2021, Number 1
The effect of post-exodontia time and age on the total cellular viability of the dental pulp
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ABSTRACT
Introduction: The human dental pulp is considered a cellular source, where fibroblasts, odontoblasts, and stem cells can obtain. Currently, cultures of these cells are used in biocompatibility studies of dental materials, regenerative medicine, and tissue engineering. Post-extraction time is essential in obtaining a high percentage of cell viability. Reports on cell viability of dental pulp stem cell cultures do not include the total pulp cell population. For this reason, this study aimed to assess the influence of post-exodontia time and patients' age and gender in obtaining viable dental pulp cells. Material and methods: Two periods of time were compared, before 24 hours and after 72 hours post-exodontia. 42 teeth of patients from 13 to 42 years were collected. The cell viability was determined using trypan blue staining, and the cell count was done under the microscope. The living cells were observed white and the dead one blue. Results: The pulps obtained before 24 hours post-exodontia had a higher percentage of viable cells than those obtained after 72 hours. Conclusions: the extraction of the dental pulp cells should be done in ≤ to 24 hours post-exodontia for optimal results. There is a relationship between the age of the patient and the viability in the pulps before 24 hours post-exodontia. Gender's patients do not influence cell viability.INTRODUCTION
In recent years, pulp tissue has gained interest as a source of cells. Fibroblasts, odontoblasts, and stem cells are found in this tissue.1-3 Fibroblasts and dental pulp-derived stem cells (DPSCs) are cultured in multiple studies in dentistry.4-10 Cell cultures testing the cytotoxicity and biocompatibility of dental materials, such as resins or endodontic types of cement.4-10 Pulp tissue is an excellent source for obtaining cells because of its easy access.11-13 The tissue is isolated from teeth extracted for orthodontic purposes, such as third molars and premolars, without causing significant harm to the patient.
New protocols are constantly updated to obtain more viable cells when isolated from the pulp. Current protocols consider low temperature, moist condition, and control of bacterial contamination.10 Post-exodontic time is also a crucial factor; however, few studies contrast this factor with cell viability.14,15 Similar viability was reported in DPSCs cultures at different times, 0, 0.5, 1, 2, and 5 hours post-exodontia.14 Cultures performed immediately, and at 24, 48, 72, 96, and 120 hours post-exodontia have also been compared.15 As a result, the shorter the time, the greater the number of cells obtained.15 The studies mentioned above were carried out in TCDC without considering other pulp cells. The anterior fact led us to investigate the total cell population of the pulp. Therefore, this study aimed to compare the cell viability of dental pulp obtained before 24 hours and after 72 hours post-exodontia.
Additionally, to determine the relationship of cell viability with the age and gender of the patients. It is hypothesized that a shorter post-extraction time will favor cell viability. Also, the dental pulps coming from younger patients present higher cell viability than adults, and, finally, that the gender of the patient does not influence cell viability. The results of this study will contribute to the optimization of cell isolation protocols, cell cultures, cytotoxicity tests used in regenerative medicine and tissue engineering.
MATERIAL AND METHODS
The present study was experimental, comparative, cross-sectional, and perspective. The teeth used were extracted for orthodontic and prophylactic reasons at the Dental Center of the University of San Martin de Porres. The study was approved and in the agreement of the Research Ethics Committee of the Faculty of Dentistry of the University of San Martin de Porres. Prior informed consent was obtained from the patients to participate in the study. Forty-two teeth, fully developed third molars and premolars, were collected from healthy patients (16 females and 26 males) between 13 and 42 years of age. The total sample was divided into two groups of 21 teeth each. In group 1, pulp extraction was performed 24 hours post-extraction. In group 2, pulp extraction was performed after 72 hours post-extraction, without exceeding 96 hours.
After the dental extractions, the teeth were placed in sterile bottles with physiological saline plus penicillin and streptomycin (PE-EST) (Gibco, USA®) and hermetically sealed, labeled, and cooled to 4 oC transported to the Oral and Molecular Biology Research Laboratory of the Faculty of Dentistry of the University of San Martin de Porres, according to the Biosafety Manual for Testing, Biomedical, and Clinical Laboratories of the Ministry of Health.16 Then, the group 2 teeth were stored at 4 oC until the corresponding time for pulp extraction.
The pulp exposes, and extraction was performed by fracturing the tooth.17 The technique employed by the principal investigator was performed based on previous training, evaluated, and measured by an expert in the field. Inside the laminar flow cabinet (BIOBAN, Angelantoni Life Science, Italy), the first thing that was performed was the washing and disinfection of the tooth. For this, the tooth was immersed in Minimum Essential Medium (MEM) (Sigma-Aldrich, UK) plus PE-EST for 20 minutes. After the rinsing time was completed, the tooth was fractured to expose the pulp. The fractured tooth was placed in a Petri dish with MEM and PE-EST, and the chambered pulp tissue was separated with the aid of a scalpel, forceps, files, and sterile curettes (Figure 1A). The isolated tissue was manually crushed and centrifuged at 1100 rpm for 10 minutes, obtaining the cell button or pellet (Figure 1B). The supernatant was then removed and resuspended in MEM and PE-EST. The cell viability was determined using 10 μL of suspension mixed with 10 μL of trypan blue (Gibco, USA). The mixture was placed in the hemocytometer for cell counting per quadrant. Live cells were observed as white and dead cells as blue (Figure 2A and 2B).
The percentage of viability was found by the following formula:18
% viability = (number viable cells / Total number of cells) ×100
The number of viable cells was found by the following formula:
Number of cells = (number viable cells × dilution factor × 104× volumen of solution) / (Number of quadrants)
The software SPSS version 21 (IBM®) was used to determine possible statistically significant differences between the mean of the two groups. The confidence interval was 95% (p = 0.05). Initially, the Shapiro-Wilk normality test was used to choose the appropriate statistical test. The nonparametric Mann-Whitney U test was used for data that did not have a normal distribution, and the parametric Student's t-test for independent samples was used for data that had a normal distribution.
RESULTS
The two groups studied used third molars and premolars corresponding to patients whose age range was between 13 and 42 years. Both groups' age and sex characteristics were similar, with no significant differences (p = 0.191 and p = 0.751, respectively) (Table 1).
In the group of dental pulps obtained before 24 hours post-extraction, the quantity and cell viability were higher than in the group of pulps obtained after 72 hours post-extraction. Both variables presented statistically significant differences (p < 0.05) (Figures 3 and 4).
In group 1, there is a relationship between the number of cells and cell viability with the patient's age. Both cell number and cell viability are higher in the group aged 13 to 27 years than those aged 28 to 42 years. The difference was statistically significant (p = 0.031 and p = 0.024, respectively) (Table 2).
In group 2, there is no relationship between the number of cells and cell viability with the patient's age. In addition, no statistically significant difference was found (p = 0.586 and p = 0.848 respectively) (Table 3).
In group 1, no relationship was found between the number of cells and cell viability with the gender of the patient. No statistically significant difference was found (p = 0.232 and p = 0.526, respectively). Similarly, in group 2, there was no relationship between the number of cells and cell viability with the sex of the patient. Again, there is no statistically significant difference (p = 0.915 and p = 0.282, respectively).
DISCUSSION
One of the objectives of the present study was to determine the effect of post-extraction time on the viability of dental pulp cells. At present, little research has addressed this relationship.11-14 It appears that 24 hours is the appropriate post-extraction time for culturing. However, it has not been established whether successful cell culture of the dental pulp can be performed at 72 hours post-extraction. The afore data led us to perform this study comparing cell viability before 24 hours and after 72 hours post-exodontia.
Woods et al11 obtained several viable cells of 0.5-2.0 × 106 of TCDC (a cellular recovery higher than 93%) after six months of freezing, verified with the trypanum blue staining method. On the other hand, we obtained a higher cell number in the group of dental pulp obtained before 24 hours post-extraction (an average of 4.1 × 106). The difference between the two results is because we evaluate the entire cell population, not a specific group of cells such as TCDC. We also did not subject the cells to a period of freezing, which can cause a decrease in cell numbers.
In the present study, group 1 obtained a cell viability of 65.64% and group 2 35.43%. Benício et al.14 established cell cultures of teeth at 0, 0.5, 1, 1, 2, and 5 hours post-exodontia. Although they did not specify the exact viability obtained at each time, they indicated that they obtained the same proliferative capacity and similar cell morphology at the different post-extraction processing times. In contrast to our study, we obtained a significant difference when comparing the cell viability of both groups, 24 and 72 hours. The discrepancy in both studies can be attributed to the longer time. The 72-hour post-exodontic time to obtain dental pulp may be an important factor in cell viability difference.
Perry et al.15 compared the cell viability of third molars stored at 0, 24, 48, 72, 96, and 120 hours before processing to obtain TCDC. Teeth that were stored in PBS and HTS at 24 hours (1.5 × 106 and 2.2 × 106 respectively) obtained higher amounts of TCDC than at 72 hours (0.6 × 106 and 0.7 × 106 respectively). These findings were similar to ours when comparing cells obtained at 24 and 72 hours. However, we obtained higher numbers of cells at 24 hours (4.1 × 106) and 72 hours (1.6 × 106) compared to that obtained by Perry et al.15 As mentioned above, this may be because we worked with the total cell population.
This study also evaluated the relationship between cell viability with the age and gender of the patients. Suchánek et al.19 mention that they used for their study the teeth of 19 women and three men, in an age range of 8 to 23 years. Viña-Almunia et al.20 used the dental pulps of 85 women and 35 men, ranging from 14 to 67 years. In both studies, they were able to isolate TCDC cultures satisfactorily. However, they did not study the viability of the cultures concerning the age and sex of the patients, nor did they mention the post-exodontic time.
We obtained pulps from 16 women and 26 men. According to the results, it could be inferred that the sex of the patient does not influence obtaining higher cell viability. The patients were divided into two age groups, from 13 to 27 years old and 28 to 42 years old. The limitation in this study was that very few samples were obtained from the older age group (3 samples) instead of the younger age group, because the most common population attending the clinic for third molar exodontia were young patients.
It is vital to assess cell viability in dental pulp cell culture assays because it serves the function of culture quality control. Therefore, we suggest using dental pulp 24 hours post-exodontia to obtain optimal cell number and cell viability. However, further research is needed to corroborate the above. Therefore, it is recommended to culture cells obtained at other time points (48 hours and more than 72 hours post-exodontia) to evaluate cell proliferation capacity. It is also recommended to consider more samples in adult age groups.
This study opens the way for further research to investigate more factors that modify cell viability. These would contribute to optimizing the protocols of cell isolation, cell culture, and cytotoxicity tests performed in regenerative medicine and tissue engineering.4-7,11-14
CONCLUSIONS
According to the results obtained in this study, it was concluded that the dental pulps that were obtained before 24 hours post-exodontia present a higher percentage of cell viability and cell population concerning the dental pulps that were obtained after 72 hours post-exodontia. Furthermore, there is an inverse relationship between the age of the patients with the cell viability and the number of cells in the group of dental pulps obtained before 24 hours post-exodontia. Cell viability and cell number are higher in patients aged 13 to 27 years, and there is no relationship between the sex of the patients and cell viability and cell number in both time groups.
It is recommended to use dental pieces before 24 hours post-extraction to obtain pulp with higher cell viability and a higher number of cells.
REFERENCES
AFFILIATIONS
1 Universidad de San Martín de Porres, Lima, Perú.
CORRESPONDENCE
Esperanza R Ayón-Haro. E-mail: eayonh@usmp.peReceived: Marzo 2019. Accepted: Julio 2019.