In vitro histomorphometric comparison of dental pulp tissue in different 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.

Materials and Methods

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^®) with a diamond disc (diameter: 22 mm and thickness: 0.4 mm; ATK^®). 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.

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 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). The histological evaluation revealed a three-layer structure consisting of a layer of odontoblasts (OB; Figs. 3A–3N, yellow arrowhead) with regularly arranged columnar cells in the contour of the pulp and a dispersed layer of cells in all DP samples. Conversely, in the subodontoblastic zone (SOB), a thin and cell-free layer zone (CFZ; Figs. 3A–3N, green arrowhead) was observed. The cell-rich zone (CRZ; Figs. 3A–3N, red arrowhead) showed a dense layer of cells per unit, especially in fibroblasts and undifferentiated mesenchymal cells that continue in the central zone of the pulp, in which the presence of blood vessels (BV), nerve fibers (NV) and connective tissue (CT) is highlighted (Figs. 3A–3N). The DP of the supernumeraries presented a limited presence of NV (Figs. 3I–3L) compared with the pulp obtained from the premolars, third molars and deciduous. The histology of the pulp in the coronal and cervical regions of deciduous teeth showed an irregular shape, making layers difficult to identify during staining with hematoxylin and eosin (Figs. 3M and 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.

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.

Teeth	Cell density (cel/mm2)
	Coronal	Cervical	Middle	Apical	Mean total
Premolar	40.90 ± 7.46	36.80 ± 9.09	25.80 ± 8.07	51.60 ± 10.12	38.78 ± 12.57a
Third molar	19.20 ± 4.42	19.40 ± 4.43	12.20 ± 5.22	30.30 ± 2.00	20.28 ± 7.70a,b,c
Supernumerary	39.70 ± 12.63	38.50 ± 10.16	24.90 ± 5.71	61.00 ± 10.08	41.03 ± 16.21b
Deciduous	47.30 ± 14.71	46.80 ± 14.74	UD	UD	47.05 ± 14.34c
Mean total	36.78 ± 14.77A,B	35.38 ± 14.12C,D	20.97 ± 8.87A,C,E	47.63 ± 15.34B,D,E

DOI: 10.7717/peerj.8212/table-2

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 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.

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