Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth.

BACKGROUND AND OBJECTIVE: It is well known that tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial-mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. MATERIAL AND METHODS: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF-soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5'-bromo-2'-deoxyuridine (BrdU) assay in an organ-culture system. RESULTS: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ-culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down-regulated when antibody against HGF receptor was present in the culture medium. CONCLUSION: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.

Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor.

The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 micrograms/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (Mr approximately 80 kD; Kd = 3.9 nM; 3.2 x 10(5) sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action.

Sequential synthesis of cartilage and bone marker proteins during transdifferentiation of mouse Meckel's cartilage chondrocytes in vitro.

Meckel's cartilage cells cultured in vitro undergo phenotypic transformation toward osteogenic cells. We examined whether these cells synthesize type X collagen and bone morphogenetic protein-2 (BMP-2). We also examined the results of Alcian blue staining and the expression of type I and type II collagen, osteocalcin and chondroitin sulfate proteoglycan (CSPG) during this transdifferentiation. Meckel's chondrocytes, isolated from day-17 mouse embryos, were inoculated at 1 x 10(4)/penicylinder and cultured in alpha-MEM for periods up to 4 weeks. Alcian blue staining and immunostaining of type II collagen and CSPG confirmed that, after cell culture for 2 weeks, the cartilaginous phenotype was expressed most intensely. Later in culture, chondrocytes underwent modification through the synthesis of bone-type proteins; nodule-forming small round cells showed ALPase activity and were immunoreactive for type I collagen and osteocalcin. Immunoreactivity for type X collagen was detected in the small round cells at the top of the nodules prior to calcification of the matrix, as well as in large hypertrophic cells. BMP-2 was also expressed first in similar small round cells after 3 weeks in culture, and it subsequently extended along the extracellular matrix in the calcified nodules. These results indicate that small round cells that are differentiating toward osteocyte-like cells from Meckel's chondrocytes express type X collagen and BMP-2 sequentially.

Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro.

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic cells, which then deposit a mineralized matrix to form calcified cartilage prior to replacement by bone. Previously, we reported that a clonal cell line, ATDC5, undergoes efficient chondrogenic differentiation through a cellular condensation stage. Here we report that the differentiated ATDC5 cells became hypertrophic at the center of cartilage nodules, when the cells ceased to grow. Formation of hypertrophic chondrocytes took place in association with type X collagen gene expression and a dramatic elevation of alkaline phosphate (ALPase) activity. After 5 weeks of culture, mineralization of the culture could be discerned as Alizarin red-positive spots, which spread throughout the nodules even in the absence of beta-glycerophosphate. Electron microscopy and electron probe microanalysis revealed that calcification was first initiated at matrix vesicles in the territorial matrix and that it advanced progressively along the collagen fibers in a manner similar to that which occurs in vivo. The infrared spectrum of the mineralized nodules indicated two absorption doublets around 1030 cm-1 and 600 cm-1, which are characteristic of apatitic mineral. Calcifying cultures of ATDC5 cells retained responsiveness to parathyroid hormone (PTH): PTH markedly inhibited elevation of ALPase activity and calcification in the culture in a dose-dependent manner. Thus, we demonstrated that ATDC5 cells keep track of the multistep differentiation process encompassing the stages from mesenchymal condensation to calcification in vitro. ATDC5 cells provide an excellent model to study the molecular mechanism underlying regulation of cartilage differentiation during endochondral bone formation.

A case of pulmonary ossified adenocarcinoma with marked osteoplastic bone metastasis.

We report the case of a 53-year-old man suffering from a pulmonary adenocarcinoma with ossification and diffuse metastatic osteoplastic lesions throughout the skeletal system. This is a rare condition in lung carcinomas. Radiographs of the chest and bones demonstrated mineral densities in the primary tumor and multiple expansive osseous lesions with a diffuse sclerotic pattern resembling multiple bone metastases from prostatic carcinoma.

Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells.

Previously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) that is identical to that of connective tissue growth factor (CTGF). In the present study, we investigated the roles of CTGF in the proliferation and migration of vascular endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occurs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junctions of mouse ribs were stained with an anti-CTGF antibody in vivo. Northern blot analysis revealed that CTGF was strongly expressed in chondrocytic cells as well as bovine aorta endothelial (BAE) cells in culture, but not in other types of cells such as osteoblastic cells. Its expression in BAE cells was greater in the growing phase than in the confluent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The addition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligonucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cells. Furthermore, the abilities to proliferate and migrate of BAE cells, which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is involved in the proliferation and migration of BAE cells.

Ultrastructural features of the developing eosinophils in bone marrow and spleen of the musk shrew, Suncus murinus.

Eosinophilopoiesis in the musk shrew, Suncus murinus, a representative of the order Insectivora, was studied by light and electron microscopy. To examine biochemical features of cytoplasmic granules, extraction with proteolytic enzymes was carried out on ultrathin sections of bone marrow. In this species, eosinophils are produced in the same manner in both spleen and bone marrow. Developing eosinophils were distinguished as belonging to four stages, recognized by ultrastructural changes in cytoplasmic organelles as well as the eosinophilic granules during maturation. Granulogenesis began by budding of vacuoles containing flocculent material from the concave face of the Golgi apparatus, in the promyelocyte to myelocyte stage. The matrix of developing granules transformed into a finely granular structure, and the large spherical granules of mature eosinophils were homogeneous without crystalline cores. It was shown by proteolytic enzyme extraction that the proteinaceous cores of mature granules were uniformly removed; there was no evidence that they contained crystalloid inclusions. These results indicate that shrew eosinophils can be regarded as cells that retain a prototype of eosinophil granules, probably like those of ancestral mammals rather than those of higher living Mammalia.

Evidence for transformation of chondrocytes and site-specific resorption during the degradation of Meckel's cartilage.

It is unknown whether cells in the midportion of Meckel's cartilage undergo transformation into other kinds of cell or whether resorption of cells occurs during development. Therefore, the midportion of Meckel's cartilage from the mouse and the rat was subdivided into anterior and posterior portions. The ultimate fates of these tissues were analyzed with a focus on resorption -related cells, death of chondrocytes by apoptosis, and transformation of the chondrocytes themselves. Cellular and extracellular features of mouse Meckel's cartilage were observed after von Kossa's staining and staining for acid phosphatase (APase) activity, as well as by light and electron microscopy. To identify resorbing cells, immunostaining specific for macrophages and staining for tartrate-resistant acid phosphatase (TRAP) were performed. The DNA nick end-labeling (TUNEL) method was used for the detection of death of chondrocytes by apoptosis. The replacement of the extracellular matrix of rat Meckel's cartilage was examined with double immunofluorescence staining for type I and type II collagens. When the anterior midportion from embryonic mice on day 18 was examined after von Kossa's staining, it was clear that the extracellular matrix had already calcified and vascularization had been initiated that reflected the calcified matrix. TRAP staining and immunostaining for macrophages revealed two types of osteoclast and macrophages that were involved in resorption of the matrix. In the posterior midportion, no vascular invasion was evident, and chondrocytes were transformed directly into fibroblastic cells by phenotypic conversion. In such cells we found reaction products specific for APase activity, suggestive of the intracellular degradation of fine collagenous fibrils. Double immunofluorescence staining showed that cartilage-specific type II collagen was replaced by type I collagen with the phenotypic transformation to fibroblastic cells. There were no significant changes in the number of TUNEL-positive apoptotic cells from day 17 of gestation to day 6 after parturition. Death of chondrocytes by apoptosis was not, therefore, involved directly in the disappearance of Meckel's cartilage. These results in the posterior midportion served as an instance of phenotypic switches in differentiated cells from chondrocytes to fibroblast-like cells. The present study indicates that there is a difference between the ultimate fate of cells in the posterior part and that of cells in the anterior part in the midportion of Meckel's cartilage in the mouse and rat.

Morphological characteristics of the life cycle of resting cartilage cells in mouse rib investigated in intrasplenic isografts.

Resting cartilages taken from 2-day-old mouse ribs were transplanted into spleens in order to carry out morphological investigations of the life cycles of their chondrocytes. The explants were isografted for periods of up to 60 days and examined at light and electron microscopic levels, using von Kossa's reaction or osmium-potassium ferrocyanide (OPF) fixation. By day 3 after transplantation, resting cartilage containing immature chondrocytes was well adapted to splenic tissue and by 7 days after transplantation these chondrocytes had changed into early hypertrophic chondrocytes containing large vacuoles, glycogen aggregates and abundant secretory organelles. It was also demonstrated by von Kossa's reaction that the initial calcification occurred in the territorial matrix during this period. In spite of the hypertrophic chondrocytes in the central zone being surrounded by an extensively calcified matrix during days 14-21 after transplantation, these cells had well-preserved organized organelles, except that Golgi-associated elements and endoplasmic reticulum revealed a tendency toward degenerative changes. With increased duration of the grafting period, from 30-60 days, the calcification zone progressed gradually, and the number of hypertrophic chondrocytes embedded in the calcified matrix decreased considerably. By day 60, degenerating hypertrophic chondrocytes of two types were distinguished:flattened cells containing large vacuoles, poorly developed Golgi apparatuses, and rough endoplasmic reticulum; and shrunken dark cells displaying terminal hypertrophy. During the present study, we observed no vascular invasion into the calcified matrix, or appearance of bone-related cells, and the morphological changes from the resting chondrocytes to cellular hypertrophy accompanied by the formation of a calcified matrix were observed at day 60. These findings indicate that resting cartilage cells of the mouse have the capacity for terminal differentiation when transplanted into the spleen.

Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression.

We examined whether Meckel's cartilage of embryonic mice, 17 days in utero, undergo the cellular transformation into the osteocyte-like phenotype under organ culture conditions. Explants were grown by our original pithole method modified Trowell-type cultures for up to 4 weeks at 37 degrees C under 5% CO2 in air. Specimens were examined using histological procedures including immunostaining and electron microscopy. In addition, the effects of beta-glycerophosphate on matrix calcification were also examined in cultures with or without beta-glycerophosphate. Addition of beta-glycerophosphate induced calcification at a higher level, but calcium mineral deposition occurred regardless of the addition of beta-glycerophosphate to the culture medium. Light and electron microscopic analyses showed that freshly isolated chondrocytes prior to cell culture had typical hypertrophic morphology, but shortly after commencement of culture, they showed morphological modifications. The cells showing chondrocytic phenotypes became basophilic elliptical cells, and eventually transformed into flattened osteocyte-like cells. Bone-like features for cellular elements were characterized by spindle-shaped cells with elongated processes accompanying bone-specific thick-banded collagen fibrils. Immunostaining showed that at 2 weeks in culture, type I and type II collagens coexisted in the matrix, but subsequently type II collagen synthesis ceased and was replaced by type I collagen synthesis. Immunofluorescent labeling for osteocalcin was noted first in the peripheral cells by 1 week, but at 3 weeks this reaction spread to the central zone in explants. Alkaline phosphatase activity (ALPase) was expressed on the cells in the central zone prior to calcium mineral deposition as shown by von Kossa's reaction at 3 weeks in culture. These results showed that Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression.

Morphogenesis of mineralized tissues induced by neonatal mouse molar pulp isografts in the spleen.

Tooth pulps dissociated intact with EDTA were isografted for up to 40 days, and examined by light and electron microscopy for hard tissue morphodifferentiation. Grafts formed tubular dentine and osteodentine. Tubular dentine, penetrated regularly by elongated odontoblast processes, resembled normal dentine and was formed when the original odontoblasts continued normal matrix secretion. Osteodentine was formed by spindle-shaped cells with large round nuclei which presumably were transformed pulp cells, and incorporated the same elements as found in cells of non-tubular dentine. Occasionally, odontoblasts were contiguous with both the regular dentine and the osteodentine. Thus in EDTA-dissociated pulps transplanted to the spleen, the original odontoblasts produce tubular dentine and other pulp cell differentiate to form osteodentine.

The development of mandibular molar tooth germs isografted in the mouse spleen.

Tooth germs taken from 13-day-old embryonic mice and isografted in the spleen of adult mice were examined by light and electron microscopy. Well-organized tooth structures from the early cap stage to fully developed and mineralized mature teeth were obtained up to day 60 after transplantation. Germs on day 2 were similar to those prior to the onset of grafting but reached the late cap stage of development on day 4. On day 6, enamel and dentine formation were initiated and inner enamel epithelium and dental papilla cells were polarized. On days 10-15, enamel-matrix secretion was completed and almost all ameloblasts had become resorptive enamel epithelium. India ink injected from the recipient caudal vein accumulated to the capillaries within the pulp throughout the newly-formed vessels. On day 20, defined root formation had begun but occasionally irregular and cellular osteodentine was formed in root areas. On day 30, transplants were covered with reduced enamel epithelium and acellular cementum was formed at the root areas together with rudimentary periodontal ligament fibres. Cellular cementum became thicker up to day 40. There was little evidence of cellular infiltration from recipient tissue up to day 60. The spleen seems to be a suitable site for transplantation of tooth germs.

Ultrastructure of the effects of calcitonin on the development of mouse tooth germs in vitro.

Mandibular first molars, from 17-day-old embryos, were cultivated in control medium or medium containing 0.1, 0.01 or 0.001 unit/ml of calcitonin (CT) for periods up to 10 days. In untreated tooth germs, cells of the dental papilla differentiated into pre-odontoblasts up to 4 days and predentine was seen on day 6. Cells treated with 0.1 unit/ml of CT differentiated into pre-odontoblasts up to 4 days, but no predentine was formed even after 10 days in culture. With 0.01 unit/ml, cells differentiated into odontoblasts, and had already secreted predentine a few days earlier than the untreated group. With 0.001 unit/ml, the developing germs were similar to the control explants during the entire 10-day cultivation period. The proportional area of rough endoplasmic reticulum to cytoplasm of the odontoblasts was low at 0.1 unit/ml of CT and high at 0.01 unit/ml compared to the untreated explants.

Immunocytochemical expression of type I and type II collagens by rat Meckel's chondrocytes in culture during phenotypic transformation.

In culture, chondrocytes of Meckel's cartilage can differentiate further to become bone-type collagen-synthesizing cells. Here, the replacement of type II collagen by type I collagen, accompanying expression of the osteocytic phenotype, was analysed by double immunofluorescence staining, histochemistry and electron microscopy. After 1 week in culture, formation of a toluidine blue-positive matrix, demonstrating the synthesis of cartilaginous proteoglycans, and the expression of type II collagen were detected. After 2 weeks, immunoreactivity specific for type II collagen was detected along the cartilaginous areas of the nodules, and type I collagen appeared in association with the immunopositive extracellular matrix around spindle-shaped cells. Electron microscopy revealed that the extracellular matrix at this stage was composed of homogeneous fine fibrils of type II collagen and thick cross-banded bundles of type I collagen: there was also continuity between the type I and II collagens. Double immunofluorescence staining of 3 week-old cultures revealed that type II collagen had been replaced by type I which was synthesized by small round cells that appeared at the top of the nodules. With further passage of time in culture, the distribution of type I collagen expanded further towards the peripheral areas from the central areas of the nodules. The present combination of ultrastructural analysis and double immunofluorescence staining shows that the transition from synthesis of cartilage-specific type II collagen to expression of type I collagen occurred sequentially in spindle-shaped cells located at the top of nodules in conjunction with the further differentiation of Meckel's cartilage cells.

Further evidence for secretion of matrix metalloproteinase-1 by Meckel's chondrocytes during degradation of the extracellular matrix.

We examined the possibility that chondrocytes in Meckel's cartilage might secrete matrix metalloproteinase-1 (MMP-1) during degradation of the extracellular matrix. Evidence for the secretion of MMP-1 was obtained by immunohistochemical staining and immunoelectron microscopy, in addition to general histochemical staining for proteoglycans. Not only staining with toluidine blue and alcian blue but also immunostaining for chondroitin sulfate proteoglycan (CSPG) revealed that levels of glycoproteins are rapidly reduced at the late stage of degradation. MMP-1 was detected continuously in cells from chondrocytes at the early stage to hypertrophic chondrocytes at the late stage. Immunoelectron microscopy revealed that the deposition of colloidal golds shifted from an intracellular localization in chondrocytes at the early stage to pericellular spaces at the late stage. The localization of tissue inhibitor of the metalloproteinase-1 (TIMP-1) at the early stage was similar to that of MMP-1, but the level of TIMP-1 decreased significantly in hypertrophic cartilage. These findings suggest that MMP-1 is present continuously in Meckel's chondrocytes but that the active form, which degrades the extracellular matrix, is the MMP-1 that accumulates in the pericellular spaces around hypertrophic chondrocytes.

Immunohistochemical and cytochemical evidences for a possible localization of leucine aminopeptidase in the Merkel cell granule.

Localization of leucine aminopeptidase (LAP) in the Merkel cell-axon complex was studied immunohistochemically and cytochemically in the labial tissues of the mouse, rat, dog, and monkey. Anti-LAP was obtained in rabbits by the injection of commercially supplied swine LAP which was confirmed as electrophoretically pure. The Merkel cells of the mouse, rat, and monkey were positively stained by treatment with anti-LAP but the Merkel cells of the dog were negative. When ultrathin sections of the hair follicle from the rat whisker pad, which contain an abundance of Merkel cells, were processed by immuno-peroxidase or by the immuno-gold method, the reaction products were predominantly deposited on the Merkel cells granules. Furthermore, an immuno-blot assay revealed that an extract of the hair follicles from murine whisker pads contained a molecule of relative molecular mass Mr = 60,000 which is similar in size to a subunit of swine LAP. Thus, it appears that Merkel cell granules of rodents and the monkey contain a protein which resembles lucine aminopeptidase.

Origin-associated features of chondrocytes in mouse Meckel's cartilage and costal cartilage: an in vitro study.

Using a cell culture method, we histochemically and immunohistochemically investigated whether chondrocytes deriving from different origins, such as Meckel's or costal cartilages, express similar phenotypic characteristics. Chondrocytes isolated enzymatically from Meckel's and costal cartilages of 17-day embryonic mice both actively proliferated and formed cartilage nodules consisting of toluidine blue-positive proteoglycans and type II collagen. Both deposited calcified cartilaginous matrix as revealed by alkaline phosphatase (ALPase) activity and alizarin red staining throughout 3 weeks in culture. Immunostaining for osteopontin (OP), osteocalcin (OC), and osteonectin (ON) revealed that chondrocytes from both cartilages were positive for their proteins, but type I collagen was detected only in cells transforming from Meckel's chondrocytes late in the culture. Electron microscopy demonstrated that although costal and Meckel's chondrocytes had typical chondrocytic features during 2 weeks in culture, Meckel's chondrocytes transformed into osteocytic cells that produced thick, banded type I collagen fibrils. In contrast, costal chondrocytes maintained typical hypertrophic morphology throughout the final stage of culture. The present study suggests that Meckel's chondrocytes derived from neural crest-ectomesenchyme retain osteogenic potential, and differ from costal chondrocytes originating from mesoderm.

Clinical experience of radiation therapy for Graves' ophthalmopathy.

The effect of radiation therapy for Graves' ophthalmopathy was evaluated. Ten patients with Graves' ophthalmopathy were treated with radiation therapy between 1992 and 1993 in Gunma University Hospital. All patients had a past history of hyperthyroidism and received 2,000 cGy to the retrobulbar tissues in 20 fractions. Nine of ten patients were treated with radiation therapy after the failure of corticosteroids. Six patients (60%) showed good or excellent responses. The exophthalmos type was more responsive to radiation therapy than the double vision type in this series. Two of five patients with the exophthalmos type demonstrated excellent responses, and their symptoms disappeared almost completely. The improvement of symptoms appeared within 3-6 months, and obvious clinical effects were demonstrated after 6 months of radiotherapy. Radiation therapy was well tolerated, and we have not observed any side effects of radiation therapy. In conclusion, radiation therapy is effective treatment for Graves' ophthalmopathy.

In vitro characterization of enamel epithelium and pulp cells in mouse tooth germs.

Mixed cell populations consisting of enamel epithelium and pulp cells obtained from 18-day embryonic mouse tooth germs were cultured in vitro. Epithelial cells and pulp cells were also cultured individually and examined. Isolated cells were cultured in alpha-MEM supplemented with 10% fetal bovine serum for up to four weeks and examined morphologically using histological procedures including immunostaining, light and electron microscopy, and electron probe microanalysis. The pulp cells proliferated and differentiated in the absence of epithelium, but the number of epithelial cells showed a strong dependence on the pulp cells. Pulp cells showing fibroblastic morphology in the mixed culture gradually became elliptical, and eventually transformed into spherical cells surrounded by a calcified extracellular matrix. Alkaline phosphatase (ALPase) activity was expressed on the pulp cells prior to calcification of the extracellular matrix, as shown by von Kossa's and alizarin-red staining. Calcification deposition, which is closely associated with thick banded type-I collagen fibrils, was shown to be composed of calcium and phosphorous using electron probe microanalysis. Type-I collagen immunoreactivity was detected on the extracellular matrix after two weeks of culturing. The present results show that the proliferation and differentiation of pulp cells, and subsequent calcification of the extracellular matrix occur in the presence or absence of epithelial elements, but that the proliferation of epithelial cells depends on the presence of pulp cells.