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.     

Calcification of rachitic cartilage to study matrix vesicle function.

Growth plate cartilage from rachitic rats was studied to assess the role of extra-cellular matrix vesicles in the reinstitution of calcification during healing. The concentration and distribution of matrix vesicles was found to be normal in rachitic growth plate, and although the rachitic cartilage matrix was largely uncalcified, an occasional vesicle did contain internal mineral. Matrix vesicles served as initial loci for mineralization when healing was brought about either by in vivo injection of phosphate or in vitro incubation of growth plates in a metastable calcifying solution. During in vitro calcification a distinct line of mineralization developed in the upper growth plate which was shown by electron microscopy to reflect mineralization by the vesicles. The appearance of this vesicle-associated calcification line was inhibited by preheating or repeated freezing and thawing, and by 30 minutes preincubation in deoxycholate, ethane-1-hydroxy-1,1-diphosphonate, or beryllium sulfate. Our results suggest that vesicle calcification is dependent on the structural and enzymatic integrity of the vesicle membrane. Enzymes that may well play a role in vesicle calcification are phosphatases (e. g., alkaline phosphatase, pyrophosphatase and ATPase), which are known to be concentrated in vesicle membranes.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method.

The ultrastructural localization of alkaline phosphatase (A1P) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl2 to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl2 and CeCl3. A1P activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper, we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4h of incubation at 25 degrees C. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degrees C.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method

Summary The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl₂ to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl₂ and CeCl₃. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.     

Roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated calcification. Effect of selective release of membrane-bound alkaline phosphatase and treatment with isosmotic pH 6 buffer

The roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated mineralization have been studied by selectively releasing the enzyme from a wide variety of matrix vesicle preparations using treatment with a bacterial phosphatidylinositol-specific phospholipase C and by demineralization of the vesicles using isosmotic pH 6 buffer. Following depletion of 50-90% of the alkaline phosphatase activity or treatment with citrate buffer, the vesicles were tested for their ability to accumulate 45Ca2+ and 32Pi from a synthetic cartilage lymph. Removal of alkaline phosphatase by phospholipase C treatment caused two principal effects, depending on the matrix vesicle preparation. In rapidly mineralizing vesicle fractions which did not require organic phosphate esters (Po) to accumulate mineral ions, release of alkaline phosphatase had only a minor effect. In slowly mineralizing vesicles preparations or those dependent on Po substrates for mineral ion uptake, release of alkaline phosphatase caused significant loss of mineralizing activity. The activity of rapidly calcifying vesicles was shown to be dependent on the presence of labile internal mineral, as demonstrated by major loss in activity when the vesicles were decalcified by various treatments. Ion uptake by demineralized vesicles or those fractionated on sucrose step gradients required Po and was significantly decreased by alkaline phosphatase depletion. Uptake of Pi, however, was not coupled with hydrolysis of the Po substrate. These findings argue against a direct role for alkaline phosphatase as a porter in matrix vesicle Pi uptake, contrary to previous postulates. The results emphasize the importance of internal labile mineral in rapid uptake of mineral ions by matrix vesicles.     

Development and fine structure of pharyngeal replacement teeth in juvenile zebrafish (Danio rerio) (Teleostei, Cyprinidae)

Teeth are commonly used model systems for the study of epithelial-mesenchymal interactions during organogenesis. We describe here the ultrastructural characteristics of developing pharyngeal replacement teeth in juvenile zebrafish, an increasingly important model organism for vertebrate development. Replacement teeth develop in close association with the dental organ of a functional tooth. Morphogenesis is well advanced prior to the start of cytodifferentiation. Fibrillar enameloid matrix is formed first, followed by the deposition of predentine. Initial mineralization of the enameloid proceeds quickly; maturation involves the presence of ruffled-bordered ameloblasts. Dentine mineralization is inotropic and is mediated by matrix vesicles. Woven-fibred attachment bone matrix is deposited before completion of dentine mineralization. Eruption of fully ankylosed teeth is a fast process and may involve degenerative changes in the pharyngeal epithelium. Mononucleated osteoclasts and clastic cells located in the pulp cavity intervene in tooth resorption prior to shedding. Structural differences with larval, first-generation zebrafish teeth include the presence of dentinal tubules and the absence of an electron-dense covering membrane. Part of these differences may relate to size differences of the teeth. Others, like the site of the replacement tooth bud, suggest that initiation may take place in already committed epithelium from the first initiation event in the larval stage.     

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.     

Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca²⁺ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro–osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.     

Cytochemical localization of alkaline and acid phosphatase in human vanishing bone disease

This report is the first cytochemical investigation of vanishing bone disease "Gorham's Disease" (Gorham and Stout 1955). The ultrastructural localization of non-specific alkaline phosphatase and of specific and non-specific acid phosphatase activity was studied in slices of tissue removed from a patient with this rare disorder. Sodium beta-glycerophosphate and phosphorylcholine chloride were used as substrates. Alkaline phosphatase was present around the plasma membranes of osteoblasts and associated with extracellular matrix vesicles in new woven bone. This is consistent with the proposed role for this enzyme (Robison 1923) and for matrix vesicles (Bonucci 1967) in the mineralization of bone (Bernard and Marvaso 1981). Concentrations of specific secretory acid phosphatase reaction product in the cytoplasm of degenerating osteoblasts may contribute to the imbalance between bone formation and resorption. Osteoclasts, while few in number, showed non-specific and specific acid phosphatase activity. The Golgi apparatus and heterophagic lysosomes of mononuclear phagocytes were rich in non-specific acid phosphatase. This was also present in the Golgi lamellae and lysosomes of endothelial cells. Acid phosphatase cytochemistry suggests that mononuclear phagocytes, multinuclear osteoclasts and the vascular endothelium are involved in bone resorption in this disease.     

Effect of vanadate, a potent alkaline phosphatase inhibitor, on 45Ca and 32Pi uptake by matrix vesicle-enriched fractions from chicken epiphyseal cartilage

Although alkaline phosphatase has been long associated with the mineralization process, its exact function remains to be elucidated. To clarify its possible role in matrix vesicle-mediated mineralization, we tested the effect of vanadate, a phosphate analogue and powerful competitive inhibitor of alkaline phosphatase activity, on calcium and phosphate uptakes by a matrix vesicle-enriched microsomal fraction. Vanadate was also tested in a hydroxyapatite-seeded ion uptake system to determine possible direct effects on mineral formation. The effect of vanadate on vesicle mineral ion uptake was complex; low dosages of vanadate (2-20 microM) were stimulatory to Ca2+ uptake, but were inhibitory to Pi. Higher dosages (greater than 67 microM) were inhibitory to both ions. The effect of vanadate on ion uptake was strongly influenced by the stage of vesicle loading; major effects were seen during the lag and early uptake phases, and minimal effects were seen in the terminal stages. Concentrations of vanadate highly inhibitory to vesicle ion uptake had minimal effects on ion accretion by a hydroxyapatite-seeded system. Inhibition of alkaline phosphatase activity by vanadate broadly paralleled inhibition of Pi and Ca2+ uptake; however, at low vanadate concentrations, inhibition of Pi uptake closely paralleled that of alkaline phosphatase. The data indicate that vanadate binds with high affinity to Pi-loading sites, blocking initial Pi uptake. Complexation between vanadate and Ca2+ may be responsible for the stimulation of Ca2+ uptake at early stages of vesicle ion loading with low levels of vanadate by enhancing binding of Ca2+ to the vesicles. It may also account for the selective inhibition of Ca2+ uptake during the rapid stage of vesicle ion loading with high levels of vanadate by reducing Ca2+ ion activity. The close parallelism between inhibition of early Pi uptake and of alkaline phosphatase activity supports the concept that alkaline phosphatase is involved in Pi transport during the early stages of matrix vesicle ion loading. However, the fact that only about half of the Pi uptake was affected by vanadate, despite the progressive inhibition of alkaline phosphatase activity, indicates that alkaline phosphatase is not solely responsible for Pi uptake by the matrix vesicle-enriched fraction.     

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.     

Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells

Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O₂) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O₂ environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs. cartilage; bone     

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

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

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Cell surface proteoglycan expression correlates with epithelial-mesenchymal interaction during tooth morphogenesis

Tooth morphogenesis and differentiation of the dental cells are guided by interactions between epithelial and mesenchymal tissues. Because the extracellular matrix is involved in these interactions, the expression of matrix receptors located at the cell surface may change during this developmental sequence. We have examined the distribution of an epithelial cell surface proteoglycan antigen, known to behave as a receptor for interstitial matrix, during tooth morphogenesis. Intense staining was seen around the cells of the embryonic oral epithelium as well as the dental epithelium at the early bud stage. With development, expression was greatly reduced in the enamel organ. Differentiation of these cells into ameloblasts was associated with the loss of expression, while the epithelial cells remaining in the stratum intermedium and stellate reticulum regained intense staining. The PG antigen was weakly expressed in the loose neural crest-derived jaw mesenchyme but it became strongly reactive in the condensed dental papilla mesenchyme when extensive morphogenetic movements took place. With development, the PG antigen disappeared from the advanced dental papilla mesenchyme but persisted in the dental sac mesenchyme, which gives rise to periodontal tissues. The PG antigen was not expressed by odontoblasts. Hence, the expression of the PG antigen changes during the epithelial-mesenchymal interactions of tooth development and is lost during terminal cell differentiation. The expression follows morphogenetic rather than histologic boundaries. The acquisition and loss of expression in epithelial and mesenchymal tissues during tooth development suggest that this proteoglycan has specific functions in the epithelial-mesenchymal interactions that guide morphogenesis.     

Fine structural and cytochemical observations of dental epithelial cells during the enameloid formation stages in red stingrays Dasyatis akajei

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid.     

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.     

Interactions between the extracellular matrix and the cell surface determine tooth morphogenesis and the cellular differentiation of the dental mesenchyme

A series of reciprocal interactions between epithelial and mesenchymal tissues control the morphogenesis and cell differentiation in the developing tooth. The molecular mechanisms operating in these interactions are, however, unknown at present. Structural components of the extracellular matrix (ECM) affect cellular behavior in the embryo and appear to be involved also in these regulatory processes. The ECM molecules exert their effects on cells through binding to specific matrix receptors on the cell surface. This review article summarizes our findings on the distribution patterns during tooth development of the ECM glycoproteins, fibronectin and tenascin, and of the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix. Based on the observed changes in these distribution patterns and on experimental evidence, roles for these molecules in epithelial-mesenchymal interactions during tooth development are suggested. Fibronectin and tenascin are enriched in the dental basement membrane at the time of odontoblast differentiation. These matrix glycoproteins may be involved in the cell-matrix interaction which controls differentiation of the dental mesenchymal cells into odontoblasts. Tenascin and syndecan are accumulated in the dental mesenchyme during bud stage of development. We have shown in tissue recombination experiments that the presumptive dental epithelium induces the expression of tenascin and syndecan in mesenchyme. We suggest that these molecules are involved in cell-matrix interactions, which regulate mesenchymal cell condensation during the earliest stages of tooth morphogenesis.