Dental Pulp Cavity

Three male and seven female systemic healthy volunteers aged 24 to 40 years (mean ± s.d., 31.1 ± 4.8 years) were recruited at the Hiyoshi Oral Health Clinics. All participants met the inclusion and exclusion criteria. Inclusion criteria were: (a) non-smoker, (b) non-denture wearer, and (c) non-brace wearer. Exclusion criteria were: (a) systemic disease, (b) received antibiotics in the last 6 months, and (c) pregnancy or breastfeeding.
Sample collection and dental examinations was conducted at the Hiyoshi Oral Health Clinics. Prior to sample collection, the participants were instructed not to brush their teeth from the previous night to the time of sampling, and were prohibited from eating or drinking for at least 1 hour prior to sampling. A minimum of 2 mL unstimulated saliva was collected by allowing saliva to accumulate on the floor of the mouth followed by spitting into a specimen tube every 60 seconds^{21 (link)}. Following the collection of unstimulated saliva, participants were asked to swish their mouth vigorously for 10 seconds with 3 mL sterilized water, and then to spit into another specimen tube. Following the collection of this mouth-rinsed water, individuals were asked to chew paraffin gum to stimulate saliva, samples of which were then collected in further specimen tubes every 60 seconds. Tongue-coating samples were collected by scraping the dorsum of the tongue with Catch-All™ Specimen Collection Swabs^{22 (link)} (Epicenter Biotechnologies, Wisconsin, USA). All samples were placed in a freezer within 10 minutes of collection and stored at −80 °C until use.
After samples collection, the numbers of present, decayed, missing and filled teeth were examined. The number of decayed, missing and filled teeth signifies teeth with caries experience, and represents the caries history of the individual. The periodontal pocket depths and bleeding on probing (BOP) at four sites (mesiobuccal, distobuccal, mesiolingual, and distolingual) of all teeth were measured using a periodontal pocket probe following sample collection. Summary of the oral health condition of the participants is shown in Table 2.Table 2

Summary of participants' information.

Sample ID	B01	B02	B03	B04	B05	B06	B07	B08	B09	B10
Age	40	39	29	28	30	24	27	32	33	29
Gender	M	F	F	F	F	F	F	M	F	M
DMFT	5	0	4	0	0	0	1	20	9	1
Flow rate of Unstimulated Saliva (g/min)	0.86	0.6	1.04	0.54	0.89	0.22	0.62	1.32	0.37	0.69
Flow rate of Stimulated Saliva (g/min)	4.33	3.09	2.75	2.37	4.81	1.45	2.75	3.71	2.73	3.45
PPD > 4 mm (%)	0	1.8	0	2.7	0	0	0	4.3	7.4	0.8
BOP (%)	2.7	15.2	6.3	50.9	2.7	3.6	0	0.9	24.1	2.5

This study was a cross-sectional descriptive-analytic study conducted on people aged 15–40 yr old living in Sanandaj, Kurdistan, western Iran in 2015. The estimated sample size was 2200 people, and finally, the required data were collected from 2000 people. We used cluster sampling method and each cluster included 10 people who were in the desired age range. The heads of the clusters were selected based on the geographical encoding of Sanandaj, obtained from the Sanandaj post office. The required data were collected using a questionnaire and clinical examinations carried out through visiting the selected households.
The questioners were four trained dental students received the necessary training on how to complete the questionnaires and perform clinical examination. To calibrate between the four questioners, prior to the initiation of data collection process, each of the four trained dental students performed a survey on 25 subjects selected from among the study population; the mean stability between their findings was 97%. The questioners visited each household at their home and briefed the household members about the research project and then completed the questionnaire through asking questions about the following items: demographic information, insurance coverage status, socioeconomic status (SES), frequency of brushing during a day, frequency of the use of dental floss during a day, and frequency of the use of fluoride mouthwash during a day. In this study, the SES of individuals was determined through questioning about their assets, which is a more appropriate way in developing countries. Using the principal component analysis (PCA) method, the studied people were divided into five quintiles including poorest, poor, moderate rich, and richest (29 ).
The DMFT score of the samples were determined based on the results of clinical examination and calculation of the number of decayed (D), filled (F), and missed (M) teeth due to caries. The data were collected by questioners through observation and direct examination of the samples’ teeth using mirror number 4 and a medisporex catheter. During the examination, the subjects under examination and the researcher sat close to the window to perform the examination under the maximum natural light. After examining each patient, the results were recorded in the questionnaire.
In this study, the statistical analysis was performed using SPSS (ver.20, Chicago, IL, USA) software at a significance level of P<0.05. Descriptive statistics (Mean, Standard Deviation (SD), and frequency distribution tables) were used to describe the collected data. Using T-test and ANOVA, the DMFT index was assessed at different levels of the independent variables. The relationship between independent variables and the DMFT index was evaluated using Pearson statistics and Kendall statistics. The variables were entered into the multiple regression model for a P<0.05 in the univariate analysis. Finally, the variables that were significant in the regression test using a stepwise backward method, remained in the model.
The study was approved by the Ethics Committee of Kurdistan University of Medical Sciences (IR.MUK.REC.1393/1). All participants provided written informed consent before participating in the study.

We investigated ten caries teeth samples (with carious fissures). Permanent masticatory human teeth were removed of European descent patients in the age of 18–25 years. A criterion for selection of the samples was the presence of infected fissures on the masticatory surface of molars and premolars. Voronezh State University Ethics Committee approved this study, approval number 001.000–2012. Teeth were prepared in the following way. Initially, the teeth were washed in flowing water, purified from plaque, and then surfaces were dried with an absorbent. Next, using a microtome, the teeth were cleaved and we got slices with the thickness of ~ 1 mm. The resulting microsections were glued to a glass plate of 2 mm thick using an acrylate adhesive. Fig 1A presents a photograph of the typical sample to be analyzed, Fig 1B shows the area of carious fissures in more detail. It should be noted that this Fig 1B is presented in grayscale, therefore the deeper the area in carious fissure the darker its image in the Fig 1B.
Raman spectra in backscattering geometry were measured at room temperature using a conventional experimental setup, which includes a close-cycle He cryostat, a TRIAX550 monochromator, and a liquid- nitrogen-cooled charge-coupled-device (CCD) detector (Princeton Instruments). The laser beam was focused on the sample using a x50 microscope objective, which allowed us to locate the laser spot on the analyzed area within 4–9 μm² limits. Scattered light from the sample was collected and directed to a monochromator and then detected by the CCD matrix (CCD-detector). To measure the Raman spectra, laser excitation power of 40 mW on the sample surface, and acquisition times of 10–20 seconds were used. To reduce the noise, spectrum averaging was set to 5–10. The exciting laser radiation was not polarized, scattered light was also detected without polarization.
The penetration depth of the laser radiation, and, consequently, the effective depth of analysis for Raman scattering can be determined from the ratio λ/2πk where k is an extinction coefficient. For argon laser with λ = 514.5 nm in the analysis of human tooth hard tissues this depth is about 40–60 nm, which makes it reasonable to state that the performed analysis affected only a thin layers of dental tissue.
Particularly, Raman spectra often show an increasing background. Background correction was performed with the method of "rubberband correction" using Bruker optics software. A “rubberband” method determines support points by finding the convex hull for each spectrum. The baselines are then piecewise linear or (smoothing) splines through the support points. Normalization of the Raman spectra was done by the same Bruker optics software.
In our work, the fluorescence spectra were obtained at room temperature on the surface of the samples by means of a standard technique in the same installation as that one of the Raman scattering spectra. In order to focus on the surface, x10 lens was used. Local changes in the fluorescence intensity of the sample surface were successfully analyzed by observing the fluorescence being excited by a 514.5 nm notch filter, which allowed us to visualize well the lighter areas of dental hard tissues with a correspondingly higher fluorescence intensity.

We recruited a convenient sample (n = 69) participant from multiple countries: 47 adults and 22 children. At each designated study site, an assigned investigator met with the participant, explained the study, before administering the questionnaire. Participants were asked if they experienced one or more of the specific dental/oral problems in the past month (e.g. bleeding gums when brushing teeth or pain in teeth while consuming hot or cold food or drink). Additional survey questions determined the individual’s perceived need for dental care (‘‘Do you currently have any cavities in your teeth?”; “Do you currently have painful gums or gums that bleed easily?”), reasons why they may not receive regular dental care. The questionnaire was administered in paper in presence of the parent/ guardian when applicable. If the participant did not have a regular dentist, they were given a list of dentists near their residence and information on accessing those services. Toothbrush and dental floss were provided to each participant in addition to oral health instructions.

This study was approved by the bioethics committee of Beijing Chaoyang Hospital of Capital Medical University (Registration number: 2020-ke-28). The single-rooted, caries free human teeth were collected and stored in 0.01% thymol solution at 4 ℃. As Fig. 2a shows, the preparation process was carried out refer to Shen Ya [24 (link)]. Fifteen teeth were selected and sectioned at 1 mm below the cementoenamel junction (CEJ) to a unified length of 4 mm cylindrical dentin block. The root segments were enlarged to F3 and each dentin block was grinded longitudinally into 2 pieces. Each tooth disk was subsequently shaped into approximately 4*4*2 mm. All samples were cleaned by using ultrasound in 2.5% NaOCl and 17% EDTA for 2 min respectively. Finally, normal saline was used to clean and sterilized by autoclaving (121 °C) for 15 min before use.Fig. 2

The schematic illustration of the preparation of dentin blocks (a) and the in vitro study (b)

Deciduous teeth were either collected locally, through the Department of Pediatric Dentistry (neurotypical controls; n = 2), or remotely, for children with AS (n = 6). Immediately following the loss of the tooth, it was placed in transportation media (DMEM/F12 50/50 mix with HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin). DPSCs were isolated and cultured according to our previously described protocol⁷² . Briefly, after mincing the dental pulp from inside the tooth cavity, 3 mg/mL dispase II and 4 mg/mL collagenase I were added to digest the tissue. Cells were then seeded on Poly-D-Lysine-coated 12-well plates with media containing DMEM/F12 1:1, 20% FBS, newborn calf serum (NCS), and 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/Strep). Confluent cultures (80%) were passaged with TrypLE Express (Gibco), and neuronal differentiation was performed only on early passage cells (less than four).
DPSC lines were seeded at 20,000 cells/cm² on Poly-D-Lysine and Matrigel-coated coverslips with DMEM/F12 1:1, 10% FBS, 10% NCS, and Pen/Strep. At 80% confluence, the neuronal differentiation protocol was followed as previously published^{71 (link)}. Briefly, epigenetic reprogramming was performed by exposing the DPSC to 10 μM 5-azacytidine (Acros Scientific) in DMEM/F12 containing 2.5% FBS and 10 ng/mL bFGF (Fisher Scientific) for 48 h. Neural differentiation was induced by exposing the cells to 250 μM IBMX, 50 μM forskolin, 200 nM TPA, 1 mM db-cAMP (Sigma Aldrich), 10 ng/mL bFGF, 10 ng/mL NGF (Invitrogen), 30 ng/mL NT-3 (Peprotech), and 1% insulin-transferrin-sodium selenite premix (ITS) (Fisher Scientific) in DMEM/F12 for 3 days. At the end of neural induction, the cells were washed with 1X PBS. Neuronal maturation was performed by maintaining the cells in Neurobasal A media (Gibco) with 1 mM db-cAMP, 2% B27, 1% N2 supplement (Gibco), 30 ng/mL NT-3, and 1X Glutamax (Gibco) for 30 days.