The oral cavity contains a variety of ecological niches with very different environmental conditions that shape biofilm structure and composition. The space between the periodontal tissue and the tooth surface supports a unique anaerobic microenvironment that is bathed in the nutrient-rich gingival crevicular fluid (GCF). During the development of periodontitis, this environment changes and clinical findings reported a sustained level of calcium ion concentration in the GCF collected from the periodontal pockets of periodontitis patients. Here, we report the effect of calcium ion supplementation on human oral microbial biofilm formation and community composition employing an established SHI medium-based in vitro model system. Saliva-derived human microbial biofilms cultured in calcium-supplemented SHI medium (SHICa) exhibited a significant dose-dependent increase in biomass and metabolic activity. The effect of SHICa medium on the microbial community composition was evaluated by 16S rRNA gene sequencing using saliva-derived microbial biofilms from healthy donors and periodontitis subjects. In this study, intracellular microbial genomic DNA (iDNA) and extracellular DNA (eDNA) were analyzed separately at the genus level. Calcium supplementation of SHI medium had a differential impact on iDNA and eDNA in the biofilms derived from healthy individuals compared to those from periodontitis subjects. In particular, the genus-level composition of the eDNA portion was distinct between the different biofilms. This study demonstrated the effect of calcium in a unique microenvironment on oral microbial complex supporting the dynamic transformation and biofilm formation.

Small-molecule compounds strongly affecting osteogenesis can form the basis of effective therapeutic strategies in bone regenerative medicine. A cell-based high-throughput screening system might be a powerful tool for identifying osteoblast-targeting candidates; however, this approach is generally limited with using only one molecule as a cell-based sensor that does not always reflect the activation of the osteogenic phenotype. In the present study, we used the MC3T3-E1 cell line stably transfected with the green fluorescent protein (GFP) reporter gene driven by a fragment of type I collagen promoter (Col-1a1GFP-MC3T3-E1) to evaluate a double-screening system to identify osteogenic inducible compounds using a combination of a cell-based reporter assay and detection of alkaline phosphatase (ALP) activity. Col-1a1GFP-MC3T3-E1 cells were cultured in an osteogenic induction medium after library screening of 1280 pharmacologically active compounds (Lopack¹²⁸⁰). After 7 days, GFP fluorescence was measured using a microplate reader. After 14 days of osteogenic induction, the cells were stained with ALP. Library screening using the Col-1a1/GFP reporter and ALP staining assay detected three candidates with significant osteogenic induction ability. Furthermore, leflunomide, one of the three detected candidates, significantly promoted new bone formation in vivo. Therefore, this double-screening method could identify candidates for osteogenesis-targeting compounds more reliably than conventional methods.

Circadian rhythms, which synchronize cellular and organismal activities with the Earth’s 24-hour light-dark cycle, are controlled by clock genes. These genes not only regulate metabolic and physiological processes but also influence osteogenesis. Despite extensive research on the genetic control of circadian rhythms, little is known about the mechanisms by which mechanical factors in the extracellular environment affect these rhythms during the osteogenic differentiation of induced pluripotent stem cells (iPSCs). Shaking culture, which promotes the formation of three-dimensional organoid-like constructs from iPSC embryoid bodies (iPSC-EBs), introduces distinct biomechanical forces compared with static adherent culture. This raises the question of how these forces affect the circadian gene expression during osteogenic differentiation. In this study, we investigated the effects of shaking cultures on the circadian rhythm of key clock genes (Clock, Bmal1, and Npas2) in iPSC-EBs. In the adherent culture, iPSC-EBs displayed rhythmic oscillations of the clock genes, which were attenuated in the shaking culture. RNA-seq analysis revealed that the yes-associated protein (YAP)-transcriptional enhanced associate domain (TEAD) transcriptional cascade was activated in the shaking culture. Further investigations using assay for transposase-accessible chromatin with sequencing and chromatin immunoprecipitation assays identified Fbxl3 as a direct target of this transcriptional cascade. Fbxl3 upregulation in the shaking culture enhanced the degradation of CRY proteins, which are essential components of the circadian feedback loop, thereby suppressing clock gene oscillations. In addition, treatment with verteporfin, a YAP-TEAD inhibitor, restored circadian gene oscillations and increased the expression of osteogenic markers in shaking culture. These findings highlight a novel mechanistic link between biomechanical cues and circadian regulation and offer potential insights for optimizing tissue engineering strategies in regenerative medicine.

Hypophosphatasia (HPP) is a rare inherited disorder caused by inactivating variants in the ALPL gene, resulting in defective mineralization of bone and teeth. Dental manifestations include early exfoliation of fully rooted primary teeth as a result of cementum dysplasia, premature loss of permanent teeth, and hypomineralization of enamel, dentin, and jawbone. However, genotype-dependent dental phenotypes and severity-based treatment strategies remain poorly defined. The aim of this study was to generate knock-in mouse models harboring clinically relevant ALPL variants and to characterize their dental and periodontal phenotypes in relation to serum tissue-nonspecific alkaline phosphatase (TNAP) activity. We analyzed mice that were compound heterozygous for a null variant and a hypomorphic variant (Alplc.1559delT/p.F327L) and mice that were heterozygous for a dominant-negative variant (Alpl+/p.R184W). Dental and alveolar phenotypes were assessed using micro-CT, histological analyses, and periodontal ligament traction experiments. Alplc.1559delT/p.F327L mice exhibited reduced serum TNAP levels, mild skeletal abnormalities, and severe dental defects, including hypomineralization of enamel, dentin, and cementum, decreased alveolar BMD, and an increased distance from the cemento–enamel junction to the alveolar bone crest. In contrast, Alpl+/p.R184W mice showed isolated dental abnormalities characterized by dentin and cementum hypomineralization and decreased alveolar bone BMD without overt skeletal involvement. Periodontal tissue fragility was increased in both models. These knock-in mouse models recapitulate genotype-dependent dental manifestations of HPP and indicate that dental phenotypes are influenced not only by circulating TNAP levels but also by the functional effects of individual ALPL variants. This platform provides a valuable framework for elucidating the mechanisms underlying genotype-dependent dental manifestations and for developing dental treatment strategies tailored to disease severity.