Comparison of Eggshell-derived Calcium Hydroxyapatite and Biodentine as Indirect Pulp-capping Agents in Primary Molars: In Vivo Study

Abstract

Aim and background

This randomized clinical trial aims to evaluate and compare the efficacy of eggshell-derived calcium hydroxyapatite (EHA) and Biodentine as indirect pulp-capping materials in primary molars.

Trial design

This randomized controlled study aimed to evaluate and compare the effectiveness of calcium hydroxyapatite (HA) derived from eggshells (ESs) and Biodentine as indirect pulp-capping materials in primary molars of school-aged children. Among the 50 primary molars screened in cooperative children (4–8 years), selected from the Department of Pedodontics, Jaipur Dental College, 40 deep carious primary molars with a history of dull, reversible pain, or mild discomfort on chewing were included in the study.

Materials and methods

A total of 40 carious primary molars were selected and randomly allocated into two equal groups. Group I received EHA, while group II was treated with Biodentine, both used as indirect pulp-capping materials. The cavities were then restored using Type IX glass ionomer cement. Radiographic evaluations were performed to measure the thickness of reparative dentine at baseline, and at 3, 6, 9, and 12 months. The collected data were analyzed statistically using paired t-tests.

Results

At the end of 3, 6, 9, and 12 months, a statistically significant increase in dentin thickness with both ES and Biodentine (p-value ≤ 0.001) was found. Within the ES group, the thickness of dentin formed was 2.20 ± 0.50 at first 3 months, 2.24 ± 0.50 mm at 6 months, 2.26 ± 0.50 at 9 months, and 2.28 ± 0.50 at 12 months. In the Biodentine group, increment in dentin deposition was 2.18 ± 0.34 mm at the first 3 months, 2.24 ± 0.34 mm at 6 months, 2.24 ± 0.33 mm at 9 months, and 2.25 ± 0.33 mm at 12 months.

Conclusion

EHA yielded better results compared to Biodentine in the as-reparative dentin thickness formation. So, EHA can be suggested as a very cost-effective, efficient substitute to Biodentine as indirect pulp-capping agent in primary molars. However, additional studies are needed to confirm these findings and support their implementation in clinical practice.

How to cite this article

Patel RM, Manohar B, Khairwa A, et al. Comparison of Eggshell-derived Calcium Hydroxyapatite and Biodentine as Indirect Pulp-capping Agents in Primary Molars: In Vivo Study. Int J Clin Pediatr Dent 2026;19(4):444–450.

Introduction

The preservation of primary teeth plays a vital role in pediatric dentistry. Ensuring their integrity is essential, as the condition of primary dentition serves as an indicator of a child's general health. Premature loss of primary teeth is most commonly attributed to dental caries. The fundamental aim of pulp therapy in deciduous teeth is to maintain their integrity and function until natural exfoliation, a prerequisite for the optimal development of the facial and skeletal complex.¹

The dental pulp performs essential biological functions, including protection, nutrition, sensation, and dentin formation. Preserving its vitality remains a primary objective in restorative management of teeth affected by caries or trauma, as it is crucial for maintaining tooth integrity and long-term oral health.² Endodontic treatment in primary teeth is generally classified into two types: Vital pulp therapy (VPT) and root canal treatment (RCT). VPT aims to address reversible damage to the pulp and to sustain its vitality and functionality.³

Vital pulp therapy encompasses three main treatment modalities: Indirect pulp capping (IDPC), indicated for teeth with deep dentinal caries and reversible pulpitis; and direct pulp capping (DPC) or pulpotomy, which are employed in cases involving pulp exposure.³

For a pulp-capping material to be considered ideal, it must support hard-tissue formation, preserve pulp vitality and functionality, promote the formation of a dentin bridge, possess suitable physical properties, ensure strong adhesion to dentin, and be easy to handle during dental procedures.⁴

Several materials are utilized for IDPC, such as mineral trioxide aggregate (MTA), Portland cement (PC), calcium hydroxide (CH), resin-modified glass ionomer (RMGI), dentin bonding agents, and bioactive molecules, such as enamel matrix derivative (Emdogain) and bone morphogenetic proteins (BMPs), including transforming growth factor-β (TGF-β).³

Various materials have been used for pulp capping, with CH long established as the standard choice. However, following its application, the pulp tissue directly beneath it often becomes severely disrupted, resulting in a zone of tissue destruction. Further below, a milder chemical reaction occurs, leading to a zone of coagulation necrosis, which is thought to stimulate the remaining healthy pulp to initiate a healing response.⁵

Despite its widespread use, calcium hydroxide has faced criticism due to its tendency to degrade over time, the presence of tunnel defects in the dentin bridge it induces, and its inadequate sealing ability. These issues may lead to persistent inflammation and, ultimately, the failure to preserve pulp vitality. Additionally, its high pH (around 12.5) can cause surface tissue damage through liquefaction necrosis.⁵

Biodentine™ is a novel bioactive cement designed as a dentin substitute. It shares similar clinical applications and mechanisms with calcium hydroxide and, due to its dentin-like mechanical properties, can be used in both coronal and root regions.⁶

Hydroxyapatite (HA) is recognized for its superior biocompatibility and bioactivity, which has led to its widespread use in coating dental implants and artificial joints. In its nano form, HA (nano-HA) shows promise in remineralizing early-stage carious lesions when incorporated into products such as toothpastes and mouth rinses. Research suggests that a 4% (by weight) nano-HA suspension can effectively enhance remineralization in initial carious lesions.⁷

The chemical and physical properties of synthetic nano-HA closely resemble those of enamel apatite, rendering it a beneficial agent in oral health applications. Its high affinity for tooth surfaces enables strong adsorption onto enamel, enhancing its remineralizing potential.⁸

Eggshells (ESs) are a rich source of calcium in the form of carbonates and oxides, making them excellent source for HA synthesis. They represent a cost-effective, renewable, and sustainable source of biologically derived HA.⁹

Eggshell is an abundant calcium source, comprising roughly 94% calcium carbonate, 1% calcium phosphate, 1% magnesium carbonate, and 4% organic material. In addition to calcium, ES also contains trace amounts of elements such as fluoride and strontium, which, although present in small quantities, have beneficial effects on bone and dental metabolism.¹⁰

Materials and Methods

Trial Design and Participants

This randomized controlled trial evaluated the efficacy of eggshell-derived calcium hydroxyapatite (EHA) vs Biodentine as IDPC agents in primary molars. A total of 40 deep carious primary molars with dull, reversible pain, or mild discomfort on chewing were selected from children aged 4–8 years at Jaipur Dental College.

Inclusion Criteria

• Teeth with advanced carious lesions not extending to the pulp tissue.

• Teeth diagnosed with reversible pulpitis based on radiographic evaluation.

• Deep carious lesions involving more than two-thirds of the dentin, close to the pulp, and without any periapical or furcation radiolucency.

• Primary molars with occlusal caries classified as ICDAS 3–4 according to the ADA Caries Classification System.

Exclusion Criteria

• Teeth with a history of symptoms indicating irreversible pulpitis.

• Teeth with extensive carious lesions reaching the pulp tissue.

• Teeth presenting with periapical or furcation radiolucency on radiographs.

• Teeth near exfoliation.

• Patients with systemic medical conditions.

The examination results were categorized according to the ADA Caries Classification System of ICDAS criteria (Table 1).

Table 1:

ICDAS criteria

Code	Description
0	Sound tooth surface: No evidence of caries after 5 sec air drying
1	First visual change in enamel: Opacity or discoloration (white or brown) is visible at the entrance to the pit or fissure seen after prolonged air drying
2	Distinct visual change in enamel visible when wet, lesion must be visible when dry
3	Localized enamel breakdown (without clinical visual signs of dentinal involvement) seen when wet and after prolonged drying
4	Underlying dark shadow from dentine
5	Distinct cavity with visible dentine
6	Extensive (more than half the surface) distinct cavity with visible dentine

Sample Size

A total of 20 participants were allocated to each group, with the sample size estimated using G*Power software in accordance with literature.

Randomization

Out of 50 primary molars screened in cooperative children aged 4–8 years attending the Outpatient Department of Pediatric and Preventive Dentistry, 40 deep carious molars associated with a history of dull, reversible pain, or mild discomfort on mastication were selected for inclusion in the study. The participants were then randomly allocated into two groups. The study employed a single-blind randomized controlled trial design, with group I receiving EHA and group II treated with Biodentine.

The present study had been approved by the Ethical Committee of Jaipur Dental College, Jaipur, Rajasthan. Patient's parents were informed about the procedure and provided written consent.

EHA Preparation

Chicken ESs were collected, thoroughly washed with distilled water, and the membranes were subsequently removed. The shells were then boiled in a distilled water–ethanol mixture to eliminate impurities and residual organic matter, followed by drying at 100°C for 24 hours (Fig. 1A).

Figs 1A to G:

Eggshell-derived calcium hydroxyapatite preparation

The dried ESs were ground using a mortar and pistal to obtain a fine particulate powder (Fig. 1B). To prepare calcium hydroxide (Ca(OH)₂), 11 gm of the ES powder was dissolved in 209 mL of hydrochloric acid (HCl, PA, Neon, Brazil) at a concentration of 0.8 mol·L, under continuous stirring at 40°C for 1 hour (Fig. 1C).

Subsequently, 80 mL of 1.09 mol·L sodium hydroxide solution (NaOH, Macron, Brazil) was added to the mixture (Fig. 1D) to induce the precipitation of calcium hydroxide. The resulting precipitate was then filtered and dried in an oven at 100°C for 24 hours. The synthesis of HA was carried out using equal-volume precursor solutions containing 0.0279 mol of calcium hydroxide (Ca(OH)₂) and 0.0167 mol of phosphoric acid (H₃PO₄). The molar ratio of calcium to phosphorus was maintained at Ca/P = 1.67, and the pH of the reaction mixture was adjusted to 9 or 11 using a 1:1 ammonium hydroxide solution to optimize HA formation (Fig. 1E).

After the addition of the precursor solutions, the resulting mixture was aged at 80°C under continuous magnetic stirring for 2 hours. For the hydrothermal synthesis, the aged precipitate was transferred into a Teflon-lined reactor, which was then placed inside a stainless steel autoclave and heated in an oven at 180°C for 6 or 12 hours.

The final product obtained through this process was EHA (Fig. 1F). The synthesized samples were subsequently characterized and identified using Fourier transform infrared (FTIR) spectroscopy (Fig. 1G).

Clinical Procedure

Following the comprehensive diagnostic evaluation, the teeth were randomly allocated into two groups, and all IDPC procedures were carried out by a single operator.

The teeth were isolated with a rubber dam, and the soft, infected peripheral dentin was carefully removed using a sharp, sterilized hand excavator, preserving the hard dentin near the pulp to avoid exposure.

Excavation was stopped when the dentin over the pulp felt firm and could only be removed in small chips (leathery or firm dentin). Complete caries removal was confirmed by the absence of discoloration, no catch or tug-back, and smooth passage of a blunt probe or explorer.

In group I, EHA was mixed with saline at a 1:1 powder-to-liquid ratio to achieve a restorative consistency. The material was applied as a liner over the cavity floor using a cement carrier and condensed with a ball-ended condenser.

In group II, Biodentine was prepared according to the manufacturer's instructions: The liquid was dispensed into a capsule containing the powder, triturated in a designated amalgamator for 30 seconds, and the resulting mixture was placed on the cavity floor as a liner, condensed with a ball-ended condenser.

The final restoration was done with glass ionomer cement (GC Gold Label Type IX, GC Corporation, Tokyo, Japan). An immediate postoperative radiograph was taken.

The treated teeth were followed up radiographically at 3, 6, 9, and 12 months to evaluate the formation of reparative dentin. Radiographs were captured using a Vatech® RVG system with an AMS® dental X-ray machine, set at a tube current of 7 mA and 230 V, with an exposure time of 440 ms.

The digital radiographs were then magnified 5×, and the thickness of reparative dentin was calculated as the difference between the dentin thickness at baseline and at each follow-up. The resultant thickness was subsequently divided by 5 to determine the actual reparative dentin formed.

Statistical Methods

The data obtained were analyzed using the independent t-test for intergroup analysis and paired t-test for intra-group analysis.

Results

This study aimed to assess and compare the radiographic outcomes of EHA and Biodentine in primary teeth treated with indirect pulp therapy (IPT) over a 12-month period (Figs 2A to J).

Figs 2A to J:

Radiographic follow-up for reparative dentine formation in ES (A to E); Biodentine group (F to J) up to 12 months

Each group initially comprised 18 primary molars. In the EHA group, two teeth were excluded—one due to restoration loss and another due to patient attrition. Similarly, in the Biodentine group, two teeth were excluded because of loss to follow-up.

Intragroup analysis using an independent t-test demonstrated a statistically significant increase in dentin thickness from baseline to each follow-up interval (3, 6, 9, and 12 months) in both the EHA and Biodentine groups (p ≤ 0.0001 for all time points). The increase in dentin thickness reflects each material's ability to stimulate tertiary dentin formation over time.

In the EHA group, mean dentin thickness increased from 2.04 mm at baseline to 2.20 mm at 3 months, while in the Biodentine group, it increased from 2.04 to 2.18 mm over the same period. These results indicate that EHA demonstrated comparable performance to Biodentine in promoting dentin deposition during the initial phase.

At 6 months, the mean dentin thickness was 2.24 mm in the EHA group and 2.22 mm in the Biodentine group. This suggests continued dentin formation in both groups, although the rate of deposition was lower compared to the first 3 months.

By 9 and 12 months, the dentin thickness in the EHA group was 2.26 and 2.28 mm, respectively. In the Biodentine group, the values were 2.24 mm at 9 months and 2.25 mm at 12 months. These findings indicate only a minimal increase in dentin deposition between the 9- and 12-month intervals in both groups.

Within the EHA group, tertiary dentine deposited at the end of 3 months was 2.20 ± 0.50 and 2.24 ± 0.50 mm at 6 months, 2.26 ± 0.50 mm at 9 months, 2.28 ± 0.50 mm at 12 months. In Biodentine group too, tertiary dentine despite at the of 3 months was 2.18 ± 0.34 mm, and at the end of 6 months it was 2.28 ± 0.34 mm, 2.24 ± 0.33 mm at the end of 9 months, and 2.25 ± 0.33 mm at the end of 12 months (Table 2).

Table 2:

Comparison of reparative dentin formation between ES and biodentin

Groups		Postoperative	3 months	6 months	9 months	12 months	Significance
ES	Mean	2.04	2.20	2.24	2.26	2.28	HS
	SD	0.48	0.50	0.50	0.50	0.50
	p-value	0.0005	0.0001	0.0001	0.0001	0.0001
Biodentine	Mean	2.04	2.18	2.22	2.24	2.25	HS
	SD	0.38	0.34	0.34	0.33	0.33
	p-value	0.0001	0.0001	0.0001	0.0001	0.0001

These findings indicate that both EHA and Biodentine are effective in inducing tertiary dentin formation, with similar performance observed over the 12-month period (Fig. 3).

Fig. 3:

Comparative trends of reparative dentin formation at different time intervals in eggshell and biodentine

Discussion

One of the most prevalent childhood illnesses that can be prevented is dental caries, which can affect a person at any moment during their lifespan. Tooth loss and oral pain are primarily caused by it. In its early stages, caries can be stopped and possibly even reversed, but it frequently does not go away on its own. If correct care is not taken, caries can worsen until the tooth is destroyed.¹¹

Maintaining the pulp of primary teeth is crucial for enabling regular exfoliation periods. Furthermore, early primary tooth loss might have unfavorable effects, such as impaction, malocclusion, and differences in arch length.¹² The conventional management of deep carious lesions typically involved the complete removal of all affected dentin, a nonselective approach that often compromised the vitality of the pulp.

In recent years, there has been a shift toward minimally invasive, biologically oriented treatment strategies, with selective (partial) caries removal increasingly advocated to minimize the risk of pulp exposure.¹³

Treatment of reversible pulpal injuries in primary and permanent teeth with the goal of preserving pulp vitality and function is known as VPT. In addition to these, it is critical to maintain the integrity of the arch by keeping primary teeth intact until their natural exfoliation period. VPT encompasses two treatment modalities: DPC or pulpotomy for cases with pulp exposure, and indirect pulp treatment (IPT) for deep dentinal cavities.¹⁴

When treating deeply carious primary teeth, indirect pulp treatment is a less invasive method that does not require extensive carious dentin excavation.¹⁵ IDPC involves leaving a portion of affected dentin over a vital pulp to avoid exposure, followed by placement of a cavity liner or sealer and restoration. This partial caries removal approach significantly reduces the risk of pulp exposure during excavation.¹⁶

Hermann introduces calcium hydroxide in 1920. In endodontics, calcium hydroxide has been utilized extensively. With a pH of roughly 12.5, it is a potent alkaline material. Calcium hydroxide splits into calcium and hydroxyl ions in an aqueous solution.¹⁷

Since then, CaH has been utilized in a number of endodontic treatments, such as root perforation, apexification, intracanal medicine, pulp capping, pulpotomy, and several root canal sealants. CaH is used in endodontics for two main reasons: First, because of its antibacterial properties; second, because it has the ability to promote mineralization repair of the pulp and periapical tissues.¹⁸

Calcium ion (Ca) availability is a key factor influencing cellular activities that promote the formation of mineralized hard tissues. Recent trends in restorative dentistry emphasize minimally invasive and biologically based interventions, with selective (partial) caries removal gaining prominence due to its ability to preserve pulp vitality and reduce the risk of iatrogenic exposure. Historically, the management of deep carious lesions involved a more aggressive approach, characterized by nonselective (complete) excavation of carious dentine, which frequently led to pulp exposure and subsequent complications.

Ravi and Subramanyam hypothesized that the resorption of primary teeth following the application of calcium hydroxide (CaH) may be attributed to the induction of odontoclastogenesis. This process is proposed to result from a CaH-induced chronic inflammatory response, which may promote the fusion of macrophages into odontoclasts either directly, or indirectly through the stimulation of stromal cells, such as odontoblasts or fibroblasts. Furthermore, the authors suggested that the pulp tissue of deciduous teeth may possess an intrinsic predisposition for progenitor cells to differentiate into odontoclasts. The resorption may be further facilitated by the degradation of the predentin layer, which exposes the underlying mineralized dentin to odontoclastic activity, thereby increasing its susceptibility to resorption.¹⁹

A new pulp-capping agent that is less limited than calcium hydroxide is therefore required for primary teeth. Thus, as substitutes for calcium hydroxide, MTA, biodentine, and a few other materials are utilized.

Biodentine™, a tricalcium silicate cement, is promoted as a bioactive dentin substitute and is used in pulp capping, demonstrating the ability to stimulate hard-tissue formation while maintaining pulp health.²⁰

Biodentine has bioactive characteristics, promotes the regeneration of hard tissue, and does not cause any indicators of mild to severe pulp inflammation. They added that because HA crystals are forming at the surface and improving the material's sealing capacity, it can successfully preserve its marginal integrity. There is very little chance of microleakage because of its excellent sealing potential.²¹

It is biocompatible and promotes reactionary dentin via odontoblast stimulation and reparative dentin through cell differentiation.²²

Biodentine promotes early reparative dentin formation, possibly through the regulation of pulp cell TGF–β1 secretion.²³

In 2011, Laurent et al. studied the effects of Biodentine™ on human pulp cells, focusing on its capacity to induce TGF-β1 release and stimulate early dental pulp mineralization. Their findings showed that direct application of Biodentine™ to the pulp promoted early reparative dentin formation, likely by modulating TGF-β1 secretion by pulp cells.²³

Biodentine is an effective IDPC material with benefits over MTA, including quicker setting, enhanced strength and hardness, and greater cost-effectiveness.²⁴ In terms of mechanical strength, Biodentine is less soluble and provides a more secure seal. Biodentine is considerably easier to work with and takes a lot less time to set than other materials, such as MTA.

Samuel et al. performed an in vitro randomized controlled trial to assess the sealing ability of Biodentine™ and MTA in primary molars using scanning electron microscopy. The study found that, although all samples showed some microleakage, Biodentine™ exhibited significantly less microleakage than MTA. These results suggest that Biodentine™ may serve as a preferable alternative to MTA due to its superior sealing properties.²⁵

Although some materials such as MTA and Biodentine are being utilized in place of calcium hydroxide, they are not economically viable. Therefore, there is a need for a novel, less expensive IDPC agent that has the same advantageous qualities as all of these materials but less drawbacks.

Chicken ES powder contains a very high amount of bioavailable calcium, which is important for the remineralization of enamel. To create a pathogen-free, pure powder and raise its alkalinity, calcination was used.²⁶ Magnesium carbonate (MgCl₃), calcium carbonate (CaCO₃), and proteins (3.3%) and water (1.6%) make up the remaining constituents, which account for 37.3% of the ES's total weight. Calcium is the primary mineral component, accounting for 95.1% of the mineral content.²⁷

Li et al. (1998) reported that HA sol is a safe biomaterial for dental pulp treatment, promoting early dentin-bridge formation and exhibiting antibacterial activity. Its antibacterial properties can be effectively utilized by applying HA as a base in carious cavities to help eliminate residual cariogenic bacteria.²⁸

In 2007, Tin Oo et al. evaluated the antibacterial effects of locally produced HA. They found that HA inhibited the growth of Streptococcus mutans at 50 mg/mL and achieved complete inhibition at 200 mg/mL, demonstrating its effectiveness against this cariogenic bacterium. These results support HA's application in dental procedures, such as pulp capping or as a base/liner material.²⁸

The use of high purity reagents makes commercially available HAs costly. In order to synthesize HA, ES is utilized as a calcium precursor. The calcium source appears to have promise in creating nanocrystalline HA, which is crucial for replacing hard tissue due to its exceptional characteristics.²⁹

For this study, dentin thickness was evaluated using digital radiography at baseline, 3, 6, 9, and 12 months, with measurements taken from the highest point of the pulp chamber floor to the cementoenamel junction (CEJ).

In 2004, Krasner and Rankow conducted an in vitro study on extracted teeth and identified consistent reference points for locating the pulp chamber floor. They demonstrated that the floor of the pulp chamber is consistently positioned at the center of the tooth, at the level of the CEJ, and that the CEJ serves as the most reliable and repeatable landmark for determining pulp chamber location. Menon et al. (2016) conducted a clinical and radiographic comparison of indirect pulp treatment using light-cured calcium silicate and mineral trioxide aggregate in primary molars, in which dentin thickness was assessed at baseline, and again at 3 months and 6 months, using digitized radiographs with the cementoenamel junction and the highest point on the floor of the pulp chamber as reference points.³⁰

The reparative dentine thickness formed in EHA in the current study was 2.20 ± 0.50 in the first 3 months, 2.24 ± 0.50 at 6 months, 2.26 ± 0.50 at 9 months, and 2.28 ± 0.50 at the 12-month follow-up. The mean dentine thickness in Biodentine was 2.18 ± 0.34 in the first 3 months, 2.22 ± 0.34 at 6 months, 2.24 ± 0.33 at 9 months, and 2.25 ± 0.33 at 12 months.

Koubi et al. (2012) found that Biodentine is a safe, effective, and well-tolerated dentine substitute, capable of restoring posterior teeth for up to 6 months.³¹

Esmaeel et al. (2023) performed an in vitro study titled “Assessment of the Power of Chicken ES Powder in Remineralization of Induced Caries-Like Lesions in Primary Teeth.” The study demonstrated that chicken ES powder possesses notable remineralization potential, facilitating the deposition of calcium, phosphorus, and magnesium ions onto demineralized dentin surfaces.³² These findings suggest that chicken ES powder may serve as a potential dentine substitute in restorative dental applications.

In our present study, a comparison between the two dentine substitute materials, Biodentine and EHA revealed similar results and efficacy in promoting reparative dentine formation.

The superior reparative dentin formation observed with EHA may result from its high phosphate content, elevated pH, and abundant bioavailable calcium. Its biocompatibility, minimal disease transmission risk, ease of handling, autoclave stability, and widespread availability make EHA a viable option for IDPC in primary molars.³³

Conclusion

Calcium HA derived from ESs demonstrated comparable performance to Biodentine in terms of reparative dentin thickness formation. Therefore, EHA can be recommended as an effective and cost-efficient alternative to Biodentine for use as an IDPC agent in primary molars.

Orcid

Sunita Ranwa https://orcid.org/0009-0000-5099-9105