Search

Our previous studies demonstrated that Meckel's chondrocytes, which are derived from ectomesenchyme, have the potential to transform into osteogenic phenotypes. The present study aimed to clarify the role of cell origin in the phenotypic transformation of chondrocytes. Cell pellets from ectomesenchyme-derived Meckel's cartilage and mesoderm-derived costal cartilage from green fluorescent protein (GFP)-transgenic mice were transplanted into the spleen for up to 4 weeks. Chondrocyte pellets from both cartilages adapted well to the splenic tissues and formed an alizarin red-positive calcified matrix, with increasing duration of transplantation. Following the production of cartilage-specific type II and type X collagens, newly-formed type I collagen appeared in the chondrocyte pellets from Meckel's cartilage during the late stage of transplantation. Although the bone-marker proteins: osteocalcin, osteopontin, osteonectin and bone morphogenetic protein-2, were detected in pellets from both Meckel's and costal cartilage, only type I collagen in Meckel's cartilage was a significant marker protein for detecting transformation. These bone-type protein-producing cells represented osteogenic cells transformed from GFP-expressing cells, rather than from recipient cells. These results indicate that neural crest-derived Meckel's cartilage displays a higher potential for phenotypic switching than mesoderm-derived costal chondrocytes under in vivo conditions.

エナメル質の基本構造をなすエナメル小柱には横紋が観察される.この横紋は概日リズムを刻んだ成長線のひとつとして知られているが,形成メカニズムについては様々な説がある.非脱灰凍結切片で基質形成期のエナメル基質を抗アメロゲニン抗体で免疫染色すると横紋様パターンを示した.この結果から,横紋は基質形成期のアメロゲニンのタンパク量に依存した石灰化パターンであり,アメロゲニンの発現は概日的に変動するのではないかと推測した.そこで,アメロゲニンの発現に周期があるか観察するために,アメロゲニンプロモーターの下流にルシフェラーゼを繋いだコンストラクト(pGL3-1730-luc)をラットエナメル芽細胞株HAT7に遺伝子導入してアメロゲニンの転写活性を計測したところ,一定の周期をもって変動することが認められた.次に,プロモーター領域のDeletion-mutant (pGL3-464, -74, -48-luc)を作製して周期性の制御に関わる領域を検索した結果, C/EBPαのbindingモチーフが有力な候補と考えられた.その転写はMsx2によって制御を受けることが知られていることから, Msx2の発現ベクターを用いた強制発現の影響を調べたところ,そのリズムが消失した.またMsx2の遺伝子欠損マウスには基質形成の横紋様パターンが見られなかった.以上の結果から, Msx2がアメロゲニンの発現周期に影響していることが推測され,さらにアメロゲニンの発現量の周期的変化によって生じた基質形成パターンが,横紋形成に関与していると考えられた., In enamel rods, periodic bands referred to as 'cross-striations' are observed, and known as incremental lines of circadian rhythm. Though it is considered that the cross-striation is involved in the biological circadian rhythm during the secretion of enamel matrix protein by ameloblasts, the developmental mechanism involved has not been examined in detail. By immunostaining amelogenin in fresh frozen sections of mouse lower incisor, we could observe fluorescent periodic bands in the enamel matrix, which were identical to the pattern of these cross-striations. Accordingly, we focused on the biological rhythm in the section of amelogenin. We examined amelogenin mRNA transcriptional activity in an ameloblast cell line (HAT7) by using real-time luminescence microscopy. The results showed that amelogenin mRNA transcriptional activity exhibited periodic rhythmicity and that Msx2 over-expression led to the disappearance of the periodic change. Further, in the lower incisors of Msx2-deficient mice, the periodic bands were not observed. Taken together, our findings suggest that the formation of cross-striations in the enamel rods was associated with the expression of amelogenin regulated by the transcriptional activity.

Similar to embryonic stem cells, induced pluripotent stem (iPS) cells can differentiate into various cell types upon appropriate induction, and thus, may be valuable cell sources for regenerative medicine. However, iPS cells have not been reported to differentiate into odontogenic cells for tooth regeneration. Here we demonstrated that neural crest-like cells (NCLC) derived from mouse iPS cells have the potential to differentiate into odontogenic mesenchymal cells. We developed an efficient culture protocol to induce the differentiation of mouse iPS cells into NCLC. We confirmed that the cells exhibited neural crest (NC) cell markers as evidenced by immunocytochemistry, flow cytometry, and real-time reverse transcription-polymerase chain reaction. Further, in recombination cultures of NCLC and mouse dental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium cultures further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration and tooth development studies.

Objective Induced pluripotent stem (iPS) cells can differentiate into a variety of cell types and can form teratomas when transplanted in vivo. The purpose of the present study was to investigate the developmental processes and features of epithelial tissues, and the existence of epithelial stem/progenitor cells in iPS cell-derived teratomas. Materials and methods iPS cell-derived teratomas formed by subcutaneous injection into immuno-deficient mice were extracted at 7-day intervals up to 21 day interval. Paraffin-embedded tissue sections were evaluated by H&E staining and immunohistochemistry for specific markers of three germ layer cell types and epithelial stem cells in oral tissues. Results During iPS cell-derived teratoma development, ectodermal cells emerged first, followed by the appearance of mesodermal and endodermal cells. The teratomas contained various types of epithelia, including epithelial masses, pseudostratified epithelium, simple epithelium and stratified epithelium. As teratoma development progressed, the epithelia matured. p63 and K14 were strongly expressed in epithelial masses and in the basal layer of pseudostratified and stratified epithelium. CD49f was expressed in all types of epithelia observed, and intense expression was observed in both the basal layer and luminal surface of pseudostratified and stratified epithelium. Conclusions iPS cell-derived teratoma is a useful model for study of epithelial development, and it may provide significant information regarding oral epithelial tissue regeneration.

Sakuraba H, Fujiwara N, Sasaki-Oikawa A, Sakano M, Tabata Y, Otsu K, Ishizeki K, Harada H. Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth. J Periodont Res 2012; 47: 81–88. © 2011 John Wiley & Sons A/S 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.

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and β-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.

Meckel's cartilage (MC), which develops as a template of the first skeleton of the mandible, undergoes site-specific fates. The anterior portion contributes to the formation of the rostral part of the mandible by endochondral ossification, while the posterior portion is involved in the formation of the ossicle bones. In contrast, the posterior part of the midportion (PM portion; located in the soft tissue) forms the sphenomandibular ligament, but its formation mechanism remains unclear. We found that chondrocytes in the PM portion have the potential to transform into other phenotypic cells under in vivo and in vitro conditions. In the present review, we focus on the results of cellular transformation based on our investigations. It has been clarified that MC chondrocytes in the PM portion transform into fibroblastic cells by phenotypic switching of the chondrocytes themselves in response to epidermal growth factor. In contrast to their conversion to fibroblastic cells in vivo , MC chondrocytes subjected to long-term culture or transplanted into ectopic sites, such as the spleen, expressed bone-type proteins accompanied by transformation into osteocytic cells. Since phenotypic transformation did not occur in mesoderm-derived costal chondrocytes, the findings suggest that neural crest-derived MC chondrocytes have high potential for transformation in response to altered stimuli.

ウナギ由来のエルカトニン(ELC)は優れた薬理学的効能から実験的ないし臨床試験的改善薬として骨粗鬆症患者に投与され良好な成績が得られている。本実験では,胎生17日マウスメッケル軟骨に対するELCの効果を細胞培養と器官培養を用いて解析した。細胞培養と器官培養のためのメッケル軟骨は以下の4種類の培養液で最長4週間まで培養した。培養液として,培養液1;イーグル培地(α-MEM),培養液2;α-MEM+β-グリセロリン酸(β-Gly),培養液3;α-MEM+ELCと培養液4;α-MEM+β-Gly+ELCを用いた。試料はアルカリフォスファターゼ(ALPase)とbromodeoxyuridine (BrdU)の取り込みを含む組織学,組織化学的方法によって解析した。ELCを含む培養液は,より効果的に石灰化を誘導することがホン・コッサ染色によって示された。BrdUの取り込みによる細胞増殖率は,培養液1と3との間では有意差が認められなかった。ALPase活性は,基質の石灰化に先駆けて促進されることが細胞培養と器官培養での組織化学と蛍光免疫染色によって確認された。ELCはβ-Glyとほぼ同等量のALPaseを発現し,さらにELCとβ-Glyを含む培養液4では相乗的にALPase活性と石灰化を誘導した。本研究の結果から,ELCはメッケル軟骨細胞のALPase活性を促進し,その後の石灰化の誘導因子として作用する可能性が示唆された。, The effects of elcatonin (ELC) on Meckel's cartilage obtained from 17-day embryonic mice were investigated using cell and organ cultures. Specimens for cell and organ cultures were cultured for 4 weeks in different media as follows: Control medium, alpha-modified Eagle's medium (α-MEM); β-Gly medium, α-MEM plus β-glycerophosphate (β-Gly); ELC medium,α-MEM plus ELC; and β-Gly & ELC medium,α-MEM plus β-Gly plus ELC. Specimens were analyzed by histological and histochemical methods, including immunohistochemistry for alkaline phosphatase (ALPase) activity and bromodeoxyuridine (BrdU) incorporation. Von Kossa staining revealed that the ELC medium inducedeffective calcification similar to β-Gly medium, while BrdU incorporation showed that there was no significant difference in the rates of cell proliferation between Control medium and ELC medium. Histochemical and immunofluorescence staining indicated that ALPase was activated prior to matrix calcification in cell and organ cultures. β-Gly & ELC medium induced synergistically the highest level of matrix calcification and ALPase activity. The present results suggest the possibility that ELC accelerates activity of ALPase in Meckel's chondrocytes and subsequently induces matrix calcification.

The tooth root is a foundation structure embedded in the alveolar bone of the jaws. Root formation starts after the completion of crown morphogenesis and proceeds slowly while coordinating the formation of the periodontal tissue that supports the tooth. Although several signaling pathways and transcription factors have been implicated in the regulation of molar crown development, relatively little is known about the regulatory mechanisms governing the transition from crown to root development. The formation of Hertwig's epithelial root sheath (HERS), consisting of 2 epithelial layers, is a key event in the initiation of root development. Recently, a new hypothesis about the mechanism of HERS formation was proposed based on the results of experiments using Fibroblast growth factor ( Fgf ) -10-deficient mice. In this review, we discuss this new hypothesis of HERS development and the signaling change for HERS formation during the transition process from crown to root development.

Chick serum (CKS) contains factors that stimulate adipocytes in Meckel's chondrocytes in vitro. In the present study, we analyzed levels of fatty acids in CKS, and further examined whether these had the potential to convert chondrocytes to adipocytes. Phenotypic changes were evaluated by light and electron microscopies, bromodeoxyuridine (BrdU) incorporation, triglyceride assays, and immunocytochemistry. We showed that CKS contained high levels of fatty acids, and a mixed medium containing 5 particular fatty acids inhibited DNA synthesis and the proliferation of chondrocytes as it facilitated their differentiation into adipocytes. The adipocytes produced were sudan-positive multilocular cells that morphologically and histochemically resembled adipocytes induced by the CKS-containing medium. Almost all lipid droplet-containing cells were positive for leptin and alpha-glycerophosphate dehydrogenase (GPDH), as evaluated by immunoperoxidase staining, and their triglyceride concentrations markedly increased during 4 to 6 days of culture. These results suggested that specific fatty acids in CKS are involved in the adipocytic conversion of Meckel's chondrocytes.

ニワトリ血清(CKS)はメッケル軟骨細胞に脂肪形成を促進する因子を含んでいる。本研究では, CKSのホルモン分析の結果から得られたような高レベルの性ホルモンが培養メッケル軟骨細胞の脂肪形成にどの様に関わっているのか検討した。培養軟骨細胞は生理的ないし薬理的濃度のテストステロンと17β-エストラジオール(E_2)を含む数種の性ホルモンで添加培養し, 脂肪滴形成能をズダン染色によって評価した。最も脂肪形成能の高かったテストステロンとE_2については, 電子顕微鏡, 免疫染色とBrdUの取込みから細胞増殖率を算定した。その結果, 生理的濃度でのテストステロンとE_2は細胞増殖を促進したが, 脂肪形成は促進しなかった。高濃度では両者のホルモンは脂肪形成を亢進した。形成された脂肪細胞はレプチン, GPDHやC/EBPα抗体による免疫染色では陽性であった。これらの結果から, テストステロンとE_2は薬理学的作用として脂肪形成能を有しているが, 生理的条件下では脂肪形成そのものより未分化型細胞での細胞増殖を刺激している可能性が示唆された。, Chick serum (CKS) contains factors that stimulate adipocyte induction in Meckel's chondrocytes in vitro. In the present study, we examined whether sex hormones as shown by the analysis of hormonel level in CKS have the capacity to induce lipogenesis in Meckel's chondrocytes in vitro. Cultures were treated with different sex hormones at physiological and pharmacological concentrations, including testosterone and 17β-estradiol (E_2). Lipogenesis was evaluated by sudan staining. Testosterone and E_2, which were the two most effective hormones, were selected for the present study and their effects were further examined by histological and immunohistochemical methods, including immunolocalization of adipocyte markers. BrdU-incorporation in cells exposed to physiological concentration of E_2 and testosterone was increased compared to control cultures, but there was no significant difference in BrdU-incorporation in cultures treated with E_2 and testosterone. However, at pharmacological concentrations, these hormones induced dose-dependent lipogenesis in Meckel's chondrocytes. Almost all the lipid droplet-containing cells were positive for leptin, α-glycerophosphate dehydrogenase (GPDH), and CCAAT-enhancer binding protein (C/EBPα), as shown by immunoperoxidase staining. Testosterone and E_2 had no capacity to induce lipogenesis in Meckel's chondrocytes at physiological concentrations, but our results suggest that they have the potential for lipogenesis as a pharmacological effect.

To elucidate the mechanism of root formation in tooth development, we examined the role of insulin-like growth factor I (IGF-I) on early root formation in mandibular first molar teeth from 5-day-old mice. Immunohistochemistry revealed the specific localization of the IGF-I receptor in Hertwig's epithelial root sheath (HERS) in the tooth root. The effect of IGF-I on root development, especially on HERS, was subsequently examined in vitro. The control culture showed normal development of HERS and the periodontium, resembling that in vivo. However, the presence of 100 ng/ml IGF-I resulted in elongation of HERS and increased cell proliferation in its outer layer. These effects were negated by the addition of antibodies specific for IGF-I. Thus, we propose that IGF-I is involved in early root formation by regulating the mitotic activity in the outer layer of HERS.

抗癌剤のエトポシトとカンプトセシンによってメッケル軟骨に誘導される細脳死かアポトーシスによるものか否かを検索した。細脳死は胎生16日のマウスから分離した培養メッケル軟骨にエトポシト(200μg/ml)とカンプトセシン(50μg/ml)によって誘導した。これらの効果はTUNEL法と光顕およひ電顕に加え、免疫組織化学的に解析した。エトポント処理した培養細胞はアポトーシスの指標となる多くのTUNEL陽性細胞を示した。微細構造的にエトポシトてのアポトーシス様の細脳死は細脳性出芽、核クロマチンの濃縮、アポトーシス小体の形成によって引き続かれた。これに対し、カンプトセシンによる細脳死はエトポシトと同様にTUNEL陽性細胞の増加を示した。これらの抗癌剤処理後のアポトーシスは時間依存的に増加し、特に、12時間後から急速に増加した。P53の免疫染色では、この蛋白は正常な培養軟骨細胞では陰性であったか、エトポノトとカンプトセシン処理群では恒常的に促進された。一方、bcl-2の反応は培養初期の正常軟骨細胞に局在したが、抗癌剤処理群では認められなかった。本研究から、エトポシトとカンプトセシンによってメッケル軟骨に誘導された細脳死は典型的なアポトーシスによる細脳死で、これらの抗癌刻は腫瘍細胞のみならす、正常な軟骨細脳にもアポトーシスを誘導することか示唆された。, Cell death induced in Meckel's chondrocytes in vitro with the anticancer drugs, etoposide and camptothecin, was examined from the viewpoint of an apoptotic event. Cell death was induced in vitro in Meckel's chondrocytes that were enzymatically isolated from 16-day gestation mouse embryos. The effects were assayed histologically and immunohistochemically by using the TUNEL procedure, and light and electron microscopy. Etoposide and camptothecin-treated cultures were followed by the strong appearance of TUNEL (TdT-mediated biotinylated dUTP nick end-labeling)-positive apoptotic cells; statistical analysis showed that these agents induced apoptosis significantly 12-24hrs after treatment. At an ultrastructural level, apoptotic cell death from etoposide was followed by the formation of cell blebs, chromatin condensation and the formation of apoptotic bodies. Immunostaining for p53 revealed that this protein was absent from intact chondrocytes but was continuously accelerated in the cells treated with etoposide and camptothecin. Bcl- 2 was immunolocalized in intact chondrocytes at an early stage of culture, but was not detected in anticancer-treated cells. In the present study, we confirmed that cell death in Meckel's chondrocytes induced by etoposide and camptothecin is typical apoptotic cell death, and that these agents also induce apoptosis in intact chondrocytes.

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.

In order to analyze the phenotypic conversion of chondrocytes, mandibular condyles of mice and rabbits were cultured under cell and organ culture systems, and then examined by a combination of morphological and biochemical procedures. In organ culture, mandibular condylar cartilage (MCC) obtained from newborn mice began to mineralize from the central zone and then progressively widened towards the peripheral zone. Electron microscopic observations showed that with the increasing duration of the organ culture, chondrocytes at the central zone converted into spindle-shaped osteoblastic cells accompanying the formation of the bone type of thick-banded collagen fibrils. To obtain a better understanding of the chondrocytic conversion, immunolocalizations for type I and type X collagens and osteocalcin (OC) were examined in mouse MCC cells in cell culture. Type X collagen and OC were expressed almost simultaneously at the late stage of culture, and type I collagen was detected along the calcified nodules after the production of these proteins. Northern blot analysis in cell cultures of rabbit MCC indicated that type II collagen and alkaline phosphatase (ALPase) messenger ribonucleic acids (mRNAs) were highly expressed at day 7, but subsequently decreased. In contrast, mRNA for type I collagen was expressed at a low level on day 7 and peaked on day 12. The present results suggest that, morphologically and biochemically, cellular modification in MCC cells under culture conditions occurs at a cellular morphological level and also at marker-gene-expression level.

To clarify whether in vitro cell death in Meckel's chondrocytes induced by heat-stress treatment involves apoptosis or necrosis, we performed morphological examinations using immunohistochemistry including BrdU-incorporation, the TUNEL method, and light and electron microscopy. Cell death was induced in Meckel's chondrocytes isolated enzymatically from 17-day embryonic mice, by exposure to temperatures 40-60°C. On exposure t0 sublethal temperatures of 40-45°C, the incorporation of BrdU was facilitated, and TUNEL-positive apoptotic cells appeared with high frequency. At the ultrastructural level, apoptotic cell death was characterized by chromatin condensation, nuclear segmentation, and the formation of apoptotic bodies. In contrast, cell death induced by lethal temperature (50-60°C) presented decreases in TUNEL-positive cells and cell incorporating BrdU. High temperature-treated cells showed apparent disruption of the membrane of cell organelles. Immunostaining for heat shock proteins (HSP) -27 and-70 revealed that these proteins are secreted continuously at lower temperatures, but have a tendency toward decrease on exposure to lethal temperatures. Our results suggest that Meckel's chondrocytes demonstrate apoptosis at sublethal temperatures, but undergo necrosis at lethal temperatures.

It is widely accepted that Meckel's cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckel's cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckel's cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckel's cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckel's cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral-type mechanism of calcification.

The localization of osteopontin (OP) was examined in Meckel's cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix.

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.

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.

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.

We examined the influence of cell density on the phenotypic conversion of Meckel's cartilage cells. The cells were isolated by enzymatic digestion and plated at a density of 0.5 × 104 or 2 × 104 cells/penicylinder and cultured under 5% CO2 in air for up to 4 weeks. The cultures were analyzed histologically by electron microscopy, histochemistry, and immunostaining. At the early stage of high-density culture, metachromatic chondrocytes appeared concomitantly with cartilage-specific type II collagen synthesis. The cells gradually transformed from large polygonal cells to multilayered small round cells, and formed nodules. During the later stage of culture, the cells exhibiting alkaline phosphatase (ALPase) activity, type I collagen, and osteocalcin as bone-type marker proteins consisted of spindle-shaped cells showing phenotypic conversion into osteocyte-like cells in the calcified nodules. In contrast, during low-density culture, most of the cells changed from fibroblastic cells to large flattened cells without cellular nodule formation, but these cells lacked chondrocytic features. Only solitary cells exhibited chondrocyte-specific features such as metachromasia, proteoglycan synthesis, and matrix calcification, but they did not undergo osteocytic transdifferentiation. These results indicate that well-organized nodule-forming cellular and extracellular components in high-density culture stimulate transdifferentiation into osteocyte-like cells.

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.

The epithelial–mesenchymal transition (EMT) is an important event in the developmental process of various organs. In periodontal development during root formation of a tooth, this EMT has been a subject of controversy. Hertwig’s epithelial root sheath (HERS), consisting of two epithelial layers, plays a role of inducing odontogenesis during root development and thereafter becomes fragmented. Some researchers have maintained that in the process of this fragmentation, some HERS cells change from epithelial to mesenchymal cells. Here, we established a HERS cell line (HERS01a) and examined its gene and protein expression. Immunohistochemical staining and real-time PCR analysis showed that HERS01a cells expressed vimentin and N-cadherin as mesenchymal markers as well as cytokeratin14, E-cadherin, and p63 as epithelial stem cell markers. In the presence of TGF-β, HERS01a cells also expressed many more mesenchymal markers, as well as snail1 and 2 as EMT markers. Taken together, our data show that HERS01a displayed unique features associated with EMT in the root formation process, and will thus be useful for analyzing the biological characteristics of HERS and the molecular mechanism underlying the EMT.

Meckel's chondrocytes, derived from the ectomesenchyme, have the potential to transform into other phenotypes. In this study, we transplanted cell pellets of Meckel's chondrocytes into isogenic mouse spleens and analyzed their phenotypic transformation into osteogenic cells using histological and immunohistochemical methods. With the increasing duration of transplantation, chondrocytes were incorporated into splenic tissues and formed a von Kossa-positive calcified matrix containing calcium and phosphoric acid, similar to that of intact bone. Type I, II, and X collagens, and the bone-marker proteins osteocalcin, osteopontin, osteonectin, and bone morphogenetic protein-2 (BMP-2) were immunolocalized in the matrix formed by the transplanted chondrocytes. Osteopontin and osteonectin were detected in the calcified matrix at earlier stages than osteocalcin and BMP-2. Type II collagen was expressed during the first week of transplantation, and type X collagen-positive cells appeared scattered during the initial stage of calcification, these collagens being later replaced by type I collagen formed by osteocyte-like cells. Electron microscopic observations revealed that chondrocytes surrounded by the calcified matrix transformed into spindle-shaped osteocytic cells accompanying the formation of bone-type thick-banded collagen fibrils. These results suggest that phenotypic switching of Meckel's chondrocytes can occur under in vivo conditions at a cellular morphological level.

Mouse, rat, and human molars begin to form their roots after the completion of crown morphogenesis. Though several signaling pathways and transcription factors have been implicated in the regulation of molar crown development, relatively little is known about the regulatory mechanisms involved in the transition from crown to root development. Tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS) from the cervical loop in the enamel organ. In this study we examined the change in epidermal growth factor (Egf) signaling during this transition process. Immunohistochemical studies showed that the expression of Egf receptors in the enamel organ disappear gradually in the process and are not observed in HERS. Here, to examine the effect of Egf on the transition, we used the organ culture method to examine the root development. In the presence of Egf, stellate reticulum (SR) cells between the inner and outer epithelial layers in the enamel organ actively proliferated and maintained the enamel organ, and the formation of HERS was not observed. On the other hand, in either the absence of Egf or the presence of the inhibitor of Egf receptors, the SR cells disappeared and HERS formation started. Subsequently, root formation proceeded in the culture period. Therefore, disappearance of SR area may be a key event that controls the timing of onset of HERS formation, and Egf may be one of regulatory factors involved in the change from cervical loop epithelium to HERS during root development.

The capacity of the dental pulp to form calcified tissue was examined in papilla cells dissociated from first molar tooth germs of the neonatal mouse and isografted in the spleen for up to 7 days. To obtain papilla cell populations without odontoblasts, pulpal mesenchyme was isolated mechanically from the enamel organ after 0.1% trypsin treatment and rolled on a membrane filter. On day 3 after transplantation, the grafted papilla cells had changed into large, spindle-shaped cells, and initial calcification with needle-like crystals began in association with the collageneous matrix surrounding those cells. On day 7 after transplantation, the spindle cells transformed into odontoblast-like cells containing well-developed secretory organelles, and irregular, but nontubular, calcified tissues were commonly observed surrounding the extracellular collagenous matrix. The calcified tissue matrix with cellular inclusions displayed a structure similar to that of osteodentin. During this period, an intense positive reaction for alkaline phosphatase (ALPase) activity was demonstrated along the cell membranes of the odontoblast-like cells aligned at the periphery of forming calcified tissue. Enzymatic activity could not be detected on the cells incorporated completely into osteodentin-like matrix. The present results show that the papilla cell population transplanted into the spleen formed osteodentin-like material, thus demonstrating the capacity of papilla cells to produce calcified tissue.

Chick serum contains a factor that stimulates adipogenesis in Meckel's chondrocytes in vitro. The present study examined whether chick serum has a capacity for adipogenic induction in vivo, by transplanting serum clots (created by drying chick serum for up to 4 weeks) into mouse spleens. Specimens were harvested for histological analyses, which included light and electron microscopy and immunohistochemistry. The transplanted serum clots induced the appearance of lipid droplet-containing cells in splenic cords and sinus. Almost all the lipid droplet-containing cells were positive for sudan staining and consisted of multilocular lipid vacuoles. Immunostaining showed that the adipocytes induced by transplantation of the serum clots initially appeared as peroxisome proliferator-activated receptor-gamma (PPARgamma)-positive cells and developed into leptin and alpha-glycerophosphate dehydrogenase (GPDH)-producing cells, in addition to type III collagen synthesis. Furthermore, double immunofluorescence staining revealed that the immunoreactivity for GPDH was detected not only in stromal cells but also in macrophages. It was thus confirmed that stromal cells and macrophages in the spleen contain lipid droplets as seen in intact white adipose cells. The present results suggest that chick serum contains factors for adipocyte induction not only in vitro but also in vivo, and that the adipogenic potential does not depend on the supplements used during the cell culture.

Chondrocyte maturation and vascular invasion of cartilage are essential in the process of endochondral ossification. Cbfal-deficient (Cbfa1-/-) mice displayed a complete absence of osteoblast and osteoclast maturation as well as severely inhibited chondrocyte maturation in most parts of the skeleton. Although chondrocyte maturation and mineralization were observed in restricted areas of Cbfa1-/- mouse skeleton, vascular invasion of calcified cartilage was never noted. To investigate the possibility of chondrocyte maturation and vascular invasion in Cbfal-/- cartilage and the role of the hematopoietic system in the process of vascular invasion, we transplanted embryonic day 18.5 (E18.5) Cbfa1-/- femurs, which are composed of immature chondrocytes, into spleens of normal mice. One week later, the transplanted femurs contained terminally differentiated chondrocytes expressing osteopontin, bone sialoprotein (BSP), and matrix metalloproteinase (MMP) 13. In the diaphyses of the transplants, the cartilage matrix was mineralized and the cartilage was invaded by vascular vessels and osteoclasts. However, chondrocyte maturation and vascular invasion were severely retarded in comparison with transplants of E14.5 wild-type femurs, in which the cartilage was rapidly replaced by bone, and neither mature osteoblasts nor bone formation were observed. In primary culture of Cbfa1-/- chondrocytes, transforming growth factor (TGF) beta1, platelet-derived growth factor (PDGF), interleukin (IL)-1beta, and thyroid hormone (T3) induced osteopontin and MMP-13 expression. These findings indicated that factors in the hematopoietic system are able to support vascular invasion of cartilage independent of Cbfal but are less effective without it, suggesting that Cbfal functions in cooperation with factors from bone marrow in the process of growth plate vascularization.

When present in the culture medium, chick serum (CKS) modulated the phenotypic change from chondrocytes of Meckel's cartilage to adipocytes in vitro, as revealed by light and electron microscopy, the incorporation of BrdU, and immunocytochemistry. CKS inhibited DNA synthesis in chondrocytes and the proliferation of these cells, while it facilitated the differentiation to adipocytes. CKS contributed to phenotypic changes in undifferentiated chondrocytes, but did not affect the characteristics of differentiated chondrocytes. Electron microscopy revealed that the lipid droplets in adipocytes were enclosed by limiting membranes that fused to yield larger lipid droplets. Immunocytochemical staining of adipocytes with stage-specific antibodies revealed the presence of immunoreactive uncoupling protein (UCP-1) and peroxisome proliferator-activated receptor (PPARgamma) in immature adipocytes, and leptin and glucose transporter (Glut-4) in mature adipocytes. The adipocytes that were formed in the present study were multilocular adipocytes that contained many small lipid droplets, but in many ways they resembled white adipocytes. CKS contains a high level of estrogen, compared with fetal bovine serum, and it is possible that estrogen might have induced the differentiation to adipocytes.

In mammals, the midportion of the soft tissue of Meckel's cartilage at the degenerating stage forms a ligament known as the sphenomandibular ligament. To clarify the mechanism of formation of this ligament by Meckel's cartilage in mouse, we examined the effects of epidermal growth factor (EGF) on the chondrocytes in terms of the proliferation and differentiation of cells and calcification of the matrix in vivo and in vitro. The effects of EGF were examined by immunohistochemical staining, with EGF-soaked beads, by electron microscopy, and by general histochemical analysis of proteoglycans and calcification. Analysis of labeling with bromodeoxyuridine (BrdU) and the rate of cell growth revealed that EGF enhanced DNA synthesis and the proliferation of Meckel's chondrocytes. Histological findings in organ culture and in cell culture, with and without the application of EGF-soaked beads, revealed that EGF inhibited the differentiation of cells to chondrocytes and induced phenotypic changes in fibroblastic cells. The inhibition of alkaline phosphatase activity that resulted from exposure to EGF was accompanied by prolonged calcification of the matrix. Whole-mount staining revealed that subcutaneous injection of EGF enhanced the disappearance of Meckel's cartilage. Our results suggest a possible mechanism whereby the midportion of Meckel's cartilage remains uncalcified and is rapidly transformed into the sphenomandibular ligament.

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.

Background We reported that when Meckel's cartilage was transplanted ectopically, chondrocytes transformed into osteocyte-like cells accompanying the extracellular calcified matrix. However, we could not determine whether the osteocyte-like cells were derived from host tissues or from Meckel's cartilage itself. Therefore, we examined whether the Meckel's cartilage chondrocytes, which have a retrogressive ultimate fate, are capable of inducing the observed calcification and further transform into osteocyte-like cells in culture. Methods Meckelian chondrocytes isolated enzymatically were plated at a low density and grown in α-MEM containing 10% FBS at 37°C under 5% CO2 in air for up to 4 weeks. Results Chondrocytes were fibroblast-like cells early in culture, but gradually transformed from polygonal cells into typical chondrocytes showing metachromasia with toluidine blue staining. After an additional week of culture, the chondrocytes transformed from large to small round cells accompanying nodule formations. Small round cells multiple-layered actively, and showed more intense alkaline phosphatase (ALPase) activity. Immunostaining identified type II collagen in the extracellular matrix at 2 weeks of culture, and type I collagen and osteocalcin were later synthesized by round cells. von Kossa's reaction showed extensive precipitation of calcification throughout the flocculent materials. Ultrastructural analysis showed that the cells surrounded by calcified matrix strongly resembled osteocytes. Conclusions The present study suggested that the Meckel's cartilage chondrocytes can express the osteocyte-like phenotype in vitro during synthesis of bone-type marker proteins such as osteocalcin or type I collagen. © 1996 Wiley-Liss, Inc.

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.

The changes in the morphological characteristics of Meckekl’s cartilage cells in culture can be divided into five stages during their transdifferentiation to cells with an osteocyte-like phenotype, as demonstrated in this study by light and electron microscopy. Chondrocytes were isolated from day-17 embryonic mice by enzymatic digestion and inoculated at a density of 1 × 104 cells. The cultures were incubated at 37 °C and cells were harvested every week for 4 weeks and processed for light and electron microscopy, as well as an immunohistochemical analysis of type I and II collagens. The primary cultures were characterized by fibroblastic cells (stage 1) around days 2–4. Polygonal cells (stage 2) expanded on a poorly formed extracellular matrix on day 7, and then they differentiated into large, round cells (stage 3) which began to form cellular nodules. The large, round cells contained many vacuoles, which were rapidly released into the pericellular space with a significant resultant decrease in cell size. Immunostaining showed that the extracellular matrix was occupied predominantly by type II collagen after 2 weeks in culture. However, the type II collagen was gradually replaced by type I collagen. Cells that formed multiple layers gradually changed into small, round cells (stage 4) at the tops of nodules and ultimately expressed an osteocytic phenotype (stage 5) after further transdifferentiation.

To examine the ultimate fate of hypertrophic chondrocytes, Meckel's cartilage bars from 18-day-old mouse embryos were transplanted into isogenic mouse spleen for 3, 7, 14 and 21 days and observed at the light and electron microscopic levels. The midportions of these Meckel's cartilage bars were used as explants; they were characterized by many hypertrophic chondrocytes containing euchromatic round nuclei, a large amount of glycogen particles, and some vacuoles. Grafted cartilage adapted well to the splenic tissue, showing intense metachromasia around the territorial matrix. Ultrastructural observations indicated that the number of large vacuoles and glycogen aggregates in the hypertrophic cells became markedly reduced with grafting time, whereas the Golgi apparatus and rough endoplasmic reticulum were well-developed. Needle-like crystals showing initial apatite deposition appeared in association with matrix vesicles; these proliferated as time elapsed after transplantation. On day 14 after transplantation, cells displaying such various structural features as pyknotic nuclei, large vacuoles, and cytoplasmic shrinkage were noted in addition to intact hypertrophic chondrocytes. Following resorption of the calcified cartilage by multinucleated giant cells, many osteoblasts appeared along the border of the calcified matrix. Some remaining hypertrophic cells in the calcified matrix had transformed into osteocyte-like cells. On day 21, the resorbed area of the calcified cartilage was invaded by many blood vessels. Hypertrophic chondrocytes, now exposed from cellular lacunae, and the osteocyte-like cells in the calcified matrix displayed involutional changes. The present study showed that, although the hypertrophic chondrocytes in Meckel's cartilage essentially underwent regressive changes, they retained the ability to stimulate endochondral ossification within the microenvironment of the spleen. In addition, some of these cells were transformed into osteocyte-like cells.

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.

We found that when the midsection of Meckel's cartilage bars obtained from mice on the eighteenth day of gestation were grafted into isogenic mouse spleen, chondrocytes induced an endochondral calcification. Concurrent with the onset of calcification throughout Meckel's cartilage matrix, periodic banded thick collagen fibrils and matrix vesicles were observed around the chondrocytes. Although most of the chondrocytes prior to grafting were hypertrophic cells, they survived for seven days in the splenic tissue and had well-developed secretory organelles. The cells which were surrounded by calcified matrix were relatively small, spherical, and showed a morphology closely resembling that of osteocytes. These findings suggest that the life span of hypertrophic chondrocytes is influenced by the microenvironment of 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 cells differentiate to form osteodentine.

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.

Organized mechanoreceptors in mucosae and vermilion borders of rat lower lips were studied by light and electron microscopy. Cholinesterase histochemistry was applied to whole-mount preparations of mucosae for the light and electron microscopic identification of mechanoreceptors. Three types of lamellated corpuscles (a simple corpuscle; a coiled, simple corpuscle; and a Meissner-like corpuscle), and a unique, organized, bush-like assembly of free nerve terminals were identified. The simple corpuscles were found exclusively in the vestibular mucosa facing the incisor teeth. In contrast, bush-like endings were confined to the vestibular mucosa and to the lateral, eminent mucosa that faced the diastema. Furthermore, coiled simple corpuscles and Meissner-like corpuscles were localized in the boundary zone between the vestibular and lateral eminent mucosae and in the vermilion border. From a functional viewpoint, it is of interest that different areas of the rat lip contain different morphological patterns of mechanoreceptors.

Lower first molar from 18-day-old mouse embryos were used. After one-day cultivation, both inner epithelia and mesenchymal cells were regarded as undifferentiated appearance and then matrix deposition did not see intermatrix space. The existence of basal lamina was confirmed, but the fine fibrils perpendicular to basal lamina was not recognized in this stage.However, these fine fibrils had a tendency to increase together with the time of cultivation and the trasformation of inner enamel epithelia. Ultimately, it would seem that fine fibrils were included in matrix formation.Basal lamina and fine fibrils seem to be a prerequisite for epithelial-mesenchymal interaction. Similary, the distance between basal lamina and mesenchymal cells suggest to play a significant role for epithelial-mesenchymal interaction.

Tooth germ derived from the mandible of newborn mice was used in this study. The enamel organs were dissected and attached directly to coverslips without using the plasma clot. By this method it was possible, for the first time as far as is known, to attain preparations in which the major part of the outgrowth from the explant consisted purely of epithelial-like cells which probably were ameloblasts. Abundant desmosomes and tonofilaments were confirmed by transmission electron microscopy.Under the scanning electron microscope this outgrowth of epithelial cells revealed characteristic intercellular connections which were divided into four types. 1) In the part nearest to the explant, cell boundaries were indicated with a large number of short microvilli. 2) In the zone beyond the first type, the intercellular connection was represented by bridge-like processes firmly combined with each other. 3) In a still further zone, the tips of bridge-like processes were free from the cell connection and extended to the adjacent cells with the advance of cell migration. 4) In the most peripheral part of the explant, the cytoplasmic processes were capable of further expansion. These cells were soon separated from the adjacent cells to migrate as free cells.

We examined electron-microscopically and histochemically the ultrastructural features of the intraodontoblastic collagen fibrils of the mouse. These collagen fibrils were most common in secreting odontoblasts (pre-odontoblasts) of the maturating stages. In such cells they were most numerous at the peripheral zone of the Golgi apparatus, and were sometimes seen in odontoblastic processes. Intraodontoblastic collagen fibrils also had morphological variations including a banded structure enclosed by limiting membranes of vacuoles, fusion with primary lysosomes, and an electron-dense material covering with a structure that was not banded. Study of acid phosphatase activity showed that these structural changes were caused by the degradation of intraodontoblastic collagen fibrils by lysosomes. The results of studies of the permeation of lanthanum nitrate and the alkaline phosphatase reaction showed that these collagen fibrils were separate from the extracellular matrix and that there was no phagocytosis of the odontoblasts.

Mandibular first molars from 17-day-old mouse embryos were cultured for 2 and 4 days in alpha-MEM supplemented with 10% newborn bovine serum containing 0.02% ethanol or 0.1, 1.0 or 10 ng/ml 1,25-(OH)2D3. After embedding, every 6th section was stained and mitotic features of inner dental epithelium (IDE) and dental papilla cells (DP) were counted under a light microscope. On the 2nd day, no significant differences were observed in the IDE and DP mitotic indexes among the control, ethanol, and the three vitamin D groups. On the 4th day, the indexes for all groups decreased. But the IDE indexes for all vitamin D groups were significantly different from those for the control (P less than 0.01) and ethanol (P less than 0.05) groups, whereas the DP index was significant (P less than 0.05) only in the 10 ng/ml vitamin D group. The present results suggest that vitamin D3 may have an influence on cell proliferation of IDE and DP in mouse embryonic molars in vitro.

The influence of the sensory nerve fibers on the differentiation of the Merkel cell was examined in the denervated labial mucosa of adult rabbits. Part of the lower labial mucosa was excised following mental nerve resection. Twenty-one and 50 days later, the regenerated mucosa was examined by electron microscopy with special reference to the distribution and maturation of Merkel cells, and compared with the normal and the denervated intact mucosa. A number of immature Merkel cells appeared in the denervated and regenerated epithelium by 21st day after the operation. The distribution density of the Merkel cells in a unit area of the mucosa did not change much but the percentage of the mature Merkel cells increased significantly until 50 days after the operation. The mature Merkel cells in the denervated and regenerated mucosa showed a normal ultrastructure, though their orientation and location were not uniform, shifting more superficially than the cells in the normal mucosa. In the controlateral intact epithelium of the denervated labial mucosa, no substantial decrease in the population density of Merkel cells was recognized, though the desquamation of Merkel cells was observed. It was conspicuous that the percentage of immature Merkel cells to the total number of the Merkel cells was significantly increased after the denervation in the intact labial mucosa. This study suggests that Merkel cells differentiate independently of sensory nerve fibers, but the latter are requisite to maintain the former in a uniform, basal disposition in the epithelium.

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 expiants 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 expiants.

Examination of barbels of the carp (Cyprinus carpio) revealed cells showing the characteristics of Merkel cells. Some ultrastructural features of these cells suggest a secretory function.

Merkel cells in the lower labial mucosa of adult rabbits were studied electron microscopically, 9, 21, 28, and 50 days after resection of the mental nerves. By day 9, nerve fibers were completely retracted from the epithelial layer of the mucosa. On and after day 21, Merkel cells were located not only in the basal layer but also in the prickle or more superficial cell layers. The ultrastructure of the migrating Merkel cells was unchanged, both as to the amount and location of the specific cored granules in the cytoplasm, until the cells reached the granular cell layer. The position of the migrating Merkel cells differed from cell to cell, and migration continued for at least 50 days. A remarkably large number of immature Merkel cells was observed in the basal and suprabasal cell layers of the denervated epithelium even by day 50. Therefore, the possibility of the reproduction of Merkel cells exists. The migrating Merkel cells, as well as the keratinocytes in the same cell layer, had degenerated drastically in the parakeratinized cell layer. This seems to indicate that the Merkel cells belong to the line of keratinocytes.