Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.
Objectives:The aim of this study was to evaluate the capacity of two rotatory instruments (controlled speed electric motor [CSEM] – 300 rpm; conventional slow handpiece [CSHP] – 18,000 rpm) to remove sound and demineralized dentin, by examining prepared cavity walls using the scanning electron microscopy (SEM) and assessing loss of mass.Materials and Methods:A total of 40 blocks of human occlusal dentin, measuring 5 mm × 5 mm × 4 mm (L × W × H), were divided into two groups according to the substrate type in which the cavity preparation was performed: D - demineralized dentin; and S - sound dentin (control group). The groups were subdivided according to the rotatory instrument used for cavity preparation (n = 10): CSEM (300 rpm); and CSHP (18,000 rpm). In half of the dentin blocks, caries lesion induction was performed for 6 weeks. The preparation of the cavities was performed on a standardizing machine, using a cylindrical tungsten carbide burr. Before and after the preparation, specimens were dehydrated in an incubator at 60°C for 30 min. The initial and final mass (in mg) of each dentin block was measured 3 times using the digital precision balance to obtain the mean weight Following cavity preparation, all specimens were hemisected and SEM was used to blindly assess each half so that the lateral walls of the prepared cavity were measured in μm, accepting the average of two measurements as the total depth of the preparation. Non-parametric Mann-Whitney analysis was performed with a 5% of significance level.Results:Regarding the weight difference (mg), no significance was detected between the groups. Regarding depth (μm), a significant difference was found between the groups, so that the CSRM showed lower cavity depth when compared with CSHP, both in sound and demineralized dentin.Conclusions:Controlled speed rotatory instruments were found to be more conservative in removing both sound and demineralized dentin, in terms of preparation and depth.
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