Bone is composed of a protein matrix containing embedded crystals of hydroxyapatite, a form of calcium phosphate. Mineralization, the incorporation of the mineral into the matrix, is an essential step for the bone to achieve the hardness and rigidity that enable the skeleton to resist gravitational and mechanical loading. Mineralization of vertebrates is generally classified into two types, physiological and pathological mineralization. Physiological mineralization is necessary for the formation of hard tissues and for their appropriate functions. In the human body, this type of mineralization occurs only in bone and teeth. On the other hand, pathological or ectopic mineralization of soft tissues is regarded as an important problem to be solved. Before presenting readers with the most recent biology of physiological and pathological mineralization, we will briefly review some basic knowledge on biomineralization from the historical aspect.

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Bone formation, including matrix mineralization by calcium phosphate crystals, is essentially organized under the control of osteoblasts. Great efforts have been conducted to establish the linkage between bio-crystals and synthetic crystals to determine the physicochemical properties of these crystals. Octacalcium phosphate (OCP) has been proposed to be a precursor of hydroxyapatite (HA), the prototype in bone and tooth apatite crystals, and suggested to have a role in apatite crystal development. Studies using synthetic OCP provided evidence that this mineral phase stimulates bone formation more than synthetic HA if implanted in bone defects during conversion from OCP to HA. Furthermore, the enhancement of bone formation was usually accompanied by osteoclastic resorption of OCP. In vitro studies showed that OCP is capable of stimulating mouse bone marrow stromal cells to differentiate into osteoblastic cells and osteoclast formation in co-culture by enhancing the expression of the receptor activator of NF-kB ligand (RANKL) in osteoblastic cells. Although the precise mechanism of the precursor precipitation and subsequent apatite formation has not been fully elucidated in normal bone mineralization, it is likely that the OCP precursor plays a role in the stimulation of bone-forming cells through interaction with the surrounding tissue environment if this mineral phase is involved in bone formation.

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Some bone diseases, such as osteoporosis and periodontitis, result from an imbalance in bone remodeling characterized by excessive bone resorption relative to bone formation. Two different pharmacological approaches can be used to treat such diseases: anti-resorptive agents that inhibit osteoclastic bone resorption, and anabolic agents that stimulate osteoblastic bone formation. Anti-resorptive agents, such as bisphosphonates and calcitonin, are currently available for the treatment of osteoporosis; however, these anti-resorptive agents are unlikely to have an anabolic effect, and the need for anabolic agents aimed at increasing bone mass has recently been a matter for concern. Natural compounds could provide the basis for developing anabolic agents. A wide variety of natural compounds has recently been found to stimulate osteoblastic bone formation. This review describes the effects of natural compounds on osteoblast proliferation, differentiation, and bone formation in vitro and in vivo.

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Runx2-deficient mice completely lack osteoblasts and bone formation. Overexpression of Runx2 in osteoblasts inhibits osteoblast maturation, leading to immature bone, which is easily resorbed, while the expression of dominant-negative Runx2 in osteoblasts increases the volume of trabecular bone by promoting the formation of mature bone, which is relatively resistant to bone resorption. Thus, RUNX2 directs mesenchymal stem cells to the osteoblast lineage, and supplies immature osteoblasts to form immature bone, but RUNX2 has to be down-regulated for terminal differentiation into mature osteoblasts, which form mature bone. Thus, the level of RUNX2 determines the maturational stage of osteoblasts, bone maturity, and the bone turnover rate. Endogenous RUNX2 protein is detected in the nuclei of preodontoblasts, immature odontoblasts, and mesenchymal cells in the dental sac at 3 days of age, and transiently detected in ameloblasts in the coronal regions at one week of age. Overexpression of Runx2 in odontoblasts inhibited their terminal differentiation. Further, dentin sialophosphoprotein (Dspp) expression was lost and nestin (NES) expression was markedly decreased, while the expressions of bone gamma-carboxyglutamate (gla) protein 1/osteocalcin (BGLAP), osteopontin (SPP1), and dentin matrix protein 1 (DMP1) were up-regulated in the odontoblasts, resulting in the formation of a bone structure. These findings indicate that RUNX2 is able to induce the transdifferentiation of odontoblasts into osteoblasts, and that RUNX2 expression has to be down-regulated during odontoblast differentiation to achieve full differentiation for dentinogenesis.

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Vascular calcification is a significant component of atherosclerosis and has important clinical implications, such as being a marker of the atherosclerotic burden and predictor of future coronary events. There are two pathophysiological processes involved in the development of vascular calcification: apoptosis and phenotypic changes to chondrocytes or osteoblasts (chondro-/osteogenic differentiation). Vascular calcification associated with phenotypic changes of vascular smooth muscle cells are facilitated by inflammation-induced ALP expression or an inorganic phosphate signaling pathway via Pit-1. These mechanisms may operate to develop vascular calcification associated with atherosclerosis and chronic kidney disease (CKD). Moreover, the functional derangement of inhibitory molecules (inhibitors of vascular calcification) such as MGP, fetuin-A, and OPG may be involved in the progression of vascular calcification under various pathological conditions.

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Bone morphogenetic proteins (BMPs) were originally identified via their unique activity in demineralized bone matrix-they induce heterotopic bone formation in skeletal muscle. BMP activity is transduced by two types of BMP-specific transmembrane serine/threonine kinase receptors and downstream transcription factors, known as Smads. Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by progressive heterotopic bone formation in skeletal muscle tissue. In patients with FOP, heterozygous mutations involving single amino-acid substitutions have been identified in the BMP type Ireceptor, ALK2. These mutations in ALK2 are scattered in an intracellular domain containing the kinase and GS domains. The mutant receptors constitutively activate downstream intracellular signaling, even in the absence of BMPs. In FOP, muscle injury induces acute heterotopic bone formation. Smad1 and Smad5 are up-regulated during muscle regeneration and induce osteoblastic differentiation of myoblasts in cooperation with mutant ALK2. Some types of BMP receptor inhibitors block the signal transduction induced by mutant ALK2 receptors in vitro and in vivo. Taken together, this work demonstrates that BMP signaling induces heterotopic bone formation in skeletal muscle. Inhibitors of BMP receptors may aid in the establishment of novel treatments to prevent heterotopic bone formation in FOP.

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Candida spp. are an opportunistic pathogen causing serious local and systemic infections, especially in immuno-compromised hosts such as the elderly and HIV-positive patients. Hinokitiol C₁₀H₁₂O₂ (β-thujaplicin) is a component of the essential oils isolated from Cupressaceae and shows antibacterial activities for various bacteria. The aim of this study was to demonstrate the preventive effects of hinokitiol on the adherence of seven species of Candida to oral epithelial cells and to establish a safe and useful method for oral hygiene. A short-time treatment (30 min) with 0.25 mM hinokitiol showed 30-70% inhibition of adherence of Candida spp. to oral epithelial cells, inhibited about 11% biofilm formation, and did not inhibit the cell growth of Candida spp. Furthermore, short treatment with 0.25 mM hinokitiol was a safe method for oral hygiene against Candida infection because it did not inhibit the cell growth of commensal bacteria, such as oral streptococci existing in normal flora, or damage the epithelial cells. However, long-time treatment and a high concentration of hinokitiol demonstrated both the adherence inhibition of Candida and damage to commensal bacteria and epithelial cells. Our data suggest an appropriate procedure to apply hinokitiol that may be beneficial for the prevention of opportunistic pathogens such as Candida spp. in the oral cavity. The clinical and daily use of hinokitiol under an appropriate procedure may be a preventive and realistic therapy for Candida infection in immune-compromised hosts.

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We examined the effect of dexamethasone on parathyroid hormone-related protein (PTHrP) mRNA expression and PTHrP production in HSC-3 cells, a human oral squamous carcinoma cell line. Dexamethasone suppressed PTHrP mRNA expression and PTHrP production in a dose-dependent manner. A time course study demonstrated a significant decrease in PTHrP mRNA from as early as 6 h after the initiation of exposure to 10^-7 M dexamethasone, and the suppressive effect was stronger at 24 h. Dexamethasone-induced suppression of PTHrP mRNA expression and PTHrP production was abolished in the presence of RU486, a glucocorticoid receptor antagonist. Dexamethasone decreased the transcriptional activity of the PTHrP promoter. These findings show that dexamethasone acts through binding to functional glucocorticoid receptors and down-regulates PTHrP gene expression via a transcriptional mechanism in HSC-3 cells.

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