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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 34  |  Issue : 4  |  Page : 282-287

Synthesis and characterization of nisin-incorporated alpha-tricalcium phosphate for pulp capping – An in vitro study


1 Department of Conservative Dentistry and Endodontics, Ragas Dental College and Hospital, Chennai, Tamil Nadu, India
2 Department of Conservative Dentistry and Endodontics, SRM Dental College and Hospital, Ramapuram, SRM Institute of Science and Technology, Chennai, Tamil Nadu, India
3 Departent of Microbiology, Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India

Date of Submission24-Apr-2022
Date of Decision05-Jul-2022
Date of Acceptance29-Jul-2022
Date of Web Publication28-Dec-2022

Correspondence Address:
Dr. Veni Ashok Baskaran
Department of Conservative Dentistry and Endodontics, Ragas Dental College and Hospital, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/endo.endo_114_22

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  Abstract 


Aim: The present study aimed to synthesize and characterize Nisin incorporated Alpha Tricalcium Phosphate (NTCP) and to evaluate nisin release from NTCP when used as a pulp capping agent.
Methods: Alpha TCP(aTCP) powder was synthesized by the wet chemical method. Nisin was incorporated into this prepared aTCP at various ratios and grouped as follows: Group 1 - 1% wt%; Group 2 -2.5wt%; Group 3-5%wt%; Group 4- 7.5%wt%; Group 5 -10%wt% ; Group 6 -Nisin 100 mg; All these samples were characterized using Fourier Transmission Infrared Spectroscopy(FTIR) and Differential Scanning Calorimetry (DSC). For evaluation of nisin release from NTCP using HPLC, around fifteen freshly extracted non-carious human third molars were collected and mounted in gypsum blocks. A standard box-type class-I cavity (4.5x 4.5 mm) was prepared with the pulpal floor ending at deep dentin, The experimental materials were mixed with propylene glycol to prepare it as pulp capping material in paste form. Subsequently pulpal floor of all the cavities were lined with the respective materials. The entire samples were kept to set for 30 minutes in ambient temperature and subsequently immersed in water and stored in an incubator at 37oC. After 14 days of immersion, dentin lying directly below the sample was cut and powdered using mortar and pestle. The powdered dentin was then subjected to HPLC analysis. The peak time of nisin release from experimental groups was recorded.
Results: FTIR results revealed Group 5 with highly appreciable corresponding bends of amine N-H, C-H stretching and phosphate peaks at 1100 and 554 close to Control TCP samples. DSC analysis showed that TCP did not change from 30oC to 80oC and sample 1, 2, 3,and 4, did not show any denaturation point. Sample 5 showed denaturation point slightly above the denaturation temperature of nisin. On HPLC analysis, samples 4 and 5 showed higher peaks of nisin release and traces of nisin release from all the groups.
Conclusion: Within the limitations of this study, it can be concluded that NTCP can be synthesized successfully without any influence on the properties of each other material. 10% NTCP provides higher release into dentinal tubules when used as a pulp capping agent.

Keywords: Alpha-tricalcium phosphate, dentin, nisin, pulp capping


How to cite this article:
Baskaran VA, Madhubala MM, Menon T, Gopal SN, Venkatesan S M. Synthesis and characterization of nisin-incorporated alpha-tricalcium phosphate for pulp capping – An in vitro study. Endodontology 2022;34:282-7

How to cite this URL:
Baskaran VA, Madhubala MM, Menon T, Gopal SN, Venkatesan S M. Synthesis and characterization of nisin-incorporated alpha-tricalcium phosphate for pulp capping – An in vitro study. Endodontology [serial online] 2022 [cited 2023 Jan 28];34:282-7. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/4/282/365807




  Introduction Top


Dental pulp is a highly specialized tissue which has the potential to repair and regenerate the lost dentin.[1] Vital pulp therapies are one of the common procedures in day-to-day clinical practice. As pulp capping is usually indicated in deep caries approximating the pulp, placement of appropriate material on the pulpal exposure site should seal and heal injured pulp by stimulating odontoblasts to form reparative dentin. The quality of reparative dentin formation depends on the bioactive material used which should have good sealing ability and enhanced antibacterial activity.[2] The material commonly used for pulp capping procedures are calcium hydroxide Ca(OH)2, mineral trioxide aggregate (MTA), and Biodentine.

Ca(OH)2 acts on the inflamed dental pulp inducing proliferation and migration of vascular cells and other associated cells.[3] However, it is reported that 89% of the dentin bridges displayed tunnel defects, failing to provide a hermetic seal against infection to the underlying pulp.[4] In addition, the dissolution of Ca(OH)2 over time causes the formation of dead space and microleakage.[5] MTA and Biodentine do not cause extensive tissue necrosis to stimulate secondary dentin formation and are more biocompatible. They also possess the enhanced capability of stem cell proliferation, migration, and adhesion abilities.[6],[7] However, some studies have shown that the prolonged contact of calcium silicate cement with dentin reduces the flexural strength of dentin collagen matrix.[8]

Many attempts to find an ideal pulp capping material have led to the use of calcium phosphate cement (CPC) materials because of their biocompatibility and chemical similarity to teeth. Calcium phosphate is a bioceramic, bioactive material in different forms such as hydroxyapatite and alpha (α)- and beta (β)-tricalcium phosphate (TCP) forms.[9] Calcium phosphate-based cement have better dentinogenic potential than Ca(OH)2 when used as pulp capping agents.[10] Among these, alpha-TCP (α-TCP) can stimulate reparative dentin formation.[10],[11],[12],[13] Several studies in the literature reveal that α-TCP gave comparable results to MTA in stimulating reparative dentin formation, and it has a lower solubility level than Ca(OH)2.[11],[13],[14] Although CPC materials exhibit excellent osteoconductivity and biocompatibility, the antibacterial action on the substrate is questionable. Recently, various newer materials have been introduced to enhance the antibacterial activities of bioactive pulp capping materials. The incorporation of antibacterial agents such as fluorine, quaternary ammonium compounds, chitosan, and silver amine on CPC has been reported to exhibit synergistic potential on antibacterial action.[15],[16]

Nisin, an antibacterial component produced by Lactococcus lactis, is an antimicrobial peptide that effectively inhibits a broad spectrum of Gram-positive bacteria and is widely used as a food preservative.[17] Tong et al. evaluated the antibacterial activity of nisin against cariogenic bacteria and found that they are effective in eradicating Streptococcus mutans and Lactobacillus acidophilus.[18] Nisin is soluble in water and active in acidic conditions. Nisin-incorporated polymer controls the growth of pathogenic bacteria and has also been shown to control Staphylococcus aureus infection in vivo.[19] There are no reports which confirm that nisin-incorporated α-TCP cement have enhanced antibacterial action when used in pulp capping procedures. Hence, the aim of this study was to primarily synthesize and optimize nisin-incorporated α-TCP (NTCP) and to evaluate nisin release from NTCP.


  Materials and Methods Top


Synthesis of NTCP

α-TCP was synthesized by a wet chemical method using analytical grade Ca(OH)2 and 85 wt% solution of H3PO4 as reagents. The pH of the reaction environment was maintained at around 4.0–5.5. The suspension of gelatinous amorphous calcium phosphate formed on mixing both the materials was next decanted and concentrated to a precipitate containing 70%–80% of water. After drying, the filter cake of α-TCP material was crushed into fractions below 0.6 mm, after which it was subjected to mechanical treatment in an agate mortar, followed by calcination at 1300°C and grinding by attrition to the grain size below 0.06 mm. Nisin was added to the prepared α-TCP at various ratios and homogenously mixed in a cyclomixer; as at following concentrations (1%, 2.5%, 5%, 7.5%, and 10%).

Grouping of the samples

The samples were grouped as follows: Group 1 (nisin 10 mg – TCP 990 mg) – 1% wt%, Group 2 (nisin 25 mg – TCP 975 mg) – 2.5% wt%, Group 3 (nisin 50 mg – TCP 950 mg) – 5% wt%, Group 4 (nisin 75 mg – TCP 925 mg) – 7.5% wt%, Group 5 (nisin 100 mg – TCP 900 mg) – 10% wt%, Group 6 – nisin (nisin 100 mg) (positive control), and Group 7 – TCP (TCP 100 mg) (negative control).

Fourier-transmission infrared spectroscopy

All the samples were ground using an agate mortar and pestle and mixed with FTIR grade potassium bromide (Sigma Aldrich, India). The mixture was made to af pellet using hydraulic pressure (10 MPa for 5 s). The resulted pellet was mounted and the infrared spectrum was recorded as transmittance for all the samples using Jasco FT IR-4700 instrument from 400 to 4000/cm in a resolution of 4/cm.

Differential scanning calorimetry

Each sample was mixed with distilled water at the proportion of 10 ml water for 10 mg sample and mixed well using a clean spatula on a glass slab to make a thick paste, which was then transferred to Tzero hermetic aluminum pans and weighed. The differential scanning calorimetry (DSC) curve was recorded using “TA DSC 25” machine from 30.00°C to 80.00°C with a ramp of 5°C/min. The curve was analyzed for denaturation endotherms to detect free nisin in the mixture.

Evaluation of nisin release in dentin using high-performance liquid chromatography

For this study, around thirty freshly extracted noncarious human third molars were collected and stored in 0.5% chloramine solution at 4°C. The study was approved by the institutional ethics board (clearance number – UM/IHEC/FRM./2019-XII), and informed consent was obtained from patients. All the samples were mounted separately in gypsum blocks to ease manipulation. A standard box-type Class I cavity (4.5 mm × 4.5 mm) was prepared with the pulpal floor ending at deep dentin, using a high-speed handpiece with a cylindrical medium grit (100 mm) diamond bur.

The respective experimental groups were mixed with propylene glycol to prepare it as a pulp capping material in paste form. Subsequently, all the groups were randomly divided into six groups (n = 5) as mentioned above (except for the TCP control group), and the pulpal floor of all the cavities was lined with the respective materials. Followed by this, all the cavities were sealed by zinc oxide eugenol restoration. The entire samples were kept to set for 30 min at ambient temperature and subsequently immersed in water and stored in an incubator at 37°C. After 14 days of immersion, the samples were retrieved, and restoration was removed using an explorer tip. The dentin lying directly below the sample was cut and frozen till used and powdered using mortar and pestle. The powdered dentin was mixed in distilled water, twice the volume of dentin powder, and left to soak for 24 h in an incubator at 37°C. Subsequently, it was placed in a vortex shaker for 30 s and then subjected to high-performance liquid chromatography (HPLC) analysis. HPLC analysis was conducted using Shimadzu LC solution analyzer, by injecting 40 μl of elutes thus prepared. The control group (nisin) showed a peak at 13.6 min, and it was taken as reference. The peak time of nisin release from experimental groups was recorded. The test was qualitative.


  Results Top


FTIR Evaluation

As shown in [Figure 1], FTIR spectrum of the nisin group clearly shows protein peaks at amide I bond at 1630/cm (C = O), amide II bond at 1500/cm, and amide III at 1300/cm. C-H stretching was seen in 2700/cm and amine N-H peak shows at 1500/cm. TCP has phosphate characteristic peaks at the FTIR spectra in the range of 1126 and 1025 (n3)/cm, as well as at 604 and 554 (n4) cm−1 indicating the presence of phosphates. Among the experimental groups, Group 5 showed highly appreciable corresponding bends of amine N-H, C-H stretching, and phosphate peaks at 1100 and 554 close to the control TCP group.
Figure 1: FTIR spectrum of samples

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Differential scanning calorimetry

AS shown in [Figure 2], in this experiment, it was clearly seen that TCP did not change from 30°C to 80°C. In addition, Groups 1, 2, 3, and 4 which contained 1%, 2.5%, 5%, and 7.5% nisin, respectively, did not show any denaturation point at all which meant that the nisin was adhering to the TCP scaffold. At 10% concentration (Group 5), the denaturation point was seen slightly above the denaturation temperature of nisin, indicating that concentrations of 10% and above were appropriate.
Figure 2: DSC curves of samples

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Evaluation of nisin release using high-performance liquid chromatography

Nisin standard showed its presence at 13.4 min, as depicted in [Figure 3]. The peak was detected for other groups at 13.1 min. The results showed that, even though there were traces of nisin released from all the groups, Groups 4 and 5 showed higher peaks when compared to other groups.
Figure 3: HPLC analysis of samples. HPLC: High-performance liquid chromatography

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  Discussion Top


FTIR analysis was performed to evaluate the presence of nisin in the mixture. While the preparation was done by solid state mixing, the knowledge of interaction between the components is crucial to use the selected vehicle for delivery. When the components were mixed, the corresponding peaks for both α-TCP and nisin were evaluated. It is hence clear that there was no strong chemical interaction between the components. This has been an important factor for drug delivery to the target and enhances the delivery of nisin into dentinal tubules. FTIR shows covalent interaction only, and no ionic interactions were seen. It has been observed that there is no irreversible bond between the two components, namely α-TCP and nisin. The incorporation of nisin into the matrix identified with the peaks at 1618 and 1587 cm could be attributed to the bands of amide I (bond stretching vibration C = O) and amide II (bending vibration of the N-H bonds), respectively, both related to the peptide bond.[20] Similar reports had been reported in the evaluation of nisin peptide bond.

DSC is used to measure the absorption of heat/heat flow as a function of temperature. In this technique, it was feasible to measure the denaturation temperature of proteins. It should be observed that when proteins denature, they absorb heat and lose their three-dimensional conformation, materialized by breaking of disulfide and ionic bridges. While such a change occurs, the absorption was seen as a downward peak in the DSC. In this study, this principle was used to determine the minimum concentration of nisin required to initiate its delivery into dentinal tubules. Shiroodi et al. observed that biocomposites added to nisin cannot be influenced by various concentrations on DSC analysis like the results of our study.[20] Similar results have been reported by other researchers as well.[21],[22],[23]

While nisin was added to α-TCP in the aqueous medium, there would be a reaction between acidic groups of nisin and basic groups of α-TCP. Such reaction would lead to immobilization of nisin on α-TCP crystals. This adhesion would be strong and cannot be easily reversed for any practical purpose. On the other hand, when the concentration of nisin increases, there would be a possibility of free nisin in the system. Only when there is a sufficient amount of free nisin, its delivery would be feasible. Hence, by testing various concentrations of nisin in α-TCP using DSC, the concentration in which free nisin is present was taken as the minimum required concentration.

PDSC curve of pure nisin would be the approximate reference point for finding the denaturation temperature. However, it cannot be considered an absolute quantity to assess the change in nature of nisin as it was incorporated into α-TCP. Mild increase in temperature was expected, as even incompletely bonded nisin can also partially denature. In this experiment, it was clearly observed that α-TCP did not change from 30°C to 80°C. In addition, Groups 1, 2, 3, and 4 made of 1%, 2%, 5%, and 7.5% nisin did not show any denaturation point at all. This means that nisin was adhering to the α-TCP scaffold. At 10% concentration (Group 5), the denaturation point was seen slightly above the denaturation temperature of nisin. This means that at 10% w/w concentration, free nisin was present in the system. Hence, for further work, concentrations of 10% and above were ideal.

As the dentin could not be obtained in identical size from all teeth due to biological variations in size and shape, and the amount of nisin released cannot be quantitated in relation to dentin mass, the release analysis by HPLC was conducted as a qualitative study. The groups were stored in water to simulate the wetness provided by live dentin, which would allow the flow of nisin from α-TCP into dentinal tubules. This methodology has also been utilized to check the elution of monomers from dental composites.[24] When nisin diffuses from α-TCP, it would start accumulating in the dentinal structure until the concentration of nisin in dentin equilibrates with that of the delivering α-TCP. Further, there is also a possibility of nisin delivery into dentinal tubules toward the pulpal side. Nisin standard showed its presence at 13.4 min, whereas nisin incorporated groups peaked at 13.1 min. This might be due to changes caused by the environment. As shown in the results of the study, there was a trace of nisin seen in all the groups indicating the release of nisin into dentinal tubules. However, groups with 10% and 7.5% nisin showed comparatively higher concentrations of nisin. It should be noted that the higher the nisin content, the lower the strength of the mix. In other words, as nisin concentration increases, the mechanical properties that favor the use of α-TCP will be compromised. Furthermore, even in positive control nisin, only traces of nisin were found in the solution, suggesting an impending saturation point. This was in accordance with studies done by Lygidakis et al. and Chanachai et al.[25],[26] This also strikes a balance between the strength and the workability of mix and nisin delivery. Hence, 10 wt% nisin in α-TCP can be chosen as the optimum concentration for further studies.

Nisin, a cationic peptide, can stably bind to cell membranes by adhering to anionic lipid II, which is a component of the membranes of Gram-positive bacteria. This leads to pore formation on the cell membrane and disruption of cell wall synthesis, resulting in the rapid efflux of small cytoplasmic compounds causing cell death.[27] As nisin has a good bactericidal action against antibiotic-resistant microbes, further studies on the evaluation of the antibacterial potential of NTCP have to be carried out to assess the utility of this material for pulp capping procedures.


  Conclusion Top


Within the limitations of this study, it can be concluded that NTCP can be synthesized successfully without any influence on the properties of each other material. Therefore, nisin provides a feasible route for surface modification of α-TCP cement with a higher release at the optimum concentration of 10% when used as a pulp capping agent.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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  [Figure 1], [Figure 2], [Figure 3]



 

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