1α,25‐Dihydroxyvitamin D₃ (1,25(OH)₂D₃), the active metabolite of vitamin D (Vit D), increases intestinal absorption of calcium and phosphate, maintaining a correct balance of bone remodeling. Vit D has an anabolic effect on the skeletal system and is key in promoting osteoblastic differentiation of human Mesenchymal Stem Cells (hMSCs) from bone marrow. MSCs can be also isolated from the immature form of the tooth, the dental bud: Dental Bud Stem Cells (DBSCs) are adult stem cells that can effectively undergo osteoblastic differentiation. In this work we investigated the effect of Vit D on DBSCs differentiation into osteoblasts. Our data demonstrate that DBSCs, cultured in an opportune osteogenic medium, differentiate into osteoblast‐like cells; Vit D treatment stimulates their osteoblastic features, increasing the expression of typical markers of osteoblastogenesis like RUNX2 and Collagen I (Coll I) and, in a more important way, determining a higher production of mineralized matrix nodules.

Vitamin D (Vit D) by means of its biological active form, 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), has a protective effect on the skeleton by acting on calcium homeostasis and bone formation. Furthermore, Vit D has a direct effect on mesenchymal stem cells (MSCs) in stimulating their osteogenic differentiation. In this work, we present for the first time the effect of 1,25(OH)₂D₃ on MSC adhesion. Considering that cell adhesion to the substrate is fundamental for cell commitment and differentiation, we focused on the expression of α_Vβ₃ integrin, which has a key role in the commitment of MSCs to the osteoblastic lineage. Our data indicate that Vit D increases α_Vβ₃ integrin expression inducing the formation of focal adhesions (FAs). Moreover, we assayed MSC commitment in the presence of the extracellular matrix (ECM) glycoprotein fibronectin (FN), which is able to favor cell adhesion on surfaces and also to induce osteopontin (OPN) expression: this suggests that Vit D and FN synergize in supporting cell adhesion. Taken together, our findings provide evidence that Vit D can promote osteogenic differentiation of MSCs through the modulation of α_Vβ₃ integrin expression and its subcellular organization, thus favoring binding with the matrix protein (FN).

Objective: Three-dimensional (3D) printing technology represents a novel method for manufacturing aligners. The aim of the present study was to assess the in-vitro cytotoxicity of 3D-printed aligners using different post-polymerisation conditions. Materials: Aligners were printed using the same 3D-print resin (TC-85DAC, Graphy, Seoul, Korea) and printer (AccuFab-L4D, Shining 3D Tech. Co., Hangzhou, China), followed by different post-curing procedures. Six aligners were post-polymerised for 14 min using the Tera Harz Cure and a nitrogen generator curing machine (THC2, Graphy, Seoul, Korea) (P1). A further six aligners were post-cured for 30 min on each side using the Form Cure machine (FormLabs Inc, Somerville, USA) (P2). The aligners were cut into smaller specimens (2 mm×2 mm) and sterilised at 121°C. The specimens were placed in 96-well plates containing Dulbecco’s Modified Eagle’s Medium (DMEM) at 37° for 7 or 14 days. The viability of MC3T3E-1 pre-osteoblasts cultured with DMEM was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The optical density of each cell culture was measured to assess cell viability, following which the data were statistically analysed using two-way and one-way ANOVA (α = 0.05). Results: The comparison of cytotoxicity revealed statistically significant differences between post-curing procedures and MTT timings ( P < 0.001). After 7 and 14 days, the cell viability of P2 was significantly reduced compared to P1 and the control groups ( P < 0.001), while P1 showed no significant differences compared to the controls. Overall, P2 post-curing exhibited moderate cytotoxicity, while P1 post-polymerisation was highly biocompatible. Conclusions: Different post-curing procedures may affect the in-vitro cytotoxicity of 3D-printed aligners. Clinicians should adhere to the manufacturer’s recommendations when using 3D-print resin.

The myokine irisin, well known for its anabolic effect on bone tissue, has been demonstrated to positively act on osteoblastic differentiation processes in vitro. Mesenchymal stem cells (MSCs) have captured great attention in precision medicine and translational research for several decades due to their differentiation capacity, potent immunomodulatory properties, and their ability to be easily cultured and manipulated. Dental bud stem cells (DBSCs) are MSCs, isolated from dental tissues, that can effectively undergo osteoblastic differentiation. In this study, we analyzed, for the first time, the effects of irisin on DBSC osteogenic differentiation in vitro. Our results indicated that DBSCs were responsive to irisin, showed an enhanced expression of osteocalcin (OCN), a late marker of osteoblast differentiation, and displayed a greater mineral matrix deposition. These findings lead to deepening the mechanism of action of this promising molecule, as part of osteoblastogenesis process. Considering the in vivo studies of the effects of irisin on skeleton, irisin could improve bone tissue metabolism in MSC regenerative procedures.

Teeth include mesenchymal stem cells (MSCs), which are multipotent cells that promote tooth growth and repair. Dental tissues, specifically the dental pulp and the dental bud, constitute a relevant source of multipotent stem cells, known as dental-derived stem cells (d-DSCs): dental pulp stem cells (DPSCs) and dental bud stem cells (DBSCs). Cell treatment with bone-associated factors and stimulation with small molecule compounds are, among the available methods, the ones who show excellent advantages promoting stem cell differentiation and osteogenesis. Recently, attention has been paid to studies on natural and non-natural compounds. Many fruits, vegetables, and some drugs contain molecules that can enhance MSC osteogenic differentiation and therefore bone formation. The purpose of this review is to examine research work over the past 10 years that has investigated two different types of MSCs from dental tissues that are attractive targets for bone tissue engineering: DPSCs and DBSCs. The reconstruction of bone defects, in fact, is still a challenge and therefore more research is needed; the articles reviewed are meant to identify compounds useful to stimulate d-DSC proliferation and osteogenic differentiation. We only consider the results of the research which is encouraging, assuming that the mentioned compounds are of some importance for bone regeneration.

Mesenchymal stem cells (MSCs) have been identified in human dental tissues. Dental pulp stem cells (DPSCs) were classified within MSC family, are multipotent, can be isolated from adult teeth, and have been shown to differentiate, under particular conditions, into various cell types including osteoblasts. In this work, we investigated how the differentiation process of DPSCs toward osteoblasts is controlled. Recent literature data attributed to the nuclear receptor related 1 (NURR1), a still unclarified role in osteoblast differentiation, while NURR1 is primarily involved in dopaminergic neuron differentiation and activity. Thus, in order to verify if NURR1 had a role in DPSC osteoblastic differentiation, we silenced it during all the processes and compared the expression of the main osteoblastic markers with control cultures. Our results showed that the inhibition of NURR1 significantly increased the expression of osteoblast markers collagen I and alkaline phosphatase. Further, in long time cultures, the mineral matrix deposition was strongly enhanced in NURR1-silenced cultures. These results suggest that NURR1 plays a key role in switching DPSC differentiation toward osteoblasts rather than neuronal or even other cell lines. In conclusion, DPSCs represent a source of osteoblast-like cells and downregulation of NURR1 strongly prompted their differentiation toward the osteoblastogenesis process.