Abstract
Background
The tyrosine-rich amelogenin peptide (TRAP) is the main enzymatic hydrolysis product of amelogenin during enamel development. The TRAP peptide has been shown to promote the remineralization of early enamel caries in vitro. The aim of this crossover blinded and randomized study was to evaluate the impact of recombinant amelogenin peptide TRAP on the remineralization of initial enamel caries lesions in situ.
Methods
The binding capacity of recombinant amelogenin peptide TRAP to hydroxyapatite (HA) and a demineralized enamel surface was analyzed by a Langmuir adsorption isotherm experiment and confocal laser scanning microscopy (CLSM). Subsequently, the remineralization effect of recombinant amelogenin peptide TRAP on initial enamel carious lesions was studied using an in situ caries model. In this randomized, crossover, and blinded study, 12 volunteers wearing intraoral removable in-situ appliances with one bovine enamel block were divided into three groups: Group A received deionized water (DDW, negative control); Group B received recombinant amelogenin peptide TRAP (100 µg/ml); Group C received sodium fluoride (2 ppm). The study took place over three periods of 14 days each. At the end of each treatment period, the percentage of surface microhardness recovery (%SHR) and the integrated change in subsurface hardness (%ΔIHC) were evaluated.
Results
Recombinant amelogenin peptide TRAP adsorbed onto HA and demineralized enamel surfaces. After in situ analysis, all samples hardened significantly compared with baseline. Fluoride had a significantly greater effect than all other treatments. %SMHR was significantly greater than the DDW for the recombinant amelogenin peptide TRAP. Considering subsurface remineralization, treatment with recombinant amelogenin peptide TRAP and fluoride promoted increases of 6.73% and 15.68%, respectively, in %ΔIHC compared with DDW.
Conclusion
Recombinant amelogenin peptide TRAP can promote the remineralization of early enamel caries lesions in situ.
Clinical relevance
This study demonstrated the potential of recombinant amelogenin peptide TRAP to assist in the remineralization of artificial carious lesions in situ. The amelogenin peptide TRAP may be a promising, non-fluorinated, biomimetic, remineralization anti-caries agent.
Clinical trial number
Not applicable.
Keywords: Caries, Peptide TRAP, Binding ability, In situ, Remineralization
Introduction
The incidence of caries has declined in most developed countries; however, it remains a highly prevalent disease and represents an important public health issue among people worldwide [1]. A previous epidemiological study confirmed that early caries, or precavitated lesions, are more prevalent than cavitated tooth surfaces [2]. Therefore, the prevention of dental caries and the remineralization of early enamel or precavitated caries are primary challenges and targets of modern dentistry. Although fluoride, which reverses the progression of lesions by remineralization, has had a significant effect on the prevalence of caries, there is still a need to seek alternative, effective anti-caries reagents. Furthermore, fluoride is potentially harmful when overexposed [3].
Biomimetic remineralization mimics enamel biomineralization, whereby the organic matrix mediates the formation of hydroxyapatite crystals through protein–inorganic material interactions. Studies have demonstrated that enamel matrix proteins (EMPs) play a crucial role in the mineralization of enamel. Amelogenin accounts for approximately 90% of all EMPs. Full-length amelogenin can be separated into three regions: the N terminus, containing 45 amino acids and a phosphate group; the central area, which is hydrophobic and mainly composed of Xxx-Yyy-Pro repeats; and the C terminus, which is charged, hydrophilic, and composed of 11 amino acids [4]. In previous studies, the key amino acids in the enamel protein sequence have been identified, which are particularly vital for crystal growth. Fan et al. reported that using an in vitro model of initial enamel caries, a hydrogel that releases amelogenin, has potential remineralizing efficacy [5]. Le Norcy et al. found that the Leucine-rich amelogenin peptide (LRAP) can self-assemble into nanospheres, which further aggregate and grow into chain structures to regulate the formation of HA crystals, thus promoting the remineralization of caries lesions [6]. We previously found that a recombinant amelogenin peptide composed of the N- and C-termini of porcine amelogenin guides the remineralization of initial enamel caries in vitro [7]. This study provided evidence suggesting that the peptide may act as a calcium ion carrier as well as a regulatory factor that directs the formation of ordered arrays of crystals. Furthermore, we have found that the solitary phosphate group at the N-terminus of recombinant amelogenin helps recombinant amelogenin to promote the remineralization process of initial enamel caries [8].
In addition to full-length amelogenin, the enamel matrix during the development stage mostly includes multitudinous enzymolysis peptide fragments. Among them, the tyrosine-rich amelogenin peptide TRAP is the main enzymatic hydrolysis product of amelogenin during enamel development [9]. TRAP, consisting of 45 amino acids at the N-terminus, contains a lectin-binding motif “PYTSYGYEPMGGW,” which may function in determining the direction of the “nanosphere” [10]. The “A-domain” sequence located in TRAP is directly involved in the self-assembly of amelogenin [11]. In addition, the only phosphorylation site (ser-16) of amelogenin is located in TRAP, which can interact with minerals, such as calcium and phosphorus during the enamel formation period, preventing premature deposition of amorphous calcium phosphate (ACP) and providing conditions for calcium ions to enter the interior of caries lesions [6]. Therefore, we speculated that the peptide TRAP, as a critical functional region of amelogenin, may assist in the remineralization of early enamel caries, which was proven in vitro [12].
In situ models have received extensive recognition as a bridge between spontaneous clinical trials and the restricted laboratory situation [13]. Using in situ models, it is possible to investigate the fundamental aespects of the caries process and more applicable issues, such as the effects of fluoride or other remineralizing agents in preventing caries in human subjects, without causing caries in the natural dentition. Thus, it is necessary to evaluate the validity of new non-fluoride anti-carious agents.
The goal of this crossover, blinded, and randomized study was to evaluate the effect of recombinant amelogenin peptide TRAP on the remineralization of initial enamel carious lesions in an in situ model.
Materials and methods
Peptide preparation
The peptide TRAP was produced by Synpeptide Co., Ltd. (Nanjing, China), synthesized using conventional solid-phase peptide and purified and identified using high performance liquid chromatograph and mass spectrometer [7]. These amino acid sequences (TRAP) corresponded to those of amelogenin P173. The synthesized recombinant TRAP is shown in Fig. 1.
Fig. 1.
Full length amelogenin P173 and the peptide TRAP amino acid sequence
Binding capacity of the TRAP peptide to HA
The peptide TRAP was dissolved in 10 mM HEPES (pH 7.4) and mixed with 5 mg of HA powder (specific surface area of 46.872 m2/g) to obtain 10–300 µg/mL with a total volume of 1 mL. After revolving at 37 °C overnight, the solution was centrifuged at 14,000 rpm for 10 min to obtain the supernatant. The concentrations of TRAP in the solution before and after incubation with HA powder were assessed using a micro BCA protein assay kit (Jimi Co., Ltd., Zhengzhou, China). The adsorption amount of TRAP on the HA crystals per square meter was obtained. Then, according to the Langmuir formula (1) [14], a linear adsorption isotherm was generated. The maximum amount of sorption locus of the peptide TRAP on the unit HA surface area and the affinity for the HA sorption locus were obtained from the optimum fitting line.
 |
1 |
Where Ceq is the concentration of peptide TRAP in the supernatant after the binding reaction with HA (mM), Q is the number of peptide TRAPs bound to the HA per unit surface area (µmol/m2), N is the maximum amount of sorption locus per unit HA surface area for peptide TRAP (µmol/m2), and K is the affinity of peptide TRAP for the HA adsorption site (mL/µmol).
Binding capacity of FITC-labeled TRAP to the enamel surface
Fresh extracted bovine incisors were selected to make bovine enamel slices without caries, cracks, or enamel deformities (approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, approval number 2021-KY-1050-002) and polished to ~ 100 μm using silicon carbide sandpaper (400, 600, 800, 1000, 1200, 1500, 1800, 2000, 2500, 3000, and 4000 #) under flowing water. The enamel blocks were demineralized with 37% phosphoric acid for 45 s, washed with deionized water, and sonicated for 5 min [15, 16].
Fluorescein isothiocyanate (FITC)-labeled TRAP was synthesized by Synpeptide Co.,Ltd. (Nanjing, China). We removed the peptide FITC-TRAP powder (1 mg/tube) from a refrigerator of − 80 °C and added three drops of dimethyl sulfoxide (DMSO), before ultrasonicating for 1 min. We then added 10 mL of deionized water to prepare a concentration of 100 µg/mL peptide FITC-TRAP solution, which was visible to the naked eye as a light yellow liquid.
Approximately 100 µL of deionized water and FITC-TRAP solution were dropped onto the surfaces of normal and acid-etched bovine enamel slices, respectively. Then, 30 min later, the enamel slices were rinsed three times with deionized water, dried naturally, and observed with a CLSM (Olympus, Tokyo, Japan) at a wave length of 488 nm [15, 16].
In situ remineralization of early enamel caries
This crossover blinded and randomized in situ study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (2021-KY-1050-002). The study consisted of three experimental phases, each lasting 14 days, with a 7-day washout period. Volunteers wore removable in situ models made of acrylic resin containing pre-demineralized bovine enamel specimens. The surface hardness (SH) was measured before and after demineralization and after in situ remineralization experiments, and the cross-section hardness (CSH) was measured after in situ remineralization.
Sample preparation
Bovine incisors were used (approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, approval number: 2021-KY-1050-002). The specimens were prepared as described previously by Chu et al. [17]. The enamel blocks were sonicated for 10 min, exposed to ethylene oxide vapor for 12 h, and tested for normal enamel surface hardness (SH0) using a microhardness tester (HV, ShiDai, HVS-1000) with a Vickers diamond indenter under a 100-g load for 15 s. Three equidistant sites, each 100 μm apart, were measured in the center of the enamel blocks, and the average value was taken for analysis. Finally, specimens with an average of 270-350VNH.
Formation of artificial caries lesions
Each bovine enamel block had only the enamel surface window exposed (4 mm × 4 mm), with two coats of acid-resistant nail polish applied to all other surfaces.Initial enamel caries lesions were obtained in the demineralization solution [18], which contained 50 mM acetic acid (pH = 4.5), 2.2 mM Ca(NO3)2, 2.2 mM KH2PO4, 5.0 mM NaN3, and 0.5 ppm NaF, at 37 °C for 3 days under continuous low-speed magnetic stirring (100 rpm). The fresh demineralization solution was replaced daily. Enamel samples were rinsed with running water after demineralization. The measurement method for SH1 after demineralization was the same as previously described. Ultimately, specimens were selected with an average of 100-180VNH.
Intraoral phase
Twelve healthy volunteers aged 18–42 years were selected based on the inclusion and exclusion criteria [19]. The inclusion criteria were as follows: having at least 22 teeth, a previous history of caries but no clinically active caries, no history of periodontal disease or other oral pathology, and a healthy, unrepaired buccal surface of the mandibular first molar. The mean saliva flow velocity unstimulated > 0.2 mL/min, and the average stimulated flow rate was ≥ 1.0 mL/min. The exclusion criteria were as follows: a history of systemic diseases that may impact dental health (e.g., diabetes, HIV/AIDS, or heart disease, or those requiring antibiotics that are likely to influence the oral flora or salivary flow rate); and the use of fluoride toothpaste ≥ 5000 ppm within the past 6 months. The volunteers lived in a city with a fluoridated water supply (0.17 mg/L–0.49 mg/L).
A movable acrylic buccal in situ appliance was constructed for each participant with a central trough (5 × 5 mm) in which a sample of bovine tooth enamel was embedded using edible wax. The enamel surface was flush with the appliance and in normal contact with saliva.
In the oral stage, the experimental process was divided into three stages, and corresponding enamel blocks were randomly distributed to three remineralization treatment groups (12 blocks/group): Group A, consisting of deionized water; Group B, consisting of recombinant amelogenin peptide TRAP (100 µg/mL); Group C, consisting of sodium fluoride (2 ppm). The volunteers were stochastically distributed throughout the three experimental phases.
We followed the experimental regime reported by Oliveira [20] (Fig. 2). Before the experiment begins, participants signed informed consent forms and received comprehensive execution training. During the experiment, participants were required to use fluoride-free toothpaste for regular oral hygiene, not use toothbrushes to brush the enamel blocks inside the appliances, and wear the appliances from 8:00 am to 8:00 pm. The in situ appliances were removed during meals, oral hygiene, and evening rest, and were stored in a humidified environment. After each meal, 1.5 mL of the experimental reagent was extracted with a syringe and dropped onto the enamel block in the appliance, where it was left for 3 min before placing back into the mouth, three times a day. Participants were informed that they should not take down the appliances for at least 30 min after each treatment. The enamel specimens were removed after each stage and stored in a humidified environment before analysis. The washout period was 7 days between each stage. The in situ remineralization study was completed after the completion of the three stages.
Fig. 2.
The experimental regime
Data analysis
Graphs were prepared using GraphPad Prism, version 8.0 (GraphPad Software, Inc., San Diego, USA). The software IBM SPSS 21.0 (IBM, Chicago, IL, USA) was used for statistical analysis. All data were tested for assumptions of normal distribution and equality of variances. Surface and subsurface hardness data were statistically analyzed using ANOVA and Kruskal–Wallis/Bonferroni tests with a significance level of 5%.
Surface hardness analysis
The measurement method for SH2 after in situ remineralization was the same as previously described. Vickers surface hardness (SH) tests were performed at three stages (SH0, sound enamel; SH1, after preformed artificial enamel caries lesions; SH2, after remineralization treatment). The percentage of surface microhardness recovery (SHR%) was calculated using the following formula: SHR% = (SH2–SH1) / (SH1–SH0) × 100%.
Cross-sectional hardness analysis
After surface microhardness measurements, 36 enamel blocks were cut longitudinally along the central line, half of which were embedded in acrylic resin, and the cross-sections were exposed. The enamel blocks were polished as previously described and tested for cross-sectional hardness (CSH) using a microhardness tester (HV, ShiDai, HVS-1000) with a Knoop diamond indenter under a 100-g load for 15 s. Two rows of indentations were made in the center of the cross-section (each row had seven test sites, corresponding to seven depths below the surface), with a distance of 100 μm, and the hardness was tested at distances of 10, 30, 50, 70, 90, 110, and 220 μm from the enamel surface. Two sites at each depth were measured and averaged. The integrated hardness (IH, KNH × µm) of the subsurface hardness of the enamel samples from each remineralization treatment group was calculated using the trapezoidal rule. The integrated change in subsurface hardness (IHC%) was calculated using the following equation: ΔIHC% = (IH treatment– IH control) / IH treatment × 100% [20]. IH treatment includes the peptide TRAP group and the NaF group, while the IH control is the DDW group.
Results
Adsorption ability of peptide TRAP on HA and bovine enamel surfaces
The shape of the isotherm adsorption line of the polypeptide TRAP on HA indicates that at a low Ceq value, the Q value continues to increase until it finally shows a transition to the platform. This isotherm can be described using the Langmuir isotherm adsorption model (Fig. 3). The affinity K of polypeptide TRAP for the HA adsorption site is 259.67 mL/µmol, and the maximum number of sorption loci N per square meter of HA combined with peptide TRAP is 11.12 × 10–2 µmol/m2. CLSM testing revealed no fluorescence images (Fig. 4A1 and A2) on the normal and demineralized bovine enamel samples in the DDW control group; sparse fluorescence spots were observed on the normal bovine tooth enamel specimens in the peptide FITC-TRAP experimental group (Fig. 4B1), while large and distinct fluorescence distributions were observed in the demineralized bovine tooth enamel specimens (Fig. 4B2).
Fig. 3.
Linear adsorption isotherms of peptide TRAP. The maxim number of adsorption sites per unit of HA surface area (N) and the affinity of peptide molecules for HA adsorption sites (K) and were calculated. R2 is the correlation coefficient obtained for linear adsorption isotherms
Fig. 4.
CLSM images of DDW-treated normal and demineralized bovine enamel surface (A1, A2), FITC-labelled TRAP-treated normal and demineralized bovine enamel surface (B1, B2)
In situ remineralization of early enamel caries
All participants completed the study with 100% completeness, and no adverse events were reported. The microhardness values of the bovine enamel samples before demineralization, after demineralization, and after in situ remineralization were measured to determine changes in mineral content. Table 1 shows that there was no prominent variation in SH before and after demineralization between the groups (P > 0.05), but there was a prominent variation in SH after remineralization, before and after demineralization, and before and after remineralization in each group (all P < 0.05). SHR% of the DDW group was 10.81, that of the peptide TRAP group was 30.37, and that of the NaF group was 48.57. The peptide TRAP group was inferior to the NaF group but superior to the DDW group. Figure 5(a–c) shows that the CSH at 30, 50, 70, and 90 μm of the enamel subsurface after in situ remineralization was significantly different between the groups (P < 0.05). Table 2 shows that the IH among groups was dominantly different (P < 0.05). The IHs of the DDW, peptide TRAP, and NaF groups were 40,553, 43462.5, and 48,469, respectively. The IHs of the peptide TRAP and NaF groups were dominantly superior to those of the DDW group. Treatment with peptide TRAP and NaF reduced lesion size by 6.73% and 15.68%, respectively, compared with the DDW group.
Table 1.
The SH for all treatment groupsin different phases of the study and SHR%
| Groups | SH0 | SH1 | SH2 | SHR% |
|---|---|---|---|---|
| DDW | 311.34 ± 23.54 | 131.93 ± 15.24* | 151.27 ± 15.02a*# |
10.81a (7.35–13.71) |
| TRAP | 311.94 ± 24.57 | 139.49 ± 22.03* | 191.14 ± 17.83b*# |
30.37b (20.88–40.58) |
| NaF | 314.16 ± 23.08 | 140.23 ± 15.93* | 228.95 ± 15.14c*# |
48.57c (37.77–72.15) |
| P | 0.954 | 0.465 | 0.000 | 0.000 |
The results are expressed as mean (standard error of the mean) and median (CI, minimum/maximum), with different letters in the same column indicating significant differences between them. The (*) and (#) in the same row indicate significant differences compared to before and after demineralization, respectively. Significance was determined using ANOVA and Kruskal-Wallis
/Bonferroni tests (P < 0.05)
Fig. 5.
KHN (median and mean) at different depths. (*) denotes significant differences between the experimental and control groups. Significance was determined using ANOVA and Kruskal-Wallis/Bonferroni tests (P< 0.05)
Table 2.
Integrated hardness (IH, KHN x µm) and integrated change of subsurface hardness (ΔIHC%)
| Groups | IH | △IHC% |
|---|---|---|
| DDW |
40,553a (39707–42579) |
- |
| TRAP |
43462.5b (41359–46147) |
6.73 ± 3.29a |
| NaF |
48,469c (45301–51290) |
15.68 ± 3.66b |
The results are expressed as mean (standard error of the mean) and median (CI, minimum/maximum), with different letters in the same column indicating significant differences between them. Significance was determined using ANOVA and Kruskal-Wallis/Bonferroni tests (p < 0.05)
Discussion
This in situ study (funded by the National Natural Science Foundation of China, No. U2004108) aimed to test the hypothesis that recombinant amelogenin TRAP induces biomimetic remineralization in early enamel caries. The study was based on our prior research exploring the ability of the recombinant amelogenin peptide TRAP to promote remineralization of early enamel caries in vitro [12]. We previously confirmed the potential of the recombinant amelogenin peptide TRAP as a key functional motif of the amelogenin protein for enamel remineralization. It was also confirmed that the peptide TRAP modulates the formation of hydroxyapatite in eroded enamel and that the newly formed crystals resemble natural enamel crystals promoting the remineralization of enamel [21]. However, previous experiments were conducted in vitro, and their impact on the remineralization of early enamel caries needs to be further verified in situ studies and eventually in clinical tests.
Langmuir isotherm adsorption experiments were conducted to analyze the adsorption capacity of the peptide TRAP on HA. The results showed that the binding between the recombinant amelogenin peptide TRAP and HA can be conveyed by a good relevance coefficient (R2 = 0.9926), indicating that the interaction between the recombinant amelogenin peptide TRAP and the HA crystal surface is a unit of interaction with a certain concentration, capacity, and affinity. CLSM technology was used to verify the binding ability of the peptide TRAP to enamel, which showed that the peptide TRAP was easily adsorbed onto enamel surfaces, particularly on the surface of demineralized enamel. Studies have shown that strong affinity with the matrix is one of the most important features of this material for promoting enamel remineralization. Previous studies have also shown that the recombinant amelogenin peptide TRAP can bind to calcium ions [12]. Therefore, it can be inferred that the recombinant amelogenin peptide TRAP (composed of N- and C-terminals) can react with calcium and phosphorus ions, bind to the surface of demineralized enamel, penetrate into the damaged interior of the enamel through the microporous channels formed on the enamel surface during demineralization, and adhere to the hydroxyapatite crystal to promote the redeposition of calcium and phosphorus ions. The undersurface enamel lesions are thus repaired by remineralization. The ability of the recombinant TRAP to bind with hydroxyapatite and demineralized enamel surfaces provides a theoretical basis for in situ experiments.
Next, we explored the potential remineralization effect of the peptide TRAP on initial caries lesions in situ. The in situ model plays a significant role in evaluating the effectiveness of caries prevention reagents. The in situ model produced in this study includes a pre-formed demineralized bovine enamel sample in vitro, where the enamel surface is not covered with nylon gauze with the appliance. The surface of the enamel sample is smooth, which is not conducive to plaque accumulation and reduces factors that affect the remineralization process. Therefore, the non-gauze model may be preferable for screening new remineralization reagents [22]. Aside from the similarity in chemical ingredients and physical characteristics between bovine and human enamel [23], bovine enamel is easier to acquire, providing a larger-scale surface area that is a more consistent enamel thickness than human enamel [20]. Therefore, bovine enamel was selected as a replacement for human tooth enamel in this study. The microhardness technology was used to analyze changes in mineral content. Research has shown that data obtained from demineralization and remineralization of enamel were strongly correlated with using transverse microradiographyand SH/CSH technologies [24, 25]. Microhardness technology has the advantages of effectiveness, sensitivity, and ease of operation and has been widely used in situ studies for the evaluation of demineralization and remineralization of enamel samples.
In this study, the TRAP and NaF peptides were used as treatment reagents, whereas DDW was used as a negative control reagent. After in situ remineralization treatment, the SH in all groups increased, indicating that saliva can improve the degree of enamel demineralization, meaning that it has the ability to remineralize. Research has shown that when saliva is used to treat caries lesions, the trend of enamel remineralization increases over time [26]. Analysis of SHR% showed that SHR% of enamel samples treated with the peptide TRAP was prominently superior to that treated with DDW, despite being slightly lower than that of NaF, consistent with the results of previous in vitro studies. CSH analysis revealed a change in subsurface hardness in samples treated with DDW, the peptide TRAP, and NaF. The samples in the peptide TRAP and NaF groups were harder than those in the DDW group at depths of 30, 50, 70, and 90 μm. Moreover, analysis of ΔIHC% showed that compared with DDW, treatment with the peptide TRAP and NaF reduced enamel lesions by 6.73% and 15.68%, respectively. ΔIHC% of enamel samples treated with the peptide TRAP was lower than that of those treated with NaF, indicating that the remineralization ability of peptide TRAP for subsurface enamel caries lesions was slightly weaker than that of NaF, consistent with previous in vitro micro-CT studies [12]. By contrast, with NaF, while it is well-known that fluoride can debase the frequency of dental caries, long-term, inordinate exposure to fluoride will detrimentally harm tooth development by interacting with growing ameloblasts or suspending the nucleation of apatite crystals [27]. Therefore, the peptide TRAP can be developed as a promising substitute for fluoride-based strategies.
At present, in-situ studies play an important role in the evaluation of the effect of anti-caries preparations. Despite the strengths of this study, it has some limitations. First, we selected bovine teeth as the study sample, which still exhibit some differences in physical and chemical characteristics compared with human teeth. Second, the factors influencing enamel demineralization and remineralization are very complex. Finally, the number of subjects in this in-situ study was limited, and its representativeness is still controversial. Therefore, further studies, including animal experiments and clinical trials, are necessary to verify the effect of the recombinant amelogenin polypeptide TRAP on the prevention and treatment of caries.
Conclusion
Based on the results of this in situ study, we demonstrated that the recombinant amelogenin peptide TRAP can affect the net rehardening of artificial carious lesions. Although not as effective as fluoride, the amelogenin peptide TRAP may still be a promising non-fluorinated biomimetic remineralization anti-caries agent.
Acknowledgements
We thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript.
Author contributions
JC, QB, YL, MW, DM and YL participated in the study concept and experimental design. JC has been involved in funding acquisition. QB, YL, MW and YL have been involved in acquisition of bovine incisors. JC and QB drafted the manuscript. JC, QB and YL contributed to manuscript editing. JC, QB, YL, MW, DM and YL revised it critically. All authors read and approved the final manuscript.
Funding
Our study was supported by the National Natural Science Foundation of China (No.U2004108). The funders had no role in study design, data collection and analysis, preparation of the manuscript, or decision to publish.
Data availability
Upon reasonable request, the corresponding author will provide the datasets used and/or analyzed in the current work.
Declarations
Ethics statement and consent to participate
This study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University: 2021-KY-1050-002. According to the decision of the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, the study has been granted an exemption from requiring written informed consent. All participant agree with this study. The owner of given us permission to experiment with bovine incisors.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Upon reasonable request, the corresponding author will provide the datasets used and/or analyzed in the current work.