Epigenetics and Its Role in Periodontal Diseases: A State‐of‐the‐Art Review

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141896/1/jper0556.pd

When epigenetics meets bioengineering—A material characteristics and surface topography perspective

The field of tissue engineering and regenerative medicine (TE/RM) involves regeneration of tissues and organs using implantable biomaterials. The term epigenetics refers to changes in gene expression that are not encoded in the DNA sequence, leading to remodeling of the chromatin and activation or inactivation of gene expression. Recently, studies have demonstrated that these modifications are influenced not only by biological cues but also by mechanical and topographical signals. This review highlights the current knowledge on emerging approaches in TE/RM with a focus on the effect of materials and topography on the epigenetic expression pattern in cells with potential impacts on modulating regenerative biology. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2065–2071, 2018.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144628/1/jbmb33953.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144628/2/jbmb33953_am.pd

Characterization of macrophages infiltrating periâ implantitis lesions

ObjectivesThe mechanisms involved in the initiation and progression of periâ implantitis lesions are poorly understood. It was the aim to determine the content and activation status of macrophages present in human periâ implantitis lesions and compare the current findings with the macrophage polarization associated with periodontitis lesions.Material and MethodsA total of 14 patients were studied in this investigation. Seven were soft tissue biopsies from dental implants affected by periâ implantitis that required explantation. Seven biopsies were from chronic periodontal disease. Immunofluorescence stains were performed using biomarkers to identify macrophages (CD68+) undergoing M1 polarization (iNOS+) and M2 polarization (CD206+), along with Hoechst 33,342 to identify DNA content. All samples were stained and photographed, and doubleâ positive cells for CD68 and iNOS or CD68 and CD206 were quantified.ResultsAll periâ implantitis biopsies examined revealed a mixed population of macrophages undergoing M1 polarization and M2 polarization. Further analysis demonstrated the coâ expression of iNOS and CD206, which indicates the presence of a heterogenic immune response on periâ implantitis lesions. Macrophage polarization in periâ implantitis lesions presents a distinct pattern than in periodontitis. We observed a significant increase in the population of M1 macrophages on periâ implantitis samples compared to periodontal disease samples.ConclusionOur results demonstrate that periâ implantitis has higher numbers of macrophages displaying a distinct macrophage M1 polarization signature compared to periodontitis lesions. This pattern may explain, in part, the distinct nature of periâ implantitis progression vs. periodontitis in humans.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154542/1/clr13568_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154542/2/clr13568.pd

Characterization of macrophage polarization in periodontal disease

AimTo explore the M1/M2 status of macrophage polarization from healthy, gingivitis, and periodontitis patient samples.Materials and methodsGingival biopsies were collected from 42 individuals (14 gingivitis, 18 periodontitis, and 10 healthy samples) receiving periodontal therapy. Histomorphology analysis was performed with haematoxylin and eosin staining. Immunofluorescence was performed using a combination of CD68 (macrophages), iNOS (M1), and CD206 (M2) in order to acquire changes in macrophage polarization at a singleâ cell resolution. Macrophages were quantified under microscopy using narrow wavelength filters to detect Alexa 488, Alexa 568, Alexa 633 fluorophores, and Hoechst 33342 to identify cellular DNA content.ResultsGingivitis and periodontitis samples showed higher levels of macrophages compared with healthy samples. Unexpectedly, periodontitis samples displayed lower levels of macrophages dispersed in the stromal tissues compared with gingivitis samples; however, it remained higher than healthy tissues. The polarization of macrophages appears to be reduced in periodontitis and showed similar levels to those observed in healthy tissues.ConclusionsOur study found that gingivitis and periodontitis differ from each other by the levels of macrophage infiltrate, but not by changes in macrophage polarization.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150506/1/jcpe13156_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150506/2/jcpe13156.pd

Entinostat is a novel therapeutic agent to treat oral squamous cell carcinoma

IntroductionAlterations of the epigenome may influence cancer initiation and progression. At the cellular level, histones are key regulators of chromatin accessibility and gene transcription; thus, the inhibition of histone deacetylase enzymes (HDACs) constitutes an attractive target for therapy. In this study, we investigated the effects of the HDAC inhibitor entinostat on oral squamous cell carcinoma (OSCC).Materials and MethodsWe tested the effects of entinostat on OSCC cell lines. Cell viability and growth were analyzed using MTT assay. Cell cycle analysis, cell apoptosis, cancer stem cell (CSC) content, and the concentration of reactive oxygen species (ROS) in OSCC tumor cells were assessed using flow cytometry. The expression of histones and cell cycle regulatory proteins was examined by Western blot.ResultsThe administration of entinostat resulted in reduced proliferation of OSCC cells, followed by cell cycle arrest at the G0/G1 phase, as well as substantial tumor apoptosis. We found an increase in ROS production and significant reductions in CSCs. We also found that entinostat caused increased acetylation histone H3 and histone H4, and changes in the expression of cell cycle‐associated proteins such as p21.ConclusionThis study indicates that entinostat is a potential novel therapeutic agent for OSCC by halting tumor proliferation, inducing cytotoxicity and intracellular ROS, and attacking the CSCs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162762/2/jop13039.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162762/1/jop13039_am.pd

Evaluation of DNA methylation of inflammatory genes following treatment of chronic periodontitis: A pilot case–control study

ObjectiveTo evaluate the influence of periodontal therapy on DNA methylation in patients with chronic periodontitis as compared to healthy individuals.Material and MethodsTwenty patients were enrolled into two groups: (i) 10 diagnosed as clinically healthy; and (ii) 10 diagnosed with chronic periodontitis. Clinical measures were recorded and gingival biopsies were harvested at baseline (both patient groups) and at 2 and 8 weeks post‐baseline for diseased individuals. Molecular DNA methylation analysis was performed by pyrosequencing for the putative inflammation‐associated genes LINE‐1, COX‐2, IFN‐γ and TNF‐α. Random‐intercept linear regression models were applied to evaluate methylation levels across groups at baseline and the methylation changes over time in the diseased and normal tissues.ResultsPeriodontal therapy did not influence gene expression methylation of TNF‐α, IFN‐γ and LINE‐1 levels at normal and periodontitis sites over time. However, it significantly reduced COX‐2 methylation levels comparable to healthy individuals at both 2 and 8 weeks post‐treatment (p < .05).ConclusionsPeriodontal therapy resets the DNA methylation status of inflammatory gene for COX‐2 in patients with periodontal disease. DNA methylation levels of TNF‐α, IFN‐γ and LINE‐1 were sustained in periodontitis sites despite therapy. Future studies should consider an expanded panel of inflammatory genes over time. (ClinicalTrials.gov NCT02835898).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138219/1/jcpe12783.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138219/2/jcpe12783_am.pd

Tensile strength essay comparing three different platelet-rich fibrin membranes (L-PRF, A-PRF, and A-PRF plus): a mechanical and structural in vitro evaluation

Predictable outcomes intended by the application of PRF (platelet-rich fibrin) derivative membranes have created a lack of consideration for their consistency and functional integrity. This study aimed to compare the mechanical properties through tensile strength and analyze the structural organization among the membranes produced by L-PRF (leukocyte platelet-rich fibrin), A-PRF (advanced platelet-rich fibrin), and A-PRF+ (advanced platelet-rich fibrin plus) (original protocols) that varied in centrifugation speed and time. L-PRF (n = 12), A-PRF (n = 19), and A-PRF+ (n = 13) membranes were submitted to a traction test, evaluating the maximum and average traction. For maximum traction, 0.0020, 0.0022, and 0.0010 N·mm−2 were obtained for A-PRF, A-PRF+, and L-PRF, respectively; regarding the average resistance to traction, 0.0012, 0.0015, and 0.006 N·mm−2 were obtained, respectively (A-PRF+ > A-PRF > L-PRF). For all groups studied, significant results were found. In the surface morphology observations through SEM, the L-PRF matrix showed a highly compact surface with thick fibers present within interfibrous areas with the apparent destruction of red blood cells and leukocytes. The A-PRF protocol showed a dense matrix composed of thin and elongated fibers that seemed to follow a preferential and orientated direction in which the platelets were well-adhered. Porosity was also evident with a large diameter of the interfibrous spaces whereas A-PRF+ was the most porous platelet concentrate with the greatest fiber abundance and cell preservation. Thus, this study concluded that A-PRF+ produced membranes with significant and higher maximum traction results, indicating a better viscoelastic strength when stretched by two opposing forces.info:eu-repo/semantics/publishedVersio

Asparaginase induces selective dose- and time- dependent cytotoxicity, apoptosis, and reduction of NFκB expression in oral cancer cells

Asparaginase is fundamental to the treatment of haematological malignancies. However, little has been studied on the effects that asparaginase could exert on solid tumours. Thus, this study aimed to evaluate the effects of asparaginase on an oral carcinoma cell line. The cytotoxicity of asparaginase in SCC- 9 (tongue squamous cell carcinoma) and HaCaT (human keratinocyte) cell lines was evaluated with MTT cell viability assay. The cells were treated with asparaginase at 0.04, 0.16, 0.63, 1.0, 1.5, 2.5, and 5.0 IU/mL. Dose- response curves and IC50 values were obtained and the Tumour Selectivity Index (TSI) was calculated. The effect of asparaginase on procaspase- 3 and nuclear factor κB (NFκB) expression was evaluated with western blot because it was reported that the overexpression of NFκB has been shown to contribute to tumour cell survival, proliferation, and migration. Caspase 3/7 staining was performed to identify cell death using flow cytometry. Effective asparaginase concentrations were lower for SCC- 9 cells when compared to HaCaT cells. The cytotoxicity results at 48 and 72 hours were significantly different for SCC- 9 cells. The TSI indicated that asparaginase was selective for the tumour cells. A decrease in procaspase- 3 and NFκB protein levels was observed in SCC- 9 cells. Furthermore, asparaginase resulted in significant apoptosis after 48 and 72 hours. Based on these results, asparaginase was cytotoxic in a dose- and time- dependent manner, induces apoptosis, and reduces NFκB expression in oral cancer cells. These results encourage further studies on the effectiveness of this enzyme as a treatment for solid tumours, especially head and neck cancer.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154950/1/cep13256.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154950/2/cep13256_am.pd

Periostin Responds to Mechanical Stress and Tension by Activating the MTOR Signaling Pathway

Current knowledge about Periostin biology has expanded from its recognized functions in embryogenesis and bone metabolism to its roles in tissue repair and remodeling and its clinical implications in cancer. Emerging evidence suggests that Periostin plays a critical role in the mechanism of wound healing; however, the paracrine effect of Periostin in epithelial cell biology is still poorly understood. We found that epithelial cells are capable of producing endogenous Periostin that, unlike mesenchymal cell, cannot be secreted. Epithelial cells responded to Periostin paracrine stimuli by enhancing cellular migration and proliferation and by activating the mTOR signaling pathway. Interestingly, biomechanical stimulation of epithelial cells, which simulates tension forces that occur during initial steps of tissue healing, induced Periostin production and mTOR activation. The molecular association of Periostin and mTOR signaling was further dissected by administering rapamycin, a selective pharmacological inhibitor of mTOR, and by disruption of Raptor and Rictor scaffold proteins implicated in the regulation of mTORC1 and mTORC2 complex assembly. Both strategies resulted in ablation of Periostin-induced mitogenic and migratory activity. These results indicate that Periostin-induced epithelial migration and proliferation requires mTOR signaling. Collectively, our findings identify Periostin as a mechanical stress responsive molecule that is primarily secreted by fibroblasts during wound healing and expressed endogenously in epithelial cells resulting in the control of cellular physiology through a mechanism mediated by the mTOR signaling cascade.This work was funded by the National Institutes of Health (NIH/NCI) P50-CA97248 (University of Michigan Head and Neck SPORE)