Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.     

Electron probe X-ray microanalysis of the composition of hyaline articular and non-articular cartilage in young and aged rats

Summary Blocks of articular cartilage were taken from tibiae of young adult (8 week) and aged adult (50–60 week) rats; xiphisternal cartilage was obtained from young adult rats. Specimens were quench-frozen in nitrogen slush, freeze-fractured and examined by low-temperature scanning electron microscopy. The results of X-ray microanalysis of frozen-hydrated bulk cartilage are semi-quantitative. The composition of chondrocyte nuclei and cytoplasm are only marginally different. Xiphisternal chondrocytes contain lipid inclusions which show an absence of element peaks and are designated as being neutral lipid. Intra- and extracellular Na, P, S, Cl, K and Ca count rates are significantly different. Cartilage from older rats contains more S and Ca, and less K and Cl in the intercellular matrix than that from young rats. Intracellular K levels are lower in aged than in young rats. The intercellular matrix of xiphisternal cartilage contains larger amounts of S, Na and K, and a smaller amount of Cl compared to that of tibial articular cartilage.     

Measurements of mineralization process in the femur growth plate and rib cartilage of the mouse using pixe in combination with a proton microprobe

The femoral bone from the 18-day pregnancy embryo and an rib cartilage of mature mice have been investigated using PIXE (proton induced X-ray emission) in combination with a proton microprobe on snap frozen cryosectioned material. The localization and the results of quantitative measurement of P, S, Cl, K, Ca, Fe and Zn have been correlated with the histochemical localization of inorganic deposits. It has been found that in calcifying and degenerating cartilage of the growth plate there is substantial loss of S; this element being indicative for sulphate groups of glycosaminoglycans. This change seems to be an important factor conditioning the process of mineralization. Zn is found in higher concentration in mineralized tissues, both in embryonal and mature cartilage as well as in the bone, and this suggests that Zn is also involved in the mineralization process. The mineralization of rib cartilage exceeds that of embryonal bone, and the Ca/P ratio is higher in the former than in the hydroxyapatite of the latter. The method described is a useful analytical tool especially for such types of studies in which elements are not easily redistributed by freezing, cutting and drying; e.g. in investigations of mineral deposits.     

Measurements of mineralization process in the femur growth plate and rib cartilage of the mouse using pixe in combination with a proton microprobe

Summary The femoral bone from the 18-day pregnancy embryo and an rib cartilage of mature mice have been investigated using PIXE (proton induced X-ray emission) in combination with a proton microprobe on snap frozen cryosectioned material. The localization and the results of quantitative measurement of P, S, Cl, K, Ca, Fe and Zn have been correlated with the histochemical localization of inorganic deposits. It has been found that in calcifying and degenerating cartilage of the growth plate there is substantial loss of S; this element being indicative for sulphate groups of glycosaminoglycans. This change seems to be an important factor conditioning the process of mineralization. Zn is found in higher concentration in mineralized tissues, both in embryonal and mature cartilage as well as in the bone, and this suggests that Zn is also involved in the mineralization process. The mineralization of rib cartilage exceeds that of embryonal bone, and the Ca/P ratio is higher in the former than in the hydroxyapatite of the latter. The method described is a useful analytical tool especially for such types of studies in which elements are not easily redistributed by freezing, cutting and drying; e.g. in investigations of mineral deposits.     

Elemental changes associated with chondrocyte differentiation in rat rib growth plate

Quantitative X-ray microanalysis was under-taken to follow the elemental changes that occur in the process of chondrocyte differentiation. For analysis at the cellular level, semi-thick freeze-dried cryosections of rat rib growth plate cartilage were used. For evaluation of the elemental concentrations at the subcellular level, thin sections of freeze-dried and low temperature vacuum embedded cartilage were analyzed. Levels of Na, P, S, Cl, K, and Ca were determined in the cells and extracellular matrix in different zones of the cartilage--resting, proliferative, and hypertrophic. Proliferative cells had a sodium concentration that was twice that of resting cells, suggesting that Na may play an important role in the regulation of DNA- and protein-synthesis in chondrocytes, A concomitant rise in Na and S concentration occurred between resting zone and proliferative zone cartilage matrix. The high concentrations of Na and K in the matrix are probably due to the high amount of sulfate in proteoglycans which may bind these cations.     

Elemental changes associated with chondrocyte differentiation in rat rib growth plate

Summary Quantitative X-ray microanalysis was under-taken to follow the elemental changes that occur in the process of chondrocyte differentiation. For analysis at the cellular level, semi-thick freeze-dried cryosections of rat rib growth plate cartilage were used. For evaluation of the elemental concentrations at the subcellular level, thin sections of freeze-dried and low temperature vacuum embedded cartilage were analyzed. Levels of Na, P, S, Cl, K, and Ca were determined in the cells and extracellular matrix in different zones of the cartilage — resting, proliferative, and hypertrophic. Proliferative cells had a sodium concentration that was twice that of resting cells, suggesting that Na may play an important role in the regulation of DNA- and protein-synthesis in chondrocytes. A concomitant rise in Na and S concentration occurred between resting zone and proliferative zone cartilage matrix. The high concentrations of Na and K in the matrix are probably due to the high amount of sulfate in proteoglycans which may bind these cations.     

Collagen/annexin V interactions regulate chondrocyte mineralization

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope. Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced Kalpha1,2 x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens. The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Summary Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope.Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced K _{1, 2}, x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens.The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

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.     

Analysis of matrix vesicles and their role in the calcification of epiphyseal cartilage.

Extracellular matrix vesicles, which have been shown to be associated with initial calcification of cartilage, were isolated, characterized, and studied with 45calcium isotope to determine whether they could form mineral in vitro. It was found that the isolated matrix vesicles contain a phosphatase, active at neutral pH, which has a very wide specificity and will hydrolyze a variety of nucleotide triphosphates, diphosphates, monophosphates, and other phosphate-containing substrate and metabolites. Acid phosphatase, beta-glucuronidase, and cathepsin D were found to be in the cell fractions, in lysosomes; these enzymes are not present in matrix vesicles and this is additional evidence for the difference between matrix vesicles and lysosomes. Matrix vesicles were found to take up 45Ca even in the presence of low levels of Ca and P1 and also to facilitate precipitation of hydroxylapatite when incubated under physiological conditions in the presence of ATP and other phosphate-containing substrates. Systematic electron probe analysis of a septum of epiphyseal cartilage indicates that matrix vesicles gradually accumulate calcium and then phosphorus and thus facilitate the advance of the calcification front. Adjoinging nonvesicular matrix in the hypertrophic zone, cell cytoplasm, and cell processes had very low levels of calcium and phosphorus in a region where matrix vesicles showed high levels of these elements. New concepts are put forward that take accounts of these findings which provide a better understanding of the sequence of mineralization in growth cartilage.     

Ultrastructural localization of acid phosphatase in cartilage of young mandibular condyles

Summary The fine structural localization of acid phosphatase was studied in cartilage of mandibular condyles of the mouse. Although the final product was found to be deposited within most chondroblasts and chondrocytes, the most abundant precipitate was observed within the hypertrophic chondrocytes in the vicinity of the mineralization front. In these cells, lead phosphate precipitates were noted along the rough endoplasmic reticulum and within lysosome-like bodies. Positive reaction to acid phosphatase was also noticed within vacuoles which were located in the matrix close to the centers of mineralization. It is conceivable that this enzyme is involved in matrix production at one stage of chondrogenesis and in the mineralization process at a later stage.This investigation was supported in part by Grant No. DE 00163 from the National Institute of Dental Research, U.S.P.H.S., and in part by Tufts University Cancer Research Center Institutional Grant IN-23-0.     

The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.

Phosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.     

Intramembranaceous ossification analyses by a proton microprobe

Summary The proton induced X-ray emission method in combination with a proton microprobe was applied to study the intramembranaceous ossification. As material sections of mouse embryo skulls from the 17th and 19th day of gestation were used. The morphology of the sample was examined by routine histochemical procedure performed on the sections adjacent to that irradiated by the proton microprobe. The measurements were made in line scan and raster scan mode. The concentrations of P, S, Cl, K, Ca, Fe and Zn were determined at each irradiated point. The average element concentrations were calculated for four parts of each section (bone, cartilage, mesenchymal tissue close to the bone and mesenchymal tissue in other places). The distributions of Ca and P (less markedly than Ca) concentrations almost exclusively correlate with localization of the bone while S, Cl and K concentrations show preference to the cartilage. The amount of inorganic material in flat bones of the 17-day embryo amounts to 14% of the dry mass. The material is characterized by a Ca/P ratio of bout 1.6 In the embryo 2 days older the amount of the inorganic phase is practically the same (15%) while the Ca/P ratio approaches 2. This suggests the presence of the precursor phase in the flat bone calcification. It is possible that octacalcium phosphate (Ca/P ratio equals to 1.72) is formed at the onset of the flat bone mineralization which transforms rapidly (in 2 days) to a more stable mineral (defective hydroxyapatite).     

Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization

Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413-420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles, and the initiation of mineralization by these vesicles.     

Intramembranaceous ossification analyses by a proton microprobe

The proton induced X-ray emission method in combination with a proton microprobe was applied to study the intramembranaceous ossification. As material sections of mouse embryo skulls from the 17th and 19th day of gestation were used. The morphology of the sample was examined by routine histochemical procedure performed on the sections adjacent to that irradiated by the proton microprobe. The measurements were made in line scan and raster scan mode. The concentrations of P, S, Cl, K, Ca, Fe and Zn were determined at each irradiated point. The average element concentrations were calculated for four parts of each section (bone, cartilage, mesenchymal tissue close to the bone and mesenchymal tissue in other places). The distributions of Ca and P (less markedly than Ca) concentrations almost exclusively correlate with localization of the bone while S, Cl and K concentrations show preference to the cartilage. The amount of inorganic material in flat bones of the 17-day embryo amounts to 14% of the dry mass. The material is characterized by a Ca/P ratio of about 1.6. In the embryo 2 days older the amount of the inorganic phase is practically the same (15%) while the Ca/P ratio approaches 2. This suggests the presence of the precursor phase in the flat bone calcification. It is possible that octacalcium phosphate (Ca/P ratio equals to 1.72) is formed at the onset of the flat bone mineralization which transforms rapidly (in 2 days) to a more stable mineral (defective hydroxyapatite).     

Sphingosine 1-phosphate activation of ERM contributes to vascular calcification

Vascular calcification is the deposition of mineral in the artery wall by vascular smooth muscle cells (VSMCs) in response to pathological stimuli. The process is similar to bone formation and is an independent risk factor for cardiovascular disease. Given that ceramide and sphingosine 1-phosphate (S1P) are involved in cardiovascular pathophysiology and biomineralization, their role in VSMC matrix mineralization was investigated. During phosphate-induced VSMC mineralization, endogenous S1P levels increased accompanied by increased sphingosine kinase (SK) activity and increased mRNA expression of SK1 and SK2. Consistent with this, mineralization was increased by exogenous S1P, but decreased by C2-ceramide. Mechanistically, exogenous S1P stimulated ezrin-radixin-moesin (ERM) phosphorylation in VSMCs and ERM phosphorylation was increased concomitantly with endogenous S1P during mineralization. Moreover, inhibition of acid sphingomyelinase and ceramidase with desipramine prevented increased S1P levels, ERM activation, and mineralization. Finally, pharmacological inhibition of ERM phosphorylation with NSC663894 decreased mineralization induced by phosphate and exogenous S1P. Although further studies will be needed to verify these findings in vivo, this study defines a novel role for the SK-S1P-ERM pathways in phosphate-induced VSMC matrix mineralization and shows that blocking these pathways with pharmacological inhibitors reduces mineralization. These results may inform new therapeutic approaches to inhibit or delay vascular calcification.     

Lipids of mineralizing epiphyseal tissues in the bovine fetus

Because lipids had been consistently detected histologically at sites of new calcification, the lipids of epiphyseal cartilage and bone in various stages of mineralization were examined. Lipids were extracted before and after demineralization and analyzed. Lipid content increased during proliferation and calcification of epiphyseal cartilage. Much less was seen in the adjacent cancellous bone; this corroborates histochemical findings. Similar phospholipid compositions were seen in the total lipids of cartilage and bone. Neutral (dipolar) phospholipids accounted for nearly 90% of the total lipid P and were almost completely extracted before demineralization. Serine- and inositol-containing phospholipids and two other, unidentified, acidic lipids could not be effectively extracted from calcifying tissues until after demineralization. Since the extraction of the acidic lipids was closely related to the degree of mineralization, it is possible that they form part of a lipoprotein-mineral complex in the calcifying matrix. Lysophospholipids were detected in all extracts, but primarily in those made after decalcification. It is concluded that acidic lipids are mainly responsible for the sudanophilia detected histologically at sites of new calcification.