Macrophage polarization in human periapical lesions in relation to histopathological diagnosis, clinical features and lesion volume: An ex vivo study.

AIM: To evaluate M1 and M2 macrophage polarization in radicular cysts and periapical granulomas through an immunohistochemical analysis and the correlation between macrophage polarization and histopathological diagnosis, clinical characteristics and lesion volume using cone-beam computed tomography. METHODOLOGY: Periapical biopsies diagnosed as radicular cysts (n = 52) and periapical granulomas (n = 51) were analysed by immunohistochemical method. Teeth with periapical lesion with no history of root canal treatment (primary lesion) and lesions persistent to root canal treatment (persistent lesions) were included. Pathological diagnosis, patients' age, gender and clinical characteristics were obtained from treatment records. A cone-beam computed tomographic periapical volume index (CBCTPAVI) score was assigned to each periapical lesion based on the volume of the lesion. Immuno-expressions of CD68 and CD163 were quantified. The CD68/CD163 ratio was adopted to represent M1 or M2 macrophage polarization. Mann-Whitney U test was used to determine the different CD68/CD163 ratio between groups of radicular cyst and periapical granuloma. Spearman's correlation test was performed to assess the correlation between the CD68/CD163 ratio and lesion volume and CBCTPAVI score. RESULTS: Radicular cysts and periapical granulomas had CD68/CD163 median of 2.05 (IQR = 1.33) and 1.26 (IQR = 0.81), respectively. A significantly higher CD68/CD163 ratio was observed in radicular cysts (p < .001). In contrast, periapical granulomas had significantly lower median of CD68/CD163 ratio. Larger lesions had a higher median of CD68/CD163 ratio, while smaller lesions had lower median of CD68/CD163 ratio (p = .007, rs = .262). CD68/CD163 ratio was significantly correlated with the CBCTPAVI score in the overall periapical lesions (p = .002, rs = .306). The higher CD68/CD163 ratio in larger lesions indicated a higher degree of M1 polarization compared to smaller lesions. Regarding the pathological diagnosis, there was a significant positive correlation between CBCTPAVI score and CD68/CD163 ratio in periapical granulomas (p < .001, rs = .453), whereas the negative correlation was observed for radicular cysts (p < .001, rs = -.471). CONCLUSIONS: Periapical granulomas are characterized by a M2-dominant macrophage polarization, while radicular cysts have significantly higher M1 macrophages. The higher degree of M1 macrophage polarization was significantly correlated with larger volume and higher CBCTPAVI scores of overall periapical lesion and periapical granuloma.

Efficacy of chitosan paste as intracanal medication against Enterococcus faecalis and Candida albicans biofilm compared with calcium hydroxide in an in vitro root canal infection model.

BACKGROUND: Enterococcus faecalis and Candida albicans are frequently found in persistent endodontic infection and could remain in dentinal tubules despite intracanal medication with calcium hydroxide (Ca(OH)2), a commonly used medication. Thus, an effective and safe antimicrobial medication against such refractory infection is necessary in endodontic retreatment, so we aimed to test the efficacy of chitosan paste against these microorganisms compared with Ca(OH)2 in root canals of extracted human teeth. METHODS: Thirty-six sterilized human root samples prepared from extracted premolars and upper maxillary incisors were infected with E. faecalis for 14 days, while 32 were infected with C. albicans for 48 h, for mature biofilm formation. The samples were assigned to 6 groups of intracanal medications: Group 1: no medication (negative control); Group 2: 20% Polyethylene glycol (PEG); Group 3: 20% Propylene glycol (PG); Group 4: Ca(OH)2; Group 5: Chitosan + PEG; and Group 6: Chitosan + PG. After 7 days, intracanal surface dentin was harvested using Protaper next, resuspended, serially diluted and spread on Brain-Heart-Infusion agar (for E. faecalis) and Yeast Extract-Peptone-Dextrose agar (for C. albicans) for colony count. Antimicrobial efficacy was determined as percentage of remaining colony forming unit (CFUs) relative to negative control and analyzed using One-way ANOVA and post-hoc Games-Howell test. The significance level was set at 0.05. RESULTS: For E. faecalis, chitosan + PG had significantly higher antibacterial activity than Ca(OH)2 (P = 0.039). Chitosan + PEG and chitosan + PG medication significantly reduced viable bacteria compared with negative control, PEG and PG (P = 0.001, 0.003, 0.024, respectively for chitosan + PEG; P = 0.002, 0.003, 0.014, respectively for chitosan + PG). For C.albicans, chitosan + PEG and chitosan + PG were not significantly different from Ca(OH)2. However, Chitosan + PEG and chitosan + PG, but not Ca(OH)2, showed a significantly lower level of remaining CFUs compared with negative control (P = 0.013 and 0.005, respectively). CONCLUSION: Chitosan paste showed better efficacy in reducing viable E. faecalis biofilm when compared to Ca(OH)2 after 7-day intracanal medication in this in vitro root canal model. It could also significantly reduce viable C. albicans, but was not significantly different from Ca(OH)2.

Evaluation of pH and calcium ion release at the simulated external root resorption cavities of teeth obturated with bioceramic sealer.

OBJECTIVE: The purpose of this study was to evaluate pH and calcium ion release at the outer dentin surface of simulated external root resorption cavities after root canals obturated with bioceramic root canal sealer compared with those medicated with calcium hydroxide. MATERIALS AND METHODS: Sixty extracted human single-rooted teeth were selected and instrumented. External root resorption cavities were prepared at the lingual surface of the root. The teeth were randomly divided into three groups: (1) Bioceramic sealer group, canals were obturated with gutta-percha and BioRoot sealer; (2) Calcium hydroxide group, canals were medicated with UltraCal XS; (3) Control group, canals were left empty. Thirty specimens were used for evaluation of pH at 7, 14, and 28 days (n = 10 per group) and the other 30 specimens were used for evaluation of calcium ion diffusion at 28 days (n = 10 per group). RESULTS: Calcium hydroxide group showed the highest median pH value at all time points (7, 14, and 28 days). Both calcium hydroxide and bioceramic sealer groups showed significantly higher median pH values compared with control (p < .001). Comparing within groups, both bioceramic sealer group and calcium hydroxide group showed significantly decreased median pH over time, while the median pH of the control did not show any significant difference among Days 7, 14, and 28. Both calcium hydroxide and bioceramic sealer groups had significantly higher calcium ion release than control. Notably, bioceramic sealer group showed significantly higher calcium ion release than the calcium hydroxide group (p < .01). CONCLUSIONS: Root canals obturated with gutta-percha and bioceramic sealer showed high calcium ion levels at the simulated external root resorption cavities but did not show an extended period of alkaline pH.

Antibacterial property of chitosan against E. faecalis standard strain and clinical isolates.

To investigate applications of chitosan as antibacterial agent for endodontic treatments, we tested its activity against Enterococcus faecalis standard strain (ATCC29212) and clinical isolates. We determined the minimum bactericidal concentration (MBC) of 6 types of chitosan against ATCC29212; the most effective types were selected for further tests. Four clinical isolates were cultured from endodontically treated-teeth and identified by biochemical assays and polymerase chain reactions. Bacterial cultures were exposed to 1,700 and 2,100 kDa chitosan at MBC for 1, 3, 5, 10, and 60 min in time-kill assays and plated on brain-heart-infusion (BHI) agar for colony counts. Both types of chitosan showed significantly lower numbers of remaining bacteria (log colony forming units per millimeter, logCFUs/mL) than negative controls (0.1% acetic acid and BHI) at 10 min, and completely eliminated the bacteria at 60 min for all strains. Thus, chitosan could be developed as alternative biocompatible antimicrobial irrigant/medication for endodontic treatments.

In vitro differentiation profile of osteoblasts derived from patients with Saethre-Chotzen syndrome.

Seathre-Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis syndrome, associated with loss-of-function mutations in the basic helix-loop-helix transcription factor, TWIST1. The biologic activity of TWIST1 has been implicated in the inhibition of differentiation of multiple cell lineages. Therefore, premature fusion of cranial sutures (craniosynostosis) in SCS may be mediated by altered differentiation of calvarial osteoblasts. In this study, we evaluated osteoblasts derived from calvarial bone of three patients with SCS and three unaffected individuals as controls to investigate the principle stages of osteoblast differentiation: (1) proliferation, (2) matrix maturation, and (3) mineralization. Using a BrdU-Hoechst flow cytometry assay, we found that the percent of proliferating cells was significantly reduced in cells derived from patients with SCS compared with those derived from controls (P < or = 0.05). In the matrix maturation stage, alkaline phosphatase (ALP) enzyme activity and the expression of extracellular matrix genes, collagen I alpha 2 (COL1A2), osteopontin (OPN), osteocalcin (OC), and the runt-related transcription factor RUNX2 were examined by enzymatic assay and real-time quantitative RT-PCR, respectively. We identified no significant differences in the expression of matrix related transcripts. However, we found significant reductions in ALP activity on days 3 and 7 and in RUNX2 expression on days 14 and 21 (P < or = 0.05). Quantitative alizarin red S mineralization assays showed a trend toward increased mineralization in osteoblasts derived from patients with SCS at days 21 and 28, although not statistically significant. Our results demonstrated that loss-of-function mutations of TWIST1 led to reduced proliferation regardless of the functional domain affected. We did not find any conclusive differences in matrix maturation or mineralization in these primary osteoblasts. It is plausible that mutations in different functional domains of TWIST1 have divergent effects on these later stages of differentiation.

Effects of FGF-2/-9 in calvarial bone cell cultures: differentiation stage-dependent mitogenic effect, inverse regulation of BMP-2 and noggin, and enhancement of osteogenic potential.

Systemically administered fibroblast growth factors (FGFs) show anabolic effects on bone formation in animals, whereas in vitro cell culture studies have demonstrated that FGFs block mineralized bone nodule formation. These apparently contradictory outcomes indicate that the nature of FGF action is complex and that the biological effect of FGFs may depend on the differentiation stage of osteoblasts, interaction with other cytokines, or the length and mode of exposure to factors. Thus, we have utilized primary calvarial bone cell populations at different maturation phases to determine their responses to 2, FGF-9, and BMP-2, the factors expressed in bone. FGF-2 and FGF-9 stimulated proliferation of the cell populations consisting of more mature osteoblasts, but not those with undifferentiated precursor cells. Continuous treatment with FGF-2/-9 inhibited expression of several osteoblast marker genes and mineralization. However, brief pretreatment with FGF-2/-9 or sequential treatment with FGF-2/-9 followed by BMP-2 led to marked stimulation of mineralization, suggesting that FGFs enhance the intrinsic osteogenic potential. Furthermore, FGF-2 and FGF-9 increased expression of other osteogenic factors BMP-2 and TGFbeta-1. Meanwhile, blocking endogenous FGF signaling, using a virally transduced dominant-negative FGF receptor (FgfR), resulted in drastically reduced expression of the BMP-2 gene, demonstrating for the first time that endogenous FGF/FgfR signaling is a positive upstream regulator of the BMP-2 gene in calvarial osteoblasts. In contrast, expression of a BMP antagonist noggin was inhibited by FGF-2 and FGF-9. Thus, collective data from this study suggest that FGF/FgfR signaling enhances the intrinsic osteogenic potential by selectively expanding committed osteogenic cell populations as well as inversely regulating BMP-2 and noggin gene expression.

Syndromic craniosynostosis: from history to hydrogen bonds.

The syndromic craniosynostoses, usually involving multiple sutures, are hereditary forms of craniosynostosis associated with extracranial phenotypes such as limb, cardiac, CNS and tracheal malformations. The genetic etiology of syndromic craniosynostosis in humans is only partially understood. Syndromic synostosis has been found to be associated with mutations of the fibroblast growth factor receptor family (FGFR1, -R2, -R3), TWIST1, MSX2, and EFNB1. Apert, Pfeiffer, Crouzon, and Jackson-Weiss syndromes are due to gain-of-function mutations of FGFR2 in either the Ig II-III linker region (Apert) or Ig III domain. Loss of function mutations of TWIST1 and gain-of-function mutations of MSX2 lead to Saethre-Chotzen and Boston-type syndromes, respectively. The mutations in Pfeiffer (FGFR1), Muenke (FGFR3), and Apert syndrome (FGFR2) are caused by the same amino acid substitution in a highly conserved region of the Ig II-III linker region of these proteins, which suggests that these receptor tyrosine kinases have an overlapping function in suture biology. In this review we will discuss the historical descriptions, current phenotypes and molecular causes of the more common forms of syndromic craniosynostosis.

Activating (P253R, C278F) and dominant negative mutations of FGFR2: differential effects on calvarial bone cell proliferation, differentiation, and mineralization.

Various activating mutations of FgfR2 have been linked to a number of craniosynostosis syndromes, suggesting that FGFR2-mediated signaling plays significant roles in intramembranous bone formation. To define (i) the roles of FGFR2-mediated signaling in osteogenesis and (ii) bone cell functions affected by abnormal signaling induced by craniosynostosis mutations, chicken calvarial osteoblasts were infected with replication competent avian sarcoma viruses expressing FgfR2 with dominant negative (DN), P253R (Apert), or C278F (Pfeiffer and Crouzon) mutation. Analyses of the infected osteoblasts revealed that attenuated FGF/FGFR signaling by DN-FgfR2 resulted in a decrease in cell proliferation and accelerated mineralization. In contrast, the C278F mutation, which causes ligand-independent activation of the receptor, significantly stimulated cell proliferation and inhibited mineralization. Interestingly, the P253R mutation, which does not cause ligand-independent activation of the receptor, showed a weaker mitogenic effect than the C278F mutation and did not inhibit mineralization. Gene expression analysis also revealed diverse effects of C278F and P253R mutations on expression of several osteogenic genes. Based on these results, we conclude that one of the major functions of FGFR2 is to mediate mitogenic signals in osteoblasts and that distinctively different cellular mechanisms underlie the pathogenesis of craniosynostosis phenotypes resulting from P253R and C278F mutations of the FGFR2 gene.

Capillary sieving electrophoresis/micellar electrokinetic capillary chromatography for two-dimensional protein fingerprinting of single mammalian cells.

We have developed a two-dimensional capillary electrophoresis method for the study of protein expression in single mammalian cells. The first-dimension capillary contains an SDS-pullulan buffer system to perform capillary sieving electrophoresis, which separates proteins based on molecular weight. The second-dimension capillary contains an SDS buffer for micellar electrokinetic capillary chromatography. After a 6-min-long preliminary separation, fractions from the first capillary are successively transferred to a second capillary, where they undergo further separation by MECC. Over 100 transfers and second-dimension separations are performed over an approximately 3.5-h-long period. We demonstrate this technology by generating protein fingerprints from single native MC3T3-E1 osteoprogenitor cells and MC3T3-E1 cells transfected with the human transcription regulator TWIST. We also present single-cell protein fingerprints from MCF-7 breast cancer cells before and following treatment to induce apoptosis.