Tooth Fractures

For breaking force experiments overall eight radulae were used (four of native and four of treated condition). For testing in dry condition, we used two native and two treated radulae, and for testing under wet condition, we also used two native and two treated radulae. As described in detail in [25 , 27 (link)], every radula was mounted on one microscope glass slide (Carl Roth, Karlsruhe, Germany) either with double sided adhesive tape (for experiments in dry condition) or by applying epoxy (RECKLI EPOXI WST, RECKLI GmbH, Herne, Germany) to both alary processus and both sides of the radular membrane (for experiments in wet condition). As mentioned above, this epoxy was chosen, because it does not infiltrate the teeth. Then teeth were carefully stroked into proposed feeding position. For experiment in dry condition the mounted radulae were left to dry for four hours at room temperature. For experiments in wet condition the epoxy was left for two days to polymerize; afterwards radulae were rehydrated by applying distilled water onto the radula. Water drops on teeth were removed by soaking it up with tissues before experiment.
Glass slides with the radulae were positioned under binocular microscope and a rounded steel needle (diameter: 0.4 mm), firmly attached to a force transducer FORT-1000 (World Precision Instruments, Sarasota, FL, USA), which was connected to an amplifier (Biopac System Inc., California, USA) and computer-based data acquisition and processing system (Acq Knowledge, Biopac Systems, Inc., version: 3.7.0.0, World Precision Instruments, Sarasota, Florida, USA), was pressed onto the individual tooth cusps by employing a remote-controlled micromanipulator (DC 3001R, World Precision Instruments Inc., Sarasota, Florida, USA). The needle was positioned on the concave part of every tooth cusps at 30° to the horizontal plane and moved onto the cusps until structural failure occurred. The forces needed to either crush or shear teeth were recorded and their maxima were calculated from the obtained force–time diagrams. Overall, we received data from 1301 individual breaking force curves (equals the quantity of broken teeth). Afterwards the broken radulae were documented with the Tabletop scanning electron microscope TM4000 Plus (Hitachi, Tokyo, Japan) and the types (e.g. crushing, rupture, breaking) and region of the structural failure (basis, stylus, cusps, etc.) were examined (Fig. 3a and b). Since the centrals and the lateral teeth I were either crushed or ripped, we were not able to measure their area of failure and to determine the breaking stress (breaking force divided by tooth cross sectional area). The lateral teeth II and the marginal teeth exhibited a rather plain breaking area, which could be measured. Here, the average breaking area was determined by documenting ten areas per tooth type and per radula using SEM. The obtained SEM images were imported into Adobe Illustrator CS 6 (Adobe Inc., San José, USA), here the breaking areas were outlined, and every outline was translated into an individual red area. By using the scale bar from SEM as reference a blue square area (in μm²) was also computed for every image. Then images were individually imported into Adobe Photoshop CS6 (Adobe Inc., San José, USA), here the quantity of blue and red pixels was read out. By accounting pixel quantity of the square with the pixel quantities for every broken tooth area, the area (in μm²) of failure and subsequently an average breaking area for every tooth type could be determined. Then breaking stress was calculated from the breaking force and the mean broken area for the corresponding tooth type.

The study was approved by the WALTHAM^® Centre for Pet Nutrition ethical review committee, owner consent was obtained and an owner survey was completed for all dogs included in the study. The study cohort comprised client owned pet dogs presented at a veterinary referral dental clinic (The Veterinary Dental Surgery, Surrey, UK). Only dogs under anaesthetic for routine dental treatment or treatment for non-periodontal complications e.g. fractured teeth or other non-infectious conditions were screened for inclusion in the study. No dogs were anaesthetised solely for the collection of plaque samples.
Dental assessments, scoring and subgingival plaque sampling were performed by a single veterinary dentist (L. Milella) to avoid variation in scoring. The periodontal health status of each dog was obtained following the Wiggs & Lobprise scoring system [6 ] and plaque samples taken from dogs regarded as having healthy teeth and gums, gingivitis or mild periodontitis ( <25% attachment loss). Dogs were excluded from the study if they had: 1) Significant veterinary oral care within the preceding 3 months; 2) Regular dental care at home i.e. dogs whose teeth are regularly brushed; 3) Systemic or oral antibiotic treatment at any time during the previous 3 months and 4) Evidence of any extra-oral bacterial infections in the past month. Veterinary observations suggest certain breeds may exhibit an atypical early onset/ aggressive form of periodontitis, though no data exists to confirm this these breeds (Greyhounds, Yorkshire Terriers, Maltese and Toy Poodles) were also excluded.
Sub-gingival plaque samples were collected using a sterile periodontal probe and placed in 350µl TE buffer (50 mM Tris pH 7.6, 1 mM EDTA pH 8.0 & 0.5% Tween 20) prior to storage at -20°C.
Healthy dogs were sampled subgingivally at eighteen sites, targeting the teeth believed to be most often affected by PD (upper 103-108 bilaterally and lower 404, 408 and 409 bilaterally), to support plaque volumes in the absence of periodontal pockets. Periodontally diseased dogs were sampled for subgingival plaque at up to twelve diseased sites (103, 104, 108, 404, 408, 409 bilaterally) during their normal periodontal treatment. In a minority of cases if a dog had sites of periodontal disease not on those teeth but more than 6 teeth were affected, the samples were taken from the affected teeth. Information on dog age, breed, size and sex was collated.

In this cross-sectional study, GT was used to evaluate Internet search trends during the COVID-19 pandemic worldwide. GT was used to collect data. When a person searches for a term using the Google search engine, GT generates data on how common the query term is. It presents normalized data about anonymous user searches in the form of relative search volumes (RSVs), varying from 0 to 100, where 100 represents the maximum value of searches in a given period.
The comparison strategy was based on standard terms among oral health policies and recommendations regarding paediatric dentistry defined by the American Academy of Pediatric Dentistry (AAPD) and related popular search queries as posted on GT [11 ]. Efforts were made to include terms that are likely to be familiar to people looking for information on dental care. Individuals may not utilise certain dental terminology such as regenerative pulp therapy. Therefore terminology such as, ‘space maintainer’ was included instead of ‘band and loop space maintainer’, and its RSV was higher. The paediatric dentistry-related queries are presented in Table 1. Each term was individually searched using GT. The monthly RSVs and the lists of paediatric dentistry-related queries were included from December 2016 to December 2021 using GT. The GT search parameters used were “Worldwide,” “December 2016–December 2021,” “All categories,” and “Web Search.” The datasets were analyzed using SPSS version 25 (IBM Corp, Armonk, NY, USA). The Shapiro–Wilk test was used to confirm the normality of distribution for each dataset per query. Two separate datasets were obtained, before and after the pandemic. One-way ANOVA was used to determine whether there was a significant difference in RSV scores between the first 2 years of COVID-19 and the first 3 years before COVID-19 worldwide. T-tests were used for bivariate comparisons. For all analyses, p < 0.05 was considered statistically significant.Table 1

Top relative search volumes of paediatric dentistry related queries

Paediatric dentistry related diseases and treatments	Search terms with top RSV's
Early childhood caries	Baby teeth caries, child teeth caries, baby tooth caries, child tooth caries
Kids toothache	Kids toothache, child toothache
Dental trauma	Avulsed tooth, fractured tooth, broken tooth, broken teeth
Cleft lip and palate	Cleft lip, cleft palate, harelip
Root canal on baby teeth	Paediatric pulpotomy, baby root canal, root canal on baby tooth
Paediatric restorative dentistry	Hall technique, stainless steel crown, fluoride treatment, atraumatic restorative treatment, kids tooth filling
Space maintainer	Band and loop space maintainer, distal shoe space maintainer, transpalatal arch, lingual holding arch
Mouthguard	Sports mouthguard, kid mouthguard, child mouthguard
Paediatric dentistry	Pedodontist, child dentist, child dentistry, paediatric dentist

At baseline, all patients had full mouth rehabilitation including: instructions in self-performed plaque control measures, full mouth supra and subgingival scaling and removal of any local irritating factors (fractured teeth, poor restorations, or fixed or removable prosthodontics) if needed. For the purpose of standardization, out of multiple oral lesions, a "marker lesion" was recorded and specified for each patient, being the most severe and painful lesion (minimum clinical score of 2) in the buccal mucosa at baseline.
Eligible patients were then randomly assigned into three study groups;
Group I (TAC patch) (n = 10) patients were instructed to apply a tacrolimus buccal mucoadhesive patch on the marker lesion as shown in Fig. 1 twice daily for 8 weeks containing [10% Tacrolimus (0.1% weight/weight [w/w]), Chitosan 2% (weight/volume [w/v]), 3% w/v hydroxypropyl methyl cellulose (HPMC) in addition to propylene glycol, acetic acid and citric acid in 100 ml distilled water [15 (link)] (Nano-Gate Company, Mokattam, Cairo, Egypt). Patients were instructed to apply slight pressure on the entire surface of the patch at the time of application either using their finger or tongue, to refrain from bringing their teeth into contact with the patch, to avoid chewing or excessive jaw movements, and to avoid eating or drinking for at least 1 h following application of the patches [19 (link)].Fig. 1

Tacrolimus mucoadhesive patch applied to the marker lesion on the buccal mucosa in Group I patient

Group II (TAC gel) (n = 10) patients received topical tacrolimus gel on marker lesion four time daily for 8 weeks containing [Tacrolimus 0.1% weight/weight [w/w]) to 2% w/w sodium Carboxy methylcellulose (CMC) as gelling agent dispersed in 25 ml of distilled water 0.1%] [20 (link)] 4 times/day.
Group III (TRI. gel) (n = 10) received topical 0.1% triamcinolone acetonide in the same gel carrier used with tacrolimus gel 4 times/day. Miconazole 2% topical antifungal (Miconaz® oral gel: Miconazole 2 g per 100 gm). (Medical Union Pharmaceuticals—MUP—Egypt) was applied only in the 4th week and 8th week of the treatment period to avoid secondary candidiasis in this group [16 (link)].
All patients in gel groups were instructed to apply a thin layer (½ ml) guided by graduation on plastic syringes loaded with the gel medication using a finger or cotton tip applicator on dried lesions after meals, considering not to eat, drink, or speak for at least 1 h afterward [16 (link)]. Patients in all groups were also instructed to apply the same intervention to other symptomatic lesions whenever needed.