The European Academy of Paediatric Dentistry (EAPD), as outlined in its 2022 policy document, reported the high success rates of pulpotomy treatments, recommending them as an effective approach for managing deep caries in primary teeth. In the present study, we found that HA demonstrated similarly high success rates, both clinically and radiographically, during the long-term follow-up of pulpotomy treatments in human primary molar teeth. The policy document also highlighted that when employed as pulpotomy medicaments, FC, FS, and MTA exhibit comparable success rates; nevertheless, due to concerns regarding the potential toxic effects of FC, clinicians are recommended to use alternative medicaments like FS and MTA, which offer similar results [30]. In this study, FC and FS materials were used as the control group, and it stood out as the first to demonstrate the long-term clinical and radiographic follow-up of HA in pulpotomy treatment of human primary molars, comparing its success with that of FC and FS materials.
In 2018, Smaïl-Faugeron et al. published their analysis comprising 87 randomized controlled trials in the Cochrane Library. They reported that the most commonly used materials in pulpotomy treatment are MTA, CH, FC, and FS. When they analyzed five studies conducted with FC and FS materials, each having a follow-up period of at least 24 months, they reported no statistically significant differences among the materials in terms of clinical and radiographic findings [31]. In 2022, Tewari et al. conducted a study in which they examined 8 systematic reviews to compare the success rates of various pulpotomy medicaments and techniques. They reported that pulpotomy medicaments or techniques analyzed, excluding CH, had a success rate of over 80% across all time periods. Additionally, they reported a higher incidence of data deficiency in follow-up periods exceeding 18 months, predominantly associated with novel medicaments or techniques. When evaluating studies comparing FC and FS with a follow-up duration exceeding 18 months, no statistically significant difference was found among the materials in terms of clinical, radiographic, and overall success. However, it was noted that this evidence was of low certainty [32].
A most recent network meta-analysis study by Guo et al. included 43 randomized controlled trials, aiming to determine the comparative effectiveness of FC, FS, NaOCl, CH, MTA, BD, and laser pulpotomy treatments in primary molars. Upon analysis of studies reporting 12th-month success rates, it was observed that both clinically and radiographically, MTA, BD, and laser pulpotomy treatments were the most successful, with FC treatments having statistically comparable but superior values compared to FS treatments both clinically and radiographically. However, as a limitation of this network meta-analysis, it was emphasized that clinical and radiographic assessments were only conducted at 6th and 12th months, and the necessity to investigate long-term effectiveness when a sufficient number of appropriate studies are available was highlighted [33].
Huth et al. stated in their study that while no statistically significant superiority was observed after 3 years, FS treatments demonstrated a better outcome in terms of both clinical and overall success rates compared to FC treatments [34]. In the study conducted by Fernández et al., it was reported that the total success rates of DFC, MTA, FS, and NaOCl materials at the two-year follow-up were 97.5%, 96.5%, 98%, and 85%, respectively [35]. No statistically significant differences were reported between the groups in terms of clinical success during any follow-up period in the study conducted by Sushynski et al. The gray MTA group demonstrated 95%, 93%, 95%, and 95% radiographic success at the 6th, 12th, 18th, and 24th-month follow-ups, respectively, while the DFC group showed 85%, 81%, 78%, and 76% success at the same follow-up intervals [36]. In the study conducted by Sonmez et al., after a 2-year clinical and radiographic follow-up, it was reported that the number of radiographic findings was significantly higher than the number of clinical findings. At the end of 24 months, the total success rates for FC, FS, CH, and MTA were reported as 76.9%, 73.3%, 46.1%, and 66.6%, respectively, with no significant difference detected among the groups [37]. Noorollahian conducted pulpotomy treatments on 60 lower second primary molars and stated that the teeth treated with 1:5 DFC showed no clinical or radiographic findings at any follow-up period, while in the white MTA group, one tooth each demonstrated clinical success but radiographically showed furcal radiolucency at the 12th and 24th month follow-ups, with total success rates reported as 96.5% and 94.4%, respectively [38]. Erdem et al. declared that the overall success rates had decreased in all groups at the 24th-month follow-up compared to the 12th-month follow-up, reported as 96%, 88%, 88%, and 68% for the MTA, FS, FC, and ZOE groups, respectively. Statistically significant differences were observed between the MTA and ZOE groups at the 24th-month follow-up, while no significant differences were reported among the other groups [39]. Nematollahi et al. carried out partial pulpotomy treatments using MTA agent and pulpotomy treatments of primary molars using FC agent, and noted that in the FC treatment group, clinical and radiographic success rates were 100% at the 6th and 12th-month follow-ups, while at the 24th-month follow-up, clinical success remained at 100%, with radiographic and total success reported as 95.2% [40]. Petel et al. reported that in their pulpotomy treatments of primary molars using pure Portland cement and FC agents, teeth treated with pure Portland cement showed 100% success clinically and radiographically throughout all follow-up periods. At the 48-month follow-up, the FC treatment group demonstrated a total success rate of 91.1% [41].
In our previous study [29], only 2 teeth in the HA group and 1 tooth in the FS group had exfoliated by the end of the first year. In our long-term examination, although there was no statistically significant difference between them, it relatively increased in all three groups. Consistent with our findings, Sushynski et al. [36], Noorollahian [38], and Petel et al. [41], with a minimum follow-up period of 2 years, similarly reported a significant reduction in the number of initially treated teeth by the final follow-up period. The mean age of the children included in our study was slightly higher than that reported in the aforementioned studies, and due to the treatment of 67 first primary molars out of the 130 primary molars treated (51.5%) [29], we believe this ratio increased in alignment with the physiological exfoliation times of these teeth.
At the end of the follow-up period > 24 months in our study, the clinical success rates for HA, FC, and FS agents were 100%, 90%, and 94.7%, respectively, with no statistically significant difference found among the groups. In studies with a 24-month follow-up period comparing FC with FS, analyses conducted by Smaïl-Faugeron et al. [31] did not report statistically significant differences between the materials, and similarly, in studies with a follow-up duration exceeding 18 months, analyses conducted by Tewari et al. [32] also did not indicate any statistically significant differences between the materials. These results are in line with our study. Additionally, in their study, Huth et al. [34] reported that despite no statistically significant superiority was found after a 3-year follow-up, FS treatments exhibited clinically better as compared to FC treatments, similar to our study.
In our previous study [29], although no significant differences were observed among the materials in terms of clinical success rates at the 12th-month follow-up, a substantial decrease in clinical findings and a notable increase in clinical success rates were observed in all three groups from the 12th month onward until the control period of retrospective analysis. The increase in clinical success rates was determined to be 12.5% in the HA and FC groups, and 7.9% in the FS group. Additionally, in clinical success rates, only the HA group exhibited increases of 1.2% during the period from the 1st-month to the 1st-year follow-up, moreover, 12.5% from the 1st-year follow-up extending to the last retrospectively analyzed period. In contrast, the other two control groups showed a decrease from the 1st-month to the 1st-year follow-up [29].
Tenderness to percussion was the most prevalent clinical findings throughout the entire first-year follow-up periods [29]; in our retrospective analysis, this observation was only noted in the FS group, with no similar finding in the HA and FC groups, and mobility was the most common clinical finding (2 FC, 1 FS), and none in the HA group. Smaïl-Faugeron et al. [31] published their analysis in the Cochrane Library and clinical pain was reported in 4 studies with 2 cases for FC and none for FS at 24th-month. In Sushynski et al. study [36], 4 teeth in the DFC group showed abscess in a two-year period. Sonmez et al. [37] reported swelling findings in 2 teeth in the FC group in a two-year study, and in the study by Petel et al. [41], sinus tract formation was observed in 2 teeth in the FC group. In our RCT with a 1-year follow-up [29] and the present retrospective study, no teeth showed spontaneous, nocturnal pain and/or swelling.
The HA, FC, and FS groups demonstrated 81.5%, 80%, and 78.9% radiographic success rates, respectively, with no statistically significant differences found among the groups. When comparing the 1st-month with > 24 months retrospective control period, the radiographic success rate had an increase of 3.1% in the HA group, while a decrease of 7.5% and 16% in the FC and FS groups, respectively. The radiographic success rates were found to be lower than the clinical success rates, throughout all follow-up periods in our RCT [29] and the present retrospective study, excluding the 1st month for the FC and FS groups. We considered teeth with advanced physiological root resorption evident on radiographs as successful since they did not exhibit radiographic and clinical findings, similar to the study by Erdem et al. [39].
In our previous RCT [29], internal root resorption was the most prevalent radiographic finding throughout the entire first-year follow-up periods, with external root resorption being the second most common radiographic finding. In our long-term retrospective examination, internal root resorption was the most common pathological radiographic finding (Fig. 2), with furcal radiolucency being the second most prevalent; external root resorption was detected in only 2 teeth.
In our initial one-year follow-up RCT [29], PCO was not observed; during our extended retrospective analysis, PCO became a prevalent radiographic feature. In line with studies by Sonmez et al. [37], Noorollahian [38], and Nematollahi et al. [40], we did not categorize PCO as a radiographic failure. Similarly, Erdem et al. [39] reported PCO as the most non-pathologic, and internal root resorption as the most pathologic radiographic findings in their second-year follow-up study. On the other hand, Nematollahi et al. [40] reported a statistically significantly higher PCO in the FC group compared to teeth treated with MTA after a two-year follow-up. In our study, however, the FC group emerged as the group with the least observed PCO, and no statistically significant difference was detected among the materials used.
Numerous studies in the literature have investigated the efficacy of HA on hDPSCs [28, 42–44]. Umemura et al. declared that in HA-containing hDPSCs cultures, dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP-1) mRNA levels significantly increased over 24 hours, serving as odontoblastic differentiation markers, while HA also elevated DSPP and DMP-1 protein levels, leading to the conclusion that HA stimulates hDPSCs towards odontoblastic differentiation. When cultured with HA for one week, hDPSCs exhibited a concentration-dependent increase in alkaline phosphatase (ALP) protein levels, a mineralization indicator, with similar results obtained when measuring ALP amounts, suggesting that HA directs hDPSCs mineralization [42]. In their in vitro study investigating the efficacy of HA on pulp cells isolated from 10 third molars, Chen et al. stated that dental pulp cells treated with 2.0 mg/ml high molecular weight HA showed an early increase in ALP activity on the 3rd day, and additionally observed that dental pulp cells treated with HA for 3 days exhibited more and larger mineralized nodules compared to control group cells. They also reported the upregulation of crucial mineralization-associated molecules such as tissue-nonspecific alkaline phosphatase (ALP), bone morphogenetic protein 7 (BMP7), and type XV collagen (Col15A1) through the HA-CD44 pathway in vitro [43]. Schmidt et al. recently investigated the efficacy of HA molecules with low, medium, and high molecular weights on hDPSCs, and revealed that while long-term culture of hDPSCs with HA slightly influenced their characteristics positively, it preserved their stem cell characteristics and differentiation potentials. They also reported that none of the HA molecules they tested exhibited cytotoxic effects on hDPSCs [44]. According to studies investigating the efficacy of HA on hDPSCs, there are few in vivo and in vitro animal studies in the literature examining the efficacy of HA on dental pulp [45, 46]. Sasaki and Kawamata-Kido concluded that, as a result of the direct pulp capping treatment they conducted with HA on the molars of female Sprague-Dawley rats, HA may influence the environment appropriate for reparative dentin formation over existing dentin walls by means of the differentiation of undifferentiated mesenchymal cells into odontoblast-like cells [45]. In their in vitro study, Bogović et al. examined the efficiency of pulp capping preparations consisting of HA, CH, and dentine adhesive on cells isolated from Sprague-Dawley rats. They reported that HA was the most efficient and the least toxic material for direct pulp capping and could be advised for such treatments [46].
In light of the numerous experimental and animal studies mentioned above demonstrating the tissue regeneration supported by HA in dental pulp tissue or at the cellular level, our study also demonstrated promising results for HA in long-term success of human primary molar pulpotomy treatments. Mahfouz et al. also reported that the success of their pulpotomy treatments using a 1:1 mixture of HA gel and ZOE cement as pulpotomy material in human primary molars, compared to FC material, was comparable based on the 12-month follow-up. However, the long-term follow-up results regarding the success of the materials have not been published to the authors’ knowledge [47]. In our retrospective analysis, the teeth treated with HA maintained clinically healthy until the physiological exfoliation time, while radiographically, there was a decrease in the number of findings, particularly internal root resorption. These results could highlight the significant healing potential of HA on human primary molar pulps in the long term (Fig. 3). However, further histological studies and comprehensive, long-term follow-up human studies are needed to better understand the effect and mechanism of HA on dental pulp of human primary or permanent teeth.
One of the limitations of the study could be considered the difficulty in tracking data in long-term studies. In our previous RCT [29], only 2 dropouts were observed within the 1-year follow-up periods, whereas in our retrospective study with a maximum follow-up period of 30 months, data for 12 previously treated teeth (3 children) were not available both clinically and radiographically. One factor contributing to the data deficiency is the lack of regular follow-up appointments due to non-compliance among families. Additionally, the interference of the Covid-19 pandemic during these follow-up periods may have further impacted data collection. Furthermore, since the HA evaluated for long-term success in the present study is a commercially available product (Gengigel®Teething, Ricerfarma, Italy) with a 0.5% HA content, there is no data regarding the long-term effects of different HA concentrations on human primary molar pulp tissue in our study. To achieve the optimal effect of HA on pulp tissue, further research is needed to determine the ideal concentration and application time of HA.