In vitro histomorphometric comparison of dental pulp tissue in different teeth

Background Dental pulp (DP) represents an accessible and valuable source promising of stem cells for clinical application. However, there are some disadvantages associated with the isolation of dental pulp stem cells (DPSCs), which include the size and weight of the pulp tissue needed to yield sufficient cells for culturing in vitro. Therefore, the objective of this study was to compare in vitro histomorphometry of DP from permanent (premolars, third molar), supernumerary and deciduous teeth of patients between 5 and 25 years old with regards to weight, length, width and the cell density in the four regions of the DP in order to obtain quantitative parameters in a tissue that represents a valuable source of stem cells. Methods DPs were obtained from 10 central incisors deciduous, 20 permanent teeth (10 premolars, 10 third molars) and 10 supernumeraries (six mesiodents and four inferior premolar shapes). The pulps were carefully removed, and the entire tissue was weighed. The pulp length and the width were measured with a digital Vernier caliper. The cellular density analysis was performed according to the four regions of the DP (coronal, cervical, medial and apical) in histological slides using photography and the ImageJ® program for quantification. Results The Pearson correlation test revealed that DP weight among different types of teeth is correlated with age in male patients. A significant positive correlation was noted between length and width of the DP with age in both genders. The mean DP weight for supernumerary and third molar teeth was greater than deciduous and premolar teeth. Finally, the histological analysis showed that the coronal and apical portions of DP in supernumerary and premolar teeth have the highest cell density. Conclusions The DP of supernumerary teeth has quantitatively the best morphometric parameters and cell density comparable with the quality of DP obtained from deciduous teeth.


INTRODUCTION
Dental pulp (DP) is an innervated, highly vascularized soft tissue that provides vitality to the tooth (Rodas-Junco et al., 2017). DP is located inside each primary or permanent tooth, and its main functions include the generation of dentin and maintenance of its biological and physiological vitality in response to traumatic injuries, physical stimulus or bacterial infections (Marrelli et al., 2018;Ravindra et al., 2015;Tatullo et al., 2015). The regenerative function of DP suggests that it contains odontogenic progenitor cells or stem cells that are involved in the regeneration process. In this context, DP has drawn attention in dental research as an accessible and valuable source of stem cells known as dental pulp stem cells (DPSCs). Moreover, DPSCs are going to be ideal for tissue engineering and regenerative medicine (Chalisserry et al., 2017;Honda, Sato & Toriumi, 2017;Ledesma-Martínez, Mendoza-Núñez & Santiago-Osorio, 2016). However, there are some disadvantages associated with the isolation of DPSCs, which might be directly related to the size and weight of DP tissue by limiting the number of stem cells isolated from it (Raoof et al., 2014). Besides, some studies have also focused on methods to correlate some morphometric parameter of the pulp tissue with the patient's age. For example, Ravindra et al. (2015) observed through intra-oral radiography that the total area of the pulp decreased with age. Other authors focus on establishing regression equations using the number of cells in DP to also predict age (Hossain et al., 2017;Von Böhl et al., 2016). Conversely, until now there are no reports that relate morphometric parameters such as weight, length, width and cellular density of DP among different types of teeth and its influence on tissue quality for the isolation of DP cells. Therefore, the objective of this study was to compare the histomorphometry of DP in temporal and permanent teeth and evaluate the cell density in four regions in this tissue with to purpose of generating quantitative parameters that would have important applications in the DPSCs isolation.

Patient recruitment and tooth storage
The DP tissue was obtained from 10 deciduous central incisors, 20 permanent teeth (10 premolars, 10 third molars) and 10 supernumeraries (six mesiodents and four inferior premolar shape). An informed patient consent was obtained from patients or parents of minors. The collection of the material was performed at the Clinics of the Master in Pediatric Dentistry and Oral Surgery, Faculty of Dentistry, Autonomous University of Yucatan. The age of patients ranged from 5 to 25 years, and a slight prevalence of females (21/40, 55.5%) was noted. The protocol was approved by the Ethics Research Committee of Hideyo Noguchi Regional Research Center, Autonomous University of Yucatán (Approval Number: CIE-06-2017). The extracted deciduous teeth exhibited one-third to two-thirds root resorption with well-defined roots. After extraction, all the teeth were rinsed for 5 min in a conical tube containing phosphate-buffered saline (PBS 1X: 138 mM NaCl, 3 mM KCl, 8.1 mM Na 2 HPO 4 and 1.5 mM KH 2 PO 4 , pH 7.4) and labeled with the donor's age and tooth type.

Tissue removal and processing
The teeth were immersed in sterile phosphate buffer saline (PBS 1X pH 7.4), stored on ice pack and transported to the cell culture lab for sample processing. After cleaning the surface of the tooth, a vertical cut of the dental organ was performed using a rotary electric micro motor (String Ò ) with a diamond disc (diameter: 22 mm and thickness: 0.4 mm; ATK Ò ). During this process, constant irrigation was maintained with cold PBS 1X pH 7.4 to reduce overheating of dental tissue (Figs. 1A-1C). Thereafter, the entire pulp tissue was carefully extracted from the cavities of the tooth using a metal clamp and it was weighed using an analytical balance (Citizen CX 200) (Fig. 1D). The pulp length and width were measured with a digital Vernier caliper with a 0.01 mm calibration.

Histomorphometry
The pulp tissues were fixed in 10% formaldehyde solution. Subsequently, the DP was dehydrated in increasing concentration of alcohol: 60%, 70%, 80% and 100%. After that, the DPs were embedded in paraffin and dissected in sections of 5 µm of thickness with a sliding microtome (Leica LM2500). The slices were then dewaxed and stained with hematoxylin and eosin. Four histology slides from each tooth were selected for analysis. Images of the pulp were captured through the digital microscope at a resolution of 1,280 × 720 (Leica DM750 camera MC170H) and 4-100× magnifications connected to a computer. A cell counting was performed manually under high-power (100× magnification) at four regions of each DP tissue: coronal, cervical, middle and apical. For each cell population, the number of cells was normalized to the total area of the pulp sample (325 µm × 402 µm). Afterwards, each image obtained from the histology slides were analyzed by ImageJ v1.49 Software.

Statistical analysis
A one-factor analysis of variance with Tukey's post hoc test was used. The Pearson correlation coefficient was calculated to determine the correlation between morphometric measurements of the DP and the relationship with the patient's age. Statistical significance was defined as p ≤ 0.05.

RESULTS
Correlation between morphometric measurements of the dental pulp and patients' age A correlation study was undertaken to examine the weight, length and width of DP with patients' age (Figs. 2A-2F). The DP weight was correlated with age in male patients, whereas significant changes (p ≤ 0.05) were not noted in females (Figs. 2A and 2B). The Pearson correlation test revealed a significant positive (p ≤ 0.05) correlation between length and width of the DP with age in both genders (Figs. 2C-2F). In general, the data showed that the DP obtained from males had a greater weight, length and width compared with that from females. The results indicate an optimal age interval in males (15-20 years) and females (20-25 years) to obtain 10 mg of DP.
Comparison of morphometric measurements of dental pulp among different types of teeth Linear data correlation and one-factor variance analyses were applied to the variables of weight, length and width (Table 1). The weight of DP was significantly increased (p ≤ 0.05) in supernumerary teeth (20.5 ± 3.56 mg) and third molars (18.7 ± 11.5 mg) from male patients compared with those from females. In contrast, the mean differences in length and width measurements of DP in the third molars were highly significant (p ≤ 0.05) compared with the other types of teeth in both genders (Table 1).
Comparison of dental pulp histology and cellular density of dental pulp tissue in different types of teeth Due the DP of the different teeth analyzed showed different morphometric measurements, we hypothesized that the density in the cell-rich zone may also be different. Thus, a histological analysis was performed in coronal, cervical, middle and apical regions of the DP to evaluate the cell density (Figs. 3A-3N  A greater cellular density was observed in the coronal region of deciduous and premolar teeth compared with supernumerary and third molars (Table 2). In contrast, a high cell density was observed in the apical region of supernumeraries and premolars compared with third molar teeth (Table 2). Together, these results indicate that the apical region of the DP in supernumerary and premolar teeth potentially represents the ideal location to obtain cells.

DISCUSSION
Knowledge of DP histomorphometry in teeth is important for identifying the tooth type that could provide the best source of cells. Several researchers have focused on obtaining DPSCs from exfoliated deciduous or permanent third molars because stem cells from these teeth exhibit a high proliferation capacity (Daud et al., 2016;Shekar & Ranganathan, 2012). It is therefore necessary to identify other source of DPs with quality cellular characteristics for the isolation of cells. In the literature, reports on morphometric and histological measurements of the DP regions among temporary and permanent teeth are quite limited. Besides, pulp weight is a parameter that few authors have considered to obtain DPSCs (Alsulaimani et al., 2016;Kellner et al., 2014;Singh et al., 2016). Our research group considers that DP weight could be important as a starting point for the isolation of DPSCs. The results of the present study show that the DP weight was greater in the supernumerary and third molars teeth in male patients compared with female patients  (Table 1). The weight observed in supernumerary (mesiodents) teeth was may be due to the dense fibrous DP, which could indicate a greater amount of organic substance in this tooth type. The third molar DP of females exhibited an increased weight compared with males likely because female DP was obtained from upper teeth with fused roots. This characteristic facilitated the procurement of a larger homogenous pulp tissue compared with males whose third molars were primarily from the mandible and with separated roots. In Méndez & Zarzoza (1999) reported a mean weight pulp of 13.10 ± 4.33 mg in eight premolars fragmented with a hammer. In our study, we used a vertical cutting method to Table 2 Cell density values in specific regions of dental pulp tissue from different teeth. All values were calculated from three histological sites for each one of the regions of DP among the different teeth expressed as the mean ± standard deviation. (a, b and c) indicate significant differences in each region of DP and the type of tooth. (A, B, C, D and E) indicate significance between different tooth and each region of DP, p ≤ 0.05. UD: denotes Not determined, this regions are not found in deciduous teeth.  obtain DP from all types of teeth. This variation in weight could be explained by the fact that the fractionation by impact could lead to the loss of pulp tissue. In addition, some premolars had separated roots, so the size of the pulp tissue may reduce. On the other hand, it is also important to note that the DP also undergoes age-related changes, and several studies have focused on aging. Recently, Kellner et al. (2014) determined that the relation between pulp vs. hard tooth tissue in third molar decreases with aging. This finding was also observed in our study (Fig. 2). Unfortunately, no reports on deciduous pulp weight were found to compare our observations.
The length of DP was three-fold higher in permanent compared with deciduous teeth (Table 1) because deciduous teeth showed a physiological reabsorption that does not occur in permanent teeth. Regarding DP width, measurements of deciduous and permanent teeth are obtained using radiological techniques, such as pericapical X-rays or orthopantomography. For example, Kazmi, Anderson & Liversidge (2017) used conventional radiology to measure the mesio-distal crown width of deciduous teeth in males and females, revealing no significant differences, which was consistent with our findings (Table 1). However, there are no reports about the in vitro length of the pulp for comparison with our results. On the other hand, the cell-rich zone contains progenitor cells that exhibit plasticity and pluripotency. For instance, Lizier et al. (2012) indicated that DPSCs are located in multiple niches, which are associated with capillaries and the nerve network of the central region in the CRZ and in the outer layer of pulp tissue (Graziano et al., 2008;Pagella et al., 2015). We observed that the apical region of the DP of the supernumerary teeth, showed a higher cell density compared to the other permanent teeth. Interestingly, in the total analysis of cell density, the supernumerary teeth have similar cellular density compared to deciduous teeth (Table 2). Although the pulp tissue of the deciduous teeth was smaller, this pulp exhibited the highest cell density compared with the other dental organs (Table 2). This finding could be explained because the coronal and apical regions are held together after a physiological resorption in deciduous teeth. Gronthos et al. (2002) showed that the DP derived from lower deciduous central incisors contains a large number of cells able to form adherent colonies similar to mesenchymal stem cells in in vitro culture. In contrast, the small number of cells available for isolation due to the size of the pulp, especially in exfoliated deciduous teeth represents a potential problem with obtaining DPSCs (Marrelli et al., 2018). Thus, one of the advantages of supernumerary teeth for the isolation of cells is that these teeth are extracted at an early age, which apparently retain embryogenic characteristics as demonstrated for another source of stem cells from the oral cavity (Dunaway et al., 2017). These results indicate that supernumerary teeth in patients between 5-20 years of age have the best morphometric parameters. However, the determination of the biological properties such as proliferation and differentiation potential of the isolated cells in the different regions of this tissue requires further studies.

CONCLUSIONS
In this study, in vitro histomorphometric comparison and cellular density of the DP from temporary and permanent teeth of patients from 5 to 25 years of age were addressed. It was shown that supernumerary DP has the best morphometric parameters and its cell density is comparable to that of deciduous tooth pulp. This phenomenon has not been described before and could have important applications in the isolation of stem cells in this tissue.