Calcium incorporation in matrix vesicles isolated from chicken epiphyseal cartilage

Summary Matrix vesicles isolated from chicken epiphyseal cartilage displayed an uptake of Ca²⁺ which was linear with time and the amount of vesicle protein. The matrix vesicles stimulated the incorporation of Ca²⁺ even at very low Ca × P, suggesting that they could bind Ca²⁺ and/or increase the local Ca × P to the metastable level. This uptake was abolished by EDTA or heating, and partially inhibited by cysteine, to the same extent as the hydrolysis of ATP. There was also a certain uptake of Ca²⁺ without added phosphate, this being stimulated by ATP up to 3 mmol, but diminishing again with higher concentrations. The presence of ATP failed to stimulate the uptake of Ca²⁺ more than an equimolar amount of phosphate in the form of inorganic KH₂PO₄. Mg²⁺ activated the hydrolysis ofp-nitrophenylphosphate at pH 10.5, and both Mg²⁺ and Ca²⁺ the hydrolysis of ATP from pH 7 to 9.5. Paradoxically, the omission of Mg²⁺ stimulated the uptake of Ca²⁺ several-fold.     

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.     

Localization of prostaglandin synthetase in chicken epiphyseal cartilage

Summary Prostaglandin synthetase activity in highspeed particulate fractions of chick epiphyseal cartilage has been characterized with respect to cofactor requirements, pH optimum, buffer-ion effects, types of prostaglandins formed, and the distribution of prostaglandin synthetase activity in zones of the epiphyseal plate.Direct homogenization of cartilage was found to be more efficacious than releasing chondrocytes by enzymatic digestion for preparation of prostaglandin synthetase, a homogenization time of 4 min yielding maximal activity. The optimal incubation medium contained 50 mM Tris buffer (pH 7.5), 2.5 mM epinephrine, 1 M hemoglobin, 3.25 mM glutathione, 200 g/ml enzyme protein, and 5 M substrate. Glutathione was effective only if present during homogenization. Rates of PGE₂ biosynthesis were linear up to 15 min and then rapidly declined, indicative of self-deactivation. The low levels of PGF₂ formed, and their decrease after 20 min incubation, suggests the possible presence of degradative enzymes.Prostaglandin synthetase was inhibited by aspirin, indomethacin, and vitamin E, but not vitamin K₁. Cation concentrations in the physiological range had only modest effects on prostaglandin biosynthesis, and then only if present during tissue homogenization. In the presence of phosphate buffer, Ca²⁺ was somewhat inhibitory. Since in the absence of phosphate Ca²⁺ had no deleterious effect, it is probably that the inhibitory effect was caused by precipitation of calcium phosphate.Hypertrophic and calcified cartilage exhibited significantly higher prostaglandin synthetase activity than the proliferating and maturing zones. The increased synthesis of prostaglandins in the low layers of the growth plate may indicate a role of these factors in chondrocyte differentiation and/or calcification.     

Correlation of lysozyme activity with proteoglycan biosynthesis in epiphyseal cartilage

Summary Pig epiphyseal cartilage (proximal ulna epiphysis) previously incubated in vitro in the presence of sodium [³⁵S]sulfate or [³H]thymidine was either analyzed by autoradiography or separated into 9 morphologically defined consecutive layers and investigated for³⁵S-incorporation into the guanidinium chloride-extractable proteoglycans and for lysozyme activity.The lowest³⁵S incorporation and lysozyme activity were determined in the zone of resting cells, but there is a consecutive increase in the rate of proteoglycan synthesis and lysozyme activity toward the diaphyseal cartilage-bone junction, with the maximum at the lower columnar cell zone and a sharp reduction of both parameters at the hypertrophic zone. The maxima of³⁵S incorporation and [³H]thymidine incorporation do not coincide.The guanidinium chloride-soluble proteoglycans exhibit macromolecular polydispersity. Fractions excluded from as well as retarded by Sepharose 2B gel could be separated and were detected in all zones.The results indicate a correlation of proteoglycan biosynthesis and lysozyme activity in epiphyseal cartilage.     

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

Summary 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.     

The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage

Summary Since calcium (Ca) deposition by isolated fetal bovine matrix vesicles is selectively supported by nucleoside triphosphate, and since the Ca deposits appear to be amorphous by transmission electron microscopy [1], attempts were made to study further the nature of these Ca deposits. Calcification of isolated matrix vesicles was allowed to occur in a calcifying medium in which either inorganic phosphate (Pi) or [γ-P]ATP was labeled with³²P.³²P in Ca P (pyrophosphate) deposits were analyzed by a Dowex 1×10 anion exchange chromatography. The results of the analysis indicate that the (³²P) radioactivity was mainly associated with Pi when Pi in the calcifying media was labeled with³²P. In contrast,³²P was found to be associated with inorganic pyrophosphate (PPi) when [γ-³²P]ATP was used. Using a specific enzyme coupling assay for PPi, the presence of PPi in the Ca deposits was demonstrated. The amounts of Pi and PPi in the Ca deposits initiated by fetal calf matrix vesicles were found to be approximately equal. To exclude the possibility that the major part of PPi of Ca P deposit existed as adsorbed form, the deposition was performed under the conditions in which Pi was omitted from calcifying medium. The results of these experiments showed that substantial amount of PPi and Ca deposits remained the same and was not correlated to the amount of Pi in these deposits. In contrast, Pi of CaP was decreased if Pi was omitted from the calcifying medium. Thus, it appears that the major portion of PPi exists as mineral rather than adsorbed form. The moles of Ca deposited were approximately equal to the sum of moles Pi and PPi deposited. Levamisole at 10 mM inhibited 70% of ATPase (specific release of³²Pi from [γ-³²P]ATP at pH 7.6) activity, 18% of Ca deposition, and 34% of Pi deposition during 3 hours of incubation. Adenosine monophosphate (AMP) or adenosine diphosphate (ADP) at 1 mM exerted earlier and greater inhibition on Ca and P (Pi and PPi) deposition than did 10 mM levamisole. Adenosine (1 mM) had little effect on Ca P deposition. Prolonged incubations which allowed enzymatic degradation of ADP and AMP substantially reduced the ADP and AMP inhibition. Levamisole was able to potentiate the AMP and ADP inhibition since it prevents further breakdown of AMP or ADP by alkaline phosphatase. Thus, we concluded that alkaline phosphate is not the major factor in thein vitro Ca P deposition by fetal bovine matrix vesicles. Instead, it appears that nucleoside trisphosphate pyrophosphohydrolase is directly involved in the early stages of deposition since both the enzyme and Ca P deposition were inhibited by AMP or ADP. The possible involvement of ATP phyophosphohydrolase in chondrocalcinosis is suggested.     

Enzymatic characterization of the matrix vesicle alkaline phosphatase isolated from bovine fetal epiphyseal cartilage

Summary Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PP_i. Optimal activities forp-nitrophenyl phosphate, ATP, and PP_i are found at pH 10.5, 10.0, and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations.p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorohydrolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg²⁺ and Hg²⁺ ions inhibit thep-nitrophenyl phosphatase activity at pH 10.5 while Mn²⁺ ions show no effect. P_i, levamisole, CN⁻, Zn²⁺, Ca²⁺ ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. P_i is a linear noncompetitive inhibitor with a K_i of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca²⁺ ions inhibit noncompetitively with a K_i of 9.3 mM. Zn²⁺ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be²⁺ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromethanediphosphonate, L-cysteine, and N-ethylmaleimide. NaCl, KCl, and Na₂SO₄ at 0.5–1.0 M inhibit the enzyme. At pH 8.5, the cleavage of PP_i by the matrix vesicle enzyme is inhibited by Mg²⁺ and Ca²⁺ ions at concentrations greater than 0.5 mM. Mg²⁺ ions in the range of 0.1–4 mM stimulate the matrix vesicle ATPase whereas higher concentrations produce inhibition. Ca²⁺ ion does not affect the ATPase activity between 0.1 and 10 mM at either pH 7.5 or 10.0. With 5′-AMP as substrate, the 3.5-fold decrease in the maximum velocity of the matrix vesicle relative to the chondrocyte enzyme may be accounted for in terms of a small excess enthalpy of activation (1160 cal/mole) partially compensated by an increased entropy of activation. Deceased May 8, 1975.     

Purification of alkaline phosphatase from extracellular vesicles of fracture callus cartilage

Summary Extracellular matrix vesicles from fracture callus cartilage were isolated by differential centrifugation and resolved by equilibrium centrifugation on a discontinuous sucrose gradient into two bands. The phosphohydrolytic activity towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was distributed similarly after differential and equilibrium centrifugation suggesting the association of this activity with the matrix vesicles. The two bands isolated by equilibrium centrifugation of the partially purified vesicular preparation demonstrated high levels of alkaline phosphatase activity. Observed with an electron microscope, the 1.07–1.14 g/cm³ band from the gradient was enriched in electron luscent matrix vesicles while the 1.27 g/cm³ band contained electron dense matrix vesicles. Enzymatic analysis of the 1.27 g/cm³ band indicated a slight contamination due to the presence of mitochondria and lysosomes while the 1.07–1.14 g/cm³ band gave no enzymatic indication of subcellular contamination. A phosphohydrolytic enzyme active towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was purified from the 1.07–1.14 g/cm³ fraction by DEAE-cellulose column chromatography. Electron micrographs of callus cartilage sections demonstrated densification of the plasma membrane and matrix vesicles following substrate incubation with-glycerophosphate or tetrasodium pyrophosphate. The histochemical and biochemical data indicate that a phosphatase, with multiple substrate specificity, is a component of fracture callus cartilage matrix vesicles.     

32Pi- and45Ca-metabolism by matrix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage by isosmotic percoll density-gradient fractionation

Summary Matrix vesicle-enriched fractions were isolated from different zones of epiphyseal cartilage by nonenzymatic methods involving tissue homogenization, differential centrifugation, and isosmotic Percoll gradient fractionation. Uptakes of both³²Pi and⁴⁵Ca were studied concomitantly over periods from 20 min to 24 h. Percoll density gradients separated epiphyseal microsomes into two alkaline phosphatase-rich fractions: a low-density noncalcifiable fraction (P-I), and a higher-density fraction (P-II) which readily mineralized. The P-II fraction was found only in calcifying regions of the growth plate. Based on chemical and physical properties and enzyme activities, both fractions were similar except that P-II contained significantly higher levels of mineral ions than did P-I, and had lower levels of alkaline phosphatase. The mineral appeared to be primarily in a noncrystalline form. Metabolism of³²Pi and⁴⁵Ca by P-II followed a complex kinetic pattern in which accumulation of large amounts of both ions was preceded by an initial limited burst of uptake and a lag-phase of variable duration. During mineral ion loading, the density of the P-II fraction progressively increased as evidenced by co-migration of⁴⁵Ca,³²Pi, and alkaline phosphatase to increasingly higher densities. During the period of early mineral deposition (1–5 h), Ca/P uptake ratios were very low (1.0–1.2) and X-ray diffraction patterns showed a predominantly amorphous pattern. This suggests that the mineral accumulated in matrix vesicles is initially some form of noncrystalline calcium monohydrogenphosphate. L-tetramisole, a potent inhibitor of alkaline phosphatase, inhibited accumulation of both⁴⁵Ca and³²Piin the absence of organic P substrates,³²Pi being preferentially inhibited over⁴⁵Ca. This finding, coupled with recent studies on the behavior of alkaline phosphatase at physiological pH, suggests that the protein is not acting as a phosphohydrolase, but rather as a Pi-binding or transport agent in vesicle-mediated calcification.     

Animal model of chondrocyte apoptosis in the epiphyseal cartilage of the neonatal bone

Apoptosis is considered to be the mechanism responsible for the death of chondrocytes during endochondral bone formation. It is also claimed that apoptosis of the chondrocytes is age related and that the apoptotic index increases with age. However, a detailed analysis of the apoptotic activity of the neonatal epiphyseal cartilage is lacking. A model that evaluates apoptosis in the femoral rat epiphyseal cartilage both quantitatively and qualitatively is reported. Apoptotic incidence in the epiphyseal cartilage reached a maximum at age 6 days, but the age in our study did not significantly affect the percentile rate of apoptotic chondrocytes (P > 0.05, Kruskal-Wallis test). Apoptosis in the zone of hypertrophic cartilage played the most important role in the growth plates homeostasis. Morphologic evidence of apoptosis was necessary in addition to positive nick end labeling of cells. Electron microscopy studies revealed atypical modes of programmed death of the growth plate chondrocytes in addition to the classical apoptotic mode.An erratum to this article can be found at     

Matrix vesicles and focal proteoglycan aggregates are the nucleation sites revealed by the lanthanum incubation method: A correlated study on the hypertrophic zone of the rat epiphyseal cartilage

Correlated studies were performed with light and electron microscopy, and backscattered electron image in conjunction with X-ray microanalysis, of lanthanum-incubated epiphyseal cartilage of the young rat. The hallmark of this procedure is the appearance of LaP electrondense deposits (not present in control sections) in precise sites of the hypertrophic zone. The ultrastructural study revealed a dual nature of these sites: “dense matrix vesicles” and “focal filament aggregates.” The dense matrix vesicles are a specific type of matrix vesicle with the intrinsic capacity of precipitating LaP mineral, as soon as they originate from the hypertrophic chondrocytes. Furthermore, the matrix vesicles were found to be heterogeneous because lanthanum-devoid, “light matrix vesicles” were also present. The focal filament aggregates, which were not recognized in unstained sections and in controls, are apparently focal concentrations of proteoglycans with high lanthanum binding capacity, although the presence in them of other components (e.g., type X collagen, C-propeptide of type II collagen) cannot be excluded. They were in close connection with the light matrix vesicles in the upper hypertrophic zone, and were loaded with a variable quantity of LaP irregular electron-dense deposits in the lower hypertrophic zone. These irregular deposits are similar to, but distinct from, calcification nodules. The lanthanum incubation method indirectly detects the matrix Ca-binding components (which bind La ions), and the calcification initiation sites (which precipitate a LaP-mineral phase). A sequence is proposed of successive steps of LaP nucleation within the focal filament aggregates, which possibly mimics calcium phosphate deposition. Such a sequence seems to require the participation not only of dense matrix vesicles, but also of the filamentous components of the focal aggregates, possibly together with the activity of alkaline phosphatase.     

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.     

Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution

Summary The ultrastructure of extracellular membrane-bound matrix vesicles (MVs), their biogenesis, and the surrounding matrix in chick tibial growth plate were studied after quick freezing and freeze substitution (FS) in an organic solvent. There were several notable differences in the ultrastructural preservation of cartilage when FS was used as compared with conventional fixation. The ultrastructural appearance of MVs after FS was extremely variable. Within the MVs, intravesicular filaments, amorphous material, and membrane-associated undercoat structures were observed. Intravesicular filaments, similar in diameter to microfilaments seen in the cytoplasm, were attached to the inside of MV membranes. This observation indicates the similarity of MV membranes and the plasma membrane. In some MVs in the proliferative zone an electron-dense material was present along the inner side of the MV membrane. In the prehypertrophic zone, crystalline material often appeared within the electron-dense material, which may be a precursor form of hydroxyapatite. The earliest crystals observed were in MVs but not in the extracellular matrix. Regarding MV formation, in addition to budding from cell surfaces and to cellular disintegration, this study also indicates that a sequential process of extrusion of preformed cytoplasmic structures may occur. Also, small MVs measuring 25–40 nm seem to arise from the disruption of large MVs. This is a previously unreported observation on MV biogenesis. FS preserves proteoglycans in the cartilage matrix as a fine, filamentous network. Initial extracellular calcification was not associated with this network.     

Regulation of epiphyseal cartilage metabolism and morphology in the chronic diabetic rat

Summary Chronic insulin deficiency, in both man and experimental animal models, has been associated with skeletal alterations, the genesis of which remains unknown. Since cartilage growth and maturation are dependent on the maintenance of adequate glycolytic activity, we evaluated cartilaginous carbohydrate metabolism and epiphyseal growth plate morphology in control, long-term (7 weeks) streptozotocin-induced diabetic and insulintreated diabetic rats. Since parathyroid hormone levels have been shown to be decreased in chronically diabetic rats, we also studied the effect of a low calcium diet (0.1%) on cartilage metabolism and morphology in the insulinopenic state. In vitro incubation of epiphyseal cartilage slices in Kreb's Ringer buffer was performed in 5 mM glucose, with either¹⁴C-6-glucose as a glycolytic marker or¹⁴C-1-glucose as a pentose phosphate pathway marker. While¹⁴C-6-glucose uptake was only marginally reduced in diabetic rat cartilage, lactate production was markedly decreased, approximating 42% of control values, and the activity of the pentose phosphate shunt increased (P<0.01). These biochemical alterations were attended by a marked reduction (P<0.005) in the width of epiphyseal growth plates obtained from rats with untreated diabetes. Both insulin replacement (P<0.001) and dietary calcium restriction (P<0.02) in diabetic animals resulted in a significant increment in the width of epiphyseal growth plates. These morphologic changes were accompanied by a significant (P<0.02) increase in cartilaginous lactate production, in the absence of altered glucose uptake. While insulin treatment corrected glycolysis, it had little effect on the augmented pentose shunt activity, implying stimulation of both these metabolic pathways. Dietary calcium restriction normalized glycolysis and corrected the accelerated activity of the pentose phosphate pathway. We conclude that chronic insulin deficiency in the growing rat is attended by alterations in cartilaginous carbohydrate metabolism which may relate not only to insulinopenia per se, but also to the relative hypoparathyroidism that characterizes the chronic experimental diabetic state. The accumulated data also suggest that these metabolic derangements may account, at least in part, for the reduced longitudinal bone growth observed in this growing animal model.     

A comparative study on the occurrence and activity of extracellular matrix vesicles in young and adult rat maxillary bone

Summary The occurrence of matrix vesicles in the maxillary bone of normal young and adult rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed that the vesicles are invested in trilaminar membranes. Occasionally, crystals were found both within the vesicles and in the surrounding matrix. Separated fractions of vesicles, membranes, and cells were studied biochemically. The amounts of vesicular protein and enzymatic activity per gram bone in the adult rats were significantly lower than in the young. This coincides with the higher number of vesicles observed in the TEM specimens of young rats when compared to that in the old ones. The specific activities of all enzymes examined in the three bone fractions obtained from both young and adult rats were similar. This indicates that no significant difference exists in the enzymatic characteristics of matrix vesicles from the maxillary bone of young and adult rats.     

In vitro precocious accumulation of calcium and matrix vesicles formation in young cartilage cells: Specific effects of corticosteroids

Summary The present study examined the effects of various corticosteroid and noncorticosteroid hormones upon the ultrastructure of chondroprogenitor cells and chondroblasts in an organ-culture system of late fetal condylar cartilage. Corticosteroids, (triamcinolone acetonide, dexamethasone, corticosterone) at concentrations of 10⁻⁶–10⁻⁸M stimulated markedly a precocious formation of matrix vesicles by chondroblasts. This stimulation was accompanied by a significant accretion of calcium complexes intra- and extracellularly in both the chondroprogenitor cell population and chondroblastsin vitro, as well as in the newly induced matrix vesicles. Nonglucocorticoid steroids (progesterone, estradiol, testosterone, cortexolone) did not evoke similar effects. Progesterone and testosterone, however, seemed to adversely affect the ultrastructure of the cartilage cells, whereas estradiol appeared to have a favorable effect on the morphology of cultured condylar cartilage.     

Matrix vesicles isolated from apical pulp of rat incisors: Crystal formation in low Ca×Pi ion-product medium containing β-glycerophosphate

Summary The ultrastructure of crystal formationin vitro associated with extracellular membranebound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-β-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultracentrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30–200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca)×inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.     

Material properties and biosynthetic activity of articular cartilage from the bovine carpo-metacarpal joint

OBJECTIVE: To determine the site variation of material properties and cellular biosynthetic activity, and to compare these at each site, of articular cartilage from the bovine carpo-metacarpal joint in order to test its usefulness as a model system for investigating the metabolic effects of mechanical stimuli. DESIGN: The mechanical properties and composition of full-depth biopsies of articular cartilage were measured at defined sites in bovine carpometacarpal joints. Metabolic activity at the same sites was assessed by incubating further biopsies in medium containing 35S-sulfate and 3H-leucine then measuring the incorporated radioisotope and cell density. These results were compared with an estimate of the distribution of forces across the joint. RESULTS: Topographical variation of water content, stiffness, cell count or incorporated radioisotope was not significant whereas collagen and glycosaminoglycan varied in different ways. A moderate correlation was found between water and collagen contents, but the correlation between water and glycosaminoglycan contents was poor. Neither compressive stiffness nor creep compliance was predicted strongly by any of the composition measurements. A negative correlation was found between metabolic activity and cell density. CONCLUSIONS: Defining the variation of tissue properties across the bovine carpometacarpal joint and the lack of variation in biosynthetic activity will enable proper matching of experimental and control groups of biopsies in studies of the effects of mechanical stimuli on the composition and mechanical properties of articular cartilage. In addition, the lack of correlation between stiffness, water and glycosaminoglycan contents is further evidence that the mechanical properties of the tissue depend significantly on factors other than these broad measures of composition.     

Increased activity of lysosomal glycohydrolases in glioma tissue and surrounding areas from human brain

Summary Increased metabolic activity represented by an increase in both anabolism and catabolism in tumours, including gliomas, is a well known phenomenon and utilised in positron emission tomography imaging of tumours. In this study lysosomal enzyme activities of some glycohydrolases were investigated in glioma tissue from human brain. Tumour tissue (ten cases) and brain tissue surrounding the tumour tissue (seven cases) from patients with a histopathological diagnosis of glioblastoma multiforme or anaplastic astrocytoma were analysed for activity of the lysosomal enzymes galactosylceramidase, glucosylceramidase, β-galactosidase, β-N-acetylglucosaminidase, β-glucuronidase, and acid phosphatase. All of the investigated lysosomal enzymes except galactosylceramidase showed increased activity compared with that in normal brain tissue. Moreover, despite sparsity of tumour cells the specimens taken from surrounding areas showed elevated activities of the same enzymes. The findings indicate an upregulation of the activity not only in tumour but also in normal cells of the surrounding area.     

Expression and distribution of key proteins of the endocannabinoid system in the human seminal vesicles

The endocannabinoid system (ECS), comprising the cannabinoid receptors (CBR), their ligands, and enzymes controlling the turnover of endocannabinoids, has been suggested to be involved in male reproductive function. As information is scarce on the expression of the ECS in human male reproductive tissues, this study aimed to investigate by means of molecular biology (RT‐PCR) and immunohistochemistry/immunofluorescence the expression and distribution of CB1 and CB2, GPR55 (an orphan G protein‐coupled receptor that recognises cannabinoid ligands) and FAAH (isoforms 1 and 2) in the human seminal vesicles (SV). The specimens expressed PCR products corresponding to CB1 (66 bp), CB2 (141 bp), GPR55 (112 bp), FAAH1 (260 bp) and FAAH2 (387 bp). Immumohistochemistry revealed dense expression of CB1, CB2 and GPR55 located to the pseudo‐stratified columnar epithelium and varicose nerves (also characterised by the expression of vasoactive intestinal polypeptide and calcitonin gene‐related peptide). Cytosolic staining for FAAH1 and FAAH2 was seen in cuboidal cells of all layers of the epithelium. No immunoreactivity was detected in the smooth musculature or nerve fibres. CB1, CB2, GPR55, FAAH1 and FAAH2 are highly expressed in the human SV. Considering their localisation, the ECS may be involved in epithelial homeostasis, secretory function or autonomic mechano‐afferent signalling.