Evaluation of the efficacy of Myristica Fragrans as a pulpotomy medicament in primary molars: A Clinical Trial

Introduction

The primary tooth has an important role in orofacial development. Primary teeth maintain dental arch length and masticatory functions until permanent teeth erupt. The health of the primary teeth is important in maintaining these functions which in turn may be affected by caries, restorative procedures, and trauma. Pulp treatment procedures for primary teeth can be divided into two main categories: vital pulp therapy (VPT) and non-vital pulp therapy. The main aim of VPT in primary teeth is to treat reversible pulpal injuries and to maintain pulp vitality/functions. Factors influencing the success of VPT are adequate blood supply, the severity of inflammation, obtaining hemostasis, disinfection of the exposure site, antibacterial properties, biocompatibility of pulp covering agents, and adequate coronal seal.¹ The success of the pulpotomy procedure depends upon the healing ability of radicular pulp tissue after surgical removal of the infected or affected coronal pulp.²

Ideally, a pulpotomy medicament should be bactericidal, easy to use, and harmless to the remaining pulp tissue and the surrounding structures. It should not interfere with physiologic root resorption and should be relatively inexpensive.²

Sweet introduced the formocresol pulpotomy technique in 1930 and it has subsequently become a popular medicament in the pulpotomy of primary teeth. Over the years, formocresol has been labeled as being mutagenic, toxic, and carcinogenic.³ The International Agency for Research on Cancer classified formaldehyde as carcinogenic for humans in June 2004.⁴

However, literature shows various materials like glutaraldehyde, calcium hydroxide, and ferric sulfate being used for pulpotomy with varying degrees of success. Each of the materials used has its limitations like toxicity, resorption, inflammation, and so on.

Mineral trioxide aggregate (MTA) was introduced in 1998 It is a very biocompatible and bioactive material with good sealing abilities and it promotes pulp tissue regeneration. However, the drawbacks include less antimicrobial efficacy, is not very economical, and a long setting time.⁵

Plant products from mother nature have been used since ancient times for the benefit of mankind. With the relevant increase in the incidence of antibiotics overuse and misuse in dentistry, phytomedicines with beneficial pharmacological properties are being tried.⁶

Information from an extensive literature review indicates that Myristica fragrans (Nutmeg) is a wonder drug from nature that has a broad spectrum of pharmacological effects. A single spice has the potential of curing a large number of diseases. Nutmeg and mace produced from M. fragrans are very effective against various animal and plant bacteria, fungi, harmful viruses, insects, and snails.⁷ The fruit and seed extracts of M. fragrans show hepatoprotective activity, anti-oxidant activity, memory enhancing activity, anti-cancer activity, aphrodisiac activity, anti-diabetic activity, anti-depressant activity, hypolipidemic and hypocholesterolemia effect, antimicrobial activity, anti-bacterial, anti-inflammatory, and anti-carcinogenic activities. They are well accepted because of the wealth of scientific literature supporting these effects.⁸

Considering these pharmacological benefits of M. fragrans, we chose it as the medicament for pulpotomy for the present study and compared it with recent medicament MTA showing a very high success rate.

Material and methods

Preparation of essential oil of Myristica fragrans and phytochemical analysis

The essential oil of M. fragrans was prepared by hydrodistillation using Clavenger’s method. Nutmeg kernels collected for the study were authenticated and certified at the Regional Institute of Ayurveda. Ground powder of Nutmeg (50g) was placed in a 500ml distillation flask with 2/3rd volume of distilled water. The heat was applied using a heating mantle and the volatile oil was carried with the steam to a cold condenser. The lighter oil rose to the top of the separator. This process was continued for about 12h until the oil inside the water ran out and stopped collecting. The volume of essential oils was determined from a calibrated trap.⁹ Phyto-chemical analysis of the extract was performed by gas chromatography-mass spectroscopy analysis (GC-MS) analysis at Auriga Research Private Limited, Bengaluru, under standard protocols.¹⁰

Clinical Procedure

The present study was conducted in the Department of Pediatric Dentistry of our Institution. The sample size was estimated using the G Power software v. 3.1.9.2. Considering the effect size to be measured (d) at 64%, the power of the study at 80%, and margin of the error at 5%, the total sample size needed was 40. Anticipating 20% attrition/loss to follow-up of the study patients, the total sample size was increased to 43, which was rounded off to 50. The study participants were further subdivided into two groups having 25 samples in each group; group A received M. fragrans and group B received MTA. (Table 1)

Subjects were included based on the selection criteria (Table 2). The study was initiated after the approval from the Institutional Ethics committee and Review Board and was registered with the Central Trial Registry of India (CTRI registration No. CTRI/2020/05/025148). Informed written consent from the parent/guardian and assent from the child was taken before recruiting.

After administering local anesthesia using 2% lignocaine hydrochloride with adrenaline 1:80000, rubber dam isolation was performed. Caries removal and un-roofing of the pulp chamber were carried out using a #330 high-speed carbide bur (SS White, USA) with air-water spray. The coronal pulp was amputated using a sterile sharp spoon excavator. The pulp chamber was flushed with normal saline. Hemostasis was achieved with a sterile cotton pellet wetted with saline applied for 4–5 mins, followed by placement of a mix of M. fragrans extract with zinc oxide powder (Deepak enterprises, Mumbai, India) into putty consistency and 2mm of this placed on the pulp stumps. The cavity was then sealed with glass ionomer cement (GC Corporation, Japan) for group A. In group B, MTA white powder (Angelus, Brazil) was mixed with distilled water following the manufacturer’s instructions into a putty consistency and a 2mm thick layer of this was placed on the pulp stumps. After the initial setting, the placement of glass ionomer cement was done.¹¹ Tooth preparation and stainless-steel crown (3M ESPE USA) placement was done in both group A and group B and post-operative radiographs were taken.¹²

Clinical and Radiographic follow-up

Follow-up was done both clinically and radiographically at 3, 6, and 12 months periods. A single calibrated outcome assessor, who was blinded to intervention allocation evaluated both the clinical signs and radiographic findings based on Zurn and Seale criteria (Table 3).¹¹

Results

Gas chromatography–mass spectrometry (GC-MS) analysis of the essential oil of M. fragrans detected over 20 chemical compounds. Volatile components of Nutmeg oil constituted 3 groups: hydrocarbons, oxygenated hydrocarbons, and aromatic hydrocarbons. Hydrocarbons were mostly terpene compounds like monoterpenes (sabinene and α–pinene); oxygenated hydrocarbons composed mainly of compounds that contain alcohol, ketone, or ester, terpene alcohol (terpinene4ol), and phenylpropenes (elemicin and safrole) and the aromatic compounds wee myristicin, isoeugenol, elemicin, and safrole which are responsible for distinctive aroma of nutmeg. This clinical study results showed no statistically significant difference in age, sex, and distribution of teeth between the two groups. All the teeth in our study were clinically asymptomatic (Table 4) throughout the follow-up periods of 3, 6, and 12 months. Radiographic images also did not show any pathological changes (Table 5).

Discussion

The present clinical study was conducted on 50 primary teeth and to prevent selection bias the teeth were randomly allocated into two groups using computerized randomization (random.org) ensuring that each patient had an equal chance of receiving one of the two medicaments. The procedure in all the selected teeth was performed with rubber dam isolation. Initial hemorrhage control after access opening was done by placing moist saline sterile cotton pellets on the radicular pulp stumps under slight pressure for 5 minutes. To ensure that the pulp was healthy, a 5-minute mark was chosen because this is the midpoint of the normal bleeding time range (1–9½ minutes).¹³ Teeth with insufficient hemostasis were not included in the study.

Stainless steel crown was cemented after the procedure to restore tooth anatomy and function, protect the remaining tooth structure, and provide a biological seal to prevent bacterial infiltration of the remaining vital pulp tissue. Farooq (2002) and Guelmann (2005) have shown that the final restoration of the pulpotomized tooth is significantly associated with the success of the pulpotomy. The stainless-steel crown is the restoration of choice or the “gold standard” because it protects the tooth from fracture (full crown coverage), minimizes the possibility of leakage, and ensures a biological seal.¹⁴

In this study, a single operator performed the procedure to avoid inter-operator discrepancy. The participants, outcome assessor, and statistician were blinded to the allocation.

Follow-up evaluation was done at 3, 6, and 12 months intervals by a well-trained evaluator unaware of medicament allocation. Masked post-treatment radiographs were used for evaluation to avoid bias that may arise from the difference in radioopacity of the medicaments used.¹⁵

In the present study, all the clinical parameters and radiographic parameters showed 100% success in both MTA and M. fragrans groups at 3, 6, and 12 months follow-up periods. The results of our study with MTA showed both clinical and radiographic success of 100% which are similar to studies done by Lin Lin (2020), Silva et al. (2019), and Vilella-Pastor (2021).^16,17,18

Mineral trioxide aggregate (MTA) showed a high effectiveness between 87.1%–100%.¹⁹ It has shown 100% clinical success in numerous studies and slightly less radiographic success rates. ¹⁹

Togaru et al. performed pulpotomy on 90 decayed primary molars with MTA and biodentine. The results of the clinical trial showed a 100% success rate with MTA at 9 months follow-up. At the 12-month follow-up, the clinical success rate decreased to 95.5% and only one tooth presented radiographic signs such as inter radicular radiolucency and periodontal ligament widening.²⁰ Bani et al. (2017) compared MTA and biodentine and evaluated the pulpotomized teeth at 12, 18, and 24 months follow-up. The results showed a clinical success rate of 96.8% for both medicaments while the success rate radiographically for the MTA group was 87.1%.²¹ According to Yildirim et al., the MTA group in a clinical trial of 140 teeth presented a high clinical success rate of 100% at 24 months follow-up and 93.9% of the teeth treated with MTA had no radiographic signs of failure.²²

The essential oil of M. fragrans has over 20 active biological constituents as shown by GC-MS analysis.^23,24

Antimicrobial activity

The compounds present in the essential oil of M. fragrans include myristicin, trimyristin, and myristic acid which show a different spectrum of activity against different microorganisms.²⁴ Groups such as COOH, COOR, NH2 , or SH have been suggested to be the functional groups responsible for the antibacterial activity which are present in myristic acid and trimyristin.²⁵ Antibacterial potential of myristicin may be due to the presence of an unsaturated side chain on the aromatic ring. The bacterial cell wall is the possible target of the compounds such as organic acids, esters, and aromatic compounds like myristicin, trimyristin, and myristic acid.^25,26 Macelignan is very effective against Streptococcus-mutans .²⁷ Shinohara et al. determined that malabaricone C isolated from Nutmeg irreversibly inhibited Arggingipain by 50% at a concentration of 0.7 μg/ml and selectively suppressed Porphyromonas gingivalis growth.²⁸ In vitro study by Yanti et al. showed a reduction in the growth of S. mutans, Actinomyces viscosus , and Streptococcus sanguinis in biofilms after 24 hours.²⁹ Shafiei et al. showed a significant decrease in the bacterial concentration of Aggregatibacter actinomycetemcomitans and P. gingivalis with the extracts of flesh, seed, and mace of M. fragrans.³⁰

Anti-inflammatory activity:

The essential oil of Nutmeg contains limonene, β-pinene, α-pinene, and sabinene which are known as 5-lipoxygenase inhibitors.³¹ Limonene is also a COX-2 selective inhibitor that has significant inhibitory effects on Prostaglandin E2 (PGE2) production. Terpene-4-ol which is 7.2% in seed oil and 23.6% in mace oil, suppresses the production of interleukin 1 beta (IL-1β) and Tumor necrosis factor- alpha (TNF-α), IL-8, IL-10, and PGE2. α-pinene reduces pro-inflammatory IL-6 in mouse colon but does not suppress IL-1β. Sabinene, eugenol, α-pinene inhibit TNF-α production.^5,8 Sabinene also inhibits IL-1β and IL-6.^31,32,33

Osteoblast proliferation stimulation activity:

M. fragrans compounds have a proven ability to stimulate osteoblastic differentiation. Machilin A present in M. fragrans stimulated osteoblast differentiation by activating p38 mitogen-activated protein (MAP) kinase. Other lignans like safrole, myristargenol, mace lignan, nectandrin B, mesodihydroguaiaretic acid, machilin F, licarin B, and licarin A from M. fragrans also showed similar actions and have anabolic activity in bone metabolism.³⁴

Analgesic activity:

Extracts from plants of the Myristicaceae family have been shown to inhibit phospholipase A2, which lowers the availability of arachidonic acid precursor for prostaglandin synthesis. The antinociceptive activity of Nutmeg extracts may be attributed to the inhibition of the release of IL-1β and IL-8 by cells or due to bradykinin and prostaglandin suppression.³⁵

M. fragrans is a resource of medicinally active compounds and has diverse pharmacological effects. The proven antimicrobial, anti-inflammatory, analgesic, and osteoblastic activity of Nutmeg may be beneficial for the healing of pulp which has shown success in this study. Conclusion This is the first clinical trial using M. fragrans in the pulpotomy of primary teeth. Results of this study showed that it is effective as a pulpotomy medicament and is comparable to MTA.

Limitations

Since this is the first clinical trial using M. fragrans as pulpotomy medicament, more studies with larger sample sizes, longer follow-up periods, diverse clinical and radiographic presentations, and well-controlled clinical conditions will be affirmative.

Conflict of interest

Nil