Enamel matrix derivative promote primary human pulp cell differentiation and mineralization.

Enamel matrix derivative (EMD) has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5-50 µg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10⁻8 M dexamethasone (DEX) for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 µg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel), and the dentinogenic markers dentin sialophosphoprotein (DSSP) and dentin matrix acidic phosphoprotein 1 (DMP1), as well as the osteogenic markers osteocalcin (OC, BGLAP) and collagen type 1 (COL1A1). Whereas, only EMD had effect on alkaline phosphatase (ALP) mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant proteins (MCP-1) in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation.

Vitamin K2 Modulates Vitamin D-Induced Mechanical Properties of Human 3D Bone Spheroids In Vitro.

Rotational culture promotes primary human osteoblasts (hOBs) to form three-dimensional (3D) multicellular spheroids with bone tissue-like structure without any scaffolding material. Cell-based bone models enable us to investigate the effect of different agents on the mechanical strength of bone. Given that low dietary intake of both vitamin D and K is negatively associated with fracture risk, we aimed to assess the effect of these vitamins in this system. Osteospheres of hOBs were generated with menaquinone-4 (MK-4; 10µM) and 25-hydroxyvitamin D3 [25(OH)D3; 0.01µM], alone and in combination, or without vitamins. The mechanical properties were tested by nanoindentation using a flat-punch compression method, and the mineralized extracellular bone matrix was characterized by microscopy. The in vitro response of hOBs to MK-4 and 25(OH)D3 was further evaluated in two-dimensional (2D) cultures and in the 3D bone constructs applying gene expression analysis and multiplex immunoassays. Mechanical testing revealed that 25(OH)D3 induced a stiffer and MK-4 a softer or more flexible osteosphere compared with control. Combined vitamin conditions induced the same flexibility as MK-4 alone. Enhanced levels of periostin (p < 0.001) and altered distribution of collagen type I (COL-1) were found in osteospheres supplemented with MK-4. In contrast, 25(OH)D3 reduced COL-1, both at the mRNA and protein levels, increased alkaline phosphatase, and stimulated mineral deposition in the osteospheres. With the two vitamins in combination, enhanced gene expression of periostin and COL-1 was seen, as well as extended osteoid formation into the central region and increased mineral deposition all over the area. Moreover, we observed enhanced levels of osteocalcin in 2D and osteopontin in 3D cultures exposed to 25(OH)D3 alone and combined with MK-4. In conclusion, the two vitamins seem to affect bone mechanical properties differently: vitamin D enhancing stiffness and K2 conveying flexibility to bone. These effects may translate to increased fracture resistance in vivo. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Fluoride reduces the expression of enamel proteins and cytokines in an ameloblast-derived cell line.

OBJECTIVE: To investigate the effects of two different fluoride concentrations on the expression of enamel proteins, alkaline phosphatase (ALP), cytokines and interleukins by an ameloblast-derived cell line. METHODS: Murine ameloblast-derived cells (LS-8), mouse odontogenic epithelia, were exposed to 1 or 5ppm sodium fluoride (NaF) (0.46 and 2.25ppm F, respectively) for 1, 3 and 7 days. The effect of NaF on the mRNA expression of enamel proteins was quantified; the secretion of cytokines, and interleukins, and the alkaline phosphatase (ALP) activity, into the cell culture medium was measured and compared to untreated controls. The effect on cell growth after 1- and 3-days in culture was measured using BrdU incorporation. RESULTS: Fluoride at 2.25ppm reduced mRNA expression of the structural enamel matrix proteins amelogenin (amel), ameloblastin (ambn), enamelin (enam), and the enamel protease matrix metallopeptidase-20 (MMP-20). Similarly several vascularisation factors (vascular endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and interferon inducible protein 10 (IP-10), was also reduced by 2.25ppm fluoride. ALP activity and proliferation were stimulated by 0.46ppm fluoride but inhibited by 2.25ppm fluoride. CONCLUSIONS: These results indicate that fluoride may impact on the expression of structural enamel proteins and the protease responsible for processing these proteins during the secretory stage of amelogenesis and go some way to explaining the mineralization defect that characterises fluorotic enamel.