Osteogenesis in human periodontal ligament stem cell sheets is enhanced by the protease-activated receptor 1 (PAR1) in vivo.

Human periodontal ligament stem cells (PDLSCs) have been studied as a promising strategy in regenerative approaches. The protease-activated receptor 1 (PAR1) plays a key role in osteogenesis and has been shown to induce osteogenesis and increase bone formation in PDLSCs. However, little is known about its effects when activated in PDLSCs as a cell sheet construct and how it would impact bone formation as a graft in vivo. Here, PDLSCs were obtained from 3 patients. Groups were divided into control, osteogenic medium and osteogenic medium + PAR1 activation by TFLLR-NH2 peptide. Cell phenotype was determined by flow cytometry and immunofluorescence. Calcium deposition was quantified by Alizarin Red Staining. Cell sheet microstructure was analyzed through light, scanning electron microscopy and histology and transplanted to Balb/c nude mice. Immunohistochemistry for bone sialoprotein (BSP), integrin ß1 and collagen type 1 and histological stains (H&E, Van Giesson, Masson's Trichrome and Von Kossa) were performed on the ex-vivo mineralized tissue after 60 days of implantation in vivo. Ectopic bone formation was evaluated through micro-CT. PAR1 activation increased calcium deposition in vitro as well as BSP, collagen type 1 and integrin ß1 protein expression and higher ectopic bone formation (micro-CT) in vivo.

Endocannabinoids Regulate Stem Cells of the Apical Papilla via a Cannabinoid Receptor and TRPV1-Independent Mechanism.

INTRODUCTION: Endogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential. METHODS: Gene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days. RESULTS: The enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group. CONCLUSIONS: SCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.

Angiotensin II Regulates Proliferation and Function of Stem Cells of Apical Papilla.

INTRODUCTION: Stem cells of apical papilla (SCAP) may be affected by inflammatory mediators released by activation with lipopolysaccharide (LPS) from infected pulpal cavities of necrotic immature teeth. Therefore, this study aimed to investigate the presence of a local renin-angiotensin system (RAS) and the role of angiotensin II (Ang II) on the modulation of SCAP in vitro. METHODS: Primary cultures of SCAP were incubated with LPS (0.1-10 µg/mL) for cell viability and quantification of the chemokine CCL2. Components of RAS were searched by gene expression of angiotensinogen (AGTN), angiotensin converting enzyme (ACE), renin, angiotensin receptor 1 (AT1) and 2 (AT2), and Mas receptor. Ang II was investigated in SCAP supernatants. Immunofluorescence was used to detect AGTN and AT1. Next, cells were treated with Ang II for viability/proliferation assessment, quantification of CCL2 and interleukin 6, and mineralization assay. Data were evaluated by analysis of variance using Tukey post hoc comparisons or the Student t test. P values <.05 were considered to be significant. RESULTS: LPS increased CCL2 production at 1 and 10 µg/mL. The gene expression of AGTN, renin, ACE, and AT1 was detected, but only ACE was increased by LPS. Ang II peptide was found in SCAP supernatants but unaltered by LPS. Both AGTN and AT1 proteins were detected by immunostaining. Ang II significantly induced SCAP proliferation, increased CCL2 production, down-regulated IL-6 release, and reduced the SCAP mineralization rate. CONCLUSIONS: A local RAS was found at the apical papilla, and Ang II was able to modulate SCAP function in vitro.

Photobiomodulation therapy and vitamin C on longevity of cell sheets of human dental pulp stem cells.

Photobiomodulation therapy (PBMT) can improve processes relevant to tissue regeneration, such as survival, proliferation, migration, and differentiation of cells, including stem cells. Thus, PBMT could be applied as auxiliary therapy for tissue regeneration. Cell sheets (CSs) induced by vitamin C (VC) can generate large amount of cells, which would also be useful for tissue regeneration. VC and PBMT cause opposite effects on cell metabolism (e.g., VC is antioxidative, and PBMT generates reactive oxygen species); however, hDPSC CSs were formed when VC and PBMT+VC were applied. Thus, this study showed that PBMT does not interfere with the formation of cell sheets induced by VC. Additionally, PBMT improved the functional differentiation of the cells isolated from the CSs. Thus, due to the clinical benefits of PBMT, the association of this therapy with cell sheets seems promising for future applications in tissue regeneration.

Photobiomodulation of mesenchymal stem cells encapsulated in an injectable rhBMP4-loaded hydrogel directs hard tissue bioengineering.

Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering.

Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue.

The initiation or progression of periodontitis might involve a local renin-angiotensin system (RAS) in periodontal tissue. The aim of this study was to further characterize the local RAS in human and rat periodontal tissues between healthy and periodontally-affected tissue. Components of the RAS were investigated using in vitro, ex vivo and in vivo experiments involving both human and Wistar rat periodontium. Although not upregulated when challenged with P. gingivalis-lipopolysaccharide, human gingival and periodontal ligament fibroblasts expressed RAS components. Likewise, healthy and inflamed human gingiva expressed RAS components, some of which were shown to be functional, yet no differences in expression were found between healthy and diseased gingiva. However, in inflamed tissue the immunoreactivity was greater for the AT1R compared to AT2R in fibroblasts. When compared to healthy tissue, ACE activity was increased in human gingiva from volunteers with gingivitis. Human-gingiva homogenates generated Ang II, Ang 1-9 and Ang 1-7 when incubated with precursors. In gingiva homogenates, Ang II formation from Ang I was nearly abolished only when captopril and chymostatin were combined. Ang 1-7 formation was significantly greater when human gingiva homogenates were incubated with chymostatin alone compared to incubation without any inhibitor, only captopril, or captopril and chymostatin. In rat gingiva, RAS components were also found; their expression was not different between healthy and experimentally induced periodontitis (EP) groups. However, renin inhibition (aliskiren) and an AT1R antagonist (losartan) significantly blocked EP-alveolar-bone loss in rats. Collectively, these data are consistent with the hypothesis that a local RAS system is not only present but is also functional in both human and rat periodontal tissue. Furthermore, blocking AT1R and renin can significantly prevent periodontal bone loss induced by EP in rats.

MicroRNA-146a and microRNA-155 show tissue-dependent expression in dental pulp, gingival and periodontal ligament fibroblasts in vitro.

MicroRNAs (miRNAs) are small non-coding RNAs showing a tissue-specific expression pattern, and whose function is to suppress protein synthesis. In this study, we hypothesized that expression of miRNAs would differ among fibroblasts from dental pulp (DPF), gingiva (GF) and periodontal ligament (PLF) in vitro. Once established by an explant technique, DPF, GF and PLF were collected for RNA isolation and subjected to a miRNA microarray. Next, cells were stimulated with E. coli lipopolysaccharide (LPS) for 24 h and then collected for RNA isolation. Expression of miR-146a and miR-155 was investigated by qPCR. Microarray screening revealed several miRNAs that showed specifically high expression in at least one of the fibroblast subtypes. These molecules are potentially involved in the regulation of extracellular matrix turnover and production of inflammatory mediators. Microarray analysis showed that both miR-146a and miR-155 were among the miRNAs expressed exclusively in GF. qPCR demonstrated significant upregulation of miR-146a only in GF after LPS stimulation, whereas basal expression of miR-155 was higher in GF than in the other cell subtypes. LPS downregulated the expression of miR-155 only in GF. Our results suggest that the expression and regulation of miR-146a and miR-155 are more pronounced in GF than in DPF and PLF.

Salivary immunity in elderly individuals presented with Candida-related denture stomatitis.

OBJECTIVES: Elderly individuals with Candida-related denture stomatitis (DS) present with a reduced defence against Candida albicans. This study evaluated levels of antimicrobial mediators in the elderly DS saliva and salivary neutrophils' activation characteristics compared with elderly and young without DS. METHODS: Salivary peroxidases (SPO) and elastase activities (ELA), nitric oxide (NO), transforming growth factor beta (TGF-ß), IL-6 and CCL3 production were determined in saliva from elderly with or without DS, and young control individuals. TLR4, CXCR1, CD11b, CD16 and CD32 expression on salivary neutrophils were evaluated. Correlations between number and apoptosis rate of salivary neutrophils, enzymatic activities and cytokine levels were determined. RESULTS: Elderly DS individuals exhibited the lowest SPO and ELA activities. Also, the activity of both enzymes was low in elderly without DS. Although both elderly groups showed higher salivary NO and TGF-ß levels compared to young control groups, elderly DS presented the highest salivary NO, TGF-ß, IL-6 and CCL3 levels. Decreased percentages of salivary TLR4(+) and CD16(+) neutrophils were detected in both elderly groups. Although these damages could influence the establishment and persistence of DS, the highest levels of salivary IL-6 and CCL3 in elderly DS could be preventing more serious complications.

Antimicrobial effects of calcium hydroxide and chlorhexidine on Enterococcus faecalis.

INTRODUCTION: Endodontic treatment is commonly based on nonspecific elimination of intraradicular microorganisms. Although some authors prefer single-visit root canal operations for endodontic treatment, several studies have shown the importance of intracanal medication between sessions to kill microorganisms that biomechanical preparations alone cannot achieve. The purpose of this study was to evaluate the efficacy of calcium hydroxide Ca(OH)(2) and chlorhexidine gel on the elimination of intratubular Enterococcus faecalis. METHODS: Human uniradicular teeth contaminated with E. faecalis were treated with Ca(OH)(2), 2% chlorhexidine gel, Ca(OH)(2) plus 2% chlorhexidine gel, or saline (0.9% NaCl) as a negative control. Samples obtained at a depth of 0 to 100 mum and 100 to 200 mum from these root canal preparations were analyzed for bacterial load by counting the number of colony-forming units (CFUs) and bacterial viability using fluorescence microscopy. RESULTS: A significant decrease in the number of CFUs and the percentage of viable E. faecalis was observed after treatment with either Ca(OH)(2) or chlorhexidine when compared with the control group. Additionally, chlorhexidine gel had a significantly higher antimicrobial efficacy as measured by the number of CFUs and the percentage of viable cells than Ca(OH)(2). No differences were observed between the antimicrobial properties of chlorhexidine gel with and without the addition of Ca(OH)(2). CONCLUSION: Both Ca(OH)(2) and chlorhexidine have antimicrobial effects on E. faecalis. Chlorhexidine had increased antimicrobial activity when compared with Ca(OH)(2.) Ca(OH)(2) combined with chlorhexidine showed similar antimicrobial activity to chlorhexidine alone.

Heat-killed Enterococcus faecalis alters nitric oxide and CXCL12 production but not CXCL8 and CCL3 production by cultured human dental pulp fibroblasts.

INTRODUCTION: Fibroblasts are the most abundant cells in dental pulp. To investigate their capacity to produce the chemokines CCL3, CXCL8, and CXCL12 as well as nitric oxide (NO), we evaluated the production of these mediators in supernatants of cultured human dental pulp fibroblasts (HDPF) stimulated by heat-killed Enterococcus faecalis (HKEF). METHODS: Primary cultures of HDPF were stimulated with medium alone or HKEF (1:1, 10:1, or 100:1 bacteria:fibroblast) for 1, 6, and 24 hours. Chemokines and NO were assessed through enzyme-linked immunosorbent assay and Griess reaction, respectively. Statistical analysis was performed by using analysis of variance and Tukey post test. RESULTS: CCL3 was not detected, whereas constitutive CXCL8 was not affected. Production of CXCL12 was increased at 1 and 6 hours, and NO was increased at the concentration of 1:1 bacteria:fibroblast at 24 hours. Viability and proliferation assays did not reveal cell number differences. CONCLUSIONS: These findings demonstrate that heat-killed E. faecalis is able to increase production of CXCL12 and NO by HDPF.

A comparison of the clinical anesthetic efficacy of 4% articaine and 0.5% bupivacaine (both with 1:200,000 epinephrine) for lower third molar removal.

OBJECTIVE: This study compared the clinical efficacy of 4% articaine (A200) and 0.5% bupivacaine (B200), both with 1:200,000 epinephrine, for lower third molar removal. STUDY DESIGN: Fifty patients underwent removal of symmetrically positioned lower third molars, in 2 separate appointments, under local anesthesia either with A200 or B200, in a double-blind, randomized, and crossover manner. Time to onset, duration of postoperative analgesia, duration of anesthetic action on soft tissues, intraoperative bleeding, and hemodynamic parameters were evaluated. RESULTS: A statistically significant difference between the time to onset of A200 (1.66 +/- 0.13 minutes) and B200 (2.51 +/- 0.21 minutes) was found (P < .05). There was no statistically significant difference in the duration of analgesia, whether the patient was subjected to osteotomy or not, regardless of the local anesthetic used (3 to 4 hours; P > .05). However, when patients received B200 they experienced a statistically significant longer period of anesthesia on the soft tissues as compared with when they had received A200 (around 5 hours and 4 hours, respectively, P < .05). The surgeon's rating of intraoperative bleeding was considered very close to minimal for both anesthetics. In the surgeries with osteotomy, the comparison between A200 and B200 showed statistically significant differences in the diastolic (64 mm Hg and 68 mm Hg, respectively, P = .001) and mean arterial pressure (86 mm Hg and 89 mm Hg, respectively, P = .031) when data from all the surgical phases were pooled. Additionally, the mouth opening at the suture removal was statistically different for A200 and B200 solutions (91.90% +/- 3.00% and 88.57% +/- 2.38% of the preoperative measure, respectively) when surgeries required bone removal (P < .05). CONCLUSIONS: In comparison with 0.5% bupivacaine, 4% articaine (both with 1:200,000 epinephrine) provided a shorter time to onset and comparable hemostasis and postoperative pain control with a shorter duration of soft tissue anesthesia in lower third molar removal.

Epinephrine concentration (1:100,000 or 1:200,000) does not affect the clinical efficacy of 4% articaine for lower third molar removal: a double-blind, randomized, crossover study.

PURPOSE: This study compared the use of 4% articaine in association with 1:100,000 (10 mug/mL; A100) or 1:200,000 (5 mug/mL; A200) epinephrine in lower third molar removal. PATIENTS AND METHODS: Fifty healthy volunteers underwent removal of symmetrically positioned lower third molars, in 2 separate appointments, under local anesthesia with either A100 or A200, in a double-blind, randomized, and crossed manner. Latency, duration of postoperative analgesia, duration of anesthetic action on soft tissues, intraoperative bleeding, and hemodynamic parameters were evaluated. RESULTS: A100 and A200 presented very similar latency (1.64 +/- 0.08 and 1.58 +/- 0.08 minutes, respectively; P > .05). Identical volumes of both anesthetic solutions were used: 2.7 mL = 108 mg of articaine plus 27 mug (A100) or 13.5 mug (A200) of epinephrine. The 2 solutions provided similar duration of postoperative analgesia regardless of bone removal (around 200 minutes; P > .05). The 2 solutions also had a similar duration of anesthetic action on soft tissues (around 250 minutes; P > .05). The surgeon's rating of intraoperative bleeding was considered very close to minimal. Transient changes in hemodynamic parameters were observed, but these were neither clinically significant nor attributable to the type of anesthetic used (P > .05). CONCLUSIONS: An epinephrine concentration of 1:100,000 or 1:200,000 in 4% articaine solution does not affect the clinical efficacy of this local anesthetic. It is possible to successfully use the 4% articaine formulation with a lower concentration of epinephrine (1:200,000 or 5 mug/mL) for lower third molar extraction with or without bone removal.