JICDRO is a UGC approved journal (Journal no. 63927)

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Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 109-117

Evaluation of the biocompatibility of silver nanoparticles, ascertaining their safety in the field of endodontic therapy

Department of conservative dentistry and Endodontics, Narayana Dental College, Nellore, Andhra Pradesh, India

Date of Submission03-May-2021
Date of Decision24-May-2021
Date of Acceptance27-May-2021
Date of Web Publication17-Jan-2022

Correspondence Address:
Dr. Kiranmayi Govula
Narayana Dental College, Nellore, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jicdro.jicdro_22_21

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The success of endodontic therapy relies on the complete elimination of bacteria from the root canals. Primary root canal infections consist of polymicrobial groups, which can efficiently eradicate through root canal treatment. However, conventional root canal treatment cannot remove persistent bacterial species from the root canals. Although significant numbers of studies that focus on developing antimicrobial agents to overcome this problem exist, most of these attempts failed to achieve desired outcomes due to the rapid degradation and fast release of antibacterial agents, causing low efficiency and safety concerns. Antimicrobials such as sodium hypochlorite and ethylenediaminetetraacetic acid are commonly used in endodontic treatment. They carry the disadvantage of antibiotic resistance. Thus nanoparticles (NPs) have been introduced in dentistry as an alternative to such materials. The dominant microorganism, or sometimes the only species present in the root canal of teeth with resistant peri-radicular lesions, is Enterococcus faecalis (E. faecalis). This bacterium is capable of tolerating starvation, high pH, and salt concentration. Its ability to penetrate dentinal tubules and create resistant species to antibiotics has made it very difficult to eradicate root canals. One of the essential characteristics of this bacterium is biofilm formation. NPs can reach the untouched portion of root canals such as cul de sacs, isthmi, fins and can implement the ideal action of irrigant such as antibacterial action. Higher-end evidence-based support is needed to ascertain the application of NPs in the field of endodontics. Hence, our systematic review encompasses, encloses, and enumerates the use of silver NPs in routine endodontic procedures.

Keywords: Biocompatibility, endodontic irrigants, intracanal medicaments, silver nanoparticles

How to cite this article:
Prasad G, Govula K, Anumula L, Kumar P. Evaluation of the biocompatibility of silver nanoparticles, ascertaining their safety in the field of endodontic therapy. J Int Clin Dent Res Organ 2021;13:109-17

How to cite this URL:
Prasad G, Govula K, Anumula L, Kumar P. Evaluation of the biocompatibility of silver nanoparticles, ascertaining their safety in the field of endodontic therapy. J Int Clin Dent Res Organ [serial online] 2021 [cited 2022 May 28];13:109-17. Available from: https://www.jicdro.org/text.asp?2021/13/2/109/335871

   Introduction Top

The bacterial biofilms on dentin surfaces and their extensions deep into the dentinal tubules cause endodontic infections. These biofilms often communicate beyond the apical foramen leading to periapical infections. Despite adequate cleaning and shaping methods, advanced intracanal irrigants, and medicaments, recontamination still became challenging. Biofilm stability and adherence are mainly due to extracellular polymeric substances that provide several resistance mechanisms against external stimuli. Even bacteria can infiltrate and penetrate the dentinal tubules to a depth of 200–1500 μm became a challenge to eradicate through traditional instrumentation and irrigation regimens as they cannot penetrate the root canal system completely. Therefore many efforts have been made to add antibacterial agents to root canal irrigants, sealers to enhance and prolong their antibacterial activity.[1]However, most antibacterial agents depend on releasing these agents into the surrounding environment, which becomes depleted over time. A promising approach for solving this problem is applying dual antibacterial agents, which release deep into the root canal system's complex anatomy. It destroys effectively away from the sealer and provides long-term antibacterial properties through a contact killing mechanism on the sealer surface.[2],[3] Hence, antibacterial silver nanoparticles (NPs), gold, calcium, magnesium, chitosan, and various polymeric materials were introduced. In such materials, NPs including gold, silver, graphene oxide, zinc oxide have been widely explored as antibacterial agents. Among them, silver NPs have been used against a wide range of bacteria. However, chemically synthesized AgNPs are highly unstable and get aggregated because they are more prone to easy oxidation.[4]

There is a lack of studies on the application of AgNPs solution in endodontic therapy. It is due to a lack of enough knowledge on the biocompatibility of AgNPs with vital tissues. The bactericidal potency of silver has been proved in many in vitro studies. The silver reduces bacterial adhesion and prevents biofilm formation, so it has a higher antibacterial potency.[5] The silver NPs showed a better antibacterial effect than broad-spectrum antibiotics against a broad range of bacteria. The exact mechanism of NPs cytotoxicity is still not so precise. Nonspecific oxidative damage is the main significant concern in NP-induced toxicity. Our systematic review focused on silver NPs' biocompatibility providing more evidence-based support.


Considering the shortage of literature evaluating the use of silver NPs in steps of root canal treatment, mainly as root canal irrigant, the systematic review aimed to analyze the biocompatibility of silver NPs. The research question “is the silver NPs biocompatible when used as root canal irrigant or medicament?”

In the problem, intervention, comparison, and outcome analysis, the population was the animals such as rats (wistar male, wistar albino, and Sprague–Dranky rats). The trial included patients with pulpal necrotized teeth and acute apical periodontitis. The intervention of this study includes silver NPs, biogenic silver NPs, and nanodiamond embedded guttapercha (NDGP). The primary outcome is analyzing the response of vital connective tissue to silver NPs dispersion and sodium hypochlorite, hemocompatibility of biogenic silver NPs, and drug delivery by nanodiamond particles coated gutta percha.

   Materials and Methods Top

Data search

The detailed search strategies for the review were developed after identifying the included studies from different database sources. The MEDLINE search used the combination of controlled vocabulary and accessible text terms. There were no language restrictions.

Search strategy

The search strategy was mentioned in detail in the search flow chart. An electronic search was conducted from 2011 till 2021 in PUBMED, DOAG, SCIMAGO, SCOPUS. The mesh terms used were NPs, nanodiamonds, nanomedicine, clinical trial, or randomized clinical trial, nano-MgO, root canal irrigants, nano-calcium hydroxide, antibacterial, chitosan, functionalized, NPs silver or silver NPs application, antimicrobial, antibacterial NPs, dentistry application. EBSCO, COCHRANE LIBRARY, LILAC, and manual search were also carried out based on references and cross-references from the included studies. The preferred language is English.

Eligibility criteria and study selection

Randomized clinical trials and in vivo studies that conducted tests on NPs as root canal sealers, intracanal medicaments, and root canal irrigants, studies that conducted or tested NPs in root canal treatment, and studies that have no restriction regarding patient's age, country, race, gender, language were included. The studies that tested NPs in conservative treatments, case reports, case series and systematic reviews were excluded.

Risk of bias and the quality of included studies

The authors assessed the quality of the included articles independently according to the GRADE methods using GRADE PRO 2008. We evaluated the quality of the body of evidence as higher by considering the overall risk of bias, directness of evidence, consistency of results, the precision of the estimates, and risk of publication bias. Two authors independently assessed the risk of bias of the included studies using SYstemic Review Center for Laboratory animal Experimentation (SYRCLE).

   Results Top

Study selection

Two authors independently searched the databases and identified 346 records, out of which 120 have been screened, and 50 were excluded. Ten were assessed for eligibility based on inclusion criteria, out of which seven were excluded based on exclusion criteria. A total of three studies were included for qualitative analysis and were represented ideally in the flowchart [Figure 1].
Figure 1: Flowchart

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Study characteristics

A single operator carried out the procedure in all the included studies. The extracted data, which includes the name of the first author, year of publication, patient's information (number), study design, follow-up time, and dropout, was mentioned in detail in [Table 1]. The results of the three included studies were described in detail in [Table 1].
Table 1: Study characteristics including Results of the including studies

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The primary outcome of the individual studies and results of individual studies

Mohammadreza Nabavizadeh et al. assessed vital connective tissue reaction in 4–5-month-old Sprague–Dawley rats. Saline, sodium hypochlorite, chlorhexidine, and silver NPs 5.7 × 10−8 ml were used in 2 h, 48 h, and 14 days. Inflammation reaction showed to 0.9% saline was mild, 5.25% Naocl showed moderate to severe inflammation reaction. Chlorhexidine showed mild-to-moderate reaction, and silver NPs showed mild-to-moderate inflammation reaction. There was a statistically significant difference in tissue inflammation responses. Naocl displayed a higher inflammatory response compared to silver NPs.

Joa˜o Eduardo Gomes-Filho et al. investigated the tissue reaction to silver NPs dispersion as an alternative irrigating solution. In this study, four polyethylene tubes filled with fibrin sponge were embedded in 47 ppm and 23 ppm of silver NPs and 2.5% Naocl. The tissue reaction observed at 7, 15, 30, 60, and 90 days under a light microscope. 23 ppm of AgNPs intensity of inflammation was reduced on the 15th day and continued reducing n 30, 60, and 90 days. 47 ppm of AgNO3 shown a moderate inflammation on 7 and 15 the day, and the intensity of inflammation reduced on 30, 60, and 90 days. Similar to the control group, Naocl showed moderate inflammation on the 7th and 15th day, where the intensity reduced on 30, 60, and 90 days. On the 15th day, there was a statistically significant difference between inflammatory cell numbers of 23 ppm AgNPs dispersion and other groups.

Md Monir Hussain et al. investigated the antibacterial activity, and in vivo cytotoxicity of biogenic silver NPs in humans and rat red blood cells (RBCs) measured the concentration of several enzymes whose levels usually get hiked if any damage in the liver and kidneys functional biomarkers. They measured serum aspartate, aminotransferase, alanine aminotransferase, creatinine of experimental rats compared with normal rats. The highest antibacterial effect is toward Gram-positive bacteria. The lowest against Escherichia coli and effect is time dependent and dose-dependent biogenic AgNPs showed excellent hemocompatibility to human and rat RBCs, broad-spectrum antibiotic propensity and excellent biocompatibility.

Risk of bias within the included studies

Animal intervention studies like randomized clinical trials are experimental studies, but they differ in many aspects. Two authors independently assessed the risk of bias in the selected three studies using the SYRCLE aim to assess methodological quality and have been adapted to aspects of bias that plays a role in animal experiments which was mentioned in [Table 2], [Table 3], [Table 4]. The following domains were evaluated and classified as low, moderate, high, or unclear risk of bias:
Table 2: SYRCLE Tool for Risk of Bias

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Table 3: SYRCLE Tool for Risk of Bias

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Table 4: SYRCLE Tool for Risk of Bias

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  • Sequence generation
  • Allocation concealment
  • Blinding of participants
  • Incomplete outcome data
  • Selective outcome reporting.

Under selection bias, sequence generation is not mentioned in all the three included studies by et al., which leads to a high risk of bias. Baseline characteristics in the three included studies, the animal characteristics were mentioned. Two of the included studies have mentioned the concealment method leading to a low risk of bias. Monir Hussain et al. have not mentioned the allocation concealment leading to a high risk of bias.

Performance bias

Random housing

Three included studies done random animal housing and housed the animals under similar temperature and conditions.


Caregivers and researchers were blinded from knowing which intervention each animal received during the experiment, hence low risk of bias.

Detection bias

Random outcome assessment: three studies did not mention the random selection methods for outcome assessment, hence under high risk of bias.


Outcome assessors were blinded in the three studies.

Attrition bias

Incomplete outcome data three studies have mentioned that all animals were analyzed, and there were no dropouts or missing.

Selective outcome reporting

The selective outcome biocompatibility of silver NPs with vital tissues was examined and reported.

Other sources of bias

All the animals were free of contamination, no funding issues, and new animals to replace the dropouts or dead animals. The three studies were free of other problems that could result in a high risk of bias hence were under low risk of bias.

   Discussion Top

Summary of the evidence

The synthesis of AgNPs is tedious porous expensive and hazardous to the environment. The advanced approaches for synthesizing AgNPs using plant extracts as the source of reducing agents are more cost-effective and eco-friendly. Several plants such as Andrographis paniculata, Phyllanthus emblica, and Centella asiatica have medicinal application and contain various secondary metabolites (alkaloids, phenols, flavonoids, tannins, so on). Plant extracts' reducing potential reduces AgNo3 to AgNPs, which exhibits anti-inflammatory, antimicrobial, antifungal, anti-angiogenesis, antiplatelet, and anticancer activities. The various application methods should be used with caution because their toxicity is concentration-dependent, as there was evidence indicating adverse effects of AgNPs in human health and the environment.[6] Many factors such as their small size, high surface area per unit mass, chemical composition, and surface property effects might be essential in analyzing the NP-induced toxicity. Ag has received much attention because of its toxicity at low ionic concentrations. The action of silver NPs is broadly similar to that of silver ions. It may be because a bacterial cell takes in silver ions after coming in contact with silver NPs, inhibiting a respiratory enzyme (s), thereby facilitating reactive oxygen species, consequently damaging the cell. Silver (weak acid) has a greater tendency to react with sulfur-or phosphorus-containing soft bases, such as R-S-R, R-SH, RS_, or PR3. Thus, sulfur-containing proteins in the membrane or inside the cells and phosphorus-containing elements like DNA are likely to be the preferential sites for silver NP binding.[7]

Some previous studies stated that oxidative stress, mitochondrial dysfunction, DNA damage, and cytokine induction might be the leading cause of their cytotoxicity. However, many in-vitro studies and some in vivo studies have proven that silver NPs are less cytotoxic to the vital tissues. Silver antimicrobial agents have been pursued as an alternative strategy for reducing bacterial adhesion and preventing biofilm formation. Silver is toxic at low ionic concentrations; hence, nonspecific oxidative stress is one of the most significant concerns in NP-induced toxicity.[8]

Further, Ag exposure is associated with specific clinical symptoms, such as argyria, which causes an irreversible gray coloration of the skin (Payne et al., 1992).[9] Allergic reactions have been noted in patients exposed topically to silver nitrate (Dunn and Edwards-Jones, 2004).[10] Some studies have reported kidney and liver toxicity (Chaby et al., 2005; Trop et al., 2006).[11],[12]

The toxicity of AgNPs has been investigated in various cell types, including BRL3A rat liver cells, PC-12 neuroendocrine cells (Hussain et al., 2006), human alveolar epithelial cells (Park et al., 2007), and germline stem cells (Braydich-Stolle et al., 2005).[13],[14],[15] The toxicity of AgNPs arises, in part, from their effect on cellular energy metabolism, as AgNPs decreases mitochondrial function. AgNp cytotoxicity has also been associated with oxidative stress (Arora et al., 2008; Carlson et al., 2008; Hussain et al., 2005).[16],[17]

AgNPs release Ag + ions in the presence of water. Thus, an AgNP solution free of Ag + ions is needed to distinguish between the cytotoxic effects of AgNPs and dissolved Ag + ions. For this purpose, AgNPs have been rinsed in pure water to remove Ag + ions before their use in toxicity assessments in various concentrations. AgNPs and Ag + ions did not exhibit a dramatic difference in cytotoxicity based upon their IC50 values after the assessments. It has been suggested that the general trend of NP cytotoxicity is similar among various types of NPs (Jin et al., 2008) and that nonspecific oxidative stress is one of the most significant concerns in NP-induced toxicity (Colvin, 2003; Nel et al., 2006; Xia et al., 2006).[18],[19] Consistent with those findings, all of the cytotoxic and genotoxic changes induced by AgNPs were efficiently prevented by NAC pretreatment, suggesting that the intrinsic toxicity of AgNPs is associated with oxidative damage-dependent pathways. Also, catalase mRNA was significantly induced in AgNO3-treated cells than in AgNP-treated cells. These findings suggest that AgNP-induced oxidative toxicity's underlying mechanisms may differ from Ag + ions, attributed primarily to oxidative stress. The expression of oxidative stress-related mRNA species was regulated differentially by AgNPs and Ag + ions. These findings suggest that AgNP-induced toxicity is an intrinsic effect of AgNPs independent of free Ag + ions. The mechanisms of AgNP action may be different from those of Ag + ions.[20]

On the other hand, silver NPs in a liquid medium, even at high concentrations, caused mainly a growth delay of E. coli. Studies showed that the interaction of these NPs with intracellular substances from the lysed cells caused their coagulation. It was most likely due to the cationic surfactant and silver NPs' synergistic effect, and a declining curve was obtained, finally leading to complete cell death. Reports on the mechanism of inhibitory action of silver ions on microorganisms show that DNA loses its replication ability and expression of ribosomal subunit proteins and some other cellular proteins enzymes upon Ag treatment. It has also hypothesized that Ag_ primarily affects the function of membrane-bound enzymes, such as those in the respiratory chain.

An in-vitro study by Abbaszadegan A et al. stated that 5.7 × 10-8 ml AgNPs were effective against Enterococcus faecalis, while it had only mild cytotoxicity to L 929 fibroblasts. Zanette C et al. also stated that NPs toxicity's intensity to the human keratinocyte HaCaT cell line is mainly concentration-dependent. Also, toxicity is related to these particles' shape and size, and surface charge. The tissue reaction is expressed as only a mild inflammation in the first 48 h, considering saline as a biocompatible material. The tissues than 5.25% Naocl significantly more tolerated AgNPs at 24 and 48 h and 14 days. Joa˜o Eduardo Gomes-Filho and Mohammadreza Nabavizadeh et al. proved that AgNPs showed a mild inflammatory response at 2 h, which increased to moderate inflammatory response until 14 days.[21]

Baker et al. proved that low silver concentration of AgNPs at 8 μg/cm2 was effective toward E. coli, Enterococcus, Staphylococcus, Pseudomonas Aeruginosa, and Candida Albicans species. The smaller the silver NPs, the more the anti-bactericidal effect. It was observed in the ivv3 study that the tissue reaction with a low concentration AgNPs produced a mild reaction showing that NPs with a good combination of concentration and size can optimize the in vivo results. Naocl induced a moderate inflammatory response in the beginning that reduced to mild after 30 days. The study concluded that AgNPs dispersion was biocompatible compared with the Naocl solution, but furthermore, studies are necessary to support this analysis, behavior of AgNPs and confirm the above observation.

Hemocompatibility was performed three times on three different days to prove that NPs' percentage hemolytic potential was low. It was less than that of the acceptable hemolytic (<5%) value, a value that is regarded as critically safe for therapeutic applications of biomaterials (Sarika et al. in 2015).[22] The biogenic AgNPs administered through intravenous route had no significant toxic effect on rat liver, kidneys. Even though the silver NPs are not toxic for the human healthy peripheral lymphocytes, they show high affinity and significant toxicity to bacteria and cancer cells (Greulich et al., 2012; Gengan et al., 2013). Recently, AgNPs have been used to diagnose and treat cancer (Ong et al., 2013).[23],[24],[25] They are used as drugs by themselves or targeted delivery vehicles of anticancer drugs or probes to detect cancer (Wei et al., 2012; Locatelli et al., 2014).[26],[27] In this study, two different types of biogenic silver NPs (i.e., Aq-bAgNPs and Et-bAgNPs) synthesized using aqueous and ethanolic extract A. paniculata stem. The antimicrobial activity of biogenic AgNPs was investigated against seven pathogenic and three non-pathogenic bacterial strains. Both et and aq AgNPs showed the highest antimicrobial activity against pathogenic gram-positive Staphylococcus aureus. The as-synthesized biogenic AgNPs have a broad-spectrum antimicrobial propensity, excellent biocompatibility, cost-effectiveness. So they recommend future therapeutic applications in endodontics.

Dong-Keun Lee et al. demonstrated the in-human validation of NDGP, a polymer that repairs root canal treatment sites following tissue disinfection. A randomized, dual-arm clinical trial was implemented, and study endpoints included confirmation of lesion healing, postoperative pain reduction, and the absence of reinfection. As conventional guttapercha may become conducive to bacterial regrowth and the bacterial remnants in the root canal space and the tissue fluid re-establish contact. For an unfavorable prognosis of retreatment cases, the leading cause could be insufficient obturation. Nanodiamond guttapercha has enhanced mechanical properties that are validated by tensile strength studies. It can improve the dense obturation, enhancing technical advancements in obturation procedures for clinicians. In nanomedicine, there are many sources for drug delivery biomaterials emerging. The NPs have been used for systematic therapy, interference, and other emerging approaches in forthcoming clinical studies.[28]

Nanodiamond has received increasing attention among the promising approaches due to their combination of unequal faceted electrostatic properties. These properties have mediated remarkably potent drug binding and sustained elution for wound repair potential regenerative medicine and tissue engineering applications. Nanodiamonds with uniform particle size and surface chemical properties can be scalably synthesized, supporting their clinical translation.

Based on the potential reduction in voids spaces or reduced incidence of coronal leakage using ND guttapercha, this study aimed to monitor and confirm the absence of apical periodontitis during this equivalence study course between the two.

In the study by Lee D-K et al., NDs were used to sequester amoxicillin for contact-mediated bacterial inhibition, which could substantially reduce the possible risk of reinfection following RCT. The antimicrobial properties of drug-incorporated NDGP could reduce reinfection incidence after the mechanical removal of debris from the root canals and sealing are complete.[29] Furthermore, recent advancements in drug optimization technologies have shown that NDs can be used for combination therapy, markedly enhancing therapeutic efficacy and reducing toxicity through the systematic and mechanism-independent design of ND-functionalized multidrug treatment. Importantly, these technology platforms must be clinically validated.

Potential biases in the review process

There is no potential bias in the included studies; hence, the inference from these studies could be helpful for further ongoing studies in the future.

Agreements and disagreements with other studies or reviews

Among the studies involved, evaluation of tissue reactions was done for a maximum period of 3 months. Further evaluation after 3 months is essential. All the tissues utilized are of animals; hence, more ex vivo studies involving human tissues are advisable for further studies that replicate the exact nature of human tissues.

   Authors' Conclusions Top

Within the limitation of these studies, it can be concluded that NPs are biocompatible with a compatible degree of inflammation resolved by the host defense mechanism. These studies included the observations for an extended range of time, about 3 months that provide considerable room for the complement system to resolve the inflammation. Intervention with NPs should be tapered accordingly to the subject's physiologic and systemic ailments for better periapical tissue prognosis.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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