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.

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection.

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.

The parathyroid gland in metal rickets. A stereological study.

Rickets can be induced by oral administration of metals. A depressive effect exerted by metal ions on the parathyroid gland has previously been suggested to be a pathogenetic factor in strontium-induced rickets. In the present investigation, rickets was induced in young rats by oral administration of strontium and manganese, respectively. The parathyroids were fixed by perfusion and the total volume of the parathyroid glands was determined by serial sectioning. Conventional stereological techniques were applied on the ultrastructural level and the densities obtained could be related to the total mass of parathyroid tissue. In both rachitic groups the volume of the parathyroid glands as well as the absolute amounts of all cellular components studied, were reduced. All these changes were, however, correlated to the decreased body weight of the experimental animals. This is well in line with the finding of unaltered serum concentrations of calcium and immunoreactive parathyroid hormone. Therefore, it is concluded that the parathyroids do not play an important role in the pathogenesis of metal rickets.

Ultrastructure of hyaline cartilage. 2. Recent developments in preparatory procedures of electron microscopy and immunocytochemistry for the classification of bone dysplasias.

Recent developments in preparatory procedures for electron microscopy and immunocytochemistry are presented as a background for the morphological diagnosis of bone dysplasia. With the new low temperature embedding techniques and the availability of monoclonal antibodies raised against the main matrix macromolecules, biopsies have proved to be an important complement to clinical and radiological workup and they may sometimes provide the final clue to the diagnosis.

Manganese rickets. A biochemical and stereologic study with special reference to the effect of phosphate.

Oral administration of manganese (Mn) to young rats results in morphologic changes in the growth plate histologically resembling rickets. Previous investigations have indicated that one important factor in the development of Mn rickets is phosphate depletion, because of the precipitation of insoluble manganese phosphate in the gut. In the present study, the morphologic and biochemical changes in rickets induced by phosphate (P) depletion per se as well as in florid and healing Mn rickets were investigated. Rachitic changes were induced in young rats by giving dietary Mn (2%) or by phosphate depletion (0.02% P) for 25 days. The rachitic changes of the proximal tibial growth plate were quantitated with the use of stereologic methods. In addition, the growth plates were dissected into one upper and one lower part and proteoglycans were separately extracted with 4 M guanidine hydrochloride and purified by ultracentrifugation. In rats given phosphate in more than equimolar amounts relative to Mn, the rachitic changes were abolished. When rats with florid Mn rickets were given standard rat food, containing 1% P, rapid healing ensued. Moreover, similar rachitic changes were obtained by phosphate depletion alone. Proteoglycans from the lowermost tissue portion of rachitic growth plates readily formed aggregates with hyaluronic acid and also contained larger chondroitin sulphate chains than controls. Upon healing, these changes tended to normalize. The data support the concept that phosphate depletion is crucial in the pathogenesis of Mn rickets. Also, it seems that the biochemical changes in different forms of rickets are of a similar kind, which further underlines the importance of proteoglycans in enchondral bone formation.

Chubby: a new autosomal recessive skeletal mutation producing dwarfism in the mouse.

The chubby (cby) mutant is a previously undescribed skeletal mutation in the mouse. Breeding experiments showed that cby is a recessive mutant with complete viability, full penetrance and fertility in both sexes. Tests for allelism showed that the cby mutant is genetically unlike the somewhat similar mutants, stumpy (stm), pituitary dwarf (dw), spondylometaphyseal chondrodysplasia (smc), brachymorphic (bm), and achondroplasia (cn). The defects seem to occur mainly in growth cartilage. Microradiography revealed increased height of the epiphyseal growth plate and irregular bone trabeculae in the metaphysis. Light microscopy showed disturbed columnar organization of proliferative chondrocytes and pronounced signs of cellular disintegration. The hypertrophic zone, however, contained normally shaped chondrocytes arranged in regular columns. In spite of the normal cellular hypertrophy the pattern of cartilage mineralization was disturbed. The electron microscopy studies revealed very high amounts of matrix vesicles and numerous larger membrane coated structures in the extracellular matrix. Biochemical analysis of the affected growth cartilage revealed a slightly modified pattern of proteoglycan subpopulations and considerably longer chondroitin sulfate chains when compared with controls. From the present study it can be concluded that the cby mutant is a genetically and morphologically distinct condition with characteristic, somewhat rickets-like stigmata. The pathogenetic mechanism underlying the condition remains to be clarified.

Ultrastructure of hyaline cartilage. I. A comparative study of cartilage from different species and locations, using cryofixation, freeze-substitution and low-temperature embedding techniques.

Important progress in the cryofixation of tissues has recently been made with the introduction of a new technique which permits a great reduction in the rates of ice-crystal growth and nucleation by rapid freezing under a pressure of 2 100 bar. Tissue pieces up to 0.5 mm in thickness can now be processed at a freezing rate sufficient to prevent the formation of detectable ice crystals at the ultrastructural level. In the present investigation this technique, in combination with freeze substitution and low temperature embedding was applied for ultrastructural and immunocytochemical studies of hyaline cartilage. No extraction of matrix proteoglycans was observed during the substitution procedure, and there are good reasons to believe that in preparations obtained by this technique the native state of the matrix components is preserved, since, for example, the collapse temperature of the macromolecules is not exceeded. Furthermore, no chemical fixatives or cryoprotectants are required. Ultrastructural differences in the hyaline cartilage of the growth-plate between normal rats and mice were observed, and also differences between cartilage at different locations, such as tracheal cartilage and growth-plate. Using this technique, further comparative ultrastructural studies enable us to obtain information about the macromolecular organisation of cartilage matrix under various normal and pathological conditions in vivo. In addition, using monoclonal antibodies to the main macromolecules of the matrix, it was found that this technique not only provides excellent tissue preservation but is also well suited for immunocytochemistry with colloidal gold as a marker.

Stereological studies on the epiphyseal growth cartilage and characterization of costal cartilage proteoglycans in the achondroplastic (cn/cn) mouse.

The achondroplastic mouse is a dwarf mouse in which the endochondral growth of the skeleton is disturbed. The main morphological characteristic of the affected growth cartilage is an underdeveloped hypertrophic zone. The pattern of matrix mineralization seems to be unaffected, even in areas where hypertrophic chondrocytes are completely absent. The present stereological results indicate a disturbance of the cn/cn cartilage already in the proliferative zone. At the electron microscopical level a clearly abnormal spatial distribution of matrix vesicles was observed in the affected growth cartilage. The vesicle concentration of the cn/cn cartilage was increased, but the mineralizing cartilage showed a decrease of vesicles similar to that in the normal tissue. Parallel biochemical characterization of the proteoglycans and glycosaminoglycans of the cn/cn costal cartilage revealed normal conditions, also in the growth region, which was sampled as a separate tissue fraction. Thus the biochemical results provide no evidence of disturbed glycosaminoglycan metabolism.

The effect of manganese ingestion, phosphate depletion, and starvation on the morphology of the epiphyseal growth plate. A stereologic study.

Oral administration of manganese to young rats results in poorly mineralized primary spongiosa and an irregularly thickened growth plate with a histologic resemblance to that in vitamin D-deficiency rickets. In the present study, the rachitic lesions were characterized by stereologic methods at the light microscopic level. With increasing doses of Mn in the diet, the animals developed rachitic lesions of increasing severity, i.e., the total height of the growth plate and the relative volume of the hypertrophic zone increased. The experimental animals developed hypophosphatemia, which was dependent on the Mn dose. The observed serum concentrations of Mn and phosphorus are compatible with the idea that MnHPO4 is precipitated in the gut, leaving only small amounts of Mn and phosphate available for absorption. Furthermore, the severity of the rachitic lesions were inversely correlated to the concentration of phosphate in serum. The most important pathomechanism in Mn rickets is phosphate depletion, which per se causes similar rachitic changes, even though Mn also seems to have other effects. Starvation caused a decrease in the height of the growth plate and in the volume fraction of the hypertrophic zone, thus changes contrary to the rachitic lesions.

The effect of strontium and manganese on freshly isolated chondrocytes.

The effect of Strontium (Sr) and Manganese (Mn) was studied on freshly isolated chondrocytes obtained from costal cartilage of young male rats. Cells isolated from rats rendered rachitic by oral exposure to Sr or Mn exhibited a decreased synthesis of glycosaminoglycans and collagen as judged by incorporation of 35SO4 and 3H proline. In vitro exposure of chondrocytes from untreated (control) animals to Sr or Mn at concentrations comparable to those found in cartilage from Sr and Mn rachitic rats, lowered the O2 consumption in both groups, but only Sr had any influence on the other parameters studied.

Proteoglycans and glycosaminoglycans in cartilage from the brachymorphic (bm/bm) mouse.

The brachymorphic (bm/bm) mouse is a disproportionate dwarf with a disturbance of the endochondral growth of the skeleton. Rib cartilage from 25-day-old affected animals and their normal siblings was analyzed for its contents and composition of proteoglycans. In addition to the previously reported undersulfation of chondroitin sulfate, it was demonstrated that one of the two types of aggregating proteoglycan and possibly the small ones are decreased in bm/bm costal cartilage, both in the growth region and in the remaining part. The molecular defect of the bm/bm condition is known to affect the synthesis of 3-phosphoadenosine 5-phosphosulfate (Sugahara and Schwartz, Proc. Natl. Acad. Sci. USA 76: 6615-6618, 1979). The above finding therefore suggests the existence of feedback mechanisms for the regulation of proteoglycan synthesis, whereby the undersulfation of glycosaminoglycans would result in decreased synthesis or increased turnover of certain proteoglycan subpopulations. Analysis of the glycosaminoglycan side chains indicated that mouse rib cartilage contains small amounts of keratan sulfate of extremely small size. The affected and control tissues, however, seemed to contain equal amounts of both glucosamine and galactosamine.

Proteoglycans and glycosaminoglycans of normal and strontium rachitic epiphyseal cartilage.

An analytical protocol for the analysis of proteoglycans and proteins extracted from small sections of cartilage has been applied to the upper and lower parts of the epiphyseal growth plate of normal rats and rats with strontium-induced rickets. Only one polydisperse proteoglycan population was found in each of the four tissue portions. In strontium-induced rachitic animals the aggregability of proteoglycan monomers with hyaluronate was considerably increased in the lower part of the growth plate. The proteoglycans in the upper portion of the epiphysis contained somewhat less of chondroitin sulfate and keratan sulfate both in normal and strontium-induced rachitic rats. The chondroitin sulfate chains were somewhat larger in samples from strontium-induced rachitic rats compared with controls while in all groups about 90 per cent of the galactosamine in chondroitin sulfate was sulfated at position four. The findings of an altered composition of proteoglycans in strontium-induced rickets demonstrate that strontium not only prevents the mineral growth but may also induce the chondrocytes to produce a matrix with a different macromolecular composition.

Proteoglycans and glycosaminoglycans of epiphyseal cartilage in florid and healing low phosphate, vitamin D deficiency rickets.

The composition of proteoglycans and glycosaminoglycans in two portions of the rat epiphyseal growth cartilage in florid and healing low phosphate, vitamin D deficiency rickets was studied. The concentration of both chondroitin sulfate and keratan sulfate was reduced with about 25 per cent in the lower (mineralizing) part of the growth plate in healing rickets compared to the lower (hypertrophic) part in florid rickets. In all samples about 85 per cent of the chondroitin sulfate chains had a sulfate ester group in the fourth position. Both in florid and healing rickets only one main population of polydisperse proteoglycans was found. In florid rickets the capacity to form aggregates with hyaluronic acid was the same in the upper and lower parts of the growth plate. With the onset of mineralization in healing rickets, however, a substantial decrease in this aggregability was demonstrated. These results give further support to the hypothesis that proteoglycans and, especially, proteoglycan aggregates, play a role in the mineralization process.

Stereological studies on the epiphyseal growth plate in strontium-induced rickets. With special reference to the distribution of matrix vesicles.

Applying stereological principles, we recently demonstrated a matrix-vesicle distribution between the zones of the epiphyseal growth plate that is at variance with the generally accepted distribution. In the present study of strontium-induced rickets we demonstrated a similar bimodal distribution, the highest matrix-vesicle volume fraction being found in the resting and hypertrophic zones and the lowest, in the proliferative zone. These volume-fraction differences are due to differences in the number of vesicles between zones, the variation in mean diameter being rather small. The disturbed mineralization in strontium-induced rickets is associated with a diminished reduction of the matrix-vesicle volume fraction in the lower part of the growth plate compared with normal animals. The findings may indicate that matrix-vesicle removal is a part of the mineralization process, but a causal connection cannot be proved on the basis of our results. Our findings are, however, compatible with the earlier-posed cell-debris theory for matrix-vesicle origin and distribution.

Structural and functional integrity of chondrocytes immediately following isolation.

Chondrocytes were isolated from costal cartilage in young rats after digestion with collagenase and hyaluronidase. The immediate survival of the cells was investigated with the use of different criteria for viability, namely structural integrity and metabolic activity. Structural integrity was studied by transmission and scanning electron microscopy, trypan blue exclusion and NADH oxidation. Metabolic activity was measured both as O2 consumption and as proline and sulphate incorporation, as indicators of collagen and proteoglycan synthesis. The cellular content of glutathione was also measured. The chondrocytes isolated were found to be structurally intact and metabolically active. Early after isolation the chondrocytes varied considerably in size similarly to the native tissue. A selective loss of the larger sized cells was observed during further incubation for 24 h.

Morphological studies of the epiphyseal growth zone in the brachymorphic (bm/bm) mouse.

Light microscopy, including immunohistochemical techniques, and electron microscopy were performed on epiphyseal growth cartilage from brachymorphic (bm/bm) mice and age-matched phenotypically normal siblings aged 5, 16 and 25 days. In the bm/bm mice light microscopy showed a disturbed columnar arrangement and numerous chondrocytes with pronounced regressive changes. The normal development of proliferative cells into hypertrophic cells was halted and thus only a rather small and ill-defined hypertrophic zone was seen. The calcifying zone was irregular and the normal lacunae were replaced by a densely staining matrix. Using immunofluorescence techniques, the presence of considerable amounts of both type II and type V collagen was demonstrated in the bm/bm mice, while the cartilage from controls contained only type II. Ultrastructurally the lacunar matrix contained bundles of fine fibrils without the typical collagen periodicity which might indicate synthesis of a defective procollagen. Our observations together with the previously demonstrated deficiency of 3'-phosphoadenosine 5'-phosphosulphate, illustrate the complexity of the growth cartilage disturbance in the bm/bm mouse. Most of our findings are at variance with those described in the literature and possible pathogenetic mechanisms for the observed alterations in the growth cartilage are discussed.

Stereological analysis of the epiphyseal growth cartilage in the brachymorphic (bm/bm) mouse, with special reference to the distribution of matrix vesicles.

The brachymorphic (bm/bm) mutation in the mouse leads to disproportional dwarfism due to a disturbance of endochondral bone formation. The morphological characteristics of bm/bm epiphyseal growth cartilage are signs of cellular degeneration and disintegration and alteration of the composition of the extracellular matrix, with an abnormal mineralization pattern. The present stereological study of the bm/bm growth plate revealed a clearly altered distribution of matrix vesicles as compared with the controls. It was also demonstrated that the bm/bm matrix vesicles have an abnormal size distribution, with an increased mean caliper diameter. The biological significance of these findings is discussed in relation to the different hypotheses on the origin of matrix vesicles and their possible role in the mineralization process. The results support the opinion that extracellular matrix vesicles, at least partly, constitute cellular debris.

Stereological studies on the epiphyseal growth plate in low phosphate, vitamin D-deficiency rickets with special reference to the distribution of matrix vesicles.

In low phosphate, vitamin D-deficiency rickets normal mineralization is reversibly arrested, and rickets is thus a suitable model for studying factors influencing the mineralization process. Partly on the basis of their distribution within the growth cartilage, the so-called matrix vesicles are considered to play an important role in the process of mineralization and it has been claimed that the distribution pattern is the same in rickets as in normal animals. With the use of modern stereological techniques, our group recently demonstrated a matrix vesicle distribution between the zones of the epiphyseal growth plate in normal rats different from that described earlier. The same bimodal distribution pattern was observed in rachitic rats in the present study, the highest volume density being found in the resting and upper hypertrophic zones and the lowest in the proliferative zone. The volume density differences are explained by differences in the number of vesicles between zones, the variation in mean caliper diameter being small. Our findings are discussed in relation to the proposed theories on matrix vesicle origin. The results seem to support the dynamic cell debris theory for matrix vesicle origin presented earlier, but the existence of subpopulations of matrix vesicles with a specialized function and origin cannot be ruled out.

Congenital spondyloepiphyseal dysplasia. Morphological and biochemical examination of skeletal tissue in an unusual case of this disorder.

This report deals with an unusual case of spondyloepiphyseal dysplasia, where the diagnosis was suspected clinically and radiologically and where cartilage and osseocartilaginous specimens were taken for ultrastructural and biochemical analyses. Unlike cases described previously the electron microscopic studies of chondrocytes showed dilatation of the rough endoplasmic reticulum cisternae. The dilated cisternae contained an amorphous substance, and the picture is suggestive of a metabolic disturbance. The results of the biochemical analyses were within the normal range and did not indicate storage of glycosaminoglycans.

Analysis of dentine glycosaminoglycans using high-performance liquid chromatography.

Puppy dentine was prepared using ultracentrifugation of tooth powder in organic density gradients. The glycosaminoglycans of the obtained tissue fraction were prepared after papain digestion and beta-elimination, using preparative chromatography on DEAE-cellulose and CPC-cellulose. These polysaccharide fractions were analyzed using highly sensitive HPLC procedures. One such HPLC procedure allowed hyaluronic acid to be determined in less than microgram amounts. The glycosaminoglycans thus prepared consisted only of chondroitin-4-sulfate, chondroitin-6-sulfate, and small amounts of highly hybridized dermatan sulfate, while the experiments failed to demonstrate even trace amounts of keratan sulfate, hyaluronic acid or heparan sulfate.