Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 µg/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

Human Dental Pulp Cells Differentiate toward Neuronal Cells and Promote Neuroregeneration in Adult Organotypic Hippocampal Slices In Vitro.

The adult mammalian central nerve system has fundamental difficulties regarding effective neuroregeneration. The aim of this study is to investigate whether human dental pulp cells (DPCs) can promote neuroregeneration by (i) being differentiated toward neuronal cells and/or (ii) stimulating local neurogenesis in the adult hippocampus. Using immunostaining, we demonstrated that adult human dental pulp contains multipotent DPCs, including STRO-1, CD146 and P75-positive stem cells. DPC-formed spheroids were able to differentiate into neuronal, vascular, osteogenic and cartilaginous lineages under osteogenic induction. However, under neuronal inductive conditions, cells in the DPC-formed spheroids differentiated toward neuronal rather than other lineages. Electrophysiological study showed that these cells consistently exhibit the capacity to produce action potentials, suggesting that they have a functional feature in neuronal cells. We further co-cultivated DPCs with adult mouse hippocampal slices on matrigel in vitro. Immunostaining and presto blue assay showed that DPCs were able to stimulate the growth of neuronal cells (especially neurons) in both the CA1 zone and the edges of the hippocampal slices. Brain-derived neurotrophic factor (BDNF), was expressed in co-cultivated DPCs. In conclusion, our data demonstrated that DPCs are well-suited to differentiate into the neuronal lineage. They are able to stimulate neurogenesis in the adult mouse hippocampus through neurotrophic support in vitro.

Cell death, cavitation and spontaneous multi-differentiation of dental pulp stem cells-derived spheroids in vitro: a journey to survival and organogenesis.

BACKGROUND INFORMATION: During embryonic development, cell death transforms the solid embryonic cell mass into a hollow structure (cavitation), which allows the surviving cells to differentiate into varied tissues and organs around the cavity. This process can be partly reproduced with embryonic stem cells. However, it is unknown if adult stem cell masses have the same ability to cavitate and then differentiate into organs. In this study, we assessed the capacity of human dental pulp stem cells (DPSCs)-derived spheroids to mimic the above-mentioned cavitation and spontaneous differentiation in vitro. RESULTS: DPSCs were able to form large-sized spheroids on matrigel in osteogenic medium. Inside the spheroids, cells in the centre showed positive stain to stem cell markers, alkaline phosphatase and STRO-1. Hypoxia and massive cell death were observed in the core of the spheroids. Cavities were formed when the spheroids were cultivated in the osteogenic medium for about 14 days. After 28 days of cultivation, the surviving cells around the cavity spontaneously differentiated into neuronal (28.8%), vascular (33.3%), osteogenic (46.7%) and cartilaginous (72.0%) tissues under the osteogenic medium only. In contrast, when DPSCs-formed cell sheets were folded into giant-sized lumps and cultivated under the same conditions, the folded cell sheets became an entire lumenal structure and failed to differentiate into neuronal, osteogenic and cartilaginous cells. Marker analysis showed that cavitation-related molecules BMP7 and FGF3 expressed on the wall of the cavity in the spheroids, suggesting that the cavitation was functional, whereas cavitation-related molecules were absent in the folded cell sheets. CONCLUSIONS: DPSC-derived spheroids can mimic the developmental process of cell survival, cavitation and spontaneous multi-differentiation on matrigel under certain conditions. This work allows for functional studies to investigate organ regeneration with human DPSCs in vitro.

Observation of the temporal crest canal in the mandibular ramus by cone beam computed tomography and macroscopic study.

PURPOSE: The mandibular ramus is regarded as a relatively safe zone for a sagittal splitting osteotomy or for harvesting bone during implant treatment. The only important anatomical structure is the mandibular canal. The mandible has some anatomical variants that need to be recognized, such as a bifid mandibular canal, a retromolar canal, and rarely a temporal crest canal (TCC). In this study, cadaver mandibles were used to evaluate the TCC in the mandibular ramus using cone beam computed tomography (CBCT). METHODS: Altogether, 90 sites on 48 mandibles from Japanese cadavers were examined in this study. The CBCT volumetric images were acquired for areas of 79 mm[Formula: see text] 71 mm. Three-dimensional observation of the images was undertaken to estimate the frequency, position of the orifices, and canal continuity. The cadaver mandibles in which the TCCs were observed were dissected from the inner surface to confirm the contents. RESULTS: Five TCCs (5.6 %) were observed in 90 observation areas. At least one TCC was confirmed in four (8.3 %) of 48 mandibles. Two types of TCC were recognized. Dissection revealed that they contained neurovascular bundles. CONCLUSION: Three-dimensional diagnosis is essential prior to surgical procedures in the mandibular ramus because unexpected blood vessels may be present that may cause bleeding or complications during the surgery.

Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: role of PHLDA1 as an apoptosis suppressor.

Hydrogen sulfide (H2S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H2S is produced by bacteria colonizing digestive organs, including the oral cavity. H2S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H2S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H2S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H2S. The susceptibility to H2S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H2S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H2S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.