The aim of this paper was to determine whether the interaction between IGF, IGFBP, and VN modulates the functions of porcine EOE cells. Enamel organs from 6-month-old porcine third molars were dissociated into single epithelial cells and subcultured on culture dishes pretreated with VN, IGF-I, and IGFBP-3 (IGF-IGFBP-VN complex). The subcultured EOE cells retained their capacity for ameloblast-related gene expression, as shown by semiquantitative reverse transcription-polymerase chain reaction. Amelogenin expression was detected in the subcultured EOE cells by immunostaining. The subcultured EOE cells were then seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells and transplanted into athymic rats. After 4 weeks, enamel-dentin-like complex structures were present in the implanted constructs. These results show that EOE cells cultured on IGF-IGFBP-VN complex differentiated into ameloblasts-like cells that were able to secrete amelogenin proteins and form enamel-like tissues in vivo. Functional assays demonstrated that the IGF/IGFBP/VN complex significantly enhanced porcine EOE cell proliferation and tissue forming capacity for enamel. This is the first study to demonstrate a functional role of the IGF-IGFBP-VN complex in EOE cells. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.

Cell scaffold-based tooth engineering research was started by 2000 at Forsyth Institute corroborated with Dr. Vacanti's team at Massachusetts General Hospital. The first work was published in 2002 in Journal of Dental Research, in which we particularly focused on cells from postnatal tooth because of its clinical application. However, making a functional tooth from postnatal cells is still ways away. Alternatively, we formulated a partial replacement of the tooth by engineering the root of the tooth. Here, we describe a new technique in which the root of the third molar is used to replace missing teeth.

The tissue in the palatal region can be divided into the hard and the soft palates, each having a specialized function such as occlusion, speech, or swallowing. Therefore, an understanding of the mechanism of palatogenesis in relation to the function of each region is important. However, in comparison with the hard palate, there is still a lack of information about the mechanisms of soft palate development. In this study, the authors investigated the contribution of cranial neural crest (CNC) cells to development of both hard and soft palates. They also demonstrated a unique pattern of periostin expression during soft palate development, which was closely related to that of collagen type I (Col I) in palatine aponeurosis. Furthermore, organ culture analysis showed that exogenous transforming growth factor-β (TGF-β) induced the expression of both periostin and Col I. These novel patterns of expression in the extracellular matrix (ECM) induced by CNC cells suggest that these cells may help to determine the character of both the hard and soft palates through ECM induction. TGF-β signaling appears to be one of the mediators of Col I and periostin expression in the formation of functional structures during soft palate development.

Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis.

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

Osteoblasts and adipocytes originate from common mesenchymal progenitor cells and although a number of compounds can induce osteoblastic and adipogenic differentiation from progenitor cells, the underlying mechanisms have not been elucidated. The present study examined the synergistic effects of dexamethasone (Dex) and bone morphogenetic protein (BMP)-2 on the differentiation of clonal mesenchymal progenitor cells isolated from rat calvaria into osteoblasts and adipocytes, as well as the effects of the timing of treatment. Cells were cultured for various periods of time in the presence of Dex and/or BMP-2. When cells were treated with Dex+BMP-2 during the early phase of differentiation, they differentiated into adipocytes. However, when cells were treated with Dex+BMP-2 during the late phase of differentiation, a synergistic effect on in vitro matrix mineralization was observed. To examine differences between the early and late phases of differentiation, ALP activity was measured in the presence of BMP-2. ALP activity increased markedly on Day 9, corresponding to the onset of the synergistic effect of Dex. Dex treatment inhibited osterix (OSX) expression in cells committed to adipogenic differentiation, but not in cells committed to osteogenic differentiation following BMP-2 treatment. The isoform2 OSX promoter region was found to be involved in the effects of Dex on cells during the early phase of differentiation. Furthermore, cells stably expressing OSX (isoform2) formed mineralized nodules even though they had been treated with Dex+BMP-2 during the early phase of differentiation. It appears that Dex modulates osteogenesis and adipogenesis in mesenchymal stem cells by regulating OSX expression.

Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.

Recently, the possibility of tooth tissue engineering has been reported. Although there are a number of available materials, information about scaffolds for tooth tissue engineering is still limited. To improve the manageability of tooth tissue engineering, the effect of scaffolds on in vivo tooth regeneration was evaluated. Collagen and fibrin were selected for this study based on the biocompatibility to dental papilla-derived cells and the results were compared with those of polyglycolic acid (PGA) fiber and beta-tricalcium phosphate (beta-TCP) porous block, which are commonly used for tooth, dentin and bone tissue engineering. Isolated porcine tooth germ-derived cells were seeded onto one of those scaffolds and transplanted to the back of nude mice. Tooth bud-like structures were observed more frequently in collagen and fibrin gels than on PGA or beta-TCP, while the amount of hard tissue formation was less. The results showed that collagen and fibrin gel support the initial regeneration process of tooth buds possibly due to their ability to support the growth of epithelial and mesenchymal cells. On the other hand, maturation of tooth buds was difficult in fibrin and collagen gels, which may require other factors.(c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

OBJECTIVES: Bacterial metabolites demineralize dental hard tissues, and soluble factors lead to tertiary dentinogenesis in the area of the dentin-pulp complex. However, it is unclear whether the oral bacteria are directly involved in the differentiation of dental pulp cells. In this study, we evaluated the effect of oral bacterial extracts on cellular differentiation in human dental pulp-derived cells (hDPC). STUDY DESIGN: The hDPC were obtained from third molar teeth, and the cells were subcultured. The sonicated extracts were obtained from Porphyromonas gingivalis (gram-negative) and Streptococcus mutans (gram-positive). The effect of bacterial extracts on cellular growth and differentiation in hDPC were tested. RESULTS: Alkaline phosphatase activity and bone sialoprotein (BSP) gene expression were increased in hDPC exposed to low concentrations of both sonicated extracts, whereas the activity was decreased upon exposure to high concentrations of sonicated extracts from P. gingivalis. CONCLUSION: This is the first evidence that oral bacteria have a positive effect on cellular differentiation in hPDC.

The effect of scaffold shape on dentin regeneration is not well understood. In this study, porous hydroxyapatite/beta-tricalcium phosphate (HAp/beta-TCP), powdered HAp/beta-TCP, and polyglycolic acid (PGA) fiber mesh were used as scaffolds and transplanted with cultured porcine dental pulp-derived cells into the backs of nude mice. Samples were harvested after 6 weeks. Newly-formed hard tissue was observed in all transplants. When porous HAp/beta-TCP was used, dentin-like hard tissue was observed on the inner wall with minimum cell inclusions and odontoblast-like cells were aligned adjacent to the hard tissue. When HAp/beta-TCP powders or PGA were used, bone-like hard tissues showed cell inclusions and cell alignment was not observed. Hard tissue from the HAp/beta-TCP block group was positive for type I collagen, osteonectin, bone sialoprotein and dentin sialoprotein (DSP), which are markers for dentin. This result was confirmed by in situ hybridization with a dsp probe. Only the aligned cells were positive with an antisense probe. On the other hand, hard tissue from other scaffolds showed incomplete expression of both bone and dentin markers and they were negative for osteonectin and DSP. These results suggest that scaffold shape affects the type of tissue regenerated by dental pulp-derived cells.