Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate (Kato, Y., Iwamoto, M., Koike, T., Suzuki, F., and Takano, Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9552-9556). Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, 45Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of [35S]sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.     

Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

The in vitro cell culture age and cell density of articular chondrocytes alter sulfated-proteoglycan biosynthesis

The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated-proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5-7 d), and high density (HD, 12-15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]-thymidine. LD cultures incorporated 35SO4 into sulfated-proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated-proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCl density gradients and by elution profiles on Sepharose CL-2B. Chondrocytes deposited into the extracellular matrix (cell-associated fraction) 35SO4-labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4-labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell-associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.     

Separation of rabbit epiphyseal chondrocytes in various stages of differentiation

Summary Separation of fractions enriched in hypertrophic cells and proliferative cells has been achieved by density gradient centrifugation of cells from collagenase digests of rabbit epiphyseal cartilage. Concentrated suspensions of cells are centrifuged on a continuous Percoll density gradient. Hypertrophic cells remain in the upper part of the gradient and proliferative zone cells move to the lower regions. The resultant fractions show differences in mean cell diameter, alkaline phosphatase activity, morphology and synthetic activity in culture. Fractions rich in hypertrophic cells contain larger cells and more alkaline phosphatase activity than those enriched in proliferative cells. In culture the hypertrophic cells flatten as large irregular polygonal cells, whereas proliferative fractions form smaller spindle-shaped cells. In micromass culture hypertrophic fractions incorporate less ³⁵S-sulphate and ¹⁴C-proline, and less tritiated thymidine than do proliferative fractions. These results suggest a general reduction in matrix and DNA synthesis with the attainment of the fully differentiated hypertrophic state, coincident with the expression of alkaline phosphatase activity and mineralisation of the cartilage matrix.     

Effects of IGF-I, EGF, and FGF on proteoglycans synthesized by fractionated chondrocytes of rat rib growth plate

The effects of insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF), or 10% newborn calf serum (NCS) on the amount and structure of the proteoglycans synthesized by fractionated chondrocytes from rat growth plate were investigated. Proliferative cells (fraction II) or resting cells (fraction III) synthesized more proteoglycans than hypertrophic cells (fraction I). Addition of IGF-I to the cultures increased proteoglycan synthesis more than addition of EGF or FGF. EGF and FGF induced synthesis of proteoglycans of smaller molecular size with a lower proportion of aggregates. The size of the constituent glycosaminoglycan chains did not differ between control and growth factor-treated cells. The present study demonstrates that proteoglycan structure and synthesis are modified by growth factors to different extents, depending on the maturation stage of the target cells.     

Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.     

Coordinate regulation of collagen and alkaline phosphatase levels in chick embryo chondrocytes

Chick embryo tibial chondrocytes release into their extracellular matrix several species of proteochondroitin sulfate and collagen as well as matrix vesicles that are rich in Ca2+ and alkaline phosphatase and that appear to play a role in the calcification of cartilage. To determine whether there was any parallel regulation of the production of these products, the rates of collagen synthesis by cultured chick embryo tibial chondrocytes were altered, and the resulting changes in proteochondroitin sulfate synthesis and alkaline phosphatase levels in the cells were measured. As the rate of collagen synthesis was increased by adding increasing amounts of ascorbic acid to the culture medium, there was a parallel increase in the level of alkaline phosphatase. Similarly, when the rate of collagen synthesis was inhibited by adding 3,4-dehydroproline to the culture medium, the levels of alkaline phosphatase fell. The alkaline phosphatase in the culture medium was associated with vesicles which appeared to be matrix vesicles. It was recovered quantitatively by filtration through membranes with a pore size of 0.1 mu and measured by solubilizing the alkaline phosphatase from the membrane with detergent and assaying with 4-methylumbelliferyl phosphate as the substrate. When the matrix vesicles from the culture medium were analyzed for collagen types, it was found that only Type X collagen was recovered in this fraction. The implications of the association of Type X collagen and the matrix vesicles, both of which are found primarily in growth plate cartilage in the zone of hypertrophied chondrocytes which is in the process of mineralization, are discussed.     

Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.     

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.     

Molecular size distribution of proteoglycan subunits and glycosaminoglycans synthesized by chondrocytes under conditions of reduced proteoglycan synthesis

The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting l-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.     

Induction and prevention of chondrocyte hypertrophy in culture

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.     

Serum-induced cytosolic calcium movements and mitogenesis in cultured preosseous chondrocytes

The differentiation of preosseous chondrocytes begins with the proliferation of resting cells and results in the expression of the hypertrophic phenotype. The effect of fetal calf serum on chondrocyte mitogenesis and intracellular Ca2+ concentration was studied in resting and hypertrophic cells in primary culture. Resting chondrocytes respond to the growth stimulus with immediate release of Ca2+ from intracellular stores and with opening of the plasma membrane Ca2+ channels. These events may be related to the elevated [3H]thymidine incorporation observed after serum exposure. In contrast, in hypertrophic chondrocytes the lower rate of DNA synthesis seems to be coupled with a lower activity of the Ca2+ signaling mechanism and, probably, with reduced intracellular calcium stores. It is proposed that expression of the Ca2+ signaling mechanism may be modulated during the differentiation of preosseous chondrocytes.     

Calcium induces differentiation of primary human salivary acinar cells

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.     

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18- 19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.     

Modification of plasma membrane of differentiating preosseous chondrocytes: Evidence for a degradative process in the mechanism of matrix vesicle formation

Chondrocytes of the growth plate are differentiating cells. Their evolution leads to matrix vesicle formation and to cartilage mineralization. This is an in vitro study of the plasma membrane of chondrocytes at two differentiation stages. Differences in protein and glycoprotein components, increased membrane fluidity, and responsiveness to PTH indicate that hypertrophic ("ossifying") chondrocytes possess a plasma membrane widely different from that of resting chondrocytes. Their plasma membrane is particularly enriched in alkaline phosphatase (Mr 70K). Purified matrix vesicles contain the 70K form of alkaline phosphatase, but a 50K species is also detectable, a signal of degradative process. In fact, proteins and glycoproteins of matrix vesicles are less numerous than those of cell plasma membranes. It is suggested that, in vivo, matrix vesicle formation may be mediated by Ca2(+)-activated neutral proteases.     

Proteoglycans synthesized by chondrocytes of human nonarthritic and osteoarthritic cartilage

Chondrocyte cultures were developed from the cell outgrowths of explanted human nonarthritic and osteoarthritic human cartilage. Two significant differences in sulfated proteoglycan synthesis were demonstrated between the chondrocytes obtained in this manner. With 35SO4 to measure newly synthesized proteoglycan, we found that chondrocytes derived from osteoarthritic cartilage secreted significantly less (P less than 0.05) high density proteoglycan into the culture medium than did chondrocytes from nonarthritic cartilage after 20 hr of radiolabeling. This reduced amount of high density proteoglycan was sustained when chondrocytes were maintained in unlabeled culture medium ("chase" medium). In addition, the osteoarthritic chondrocytes secreted an increased amount of low density proteoglycan when compared with their nonarthritic counterparts. The elution profile of secreted high density proteoglycan isolated from the osteoarthritic chondrocyte culture medium was assessed by gel filtration on Sepharose CL-2B and revealed the presence of two proteoglycan subpopulations (Kav, 0.25, 0.58), whereas only one proteoglycan series (Kav, 0.37) was seen in the high density fraction of nonarthritic chondrocyte culture medium. Similar gel filtration profiles were also obtained when chondrocytes were maintained in chase medium. The results of this study demonstrated that stable differences in proteoglycan synthesis, but not in intracellular processing, exist between nonarthritic and osteoarthritic chondrocytes. The findings are noteworthy in that these differences were not previously apparent when organ-cultured cartilage was used to assess putative alterations in proteoglycans between the two groups.     

Effects of zinc on cell proliferation and proteoglycan characteristics of epiphyseal chondrocytes

Zinc has been postulated as an important nutritional factor involved in growth promotion; however, the cellular mechanisms involved in the effects of zinc on linear growth remain to be elucidated. This study was conducted to evaluate the effects of zinc on the proliferation rate of epiphyseal growth plate chondrocytes and on the structural characteristics of the proteoglycans synthesized by these cells. For these purposes, hypertrophic and proliferating chondrocytes were isolated from the tibiae of 1- and 5-week-old chickens, respectively. Chondrocytes were cultured under serum-free conditions and primary cultures were used. The results showed that zinc stimulated proliferation by 40-50% above the baseline in the case of proliferating chondrocytes, but it had no effect on hypertrophic chondrocytes. Zinc had neither any effects on mean charge density of proteoglycans synthesized by hypertrophic chondrocytes nor in their hydrodynamic size. In contrast, zinc induced an increase in mean charge density and a decrease of hydrodynamic size of proteoglycans synthesized by proliferating chondrocytes. In both cell types zinc had no effect on the composition and hydrodynamic size of the glycosaminoglycan chains. The increased ability of proliferating chondrocytes cultured in the presence of zinc to synthesize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) could be explained by the induction of enzymes participating in the sulfation pathway of proteoglycans. Therefore, the increase in mean charge density of proteoglycans observed in this study may be explained by an increase of the degree of sulfation of proteoglycan molecules. We speculate that the effect of zinc on linear growth may be explained at a cellular level by: a) an increase in proliferation rates of proliferating chondrocytes, and b) increased synthesis of highly charged proteoglycan molecules which decreases mineralization.     

Ultrastructural localization of alkaline phosphatase in the hypertrophic chondrocyte of the frog.

The ultrastructural localization of alkaline phosphatase was studied in the hypertrophic chondrocyte of the frog (Rana temporaria) by incubating sections of glutaraldehyde fixed tissue in a medium containing sodium beta glycerophosphate and calcium chloride. Control specimens were incubated in substrate free medium. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase) is a high molecular weight glycoprotein that hydrolyses phosphorylated metabolites much as acid phosphatase does except that its action is optimal at an alkaline pH. The results of this investigation showed that alkaline phosphatase activity was present within the cytoplasm and around the plasma membrane of frog hypertrophic chondrocytes. Although only a small proportion of frog hypertrophic chondrocytes demonstrated enzyme activity, there was evidence that this was concentrated within Golgi lamellae and vesicles leaving other organelles unreactive. The finding of alkaline phosphatase activity within Golgi lamellae of hypertrophic chondrocytes is regarded as unusual although postitive reactions within chondrocyte lysosomes have previously been reported (Doty and Schofield, 1976).