Ongoing research persists in advancing endodontic dental materials for pediatric dentistry, specifically aiming to identify an optimal material for primary dentition. Although HA has not been incorporated into any commercially available endodontic material to date, it holds potential as a candidate. However, its utilization may necessitate supplementation with other dental materials due to inherent structural characteristics [23, 24]. In this study, various concentrations of HA were introduced into ZOE materials. A comprehensive analysis was then conducted to assess the effects of applying these HA-added ZOE materials to the dental pulp of rats. The study revealed histological success, particularly at lower concentration.

The rheological examination of HA-added materials is commonly undertaken due to their predominant application in gel form. In addition, researchers have explored mixed stiff HA/collagen and HA/gelatin scaffolds [9, 25]. There is no study evaluating the mechanical properties of a dental material containing hyaluronic acid, used as a base, similar to our research.

Per ISO 3107:2011, Type 2 ZOE materials must display a minimum compressive strength of 5 MPa. Manappallil suggested acceptable values, recommending 6 MPa to 28 MPa for Type 1 temporary cements and 5 MPa to 55 MPa for Type 2 bases and temporary restorations [26]. In a study by Karimy et al., commercially available ZOE showed compressive strengths ranging from 5.67 to 8.5 MPa, and Anderson et al. reported variations from 4.1 to 21.61 MPa [27, 28]. This study evaluated compressive strength values of 0.5% to 3% concentrations of HA added to two commercially available ZOE products. Both 0.5% HA-added IRM® or Kalzinol® and Gengigel Teething® (0.54% HA)-added IRM® or Kalzinol® met these standards, suggesting that concentrations exceeding 0.5% HA did not compromise ZOE material compressive strength.

In a study evaluating the mechanical properties of ZOE mixtures with varying sizes and shapes of zinc oxide particles, samples with a small average particle size, homogeneous distribution, and an oxygen-rich surface demonstrated the highest compressive strength and hardness. This performance was attributed to the large particle surface area, facilitating enhanced interfacial bonding [29]. Materials incorporating HA exhibited diverse viscoelastic properties, showing behavior ranging from dilute to entangled solutions based on HA concentration and molecular weight. Recent attempts have been made to enhance the mechanical stability of hydrogels [30]. In this study, a significant reduction in hardness was observed in both HA-added IRM® and Kalzinol® materials, potentially impacting material durability in clinical settings. Further studies are required to delve deeper into this aspect.

After in vitro experiments, 3% HA-added ZOE samples were excluded from the animal experiment group due to poor results in compressive strength and Vickers hardness tests. However, 1% HA-added ZOE was retained for experimentation. Overall, significant differences were observed in pulp vitality within the 7-day groups and in odontoblast layer continuity and pulp vitality within the 30-day groups.

Many studies have aimed to assess the impact of different materials on rat pulp histologically. Sharma et al. focused on the reparative effects of keratin hydrogel in rat molars after partial pulpotomy [21]. Several histological evaluation criteria, such as inflammatory cell response, necrosis, vitality, and mineralization, aligned with those in our study. Lopes et al. conducted a comparative analysis of MTA and ferric sulfate in pulpotomy, with MTA demonstrating superior histological features, higher interleukin-6 expression, and similar inflammatory cell counts [22]. Our study similarly investigated the inflammatory response, revealing no significant differences between the groups. Cengiz et al. evaluated sodium alendronate as an alternative for pulpotomy, showing similar pulp vitality preservation and hard tissue formation as calcium hydroxide, with no significant difference in inflammatory response and vascularization [20]. In our study, we considered the lateral exposure site for the evaluation of hard tissue formation, the roots for assessing canal obliteration, and the entire tooth in cross-sectional evaluations for other histological assessments.

There are numerous studies assessing the effects of diverse application of HA on dental pulp in animals. Bogovic’s investigation into the direct use of HA in pulp capping suggested a higher potential for reparative dentin formation in the HA group with superior cell viability and minimal apoptosis and necrosis [31]. Umemura et al. concluded that HA induces odontoblastic differentiation without affecting cell proliferation, utilizing high-molecular-weight HA for induced mineralization [32]. Chrepa et al. explored cell scaffolds containing Restylane (HA) or Matrigel, with Restylane demonstrating successful outcomes in terms of survival, mineralization, and odontoblastic activity [33]. Chen et al. evaluated high-molecular-weight HAs for regenerative pulp therapies, showcasing their potential as pulp capping and filling materials [23].

On the other hand, Ildes et al. examined the efficacy of HA as a pulpotomy medicament in human primary molars, comparing it with FC and ferric sulfate treatments over a 12-month period, both clinically and radiographically. Their findings revealed no statistically significant differences among the groups [11]. Similarly, Mahfouz et al. investigated a 1:1 mixture of high-molecular-weight HA gel and ZOE cement for pulpotomy in primary molars, finding its success comparable to FC pulpotomy after 12 months [34]. These in vivo short-term studies collectively emphasized the versatile and promising role of HA in various aspects of dental research, ranging from reparative dentin formation to odontoblastic differentiation and regenerative endodontic treatments.

In our study, the 7-day group showed that odontoblastic layer continuity and inflammation were lowest in 0.5% HA groups among the treatment groups, without any statistically significant difference. The 30-day group results showed that these parameters were still higher especially in Gengigel teething®-added group which has similar 0.5% HA content.

In the 7-day group, a noticeable reduction and rarefaction of odontoblasts were evident. This impact was observed in both coronal and root odontoblasts. Specifically, within the coronal pulp of the 7-day group, discernible bacterial clusters, signs of inflammation, congestion, and the presence of micro-abscesses were noted (Fig. 5a–d). Notably, pulp vitality exhibited a significant difference, underscoring the impact of the applied formulations on short-term outcomes. Furthermore, necrosis was identified in the coronal area of this group, leading to the loss of odontoblasts within the necrotic regions.

Congestion and inflammation examples in 7-day group: a (H&E, × 20), b (H&E, × 20), c (H&E, × 10), d (H&E, × 4); d dentin, p pulp, na necrosis area, if inflammation infiltration area, bv blood vessels, rd reparative dentin, m material, g gingiva

The 30-day group revealed distinctions in hard tissue formation, intracanal calcification, odontoblast layer continuity, and pulp vitality among the treatment groups. The combination of Gengigel Teething® with ZOE demonstrated superior outcomes in certain parameters, such as odontoblast layer continuity, suggesting a potential positive influence of the combination approach. However, the lack of statistical significance in some parameters implies the need for further investigation and potentially larger sample sizes to draw robust conclusions. In contrast, Palma et al. examined the effectiveness of lyophilized hydrogel HA scaffolds for dental pulp regeneration in immature dog teeth. The experimental groups comprised either blood clotting or two different formulations of a chitosan hydrogel as scaffolds. Despite the initial expectations, the addition of chitosan scaffolds to blood in regenerative procedures in dogs did not yield significant improvements in the formation of new mineralized tissues along the root canal walls or provide histological evidence of the regeneration of a pulp-dentin complex [35].

In the 30-day group, calcifications and pulp obliterations were observed (Fig. 6). Notably, the infection and necrosis detected in the coronal region extended to the root in certain cases. In addition, in some teeth, osteoclastic regenerative changes were noted, possibly attributed to a foreign body reaction related to perforation during the experimental procedure. These features were possibly due to the accelerated biological response in rat teeth as compared to humans [6, 36, 37].

Obliteration in 30-day group (H&E, × 10)

Reinforced zinc oxide eugenol cements remain popular due to their cost-effectiveness compared to calcium silicate-based materials. Therefore, considering its clinical preference and continued relevance, we selected ZOE for the in vivo animal experiment section of our research. However, direct application onto dental pulp has been linked to various toxic effects, including chronic inflammation and an increased risk of internal resorption [1].

For the past 50 years, studies have focused on tissue reactions following direct pulp capping, pulpotomy, and pulp exposure in rat teeth [8, 9, 20,21,22, 36,37,38]. Numerous studies have indicated that histological healing of the pulp in rat teeth treated with calcium hydroxide in pulp capping treatments is similar to that observed in human teeth [8, 39]. Therefore, researchers suggest that studies on rat teeth could be utilized as an appropriate model.

During the study, there were several factors complicating work on rat molars. Factors such as narrow mouth opening of rats, very small dimensions of teeth and pulps, and the anatomical position of teeth being far back in the oral cavity (long diastema) caused technical difficulties when working on rat teeth. Limitations of this study include the restricted number of animals and teeth used during the operation. We believe that increasing the number of rats could render some of the findings statistically significant. Conversely, the limited number of molar teeth in each rat prevented us from investigating different HA-added groups for each rat. We chose to study rat molars, despite the higher regenerative potential of incisors in rats. Molar teeth were preferred to observe realistic histological responses, as they possess a structure containing pulp tissue that closely resembles human molars in terms of anatomy, physiology, histology, and biology. However, it remains challenging to make direct comparisons with primary molars in humans, which have distinct physiological resorptive features.

While rapid inflammatory responses in rats raise questions about the optimal timing for realistic pulpal evaluation, future studies can adjust evaluation times and concentrations. Control groups, such as calcium hydroxide or MTA, may be employed for comparison. In addition, incorporating various counterstains or immunohistochemical markers can enhance the assessment of odontogenic, angiogenic, and neurogenic potential.

Furthermore, the success of the performed treatment is influenced not only by the base material placed on the pulp but also by the shear bond between the subsequently applied restoration and the base material. Studies have examined the shear bond of materials such as MTA and Biodentine with glass ionomer cement applied over them [40]. In the in vitro and in vivo experiments, alongside ZOE, groups such as MTA and Biodentine could have been selected, and the bond between them and GIC used as the restoration could have been evaluated. In vitro study limitations include challenges in determining the released amount of HA using chromatographic methods, measuring material setting times and pH levels, and employing imaging techniques to identify variations in surface morphology.

In previous studies, the direct effect of hyaluronic acid on the pulp was investigated solely through gel form preparations applied in pulp capping and pulpotomy treatments, with additional filling materials applied over the hyaluronic acid. A pulpotomy material containing hyaluronic acid has not been developed or tried. In this regard, our study bears the distinction of being a first. Confirmation of the designed material’s effectiveness not only eliminates a step in the multi-stage pulpotomy treatment, reducing time, but also suggests that HA, a tissue-friendly substance, could replace other agents, reducing toxicity risks. The data from this study may contribute to the development of a new pulpotomy material in the future.

In conclusion, our exploration for a more optimal concentration of HA added to ZOE and the results from in vivo experiments have shed light on the intricate dynamics of ZOE material incorporating HA. Significantly, our study stood out as the first inquiry into the influence of ZOE material containing HA on dental pulp, providing valuable insights into the short- and medium-term effects of various pulpotomy approaches. This contribution advances the broader investigation of dental pulp regeneration and treatment efficacy. The outcomes of our study suggest that HA holds promise as a material for pulpotomy and regenerative endodontic treatments. These findings indicate that the use of such materials in dental procedures, especially in pediatric cases, should be approached with careful consideration. Further research is essential to better understand the long-term clinical implications of these materials in dental applications.