University of Birmingham The Relationship of Surface Characteristics and Antimicrobial Performance of Pulp Capping Materials Farrugia, Cher; Lung, Christie Y K; Schembri Wismayer, Pierre; Arias-Moliz, Maria Teresa; Camilleri, Josette DOI: 10.1016/j.joen.2018.04.002 License: Creative Commons: Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) Document Version Peer reviewed version Citation for published version (Harvard): Farrugia, C, Lung, CYK, Schembri Wismayer, P, Arias-Moliz, MT & Camilleri, J 2018, 'The Relationship of Surface Characteristics and Antimicrobial Performance of Pulp Capping Materials', Journal of Endodontics. https://doi.org/10.1016/j.joen.2018.04.002 Link to publication on Research at Birmingham portal General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. • Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. When citing, please reference the published version. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact [email protected] providing details and we will remove access to the work immediately and investigate. Download date: 08. Aug. 2020
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University of Birmingham
The Relationship of Surface Characteristics andAntimicrobial Performance of Pulp CappingMaterialsFarrugia, Cher; Lung, Christie Y K; Schembri Wismayer, Pierre; Arias-Moliz, Maria Teresa;Camilleri, JosetteDOI:10.1016/j.joen.2018.04.002
Citation for published version (Harvard):Farrugia, C, Lung, CYK, Schembri Wismayer, P, Arias-Moliz, MT & Camilleri, J 2018, 'The Relationship ofSurface Characteristics and Antimicrobial Performance of Pulp Capping Materials', Journal of Endodontics.https://doi.org/10.1016/j.joen.2018.04.002
Link to publication on Research at Birmingham portal
General rightsUnless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or thecopyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposespermitted by law.
•Users may freely distribute the URL that is used to identify this publication.•Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of privatestudy or non-commercial research.•User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?)•Users may not further distribute the material nor use it for the purposes of commercial gain.
Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document.
When citing, please reference the published version.
Take down policyWhile the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has beenuploaded in error or has been deemed to be commercially or otherwise sensitive.
If you believe that this is the case for this document, please contact [email protected] providing details and we will remove access tothe work immediately and investigate.
Moliz MT: Microbiology, protocols, writing and editing manuscript; Camilleri J.:
Characterization, writing, editing, finalizing manuscript and originator of idea.
Corresponding author:
J. Camilleri School of Dentistry College of Medical and Dental Sciences Institute of Clinical Sciences University of Birmingham Birmingham United Kingdom [email protected]
Table 1A. Viable counts expressed as mean Log10 (CFU + 1/mL) (standard deviation) of the polymicrobial suspension after being in contact with the materials.
Log10
Positive control 4.42 (0.08)a
LCS 3.45 (0.20)b
LCS+TCS 3.25 (0.32)b
LCS+TCS+TO 3.38 (0.19)b
Theracal 3.31 (0.35)b
Biodentine 0c
Read vertically, different superscript letters indicate statistical differences by Tukey test after ANOVA showed significant values.
Table 1B. Mean of the total biovolume (µm3) and the red (dead) percentage of the bacteria grown on the materials surface after 7 days.
SD: standard deviation. Read vertically, different superscript letters indicate statistical differences by Tukey test after ANOVA showed significant values.
Highlights
• Light curable pulp capping materials are essential to improve the bonding to overlying resin-
based restorations thus reducing micro leakage
• The surface characteristics of a material affect its antimicrobial properties.
• The experimental light curable materials exhibited comparable antimicrobial properties to other
light curable pulp capping agents.
Highlights (for review)
Clinical Significance
Materials which posses antimicrobial properties are ideal for pulp capping. The surface
characteristics may effect the bacterial attachment.
Figure 1a: Secondary electron scanning electron micrographs of test materials showing surface microstructure (Mag: 2K X and inset for Biodentine 10K X)
Figure 1b: X-‐ray diffraction plots of test materials obtained by scanning the material surface at a 3° angle showing the surface constitution of the materials.
2. The experimental setup seems to be useful to test the surface of a material exposed to the oral
environment. Nevertheless, it is not the ideal setup to evaluate the anaerobic environment present between
infected dentin and composite. Perhaps dentine disks could be a better choice to test the interaction with
bacteria. Moreover, the evaluation of an anaerobic environment could be useful to reproduce clinical
conditions. The same microorganism shows different behavior in presence of diverse O2 partial pressure
conditions. Reply: In this study, a first step on the antimicrobial activity of 2 already launched and 1 prototype pulp
capping materials was studied by exposing them to a polymicrobial bacteria suspension. We agree with the
reviewer´s first comment and further research on the interaction between the materials, dentine and the
bacteria should be performed.
Regarding the incubation conditions, the reviewer is right and streptococci has been shown to exhibit
different behaviours under different environment conditions (oxygen, time of incubation, etc). In fact, a more
efficient growth is experienced by the organism with deprived oxygen conditions. In this study, we chose an
anaerobic atmosphere to mimic the conditions under restorations where the availability of oxygen is
generally very low. This was highlighted further in the manuscript.
Mjör IA. The location of clinically diagnosed secondary caries. Quintessence Int, 1998;29:313-7.
Ahn S, Ahn S, Browngardt CM, Burne RB. Changes in Biochemical and Phenotypic Properties
of Streptococcus mutans during Growth with Aeration. Appl Environ Microbiol. 2009; 75: 2517–2527.
3. No detailed explanation of sample preparation is present in the text. A table with the description of
the materials used in the study should be included in the paper (in particular for the resin-based
experimental materials).
Reply: These materials have already been described in other published work. A reference has been
added to direct the reader to the description of the materials used. 4. Moreover, the M&M section doesn't include any information about the curing procedures (i.e light
source, wavelength, time, distance from the light source). It has been therefore demonstrated that the light
curing procedure is a very important variable in the study of the biological behavior of resin-based
materials.
Reply: The curing procedures have been addressed in the Material and Methods section of the manuscript. 5. The description of the specimens preparation is not clear. Authors stated: "An area of established
dimensions on one side of the wells of a 96-well microtiter plate (Nunclon Delta Surface; Nunc, Roskilde,
Denmark) was coated with an equal amount of the materials". The Reviewer can't understand if the
material tested was placed on the bottom of the wells (atp test) or on the vertical wall. In the second case,
Authors should better describe the procedure and explain how did they determine the area and the
thickness of the specimens.
Reply: In the direct contact test (DCT), the materials were placed on the vertical wall of the wells. In order
to ensure the equal coating of the sealer, the area of the wall well that was coated was delimited by
measuring two points of the edge of the well separated by 4 mm. The materials were placed on this area
(from the edge of the well until the beginning of the round bottom) with a thickness of 1 mm, using a sterile
spatula and a calibre.
These details have been included in the Material and Methods section.
6. No washing procedure was performed after the polymerization of the resin specimens. This
procedure is very important since it has been shown that resins specimens often release unpolymerized
monomers both in the biofilm and in the medium, thus influencing the results of the experiment. Authors
speculations on the behavior and composition of the biofilm obtained on the specimens surfaces are
probably influenced by this relevant bias.
Reply: A previous study that evaluated the antimicrobial activity of 24 hour leachates of these materials
showed that at this time the antimicrobial activity is not dependant on the leachates but might be influenced
by their pH. However, in this study the results in the ATP assay and the CLSM showed that the Theracal
and the experimental material exerted a higher antimicrobial activity compared to Biodentine after 7 days of
exposure to the bacteria suspension. This higher antimicrobial activity could be related to the toxicity of the
unpolymerized monomers release of both after longer time of exposure. A comment on this and two new
references have been included in the Discussion.
Arias-Moliz MT, Farrugia C, Lung CYK, Wismayer PS, Camilleri J. Antimicrobial and biological activity of
Jeanneau C, Laurent P, Rombouts C, Giraud T, About I. Light-cured Tricalcium Silicate Toxicity to the
Dental Pulp. J Endod. 2017 Dec;43(12):2074-2080.
7. How did Authors avoid specimens contamination? No sterilization procedures were described in
the M&M section. A microbiological check on the final biofilms seems to be mandatory. Reply: The reviewer is right and no information regarding the sterilization procedures and the negative
controls were mentioned. All samples in the antimicrobial activity tests were exposed to ultraviolet light for 1
hour for sterilization. Negative controls were included in the DCT and in the ATP and CLSM tests to check
their sterility. This has been included in the Material and Methods and in the Results sections.
8. SEM microphotographs quality is not excellent. Some overexposure problems occurred.
Reply: The scanning electron micrographs are taken in secondary electron mode to be able to assess the
material surface well. The material surfaces are obviously rough as the materials are hand mixed and
applied by hand. The resins are non conductive and thus the current quality of the imaging.
9. Why did Authors applied the Anscombe transformation only to CLSM data? How the normality
(needed to use ANOVA) of the other sets of data was checked?