PRIASE 2021 guidelines for reporting animal studies in Endodontology: explanation and elaboration.

Laws and ethics require that before conducting human clinical trials, a new material, device or drug may have to undergo testing in animals in order to minimize health risks to humans, unless suitable supporting grandfather data already exist. The Preferred Reporting Items for Animal Studies in Endodontology (PRIASE) 2021 guidelines were developed exclusively for the specialty of Endodontology by integrating and adapting the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines and the Clinical and Laboratory Images in Publications (CLIP) principles using a validated consensus-based methodology. Implementation of the PRIASE 2021 guidelines will reduce potential sources of bias and thus improve the quality, accuracy, reproducibility, completeness and transparency of reports describing animal studies in Endodontology. The PRIASE 2021 guidelines consist of a checklist with 11 domains and 43 individual items and a flowchart. The aim of the current document is to provide an explanation for each item in the PRIASE 2021 checklist and flowchart and is supplemented with examples from the literature in order for readers to understand their significance and to provide usage guidance. A link to the PRIASE 2021 explanation and elaboration document and PRIASE 2021 checklist and flowchart is available on the Preferred Reporting Items for study Designs in Endodontology (PRIDE) website (http://pride-endodonticguidelines.org/priase/).

PRIASE 2021 guidelines for reporting animal studies in Endodontology: a consensus-based development.

Animal testing is crucial in situations when research on humans is not allowed because of unknown health risks and ethical concerns. The current project aims to develop reporting guidelines exclusively for animal studies in Endodontology, using an established consensus-based methodology. The guidelines have been named: Preferred Reporting Items for Animal Studies in Endodontology (PRIASE) 2021. Nine individuals (PD, VN, AK, PM, MN, JF, EP, JJ and SJ), including the project leaders (PD, VN) formed a steering committee. The steering committee developed a novel checklist by adapting and integrating their animal testing and peer review experience with the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines and also the Clinical and Laboratory Images in Publications (CLIP) principles. A PRIASE Delphi Group (PDG) and PRIASE Online Meeting Group (POMG) were also formed. Thirty-one PDG members participated in the online Delphi process and achieved consensus on the checklist items and flowchart that were used to formulate the PRIASE guidelines. The novel PRIASE 2021 guidelines were discussed with the POMG on 9 September 2020 via a Zoom online video call attended by 21 individuals from across the globe and seven steering committee members. Following the discussions, the guidelines were modified and then piloted by several authors whilst writing a manuscript involving research on animals. The PRIASE 2021 guidelines are a checklist consisting of 11 domains and 43 individual items together with a flowchart. The PRIASE 2021 guidelines are focused on improving the methodological principles, reproducibility and quality of animal studies in order to enhance their reliability as well as repeatability to estimate the effects of endodontic treatments and usefulness for guiding future clinical studies on humans.

Preferred Reporting Items for Animal Studies in Endodontology: a development protocol.

The regulated use of animals in endodontic research is often necessary to investigate the biological mechanisms of endodontic diseases and to measure the preclinical efficacy, biocompatibility, toxicology and safety of new treatments, biomaterials, sealers, drugs, disinfectants, irrigants, devices and instruments. Animal testing is most crucial in situations when research on humans is not ethical, practical or has unknown health risks. Currently, there is a wide variability in the quality of manuscripts that report the results of animal studies. Towards the goal of improving the quality of publications, guidelines for preventing disability, pain, and suffering to animals, and enhanced reporting requirements for animal research have been developed. These guidelines are referred to as Animals in Research: Reporting In Vivo Experiments (ARRIVE). Henceforth, causing any form of animal suffering for research purposes is not acceptable and cannot be justified under any circumstances. The present report describes a protocol for the development of welfare and reporting guidelines for animal studies conducted in the specialty of Endodontology: the Preferred Reporting Items for Animal Studies in Endodontology (PRIASE) guidelines. The PRIASE guidelines will be developed by adapting and modifying the ARRIVE guidelines and the Clinical and Laboratory Images in Publication (CLIP) principles. The development of the new PRIASE guidelines will include a five-step consensus process. An initial draft of the PRIASE guidelines will be developed by a steering committee. Each item in the draft guidelines will then be evaluated by members of a PRIASE Delphi Group (PDG) for its clarity using a dichotomous scale (yes or no) and suitability for its inclusion using a 9-point Likert scale. The online surveys will continue until each item achieves this standard, and a set of items are agreed for further analysis by a PRIASE Face-to-face Consensus Meeting Group (PFCMG). Following the consensus meeting, the steering committee will finalize and confirm the PRIASE guidelines taking into account the responses and comments of the PFCMG. The PRIASE guidelines will be published and disseminated internationally and updated periodically based on feedback from stakeholders.

A protocol for developing reporting guidelines for laboratory studies in Endodontology.

Laboratory-based research studies are the most common form of research endeavour and make up the majority of manuscripts that are submitted for publication in the field of Endodontology. The scientific information derived from laboratory studies can be used to design a wide range of subsequent studies and clinical trials and may have translational potential to benefit clinical practice. Unfortunately, the majority of laboratory-based articles submitted for publication fail the peer-review step, because unacceptable flaws or substantial limitations are identified. Even when apparently well-conducted laboratory-based articles are peer-reviewed, they can often require substantial corrections prior to the publication. It is apparent that some authors and reviewers may lack the training and experience to have developed a systematic approach to evaluate the quality of laboratory studies. Occasionally, even accepted manuscripts contain limitations that may compromise interpretation of data. To help authors avoid manuscript rejection and correction pitfalls, and to aid editors/reviewers to evaluate manuscripts systematically, the purpose of this project is to establish and publish quality guidelines for authors to report laboratory studies in the field of Endodontology so that the highest standards are achieved. The new guidelines will be named-'Preferred Reporting Items for Laboratory studies in Endodontology' (PRILE). A steering committee was assembled by the project leads to develop the guidelines through a five-phase consensus process. The committee will identify new items as well as review and adapt items from existing guidelines. The items forming the draft guidelines will be reviewed and refined by a PRILE Delphi Group (PDG). The items will be evaluated by the PDG on a nine-point Likert scale for relevance and inclusion. The agreed items will then be discussed by a PRILE face-to-face consensus meeting group (PFCMG) formed by 20 individuals to further refine the guidelines. This will be subject to final approval by the steering committee. The approved PRILE guidelines will be disseminated through publication in relevant journals, presented at congresses/meetings, and be freely available on a dedicated website. Feedback and comments will be solicited from researchers, editors and peer reviewers, who are invited to contact the steering committee with comments to help them update the guidelines periodically.

Assessment of bioactive and bio-adhesive therapies to enhance stem cell attachment to root surface dentine.

AIM: To compare bioactive and bio-adhesive therapies to enhance stem cell attachment to the root dentine of human teeth. METHODOLOGY: Dentine slabs (n = 72) were cut from the lower 3 mm of the roots of extracted human permanent teeth. The root dentine slabs were untreated, or coated with bio-adhesive, or human recombinant transforming growth factor-beta1 (hrTGF-B1), or human recombinant bone morphogenic protein-2 (hrBMP-2). The dentine slabs were placed with the root surface in contact with confluent periodontal stem cell (PSC) cultures using aseptic techniques. The cells and dentine slabs were submerged in culture media for 4, 24 and 72 h. The specimens were fixed in formalin, dehydrated and processed for scanning electron microscopy (SEM). RESULTS: SEM micrographs at x2000 magnification revealed PSC extensive adherence to root dentine for all of the bio-adhesive and bioactive treatments. The addition of bioactive molecules did not improve PSC attachment. Few cells attached to the negative control treatments. CONCLUSIONS: Bio-adhesive and bioactive growth factors were not needed to promote PSC attachment to the root dentine of human teeth, because it already appears to have good natural properties to promote PSC attachment. This suggests PSC can be used for the clinical replantation of avulsed teeth without the need for bio-adhesive and bioactive treatments.

Comparison of operative procedure variables on pulpal viability in an ex vivo model.

AIM: To measure and compare the responses of pulp tissue to cavity preparation and restoration variables using a novel tooth slice culture model. METHODOLOGY: Experimental cavities (265) were continuously cut, under carefully controlled conditions, into the dentine of the labial aspect of 28-day-old Wistar rat incisors, and slices of these teeth maintained in organ culture for up to 2 weeks. The experimental variables examined were: the preparation method, remaining dentine thickness, coolant, drill speed, conditioning with EDTA and filling materials. The reactions of the dentine-pulp complex to the experimental variables were measured using pathohistometric analysis and the correlations between variables were determined using analysis of variance statistical tests. RESULTS: In rank order of surgically induced restorative pulpal injury, from the most to the least injurious were: remaining dentine thickness, absence of coolant during cavity preparation, bur speed, cavity conditioning treatments and the filling material. CONCLUSIONS: To reduce pulp injury and to promote pulpal repair activity, the correct use of appropriate materials are important. However, of relatively greater importance is the operative technique adopted, the need to avoid the excess removal of dentine and to minimize trauma during preparation.

Oral health in Florida nursing homes.

OBJECTIVES: The purpose of this study was to measure the oral health and hygiene status among 265 South Florida nursing home residents aged between 45 and 98 years. METHODS: The oral health and hygiene status of the residents were assessed by noting the presence of calculus, caries, gingivitis, cheilitis, apthous ulcer, dry mouth and red or white lesions. RESULTS: The incidence of nursing home residents with calculus was 79.6% and the remaining 20.4% were edentulous. More than half of residents had oral problems (50.6%) the commonest was gingivitis (36.6%), followed by caries (26%) and tooth fracture (15.9%). Almost half the residents wore dentures (47.2%). Statistical analysis was conducted using analysis of variance (P-values). Ageing of the residents was statistically correlated to a worsening of oral hygiene status (P<0.0066), absence and presence of one or two dentures (P<0.0034) and a loss of teeth (P<0.0001). CONCLUSIONS: The ageing of residents is correlated to increasing oral health problems and the loss of teeth. Oral health neglect affects almost all of the nursing home residents. Care providers should receive education and training from dental hygienists to improve the standard of oral hygiene and health of the elderly.

Comparison of pulp responses to resin composites.

Diverse reports have described how various types of adhesive systems cause disastrous pulp necrosis, chronic severe inflammation or failure to stimulate any pulp reactions. This article reports on the effects of five common adhesive systems and how they compare in terms of pulp injury as measured by odontoblast survival or dentin regeneration and reactionary dentin formation. One hundred and thirty Class V pulp, non-exposed cavities were prepared in non-human primate teeth and were restored with five different adhesive systems. After a period of time between 3 and 172 days, the teeth were extracted, fixed, processed and examined histomorphometrically. Bacterial microleakage was detected with McKays stain and inflammation was categorized according to the International Organization for Standardization (ISO) criteria. The number of odontoblasts and the area of reactionary dentin were measured. Pulp reactions of all adhesive systems were generally minimal, although some systems permitted bacterial microleakage in 33% of restorations, and some other systems were associated with pulp inflammation in 22% of restorations. These observations suggest that adhesive systems provide acceptable biocompatibility, however, there is strong potential for improvement.

Remaining dentine thickness and human pulp responses.

AIM: To evaluate pulp responses as a function of remaining dentine thickness (RDT) of 98 class V cavity preparations in 49 teeth of 31 patients aged 10-16 years. METHODOLOGY: Shallow cavities were restored with amalgam, deeper cavities or pulp exposures were restored with amalgam lined with calcium hydroxide or with zinc oxide eugenol. Teeth were extracted after 3-89 days for orthodontic reasons. Following processing for light microscope analysis, the number of odontoblasts, pulp inflammation, and repair was recorded. RESULTS: In comparison with independent odontoblasts, the numbers of odontoblasts were reduced by 13.6% beneath a RDT of 2.5-0.5 mm, 33.7% beneath a RDT of 0.5-0.01 mm and 99.0% beneath pulp-exposed cavities. Reparative dentine was observed following pulp exposure and reactionary dentine was observed with a mean RDT of 0.77 mm (2.5-0.01 mm). Reactionary dentine secretion was influenced by RDT and restorative materials. Pulp inflammation was not influenced by RDT in the present study. CONCLUSIONS: Cavity RDT mediates a powerful influence on underlying pulp tissue vitality but it has little effect on reactionary dentine secretion and inflammatory activity. Gross tissue injury explains the poor pulp capping prognosis following exposure and underlies the need to avoid this type of injury. Following restoration, a RDT of 0.5 mm or greater is necessary to avoid evidence of pulp injury.

Histomorphometric analysis of odontoblast-like cell numbers and dentine bridge secretory activity following pulp exposure.

AIM: The purpose of this study was to collect quantitative information about the numbers and dentine bridge secretory activity of odontoblast-like cells following dental pulp exposure. METHODOLOGY: The numbers and secretory activity of odontoblast-like cells were measured histomorphometrically between 7 days and 2 years in 161 pulp-exposed nonhuman primate teeth. The area of dentine bridges and the dimensions of cavity preparations were measured. The density of odontoblast-like cells and subjacent reorganizing tissue cells were measured beneath dentine bridge formation. The presence of operative dentine debris and tunnel defects in bridges was noted. Pulp inflammation was categorized according to ISO standards. Bacteria were detected using McKay's stain. RESULTS: The area of dentine bridges was mediated by the density and secretory activity of odontoblast-like cells over time. The cell density of subjacent reorganizing tissue was found to be strongly associated with that of odontoblast-like cells. Bacterial microleakage was found to impede dentine bridge secretion by odontoblast-like cells. CONCLUSIONS: Pulp reparative activity occurs naturally beneath capping materials in the absence of bacterial microleakage. The outcome of pulp-capping treatments could be beneficially influenced by concentrating attention on limiting the width of pulp exposure, minimizing pulp injury by limiting the creation of operative debris and placing materials which prevent bacterial microleakage.

Odontoblast morphology and dental repair.

OBJECTIVES: To investigate the changes in morphology and activity of pulp odontoblasts in response to cavity restoration variables and patient factors. METHODS: Class V non exposed cavities were prepared in the intact 1st or 2nd premolar teeth of 27 patients, aged between 9 and 17 years-old. Following tooth extraction, the area of reactionary dentine and the area of the odontoblasts were measured using computerised histomorphometry. RESULTS: The cytoplasm to nucleus ratio of the odontoblasts was found to increase beneath cut dentinal tubules, following the secretion of reactionary dentine. However, none of the patient or preparation variables were found to be correlated with changes in the odontoblast cytoplasm to nucleus ratio. CONCLUSIONS: Morphological changes in human odontoblasts is directly related to their capacity to repair dentine injuries and provide pulp protection. Changes in odontoblast morphology reflect secretory activity.

Comparison of pulp responses following restoration of exposed and non-exposed cavities.

OBJECTIVES: The purpose of this study is to compare and contrast differences of pulp responses between non-exposed and exposed cavity preparations in terms of inflammation, frequency of bacterial microleakage, odontoblast and odontoblastoid cell numbers, and tertiary dentine formation. METHODS: Class V non-exposed cavities (n=161) and exposed cavities (n=161 teeth) were prepared in non-human primate teeth. Cavities were restored with calcium hydroxide [Ca(OH)(2)], resin modified glass ionomer, or resin composite. Following extraction (7-730 days), bacteria were detected with McKays stain and pulp reactions were categorized according to ISO guidelines. Teeth were analyzed histomorphometrically and statistically using analysis of variance tests. RESULTS: Exposed cavities in comparison with non-exposed cavities were found to have more severe inflammation (p=0.0001), greater quantities of tertiary dentine (p=0.0001), and an increased frequency of bacterial microleakage (p=0.0034). The density of odontoblastoid cells beneath pulp exposed tertiary dentine was found to be 47.8% of odontoblast cell density beneath non-exposed dentine (p=0.0001). CONCLUSIONS: The restoration of exposed cavity preparations is associated with more traumatic pulp injury and repair responses. Consequently, efforts should be made to minimize iatrogenic dentine removal during cavity preparation and the creation of pulp exposures whenever possible.

Analysis of incisor pulp cell populations in Wistar rats of different ages.

Pulp cell function and viability is important for maintaining tooth vitality throughout life. However, the effects of ageing on pulpal cell populations and pulp tissue remodelling are still unclear. The aim here was to quantify age-related cell-density changes in the pulp of rat incisor teeth, using histomorphometric analysis and ANOVA. Mandibular (n=35) and maxillary (n=34) incisors were carefully extracted from 20 Wistar rats aged between 1 and 18 months, fixed and processed for light microscopy. Cell counts were performed in mature and immature regions at both labial and lingual aspects of all teeth. Odontoblast and subodontoblast cell densities were reported per mm of pulp-dentine border and core fibroblast density per mm2 pulp tissue. Irrespective of age, odontoblast and subodontoblast densities were lower in the immature than the mature regions of both maxillary and mandibular incisors (P<0.001). However, in both regions odontoblast and subodontoblast densities decreased significantly with increasing age (P<0.0001). The age-related reduction in odontoblasts was significantly greater in mature than immature regions (P<0.02) but not influenced by other variables. In contrast, the age-related reduction in subodontoblasts was significantly different between mandibular and maxillary teeth (P=0.012) but not influenced by site. Unlike odontoblasts and subodontoblasts, core fibroblasts showed small but significant increases with increasing age (P<0.0001). These age-related reductions in the density of odontoblasts and subodontoblasts may partly explain the slower rate of secondary dentine secretion and decreased pulp repair activity associated with ageing.

Hierarchy of variables correlated to odontoblast-like cell numbers following pulp capping.

OBJECTIVES: Following tooth pulp exposure, pulpal repair is accomplished by dentine bridge secretion by odontoblast-like cells. However, little information is available about the hierarchy of variables, which influence odontoblast-like cell numbers. The purpose of this study was to examine correlations between pulp capping events and odontoblast-like cell numbers. METHODS: Two hundred and fifty standardised pulp exposed cavities were prepared in non-human primate teeth according to ISO usage guidelines. Exposed pulps were capped with Calcium hydroxide [Ca(OH)(2)], and multi-step and self-etching primer composite resins. Teeth were collected from 3 to 60-days to observe pulp reactions. Statistical analysis was evaluated using analysis of variance. RESULTS: The hierarchy of variables correlated to odontoblast-like cells were the dentine bridge area (P = 0.0001), time since pulp exposure (P = 0.0001), odontoblast numbers opposite the exposure site (P = 0.0002), and pulp capping materials (P = 0.0313). Other pulp capping variables were found to be less likely to be correlated with odontoblast-like cell numbers. CONCLUSIONS: The area of dentine bridge formation is directly related to the numbers of odontoblast-like cells, cell activity is time dependent, and the cell numbers are much lower than original odontoblast cells. The time-lag between the appearance of odontoblast-like cells at the site of pulp exposure, and the limited numbers of these cells, explain why pulpal repair is difficult to achieve successfully following pulp exposure.

Pulpal inflammatory responses following non-carious class V restorations.

The effects of inflammatory activity following surgical intervention can injure pulp tissues; in severe cases it can lead to pulpal complications. With this article, the authors report on the effects of cavity preparation and restoration events and how they can interact together to reduce or increase the severity of pulpal inflammatory activity in 202 restored Class V cavities. Although some inflammatory activity was observed in the absence of bacteria, the severity of pulpal inflammatory activity was increased when cavity restorations became infected. Zinc oxide eugenol and resin-modified glass ionomer cement prevented bacterial microleakage in cavity restorations, with no severe inflammatory activity observed with these materials. Bacteria were observed in cavities restored with enamel bonding resin and adhesive bonded composites and were associated with severe grades of inflammatory activity. The cavity remaining dentin thickness influenced the grade of inflammatory activity. In the absence of infection, the grade of inflammatory activity decreased after 20 weeks post-operatively. In the presence of infection, the grade of pulpal inflammation remained stable until a minimum of 30 weeks had elapsed.

Transdentinal stimulation of reactionary dentinogenesis in ferrets by dentine matrix components.

OBJECTIVES: Trans-dentinal stimulation of reactionary dentinogenesis may be mediated through cellular signalling by bio-active components released from the dentine matrix during injury. Understanding of these processes will be important to guide dentinal repair activity following restorative surgery. The purpose of this study was to investigate the effects of implanting isolated dentine matrix proteins within cavity preparations on dentinal repair activity and odontoblast survival using a controlled experimental animal model. METHODS: Forty-five ferret canine teeth each had a standardised non-exposed cylindrical Class V cavity cut into the buccal dentine. Ten cavities were restored with zinc oxide eugenol, as a control. Two different lyophilised preparations of dentine matrix components were implanted on the axial floor of the remaining 35 cavities prior to filling with zinc oxide eugenol. After post-operative periods of 2, 7, 14, 28, and 90 days, the teeth were extracted and examined histomorphometrically and the data analysed statistically by analysis of variance tests. RESULTS: The odontoblasts beneath the restored cavities responded to the presence of the two dentine matrix preparations by increasing the mean area of reactionary dentine secreted by 433 and 578%, and the numbers of odontoblasts remained stable. CONCLUSION: Dentine matrix components can stimulate reactionary dentinogenesis in non-exposed cavity preparations. It will now be important to identify how this may be harnessed as a part of routine restorative surgery to optimise treatment outcomes with a biological basis.

Restorative pulpal and repair responses.

BACKGROUND: Each year, about 90 million new restorations are placed in the United States and 200 million are replaced. Controversy surrounds the pulpal reactions and frequency of bacterial microleakage associated with common restorative materials. The authors investigated and compared pulpal reactions to different types of restorative materials. METHODS: Two hundred seventy-two teeth with standardized rectangular Class V unexposed cavities were restored with resin-based composite bonded to dentin; resin-based composite bonded to enamel; resin-modified glass ionomers, or RMGI; amalgam lined with zinc polycarboxylate, or ZnPC; amalgam lined with calcium hydroxide, or Ca(OH)2; or zinc oxide-eugenol, or ZnOE. Teeth were extracted for orthodontic reasons between 20 and 381 days later. The authors categorized pulpal responses according to standards set by the Federation Dentaire Internationale and the International Organization for Standardization. Bacteria were detected using Brown-Brenn-stained sections. Pulpal responses were evaluated using histomorphometric analysis and analysis of variance statistics. RESULTS: The results showed that RMGI was the best material for preventing bacterial microleakage, and resin-based composite bonded to enamel was the worst. In regard to minimizing pulpal inflammatory activity, ZnOE was the best material and resin-based composite bonded to enamel was the worst. In terms of maximizing odontoblast survival beneath deep cavity preparations, Ca(OH)2, was the best material and RMGI was the worst. CONCLUSIONS: The results show that bacterial microleakage, pulpal injury and repair responses varied widely with different restorative materials. CLINICAL IMPLICATIONS: The authors recommend that RMGI be used to restore teeth with cavities that are shallow to moderate in depth, with the floor of deep cavities being lined with Ca(OH)2 before the teeth are restored with RMGI.