immunofluorescent study

Monospecific antibodies to cartilage proteoglycan monomer and link protein were employed with immunofluorescence microscopy to determine the tissue distribution of these constituents during matrix-induced endochondral bone development. Subcutaneous implantation of demineralized diaphyseal bone matrix resulted in new endochondral bone formation. On Day 3, the implant consisted of mesenchymal tissue which did not contain any demonstrable cartilage-related proteoglycan or link protein. With the onset of early chondrogenesis on Day 5, cartilage proteoglycan monomer and link protein were first localized together in the cartilage matrix, particularly around chondrocytes in territorial sites. Progressively more staining around cells was observed at Days 7 and 9. On Day 9, when mineralization was first observed, there was no evidence of a net loss of these molecules prior to mineralization of the cartilage matrix. On Day 11 and thereafter, bone formation was observed by appositional growth on calcified cartilage spicules. Whereas the osteoblasts and bone matrix were devoid of any staining for cartilage proteoglycan and link components, the residual, partly mineralized cartilage spicules still reacted with antibodies to cartilage proteoglycan monomer and link protein in territorial sites, but in reduced amounts, indicating a loss of these molecules associated with a loss of hypertrophic chondrocytes. Since mineral prevented the access of Fab' antibody subunits, demineralization after fixation was routinely employed. The results reveal that cartilage proteoglycan monomer and link protein are present around chondrocytes in hyaline cartilage during the early stages of endochondral bone formation and that there is no net loss of these molecules prior to mineralization of this cartilage matrix as was previously thought.     

Fixation,decalcification, and tissue processing effects on articular cartilage proteoglycans

Summary Neutral buffered 4% formaldehyde fixation for 48h preserved well the proteoglycan content of bovine articular cartilage. Neither subsequent demineralization in 10% EDTA, nor light microscopic tissue processing, reduced the hexosamine or uronic acid content of the tissue. Fixation in alcoholic solutions increased Safranin O binding as well as periodic acid Schiff reaction of the cartilage matrix as measured by microspectrophotometry. It is suggested that the enhanced staining of cartilage was due to better preservation of the glycoprotein oligosaccharides. Quaternary ammonium salts in the fixative suppressed the staining of cartilage matrix with Safranin O.     

The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.     

Synthetic Orcein as a Stain for Chick Embryo Cartilage Matrix

Chick embryo tissues fixed in Bouin's fluid, in 10% formol saline or in 10% formol saline with subsequent mordanting in saturated picric acid containing 3% HgCl₂, were examined as 5 μ paraffin sections after staining with 1% synthetic orcein in 80% ethanol containing 1% HCl (conc.). Orcein defined the young elastic fibres formed in the truncus arteriosus, aorta and other large arteries after the 5th day of embryonic development but also reacted with the matrix of cartilage in all parts of the skeleton from the 3rd day onward. It is thought that a glycoprotein or proteoglycan shared by these two tissues could account for their mutual affinity for orcein.     

A simplified aluminum stain in paraffin sections of bone from hemodialysis patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Degradation of proteoglycan in articular cartilage

Adult rabbit articular cartilage was labelled in vivo over 48 h with [³⁵S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [³⁵S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the [roduction of non-aggregating species which diffuse readily from the tissue.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer

Summary A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with ³⁵S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of ³⁵S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.The IBAS program used in this work is available from Dr. J.J. Parkkinen or through Bitnet or EARN mail: MLAMMI at FINKUO     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

A Detailed Analysis of the Effects of Various Fixatives on Animal Tissue with Particular Reference to Muscle Tissue

Nine different fixatives (Carnoy's, Susa, Baker's formalin, 5% formalin, 10% formalin, 10% formol saline, Bouin, Zenker, and 2.5% glutaraldehyde) were compared by two methods. Gelatin-albumin gels were used to study volum changes after fixation and after various stages of subsequent processing. The appearance and hardness of the gels were also noted. The fixatives either shrunk or swelled the gels, but dehydration and clearing shrunk the gels in all cases. Sampkes of muscle tissue from one location in beef longissimus dorsi muscle were also placed in the different fixatives and processed. Various features were noted for each fixative, including the ease with which the paraffin wax blocks were cut and the staining ability of the sections in Mallory's triple stain. The diameters of the muscle fibers were measured from transverse sections of these samples and compared with the mean diameter of muscle fibera in a frozen unfixed section of muscle tissue. It was found that the fixatives had the same shrinkage effects on both the gels and the muscle samples. Analysis of variance tests showed that the various fuatives caused different degrees of shrinkage. Statistical details are given for the amounts of shrinkage caused by each fixative. Both the general histological picture and the amount of shrinkage were considered when deciding the bcst fixative. Carnoy was found to be the best of the fixatives investigated.     

Multiparametric MRI of Epiphyseal Cartilage Necrosis (Osteochondrosis) with Histological Validation in a Goat Model

Purpose

To evaluate multiple MRI parameters in a surgical model of osteochondrosis (OC) in goats.

Methods

Focal ischemic lesions of two different sizes were induced in the epiphyseal cartilage of the medial femoral condyles of goats at 4 days of age by surgical transection of cartilage canal blood vessels. Goats were euthanized and specimens harvested 3, 4, 5, 6, 9 and 10 weeks post-op. Ex vivo MRI scans were conducted at 9.4 Tesla for mapping the T₁, T₂, T_1ρ, adiabatic T_1ρ and T_RAFF relaxation times of articular cartilage, unaffected epiphyseal cartilage, and epiphyseal cartilage within the area of the induced lesion. After MRI scans, safranin O staining was conducted to validate areas of ischemic necrosis induced in the medial femoral condyles of six goats, and to allow comparison of MRI findings with the semi-quantitative proteoglycan assessment in corresponding safranin O-stained histological sections.

Results

All relaxation time constants differentiated normal epiphyseal cartilage from lesions of ischemic cartilage necrosis, and the histological staining results confirmed the proteoglycan (PG) loss in the areas of ischemia. In the scanned specimens, all of the measured relaxation time constants were higher in the articular than in the normal epiphyseal cartilage, consistently allowing differentiation between these two tissues.

Conclusions

Multiparametric MRI provided a sensitive approach to discriminate between necrotic and viable epiphyseal cartilage and between articular and epiphyseal cartilage, which may be useful for diagnosing and monitoring OC lesions and, potentially, for assessing effectiveness of treatment interventions.     

Does protein kinase R mediate TNF-α- and ceramide-induced increases in expression and activation of matrix metalloproteinases in articular cartilage by a novel mechanism?

We investigated the role of the proinflammatory cytokine TNF-α, the second messenger C₂-ceramide, and protein kinase R (PKR) in bovine articular cartilage degradation. Bovine articular cartilage explants were stimulated with C₂-ceramide or TNF-α for 24 hours. To inhibit the activation of PKR, 2-aminopurine was added to duplicate cultures. Matrix metalloproteinase (MMP) expression and activation in the medium were analysed by gelatin zymography, proteoglycan release by the dimethylmethylene blue assay, and cell viability by the Cytotox 96^® assay. C₂-ceramide treatment of cartilage explants resulted in a significant release of both pro- and active MMP-2 into the medium. Small increases were also seen with TNF-α treatment. Incubation of explants with 2-aminopurine before TNF-α or C₂-ceramide treatment resulted in a marked reduction in expression and activation of both MMP-2 and MMP-9. TNF-α and C₂-ceramide significantly increased proteoglycan release into the medium, which was also inhibited by cotreatment with 2-aminopurine. A loss of cell viability was observed when explants were treated with TNF-α and C₂-ceramide, which was found to be regulated by PKR. We have shown that C₂-ceramide and TNF-α treatment of articular cartilage result in the increased synthesis and activation of MMPs, increased release of proteoglycan, and increased cell death. These effects are abrogated by treatment with the PKR inhibitor 2-aminopurine. Collectively, these results suggest a novel role for PKR in the synthesis and activation of MMPs and support our hypothesis that PKR and its activator, PACT, are implicated in the cartilage degradation that occurs in arthritic disease.     

A Comparative Study of some Dehydration and Clearing Agents

An experiment to determine the advantages of diozan, iso-butyl alcohol, tertiary butyl alcohol, and ethyl alcohol as dehydrants and chloroform, toluol, xylene, benzol, methyl benzoate, methyl salicylate, and acetone as clearers is described. Materials fixed in Bouin's fluid, Zenker formol, and 10% neutral formalin were dehydrated, embedded, sectioned, and stained. Bouin's fluid produces less hardening, shrinkage and distortion than the other fixatives employed. Slow dioxan is the best method of dehydration. All the picric acid need not be removed from tissues to be embedded in paraffin. Tissue blocks not more than 4 mm. thick may be dehydrated and impregnated with paraffin by slow dioxan in 13 hours, fast dioxan in 10 hours, iso-butyl alcohol and tertiary butyl alcohol in 14 hours, and ethyl alcohol-chloroform in 17 hours without incurring any distortion due to rapidity of dehydration and infiltration.     

Delayed aggregation of proteoglycans in adult human articular cartilage

The biosynthesis and macromolecular organization of proteoglycans was studied in explants of adult human articular cartilage. In a series of pulse-chase experiments, labelling with (³⁵S)sulphate, it was shown that the proteoglycan monomer is synthesized as a precursor that has a low affinity for hyaluronic acid. These findings suggest a possible mechanism by which the rate of incorporation of proteoglycans into the extraceHular matrix may be controlled.     

Proteoglycan 4 downregulation in a sheep meniscectomy model of early osteoarthritis

Osteoarthritis is a disease of multifactorial aetiology characterised by progressive breakdown of articular cartilage. In the early stages of the disease, changes become apparent in the superficial zone of articular cartilage, including fibrillation and fissuring. Normally, a monolayer of lubricating molecules is adsorbed on the surface of cartilage and contributes to the minimal friction and wear properties of synovial joints. Proteoglycan 4 is the lubricating glycoprotein believed to be primarily responsible for this boundary lubrication. Here we have used an established ovine meniscectomy model of osteoarthritis, in which typical degenerative changes are observed in the operated knee joints at three months after surgery, to evaluate alterations in proteoglycan 4 expression and localisation in the early phases of the disease. In normal control joints, proteoglycan 4 was immunolocalised in the superficial zone of cartilage, particularly in those regions of the knee joint covered by a meniscus. After the onset of early osteoarthritis, we demonstrated a loss of cellular proteoglycan 4 immunostaining in degenerative articular cartilage, accompanied by a significant (p < 0.01) decrease in corresponding mRNA levels. Early loss of proteoglycan 4 from the cartilage surface in association with a decrease in its expression by superficial-zone chondrocytes might have a role in the pathogenesis of osteoarthritis.     

The electrophoretic heterogeneity of bovine nasal cartilage proteoglycans.

1. Proteoglycans were extracted from bovine nasal cartilage with 2.0M-CaC2 or with 0.15M-KCl followed by 2.0M-CaC2.. Proteoglycan fractions were prepared from the extracts by density-gradient centrifugation in CsCl under 'associative' and 'dissociative' conditions. 2. The heterogeneity of the proteoglycan fractions was investigated by large-pore-gel electrophoresis. It was concluded that extracts made with 2.0M-CaCl2 or sequential 2.0M-CaCl2 contain two major species of proteoglycan 'subunit' of different hydrodynamic size, together with proteoglycan aggregates. Both 'subunits' have mobilities that are greater than those of proteoglycans obtained from pig articular cartilage McDevitt & Muir (1971) Anal. Biochem. 44, 612-622] and are therefore probably smaller in size than the latter. 3. Proteoglycan fractions isolated from cartilage extracted lith 0.15M-KCl separated into two main components on large-pore-gel electrophoresis with mobilities greater than those of proteoglycans extracted with 2.0M-CaCl2. Proteoglycans extracted at low ionic strength from bovine nasal cartilage are of similar hydrodynamic size to those extracted from pig articular cartilage under the same conditions [McDevitt & Muir (1971) Anal. Biochem. 44, 612-622]. 4. The role of endogenous proteolytic enzymes in producing proteoglycan heterogeneity, particularly in low-ionic-strength cartilage extracts is discussed. 5. Hyaluronic acid and 'link proteins' were present in the proteoglycan fraction separated from KCl extracts as well as in the fraction separated from CaCl2 extracts. Hyaluronic acid can only be identified in proteoglycan fractions by large-pore-gel electrophoresis after proteolysis and further purification of the fraction. 6. Collagen was extracted by both salt solutions and was tentatively identified as type II. Small amounts of collagen appear to be associated with the proteoglycan-aggregate fraction from the high-ionic-strength extract but not with the corresponding fraction from the KCl extract.