Karina Gonzales Silvério

Impact of smoking on inflammation: overview of molecular mechanisms

BackgroundInflammation is a critical component of normal tissue repair, as well as being fundamental to the body’s defense against infection. Environmental factors, such as smoking, have been reported to modify the host response and hence modify inflammation progression, severity and outcome. Therefore, a comprehensive understanding of the molecular mechanisms by which smoking affects inflammation is vital for preventive and therapeutic strategies on a clinical level.AimThe purpose of the present article is to review the potential biological mechanisms by which smoking affects inflammation, emphasizing recent developments.ResultsSmoking is reported to effect a number of biological mediators of inflammation through its effect on immune-inflammatory cells, leading to an immunosuppressant state. Recent evidence strongly suggests that the molecular mechanisms behind the modulation of inflammation by smoking mainly involve the nuclear factor-kappa B (NF-kB) family, through the activation of both an inhibitor of IkB kinase (IKK)-dependent and -independent pathway. In addition to NF-kB activation, a number of transcriptional factors including GATA, PAX5 and Smad 3/4, have also been implicated.ConclusionMultiple mechanisms may be responsible for the association of smoking and inflammation, and the identification of potential therapeutic targets should guide future research.

Mesenchymal Stem Cell Properties of Periodontal Ligament Cells From Deciduous and Permanent Teeth

BACKGROUND Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105(+) enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed. METHODS PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105(+) CD34(-) CD45(-) cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method. RESULTS Magnetic cell sorting isolated cell populations containing 23.87% (+/- 11.98%) and 11.68% (+/- 6.27%) of CD105(+) expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105(+) cells coexpressing CD166 surface antigen in PePDL, whereas immunostaining and real-time polymerase chain reaction analysis demonstrated that both cell subsets expressed Stro-1 and Oct-4. DePDL-CD105(+) subsets were more proliferative compared to PePDL subsets, and both cell populations showed multipotential capabilities to differentiate in vitro to osteoblast/cementoblast- and adipocyte-like cells. However, a higher expression of adipogenic-related genes was observed in DePDL cells, whereas PePDL-CD105(+) cell subset presented a more homogeneous osteoblast/cementoblast response. CONCLUSION These findings demonstrate that highly purified mesenchymal progenitor cell subsets can be obtained from the PDLs of both deciduous and permanent teeth, and further indicate phenotype dissimilarities that may have an impact on their clinical applications.

Wnt/β‐catenin pathway regulates bone morphogenetic protein (BMP2)‐mediated differentiation of dental follicle cells

BACKGROUND AND OBJECTIVE Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/βcatenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway. MATERIAL AND METHODS SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/β-catenin pathway activation assessed by western blotting, β-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of β-catenin. RESULTS WNT3A induced β-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/β-catenin signaling. CONCLUSION These data suggest that stabilization of β-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.

Tooth-derived stem cells: Update and perspectives

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context, stem cells (SCs) present the characteristics of self-renewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation (CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs (TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.

Wnt/β-catenin pathway regulates Bmp2-mediated differentiation of dental follicle cells

BACKGROUND AND OBJECTIVE Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/βcatenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway. MATERIAL AND METHODS SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/β-catenin pathway activation assessed by western blotting, β-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of β-catenin. RESULTS WNT3A induced β-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/β-catenin signaling. CONCLUSION These data suggest that stabilization of β-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.

Inflammatory and bone-related genes are modulated by aging in human periodontal ligament cells.

Periodontal ligament cells (PDLC) play a major role in periodontal tissues homeostasis and destruction. Most age-associated diseases seem to be closely related to an underlying chronic inflammatory state. Thus, the present study aimed at evaluating in PDLC the effect of aging on the basal levels of inflammatory and bone-related genes. Primary PDLC cultures were obtained from subjects aged 15-20 years (control- n=5), and subjects aged more than 60 years (test- n=5). Proliferation, cell viability and total secreted protein assays were performed, and mRNA levels were quantitatively assessed for interleukin (IL)-1beta, IL-4, IL-6 and IL-8, and for receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) by real time PCR. Data analysis demonstrated that aging negatively influenced cell proliferation, whereas cell viability and total secreted protein were not affected (p>0.05). Gene expression analysis showed that mRNA levels for RANKL and IL-8 were not affected by aging (p>0.05) whereas, mRNA levels for IL-4 was significantly lower in aged cells (p<0.05) and OPG, IL-1beta and IL-6 mRNA levels were higher (p<0.05). Data analysis suggests that aging decreased the ability of PDLC to proliferate and modulated the expression of important inflammatory and bone-related genes in periodontal ligament cells, favoring a proinflammatory and an antiresorptive profile.

Stem Cells: Potential Therapeutics for Periodontal Regeneration

This paper is concerned about dental-derived stem cells and their characterization in vitro and in vivo. Aditionally, since conventional periodontal techniques remain insufficient to attain complete and reliable periodontal regeneration, the potential of dental-derived stem cells in promoting periodontal tissue regeneration is also reviewed.

Influence of Aging on Biological Properties of Periodontal Ligament Cells

The majority of patients eligible for periodontal regenerative therapies are aged subjects. Since periodontal ligament cells (PDLC) are essential for periodontal regeneration, the aim of the present study was to determine the effect of cellular aging on PDLC, including genes associated with extracellular matrix metabolism and growth-associated factors. PDLC cultures were obtained from subjects aged 15 to 20 years and subjects aged more than 60 years. Proliferation, cell viability, mineralization assays, and mRNA levels were assessed for type I and III collagen, platelet-derived growth factor (PDGF)-1, basic fibroblast growth factor (bFGF), metalloproteinase (MMP)-2 and-8, and tissue inhibitor of metalloproteinases (TIMP)-1 and-2. Data analysis demonstrated that aging negatively influenced cell proliferation and mineral nodule formation (p < 0.05). Gene expression analysis further showed that mRNA levels for bFGF, PDGF-1, and TIMP-2 were not affected by aging (p > 0.05). In addition, mRNA levels for type I and III collagen were significantly lower in aged cells (p < 0.05), whereas MMP-2 and-8 and TIMP-1 mRNA levels were higher (p < 0.05). Within the limits of the present study, data analysis suggests that aging modulates important biological properties of periodontal ligament cells, diminishes the potential for mineral nodule formation, and favors extracellular matrix degradation.

Autologous periodontal ligament cells in the treatment of class II furcation defects: a study in dogs: Use of PDL cells for tissue engineering

AIM The goal of this study was to histologically investigate the use of periodontal ligament cells (PDL cells) in tissue engineering to regenerate class II furcation defects. MATERIAL AND METHODS PDL cells were obtained from the mandibular tooth extracted from each dog (seven), cultured in vitro and phenotypically characterized with regard to their biological properties. Following, bilateral class II furcation lesions were created at maxillary 3rd premolars and were randomly assigned to the test group [PDL cells+guided tissue regeneration (GTR)] or the control group (GTR). After 3 months, the animals were euthanized to evaluate the histometric parameters. RESULTS In vitro, PDL cells were able to promote mineral nodule formation and to express bone sialoprotein, type I collagen and alkaline phosphatase. Histometrically, data analysis demonstrated that the cell-treated group presented a superior length of new cementum (6.00 ± 1.50 and 8.08 ± 1.08 mm), a greater extension of periodontal regeneration (3.94 ± 1.20 and 7.28 ± 1.00 mm), a lower formation of connective tissue/epithelium (2.15 ± 1.92 and 0.60 ± 0.99 mm), a larger area of new bone (7.01 ± 0.61 and 9.02 ± 2.30 mm(2)) and a smaller area of connective tissue/epithelium (5.90 ± 1.67 and 4.22 ± 0.95 mm(2)), when compared with control group. CONCLUSION PDL cells in association with GTR may significantly promote periodontal regeneration in class II furcation defects in dog.

Characterization of Highly Osteoblast/Cementoblast Cell Clones From a CD105-Enriched Periodontal Ligament Progenitor Cell Population

BACKGROUND It is known that periodontal ligament (PDL) harbors a heterogeneous progenitor cell population at different stages of lineage commitment. However, characterization of PDL stem cells committed to osteoblast/cementoblast (O/C) differentiation remains to be elucidated. The present study is carried out to isolate single cell-derived, cluster of differentiation (CD)105-positive PDL clones and to characterize the clones that present high potential to differentiate toward O/C phenotype in vitro. METHODS Isolation of single cell-derived colonies (clones) from a CD105-enriched PDL progenitor cell population was performed by the ring-cloning technique. Cell clones were evaluated for their O/C differentiation potential, metabolic activity, and expression of STRO-1 protein. Additionally, the clones that showed potential to O/C differentiation were characterized by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for expression of runt-related transcriptor factor 2 (RUNX2), alkaline phosphatase, CD105, and CD166 during osteogenic induction. RESULTS Six PDL-CD105(+) clones were obtained, three being highly O/C clones (C-O) and three others that did not have the ability to produce mineralized matrix in vitro (C-F). The C-O group showed lower metabolic activity compared with the C-F group, and both cell groups were positively immunostained for STRO-1. qRT-PCR analysis demonstrated an increased expression of transcripts for RUNX2 and CD166 during the maturation of C-O cells toward O/C phenotype. CONCLUSIONS These results provide evidence that PDL-CD105(+) purified progenitor cells comprise a heterogeneous cell population that presents a cell subset with high O/C potential and, further, that surface antigen CD166 is modulated during the O/C maturation of this cell subset.