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A previous study suggested that fibroblast growth factor (FGF) signaling plays an important role in dentin formation during tooth development. In this study, to examine dentin formation after tooth eruption involving secondary and tertiary dentin, we analyzed the expression patterns and expressing cells of Fgfr1, -2c, and -3c in mouse maxillary first molars (M1). Since it is difficult to recover the mRNAs from mineralized tissues, we tested methods for extraction after fixation and decalcification of teeth. We successfully obtained consistent results with quantitative real-time PCR (qPCR) using β-actin transcripts for validation. qPCR for Dentin sialo phosphoprotein (Dspp), Fgfr1, -2c, and -3c transcripts was performed on mice at ages of 2–20 weeks. The results showed that the highest expression levels of Dspp and Fgfr2c occurred at 2 weeks old followed by lower expression levels after 4 weeks old. However, the expression levels of Fgfr1 and Fgfr3c were constant throughout the experimental period. By in situ hybridization, Dspp, Fgfr1, and Fgfr3c transcripts were detected in odontoblasts at ages of 2 and 4 weeks. In addition, Dspp and Fgfr1 transcripts were detected in odontoblasts facing reactionary dentin at 8 weeks old. These results suggest that FGF-FGFR signaling might be involved in the regulation of odontoblasts even after tooth eruption, including secondary and tertiary dentin formation. Moreover, our modified method for extracting mRNA from mineralized tissues after fixation and decalcification successfully produced consistent results.

Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.

The transition muscle between the palatopharyngeus (PP) and the superior constrictor of the pharynx (SCP) encircles the pharyngeal isthmus from behind and is designated as the palatopharyngeal sphincter (PPS). The PPS is inferred to play important roles for velopharyngeal closure, but its existence remains controversial and its roles have been regarded as being played by the SCP. The present study aimed to clarify the anatomical status and functional implications of the PPS. Macroscopic and microscopic examinations were performed on 39 and 4 cadavers, respectively. In the former, the bilateral PPSs and their adjacent structures were exposed from outside and/or inside. In the latter, the velums embedded in paraffin were cut into frontal or sagittal sections and alternately processed with HE and Azan stains. The PPS originated from the nasal aspect of the lateral half of the palatine aponeurosis and the inferior margin of the medial pterygoid plate and was distinguishable from the PP descending in and along the palatopharyngeal arch and the cranialmost portion of the SCP in its origin. It passed dorsally on the lateral side of the levator veli palatini and traversed around the salpingopharyngeal fold running longitudinally. It then entered below the SCP and ran toward the pharyngeal raphe with SCP muscle fibers intermingled. The PPS is a muscle distinct from the SCP. Its contraction produces Passavant’s ridge and conceivably enhances the efficiency of velopharyngeal closure by pressing the salpingopharyngeal fold and the musculus uvulae ridge against the velum.

Glycogen, the largest cytosolic macromolecule, is soluble because of intricate construction generating perfect hydrophilic-surfaced spheres. Little is known about neuronal glycogen function and metabolism, though progress is accruing through the neurodegenerative epilepsy Lafora disease (LD) proteins laforin and malin. Neurons in LD exhibit Lafora bodies (LBs), large accumulations of malconstructed insoluble glycogen (polyglucosans). We demonstrated that the laforin-malin complex reduces LBs and protects neuronal cells against endoplasmic reticulum stress-induced apoptosis. We now show that stress induces polyglucosan formation in normal neurons in culture and in the brain. This is mediated by increased glucose-6-phosphate allosterically hyperactivating muscle glycogen synthase (GS1) and is followed by activation of the glycogen digesting enzyme glycogen phosphorylase. In the absence of laforin, stress-induced polyglucosans are undigested and accumulate into massive LBs, and in laforin-deficient mice, stress drastically accelerates LB accumulation and LD. The mechanism through which laforin-malin mediates polyglucosan degradation remains unclear but involves GS1 dephosphorylation by laforin. Our work uncovers the presence of rapid polyglucosan metabolism as part of the normal physiology of neuroprotection. We propose that deficiency in the degradative phase of this metabolism, leading to LB accumulation and resultant seizure predisposition and neurodegeneration, underlies LD.

Cement lines represent mineralized, extracellular matrix interfacial boundaries at which bone resorption by osteoclasts is followed by bone deposition by osteoblasts. To determine the contribution of cement lines to bone quality, the osteopetrotic c-Src mouse model-where cement lines accumulate and persist as a result of defective osteoclastic resorption-was used to investigate age-related changes in structural and mechanical properties of bone having long-lasting cement lines. Cement lines of osteopetrotic bones in c-Src knockout mice progressively mineralized with age up to the level that the entire matrix of cement lines was lost by EDTA decalcification. While it was anticipated that suppressed and abnormal remodeling, together with the accumulation of cement line interfaces, would lead to defective bone quality with advancing age of the mutant mice, unexpectedly, three-point bending tests of the long bones of 1-year-old c-Src-deficient mice indicated significantly elevated strength relative to age-matched wild-type bones despite the presence of numerous de novo microcracks. Among these microcracks in the c-Src bones, there was no sign of preferential propagation or arrest of microcracks along the cement lines in either fractured or nonfractured bones of old c-Src mice. These data indicate that cement lines are not the site of a potential internal failure of bone strength in aged c-Src osteopetrotic mice and that abundant and long-lasting cement lines in these osteopetrotic bones appear to have no negative impacts on the mechanical properties of this low-turnover bone despite their progressive hypermineralization (and thus potential brittleness) with age.