Publications by authors named "Paul R Cooper"

89 Publications

Potential of Lyophilized Platelet Concentrates for Craniofacial Tissue Regenerative Therapies.

Molecules 2021 Jan 20;26(3). Epub 2021 Jan 20.

Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.

Objective: The use of platelet concentrates (PCs) in oral and maxillofacial surgery, periodontology, and craniofacial surgery has been reported. While PCs provide a rich reservoir of autologous bioactive growth factors for tissue regeneration, their drawbacks include lack of utility for long-term application, low elastic modulus and strength, and limited storage capability. These issues restrict their broader application. This review focuses on the lyophilization of PCs (LPCs) and how this processing approach affects their biological and mechanical properties for application as a bioactive scaffold for craniofacial tissue regeneration.

Materials And Methods: A comprehensive search of five electronic databases, including Medline, PubMed, EMBASE, Web of Science, and Scopus, was conducted from 1946 until 2019 using a combination of search terms relating to this topic.

Results: Ten manuscripts were identified as being relevant. The use of LPCs was mostly studied in in vitro and in vivo craniofacial bone regeneration models. Notably, one clinical study reported the utility of LPCs for guided bone regeneration prior to dental implant placement.

Conclusions: Lyophilization can enhance the inherent characteristics of PCs and extends shelf-life, enable their use in emergency surgery, and improve storage and transportation capabilities. In light of this, further preclinical studies and clinical trials are required, as LPCs offer a potential approach for clinical application in craniofacial tissue regeneration.
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http://dx.doi.org/10.3390/molecules26030517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863735PMC
January 2021

Potential for direct application of blue light for photo-disinfection of dentine.

J Photochem Photobiol B 2021 Feb 9;215:112123. Epub 2021 Jan 9.

Institute of Clinical Sciences, School of Dentistry, University of Birmingham, 5 Mill Pool Way, Edgbaston, Birmingham B5 7EG, UK; Department of Oral Sciences, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand.

The direct application of light for photo-disinfection potentially provides a safe and novel modality to inhibit or eliminate cariogenic bacteria residing upon and within dentine. This study aimed to both; characterize the pattern of transmission of 405 nm light through molar dentine at different tooth locations, as well as, determine the irradiation parameters that are antibacterial for Streptococcus mutans under various growth conditions, including lawns, planktonic cultures, and biofilms. To determine the amount of light (405 nm) transmitted at different anatomical tooth locations; irradiance values were recorded after blue light (470-4054 mW/cm) had traversed through occlusal, oblique, and buccal dentine sections; and three thicknesses - 1, 2 and 3 mm were investigated. To determine tubular density; scanning electron micrographs from 2 mm outer (dentine-enamel junction) and inner (pulp) dentine sections were analysed. For photo-disinfection studies; S. mutans was irradiated using the same 405 nm wavelength light at a range of doses (110-1254 J/cm) in both biofilm and planktonic cultures. The inhibitory effect of the irradiation on bacterial lawns was compared by measuring zones of inhibition; and for planktonic cultures both spectrophotometric and colony forming unit (CFU) assays were performed. A live/dead staining assay was utilised to determine the effect of irradiation on bacterial viability in mature biofilms. Data indicated that increasing dentine thickness decreased light transmission significantly irrespective of its orientation. Occlusal and oblique samples exhibited higher transmission compared with buccal dentine. Oblique dentine 405 nm light transmission was comparable with that of occlusal dentine independent of section thickness. An increased tubule density directly positively correlated with light transmission. Irradiation at 405 nm inhibited S. mutans growth in both biofilm and planktonic cultures and a dose response relationship was observed. Irradiation at doses of 340 and 831 J/cm led to significant reductions in bacterial growth and viability; as determined by CFU counting and live/dead staining. Data suggests that phototherapy approaches utilising a 405 nm wavelength have therapeutic potential to limit cariogenic bacterial infections both at the surface and within dentine.
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http://dx.doi.org/10.1016/j.jphotobiol.2021.112123DOI Listing
February 2021

Inflammasome dysregulation in human gingival fibroblasts in response to periodontal pathogens.

Oral Dis 2020 Dec 23. Epub 2020 Dec 23.

School of Dentistry, University of Birmingham, Birmingham, UK.

Objective: Uncontrolled production of Interleukin-1β (IL-1β), a major proinflammatory cytokine, is associated with tissue destruction in periodontal disease. IL-1β production is controlled by inflammasomes which are multiprotein regulatory complexes. The current study aimed to elucidate potential regulatory pathways by monitoring the effects of periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) on inflammasomes and their regulators in human gingival fibroblasts (HGFs) in vitro.

Methods: HGFs were exposed to Fn and Pg alone or in combination for 24 hr at a multiplicity of infection of 100, ±30 min exposure with 5 mM adenosine triphosphate (ATP) incubation. Gene expression of NLRP3 and AIM2, inflammasome regulatory proteins POP1, CARD16 and TRIM16, and inflammasome components ASC and CASPASE 1, and IL-1β, were evaluated by RT-PCR. Pro- and mature IL-1β levels were monitored intracellularly by immunocytochemistry and extracellularly by ELISA.

Results: Fn + ATP significantly upregulated NLRP3, AIM2, IL-1β, ASC, and CASPASE 1; however, it downregulated POP1 and TRIM16. Pg + ATP downregulated NLRP3, ASC, POP1, but upregulated IL-1β and CARD16. Pg + Fn+ATP significantly upregulated AIM2, IL-1β and CARD16, and downregulated POP1, TRIM16, and CASPASE 1. Pg + ATP exposure significantly increased pro- and mature IL-1β production.

Conclusion: Bacterial exposure with ATP may deregulate IL-1β by dysregulating inflammasomes and their regulators in HGFs.
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http://dx.doi.org/10.1111/odi.13760DOI Listing
December 2020

The anti-tumour activity of DNA methylation inhibitor 5-aza-2'-deoxycytidine is enhanced by the common analgesic paracetamol through induction of oxidative stress.

Cancer Lett 2021 Mar 5;501:172-186. Epub 2021 Jan 5.

School of Dentistry, Institute of Clinical Sciences, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B5 7EG, UK; Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B15 2TT, UK. Electronic address:

The DNA demethylating agent 5-aza-2'-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects and its use in solid tumours is debated. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E pathway, inadvertently providing cancer cells with survival potential, while the addition of paracetamol offsets this effect. Secondly, in the presence of paracetamol, DAC treatment leads to glutathione depletion and finally to accumulation of ROS and/or mitochondrial superoxide, both of which have the potential to restrict tumour growth. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. The sensitizing effect of paracetamol supplementation is specific to DAC but not its analogue 5-azacitidine. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy.
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http://dx.doi.org/10.1016/j.canlet.2020.12.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845757PMC
March 2021

Cytomorphometric Analysis of Inflammation Dynamics in the Periodontium Following the Use of Fixed Dental Prostheses.

Molecules 2020 Oct 12;25(20). Epub 2020 Oct 12.

Department of Therapeutic Stomatology, Faculty of Stomatology, Yerevan State Medical University, Str. Koryun 2, Yerevan 0025, Armenia.

Cytomorphometry is used in the sampling of biological materials and diagnostic procedures. The use of cytological studies in periodontal diseases is not well described in the literature. Our study aimed to quantitatively assess the inflammation dynamics using cytomorphometric analysis of the periodontium before and after the use of fixed dental prostheses. Following ethics approval, a total of 105 subjects were divided in 3 groups as gingivitis ( = 23), periodontitis ( = 58), and healthy periodontium (control) ( = 24). The fixed dental prostheses (crowns and fixed partial dentures) were fabricated from cobalt-chrome metal-ceramic prostheses using the conventional method (C/M-CoCr), cobalt-chrome metal-ceramic prostheses by the computer-aided design and computer-aided manufacturing (CAD/CAM) technique (C/C-CoCr), and zirconia-based ceramic prostheses by the CAD/CAM technique (C/C-Zr) among subjects with gingivitis and periodontitis. The gingival crevicular fluid (GCF) was obtained from subjects before and after the use of the prostheses. The total count of epithelial cells and the connective tissue cells or polymorphonuclear neutrophils (PMNs) in GCF were studied using cytomorphometric analysis. The Statistical Package Tor the Social Sciences (SPSS), Version 20 (IBM Company, Chicago, IL, USA) was used to analyze the results and the significance level was set at = 0.05. The data for before and after the use of the prostheses were compared using independent -Tests. Similarly, the results after the use of prostheses in gingivitis, periodontitis, and control in each type of prostheses were compared using One-way ANOVA with post hoc using Scheffe. The total epithelial cells and the PMNs were determined along with the epithelium/leukocyte index. Regardless of the prostheses type used, no significant change in the parameters was identified among patients with a healthy periodontium, before and after prosthetic treatment. In all study groups, a statistically increase ( value < 0.05) was observed in the oral epithelial cell counts and a statistically decrease ( < 0.05) in the PMNs count following the use of the fixed prostheses. Data on cytomorphometric analysis could enable the selection of the most appropriate prostheses for use in patients with periodontal pathologies. When choosing prostheses, changes in the composition of GCF could be considered as a useful criterion for their use.
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http://dx.doi.org/10.3390/molecules25204650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587339PMC
October 2020

Pulp Innate Immune Defense: Translational Opportunities.

J Endod 2020 Sep;46(9S):S10-S18

Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand. Electronic address:

Introduction: The improvement of regenerative endodontic procedures requires an understanding of the key clinical questions combined with a fundamental biological knowledge of how the dental tissues behave during health, disease, and repair. Therefore, partnerships between clinicians and basic scientists are essential to drive the field forward and improve patient outcomes.

Methods: This review aimed to provide a background to dentin-pulp biology and the interaction between infection, inflammation, and regeneration.

Results: We have highlighted how the release of neutrophil extracellular traps (NETs) within the pulp are double-edged; while they aim to limit the bacterial infection, they may actually exacerbate cell death and chronic inflammation. Aberrant levels of these structures may occur because of ineffective host immunologic processes, viral infections, or impaired clearance caused by bacterial virulence factors. We also postulate a proinflammatory link in the pulp between NETs and the inflammasome activated by pathogen-associated molecular patterns and damage-associated molecular patterns. Subsequently, we discuss areas potentially fruitful for future clinical exploitation involving NET inhibitors, inflammasome modulators, phototherapies, and novel epigenetic approaches.

Conclusions: Sustained scientist-clinician research partnerships along with an increased understanding of the association between inflammation and regeneration within the dentin-pulp complex will lead to future patient benefit.
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http://dx.doi.org/10.1016/j.joen.2020.06.019DOI Listing
September 2020

Author Correction: Dentinogenic effects of extracted dentin matrix components digested with matrix metalloproteinases.

Sci Rep 2020 09 15;10(1):15342. Epub 2020 Sep 15.

Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-72251-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490343PMC
September 2020

Dysregulation of Inflammasomes in Human Dental Pulp Cells Exposed to Porphyromonas gingivalis and Fusobacterium nucleatum.

J Endod 2020 Sep 18;46(9):1265-1272. Epub 2020 Jun 18.

School of Dentistry, University of Birmingham, Birmingham, United Kingdom; Faculty of Dentistry, University of Otago, Dunedin, New Zealand.

Introduction: Interleukin-1β (IL-1β) is a major proinflammatory cytokine that plays a significant role in pulpal inflammation. The regulation of IL-1β as well as different cytokines and chemokines is controlled by multiprotein complexes named inflammasomes, which are known to be involved in pulpal inflammation. The goal of this study was to evaluate the effects of well-established endodontic bacteria and periodontal pathogens Fusobacterium nucleatum and Porphyromonas gingivalis on NLRP3 and AIM2 inflammasomes; the inflammasome regulatory proteins POP1, CARD16, and TRIM16; inflammasome components ASC and caspase-1; and IL-1β levels in human dental pulp cells (HDPCs) in vitro.

Methods: HDPCs were exposed to either F. nucleatum or P. gingivalis or to the combination of both with an additional 30 minutes of 5 mmol/L adenosine triphosphate (ATP) incubation for 24 hours. Escherichia coli lipopolysaccharide exposure was used as a control. Gene expression of NLRP3, AIM2, POP1, CARD16, TRIM16, ASC and caspase-1, and IL-1β were evaluated by reverse transcription polymerase chain reaction. The presence and levels of pro- and mature IL-1β were monitored by immunocytochemistry and the release with enzyme-linked immunosorbent assay.

Results: Up-regulation of NLRP3 and AIM2 was detected in all exposure groups. IL-1β was up-regulated in all groups, except for the F. nucleatum + ATP group. CARD16 was significantly down-regulated by F. nucleatum or P. gingivalis with or without ATP; however, POP1 was down-regulated only in P. gingivalis and E. coli LPS + ATP groups. P. gingivalis alone significantly increased intracellular pro- and mature IL-1β levels.

Conclusions: P. gingivalis and F. nucleatum in the presence of ATP may play a significant role in IL-1β-induced pulpal inflammation by dysregulating inflammasomes and their regulators.
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http://dx.doi.org/10.1016/j.joen.2020.06.008DOI Listing
September 2020

Effects of Porphyromonas gingivalis and Fusobacterium nucleatum on inflammasomes and their regulators in H400 cells.

Mol Oral Microbiol 2020 08 26;35(4):158-167. Epub 2020 Jun 26.

School of Dentistry, University of Birmingham, Birmingham, UK.

Introduction: Inflammasomes are multiprotein complexes that regulate immune processes in response to infections and tissue damage. They modulate Interleukin-1beta (IL-1β) expression, a major proinflammatory cytokine. The inflammasome/IL-1β pathway is involved in head and neck squamous cell carcinoma (HNSCC) progression and the periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) have been reported to cause chronic inflammation in HNSCC. The aim of this study was to characterise the role of these pathogens in regulating inflammasome activity and the IL-1β response in HNSCC in vitro.

Methods: An HNSCC cell line (H400) was exposed to Fn and Pg individually or in combination for 24h, ± incubation for 30 min with 5 mM adenosine triphosphate (ATP). Transcript levels of inflammasomes, NLRP3 and AIM2; inflammasome-regulatory proteins, POP1, CARD16 and TRIM16; and inflammasome-component, ASC and caspase 1 and IL-1β, were assayed by RT-PCR. Expression of IL-1β was by immunocytochemistry and ELISA.

Results: NLRP3 expression was significantly upregulated in response to Pg, Fn + Pg, Pg + ATP and Fn + Pg + ATP. AIM2 was significantly upregulated by Fn, Pg and Fn + Pg + ATP exposure. All conditions significantly upregulated IL-1β gene expression. POP1 expression was significantly downregulated by Pg or Fn exposure but not by Fn + Pg. Intracellular pro- and mature IL-1β were significantly higher following Fn and Pg + ATP exposure.

Conclusion: Pg alone increased IL-1β by upregulating AIM2, NLRP3 and downregulating POP1. Fn promoted IL-1β by increasing AIM2 and downregulating POP1. Pg + ATP with or without Fn upregulated NLRP3, IL-1β by downregulating POP1. Periodontal pathogens may contribute to HNSCC pathogenesis by increasing the IL-1β response due to inflammasome dysregulation.
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http://dx.doi.org/10.1111/omi.12302DOI Listing
August 2020

Inflammasomes and their regulation in periodontal disease: A review.

J Periodontal Res 2020 Aug 20;55(4):473-487. Epub 2020 Jan 20.

Oral Biology, School of Dentistry, University of Birmingham, Birmingham, UK.

Interleukin-1β (IL-1β), which is secreted by host tissues leading to periodontal tissue inflammation, is a major pro-inflammatory cytokine in the pathogenesis of periodontal disease. The conversion of pro-IL-1β into its biologically active form is controlled by multiprotein complexes named as inflammasomes, which are key regulator of host defense mechanisms and inflammasome involved diseases, including the periodontal diseases. Inflammasomes are regulated by different proteins and processes, including pyrin domain (PYD)-only proteins (POPs), CARD-only proteins (COPs), tripartite motif family proteins (TRIMs), autophagy, and interferons. A review of in vitro, in vivo, and clinical data from these publications revealed that several inflammasomes including (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) have been found to be involved in periodontal disease pathogenesis. To the best of our knowledge, the current article provides the first review of the literature focusing on studies that evaluated both inflammasomes and their regulators in periodontal disease. An upregulation for inflammasomes and a downregulation of inflammasome regulator proteins including POPs, COPs, and TRIMs have been reported in periodontal disease. Although interferons (types I and II) and autophagy have been found to be involved in periodontal disease, their possible role in inflammasome activation has not evaluated yet. Modulating the excessive inflammatory response by the use of inflammasome regulators may have potential in the management of periodontal disease.
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http://dx.doi.org/10.1111/jre.12733DOI Listing
August 2020

The role of calcium ion release on biocompatibility and antimicrobial properties of hydraulic cements.

Sci Rep 2019 12 13;9(1):19019. Epub 2019 Dec 13.

School of Dentistry, Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, B5 7EG, Birmingham, United Kingdom.

Tricalcium silicate (TCS)-based materials produce calcium hydroxide as a byproduct of their hydration reaction. The present study investigated whether calcium ion release (CIR) affects their biological and antimicrobial properties when used as pulp protection materials. The effect of incorporation of micro-silica and calcium phosphate monobasic to radiopacified TCS-based materials was investigated. The commercial TCS-based Biodentine, Bio-C Pulpo, TotalFill Root Repair Material, TheraCal LC and a base/liner- ACTIVA BioACTIVE (Activa) were also evaluated. The hydration and CIR were monitored and correlated with biocompatibility and antimicrobial assessment of eluates. Overall, the additives altered the hydration and leaching profile of the prototype cements. The micro-silica inclusion resulted in a decreased long-term calcium hydroxide formation which was associated with neutralised cytotoxicity and antibacterial activity. Calcium phosphate did not alter the leaching profile, although a stronger antibacterial effect was induced. The commercial materials also had different CIR profiles. The water-based ones had higher CIR, and this was associated with stronger antimicrobial effect but not enhanced biological activity. Both TheraCal LC and Activa exhibited poor degree of conversion, low CIR, acceptable biocompatibility and moderate antibacterial activity. A positive correlation of CIR with antibacterial effectiveness was observed (0.3 < r < 0.49; p = 0.021, p = 0.011 for the two test bacterial cultures). No relation was shown between CIR and cytotoxicity (0.3 < r < 0.49; p = 0.150, p = 0.068 for the two cell cultures studied). The additives modified the CIR. The antimicrobial properties were dependent on the CIR; the cytotoxicity of the materials was unaffected.
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http://dx.doi.org/10.1038/s41598-019-55288-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910940PMC
December 2019

Under the spotlight: mechanisms of photobiomodulation concentrating on blue and green light.

Photochem Photobiol Sci 2019 Aug 11;18(8):1877-1909. Epub 2019 Jun 11.

College of Medical and Dental Sciences, University of Birmingham, UK.

Photobiomodulation (PBM) describes the application of light at wavelengths ranging from 400-1100 nm to promote tissue healing, reduce inflammation and promote analgesia. Traditionally, red and near-infra red (NIR) light have been used therapeutically, however recent studies indicate that other wavelengths within the visible spectrum could prove beneficial including blue and green light. This review aims to evaluate the literature surrounding the potential therapeutic effects of PBM with particular emphasis on the effects of blue and green light. In particular focus is on the possible primary and secondary molecular mechanisms of PBM and also evaluation of the potential effective parameters for application both in vitro and in vivo. Studies have reported that PBM affects an array of molecular targets, including chromophores such as signalling molecules containing flavins and porphyrins as well as components of the electron transport chain. However, secondary mechanisms tend to converge on pathways induced by increases in reactive oxygen species (ROS) production. Systematic evaluation of the literature indicated 72% of publications reported beneficial effects of blue light and 75% reported therapeutic effects of green light. However, of the publications evaluating the effects of green light, reporting of treatment parameters was uneven with 41% failing to report irradiance (mW cm) and 44% failing to report radiant exposure (J cm). This review highlights the potential of PBM to exert broad effects on a range of different chromophores within the body, dependent upon the wavelength of light applied. Emphasis still remains on the need to report exposure and treatment parameters, as this will enable direct comparison between different studies and hence enable the determination of the full potential of PBM.
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http://dx.doi.org/10.1039/c9pp00089eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685747PMC
August 2019

Neutrophil Extracellular Traps Exert Potential Cytotoxic and Proinflammatory Effects in the Dental Pulp.

J Endod 2019 May 29;45(5):513-520.e3. Epub 2019 Mar 29.

Oral Biology, Birmingham Dental School and Hospital, College of Medical and Dental Sciences, Birmingham, United Kingdom. Electronic address:

Introduction: Neutrophil extracellular traps (NETs) are an important innate immune mechanism aimed at limiting the dissemination of bacteria within tissues and localizing antibacterial killing mechanisms. There is significant interest in the role of NETs in a range of infectious and inflammatory diseases; however, their role in diseased pulp has yet to be explored. Our aim was to determine their relevance to infected pulp and how their components affect human dental pulp cell (HDPC) responses.

Methods: Diseased pulp tissue was stained for the presence of extracellular DNA and elastase to detect the presence of NETs. Bacteria known to infect pulp were also assayed to determine their ability to stimulate NETs. Coculture studies and NET component challenge were used to determine the effect of extracellular NET release on HDPC viability and inflammatory response. NET-stimulated HDPC secretomes were assessed for their chemotactic activity for lymphocytes and macrophages.

Results: Data indicate that NETs are present in infected pulp tissue and whole NETs, and their histone components, particularly H2A, decreased HDPC viability and stimulated chemokine release, resulting in an attraction of lymphocyte populations.

Conclusions: NETs are likely important in pulpal pathogenesis with injurious and chronic inflammatory effects on HDPCs, which may contribute to disease progression. Macrophages are chemoattracted to NET-induced apoptotic HDPCs, facilitating cellular debris removal. NETs and histones may provide novel prognostic markers and/or therapeutic targets for pulpal diseases.
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http://dx.doi.org/10.1016/j.joen.2019.02.014DOI Listing
May 2019

Differential responses of myoblasts and myotubes to photobiomodulation are associated with mitochondrial number.

J Biophotonics 2019 06 20;12(6):e201800411. Epub 2019 Feb 20.

School of Dentistry, College of Medical and Dental Sciences, Institute of Clinical Sciences, University of Birmingham, Birmingham, UK.

Objective: Photobiomodulation (PBM) is the application of light to promote tissue healing. Current indications suggest PBM induces its beneficial effects in vivo through upregulation of mitochondrial activity. However, how mitochondrial content influences such PBM responses have yet to be evaluated. Hence, the current study assessed the biological response of cells to PBM with varying mitochondrial contents.

Methods: DNA was isolated from myoblasts and myotubes (differentiated myoblasts), and mitochondrial DNA (mtDNA) was amplified and quantified using a microplate assay. Cells were seeded in 96-wellplates, incubated overnight and subsequently irradiated using a light-emitting diode array (400, 450, 525, 660, 740, 810, 830 and white light, 24 mW/cm , 30-240 seconds, 0.72-5.76J/cm ). The effects of PBM on markers of mitochondrial activity including reactive-oxygen-species and real-time mitochondrial respiration (Seahorse XFe96) assays were assessed 8 hours post-irradiation. Datasets were analysed using general linear model followed by one-way analysis of variance (and post hoc-Tukey tests); P = 0.05).

Results: Myotubes exhibited mtDNA levels 86% greater than myoblasts (P < 0.001). Irradiation of myotubes at 400, 450 or 810 nm induced 53%, 29% and 47% increases (relative to non-irradiated control) in maximal respiratory rates, respectively (P < 0.001). Conversely, irradiation of myoblasts at 400 or 450 nm had no significant effect on maximal respiratory rates.

Conclusion: This study suggests that mitochondrial content may influence cellular responses to PBM and as such explain the variability of PBM responses seen in the literature.
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http://dx.doi.org/10.1002/jbio.201800411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065641PMC
June 2019

Dental Pulp Stem Cell Mechanoresponsiveness: Effects of Mechanical Stimuli on Dental Pulp Stem Cell Behavior.

Front Physiol 2018 26;9:1685. Epub 2018 Nov 26.

Stem Cells Unit, Biomedical Section, Tecnologica Research Institute and Marrelli Health, Crotone, Italy.

Dental pulp is known to be an accessible and important source of multipotent mesenchymal progenitor cells termed dental pulp stem cells (DPSCs). DPSCs can differentiate into odontoblast-like cells and maintain pulp homeostasis by the formation of new dentin which protects the underlying pulp. DPSCs similar to other mesenchymal stem cells (MSCs) reside in a niche, a complex microenvironment consisting of an extracellular matrix, other local cell types and biochemical stimuli that influence the decision between stem cell (SC) self-renewal and differentiation. In addition to biochemical factors, mechanical factors are increasingly recognized as key regulators in DPSC behavior and function. Thus, microenvironments can significantly influence the role and differentiation of DPSCs through a combination of factors which are biochemical, biomechanical and biophysical in nature. Under conditions, it has been shown that DPSCs are sensitive to different types of force, such as uniaxial mechanical stretch, cyclic tensile strain, pulsating fluid flow, low-intensity pulsed ultrasound as well as being responsive to biomechanical cues presented in the form of micro- and nano-scale surface topographies. To understand how DPSCs sense and respond to the mechanics of their microenvironments, it is essential to determine how these cells convert mechanical and physical stimuli into function, including lineage specification. This review therefore covers some aspects of DPSC mechanoresponsivity with an emphasis on the factors that influence their behavior. An in-depth understanding of the physical environment that influence DPSC fate is necessary to improve the outcome of their therapeutic application for tissue regeneration.
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http://dx.doi.org/10.3389/fphys.2018.01685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275199PMC
November 2018

Epigenetic Approaches to the Treatment of Dental Pulp Inflammation and Repair: Opportunities and Obstacles.

Front Genet 2018 7;9:311. Epub 2018 Aug 7.

Division of Restorative Dentistry & Periodontology, Dublin Dental University Hospital, Trinity College Dublin, University of Dublin, Dublin, Ireland.

Concerns over the cost and destructive nature of dental treatment have led to the call for novel minimally invasive, biologically based restorative solutions. For patients with toothache, this has resulted in a shift from invasive root-canal-treatment (RCT) toward more conservative vital-pulp-treatment (VPT) procedures, aimed to protect the pulp and harness its natural regenerative capacity. If the dental pulp is exposed, as long as the infection and inflammation can be controlled, conservative therapies can promote the formation of new tertiary dentine in a stem cell-led reparative process. Crucially, the volume and quality of new dentine is dependent on the material applied; however, currently available dental-materials are limited by non-specific action, cytotoxicity and poor clinical handling. Looking to the future, an improved understanding of the cellular regulators of pulpal inflammation and associated repair mechanisms is critical to predict pulpal responses and devise novel treatment strategies. Epigenetic modifications of DNA-associated proteins and the influences of non-coding RNAs have been demonstrated to control the self-renewal of stem cell populations as well as regulate mineralised tissue development and repair. Notably, the stability of microRNAs and their relative ease of sampling from pulpal blood highlight their potential for application as diagnostic inflammatory biomarkers, while increased understanding of their actions will not only enhance our knowledge of pulpal disease and repair, but also identify novel molecular targets. The potential therapeutic application of epigenetic modifying agents, DNA-methyltransferase-inhibitors (DNMTi) and histone-deacetylase-inhibitors (HDACi), have been shown to promote mineralisation and repair processes in dental-pulp-cell (DPC) populations as well as induce the release of bioactive dentine-matrix-components. Consequently, HDACis and DNMTis have the potential to enhance tertiary dentinogenesis by influencing the cellular and tissue processes at low concentrations with minimal side effects, providing an opportunity to develop a topically placed, inexpensive bio-inductive restorative material. The aim of this review is to highlight the potential role of epigenetic approaches in the treatment of the damaged dental pulp, considering the opportunities and obstacles, such as off-target effects, delivery mechanisms, for the therapeutic use of miRNA as an inflammatory biomarker or molecular target, before discussing the application of HDACi and DNMTi to the damaged pulp to stimulate repair.
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http://dx.doi.org/10.3389/fgene.2018.00311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090030PMC
August 2018

Dentinogenic effects of extracted dentin matrix components digested with matrix metalloproteinases.

Sci Rep 2018 Jul 16;8(1):10690. Epub 2018 Jul 16.

Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.

Dentin is primarily composed of hydroxyapatite crystals within a rich organic matrix. The organic matrix comprises collagenous structural components, within which a variety of bioactive molecules are sequestered. During caries progression, dentin is degraded by acids and enzymes derived from various sources, which can release bioactive molecules with potential reparative activity towards the dentin-pulp complex. While these molecules' repair activities in other tissues are already known, their biological effects are unclear in relation to degradation events during disease in the dentin-pulp complex. This study was undertaken to investigate the effects of dentin matrix components (DMCs) that are partially digested by matrix metalloproteinases (MMPs) in vitro and in vivo during wound healing of the dentin-pulp complex. DMCs were initially isolated from healthy dentin and treated with recombinant MMPs. Subsequently, their effects on the behaviour of primary pulp cells were investigated in vitro and in vivo. Digested DMCs modulated a range of pulp cell functions in vitro. In addition, DMCs partially digested with MMP-20 stimulated tertiary dentin formation in vivo, which exhibited a more regular tubular structure than that induced by treatment with other MMPs. Our results indicate that MMP-20 may be especially effective in stimulating wound healing of the dentin-pulp complex.
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http://dx.doi.org/10.1038/s41598-018-29112-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048071PMC
July 2018

Development and Application of High-Content Biological Screening for Modulators of NET Production.

Front Immunol 2018 5;9:337. Epub 2018 Mar 5.

School of Dentistry, Institute of Clinical Sciences, University of Birmingham, Birmingham, United Kingdom.

Neutrophil extracellular traps (NETs) are DNA-based antimicrobial web-like structures whose release is predominantly mediated by reactive oxygen species (ROS); their purpose is to combat infections. However, unbalanced NET production and clearance is involved in tissue injury, circulation of auto-antibodies and development of several chronic diseases. Currently, there is lack of agreement regarding the high-throughput methods available for NET investigation. This study, therefore, aimed to develop and optimize a high-content analysis (HCA) approach, which can be applied for the assay of NET production and for the screening of compounds involved in the modulation of NET release. A suitable paraformaldehyde fixation protocol was established to enable HCA of neutrophils and NETs. Bespoke and in-built bioinformatics algorithms were validated by comparison with standard low-throughput approaches for application in HCA of NETs. Subsequently, the optimized protocol was applied to high-content screening (HCS) of a pharmaceutically derived compound library to identify modulators of NETosis. Of 56 compounds assessed, 8 were identified from HCS for further characterization of their effects on NET formation as being either inducers, inhibitors or biphasic modulators. The effects of these compounds on naïve neutrophils were evaluated by using specific assays for the induction of ROS and NET production, while their modulatory activity was validated in phorbol 12-myristate 13-acetate-stimulated neutrophils. Results indicated the involvement of glutathione reductase, Src family kinases, molecular-target-of-Rapamycin, and mitogen-activated-protein-kinase pathways in NET release. The compounds and pathways identified may provide targets for novel therapeutic approaches for treating NET-associated pathologies.
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http://dx.doi.org/10.3389/fimmu.2018.00337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844942PMC
March 2019

Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria.

Infect Immun 2017 12 17;85(12). Epub 2017 Nov 17.

Periodontal Research Group, Birmingham Dental School and Hospital, Institute of Clinical Sciences, The University of Birmingham and Birmingham Community Health NHS Trust, Edgbaston, Birmingham, United Kingdom

Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., and , stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated.
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http://dx.doi.org/10.1128/IAI.00297-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695129PMC
December 2017

Inflammation and Regeneration in the Dentin-pulp Complex: Net Gain or Net Loss?

J Endod 2017 Sep;43(9S):S87-S94

Oral Biology, School of Dentistry, College of Medical and Dental Sciences, Edgbaston, Birmingham, UK.

The balance between the immune/inflammatory and regenerative responses in the diseased pulp is central to the clinical outcome, and this response is unique within the body because of its tissue site. Cariogenic bacteria invade the dentin and pulp tissues, triggering molecular and cellular events dependent on the disease stage. At the early onset, odontoblasts respond to bacterial components in an attempt to protect the tooth's hard and soft tissues and limit disease progression. However, as disease advances, the odontoblasts die, and cells central to the pulp core, including resident immune cells, pulpal fibroblasts, endothelial cells, and stem cells, respond to the bacterial challenge via their expression of a range of pattern recognition receptors that identify pathogen-associated molecular patterns. Subsequently, recruitment and activation occurs of a range of immune cell types, including neutrophils, macrophages, and T and B cells, which are attracted to the diseased site by cytokine/chemokine chemotactic gradients initially generated by resident pulpal cells. Although these cells aim to disinfect the tooth, their extravasation, migration, and antibacterial activity (eg, release of reactive oxygen species [ROS]) along with the bacterial toxins cause pulp damage and impede tissue regeneration processes. Recently, a novel bacterial killing mechanism termed neutrophil extracellular traps (NETs) has also been described that uses ROS signaling and results in cellular DNA extrusion. The NETs are decorated with antimicrobial peptides (AMPs), and their interaction with bacteria results in microbial entrapment and death. Recent data show that NETs can be stimulated by bacteria associated with endodontic infections, and they may be present in inflamed pulp tissue. Interestingly, some bacteria associated with pulpal infections express deoxyribonuclease enzymes, which may enable their evasion of NETs. Furthermore, although NETs aim to localize and kill invading bacteria using AMPs and histones, limiting the spread of the infection, data also indicate that NETs can exacerbate inflammation and their components are cytotoxic. This review considers the potential role of NETs within pulpal infections and how these structures may influence the pulp's vitality and regenerative responses.
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http://dx.doi.org/10.1016/j.joen.2017.06.011DOI Listing
September 2017

Regenerative Endodontics: Burning Questions.

J Endod 2017 Sep 1;43(9S):S1-S6. Epub 2017 Aug 1.

School of Dentistry, University of Birmingham, Birmingham, United Kingdom.

Pulp regeneration and its clinical translation into regenerative endodontic procedures are receiving increasing research attention, leading to significant growth of the published scientific and clinical literature within these areas. Development of research strategies, which consider patient-, clinician-, and scientist-based outcomes, will allow greater focus on key research questions driving more rapid clinical translation. Three key areas of focus for these research questions should include cells, signaling, and infection/inflammation. A translational pathway is envisaged in which clinical approaches are increasingly refined to provide regenerative endodontic protocols that are based on a robust understanding of the physiological processes and events responsible for the normal secretion, structure, and biological behavior of pulpal tissue.
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http://dx.doi.org/10.1016/j.joen.2017.06.002DOI Listing
September 2017

Role of Piezo Channels in Ultrasound-stimulated Dental Stem Cells.

J Endod 2017 Jul 17;43(7):1130-1136. Epub 2017 May 17.

School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK. Electronic address:

Introduction: Piezo1 and Piezo2 are mechanosensitive membrane ion channels. We hypothesized that Piezo proteins may play a role in transducing ultrasound-associated mechanical signals and activate downstream mitogen-activated protein kinase (MAPK) signaling processes in dental cells. In this study, the expression and role of Piezo channels were investigated in dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) after treatment with low-intensity pulsed ultrasound (LIPUS).

Methods: Cell proliferation was evaluated by bromodeoxyuridine incorporation. Western blots were used to analyze the proliferating cell nuclear antigen as well as the transcription factors c-fos and c-jun. Enzyme-linked immunosorbent assay and Western blotting were used to determine the activation of MAPK after LIPUS treatment. Ruthenium red (RR), a Piezo ion channel blocker, was applied to determine the functional role of Piezo proteins in LIPUS-stimulated cell proliferation and MAPK signaling.

Results: Western blotting showed the presence of Piezo1 and Piezo2 in both dental cell types. LIPUS treatment significantly increased the level of the Piezo proteins in DPSCs after 24 hours; however, no significant effects were observed in PDLSCs. Treatment with RR significantly inhibited LIPUS-stimulated DPSC proliferation but not PDLSC proliferation. Extracellular signal-related kinase (ERK) 1/2 MAPK was consistently activated in DPSCs over a 24-hour time period after LIPUS exposure, whereas phosphorylated c-Jun N-terminal kinase and p38 mitogen-activated protein kinase MAPK were mainly increased in PDLSCs. RR affected MAPK signaling in both dental cell types with its most prominent effects on ERK1/2/MAPK phosphorylation levels; the significant inhibition of LIPUS-induced stimulation of ERK1/2 activation in DPSCs by RR suggests that stimulation of DPSC proliferation by LIPUS involves Piezo-mediated regulation of ERK1/2 MAPK signaling.

Conclusions: This study for the first time supports the role of Piezo ion channels in transducing the LIPUS response in dental stem cells.
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http://dx.doi.org/10.1016/j.joen.2017.02.022DOI Listing
July 2017

Porphyromonas gingivalis gingipains cause defective macrophage migration towards apoptotic cells and inhibit phagocytosis of primary apoptotic neutrophils.

Cell Death Dis 2017 03 2;8(3):e2644. Epub 2017 Mar 2.

School of Life & Health Sciences and Aston Research Centre for Healthy Ageing, Aston University, Birmingham B4 7ET, UK.

Periodontal disease is a prevalent chronic inflammatory condition characterised by an aberrant host response to a pathogenic plaque biofilm resulting in local tissue damage and frustrated healing that can result in tooth loss. Cysteine proteases (gingipains) from the key periodontal pathogen Porphyromonas gingivalis have been implicated in periodontal disease pathogenesis by inhibiting inflammation resolution and are linked with systemic chronic inflammatory conditions such as rheumatoid arthritis. Efficient clearance of apoptotic cells is essential for the resolution of inflammation and tissue restoration. Here we sought to characterise the innate immune clearance of apoptotic cells and its modulation by gingipains. We examined the capacity of gingipain-treated macrophages to migrate towards and phagocytose apoptotic cells. Lysine gingipain treatment of macrophages impaired macrophage migration towards apoptotic neutrophils. Furthermore, lysine gingipain treatment reduced surface expression levels of CD14, a key macrophage receptor for apoptotic cells, which resulted in reduced macrophage interactions with apoptotic cells. Additionally, while apoptotic cells and their derived secretome were shown to inhibit TNF-α-induced expression by P. gingivalis lipopolysaccharide, we demonstrated that gingipain preparations induced a rapid inflammatory response in macrophages that was resistant to the anti-inflammatory effects of apoptotic cells or their secretome. Taken together, these data indicate that P. gingivalis may promote the chronic inflammation seen in periodontal disease patients by multiple mechanisms, including rapid, potent gingipain-mediated inflammation, coupled with receptor cleavage leading to defective clearance of apoptotic cells and reduced anti-inflammatory responses. Thus, gingipains represent a potential therapeutic target for intervention in the management of chronic periodontal disease.
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http://dx.doi.org/10.1038/cddis.2016.481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386511PMC
March 2017

Development and application of LED arrays for use in phototherapy research.

J Biophotonics 2017 Nov 6;10(11):1514-1525. Epub 2017 Feb 6.

School of Dentistry, College of Medical and Dental Sciences, Institute of Clinical Sciences, University of Birmingham, 5 Mill Pool Way, Edgbaston, Birmingham, B5 7EG, UK.

Lasers/LEDs demonstrate therapeutic effects for a range of biomedical applications. However, a consensus on effective light irradiation parameters and efficient and reliable measurement techniques remain limited. The objective here is to develop, characterise and demonstrate the application of LED arrays in order to progress and improve the effectiveness and accuracy of in vitro photobiomodulation studies. 96-well plate format LED arrays (400-850 nm) were developed and characterised to accurately assess irradiance delivery to cell cultures. Human dental pulp cells (DPCs) were irradiated (3.5-142 mW/cm : 15-120 s) and the biological responses were assessed using MTT assays. Array calibration was confirmed using a range of optical and analytical techniques. Multivariate analysis of variance revealed biological responses were dependent on wavelength, exposure time and the post-exposure assay time (P < 0.05). Increased MTT asbsorbance was measured 24 h post-irradiation for 30 s exposures of 3.5 mW/cm at 470, 527, 631, 655, 680, 777, 798 and 826 nm with distinct peaks at 631 nm and 798 nm (P < 0.05). Similar wavelengths were also effective at higher irradiances (48-142 mW/cm ). LED arrays and high throughput assays provide a robust and reliable platform to rapidly identify irradiation parameters which is both time- and cost-effective. These arrrays are applicable in photobiomodulation, photodynamic therapy and other photobiomedical research.
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http://dx.doi.org/10.1002/jbio.201600273DOI Listing
November 2017

IFN-γ regulates human dental pulp stem cells behavior via NF-κB and MAPK signaling.

Sci Rep 2017 01 18;7:40681. Epub 2017 Jan 18.

State Key Laboratory of Military Stomatology, Department of Operative Dentistry &Endodontics, School of Stomatology, the Fourth Military Medical University, Xi'an Shaanxi, China.

During caries, dental pulp expresses a range of pro-inflammatory cytokines in response to the infectious challenge. Interferon gamma (IFN-γ) is a dimerized soluble cytokine, which is critical for immune responses. Previous study has demonstrated that IFN-γ at relative high concentration (100 ng/mL) treatment improved the impaired dentinogenic and immunosuppressive regulatory functions of disease-derived dental pulp stem cells (DPSCs). However, little is known about the regulatory effects of IFN-γ at relative low concentration on healthy DPSC behavior (including proliferation, migration, and multiple-potential differentiation). Here we demonstrate that IFN-γ at relatively low concentrations (0.5 ng/mL) promoted the proliferation and migration of DPSCs, but abrogated odonto/osteogenic differentiation. Additionally, we identified that NF-κB and MAPK signaling pathways are both involved in the process of IFN-γ-regulated odonto/osteogenic differentiation of DPSCs. DPSCs treated with IFN-γ and supplemented with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) or SB203580 (a MAPK inhibitor) showed significantly improved potential for odonto/osteogenic differentiation of DPSCs both in vivo and in vitro. These data provide important insight into the regulatory effects of IFN-γ on the biological behavior of DPSCs and indicate a promising therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.
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http://dx.doi.org/10.1038/srep40681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241669PMC
January 2017

An in vitro screening assay for dental stain cleaning.

BMC Oral Health 2017 Jan 9;17(1):37. Epub 2017 Jan 9.

Oral Biology, School of Dentistry, University of Birmingham, 5 Mill Pool Way, Edgbaston, Birmingham, B5 7EG, UK.

Background: The present study aimed to develop an in vitro model for stain removal from natural enamel for the assessment and comparison of oral hygiene products.

Methods: Bovine teeth (n = 8 per group) were ground/polished to provide flat enamel specimens and ferric-tannate deposits were precipitated onto the enamel surfaces. The ferric-tannate stained enamel specimens were brushed using an in vitro tooth-brushing simulator with slurries containing commercially available toothpaste products, dental abrasive particles, and sodium tripolyphosphate (STP) solutions of different concentrations. The colour of the enamel surfaces was measured using a spectrophotometer before and after stain application as well as after the brushing treatments.

Results: Differences in stain removal efficacy were found between the toothpastes categorised as whitening and non-whitening comprising of different types of dental abrasives (hydrated silica and alumina). A mean value of 27% for stain removal was detected for the three non-whitening toothpastes and 59% of stain removal was detected for the three whitening toothpastes after 1000 strokes. Compared with the slurry with Zeodent 113 abrasive alone, the addition of STP provided better performance for stain removal under the same brushing conditions (mean value of 62% for Zeodent 113 abrasive alone and 72% with the addition of 5% (w/w) STP after 1000 strokes). No difference was evident between the STP concentration of 5% (w/w) and 10% (w/w).

Conclusions: The ferric-tannate/bovine enamel model reported here provides good stain retention, is rapidly and easily prepared, and is shown to be progressively and reproducibly sensitive to toothbrushing using different toothpastes and surfactant/chelating agent solutions. Importantly, it provides good discrimination between various oral hygiene products. The stain removal assay reported here has considerable potential to enable comparative assessments of different toothpaste types in terms of their cleaning capabilities.
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http://dx.doi.org/10.1186/s12903-016-0328-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223583PMC
January 2017

Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps.

Methods Mol Biol 2017 ;1537:481-497

Institute of Clinical Sciences, College of Medical and Dental Sciences, The School of Dentistry, University of Birmingham, 5 Mill Pool Way, Edgbaston, Birmingham, B5 7EG, UK.

Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.
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http://dx.doi.org/10.1007/978-1-4939-6685-1_29DOI Listing
January 2018

The systematics of the Mongolepidida (Chondrichthyes) and the Ordovician origins of the clade.

PeerJ 2016 16;4:e1850. Epub 2016 Jun 16.

School of Geography, Earth and Environmental Sciences, University of Birmingham , Birmingham , United Kingdom.

The Mongolepidida is an Order of putative early chondrichthyan fish, originally erected to unite taxa from the Lower Silurian of Mongolia. The present study reassesses mongolepid systematics through the examination of the developmental, histological and morphological characteristics of scale-based specimens from the Upper Ordovician Harding Sandstone (Colorado, USA) and the Upper Llandovery-Lower Wenlock Yimugantawu (Tarim Basin, China), Xiushan (Guizhou Province, China) and Chargat (north-western Mongolia) Formations. The inclusion of the Mongolepidida within the Class Chondrichthyes is supported on the basis of a suite of scale attributes (areal odontode deposition, linear odontocomplex structure and lack of enamel, cancellous bone and hard-tissue resorption) shared with traditionally recognized chondrichthyans (euchondrichthyans, e.g., ctenacanthiforms). The mongolepid dermal skeleton exhibits a rare type of atubular dentine (lamellin) that is regarded as one of the diagnostic features of the Order within crown gnathostomes. The previously erected Mongolepididae and Shiqianolepidae families are revised, differentiated by scale-base histology and expanded to include the genera Rongolepisand Xinjiangichthys, respectively. A newly described mongolepid species (Solinalepis levis gen. et sp. nov.) from the Ordovician of North America is treated as family incertae sedis, as it possesses a type of basal bone tissue (acellular and vascular) that has yet to be documented in other mongolepids. This study extends the stratigraphic and palaeogeographic range of Mongolepidida and adds further evidence for an early diversification of the Chondrichthyes in the Ordovician Period, 50 million years prior to the first recorded appearance of euchondrichthyan teeth in the Lower Devonian.
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http://dx.doi.org/10.7717/peerj.1850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918221PMC
June 2016

The dark art of light measurement: accurate radiometry for low-level light therapy.

Lasers Med Sci 2016 May 10;31(4):789-809. Epub 2016 Mar 10.

Biomaterials Unit, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chads Queensway, Birmingham, UK, B4 6NN.

Lasers and light-emitting diodes are used for a range of biomedical applications with many studies reporting their beneficial effects. However, three main concerns exist regarding much of the low-level light therapy (LLLT) or photobiomodulation literature; (1) incomplete, inaccurate and unverified irradiation parameters, (2) miscalculation of 'dose,' and (3) the misuse of appropriate light property terminology. The aim of this systematic review was to assess where, and to what extent, these inadequacies exist and to provide an overview of 'best practice' in light measurement methods and importance of correct light measurement. A review of recent relevant literature was performed in PubMed using the terms LLLT and photobiomodulation (March 2014-March 2015) to investigate the contemporary information available in LLLT and photobiomodulation literature in terms of reporting light properties and irradiation parameters. A total of 74 articles formed the basis of this systematic review. Although most articles reported beneficial effects following LLLT, the majority contained no information in terms of how light was measured (73%) and relied on manufacturer-stated values. For all papers reviewed, missing information for specific light parameters included wavelength (3%), light source type (8%), power (41%), pulse frequency (52%), beam area (40%), irradiance (43%), exposure time (16%), radiant energy (74%) and fluence (16%). Frequent use of incorrect terminology was also observed within the reviewed literature. A poor understanding of photophysics is evident as a significant number of papers neglected to report or misreported important radiometric data. These errors affect repeatability and reliability of studies shared between scientists, manufacturers and clinicians and could degrade efficacy of patient treatments. Researchers need a physicist or appropriately skilled engineer on the team, and manuscript reviewers should reject papers that do not report beam measurement methods and all ten key parameters: wavelength, power, irradiation time, beam area (at the skin or culture surface; this is not necessarily the same size as the aperture), radiant energy, radiant exposure, pulse parameters, number of treatments, interval between treatments and anatomical location. Inclusion of these parameters will improve the information available to compare and contrast study outcomes and improve repeatability, reliability of studies.
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http://dx.doi.org/10.1007/s10103-016-1914-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851696PMC
May 2016

Ultrasound Stimulation of Different Dental Stem Cell Populations: Role of Mitogen-activated Protein Kinase Signaling.

J Endod 2016 Mar 30;42(3):425-31. Epub 2016 Jan 30.

School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom. Electronic address:

Introduction: Mesenchymal stem cells (MSCs) from dental tissues may respond to low-intensity pulsed ultrasound (LIPUS) treatment, potentially providing a therapeutic approach to promoting dental tissue regeneration. This work aimed to compare LIPUS effects on the proliferation and MAPK signaling in MSCs from rodent dental pulp stem cells (DPSCs) compared with MSCs from periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs).

Methods: Isolated MSCs were treated with 1-MHz LIPUS at an intensity of 250 or 750 mW/cm2 for 5 or 20 minutes. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) staining after 24 hours of culture following a single LIPUS treatment. Specific ELISAs were used to determine the total and activated p38, ERK1/2, and JNK MAPK signaling proteins up to 4 hours after treatment. Selective MAPK inhibitors PD98059 (ERK1/2), SB203580 (p38), and SP600125 (JNK) were used to determine the role of activation of the particular MAPK pathways.

Results: The proliferation of all MSC types was significantly increased after LIPUS treatment. LIPUS at a 750-mW/cm2 dose induced the greatest effects on DPSCs. BMSC proliferation was stimulated in equal measures by both intensities, whereas 250 mW/cm2 LIPUS exposure exerted maximum effects on PDLSCs. ERK1/2 was activated immediately in DPSCs after treatment. Concomitantly, DPSC proliferation was specifically modulated by ERK1/2 inhibition, whereas p38 and JNK inhibition exerted no effects. In BMSCs, JNK MAPK signaling was LIPUS activated, and the increase in proliferation was blocked by specific inhibition of the JNK pathway. In PDLSCs, JNK MAPK signaling was activated immediately after LIPUS, whereas p-p38 MAPK increased significantly in these cells 4 hours after exposure. Correspondingly, JNK and p38 inhibition modulated LIPUS-stimulated PDLSC proliferation.

Conclusions: LIPUS promoted MSC proliferation in an intensity and cell-specific dependent manner via activation of distinct MAPK pathways.
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http://dx.doi.org/10.1016/j.joen.2015.12.019DOI Listing
March 2016