Optimizing Methods for Bovine Dental Pulp Decellularization

IntroductionThis study aimed to characterize the decellularization effects of different treatment protocols on the bovine dental pulp extracellular matrix (ECM) for tissue regeneration.MethodsSeven different decellularization protocols consisting of trypsin/EDTA (for 1 hour, 24 hours, or 48 hours), sodium dodecyl sulfate (SDS, for 24 hours or 48 hours), Triton X-100 (for 1 hour), and deoxyribonuclease treatments were tested on bovine dental pulp tissue. The posttreatment samples were evaluated for remaining DNA and cellular contents, structural durability, immunofluorescence analysis, and in vivo immune responses.ResultsA complete decellularization process in all of the experimental groups was observed. The protocol that included 1 hour of Triton X-100 treatment and 12 hours of trypsin/EDTA treatment with no SDS treatment (P7 [12E-0S-1T]) showed the highest retention of glycosaminoglycan and the absence of nuclei in 4,6-diamidino-2-phenylindole. All groups showed significantly lower DNA content compared with native pulp tissue (P < .05), whereas compared with other protocols, protocols 1 (1 hour of EDTA/trypsin, 24 hours of SDS, and 1 hour of Triton X-100) and 4 (1 hour of EDTA/Trypsin, 48 hours of SDS, and no Triton X-100) resulted in the highest DNA contents (P < .05). Based on these results, P7 was further evaluated by immunofluorescence and in vivo immunogenicity. P7 specimens preserved collagen type I, whereas mononuclear cell infiltration along with neovascularization was observed in vivo.ConclusionsAll tested treatments displayed the potential ability to decellularize pulp tissue and are viable options for a xenogeneic dental pulp ECM scaffold. The P7 (12E-0S-1T) protocol resulted in decellularized ECM with minimal organic matrix/ultrastructural detriments and an acceptable host immune response.     

Electrochemical behaviour of certain biomimetic copper(II) complexes in aqueous and aqueous micellar solutions

Voltammetric studies of biomimetic Cu(II) complexes of bis(pyrid-2-yl)dithia (N₂S₂) and -trithia (N₂S₃) and bis(benzimidazol-2-yl)dithia ligands in aqueous and sodium dodecylsulphate (SDS, anionic), cetyltrimethylammonium bromide (CTAB, cationic) and Triton X-100 [α-[p-(1,1,3,3-tetramethylbutyl)phenyl]-w-hydroxypolyoxyethylene(9.5), nonionic] micellar solutions have been carried out on a glassy carbon electrode. The electronic spectral properties of Cu(II) complexes in micellar solutions are consistent with their incorporation into hydrophobic microenvironments. For most of the complexes, the adsorption of Cu(I) species observed in aqueous solution is absent and the reversibility of the Cu(II)/Cu(I) couple is increased in SDS and Triton X-100 micellar solutions. From the dependence of I_pc values on SDS concentration, the micellar binding constant K_M has been estimated. Ligand enlargement and the structure of the Cu(II) complexes and Cu(I) forms of complexes enhance the binding with micelles. Compared with aqueous isotropic solutions, the E_1/2 values in micellar solutions either decrease or increase depending upon the nature of the surfactant and the hydrophobicity and structure of the complexes. The ratio of equilibrium binding constants K₊/K₂₊ of Cu(I) and Cu(II) species does not follow the same trend in SDS, CTAB and Triton X-100 solutions, indicating that the interaction of the cationic complexes depends on a delicate balance between hydrophobic and electrostatic interactions. Interestingly, for complexes with the CuN₂S₂ chromophore the Cu(I)/Cu(0) couple is also observed in Triton X-100 micelles.     

Antimicrobial and antiplaque effects of a chlorhexidine and Triton X-100 combination

Abstract – This study aimed to assess the in vitro antimicrobial effect and in vivo plaqueinhibiting capacity of chlorhexidine (CH) combined with the nonionic detergent Triton X-100. Synergistic inhibition was observed by the combination of Triton X-100 and CH on in vitro growth of S. sobrinus OMZ 176 and of S. sanguis 10556. In the clinical experiments, 10 subjects rinsed twice dally with 10 ml aqueous solutions of 11.6 mM Triton X-100, and 0.55 mM CH, and with a combination of the two agents. All mechanical oral hygiene was suspended during the test periods. Sucrose enhanced plaque accumulations were recorded after 4 days of rinsing. The combination of CH-Triton X-100 decreased plaque accumulations significantly compared to placebo, but was less effective than CH alone ( P <0.05). Thus no beneficial clinical effect was gained by combining the nonionic agent Triton X-100 with the cationic agent CH. The results also clearly demonstrate that additions which increase the antibacterial activity of CH do not necessarily coincide with improved in vivo antiplaque efficacy.     

Automutanolysin disrupts clinical isolates of cariogenic streptococci in biofilms and planktonic cells

Introduction:  Dental caries remains one of the most common chronic infectious diseases throughout the world. The formation of dental plaque is one of the caries risk factors. As a consequence, the removal of plaque may reduce the incidence of caries development. We identified an autolysin produced by Streptococcus mutans named auto-mutanolysin (Aml). Aml selectively lyses S. mutans and Streptococcus sobrinus . The specificity towards these cariogenic bacteria suggests that Aml may be used to prevent dental caries. Here, with the aim towards therapeutic application, we investigated the lytic activity of Aml against clinical isolates of S. mutans and S. sobrinus using planktonic cells and biofilms.
Methods:  Planktonic cell suspensions and biofilms of clinically isolated streptococci were treated with Aml in the absence or the presence of Triton X-100. The lytic activity of Aml was monitored as the change in turbidity. The disruption of biofilms was evaluated by detecting the released DNA by polymerase chain reaction and observing the alteration of optical density of treated biofilms.
Results:  Triton X-100 enhances the lytic ability of Aml. Using planktonic cells, Aml had various lysis levels against clinical strains. Repeated Aml treatment showed disruption of the biofilm using the representative clinical strains.
Conclusion:  Our study demonstrates that Aml has an ability to lyse planktonic and biofilm cells of clinically isolated mutans streptococci in the presence of Triton X-100. These results suggest the possibility of using Aml as an alternative or additional approach for caries prevention.     

Antimicrobial and antiplaque effects of a chlorhexidine and Triton X-100 combination

This study aimed to assess the in vitro antimicrobial effect and in vivo plaque-inhibiting capacity of chlorhexidine (CH) combined with the nonionic detergent Triton X-100. Synergistic inhibition was observed by the combination of Triton X-100 and CH on in vitro growth of S. sobrinus OMZ 176 and of S. sanguis 10556. In the clinical experiments, 10 subjects rinsed twice daily with 10 ml aqueous solutions of 11.6 mM Triton X-100, and 0.55 mM CH, and with a combination of the two agents. All mechanical oral hygiene was suspended during the test periods. Sucrose enhanced plaque accumulations were recorded after 4 days of rinsing. The combination of CH-Triton X-100 decreased plaque accumulations significantly compared to placebo, but was less effective than CH alone (P less than 0.05). Thus no beneficial clinical effect was gained by combining the nonionic agent Triton X-100 with the cationic agent CH. The results also clearly demonstrate that addition which increase the antibacterial activity of CH do not necessarily coincide with improved in vivo antiplaque efficacy.     

Developmental expression of vascular endothelial growth factor in the masseter muscle of rats

Developmental changes in vascular endothelial growth factor (VEGF) in rat masseter after birth were investigated. VEGF was extracted efficiently and reproducibly from muscle homogenate with low concentrations of non-ionic detergents, such as Triton X-100, Nonidet P-40, and Tween 20. The amount of VEGF measured by enzyme-linked immunosorbent assay (ELISA) increased markedly by approximately 9-fold, from day 8 to 35 after birth. The increase in VEGF was closely correlated with the development of the capillary network, as shown by the capillary to muscle fibre ratio (C/F ratio). Immunoblotting revealed that the predominant molecular species of VEGF concentrated with heparin-sepharose beads was VEGF(188). These results suggest that VEGF plays an important part in the development and maintenance of the capillary network in the rat masseter.     

Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms

The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W), suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS; pH 5.0) and homogenization-mechanical cells disruption in NAES- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg) of crude preparation of RNA per 100 mg of total cell (or biofilm) dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg) of purified RNA per 100 mg of total cell (or biofilm) dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses.     

A comparative kinetic investigation of ethylcinnamate electrohydrodimerization on mercury and liquid gallium

Electrohydrodimerization (EHD) of ethylcinnamate (EC) on mercury takes place both from hydrotropic solutions of tetraethylammonium-p-toluenesulphonate (TEA-PTS) and from dilute aqueous solutions of the strong surfactant Triton X-100. In both cases the one-electron reduction wave due to hydrodimer formation satisfies the requirements for a rate-determining homogeneous coupling of the electrochemically generated anion radicals. The kinetics of EC EHD on liquid gallium is identical with that on mercury at the same temperature of 31°C in hydrotropic solutions of TEA-PTS; conversely, in aqueous Triton X-100 it is faster than on mercury and satisfies the requirements for a rate-determining heterogeneous radical-radical coupling. This behaviour is explained by a stronger adsorptivity of EC and its intermediate reduction products on gallium than on mercury, so that Triton X-100 does not succeed in displacing these species completely from the electrode surface. The general trend consisting in a higher adsorptivity of organic compounds with conjugated double bonds and/or aromatic rings on gallium than on mercury is explained on the basis of the difference in hydrophilicity between these two metals.     

The relative amounts of free and latent acid hydrolases in homogenates of human gingiva.

Acid phosphatase, β-glucuronidase, cathepsin D and alkaline phosphatase were determined quantitatively in cytoplasmic extracts of human gingiva. Acid phosphatase showed maximal activity at pH 4.8 and a K_m value of 0.035 mM. β-Glucuronidase had an optimum pH at 3.6 with a K_m value of 0.75 mM. The optimum pH for gingival alkaline phosphatase was 9.0.Samples of cytoplasmic extract were subjected to several disruptive procedures: treatment with Triton X-100, freezing and thawing, and treatment with acid pH. The extent of disruption was different with the above treatments; only Triton X-100 was able fully to activate the enzyme release.A variable degree of latency (25–40 per cent) was found when measuring free to total activities of the three lysosomal enzymes. Alkaline phosphatase showed a slight but constant degree of latency.     

Effect of sodium hypochlorite irrigation with or without surfactants on the bond strength of an epoxy-based sealer to dentin

Objectives

This study evaluated the effect of sodium hypochlorite (NaOCl) irrigation with or without surfactants on the bond strength of an epoxy-based sealer to the root canal dentin.

Materials and methods

Eighty decoronated single-rooted human mandibular premolars were instrumented using the rotary system. The roots were subsequently rinsed with 5 ml 17 % EDTA for 1 min and then randomly divided into 3 test groups (n = 20) and 1 control group (n = 20) according to the type of irrigation with experimental 5 % NaOCl (Wizard, RehberKimya, Istanbul, Turkey) solutions: Group 1: NaOCl-0.1 % benzalkonium chloride; Group 2: NaOCl-0.1 % Tween 80; Group 3: NaOCl-0.1 % Triton X-100; control group: NaOCl without any surfactants. Five samples from each group were prepared for scanning electron microscopy to examine the surface of root canal dentin. The 15 samples remaining in each group were obturated with gutta-percha and AH Plus (Dentsply DeTrey GmbH, Konstanz, Germany) using the cold lateral compaction technique. A push-out test was used to measure the bond strength between the sealer and root canal dentin. Data were analyzed using two-way analysis of variance and Tukey’s post hoc tests (P = 0.05).

Results

The NaOCl-0.1 % Triton X-100 group demonstrated the highest mean bond-strength values in all root thirds among the groups (P < 0.05). However, the bond strength of the sealer in the NaOCl-0.1 % benzalkonium chloride and NaOCl-0.1 % Tween 80 groups did not differ from that in the control group (P > 0.05). Additionally, the bond-strength values decreased in the corono-apical direction for all groups (P < 0.05).

Conclusions

NaOCl solution with Triton X-100 can provide higher bond strength of the epoxy resin-based sealer to root dentin compared to NaOCl solution wiithout any surfactant.

Clinical relevance

The bond strength of sealer to dentin can be improved by the addition of the surfactants to NaOCl solution.

     

3种氟素制剂对致龋链球菌生长抑制作用的比较研究

目的 :比较 380 g/LAg(NH3 ) 2 F、10 0 g/L (NH4)MoO2 F4和APF -LaCl3 对变形链球菌In gbritt(C)和远缘链球菌KI(g)的生长抑制作用。方法 :涂布 1.5× 10 8/mLIngbritt(C)和KI(g)菌液于心脑浸液琼脂培养基 ,用直径 6mm无菌滤纸片分别蘸 10 0 g/L(NH4)MoO2 F4、380 g/LAg(NH3 ) 2 F、APF、LaCl3 、APF +LaCl3 及生理盐水贴于培养基上 ,37℃微需氧培养 4 8h ,测量抑菌圈直径 ,比较分析。结果 :380g/LAg(NH3 ) 2 F ,10 0 g/L(NH4)MoO2 F4,9g/LAPF对Ingbritt(C)和KI(g)均有抑制作用 ,其中 380 g/LAg(NH3 ) 2 F最强 ,9g/LAPF最弱 ;而LaCl3 、APF +LaCl3 对Ingbritt(C)和KI(g)生长无抑制作用。 结论 :380 g/LAg(NH3 ) 2 F、10 0 g/L(NH4)MoO2 F4和APF -LaCl3 3者中 ,380g/LAg(NH3 ) 2 F抑菌作用最强 ,APF -LaCl3 最弱     

Factors influencing the growth and viability of Actinobacillus actinomycetemcomitans

The metabolic requirements for the routine growth of Actinobacillus actinomycetemcomitans were investigated by the addition of nutrients to conventional bacteriological and tissue culture media. Commonly used tissue culture media required fetal bovine serum as an additive to sustain bacterial growth rates comparable to those obtained with bacteriological media. The addition of increasing concentrations of yeast extract to bacteriological medium increased the growth rate of several A. actinomycetemcomitans strains. In an attempt to identify the components of yeast extract that enhanced the growth of A. actinomycetemcomitans , a number of vitamins, essential and non-essential amino acids were tested for their role in promoting growth. The addition of L-cystine resulted in bacterial growth rates comparable to those with yeast extract. Thiamine increased the growth of several A. actinomycetemcomitans strains but did not result in growth rates comparable to those with yeast extract. The addition of physiological concentrations of steroid hormones to bacteriological medium enhanced the growth of A. actinomycetemcomitans. Additional iron compounds and fat-soluble vitamins had no influence on A. actinomycetemcomitans growth. However, the requirement of iron for bacterial growth remains unclear. The optimal pH range for growth of A. actinomycetemcomitans was between pH 7.0–8.0 in a medium containing 0.5–1% Nacl. Several interesting observations on the viability of A. actinomycetemcomitans were made. A rapid reduction of A. actinomycetemcomitans viability occurred following suspension in distilled water. The presence of the detergent Triton X-100 at concentrations above 2% (v/v) also decreased the viability of A. actinomycetemcomitans within 10 min.     

PERIOSTIN regulates MMP-2 expression via the αvβ3 integrin/ERK pathway in human periodontal ligament cells

ObjectiveDuring orthodontic tooth movement, activation of the vascular system in the compressed periodontal ligament (PDL), which becomes hypoxic, is essential for periodontal tissue remodelling. PERIOSTIN, an extracellular matrix protein, is expressed in PDL and its concentration is increased on the compressive side during orthodontic tooth movement. PERIOSTIN promotes angiogenesis through upregulation of matrix metalloproteinase (MMP)-2, which has been shown to be expressed via αvβ3 integrin/extracellular signal-related kinase (ERK) signalling pathway and vascular endothelial growth factor (VEGF). Therefore, we hypothesized that hypoxia-induced PERIOSTIN promotes MMP-2 expression via αvβ3 integrin/ERK signalling and VEGF in PDL cells.MethodsHuman PDL cells were cultured in condition medium containing desferrioxamine (DFO) to mimic hypoxia. The total RNA, cell lysates or supernatant were collected, and MMP2 and VEGF expression, PERIOSTIN expression and ERK phosphorylation, and MMP-2 activity were analysed by real-time RT-PCR, western blot analysis, and zymography, respectively. A recombinant human PERIOSTIN or PERIOSTIN siRNA was applied to the cells, then the total RNA was extracted to measure MMP-2 and VEGF expression. The cells were treated with αvβ3 integrin-blocking antibody or ERK inhibitor followed by PERIOSTIN stimulation. MMP-2 expression was measured by real-time RT-PCR.ResultsPERIOSTIN was upregulated in a time-dependent manner in human PDL cells treated with DFO, a chemical hypoxia mimic. MMP-2 and VEGF expression, and MMP-2 activity were increased by DFO or PERIOSTIN treatment, and decreased by PERIOSTIN silencing. PERIOSTIN treatment also induced ERK phosphorylation, and PERIOSTIN-induced MMP-2 was reduced by αvβ3 integrin-blocking antibody or ERK inhibitor.ConclusionThese data suggest that PERIOSTIN upregulates MMP-2 expression via the αvβ3 integrin/ERK signalling pathway and VEGF expression in human PDL cells.     

Cytotoxicity of two bonding adhesives assessed by three-dimensional cell culture

OBJECTIVE: To determine the toxicity of orthodontic adhesives assessed on in vitro three-dimensional reconstructed human oral epithelium (RHOE). MATERIALS AND METHODS: Two adhesive primers, Transbond XT (3M, Monrovia, Calif) and Excite (Vivadent, Schaan, Liechtenstein), were tested. After topical exposure, the cell cultures were fixed, cut, and stained for light microscopy (LM) and transmission electron microscopy (TEM). Detection of cytotoxicity by measuring lactate dehydrogenase (LDH) activity was performed. Toxicity was assessed by evaluating the morphological changes with LM and TEM. Copper wires and Triton X-100 served as positive controls, and native cell cultures as negative control. RESULTS: Morphological evaluation of the native cell cultures revealed no toxic reactions. The LDH assay revealed the following mean values for viability: native cell line (negative control), 1.51; Triton X-100 (positive control), 3.06; Transbond XT polymerized, 1.15; Excite polymerized, 1.11; Transbond XT primer, 2.67; and Excite primer, 0.04. Acute toxicity was observed for Triton X-100 and Transbond XT primer (P < .001). Histological evaluation of the RHOE showed toxicity for both primers and mild changes after topical application of polymerized adhesives. The biocompatibility ranking of the adhesive primers was the same after histological analysis and LDH assay except for Excite noncured. CONCLUSIONS: RHOE proved to be a valuable model for topical exposure. The toxicity of both uncured primers was demonstrated.     

Pulp ECM-derived macroporous scaffolds for stimulation of dental-pulp regeneration process

ObjectiveRecent studies suggest xenogeneic extracellular matrices as potential regenerative tools in dental pulp regeneration. This study aimed to fabricate and characterize a novel three-dimensional macroporous pulp-derived scaffold that enables the attachment, penetration, proliferation and differentiation of mesenchymal stem cells.MethodBovine pulp was decellularized and characterized with histological and DNA content methods. This scaffold was prepared using finely milled lyophilized decellularized pulp extracellular matrix (ECM) digested with pepsin. Three different concentrations of ECM (1.50, 2.25 and 3.00 mg/ml) were freeze-dried and were tested with/without chemical crosslinking. The specimens were subjected to physicochemical characterization, cell viability and quantitative real time polymerase chain reaction assessments with human bone marrow mesenchymal stem cells (hBMMSCs). All scaffolds were subcutaneously implanted in rats for two weeks and histological and immunostaining analyses were performed.ResultsHistological and DNA analysis confirmed complete decellularization. All samples demonstrated more than 97% porosity and 1.50 mg/ml scaffold demonstrated highest water absorption. The highest cell viability and proliferation of hBMMSCs was observed on the 3.00 mg/ml crosslinked scaffolds. The gene expression analysis showed a significant increase of dmp-1 and collagen-I on 3.00 mg/ml crosslinked scaffolds compared to the other scaffolds. Histological examination of subcutaneous implanted scaffolds revealed low immunological response, and enhanced angiogenesis in cross-linked samples compared to non-crosslinked samples.SignificanceThe three-dimensional macroporous pulp-derived injectable scaffold developed and characterized in this study displayed potential for regenerative therapy. While the scaffold biodegradability was decreased by crosslinking, the biocompatibility of post-crosslinked scaffold was significantly improved.     

Extracellular deoxyribonuclease production by periodontal bacteria

Palmer LJ, Chapple ILC, Wright HJ, Roberts A, Cooper PR. Extracellular deoxyribonuclease production by periodontal bacteria. J Periodont Res 2012; 47: 439–445. © 2011 John Wiley & Sons A/S Background and Objective: Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host’s immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. Material and Methods: To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA‐supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Results: Twenty‐seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. Conclusion: The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.     

Multiple forms of acid phosphatase in rat secretory enamel organ

Abstract – Acid phosphatase activity was studied biochemically in homogenates of secretory enamel organs from the rat. Incubations with crude homogenate failed to show distinct pH optima or kinetics characteristic for single enzymes. Crude homogenate activity was strongly inhibited by concentrations higher than 1 mM of NaF and Na-tartrate, and higher than 10 mM of ZnSO₄ and p -bromotetramisole oxalate. lOmM MgCl₂ gave a slight stimulation. CaCl₂, KCl and EDTA were uneffective. Electrophoretic separation of the crude homogenate acid phosphatase on Triton X-100 containing polyacrylamide gel demonstrated the presence of atleast three multiple forms of the enzyme. Two of them showed distinct pH optima at pH 4.4. The third one showed a broad pH plateau in the acid pH range. Kinetic studies of the three forms indicated single enzyme reactions. Two forms had electrophoretic mobilities similar to alkaline phosphatase. One form could be solubilized only after Triton X-100 treatment. All forms were strongly sensitive to 10 mM NaF when added to the reaction mixture. The sensibility to 10 mM ZnSO₄, CuSO₄, Na-tartrate and p -bromotetramisole oxalate differed between the different forms.     

The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.     

Effect of Triton X-100 on electron transfer kinetics at the interface between two immiscible electrolyte solutions: a scanning electrochemical microscopy study

Adsorption of the nonionic surfactant, Triton X-100, at the interface between two immiscible electrolyte solutions (ITIES) and its effect on the oxidation of decamethyl ferrocene in 1,2-dichloroethane by aqueous Ru(CN)₆^3? has been investigated using scanning electrochemical microscopy. As the concentration of Triton X-100 in the aqueous phase was increased from 0 to 2.5×10^?4 M, the rate constant for electron transfer (ET) across the ITIES decreased in a manner consistent with the surfactant blocking the area available for the reaction. The decrease in rate constant with increasing surfactant concentration was successfully analyzed in terms of Langmuirian adsorption of the surfactant, with an equilibrium constant of (2.72±0.06)×10⁴ M^?1. The overall behavior observed indicates that the ET reaction occurs primarily at the portion of the ITIES free from surfactant.     

Characterization and evaluation of ascorbic acid-induced cell sheet formation in human periodontal ligament stem cells: An in vitro study

ObjectivesPeriodontal ligament-derived stem cells (PDLSCs) are regarded as a viable option for periodontal regeneration using cell sheet technology. The objective of the present in vitro study was to characterize human PDLSCs based on their phenotypic and biological properties and to evaluate the ascorbic acid (AA or vitamin C)-induced cell sheet by analyzing the molecular markers.MethodsPDLSCs were established from premolars, and their morphology, viability, proliferation, phenotypic marker expression, and ability to differentiate into osteocytes and adipocytes were analyzed. PDLSCs were then induced to form cell sheets using 100 μM AA, and gene expression was examined by real-time polymerase chain reaction.ResultsPDLSCs showed fibroblastic morphology with >95% viability. The cells were highly proliferative and positive for surface antigens CD29, CD73, and CD90 but negative for CD34 and CD45. They were capable of differentiating into osteocytes and adipocytes. Induction with 100 μM AA transformed PDLSCs into two-to three-layered cell sheets. There was no significant upregulation in ALP and RUNX2 expression in the AA-induced cell sheet. However, the expression levels of late osteoblast differentiation marker (bone gamma-carboxy glutamate protein); cementogenic markers (cementum attachment protein and CP23), and genes encoding extracellular matrix (ECM) proteins [collagen type 1 alpha 1 and integrin beta 1) were higher in AA-induced cell sheets by PDLSCs.ConclusionsThe stimulating effect of AA on cell sheet formation by PDLSCs was confirmed by the expression of typical markers involved in osteogenesis/cementogenesis and ECM secretion, which makes this procedure a prospective option for periodontal tissue regeneration applications.