ABSTRACT
Objective @# To investigate the effect of miR-214 on the osteogenic differentiation of dental follicle cells (DFCs).@*Methods@#Purified DFCs were cultured in vitro by bidirectional differential passage, with the untransfected DFCs as the control group (DFCs group). The expression of miR-214-3p in DFCs was upregulated and downregulated by transfection of miR-214-3p(miR-214 mimics group) or miR-214-3p inhibitors(miR-214 inhibitor group) into DFCs. The expression levels of miR-214, alkaline phosphatase (ALP), osteonectin (OSN) and runt-related transcription factor-2(RUNX-2) were detected by qRT-PCR after 7 days of osteogenesis induction, the protein expression levels of RUNX-2 and β-catenin were detected by western blot, and the formation of mineralized nodules was observed with alizarin red staining after 14 days of osteogenesis induction. @*Results @# Compared with the DFCs group, in the miR-214 mimics group, the expression of miR-214 was upregulated after 7 days of osteogenesis induction. The mRNA expression of ALP, OSN and RUNX-2 in the miR-214 mimics group was lower than that in the DFCs group, but only ALP in the two groups was statistically significant (P > 0.05); the mRNA expression of ALP, OSN and RUNX-2 in the miR-214 inhibitor group was higher than that in the DFCs group, and the difference was statistically significant (P < 0.05). The protein expression of RUNX-2 and β-catenin in the miR-214 mimics group was lower than that in the miR-214 inhibitor group. The number of calcified nodules in the miR-214 mimics group was significantly less than that in the DFCs group, while that in the miR-214 inhibitor group was significantly higher than that in the DFCs group. @*Conclusion@#The upregulation of miR-214 can downregulate the expression of β-catenin, can inhibit the expression of ALP, OSN and RUNX-2 related to osteogenesis, and can inhibit osteogenic differentiation. The downregulation of miR-214 demonstrated the opposite results; miR-214 may downregulate the expression of β-catenin and inhibit the osteogenic differentiation of DFCs.
ABSTRACT
Objective@#To compare the in vitro biocompatibility of bone marrow mesenchymal cells on polyetheretherketone (PEEK) and titanium (Ti) surfaces.@*Methods @#PEEK and Ti foils with thicknesses of 1 mm and diameters of 10 mm were prepared. First, bone marrow mesenchymal cells were separated and purified by the whole bone marrow adherent culture method in vitro. Then, osteogenesis-induced bone marrow mesenchymal cells were cultivated on the surfaces of the PEEK and Ti foils. Scanning electron microscopy (SEM), the Alamar Blue test, an alkaline phosphatase (ALP) kit and Alizarin Red staining were used to analyze calcium nodules and compare the adhesion, proliferation and osteogenic differentiation ability of bone marrow mesenchymal cells on the surfaces of the PEEK and Ti foils.@*Results @# ① The morphology of the bone marrow mesenchymal cells cultured on the PEEK and Ti foils at 1 h, 4 h and 24 h showed no significant differences. ② In the 1 h, 3 h, 1 d and 3 d cultures of the bone marrow mesenchymal cells inoculated on the surfaces of the foils, the number of living cells in the PEEK group was greater than that in the Ti group (P < 0.05). ③ In the 7 d and 14 d osteogenesis-induced cultures of the inoculated bone marrow mesenchymal cells, the ALP activity of the PEEK group cells was significantly greater than that of the Ti group cells (P < 0.05). ④ Semiquantitative analysis after Alizarin Red staining showed that the mineralization degree of the bone marrow mesenchymal cells induced by osteoblasts was greater in the PEEK group than in the Ti group (P < 0.05). @*Conclusion@#PEEK has better in vitro biocompatibility than Ti and can better promote cell adhesion, proliferation and osteogenic differentiation compared with Ti, and so it is expected to become a new dental implant material.
ABSTRACT
PURPOSE: Zinc (Zn) is an essential trace element for bone mineralization and osteoblast function. We examined the effects of Zn deficiency on osteoblast differentiation and mineralization in MC3T3-E1 cells. METHODS: Osteoblastic MC3T3-E1 cells were cultured at concentration of 1 to 15 µM ZnCl2 (Zn− or Zn+) for 5, 15 and 25 days up to the calcification period. Extracellular matrix mineralization was detected by staining Ca and P deposits using Alizarin Red and von Kossa stain respectively, and alkaline phosphatase (ALP) activity was detected by ALP staining and colorimetric method. RESULTS: Extracellular matrix mineralization was decreased in Zn deficiency over 5, 15, and 25 days. Similarly, staining of ALP activity as the sign of an osteoblast differentiation, was also decreased by Zn deficiency over the same period. Interestingly, the gene expression of bone-related markers (ALP, PTHR; parathyroid hormone receptor, OPN; osteopontin, OC; osteocalcin and COLI; collagen type I), and bone-specific transcription factor Runx2 were downregulated by Zn deficiency for 5 or 15 days, however, this was restored at 25 days. CONCLUSION: Our data suggests that Zn deficiency inhibits osteoblast differentiation by retarding bone marker gene expression and also inhibits bone mineralization by decreasing Ca/P deposition as well as ALP activity.
Subject(s)
Alkaline Phosphatase , Calcification, Physiologic , Collagen , Extracellular Matrix , Gene Expression , Methods , Miners , Osteoblasts , Osteocalcin , Osteopontin , Receptor, Parathyroid Hormone, Type 1 , Transcription Factors , ZincABSTRACT
Bone morphogenetic protein-7(BMP-7), a member of the transforming growth factor superfamily, stimulates osteoblast differentiation and bone formation. There are lots of evidences supporting a direct participation of periodontal ligament(PDL) cells on periodontal tissue regeneration. The purpose of this study was to evaluate the effect of recombinant human(rh) BMP-7 on primary rat PDL cells in vitro, with special focus on the ability of bone formation. The PDL cells were cultured with rhBMP-7 at the concentration of 0, 10, 25, 50, 100 and 200ng/ml for MTT assay. We evaluated the alkaline phosphatase activity at 3 and 5 days of incubation and the ability to produce mineralized nodules of rat PDL cells at 14 days of cell culture in concentration of 0, 10, 25, 50 and 100ng/ml. The cell activity was not reduced in cells treated with BMP-7 at 10~100ng/ml, whereas the cell activity was reduced in the concentration of 200ng/ml than the control at day 1 and 3(p<0.01). At 3 and 5 day, alkaline phosphatase activity was significantly increased in cells treated with BMP-7 at 50ng/ml and 100ng/ml(p<0.05). The area of mineralized bone nodule was greater in cells treated with BMP-7 at 50 and 100 ng/ml than the control(p<0.01). These results suggest that rhBMP-7 stimulate rat PDL cells to differentiate toward osteoblast phenotype and secretion of the extracellular matrix of rat PDL cells.