Occlusal Trauma and Bisphosphonate-Related Osteonecrosis of the Jaw in Mice.

Osteonecrosis of the jaw (ONJ) is a serious adverse event that is associated with antiresorptive agents, and it manifests as bone exposure in the maxillofacial region. Previous clinical reports suggest that mechanical trauma would trigger ONJ in a manner that is similar to tooth extractions. To the best of our knowledge, there have been few detailed pathophysiological investigations of the mechanisms by which occlusal/mechanical trauma influences ONJ. Here, we developed a novel mouse model that exhibits ONJ following experimental hyperocclusion and nitrogen-containing bisphosphonate (N-BP) treatment. This in vivo model exhibited ONJ in alveolar bone, particularly in the mandible. Moreover, the experimental hyperocclusion induced remarkable alveolar bone resorption in both mouse mandible and maxilla, whereas N-BP treatment completely prevented alveolar bone resorption. In this study, we also modeled trauma by exposing clumps of mesenchymal stem cells (MSCs)/extracellular matrix complex to hydrostatic pressure in combination with N-BP. Hydrostatic pressure loading induced lactate dehydrogenase (LDH) release by calcified cell clumps that were differentiated from MSCs; this LDH release was enhanced by N-BP priming. These in vivo and in vitro models may contribute further insights into the effect of excessive mechanical loading on ONJ onset in patients with occlusal trauma.

The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.

Lignin-facilitated growth of Ag/CuNPs on surface-activated polyacryloamidoxime nanofibers for superior antibacterial activity with improved biocompatibility.

INTRODUCTION: Nanofibers are one of the role-playing innovations of nanotechnology. Their high surface-to-volume ratio allows them to be actively functionalized with a wide range of materials for a variety of applications. The functionalization of nanofibers with different metal nanoparticles (NPs) has been studied widely to fabricate antibacterial substrates to battle antibiotic-resistant bacteria. However, metal NPs show cytotoxicity to living cells, thereby restricting their application in biomedicine. OBJECTIVES: To minimize the cytotoxicity of NPs, biomacromolecule lignin was employed as both a reducing and capping agent to green synthesize silver (Ag) and copper (Cu) NPs on the surface of highly activated polyacryloamidoxime nanofibers. The activation of polyacrylonitrile (PAN) nanofibers via amidoximation was employed for enhanced loading of NPs to achieve superior antibacterial activity. METHODOLOGY: At first, electrospun PAN nanofibers (PANNM) were activated to produce polyacryloamidoxime nanofibers (AO-PANNM) by immersing PANNM in a solution of Hydroxylamine hydrochloride (HH) and Na2CO3 under controlled conditions. Later, Ag and Cu ions were loaded by immersing AO-PANNM in different molar concentrations of AgNO3 and CuSO4 solutions in a stepwise manner. The reduction of Ag and Cu ions into NPs to fabricate bimetal-coated PANNM (BM-PANNM) was carried out via alkali lignin at 37 °C for 3 h in a shaking incubator with ultrasonication every 1 h. RESULTS: AO-APNNM and BM-PANNM hold their nano-morphology except for some changes in fiber orientation. XRD analysis demonstrated the formation of Ag and CuNPs as evident from their respective spectral band. Maximum 8.46 ± 0.14 wt% and 0.98 ± 0.04 wt% Ag and Cu species were loaded on AO-PANNM, respectively as revealed by ICP spectrometric analysis. The hydrophobic PANNM turned into super hydrophilic, having WCA of 14 ± 3.32° after amidoximation which further reduced to 0° for BM-PANNM. However, the swelling ratio of PANNM reduced from 13.19 ± 0.18 g/g to 3.72 ± 0.20 g/g for AO-PANNM. Even at the third cycle test against S. aureus strains, 0.1Ag/Cu-PANNM, 0.3Ag/Cu-PANNM, and 0.5Ag/Cu-PANNM displayed bacterial reduction of 71.3 ± 1.64 %, 75.2 ± 1.91 %, and 77.24 ± 1.25 %, respectively. On 3rd cycle test against E. coli, above 82 % bacterial reduction was noticed for all BM-PANNM. Amidoximation increased COS-7 cell viability up to 82 %. The cell viability of 0.1Ag/Cu-PANNM, 0.3Ag/Cu-PANNM, and 0.5Ag/Cu-PANNM was found to be ∼68 %, ∼62, and 54 %, respectively. In LDH assay, almost no release of LDH was detected, suggesting the compatibility of the cell membrane in contact with BM-PANNM. The improved biocompatibility of BM-PANNM even at higher loading (%) of NPs must be ascribed to the controlled release of metal species in the early stage, antioxidant, and biocompatible lignin capping of NPs. CONCLUSIONS: BM-PANNM displayed superior antibacterial activity against E. coli and S. aureus bacterial strains and acceptable biocompatibility of COS-7 cells even at higher loading (%) of Ag/CuNPs. Our findings suggest that BM-PANNM can be used as a potential antibacterial wound dressing and other antibacterial applications where sustained antibacterial activity is needed.

Synthesized bioactive lignin nanoparticles/polycaprolactone nanofibers: A novel nanobiocomposite for bone tissue engineering.

The use of artificial biomaterial with enhanced bioactivity for osteostimulation is a major research concern at present days. In this research, antibacterial and osteostimulative core-shell lignin nanoparticles (LgNP) were synthesized from alkali lignin using tetrahydrofuran (THF) as solvent via a simultaneous pH and solvent shifting technology. Later, LgNP-loaded polycaprolactone (PCL) composite nanofibers were fabricated via the electrospinning technique. The addition of LgNP significantly increased the diameter of the nanofibers, ranging from 400 to 2200 nm. The addition of LgNP reduced the mechanical performance, crystallinity, and porosity of the nanofibers while improving surface wetting and swelling properties of the inherently hydrophobic PCL polymer. The prepared nanofibers showed excellent bactericidal efficacy against major bone infectious Gram-positive Staphylococcus aureus bacterial strains. The incorporation of LgNP imparted superior antioxidant activity and boosted the biodegradation process of the nanofibers. The deposition of biomineral apatite with platelet-like clustered protrusions having a Ca/P ratio of 1.67 was observed while incubating the scaffold in simulated body fluid. Based on the results of the LDH and WST-1 assay, it was demonstrated that the composite nanofibers are non-toxic to pre-osteoblastic cell line (MC3T3-E1) when they are placed in direct contact with the LgNP/PCL scaffold nanofibers. The MC3T3-E1 cells exhibited excellent proliferation and attachment on the prepared composite scaffold via filopodial and lamellipodial expansion with cell-secreted Ca deposition. According to the alkaline phosphatase activity test, LgNP/PCL nanofiber scaffolds significantly improved osteogenic differentiation of MC3T3-E1 cells compared to neat PCL nanofibers. Overall, our findings suggest that LgNP/PCL nanofiber scaffold could be a promising functional biomaterial for bone tissue engineering.

Developmental impairments of craniofacial bone and cartilage in transgenic mice expressing FGF10.

Mutations in a common extracellular domain of fibroblast growth factor receptor (FGFR)-2 isoforms (type IIIb and IIIc) cause craniosynostosis syndrome and chondrodysplasia syndrome. FGF10, a major ligand for FGFR2-IIIb and FGFR1-IIIb, is a key participant in the epithelial-mesenchymal interactions required for morphogenetic events. FGF10 also regulates preadipocyte differentiation and early chondrogenesis in vitro, suggesting that FGF10-FGFR signaling may be involved in craniofacial skeletogenesis in vivo. To test this hypothesis, we used a tet-on doxycycline-inducible transgenic mouse model (FGF10 Tg) to overexpress Fgf10 from embryonic day 12.5. Fgf10 expression was 73.3-fold higher in FGF10 Tg than in wild-type mice. FGF10 Tg mice exhibited craniofacial anomalies, such as a short rostrum and mandible, an underdeveloped (cleft) palate, and no tympanic ring. Opposite effects on chondrogenesis in different anatomical regions were seen, e.g., hyperplasia in the nasal septum and hypoplasia in the mandibular condyle. We found an alternative splicing variant of Fgfr2-IIIb with a predicted translation product lacking the transmembrane domain, and suggesting a soluble form of FGFR2-IIIb (sFGFR2-IIIb), differentially expressed in some of the craniofacial bones and cartilages. Thus, excessive FGF10 may perturb signal transduction of the FGF-FGFR, leading to craniofacial skeletal abnormalities in FGF10 Tg mice.

Incisor enamel formation is impaired in transgenic rats overexpressing the type III NaPi transporter Slc20a1.

Inorganic phosphate (Pi) is required in many biological processes, including signaling cascades, skeletal development, tooth mineralization, and nucleic acid synthesis. Recently, we showed that Pi transport in osteoblasts, mediated by Slc20a1, a member of the type III sodium-dependent phosphate transporter family, is indispensable for osteoid mineralization in rapidly growing rat bone. In addition, we found that bone mineral density decreased slightly with dysfunction of Pi homeostasis in aged transgenic rats overexpressing mouse Slc20a1 (Slc20a1-Tg). Bone and tooth share certain common molecular features, and thus, we focused on tooth development in Slc20a1-Tg mandibular incisors in order to determine the role of Slc20a1 in tooth mineralization. Around the time of weaning, there were no significant differences in serologic parameters between wild-type and Slc20a1-Tg rats. However, histological analysis showed that Slc20a1-Tg ameloblasts formed clusters in the papillary layer during the maturation stage as early as 4 weeks of age. These pathologies became more severe with age and included the formation of cyst-like or multilayer ameloblast structures, accompanied by a chalky white appearance with abnormal attrition and fracture. Hyperphosphatemia was also observed in aging Slc20a1-Tg rats. Micro-computed tomography and electron probe microanalysis revealed impairments in enamel, such as delayed mineralization and hypomineralization. Our results suggest that enamel formation is sensitive to imbalances in Pit1-mediated cellular function as seen in bone, although these processes are under the control of systemic Pi homeostasis.

Mineralized tissue cells are a principal source of FGF23.

While fibroblast growth factor (FGF) 23 is known as a phosphaturic factor in inherited and/or acquired hypophosphatemic disorders, it also serves an endocrine role in normal phosphate homeostasis. FGF23 acts negatively on the NaPi2a cotransporter and 25-hydroxy D(3)-1 alpha-hydroxylase with a resultant decrease in renal phosphate (Pi) reabsorption, while osteoblasts appear to be a primary source of FGF23 whose expression is counter-upregulated by 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Here we have shown the distribution of FGF23 in normal rat bone and tooth, and its expression profile in fetal rat calvaria (RC) cell cultures. FGF23 mRNA was detectable in multiple fetal and adult tissues but levels were much higher in adult calvaria, femur and incisor, compared to the other tissues tested. Immunoreactive FGF23 was predominantly localized to osteoblasts, cementoblasts, and odontoblasts, with sporadic labeling in some chondrocytes, osteocytes and cementocytes. Notably, osteoclasts were also found to be a possible source of FGF23. Fetal bone and tooth germ cells labeled much less intensely than young adult osteoblasts and odontoblasts. In the RC cell model, FGF23 was expressed during osteoblast development. During matrix mineralization induced by beta-glycerophosphate (beta GP), FGF23 expression was transiently upregulated and then decreased to levels lower than in their non-beta GP-treated counterparts. 1,25(OH)(2)D(3) markedly increased FGF23 expression concomitant with the inhibition of beta GP-induced mineralization. Our data suggest that FGF23 expression in bone is closely correlated with bone formation in vitro and vivo, and points towards an important role(s) for FGF23 in young adult but not fetal mineralized tissues as a systemic factor for Pi homeostasis.