Biomineralization is regulated by inorganic pyrophosphate (PPi), 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 PPi, 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 PPi) resulted in increased cementum formation, suggesting PPi 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 ( Enpp1asj/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; Enpp1asj/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 Enpp1asj/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 PPi 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 PPi conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory bone destruction in which tumor necrosis factor alpha (TNF-α) plays a key role. Bovine lactoferrin (bLF) is a multifunctional protein with anti-inflammatory and immunomodulatory properties. This study aimed to clarify the inhibitory effects of bLF on the pathological progression of RA. The mannan-induced arthritis model in SKG mice (genetic RA model) was used. Orally applied liposomal bLF (LbLF) markedly reduced ankle joint swelling and bone destruction. Histologically, pannus formation and osteoclastic bone destruction were prevented in the LbLF-treated animals. Moreover, orally administered LbLF improved the balance between Th17 cells and regulatory T cells isolated from the spleen of mannan-treated SKG mice. In an in vitro study, the anti-inflammatory effects of bLF on TNF-α-induced TNF-α production and downstream signaling pathways were analyzed in human synovial fibroblasts from RA patients (RASFs). bLF suppressed TNF-α production from RASFs by inhibiting the nuclear factor kappa B and mitogen-activated protein kinase pathways. The intracellular accumulation of bLF in RASFs increased in an applied bLF dose-dependent manner. Knockdown of the lipoprotein receptor-related protein-1 (LRP1) siRNA gene reduced bLF expression in RASFs, indicating that exogenously applied bLF was mainly internalized through LRP-1. Immunoprecipitated proteins with anti-TNF receptor-associated factor 2 (TRAF2; an adapter protein/ubiquitin ligase) included bLF, indicating that bLF binds directly to the TRAF2-TRADD-RIP complex. This indicates that LbLF may effectively prevent the pathological progression of RA by suppressing TNF-α production by binding to the TRAF2-TRADD-RIP complex from the RASFs in the pannus. Therefore, supplemental administration of LbLF may have a beneficial effect on preventive/therapeutic reagents for RA.

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 ( IbspKAE/KAE) mice and OCCM.30 murine ( IbspKAE) cementoblasts. The RGD domain was not critical for acellular or cellular cementum formation in IbspKAE/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 IbspKAE/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.