Immunohistochemical localization of tenascin-C in rat periodontal ligament with reference to alveolar bone remodeling.

We investigated the immunohistochemical localization of tenascin-C in 8-week-old rat periodontal ligaments. Tenascin-C immunoreactivity was detected in zones along with cementum and alveolar bone, and more intensely on the resorption surface of alveolar bone than on the formation surface. On the resorbing surface, tenascin-C immunoreactivity was detected in Howship's lacunae without osteoclasts, and in the interfibrous space of the periodontal ligaments, indicating that this molecule works as an adhesion molecule between bone and fibers of periodontal ligaments. Upon experimental tooth movement by inserting elastic bands (Waldo method), the physiological resorption surface of alveolar bone under compressive force showed enhanced bone resorption and enhanced tenascin-C immunoreactivity. However, on the physiological bone formation surface under compressive force, bone resorption was seen only occasionally, and no enhanced tenascin-C immunoreactivity was noted. In an experiment involving excessive occlusal loading to rat molars, transient bone resorption occurred within interradicular septa, but no enhanced tenascin-C immunoreactivity was seen in the periodontal ligaments. These results indicate that tenascin-C works effectively on the bone resorbing surface of physiological alveolar bone remodeling sites, rather than on the non-physiological transient bone resorbing surface. Fibronectin immunoreactivity was distributed evenly in the periodontal ligaments under experimental conditions. Co-localization of tenascin-C and fibronectin immunoreactivity was observed in many regions, but mutually exclusive expression patterns were also seen in some regions, indicating that fibronectin might not be directly involved in alveolar bone remodeling, but may play a role via interaction with tenascin-C.

CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in the dental stem cell niche.

Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche.

An Immunohistochemical Study of Matrix Components in Early-Stage Vascular Canals Within Mandibular Condylar Cartilage in Midterm Human Fetuses.

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15-16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle-shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin-C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin-C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate-resistant acid phosphatase (TRAP)-positive cells were present. Meanwhile, TRAP-positive, multinucleated chondroclasts and flattened, osteoblast-like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles.

Distribution of matrix proteins in perichondrium and periosteum during the incorporation of Meckel's cartilage into ossifying mandible in midterm human fetuses: an immunohistochemical study.

Immunohistochemical localization of versican and tenascin-C were performed; the periosteum of ossifying mandible and the perichondrium of Meckel's cartilage, of vertebral cartilage, and of mandibular condylar cartilage were examined in midterm human fetuses. Versican immunoreactivity was restricted and evident only in perichondrium of Meckel's cartilage and vertebral cartilage; conversely, tenascin-C immunoreactivity was only evident in periosteum. Therefore, versican and tenascin-C can be used as molecular markers for human fetal perichondrium and fetal periosteum, respectively. Meckel's cartilage underwent endochondral ossification when it was incorporated into the ossifying mandible at the deciduous lateral incisor region. Versican immunoreactivity in the perichondrium gradually became weak toward the anterior primary bone marrow. Tenascin-C immunoreactivity in the primary bone marrow was also weak, but tenascin-C positive areas did not overlap with versican-positive areas; therefore, degradation of the perichondrium probably progressed slowly. Meanwhile, versican-positive perichondrium and tenascin-C-positive periosteum around the bone collar in vertebral cartilage were clearly discriminated. Therefore, the degradation of Meckel's cartilage perichondrium during endochondral ossification occurred at a different rate than did degradation of vertebral cartilage perichondrium. Additionally, the perichondrium of mandibular condylar cartilage showed tenascin-C immunoreactivity, but not versican immunoreactivity. That perichondrium of mandibular condylar cartilage has immunoreactivity characteristic of other periosteum tissues may indicate that this cartilage is actually distinct from primary cartilage and representative of secondary cartilage.

Short-term serum-free culture reveals that inhibition of Gsk3ß induces the tumor-like growth of mouse embryonic stem cells.

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3ß. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3ß, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3ß in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3ß governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.

Functional analysis of CTRP3/cartducin in Meckel's cartilage and developing condylar cartilage in the fetal mouse mandible.

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage.

Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions.

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.

Expression, localisation and synthesis of versican by the enamel organ of developing mouse molar tooth germ: an in vivo and in vitro study.

OBJECTIVE: Versican is a large, aggregating chondroitin sulphate proteoglycan. In dental tissue, versican expression occurs primarily in mesenchymal tissue but rarely in epithelial tissue. We investigated the expression, localisation and synthesis of versican in the enamel organ of the developing tooth germ. DESIGN: To elucidate versican localisation in vivo, in situ hybridisation and immunohistochemistry were conducted in foetal ICR mice at E11.5-E18.5. Epithelium and mesenchyme from the lower first molars at E16.0 were enzymatically separated and versican mRNA expression was investigated by semi-quantitative RT-PCR. Organ culture of the separated samples combined with metabolic labelling with [(35)S], followed by gel filtration, was performed to analyse secreted proteoglycans. RESULTS: Versican mRNA was first expressed in the thickened dental epithelium at E12.0 and continued to be expressed in the enamel organ until the bell stage. Versican immunostaining was detected in the stellate reticulum areas from the bud stage to the apposition stage. The enamel organ at E16.0 expressed versican mRNA at a level comparable to that in dental mesenchyme. Furthermore, when compared to dental mesenchyme, about 1/2-3/4 of the [(35)S]-labelled versican-like large proteoglycan was synthesised and released into tissue explants by the enamel organ. CONCLUSIONS: The dental epithelium of developing tooth germ is able to synthesise significant amounts of versican.

An in situ hybridization study of Runx2, Osterix, and Sox9 in the anlagen of mouse mandibular condylar cartilage in the early stages of embryogenesis.

Mandibular condylar cartilage is the best-studied mammalian secondary cartilage, differing from primary cartilage in that it originates from alkaline phosphatase-positive progenitor cells. We previously demonstrated that three transcription factors related to bone and cartilage formation, namely Runx2, Osterix and Sox9, are simultaneously expressed in the anlage of mandibular condylar cartilage (condylar anlage) at embryonic day (E)14. In this study, expression of these transcription factors was investigated in the anlagen of mandibular bone (mandibular anlagen) from E11.0 to 14.0. Runx2 mRNA was first expressed in the mandibular anlage at E11.5. Osterix mRNA was first expressed at E12.0, and showed a different expression pattern from that of Runx2 from E12.5 to E14.0, confirming that Osterix acts downstream of Runx2. Sox9 mRNA was expressed in Meckel's cartilage and its anlagen throughout the experimental period, but not clearly in the mandibular anlagen until E13.0. At E13.5, the condylar anlage was morphologically identified at the posterior end of the mandibular anlage, and enhanced Sox9 mRNA expression was detected here. At this stage, Runx2 and Osterix mRNA were simultaneously detected in the condylar anlage. These results indicate that the Sox9 mRNA-expressing condylar anlage is derived from Runx2/Osterix mRNA-expressing mandibular anlage, and that upregulation of Sox9 in this region acts as a trigger for subsequent condylar cartilage formation.

An immunohistochemical and ultrastructural study of the pericellular matrix of uneroded hypertrophic chondrocytes in the mandibular condyle of aged c-src-deficient mice.

OBJECTIVE: Previous studies indicate that hypertrophic chondrocytes can transdifferentiate or dedifferentiate and redifferentiate into bone cells during the endochondral bone formation. Mandibular condyle in aged c-src-deficient mice has incremental line-like striations consisting of cartilaginous and non-cartilaginous layers, and the former contains intact hypertrophic chondrocytes in uneroded lacunae. The purpose of this study is to determine the phenotype changes of uneroded hypertrophic chondrocytes. DESIGN: Immunohistochemical and ultrastructural examinations of the pericellular matrix of hypertrophic chondrocytes in the upper, middle, and lower regions of the mandibular condyle were conducted in aged c-src-deficient mice, using several antibodies of cartilage/bone marker proteins. RESULTS: Co-localisation of aggrecan, type I collagen, and dentin matrix protein-1 (DMP-1) or matrix extracellular phosphoprotein (MEPE) was detected in the pericellular matrix of the middle region. Ultrastructurally, granular substances in the pericellular matrix of the middle region were the remains of upper region chondrocytes, which were mixed with thick collagen fibrils. In the lower region, the width of the pericellular matrix and the amount of collagen fibrils were increased. Versican, type I collagen, DMP-1, and MEPE were detected in the osteocyte lacunae. Additionally, DMP-1 and MEPE were detected in the pericellular matrix of uneroded hypertrophic chondrocytes located in the lower, peripheral region of the mandibular condyle in younger c-src-deficient mice, but not in the aged wild-type mice. CONCLUSIONS: These results indicate that long-term survived, uneroded hypertrophic chondrocytes, at least in a part, acquire osteocytic characteristics.

Cessation of Fgf10 signaling, resulting in a defective dental epithelial stem cell compartment, leads to the transition from crown to root formation.

Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as ;apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of inner enamel epithelium growth and the more-active proliferation of the outer enamel epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.

Cartducin, a paralog of Acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes.

We previously reported that CORS26 gene, isolated from C3H10T1/2 cells treated with transforming growth factor-beta1, was predominantly expressed in cartilage. Because the gene product is a kind of secretory protein produced by cartilage tissue, we named it "cartducin". Cartducin shares a similar modular organization to adipocyte-derived hormone, adiponectin. In this study, we investigated cartducin function during chondrogenesis and cartilage development. In situ hybridization analysis showed that cartducin transcripts were restricted to the proliferating chondrocytes in the growth plate cartilage. Whole-mount in situ hybridization revealed that the first significant induction of cartducin expression occurred in the sclerotome, which contains a chondrogenic cell lineage between days 9.5 and 10.5 postcoitus (p.c.) during mouse embryogenesis. Chondrogenic differentiation by combined treatment with bone morphogenetic protein-2 and insulin induced cartducin expression along with type II and IX collagen expression in chondrogenic progenitor N1511 cells. To elucidate the direct action of cartducin on the cells, recombinant cartducin protein was expressed in and purified from Escherichia coli. The recombinant cartducin potentially forms homo-oligomers and promoted the proliferation of chondrogenic progenitor N1511 cells, and chondrocytic HCS-2/8 cells in a dose-dependent manner. On the other hand, cartducin did not affect the production of sulfated glycosaminoglycan (sGAG) in these cells. These findings indicate that cartducin is a novel growth factor and plays important roles in regulating both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes.

Stratum intermedium lineage diverges from ameloblast lineage via Notch signaling.

The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the inner enamel epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.

Reuptake of extracellular amelogenin by dental epithelial cells results in increased levels of amelogenin mRNA through enhanced mRNA stabilization.

Amelogenin is an extracellular matrix protein secreted by ameloblasts and is a major component of enamel matrix. Recently, in addition to their role in enamel formation, the biological activity of enamel proteins in the process of cell differentiation has recently become widely appreciated. In this study, we examined the biological activity of amelogenin on ameloblast differentiation. Recombinant mouse amelogenin (rm-amelogenin) enhanced the expression of endogenous amelogenin mRNA in a cultured dental epithelial cell line (HAT-7), despite a lack of increased amelogenin promoter activity. To solve this discrepancy, we analyzed the effects of rm-amelogenin on the stability of amelogenin mRNA. The half-life of amelogenin mRNA is extremely short, but in the presence of rm-amelogenin its half-life was extended three times longer than the control. Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells. It follows from our results that exogenous amelogenin increases amelogenin mRNA levels through stabilization of mRNA in the cytoplasm of HAT-7 cells. Here we speculated that during differentiation, dental epithelial cells utilize a unique mechanism for increasing the production of amelogenin, the reuptake of secreted amelogenin.