Response of intra canal medicaments on viability and survival of SHEDs

INTRODUCTION

SHEDs were discovered in the dental pulp of human exfoliated primary teeth and expressed the same characteristics as Mesenchymal Stem Cells (MSCs), such as fibroblastic traits, clonogenicity, cell surface antigen presentation, proliferative ability, and multidifferentiation capacity [1]. SHEDs have recently been examined for their potential application in tissue engineering for bone tissue regeneration and cell-based therapies for a variety of refractory medical conditions, including hypoxic-ischemic brain damage, systemic lupus erythematosus, spinal cord injury, and diabetes. Additionally, exosomes generated from Dental Pulp Stem Cells (hDPSCs) are regarded as beneficial [2, 3, 4].

In comparison to other human tissues, exfoliating primary teeth have the distinct advantages of being less ethically contentious, being a readily accessible source, being simple and minimally invasive to collect, and retaining high stem cell potential such as cell growth, multipotency, and immunoregulatory functions even after cryopreservation. Numerous associations have been developing a SHED bank for allogeneic and autologous cell treatment. As a result, they are regarded as a viable supply of cells for tissue regeneration. The following clinical approaches are used in regenerative endodontics: minimal instrumentation, root canal disinfection, intracanal medicament application, canal bleeding induction and capping followed by a hermetic coronal seal [6].

Microbes can have a detrimental effect on the growth and regeneration capacity of stem cells. Because Regenerative Endodontic Procedures (REP) advocate for little root canal instrumentation, they rely primarily on intracanal antimicrobial medicaments to maintain a sterile environment conducive to pulpal revival [7]. Calcium hydroxide is the disinfectant of choice for root canals [8]. Triple antibiotic paste and double antibiotic paste are more efficient disinfectants for root canals, according to studies [9, 10]. Intracanal medications affect mesenchymal stem cell survival and multiplication. In a study, it was discovered that triple and double antibiotic paste were detrimental to the viability of stem cells in the apical papilla [11].

However, a search of the literature reveals no research examining the response of intra canal medicaments to the growth and viability of SHEDs. As a result, the goal of this trial was to ascertain the influence of different intracanal medicaments on SHED proliferation and survival.

MATERIALS AND METHOD

The current in vitro experimental trial adhered to the Nagendrababu V et al. [12], 2021 standards for research reporting. The sample size for this experiment was estimated using the “resource equation” method [13]. All analyses were carried out in three separate trials, with triplicates of each test specimen and reaction. Human exfoliating caries-free primary teeth indicated for orthodontic extraction in 7–14 year-old healthy children whose guardian/parents signed informed consent for participating in the present study were the source for the SHEDs. The institutional ethics committee gave its approval to this trial (IREB/2019/PHD/PEDO/01).

Under sterile conditions, the pulp was collected and transferred to the laboratory (National Institute of Immunohematology, Parel, Mumbai) for further processing. Hank’s Balanced Salt Solution was used to disinfect the pulp tissues (Gibco, USA). Following that, collagenase-A (3 mg mL) was used for enzymatic action at 37 ${}^{\circ }$C for 60 minutes. Cells (1.5 $\times$ 10${}^4$ cells/cm${}^2$) were collected and cultured for 72 hours in 25-cm${}^2$ plastic culture flasks in complete media (5% CO${}_2$ at 37 ${}^{\circ }$C). The red blood cells were eliminated, as were any other non-adherent cells. To stimulate growth, fresh media was added. Passage zero (P${}_0$) was defined as the density of adherent cells at 80% growth.

To enable future passaging, SHEDs were rinsed in Phosphate Buffer Saline (PBS) and isolated by culturing for 2-5 minutes at 37 ${}^{\circ }$C with 0.25% trypsin. The trypsin action was deactivated by adding a culture medium. SHEDs were centrifuged at 500 g for 5 minutes before being seeded into 75-cm${}^2$ flasks with 5 $\times$ 10${}^3$ cells cm${}^2$. Cells from passage 4 were used in this study. SHEDs phenotypes were assessed using a flow cytometer before the experiments, with specific antibodies for Cluster of Differentiation CD 90, CD 105, CD 73, CD 34, CD 45, and Human Leucocyte Antigen-DR Isotype (HLA-DR) (BD Biosciences, Pharmingen) [14].

To examine the proliferation and survival of SHEDs, we performed MTT, Apoptosis assay and ALP assay. For the MTT assay, calcium hydroxide (Ca), doxycycline (Dy), potassium iodide (PI), triamcinolone (Tr), and glutaraldehyde (Gt) (Sigma Aldrich, USA) were used. Various medicament was evaluated at concentrations of 0 (control), 10 g/mL, 25 g/mL, 50 g/mL and 100 g/mL. The SHEDs were exposed to the medicaments for 24 hrs, 48 hrs and 72 hrs.

RESULTS

Cell viability assay

The medicaments reported variable cytotoxicity at different concentrations and time intervals. Calcium hydroxide showed a significant decrease in cell viability at 50 $\mu$g/mL or higher after all time intervals of incubation. Calcium hydroxide reported cytotoxicity at a concentration of 500 $\mu$g/mL and 1000 $\mu$g/mL after 24 hrs, 48 hrs and 72 hrs interval (Fig. 1). Doxycycline reported a statistically significant reduction in cell survival at a concentration of 50 $\mu$g/mL or more after 24-hrs, 48-hrs and 72-hrs incubation. Cytotoxicity was seen at a concentration of 250 $\mu$g/mL or more at all time intervals (Fig. 2).

Fig. 1.MTT analysis for SHEDs cultured in calcium hydroxide at various dilutions after 24 hrs, 48 hrs and 72 hrs incubation.

Fig. 2.MTT analysis for SHEDs cultured in doxycycline at various dilutions after 24hrs, 48 hrs and 72 hrs incubation.

Potassium iodide (Fig. 3), triamcinolone (Fig. 4) and glutaraldehyde (Fig. 5) showed a statistically substantial decrease in cell viability at concentration level of 50 $\mu$g/mL or more at all time intervals. Triamcinolone reported cytotoxicity at a concentration of 100 $\mu$g/mL or more after 24-hrs, 48-hrs and 72-hrs incubation. In the present study, SHEDs reported the highest cell survivability when cultured in calcium hydroxide followed by potassium iodide, doxycycline, triamcinolone and glutaraldehyde at a concentration of 25 $\mu$g/mL, 10 $\mu$g/mL, 10 $\mu$g/mL, 25 $\mu$g/mL and 25 $\mu$g/mL respectively after a time interval of 72 hours of treatment. Hence the subsequent experiment involving these test specimens was performed with these dilutions only.

Fig. 3.MTT analysis for SHEDs cultured in potassium iodide at various dilutions after 24hrs, 48 hrs and 72 hrs incubation.

Fig. 4.MTT analysis for SHEDs cultured in triamcinolone at various dilutions after 24hrs, 48 hrs and 72 hrs incubation.

Fig. 5.MTT analysis for SHEDs cultured in glutaraldehyde at various dilutions after 24hrs, 48 hrs and 72 hrs incubation.

Detection of apoptosis and necrosis by flow cytometry

The mean $\pm$ SD variation among various medicaments, were statistically significant for % of live cells (p = 0.045), early (p = 0.001) and late (p = 0.036) apoptotic cells when compared to control. The highest % of live cells were found in calcium hydroxide (97.50 $\pm$ 1.46), followed by triamcinolone (95.00 $\pm$ 2.01), negative control (94.50 $\pm$ 1.79), glutaraldehyde (93.7 $\pm$ 1.55), potassium iodide (93.20 $\pm$ 0.36), doxycycline (92.00 $\pm$ 1.70) and positive control (15.40 $\pm$ 10.36) (Fig. 6).

Fig. 6.Representative 2D flow cytometry dot plot of data derived from FITC-AnV and 7-AAD stained SHEDs cultured in various intra canal medicaments.

Alkaline phosphatase assay

The difference in ALP activity between all test specimens was statistically significant at all the time intervals—7 days, 14 days and 21 days (p = 0.001). The highest amount of ALP activity was observed in SHEDs incubated in calcium hydroxide (1375.4 $\pm$ 321.89), followed by control (1150.7 $\pm$ 222.32), potassium iodide (1050.2 $\pm$ 155.12), triamcinolone (1030.7 $\pm$ 122.98), glutaraldehyde (1025.3 $\pm$ 164.26) & doxycycline (900.3 $\pm$ 64.21) after 72 hours of incubation. Pair-wise comparison of ALP activity among all test specimens was statistically significant than the control specimen at all time intervals (Fig. 7).

Fig. 7.ALP activity of SHEDs cultured in various intra canal medicaments.

DISCUSSION

The goal of this trial was to examine the effect of several antibacterial intracanal medicaments at varying concentrations on the viability and survival of SHEDs over time. The results suggested that SHEDs were more viable when exposed to all medicaments at lower concentrations for 48 or 72 hours, but were tolerated at elevated doses when exposed for 24 hours. Both the concentration and duration of intracanal medication exposure affected the survivability of SHEDs.

MSCs population is expected to be positive for CD 105, CD 73, and CD 90 as per the phenotype requirements of International Society for Cell Therapy (ISCT). Furthermore, such cells should not express CD 14, CD 45, CD 34, CD 19, HLA class II, CD 79a or CD11b [19]. The present study used CD 45, HLA-DR, and CD 45 as negative markers, while CD 105, CD 73, and CD 90 were used as positive markers.Phenotypical analysis of SHED in the present study showed high positivity for positive markersCD 73 (93.9 %), CD 90 (94.60 %) and low positivity was observed for CD 105 (35.30 %) which is commonly expressed by endothelial progenitors. These results were consistent with other studies [20, 21, 22].

Tetrazolium compounds are employed to produce a quantifiable colorimetric assay to evaluate human cell proliferation and survival. Hence, the MTT assay has been used extensively to the extent that it is now the typical technique for assessing cell viability [16]. Therefore, the present study used the same technique for determining cell viability. Cells that have been constantly exposed to medications for up to 72 hours are analyzed to assess the medication’s optimum efficiency and efficacy [23]. As a result, the MTT assay was assessed at intervals of 24, 48, and 72 hours.

Calcium hydroxide is a disinfectant that is approved for intracanal use during regenerative endodontic operations [24]. Calcium hydroxide increases the proliferation, osteogenic differentiation, and mineralization of Dental Pulp Stem Cells (DPSCs) via the mitogen-activated protein kinase pathway [25]. This could be the probable reason for calcium hydroxide exposure up to 25 g/mL, not significantly affecting the viability (after 72 hrs incubation), ALP activity (after 21-days) and apoptosis of SHEDs in the present trial. This finding corroborated with a study showing calcium hydroxide as non-toxic to apical papilla stem cells (SCAPs) and promotes cell survival and proliferation [26]. However, cytotoxicity testing of calcium hydroxide against SCAPs at its minimal bactericidal concentration revealed that it was more harmful than antibacterial combinations [27]. This fact was supported by a study that highlighted that both dosage and exposure time contribute to calcium hydroxide cytotoxic response [28]. However according to Neelakantan et al. [29] calcium hydroxide inhibits microbial growth for 48 hrs; after which, it declines.

Doxycycline has previously been shown to be an effective intracanal antibacterial irrigant and medicament [30, 31]. However, the amounts of doxycycline utilized in these trials were far higher than those identified in our research to be cytotoxic. The present trial reported cytotoxicity with doxycycline at a concentration of 250 $\mu$g/mL after 24 hrs incubation. This result contradicted the findings of Bhandi and colleagues [28], who reported that doxycycline induced considerable cytotoxicity after 24 hours and 48 hours at a concentration level of 25 g/mL and 10 g/mL respectively. These were the lowest concentrations observed for any of the medicaments examined. Due to its increased tolerance for brief exposure, it is well suited for use as an intracanal irrigant. However, the concentration must be kept low due to the substance’s substantivity, which lasts up to three weeks following 10 minutes of exposure [30]. The present trial found doxycycline with the least number of live cells which was in accordance with the study conducted by Bhandi and et al. [28] who reported the highest apoptosis of DPSCs with doxycycline. The likely explanation is that prior research has established that the second-generation tetracycline derivative doxycycline disrupts mitochondrial proteostasis and physiology, decreases proliferation of a variety of cell types, and promotes apoptosis [30].

Potassium iodide has been utilized as a disinfectant and antibacterial agent. It is a disinfecting irrigant solution that is highly effective [31]. The SHEDs survival significantly decreased at a dose of 50 $\mu$g/mL post 24 hrs incubation. The number of live SHEDs cultured in potassium iodide was less when compared to control. The ALP activity of SHEDs cultured in potassium iodide was significantly low when compared to control. However, the present trial found potassium iodide better than doxycycline. This finding is in accordance with a trial conducted by Bhandi and co-workers [28].

Triamcinolone, a corticosteroid, has been used in combination with tetracycline as an initial dressing for individuals with endodontic discomfort undergoing pulp capping procedures [32]. The present trial reports triamcinolone with cytotoxicity at concentration of 100 $\mu$g/mL or more after 24-hrs incubation. This finding is further confirmed by the results of the study conducted by Bhandi et al. [28] The number of live cells was suggestively greater than the control. However, the ALP activity was significantly lower than the control. Triamcinolone’s role in the hepatogenic development of stem cells, such as the differentiation and proliferation of macrophages, adipocytes, and osteocytes, may explain these data [33]. These findings were in accordance with the study conducted by Wemes and co-workers [34] that evaluated the response of corticosteroids and found a dose-dependent association on the viability of MSCs.

Glutaraldehyde has been utilized as an irrigant and as an intracanal medicament in the past [35]. The present study reported a significant reduction in SHEDs survival at a concentration of 50 $\mu$g/mL or more after 24 hrs incubation. The number of live cells and ALP activity of SHEDs cultured in glutaraldehyde was significantly less when compared to control. These facts were further confirmed by the study conducted by Bhandi and co-workers [28]. Shi J observed that the presence of glutaraldehyde in mouse lymphoma cells could cause considerable cytotoxic and mutagenic consequences [36].

The study’s shortcoming was that it examined the impact of the medications up to 72 hours after administration for survival and up to 21 days for ALP activity. Clinical recommendations currently recommend that the medication be left in place for 1–4 weeks [37]. It is critical to understand which medications can be safely and efficiently employed in regeneration therapies without compromising SHEDs viability or regenerative capacity. Hence the present trial assessed MTT assay, apoptosis and necrosis and ALP activity of intra canal medicaments. ALP plays a role in cell growth modulation and serves as an early marker of osteogenic proliferation and differentiation [18].

CONCLUSION

The current research reveals that potassium iodide and glutaraldehyde were significantly less likely of all the medications examined to adversely affect the viability and survival of SHEDs. Calcium hydroxide, doxycycline and triamcinolone does affect the SHEDs viability and survival at a concentration of 250 $\mu$g/mL or higher. After 14 and 21 days of incubation, calcium hydroxide exhibited the maximum ALP activity of all the medicaments examined.

There is also a need for us to concentrate on the long-term impact of these medicaments on SHEDs due to their presence in the dentinal tubules after the medicament is withdrawn from the root canal. Additional research is necessary to determine the antibacterial effect of potassium iodide and glutaraldehyde alone or in combination with other antimicrobial agents.

FUNDING

This research received no external funding.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.