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 Table of Contents  
Year : 2017  |  Volume : 14  |  Issue : 4  |  Page : 198-207

Cell viability and apoptotic changes of dental pulp stem cells treated with propolis, chitosan, and their nano counterparts

1 Endodontic Department, Faculty of Dentistry, Ain Shams University, Cairo, Egypt
2 Endodontic Department, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt

Date of Submission14-May-2017
Date of Acceptance30-Jul-2017
Date of Web Publication21-Dec-2017

Correspondence Address:
Abeer A Elgendy
Endodontic Department, Faculty of Dentistry, Ain Shams University, Cairo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tdj.tdj_27_17

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Biocompatibility of materials used in regenerative endodontics is of prime importance. Thus, this study was conducted to evaluate and compare cytotoxicity and apoptotic changes of propolis, chitosan, and their nano counterparts on dental pulp stem cells.
Materials and methods
Aqueous and ethanolic extract of propolis, chitosan, propolis nanoparticles, and chitosan nanoparticles were prepared. Dental pulp stem cells were isolated from human third molars and characterized. Cultured cells were incubated with each extract of each material used for 24 and 72 h. Thereafter, cellular viability was assessed using MTT assay and DNA fragmentation using DNA fragmentation laddering assay. Control samples containing only medium were treated similarly. Differences in mean values of cell viability and DNA fragmentation between materials were assessed by using the one-way analysis of variance and Tukey's test.
Nanoparticles of both materials expressed higher cell viability and lower DNA fragmentation in comparison to their original particles counterpart. Chitosan nanoparticles recorded a lower cytotoxicity and DNA fragmentation after 24 h, which increased by time, meanwhile that of propolis nanoparticles were decreased by time. Type of vehicle was a factor affecting the results of chitosan nanoparticles, while time was the factor affecting propolis nanoparticles.
Both propolis and chitosan nanoparticles displayed an acceptable biocompatibility and can be used in endodontic regeneration purposes.

Keywords: apoptosis, aqueous, cell viability, chitosan, propolis, vehicle

How to cite this article:
Elgendy AA, Fayyad DM. Cell viability and apoptotic changes of dental pulp stem cells treated with propolis, chitosan, and their nano counterparts. Tanta Dent J 2017;14:198-207

How to cite this URL:
Elgendy AA, Fayyad DM. Cell viability and apoptotic changes of dental pulp stem cells treated with propolis, chitosan, and their nano counterparts. Tanta Dent J [serial online] 2017 [cited 2019 Jan 19];14:198-207. Available from: http://www.tmj.eg.net/text.asp?2017/14/4/198/221379

  Introduction Top

Regenerative endodontic treatment aims to replace the lost or damaged structures such as dentin, root structures, and pulp dentin complex cells [1],[2]. The endogenous adult stem cell niche within dental pulp tissue, commonly referred to as dental pulp stem cells (DPSCs), is of a neural crest origin and plays a crucial role in the homeostasis and self-renewal of dental pulp tissue [3]. The simplest method for pulp tissue regeneration is pulp regeneration over the infected or necrotic tissue. Stem cells of the pulp, periodontal tissue, and fibroblasts do not adhere or proliferate in infected root canal system [4]. Therefore, one of the essential elements for a successful endodontic regeneration protocol is the creation of a bacteria-free biological environment inside the root canal space.

Several chemicals and therapeutic agents have been suggested for preservation and stimulation of dental stem cells [5],[6],[7],[8]. These agents were used because of their known bactericidal and bacteriostatic effects. But owing to the potential side effects, safety concerns, and ineffectiveness of conventional allopathic formulations [9],[10], most researchers have shifted focus on modern natural substances in endodontic treatment with fewer side-effect, greater antibacterial activity, biocompatibility, anti-inflammatory, and antioxidant properties [11]. One such modern substance is propolis.

Propolis is a resinous substance that honey bees collect from different plant species. The most important pharmacologically active constituents in propolis are flavonoids, phenolics, and aromatics. It is believed that flavonoids account for much of the biologic activity in propolis [11]. Propolis exhibits a wide range of biologic activities, including antimicrobial, anti-inflammatory, antioxidant, anesthetic, and cytotoxic properties [12]. Ethanolic extract of propolis can promote bone regeneration and induce hard tissue bridge formation in pulpotomy or pulp capping. Propolis being a good antimicrobial and anti-inflammatory agent can serve as a better intracanal irrigant and intracanal medicament [13],[14].

Al-Qathami and Al-Madi [15] indicated that propolis showed antimicrobial activity equal to that of sodium hypochlorite. As well, Kousedghi et al. [16] compared the antimicrobial activity of propolis and calcium hydroxide and claimed that propolis is more effective against Enterococcus faecalis, Lactobacillus spp., and Peptostreptococcus spp. than calcium hydroxide in agar culture. Also, Verma et al. [17]confirmed antimicrobial effectiveness of 25% water-soluble extract of propolis in the root canals of primary teeth.

Chitosan (CS) is another modern substance, which is a nontoxic cationic biopolymer usually obtained by alkaline deacetylation from chitin, which is the principal component of crustacean exoskeletons [18]. CS treatment improves the resistance of the dentinal surface to degradation by collagenase [19], and significantly enhances the bond strength to dentin with or without phosphoric acid pretreatment [20]. Furthermore, it presents with biocompatibility, chelating capacity, and also antimicrobial effects against a broad range of Gram-positive and Gram-negative bacteria as well as fungi [21],[22],[23]. Additionally, previous in-vitro studies have demonstrated the significant antibiofilm efficacy of chitosan nanoparticles (CNPs) [21],[24].

Intracanal medicaments must be used at concentrations that are bactericidal while having minimal effects on cell viability. Two recent studies suggested that intracanal medicament concentrations currently used in regenerative endodontics had detrimental effects on the survival of human stem cells of the apical papilla and human dental pulp cells [8],[25], which may adversely affect the successful outcome of endodontic regeneration.

Other factor that may influence the therapeutic action of medicaments is the vehicle used [26],[27]. However, there appears to be no sound scientific rationale adopted for the choice of the vehicle [28],[29]. Several authors [30],[31],[32] have proposed associations of calcium hydroxide with different vehicles to maximize its qualities. Fava and Saunders [33] concluded that the vehicle with which calcium hydroxide is mixed to form the paste used in endodontics affects the physical and chemical properties of the compound and hence its clinical applications. Also, vehicles contribute to calcium hydroxide reduction of root canal dentin microhardness as constituent of endodontic pastes [34]. Gupta et al. [35] compared the antibacterial efficacy of propolis in two vehicles and reported that, though not significant, there was a difference between the activities of the two propolis solutions as the nature of the vehicle used has an impact on the action of propolis.

'Nanotechnology: It's a small world' [36]. One nanometer is equal to one billionth of a meter [37]. In this scale, particles have properties much different from those of the same material in mass scale. This concept led to the development of lighter but stronger materials [38] with unique physical, chemical, and biological properties [39]. Nanotechnology is a new approach in dentistry. By providing novel methods, it increases efficacy, accuracy, and speed of treatment while decreasing costs. However, similar to other technologies, nanotechnology can cause problems as well if not appropriately employed [40].

The antimicrobial effect of propolis and CS was investigated in many studies, but not yet their biocompatibility on DPSCs. Therefore, this study aimed to evaluate and compare the cytotoxic effect and apoptotic changes of propolis, CS, and two concentrations of their nano counterparts on DPSCs with two vehicles for complete risk assessment of these compounds.

  Materials and Methods Top

Agent preparation

The materials used in this study were propolis (Emtenan Company, Cairo, Egypt), propolis nanoparticles (PNPs) [obtained by milling of propolis in a ball-mill machine for 24 h (Raymer Engineering, Mumbai, India)], CS (NanoTech Egypt, 6th October City, Egypt), and CNPs were prepared according to the procedure developed by Grenha et al. [41] based on the ionotropic gelation of CS with TPP [42].

Each material was supplied in its original particle size and in its nanoform. The nanoform of each material was dissolved in distilled water (10 mg, 20 mg/ml) and ethanol (10 mg, 20 mg/ml). However, the original particle sizes of each material were prepared by dissolving 20 mg/ml in distilled water and ethanol. The extracts obtained were incubated at 37°C for 5 days to allow the soluble materials to leach from the samples into the medium. Then the extracts were sterile filtered using Millex-GS Sterile Filter (Millipore S.A.S., Mölsheim, France). Control samples containing only medium were treated similarly.

Isolation and identification of dental pulp stem cells

DPSCs were isolated, immediately, from the extracted third molars of human dental patients between 18 and 25 years of age after obtaining informed consent. After cleaning the surfaces of the freshly extracted tooth, the pulp chamber was exposed by using a sterile fissure bur to cut at the cement–enamel junction. The pulp tissue was gently separated from the crown and root, and cell dissociation was achieved through enzymatic digestion in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase dissolved in Dulbecco's modified Eagle's medium for 1 h at CO2 incubator. Then, single-cell suspensions were separated by passing the cells through a 70-mm cell strainer (Falcon; BD Labware, Franklin Lakes, New Jersey, USA). The suspensions of cells were cultured into 25 cm 2 plastic flask with DPSC medium (MEM; Gibco BRL, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Gibco BRL), 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco BRL), and amphotericin B (Biochrom AG, Berlin, Germany) for primary culture and then incubated at 37°C with 5% CO2; the medium was refreshed every 2 days, and 80% confluence was obtained for optimal cell harvesting [43]. DPSCs were identified by their shape [Figure 1] and CD surface markers, which were detected by flow cytometry.
Figure 1: Stages of dental pulp stem cell. (a) Rounded cells early in culture (3 days); (b) typical spindle shape cells late in culture (10 days).

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Flow cytometry

DPSCs were washed and suspended in PBS. Cluster of differentiation (CD14, CD73, and CD146) [Figure 2]: all monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA) were added directly to cells and kept for 1 h in 4°C. The cells were then incubated with antimouse immunoglobulin G fluorescein conjugated secondary antibody (Millipore Corp., Temecula, California, USA) for 45 min on ice. Cell suspensions were washed twice and analyzed on a FACScaliber flow cytometer. The 'stemness' of the putative DPSCs was confirmed by positive results obtained from these analyses.
Figure 2: Cytometric graph represents positivity of CD14, CD73, CD146 markers.

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Cell culture

Confluent monolayers of cells were detached with 0.5% (w/v) trypsin–EDTA for subcultures. Cells were diluted in fresh medium and seeded into 96-well plates (1.0 × 104 cells/well). After incubation for 24 h, the medium was aspirated from all wells and replaced with 100 μl/well of material extract or control medium. Cells were incubated for another 24 and 72 h (eight wells/material/vehicle/incubation period). All cell manipulations were carried out under aseptic conditions (laminar, BIO-CL-130; EHRET Gmbh & Co. KG, Emmendingen, Germany).

DPSCs were harvested for assessment of the following: proliferation rate by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) cell proliferation assay kit (Trevigen Inc., Gaithersburg, Maryland, USA) and DNA fragmentation using quantitation of DNA fragmentation with diphenylamine (Diana Boraschi and Giovanni Maurizi Dompé Research Center, L'Aquila, Italy).

Cell proliferation assay

Cell proliferation and survival was determined in this study with the colorimetric assay developed by Mosmann [44] and modified by Edmondson et al. [45]using the MTT cell proliferation kit (Trevigen Inc.) as per manufacturer's protocol.

MTT dye was prepared as 0.5 mg/ml in PBS at 37°C just before use. A total of 10 μl MTT dye was added to each well and incubated at 37°C in air containing 5% CO2 and at 95% relative humidity for 2–12 h to allow for intracellular reduction of the soluble yellow MTT to the insoluble purple formazan dye. Detergent reagent was added to each well to solubilize the formazan dye prior to measuring the absorbance of each sample in a microplate reader at 550–600 nm (Dynatech-MRX; Dynatech Laboratories Inc., Alexandria, Virginia, USA). All assays were repeated at least twice to ensure reproducibility. The absorption value obtained with the untreated DPSC as a control was deemed to indicate 100% viability. The percentage of viable cells was determined using the following formula:

Where A is the viable cells in the experimental well and B is the viable cells in the control well.

DNA fragmentation laddering assay

The principle of DNA fragmentation laddering assay depend on endogenous endonucleases; a group of enzymes that cleave double-stranded DNA in the linker region between nucleosomes, generate mononucleosome and oligonucleosome of 180 bp or multiples is one of the characteristics of apoptosis. To assess endonuclease-dependent ladder-like DNA fragmentation by gel electrophoresis, genomic DNA was extracted from DPSC using Wizard Genomic DNA purification kit (Promega Corporation, Madison, Wisconsin, USA) according to the manufacturer's instructions. DNA was then loaded onto agarose gel (15 μg/lane). DNA laddering was determined by constant voltage mode electrophoresis (in a large submarine at 4 V/cm, for 2 h) on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. Gels were visualized by Biometra Gel Documentation System (Biometra, Göttingen, Germany).

Percent of DNA fragmentation

To the extracted DNA diphenylamine, which binds to deoxyribose, was added then the absorbance should be measured at 600 nm using a UV double-beam spectrophotometer. The proportion of fragmented DNA should be calculated from absorbance reading at 600 nm using the formula:

Where ODS is the optical density of supernatant and ODP is the optical density of pellet [46].

Statistical analysis

The data were collected, tabulated, and the mean and SD were calculated for each group. Statistical analysis was performed by SPSS software (version 17; SPSS Inc., Chicago, Illinois, USA). Comparisons between groups were performed by using one-way analysis of variance, followed by Tukey's honestly significant difference test at P of 0.05.

  Results Top

MTT assay

The highest cell viability%, insignificantly different from that of control, was recorded for 10 and 20% ethanolic extract of CNPs at 24 and 72 h. As well as for 10 and 20% ethanolic and aqueous extract of propolis nanoparticles (EEPNPs and AEPNPs, respectively) at 72 h. Original particles of both materials significantly decreased the cell viability% compared with the control [Table 1].
Table 1: Mean±SDs of cell viability% using MTT assay of different materials tested in different time intervals

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Effect of material

Not concerning the type of vehicle used, CNPs recorded a significantly higher cell viability% than PNPs at 24 h (P < 0.001). But at a longer time interval (72 h), PNPs recorded a higher cell viability% than CNPs significantly in aqueous extract and insignificantly in ethanolic extract.

Effect of time

Time was a factor affecting the cell viability% in both materials. Regardless of particle size, mean values of cell viability% were decreased by time with CS (original and nanoparticles) both in aqueous (significantly) and ethanolic extract (insignificantly). Cell viability% was also decreased significantly from 24 to 72 h with both extracts of propolis original particles. Meanwhile, with both extracts of PNPs, mean values of cell viability% were significantly increased by time.

Effect of particle size

Both concentrations of nanoparticles of both materials recorded a statistically significantly higher mean values of cell viability% than their original particle counterpart at the two time intervals tested (P < 0.05). Nevertheless, concentration of nanoparticles was not a factor affecting the cell viability%.

Effect of vehicle

Regarding nanoparticles, mean values of cell viability% of ethanolic extract of CS were significantly higher than that of aqueous extract at both time intervals. On the other hand, mean values of cell viability% of ethanolic extract of propolis were insignificantly lower than that of aqueous extract at both time intervals. Regarding original particles, type of vehicle used was not a factor affecting cell viability%.

DNA fragmentation

Effect of material

Regardless of particle size and type of vehicle, both propolis and CS produced a significantly higher DNA fragmentation rate than that of control at both time intervals. Regarding nanoparticles, both aqueous and ethanolic extract of CS reported an initial significant lower rate of DNA fragmentation after 24 h than that of propolis. This rate increased by time to be significantly higher than that of propolis after 72 h [Table 2] and [Figure 3].
Table 2 Mean±SDs of DNA fragmentation percentage using laddering assay of different materials tested in different time intervals

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Figure 3: Agarose gel electrophoresis showing DNA fragmentation. Lane M, DNA marker with 100 bp; NPs, nanoparticles.

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Effect of time

Time was also a factor affecting DNA fragmentation rate, which significantly increased by time from 24 to 72 h with original particles of both materials in both vehicles (P < 0.001). Concerning nanoparticles, DNA fragmentation rate produced by aqueous and ethanolic extract of CS was also increased significantly from 24 to 72 h. Meanwhile, DNA fragmentation rate was significantly decreased by time with aqueous and ethanolic extract of propolis (P < 0.001).

Effect of particle size

In spite of materials used, the significantly highest DNA fragmentation rate was recorded with the original particle size. This was followed by 20% concentration nanoparticles and the lowest DNA fragmentation was recorded with 10% concentration nanoparticles. The difference between the two concentrations of CNPs was statistically insignificant, although statistically significant in PNPs.

Effect of vehicle

Concerning nanoparticles of both materials, ethanolic extract showed lower mean values of DNA fragmentation rate than that of aqueous extract at both time intervals except, ethanolic extract of propolis at 24 h where it showed higher mean values of DNA fragmentation rate than that of aqueous extract.

  Discussion Top

Root canal disinfection requires the use of canal irrigation and medication. Naturally occurring materials have been proposed for root canal disinfection as propolis, which was proved to be effective against resistant endodontic pathogens [47],[48]. Also CS was proved to be used for treatment of dentinal tubule infection, in cases of direct pulp capping [49] and in tissue regeneration [50]. CS was shown to remove the smear layer when used as root canal irrigation [51],[52]. Recently, there is a growing interest for using dental materials in its nanoform in order to modify the material's physical properties [53],[54]. However, with the increased production and use of nanoparticles, the cytotoxic adverse effect of these materials in its nanoform is still unclear.

In the present study, cytotoxicity was evaluated using MTT assay, a method that was first developed by Mossman in 1983 [44]. It is a standardized method that indicates the effect on cell viability, depending on the conversion of the water-soluble methylthiazol tetrazolium to an insoluble purple formazan where its concentration can then be determined spectrophotometrically. Apoptosis is characterized by a number of intercellular phenomena, such as membrane blebbing, chromatin condensation, and nuclear DNA fragmentation. This method based on the difference in DNA molecular weight. The high molecular weight DNA fraction presents in the nuclei of normal cells, while low molecular weight DNA fraction, consists of fragmented DNA, presents in the nuclei of apoptotic cells. The individual fractions are separated by centrifugation [55]. The apoptotic cells could be differentiated from the necrotic cells by the so-called 'ladder' composed of histone octamers formed during the electrophoretic separation of DNA from the apoptotic cells. DNA from necrotic cell nuclei is degraded according to a different, unorganized pattern [56]. Therefore, studying the cytotoxic effect and apoptotic changes of these disinfectants on DPSCs was of great importance.

Different solvents were used to solubilize propolis as ethanol, methanol, and water [57]. Results of the current study indicated that EEPNPs was more cytotoxic than AEPNPs, although nonsignificantly different. EEP is one of the richest sources of phenolic acids and flavonoids and revealed various biological activities, including immunomodulatory, chemopreventive, and antitumour effects [58]. Nevertheless, the cytotoxic effect of 13 phenolic components of propolis was reported [59]. It was proved that ethanol–water solvents were more effective in extracting phenolic compounds than water [60]. Accordingly, the cytotoxic effect of the EEPNPs could be attributed to the effect of ethanol [61]. It was reported that ethanol decreased the coenzyme-Q10, which has a membrane stabilizing effects and is important for cellular mitochondrial respiration [62],[63]. Furthermore, alcohol is known to induce many proinflammatory cytokines and mediators favoring the toxic effect and it is known that tumor necrosis factor α is the major mediator in the alcohol-mediated liver diseases [64].

Regarding to the apoptosis induced by propolis in this study, there are suggested explanations in previous studies [65],[66]. In general, apoptosis may occur through two main specific signaling pathways, the extrinsic and intrinsic pathway. The extrinsic pathway is induced by an external signal stimulated by receptors such as death receptors; this pathway is generally triggered in association of activation of caspase 8 (initiator) and caspase 3 (effector). The intrinsic pathway is mediated by mitochondria and releasing of proapoptotic proteins including cytochrome c into the cytoplasm [66],[67] that activate caspase 9 signaling that triggers caspase 3 activation. Caspase 3 has been suggested to be a key role of the apoptotic machinery [67],[68]. It was reported in previous study that cancer cells treated with Sonoran propolis exhibited morphological changes and a characteristic DNA fragmentation pattern related to apoptosis [69]. Also, it was found that propolin C, one of components of propolis, potently inhibits the proliferation of human melanoma cells through inducing a cytotoxic effect and triggering apoptosis. Propolin C exhibited a dose and time-dependent inhibition of cellular proliferation in the human melanoma cells. Propolin C can induce the activation of caspase 8, Bid, and caspase3, finally producing DNA fragmentation [70].

In addition, Xuan et al. [71] evaluated the Brazilian propolis and concluded that propolis with high concentrations may be an apoptosis-inducing agent. However, other study found that Brazilian red propolis displayed 100% cytotoxicity against the human pancreatic cancer PANC-1 cell line. This displayed cytotoxicity was a time-dependent manner through a nonapoptotic pathway that did not lead to fragmentation of DNA but was accompanied by necrotic type morphological changes [72].

In the present study both EEPNPs and AEPNPs induced a short-term cytotoxicity and apoptosis (at 24 h), that were significantly higher than CS and that of control. This was inconsonance with previous studies which confirmed the cytotoxic effect of EEP against different tumor and cancer cells. [59],[72],[73],[74],[75]. In addition, a recent study by Elbaz et al. [76] evaluated the cytotoxic effect of the Egyptian propolis in vitro and confirmed its cytotoxic and apoptotic effect on HepG2 cells that significantly decreased in number in different proliferative phases.

Cytotoxicity and apoptosis induced by both extracts of PNPs were decreased by time. This might be attributed to its antioxidant property. Antioxidant activity is defined as the ability of a compound to inhibit oxidative degradation like lipid peroxidation [77]. Reactive oxygen species (ROS) and free radicals are by-products of normal cellular metabolism in aerobic life. When ROS are overproduced, it induce oxidative stress that inhibits normal functions of cellular lipids, proteins, DNA, and RNA. Previous reports showed that apoptosis occurs with increased levels of intracellular ROS [78]. Propolis is characterized by the presence of antioxidants as benzyl caffeate and phenethyl caffeate, which were related to oxygen radical absorption capacity [79]. Also, it was reported that propolis has an antioxidant and radical scavenging mechanism [80].

CNPs was tested in the current study as an aqueous and ethanolic extract, however, the cytotoxicity of the aqueous extract was more obvious and time dependant. This could be inferred to the insolubility of CS in ethanol as it was proved that CS is considered soluble in aqueous solutions of organic or inorganic acids [81],[82].

Referring to the time factor, both aqueous and ethanolic extracts of CNPs had low cytotoxicity at 24 h time interval that increased by time. This was in accordance with Amarnath et al. [83] who reported that cytotoxicity of aqueous ethanolic extract of Acalypha indica (ETAI) loaded chitosan–casein (CS–CT) microparticles revealed insignificant differences with free ETAI in the first 24 h, while higher cytotoxicity was demonstrated by CS–CT–ETAI microparticles following 72 h of incubation on human prostate cancer cell line. In addition, it was found that either calcium phosphate cements alone or containing CNPs were cytotoxic for human dental pulp cells at 1, 7, and 14 days of cultivation [84].

Similar results showed low toxicity of CNPs to breast cancer cells (almost 98% viability) using MTT assay [85]. Also, stem cells from apical papilla treated with CNPs and cultured for 24 h revealed a morphology that is similar to that of control group [86].

The resultant cytotoxic effect of CNPs could be explained by its potent cytotoxic effect on the microbial cell as the nanoparticles exert its antibacterial activity mainly through the reaction of positive charged CS with negative charged molecules at the bacterial cell surface [86],[87], which affect cell permeability and end with binding of CS with bacterial DNA to inhibit RNA synthesis [88],[89],[90].

Most of the previous toxicity studies have demonstrated an inverse relationship between particle size and toxicity [91],[92],[93]. Decrease in the particle size from macroscopic to nanoscopic scale leads to changes in physicochemical properties of the materials results in particles with smaller size that may elicit different biological responses including toxicity [94],[95],[96]. What is surprising in the present study that, the original microparticle size produced the most cytotoxic effect using either propolis or CS. This could be explained by particle concentration as nanoparticle toxicity has been shown to function in a concentration-dependent manner [92],[93].

The nanoparticles have a tendency to diffuse, settle, and agglomerate in dispersion media. The extent of these processes depends on the nanoparticle size, shape, charge, and density, as well as the viscosity and density of solution [97]. These properties influence the transportation rate of nanoparticles to adherent cells on the culture plate and therefore affect the effective dose within cells [97]. Probably, the transportation rat of the original microparticles kept these particles in direct contact more than their nanoform.

In agreement to our results, Zhang et al. [98]reported that oleyl-CNPs exhibited no cytotoxicity on A549 cells. CS–CT phosphopeptide nanoparticles with concentration below 500 μg/ml were also nontoxic to Caco-2 cells [99]. Similarly, Omar Zaki et al. [100]concluded that medium-sized and large CNPs were relatively nontoxic at lower concentrations.

  Conclusion Top

Within the limitation of this study it was concluded that CNPs was less cytotoxic and induced less apoptotic changes on DPSCs than PNPs in short-term interval, whereas after 72 h it was higher than propolis indicating proliferating effect of PNPs. While vehicle was the prime factor affecting the cytotoxicity of CS, time was the prime factor affecting the cytotoxicity of propolis. Nanoparticles of both materials, regardless the vehicles or concentrations used, were less cytotoxic and induced less apoptotic changes than their original particles counterpart.

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Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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