Phospholipids have been identified in enamel and dentin. Before demineralization, a group of phospholipids extracted by lipid solvents was associated with cell membranes and is therefore closely related to cell growth and intracellular regulations. After demineralization, a second group of phospholipids, associated with the extracellular matrix, was extracted; this group is probably linked to the mineralized phase. Using imidazole-osmium tetroxide fixation of rat incisors, we stained cellular unsaturated fatty acids, so that we could visualize the membrane domains, coated pits, and endocytic inclusions. Filipin, a probe for cholesterol, varied in density along the plasma membrane of secretory ameloblasts, and allowed us to visualize membrane remnants inside the forming enamel. With respect to phospholipids located in the extracellular matrix, the malachite-green-glutaraldehyde (MGA) method or iodoplatinate (IP) reaction retains and visualizes enamel and dentin phospholipids. In predentin, aggregates appearing as granules and filaments, or liposome-like structures, were located in the spaces between collagen fibrils. In dentin, organic envelopes coating the crystals, also named “crystal-ghost” structures, outlined groups of collagen fibrils. Histochemical data provided evidence that phospholipids are co-distributed or interact with proteoglycans. Radioautography after IP reaction established that [³H] choline was detected in dentin as early as 30 min after the intravenous injection of the labeled precursor, before any labeling was seen in odontoblasts and predentin. This suggests that blood-serum-labeled phospholipids pass between odontoblasts, cross the distal permeable junctional complex, and diffuse in dentin prior to any cellular uptake and phospholipid synthesis. Pharmacologically and genetically induced pathology also supports the suggestion that phospholipids play an important role in the formation and mineralization of dental tissues.

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Recently, pepsin-digested collagen (so-called atelocollagen) has been used for tissue engineering as one of the most useful scaffolds for bone formation. Its excellent biocompatibility, due to its biological characteristics such, as absorbable properties and low antigenicity, has elevated collagen to become a primary resource in medical applications. Platelet-rich plasma (PRP) is an autologous vehicle that delivers a highly concentrated dose of platelets containing osteopromotive factors, such as platelet-derived growth factor (PDGF), and transforming growth factor beta-1 and beta-2 (TGF-β1 and TGF-β2). These growth factors are thought to help to induce local mesenchymal and epithelial cells to migrate, divide, and increase collagen synthesis. In this paper, the characteristics of bovine collagen, PRP and animal studies using them for bone formation are reviewed.

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The present study examined the effect of melatonin on prostaglandin E₂-induced pro-inflammatory cytokine production in human gingival fibroblasts. Human gingival fibroblasts were cultured in melatonin in the presence/absence of prostaglandin E₂ for 1h. The levels of mRNA for IL-1β, IL-8 and osteoprotegerin/osteoclastogenesis inhibitory factor were quantitatively evaluated by reverse transcriptase-PCR using a LightCycler method. While melatonin did not significantly alter the levels of osteoprotegerin/osteoclastogenesis inhibitory factor mRNA, either in the presence or absence of prostaglandin E₂, it markedly decreased the levels of prostaglandin E₂-stimulated IL-1β and IL-8 mRNA. These results suggest that melatonin has the potency to reduce the production of pro-inflammatory cytokines from prostaglandin E₂-activated human gingival fibroblasts in the inflamed periodontal tissues and may act as a new potent drug for the treatment for periodontal disease.

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This study examined the modulating effect of PGE₂ on LPS-induced IL-8 production by human gingival fibroblasts in vitro. The expression of IL-8 mRNA, after stimulation with PGE₂, was determined by RT-PCR. IL-8 production by HGFs was shown to be related to the time of incubation and the dose of LPS. A slight increase in IL-8 levels was observed when cells were exposed to PGE₂ alone compared to controls. When HGFs were pre-stimulated with PGE₂ and then exposed to LPS, IL-8 production was increased 2-3 fold compared to non-treated cells. Herbimycin A completely abolished the secretion of IL-8. These results indicate that PGE₂ can modulate cytokine secretion from HGFs and increase IL-8 production from cells exposed to LPS. IL-8 production induced by LPS was dependent on the tyrosine phosphorylation pathway.

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Odontogenesis and osteogenesis are two distinct yet closely related processes that share common signalling pathways including regulatory control by Cbfa1. Although Cbfa1 is well-recognized as the master regulator of osteoblastic differentiation, little is known of its precise role during odontogenesis. We sought to clarify this by examining morphogenesis of teeth and matrix protein gene expression in cbfa1-knock-out mice at ed 17.5 and day 0 of birth, and comparing it with presumptive mandible bone-forming site. Results demonstrated morphological and functional differentiation differences between these two processes: incisor tooth germ showed morphological and functional differentiation of odontoblasts with expression of osteopontin and osteocalcin whereas the presumptive mandible bone-forming site showed lack of osteoblastic differentiation, and no expression of these two matrix proteins. Stage-specific and cytodifferentiation differences were also observed between incisor and molar tooth germs: incisor tooth germ progressed to the bell stage with odontoblast and ameloblast differentiation, and expression of tooth-specific proteins, amelogenin and dentine sialophosphoprotein, whereas molar tooth germ showed marurational arrest at bud to cap stage. Present findings suggest that 1. The transcription factor Cbfa-1 is associated with morphogenesis of teeth and matrix protein gene expression. 2. Compared to the incisor tooth germ, the molar tooth germ is more strongly subjected to control by Cbfa1, and 3. In Cbfa1-knockout mice, the odontoblast-like cells in the incisor and the spindle cells in the presumptive mandible forming region showed different patterns of gene expression of matrix proteins common to teeth and bones.

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Messenger RNA analysis of bone marrow is a potential examination to diagnose the quality of maxillofacial bone. Lysyl hydroxylases (LHs) are responsible for collagen cross-linking and determine bone quality. To investigate mRNA expression patterns of LHs in bone marrow with potentially different bone quality, we quantified the mRNA expression of LH isoforms (LH1, LH2, and LH3), type I collagen (COLI), and alkaline phosphatase (ALP) in the mandibular and femoral bone marrows of three senescence-accelerated mouse (SAM) strains; the osteoporosis model SAMP6, mandibular osteoarthritis model SAMP8, and their control SAMR1. Total RNAs directly isolated from the bone marrows were used for reverse transcription and the products were then used to quantify mRNA expressions of the molecules using a real-time PCR assay. The expression levels of all the molecules varied in the mandibular and femoral bone marrows. The expression levels of COLI, ALP, and LH2 were higher in the mandibular than in the femoral bone marrow. In the mandibular bone marrow, the expression levels of COLI were significantly different among the three SAM strains. In both bone marrows, an association was observed between COLI and LH1 expressions, and between ALP and LH2 expressions. This study indicates that gene expression patterns of LH1 and LH2 in bone marrows are associated with those of developmental osteoblast markers and that the expression patterns of LH2 and the markers are different in mandibular and femoral bone marrows. Quantitative mRNA analysis of LHs in combination with developmental osteoblast markers in bone marrow may be useful to investigate their relationship with the quality of maxillofacial bone.

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