Bone sialoprotein (gene: Ibsp; protein: BSP) is a multifunctional extracellular matrix protein present in bone, cementum, and dentin. Accumulating evidence supports BSP as a key regulator of mineralized tissue formation via evolutionarily conserved functional domains, including a C-terminal integrin-binding Arg-Gly-Asp (RGD) domain implicated in extracellular matrix–cell signaling. Ablation of Ibsp in mice ( Ibsp^−/−) results in impaired bone growth and mineralization and defective osteoclastogenesis, with effects in the craniofacial region including reduced acellular cementum formation, detachment of the periodontal ligament (PDL), alveolar bone hypomineralization, and severe periodontal breakdown. We hypothesized that BSP-RGD plays an important role in cementum and alveolar bone formation and mineralization, as well as periodontal function. This hypothesis was tested by replacing the RGD motif with a nonfunctional Lys-Ala-Glu (KAE) sequence in ( Ibsp^KAE/KAE) mice and OCCM.30 murine ( Ibsp^KAE) cementoblasts. The RGD domain was not critical for acellular or cellular cementum formation in Ibsp^KAE/KAE mice. However, PDL volume and thickness were increased, and significantly more tartrate-resistant acid phosphatase–positive osteoclasts were found on alveolar bone surfaces of Ibsp^KAE/KAE mice versus wild type mice. PDL organization was disrupted as indicated by picrosirius red stain, second harmonic generation imaging, dynamic mechanical analysis, and decreased asporin proteoglycan localization. In vitro studies implicated RGD functions in cell migration, adhesion, and mineralization, and this was confirmed by an ossicle implant model where cells lacking BSP-RGD showed substantial defects as compared with controls. In total, the BSP-RGD domain is implicated in periodontal development, though the scale and scope of changes indicated by in vitro studies indicate that other factors may partially compensate for and reduce the phenotypic severity of mice lacking BSP-RGD in vivo.

Factors regulating the ratio of pyrophosphate (PP_i) to phosphate (P_i) modulate biomineralization. Tissue-nonspecific alkaline phosphatase (TNAP) is a key promineralization enzyme that hydrolyzes the potent mineralization inhibitor PP_i. The goal of this study was to determine whether TNAP could promote periodontal regeneration in bone sialoprotein knockout mice ( Ibsp^−/− mice), which are known to have a periodontal disease phenotype. Delivery of TNAP was accomplished either systemically (through a lentiviral construct expressing a mineral-targeted TNAP-D₁₀ protein) or locally (through addition of recombinant human TNAP to a fenestration defect model). Systemic TNAP-D₁₀ delivered by intramuscular injection at 5 d postnatal (dpn) increased circulating alkaline phosphatase (ALP) levels in Ibsp^−/− mice by 5-fold at 30 dpn, with levels returning to normal by 60 dpn when tissues were evaluated by micro–computed tomography and histology. Local delivery of recombinant human TNAP to fenestration defects in 5-wk-old wild type (WT) and Ibsp^−/− mice did not alter long-term circulating ALP levels, and tissues were evaluated by micro–computed tomography and histology at postoperative day 45. Systemic and local delivery of TNAP significantly increased alveolar bone volume (20% and 37%, respectively) and cementum thickness (3- and 42-fold) in Ibsp^−/− mice, with evidence for periodontal ligament attachment and bone/cementum marker localization. Local delivery significantly increased regenerated cementum and bone in WT mice. Addition of 100-μg/mL bovine intestinal ALP to culture media to increase ALP in vitro increased media P_i concentration, mineralization, and Spp1 and Dmp1 marker gene expression in WT and Ibsp^−/− OCCM.30 cementoblasts. Use of phosphonoformic acid, a nonspecific inhibitor of sodium P_i cotransport, indicated that effects of bovine intestinal ALP on mineralization and marker gene expression were in part through P_i transport. These findings show for the first time through multiple in vivo and in vitro approaches that pharmacologic modulation of P_i/PP_i metabolism can overcome periodontal breakdown and accomplish regeneration.

Biomineralization is regulated by inorganic pyrophosphate (PP_i), a potent physiological inhibitor of hydroxyapatite crystal growth. Progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) act to increase local extracellular levels of PP_i, inhibiting mineralization. The periodontal complex includes 2 mineralized tissues, cementum and alveolar bone (AB), both essential for tooth attachment. Previous studies demonstrated that loss of function of ANK or ENPP1 (reducing PP_i) resulted in increased cementum formation, suggesting PP_i metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of Ank, Enpp1, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in Ank knockout ( Ank KO), Enpp1 mutant ( Enpp1^asj/asj), and double KO (dKO) mice. Genetic ablation of Ank, Enpp1, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 ( Ank KO: 8-fold, 3-fold; Enpp1^asj/asj: 7-fold, 3-fold; dKO: 11-fold, 4-fold, respectively) associated with increased fluorochrome labeling and expression of mineralized tissue markers, dentin matrix protein 1 ( Dmp1/DMP1), osteopontin ( Spp1/OPN), and bone sialoprotein ( Ibsp/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and Ank KO at POD30. No differences were noted in AB volume between genotypes, but osteoblast/osteocyte markers were increased in all KOs, partially mineralized osteoid volume was increased in dKO versus controls at POD15 (3-fold), and mineral density was decreased in Enpp1^asj/asj and dKOs at POD30 (6% and 9%, respectively). Increased numbers of osteoclasts were present in regenerated AB of all KOs versus controls. These preclinical studies suggest PP_i modulation as a potential and novel approach for cementum regeneration, particularly targeting ENPP1 and/or ANK. Differences in cementum and AB regeneration in response to reduced PP_i conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.

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.

Extending life expectancy is a major public health priority. Oral frailty is closely associated with systemic health. However, the association between oral functions and systemic health, particularly nailfold capillary parameters, remains unclear. We analyzed data from 311 participants (mean age: 55.66 ± 12.54 years) to examine the associations between oral function parameters (number of remaining teeth, tongue pressure, and oral mucosal moisture) and nailfold capillary parameters (number, length, width, and turbidity). The results showed that the number of remaining teeth was significantly associated with capillary number. Additionally, oral mucosal moisture was significantly associated with capillary turbidity and width; notably, these two capillary parameters were moderately correlated with each other. These associations did not remain statistically significant after adjusting the P -values using the Holm–Bonferroni correction for multiple comparisons, and should therefore be interpreted as preliminary, hypothesis-generating findings. Overall, these findings may contribute to the growing body of evidence linking oral and systemic health and provide preliminary insights for future research on potential diagnostic applications. Examining oral functions and dental findings in the context of interconnected disease pathways, rather than individual diseases may improve our understanding of the relationship between oral and systemic health.