This study aimed to develop an experimental pulp-capping material by incorporating a collagen–hydroxyapatite nanocomposite (cHAP) derived from fish scales, with chlorhexidine (CHX) added as an antimicrobial agent. The material was evaluated for microhardness, degree of monomer conversion (DC), and antibacterial activity.

The addition of cHAP, either alone or in combination with CHX, numerically increased the microhardness of the pulp-capping material. However, the increase was not statistically significant compared to the control group. This suggests that while cHAP may influence the physical properties of the material, its effect on microhardness is minimal. Consequently, the first hypothesis of this study, which proposed that the addition of cHAP would increase the microhardness of the material, was rejected. The degree of monomer conversion, which reflects polymerization efficiency, was unaffected by cHAP alone. However, a notable improvement was observed when cHAP was combined with CHX. This suggests that CHX may enhance polymerization of the material. Consequently, the second hypothesis, which posited that cHAP would improve DC, was also rejected, as the material's conversion was only enhanced when both cHAP and CHX were present. As for the antibacterial properties, the results showed that the incorporation of cHAP, either alone or with CHX, improved the antibacterial activity of the material, particularly against E. faecalis. The combination of cHAP with CHX demonstrated significant antibacterial activity against both E. faecalis and S. mutans, while cHAP alone showed efficacy only against E. faecalis. These findings support the third hypothesis, which proposed that the inclusion of cHAP, either with or without CHX, would enhance the antibacterial properties of the material. However, since the antibacterial effect was more pronounced when both components (cHAP and CHX) were combined, the hypothesis was partially accepted.

Previous studies have shown that fish scales contain various elements, including Na, P, Cl, and Ca [27, 28]. EDX analysis conducted in our study confirmed the presence of these elements. The EDX analysis also demonstrated that the Cl ratio of the groups containing CHX was high, as expected. According to studies involving XRD, CHX has a crystalline structure, showing peaks in the 2θ range of 15–30° [29], which was also observed in the current study. This consistency with prior findings confirms the successful inclusion of CHX in the material matrix. The XRD peaks associated with the fish scale-derived HAp overlapped with the peaks from the pulp-capping material used (TheraCal LC), indicating that the cHAP component did not introduce new crystalline phases that could disrupt the overall structure of the pulp-capping material. Instead, it contributed to a more integrated composite structure.

Similarly, the FT-IR spectra of the experimental groups containing both CHX and cHAP exhibited a pattern similar to that of the control group. However, distinct peaks were observed in the samples containing cHAP, particularly at specific wavelengths associated with collagen and hydroxyapatite components (e.g., peaks around 550 cm−1, 1014 cm−1, 1633 cm−1, and 2920 cm−1). These findings suggest that while the incorporation of CHX and cHAP did not dramatically alter the overall molecular structure, it influenced the characteristics of the composite material, as evidenced by shifts in the intensity of certain peaks. This is an important indication that the composite material retained its essential characteristics while incorporating antimicrobial properties from CHX.

Theracal LC, a light-curing resin-modified calcium silicate-based capping material, was utilized in this study. As with all resin-based materials, some residual monomers may remain due to incomplete polymerization [30]. These residual monomers can potentially cause pulp irritation, highlighting the importance of high polymerization rates in resin-based materials to minimize the presence of unreacted monomers. Ensuring efficient polymerization is crucial for the success of resin-based pulp-capping agents, as it directly impacts the material's biocompatibility and performance. Previous studies have reported DC values for resin-based pulp-capping materials ranging from 10 to 40%, depending on sample thickness [31, 32]. In the present study, the DC values of the groups ranged from 40 to 50%, indicating a more efficient polymerization process. The DC was significantly increased in the cHAP-CHX group. This improvement in monomer conversion may be explained by enhanced light absorption during the light-curing process, potentially due to an increase in the crystallinity of the material. The crystalline structure of hydroxyapatite and CHX may have increased the material's ability to absorb light more effectively during the light-curing process, leading to a more efficient polymerization by allowing greater light penetration. This result aligns with the previous findings [26]. The improved monomer conversion observed in the cHAP-CHX group suggests that the addition of cHAP and CHX did not impair the polymerization properties of the material. Instead, their combined effects may have enhanced the overall performance of the pulp-capping material, promoting better conversion of monomers into polymerized material and potentially reducing the occurrence of residual monomers that could cause pulp irritation.

The Vickers hardness test results revealed that the addition of CHX alone to the capping material significantly reduced its microhardness. This decrease may be attributed to the softer nature of CHX particles compared to the resin-based material. Chlorhexidine, as a relatively soft substance, likely did not contribute to strengthening the material matrix, leading to a reduction in the overall hardness. This observation is consistent with the previous research [26]. In contrast, the addition of cHAP, either alone or in combination with CHX, did not significantly affect the microhardness of the material. This could be due to the low percentage of functional components (cHAP and CHX) in the formulation, which may not have been sufficient to substantially affect the overall microhardness of the material. Additionally, the structural similarity between cHAP and the pulp-capping material may have contributed to the lack of significant change in hardness.

An ideal pulp capping material should exhibit antibacterial activity, as this is essential for protecting the dental pulp from infection and promoting healing. Calcium silicate-based materials are known to have better antimicrobial activity than calcium hydroxide-based materials [33]. Hydraulic calcium silicate-based materials (HCSMs) gradually release hydroxyl ions, leading to a sustained alkalinization, which contributes to prolonged antimicrobial action while minimizing abrupt pH shifts that could damage surrounding tissues. In contrast, Ca(OH)₂ raises the pH rapidly but loses its effectiveness over time as it dissolves or washes out [33, 34]. The antimicrobial effectiveness of each material varies depending on factors, such as the microorganism targeted, duration of exposure, and the amount of drug released. The antibacterial mechanism of hydraulic calcium silicate cements is attributed to the release of calcium ions (Ca2+) and hydroxyl ions (OH−) during hydration, which increases the material’s alkalinity [35, 36]. In the current study, the antibacterial activity of the materials was tested against S. mutans, a caries-associated bacterium, and E. feacalis, a microorganism commonly detected in endodontic infections. The collagen–hydroxyapatite nanocomposite derived from fish scales was incorporated into the material’s composition, considering that it could improve the antibacterial and mineralizing properties of the material by increasing the release of calcium and hydroxyl ions, thereby improving its alkanity.

Although previous studies have reported the antimicrobial effectiveness of TheraCal LC [31, 33], it did not create inhibition zones against E. faecalis and S. mutans in the present study, indicating a lack of antimicrobial activity of this material against these microorganisms. This discrepancy could be explained by differences in the antibacterial testing methods or the bacterial strains used. Our study also found that the addition of cHAP at a low concentration (1%) did not enhance the antibacterial activity of the material. However, when the cHAP concentration was increased, antibacterial activity against E. feacalis improved, though there was no significant effect on S. mutans. This finding supports the aforementioned argument that the antibacterial activity of a material may vary depending on the microorganism and concentration. The highest antibacterial activity was observed when CHX, a well-known broad-spectrum antibacterial agent, was added to the material alone or in combination with cHAP. Thus, an experimental pulp-capping material with antimicrobial activity against both E. feacalis and S. mutans was achieved.

Chlorhexidine diacetate is a soluble compound, and its incorporation into materials alone may compromise mechanical strength over time. However, when combined with cHAP, this potential drawback can be mitigated, enhancing the material’s overall properties. Based on the results of the present study, the resin-based pulp-capping material containing both CHX and cHAP nanostructure derived from fish scales demonstrated the most favorable balance of properties among the tested materials. Several studies have investigated the impact of chlorhexidine (CHX) on the degree of monomer conversion (DC) in dental materials, yielding varied results. One study examined the effect of CHX content on DC and E-modulus in experimental adhesive blends. The findings indicated that increasing concentrations of CHX had minimal adverse effects on DC but decreased the E-modulus by 27–48% compared to controls [37]. Another study evaluated the antimicrobial activity, DC, and Knoop hardness (KH) of experimental infiltrants incorporating CHX. The study concluded that DC was not affected by the monomer blend composition or CHX concentration [38]. A study investigated the effect of various CHX concentrations on the DC of an experimental adhesive resin. The results showed that 1% CHX did not significantly alter the DC, while 5% CHX led to a significant decrease in DC [39]. In brief, the impact of CHX on DC varies depending on its concentration and the specific dental material involved. While some studies report minimal effects, others indicate a reduction in DC with higher CHX concentrations. Therefore, incorporating CHX into dental materials requires careful consideration of its concentration to balance antimicrobial benefits with potential alterations in polymerization efficiency.

In summary, the findings of this study suggest that incorporating fish scale-derived cHAP and CHX into pulp-capping materials holds promise for developing novel materials with enhanced antibacterial effectiveness. However, as this was an in vitro study, several factors, such as saliva in the oral cavity, chewing forces, intraoral thermal and pH changes, and the effect of pulpal responses, were not accounted for. Additionally, in this study, the properties of a resin-based calcium silicate material were evaluated after the addition of cHAP and CHX, but no comparison was made with other commercially available pulp-capping materials (such as MTA or calcium hydroxide). Further in vivo and in vitro studies are warranted to validate these findings and confirm their clinical relevance.