Preparation and characterization of biomineralizing dentin bonding primer based on bioactive amphiphilic raspberry-like nanoparticles

Abstract

Background

Incomplete resin coverage of demineralized collagen can compromise dentin bond durability. This study developed a biomineralizing dentin bonding primer incorporating bioactive amphiphilic raspberry-like nanoparticles (BRPs) to enhance biomineralization and dentin bond strength.

Methods

BRPs were characterized using scanning electron microscopy (SEM) and nitrogen (N₂) adsorption experiments. The ions release from BRPs was measured using inductively coupled plasma mass spectrometry (ICP-MS), while the mineralization was evaluated using SEM and X-ray diffraction (XRD). Subsequently, an etch-and-rinse bonding system was prepared, with primer containing 1%, 5%, or 10% BRPs (w/w). XRD and SEM assessed the biomineralization of each primer group after one-month immersion in simulated body fluid (SBF). Then, 48 dentin plane samples were prepared from extracted non-carious human third molars and bonded with each primer. Stick-shaped samples (n = 30 for each group) were fabricated to evaluate the micro-tensile bond strength (µTBS) after 24 h and 6 months of aging. Degree of conversion (DC) was analyzed via attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR).

Results

BRPs (~ 100 nm) released Ca²⁺ and SiO₃^2˗ in SBF, with surface areas of 83.19 ± 0.52 m²/g. After SBF immersion, SEM detected the formation of mineral clusters on BRPs, and XRD analysis confirmed the presence of distinct characteristic peaks corresponding to hydroxyapatite (HAP). Regarding the mineralization of primers, only 5% BRPs-Primer and 10% BRPs-Primer formed needle-like mineral clusters, with distinct HAP characteristic peaks. For the 24-h µTBS, the 1% and 5% BRPs-Primers were comparable to Control-Primer (P > 0.05), but 10% BRPs-Primer showed a significant reduction (P < 0.05). After 6-month aging, 5% BRPs-Primer maintained µTBS compared to its respective 24-h measurements (P > 0.05), whereas µTBS significantly declined in both Control-Primer and 1% BRPs-Primer (P < 0.05). The 5% BRPs-Primer also exhibited a comparable DC to the Control-Primer (P > 0.05), and showed good nanoparticle dispersion and effective penetration into dentinal tubules.

Conclusion

BRPs demonstrated superior mineralization ability. Incorporating 5% BRPs into dentin primer achieved effective biomineralization, excellent initial bond strength, and long-term bond stability after 6-month aging.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12903-025-06698-5.

Background

Resin-based composite bonding technology has emerged as the primary treatment modality for direct dental restoration owing to its commendable functional and aesthetic attributes. The optimal dentin bonding process entails the utilization of phosphoric acid or acid monomers to remove hydroxyapatite (HAP) from the dentin, thereby inducing dentin demineralization. This process enables the resin monomers to penetrate the demineralized dentin, creating a hybrid layer and establishing micro-mechanical retention [1, 2]. However, in clinical practice, the resin monomers cannot completely encapsulate the demineralized collagen, leading to the exposure of collagen fibers within the dentin bonding hybrid layer and ultimately compromising dentin bond strength [3]. Simultaneously, the hydrolysis of demineralized collagen fibers and resin monomers contributes to microleakage, tooth sensitivity, and secondary dental caries, which are the primary factors affecting the long-term stability of dentin bonding [4–7]. Therefore, improving the durability of the dentin-bonding hybrid layer holds significant clinical implications.

Remineralization of the dentin bonding interface has become a pivotal research focus to enhance its mechanical strength and reduce the activity of hydrolases. To achieve the remineralization of demineralized dentin collagen within the bonding hybrid layer, previous studies have developed ion-releasing dentin bonding systems incorporating mineral fillers [8, 9]. In previous investigations, the fillers of ion-releasing dentin bonding systems primarily consist of calcium silicate, bioactive glass (BG), and calcium phosphate [10]. The incorporation of hydrated Portland cement, 45S5 BG, and other silicate materials into dentin bonding systems could effectively promote remineralization at bonding interfaces [11–13]. However, a significant drawback was the size of these filler particles. At the micron scale, they often failed to integrate well with the resin adhesive, leading to a weaker bond [14].

With advances in technology, the remineralization of the dentin bonding interface can be achieved through the incorporation of nanoscale inorganic fillers into the dentin bonding system, such as nano-BG and nano-amorphous calcium phosphate (NACP) [15–17]. However, a significant technical hurdle arises from the inherent chemical antagonism between the hydrophilic inorganic fillers and the hydrophobic resin matrix. This leads to their heterogeneous dispersion and a strong propensity for agglomeration within the organic monomers. Consequently, delamination occurs between the fillers and organic resin polymers [18]. Furthermore, the delamination between the inorganic fillers and the hydrophobic resin monomers results in an uneven stress distribution, compromising the dentin bonding system’s mechanical strength and bond strength [10, 18].

In order to overcome incompatibility and agglomeration challenges of bioactive fillers within the monomer matrix of the dentin bonding system, we synthesized nanoparticles with amphiphilic characteristics as the fillers. The bioactive amphiphilic raspberry-like nanoparticles (BRPs) were synthesized by the Pickering emulsion method. The BRPs possessed a composite structure resembling raspberries, with the bioactive component embedded in the hydrophobic matrix component [19]. The hydrophobic matrix component ensures outstanding compatibility with the resin matrix, facilitating a homogeneous and stable dispersion while preventing the agglomeration that plagued previous nanofiller systems. The bioactive component remains capable of exerting its remineralization effect at the target interface. This design effectively acts as a molecular bridge, reconciling the chemical incompatibility between the bioactive mineral phase and the organic resin phase.

Consequently, the objective of this study was to facilitate remineralization at the hybrid layer of dentin bonding and to maintain the excellent bond strength of dentin bonding by developing a novel biomineralizing dentin bonding primer incorporating BRPs. The null hypothesis was that (1) the BRPs-Primer could not exert the mineralization effect, and (2) the BRPs would have no effect on maintaining the durability of the dentin bond strength.

Materials and methods

Preparation of BRPs

The raspberry-like composite nanoparticles (RPs) were synthesized using the Pickering emulsion method, followed by surface modification with Ca (OH)₂ to obtain BRPs particles [19, 20]. The specific preparation procedure was as follows: Ludox silica TM40 (Sigma-Aldrich, St. Louis, MO, USA), featuring particles measuring about 25 nm in diameter, was thoroughly mixed into deionized water. Subsequently, 3-(trimethoxysilyl) propyl methacrylate (TPM; Alfa Aesar Chemicals Co., Ltd., Shanghai, China) was added as the silane coupling agent. The mixture was stirred to facilitate hydrolysis, forming a homogeneous emulsion under nitrogen (N₂) assistance. Then potassium persulfate (KPS; Alfa Aesar Chemicals Co., Ltd.) was introduced to initiate polymerization at a temperature of 70 ℃. Following centrifugation and three rounds of washing with ethanol and ultrapure water, the RPs were obtained. Subsequently, the dispersion of RPs was combined with a saturated aqueous solution of Ca (OH)₂ and stirred at room temperature for 3 days. After three additional rounds of washing and centrifugation steps, the BRPs were lyophilized and stored for further utilization (Fig. 1).

Fig. 1.

Flow chart of the experiment.TM40 refers to Ludox silica TM40 (Sigma-Aldrich, St. Louis, MO, USA), featuring particles measuring about 25 nm in diameter; TPM refers to 3-(trimethoxysilyl) propyl methacrylate (Alfa Aesar Chemicals Co., Ltd., Shanghai, China); KPS refers to potassium persulfate (Alfa Aesar Chemicals Co., Ltd.); BRPs refers to bioactive amphiphilic raspberry-like nanoparticles; SBF refers to simulated body fluid; ICP-MS refers to inductively coupled plasma mass spectrometer (Thermo Scientific, Waltham, MA, USA); SEM refers to scanning electron microscopy (SU8010, Hitachi, Tokyo, Japan); XRD refers to X-ray diffraction (D/MAX 2500, Rigaku, The Woodlands, TX, USA); ATR-FTIR refers to Attenuated total internal reflection Fourier transform infrared spectroscopy (Nexue, Plymouth, MI, USA); DC refers to degree of conversion

Surface morphology properties and ions release profiles of BRPs

Scanning electron microscopy (SEM; SU8010, Hitachi, Tokyo, Japan) was utilized to analyze the morphology of the BRPs. The BRPs were first dispersed in absolute ethanol (EtOH; Sigma-Aldrich) to form a homogeneous suspension, which was then deposited onto a silicon wafer for subsequent SEM analysis. The specific surface area, pore structure of the BRPs were obtained via N₂ adsorption measurements using a Nova 4200e adsorption instrument (Quantachrome, Boynton Beach, FL, USA) conducted at 77.3 K. The specific surface area was calculated from the Brunauer-Emmett-Teller (BET) plot using adsorption points in the P/P₀ range of 0.02 to 0.25 (NOVAWin software). Pore volumes were derived from N₂ adsorption at P/P₀ = 0.99. The average pore diameter was determined by dividing four times the total pore volume by the surface area. Micro-sized BG 45S5 particles (with a size less than 10 μm, prepared via the sol-gel method) were also evaluated as a control. Each test was performed in triplicate.

The BRPs were immersed in a solution of simulated body fluid (SBF, consisting of 136.8 mM NaCl, 3.0 mM KCl, 4.2 mM NaHCO₃, 1.0 mM K₂HPO₄·3H₂O, 2.5 mM CaCl₂, 1.5 mM MgCl₂·6H₂O, and 0.5 mM Na₂SO₄) at a concentration of 1.0 mg/mL. After various time immersions (1 h, 6 h, 12 h, 18 h, and 24 h), supernatants were collected following centrifugation, and the concentrations of released Ca²⁺ and SiO₃²⁻ were quantified through an inductively coupled plasma mass spectrometer (ICP-MS) supplied by Thermo Scientific (Waltham, MA, USA).

Mineralization properties of BRPs

The 50 mg BRPs were compressed into circular slices with a diameter of 1.0 cm using a tablet press and then immersed in SBF solution at a concentration of 1.0 mg/mL. After soaking in the water bath at 37 ℃ for one and two weeks, X-ray diffraction (XRD) analysis was performed using a D/MAX 2500 instrument (Rigaku, The Woodlands, TX, USA). XRD measurements were conducted at an operating voltage of 40 kV and a current of 200 mA. The diffraction data were collected within a 2θ range of 10° to 70°, with a scan speed of 4°/minute. The resulting data were analyzed using Origin 2020b software (Northampton, MA, USA), and the full width at half maximum (FWHM) of the diffraction peak at the 32° position was measured. Then, the size of HAP crystals (D) was calculated by the Scherrer formula:

κ: the shape factor

λ: the wavelength of X - rays

β: the half - width at half - maximum (FWHM) of the diffraction peak after subtracting the instrument broadening, in units of radians.

θ: the Bragg diffraction angle.

Furthermore, the morphological characteristics of the aforementioned BRPs samples were examined using SEM (SU8010, Hitachi), along with an energy-dispersive spectrometer (EDS) to analyze the elemental makeup before and after mineralization.

Preparation of the biomineralizing dentin bonding primer

The three-step etch-and-rinse dentin bonding system was prepared using monomers acquired from Sigma-Aldrich, consisting of a primer and an adhesive.

Preparation of the adhesive (Control-Adhesive): 3.0 g of urethane dimethacrylate (UDMA), 3.0 g of bisphenol A diglycidylmethacrylate (Bis-GMA), 2.5 g of trimethylene glycol dimethacrylate (TEGDMA), and 1.3 g of hydroxyethyl methacrylate (HEMA) were weighed and mixed using magnetic stirring at 60 °C. Subsequently, under dark conditions, 0.1 g of ethyl dimethylamino benzoate (EDMAB) and 0.1 g of camphoroquinone (CQ) were added fractionally while maintaining magnetic stirring at room temperature. Then, the mixture of the above monomers was further dispersed by ultrasonic treatment and a rotary mixer (Thinky Mixer, IS-310, Tokyo, Japan) operating at 2000 rpm. Finally, the Control-Adhesive was completed and kept in a 4 °C refrigerator under dark conditions (Table 1).

Table 1.

Chemical monomers of the dentin bonding system used in this research

Dentin bonding system	Composition
Control-Adhesive	3.0 g UDMA, 3.0 g Bis-GMA, 2.5 g TEGDMA, 1.3 g HEMA, 0.1 g CQ, 0.1 g EDMAB
Control-Primer	5.0 g Control-Adhesive, 5.0 g EtOH
BRPs-Primer	Control-Primer with 1wt% BRPs, 5wt% BRPs, or 10wt% BRPs

All monomers were purchased from Sigma-Aldrich

UDMA Urethane dimethacrylate, Bis-GMA Bisphenol A diglycidylmethacrylate, TEGDMA Trimethylene glycol dimethacrylate, HEMA Hydroxyethyl methacrylate, EtOH Absolute ethyl alcohol, CQ Camphoroquinone, EDMAB Ethyl dimethylamino benzoate

Preparation of the primer (Control-Primer): In the absence of light, 5.0 g of the Control-Adhesive was mixed with 5.0 g of EtOH, as detailed in Table 1. The mixture underwent magnetic stirring and ultrasonic dispersion, followed by final mixing at a speed of 2000 rpm using the rotary mixer (Thinky Mixer) to complete the preparation of the Control-Primer. Subsequently, the Control-Primer was kept in a 4 °C refrigerator under dark conditions.

Preparation of biomineralizing bonding primer containing BRPs (BRPs-Primer): Under dark conditions, 1wt% BRPs, 5wt% BRPs, and 10wt% BRPs were respectively added to the Control-Primer (Table 1). The mixture was subjected to magnetic stirring for 24 h and ultrasonic intermittent dispersion for 1 h before being finally mixed at a speed of 2000 rpm using the rotary mixer (Thinky Mixer). Subsequently, the resulting formulations of 1% BRPs-Primer, 5% BRPs-Primer, and 10% BRPs-Primer were stored in a refrigerator at 4 °C under dark conditions.

Mineralization properties of BRPs-Primer

Circular Teflon molds (5.0 mm diameter, 1.5 mm thickness) were filled with Control-Primer, 1% BRPs-Primer, 5% BRPs-Primer, and 10% BRPs-Primer. The samples were cured using an LED light device (Bluephase Style, Ivoclar, Liechtenstein) for 20 s on each side at an intensity of 1100 mW/cm². After immersion in SBF, which was renewed every 72 h for one month at 37 ℃ water bathing, the surface mineralization of the sheet samples was analyzed by XRD (D/MAX 2500, Rigaku, The Woodlands, TX, USA) at an operating voltage of 40 kV and a current of 200 mA. The diffraction data were collected within a 2θ range of 20° to 70°, with a scan speed of 4°/minute. The resulting data were analyzed using Origin 2020b software. The surface morphology of each primer sample was observed using SEM (SU8010, Hitachi) after gold coating under vacuum conditions at a pressure of 50 mTorr for 1 min.

The dentin bond strength test of BRPs-Primer

The micro-tensile bond strength (µTBS) was evaluated using a micro-tensile testing device (model T-61010 K, Bisco, Anaheim, CA, USA). For this study, forty-eight non-carious third molars were collected from patients aged 20 to 40 due to therapeutic requirements at the Dental Department of Beijing Friendship Hospital, Capital Medical University, with ethical approval (approval number: BFHHZS20240030). Then the teeth were stored in 0.2% thymol solution at 4 °C refrigerator for not more than 1 month.

To prepare flat dentin plane samples, the occlusal enamel was removed at the mid-coronal level using a slow-speed diamond saw (SYJ-150, Shenyang Kejing Automation Equipment Co., Shenyang, China). These dentin samples were randomly divided into four groups (12 teeth for each group): Control-Primer group, 1% BRPs-Primer group, 5% BRPs-Primer group, and 10% BRPs-Primer group. The dentin samples were treated for 15 s using 37% phosphoric acid gel (Eco-Etch, Ivoclar). They were rinsed with ultrapure water for 30 s and gently dried with lint-free paper while ensuring the dentin surface remained moist. The dentin samples of the above four groups were coated with the Control-Primer, 1% BRPs-Primer, 5% BRPs-Primer, and 10% BRPs-Primer for 20 s, respectively, and then were dried with the dental air syringe. Thereafter, the Control-Adhesive was applied to the dentin samples for 20 s and evenly blown with the dental air syringe. Subsequently, the dentin bonding samples were cured for 20 s using an LED light curing device (Bluephase Style, Ivoclar) at 1100 mW/cm². The crown sections of each group were restored using incremental layers of composite resin (IPS Empress Direct, Ivoclar) with a total restoration thickness of approximately 4 mm while ensuring that each layer did not exceed more than 2 mm.

The bonding samples of each group were randomly divided into two subgroups: 24-h group and 6-month group, with 6 bonded teeth assigned to each subgroup. Then, the samples were immersed in ultrapure water at a temperature of 37 ℃ for a duration of 24 h and then cut into stick-shaped samples with a bonding area (A) of 0.9 mm * 0.9 mm on the vertical bonding surface with the slow-speed diamond saw (SYJ-150, Shenyang Kejing Automation Equipment Co.). Stick-shaped samples were selected from 4 slabs near the center of each tooth, and defective samples with bubbles or cracks were excluded under stereomicroscopy. Finally, five intact samples were selected per tooth (n = 30 for each subgroup). Subsequently, the intact strip samples were immersed in SBF at 37 ℃, with the SBF being refreshed at 72-h intervals. After 24 h or 6 months of immersion, stick-shaped samples were subjected to micro-tensile testing using a micro-tensile tester (T-61010 K, Bisco) at a crosshead speed of 1 mm/min. The peak load force (F) at the fracture point was recorded. The equation for calculating the value of µTBS was .

The degree of conversion of BRPs-Primer

Attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR; Nexue, Plymouth, MI, USA) was employed to assess the degree of conversion (DC). In this study, 10 µL of each primer (Control-Primer, 1% BRPs-Primer, 5% BRPs-Primer, and 10% BRPs-Primer) was evenly coated onto the ATR-FTIR test well surface. After being gently blown using a dental air syringe, the samples underwent polymerization using an LED light-curing device (Bluephase Style, Ivoclar) for 20 s at a distance of 1 mm, with an intensity of 1100 mW/cm². ATR-FTIR spectra were acquired before and after photopolymerization. The vibrational signals at 1638 cm⁻¹ (aliphatic C = C bonds) and 1608 cm⁻¹ (aromatic C-C bonds) were evaluated, and the DC was computed using the formula below [21]:

.

Where V_after1 and V_after2 were, respectively, the absorbance values at 1638 cm⁻¹ and 1608 cm⁻¹ after light polymerization, and V_before1 and V_before2 were, respectively, the absorbance values at 1638 cm⁻¹ and 1608 cm⁻¹ before light polymerization. Each group’s measurements were conducted in six replicates (n = 6 for each group).

The dispersive capacity of BRPs-Primer

5% BRPs-Primer and 5% 45S5-Primer were prepared by dispersing 5 wt% of BRPs or micro-sized BG 45S5 particles (with a size less than 10 μm, synthesized via the sol-gel method) into the Control-Primer as described in Sect. 2.4. After standing for 1 h, optical photographs were taken to evaluate their dispersion performance. In addition, three dentin slice samples were prepared and demineralized with 37% phosphate gel (Eco-Etch, Ivoclar), followed by the application of a coating of 5% BRPs-Primer for 20 s. The dentin samples were then observed by SEM (SU8010, Hitachi) to analyze the distribution of BRPs on the dentin bonding interface.

Statistical analysis

Statistical analyses of µTBS values and DC values were conducted using SPSS 26.0 software (SPSS Inc., IL, USA). Before applying the parametric statistical methods, each dataset was checked for normality and homoscedasticity. If any of these assumptions were violated, the datasets would undergo a nonlinear transformation to meet these criteria, and then further analyses were conducted. For µTBS values, comparisons among different bonding groups were assessed by one-way ANOVA with LSD post-hoc tests. Comparisons between different storage times were analyzed by independent samples t-test. For the DC values, comparisons among different bonding groups were evaluated by one-way ANOVA with LSD post-hoc tests. The significance level was set at α = 0.05.

Results

Surface morphology properties and ions release of BRPs

The SEM observation revealed that the particle size of BRPs was approximately 100 nm, displaying dendritic clusters with small surface protrusions (Fig. 2A). BRPs exhibited a specific surface area of 83.19 ± 0.52 m²/g, which was approximately 80 times greater than that of 45S5. Furthermore, both the total pore volume and average pore diameter of BRPs were higher than those of 45S5 (Table 2). Following a 24-h immersion in SBF, BRPs exhibited a gradual release of Ca²⁺ and SiO₃²⁻ mineral ions. Notably, SiO₃²⁻ demonstrated a rapid initial release within the first 6 h, indicating the good mineralization performance of BRPs (Fig. 2B).

Fig. 2.

The surface morphology and ions release of BRPs. A displays the surface morphology of dendritic clusters on BRPs (indicated by arrows) under SEM observation. B demonstrates the ions release profile of BRPs during a 24-h immersion in SBF

Table 2.

The surface area, total pore volume, and average pore diameter of BRPs and 45S5 (Mean ± SD)

Sample	Surface area (m2/g)		Total pore volume (cm3/g)	Average pore diameter (nm)
BRPs		83.19 ± 0.52	0.550 ± 0.004	26.47 ± 0.08
45S5		1.1 5 ± 0.33	0.005 ± 0.001	18.86 ± 0.05

Mineralization properties of BRPs

Before mineralization, the BRPs exhibited a granular morphology (Fig. 3A). Following an immersion in SBF for 1 week, flaky and needle-shaped new minerals formed on the surface of BRPs (Fig. 3B). With an extended immersion time of 2 weeks, these new minerals clustered and covered the entire surface of BRPs (Fig. 3C). The EDS analysis showed that the BRPs mainly consisted of carbon (C), oxygen (O), calcium (Ca), silicon (Si), with no detectable presence of phosphorus (P) before mineralization (Fig. 3A₁−3A₃). After being immersed in SBF for both 1 and 2 weeks, the BRPs samples exhibited a consistent distribution of Ca and P elements on the surface, which exhibited an increasing trend with prolonged immersion time. Notably, the P element underwent a transformation from absence to clear manifestation (Fig.3A₃−3C₃). The Ca/P ratio also gradually increased over time and reached a value of 1.85 at 2 weeks (Fig. 3C₁), which closely resembled that of HAP (Ca/P = 1.75).

Fig. 3.

The mineralized morphology and elemental analysis of BRPs after immersing in SBF for 1 and 2 weeks observed by SEM along with EDS. A, B, C show the surface morphologies of BRPs evaluated by SEM after 1 and 2 weeks of immersion in SBF. A₁– C₁ present the element composition and the calculated Ca/P ratio of the corresponding white boxes tested by energy dispersive spectrometer (EDS). A₂– C₂and (A₃– C₃) display the Ca and P element distribution of BRPs after 1 and 2 weeks of immersion in SBF, as tested by EDS

XRD patterns showed no distinct crystalline peaks for BRPs before mineralization. However, after soaking in SBF for 1 week, characteristic diffraction peaks corresponding to HAP were detected at angles of 26°, 32°, 40°, 46°, 49° and 53°. Furthermore, the intensity of these HAP characteristic diffraction peaks significantly enhanced after 2 weeks of immersion (Fig. 4). The size of the formed HAP was determined using the Scherrer formula: after 2 weeks of immersion in SBF, the HAP size was found to be 8.13 nm, compared to 6.01 nm after 1 week of immersion. These findings indicated that the excellent biomineralization activity of BRPs in forming HAP.

Fig. 4.

The XRD diffractograms of BRPs after immersing in SBF for 1 and 2 weeks. The characteristic peaks of hydroxyapatite (HAP) were indicated by *

Mineralization properties of BRPs-Primer

The surface morphology of each bonding primer group was examined using SEM after 1 month of mineralization in SBF, as shown in Fig. 5. Interestingly, the Control-Primer group (Fig. 5A₂) exhibited no discernible mineral formation. Similarly, while BRPs were observed on the sample surface in the 1% BRPs-Primer group (Fig. 5B₂), there was no evident mineral formation. In contrast, the 5% BRPs-Primer group exhibited the formation of fine needle-like new minerals (Fig. 5C₂), and the 10% BRPs-Primer group demonstrated more pronounced new minerals, characterized by small flake-like minerals (Fig. 5D₂).

Fig. 5.

The mineralization effect of BRPs-Primer following a one-month immersion in SBF. A₁-D₁ display the low-power SEM morphology of the mineralized samples for each group, while ( A₂-D₂) demonstrate the corresponding high-power SEM morphology for each group. The arrows indicate newly formed needle-like minerals

XRD analysis revealed the absence of distinct crystal peaks in both the Control-Primer group and the 1% BRPs-Primer group after immersing in SBF for 1 month. Notably, both the 5% BRPs-Primer sample and 10% BRPs-Primer sample resulted in the emergence of the characteristic peak corresponding to HAP at 2θ = 32° (211) after immersion in SBF for 1 month. Furthermore, an enhanced presence of HAP characteristic peaks was detected when the content of BRPs increased to 10wt% (Fig. 6). These findings suggested that the dentin bonding primer with a content of at least 5wt% BRPs could initiate mineralization. These XRD results were consistent with those obtained from SEM analysis.

Fig. 6.

The XRD patterns of different BRPs-Primer groups after being immersed in SBF for 1 month. The characteristic peaks of HAP were indicated by *

The dentin bond strength of BRPs-Primer

The results of the microtensile test (Table 3) demonstrated that the 24-h µTBS of the 1% BRPs-Primer group and the 5% BRPs-Primer group were 43.81 ± 5.55 MPa and 43.07 ± 5.42 MPa, respectively. Statistical analysis confirmed that neither of these values differed significantly from the Control-Primer group (P > 0.05). However, the 24-h µTBS for the 10% BRPs-Primer group dropped to 38.87 ± 4.86 MPa, which was significantly lower than that of the Control-Primer group (P < 0.05).

Table 3.

The micro-tensile bond strength (µTBS) of different primer groups to dentin after SBF immersion for varying periods (Mean ± SD, MPa)

Group	Time
	24 h	6 months
Control-Primer	44.16 ± 4.99 A, a	38.48 ± 5.01 A, b
1% BRPs-Primer	43.81 ± 5.55 A, a	39.80 ± 5.22 AB, b
5% BRPs-Primer	43.07 ± 5.42 A, a	41.09 ± 4.56 B, a
10% BRPs-Primer	38.87 ± 4.86 B, a	36.93 ± 4.08 AC, a

Following a 6-month aging period (Table 3), the µTBS values of the 1% BRPs-Primer group and the 10% BRPs-Primer group exhibited no significant differences relative to the Control-Primer group (P > 0.05). In contrast, the µTBS of the 5% BRPs-Primer group was 41.09 ± 4.56 MPa, which was higher than that of the Control-Primer group, with statistical significance (P = 0.035, P < 0.05).

When comparing µTBS values at 6 months to those at 24 h, both the Control-Primer group and 1% BRPs-Primer group demonstrated a significant reduction (P < 0.05). In contrast, the 5% BRPs-Primer group and 10% BRPs-Primer group did not exhibit a statistically significant decrease (P > 0.05).

Comparisons among different primer groups were assessed by one-way ANOVA and LSD post-hoc tests, and statistical differences among columns (P < 0.05) are indicated by capital letters. Comparisons between different storage times were assessed by independent samples t-test, and statistical differences between rows (P < 0.05) are represented by lowercase letters.

The DC and dispersive capacity of BRPs-Primer

The DC of the 1% BRPs-Primer and the 5% BRPs-Primer were measured at 43.24 ± 3.05% and 42.17 ± 2.63%, respectively, which were slightly lower than that of the Control-Primer (44.54 ± 2.57%). However, this difference did not reach statistical significance (P > 0.05). In contrast, the DC value of the 10% BRPs-Primer group was recorded at 40.78 ± 3.21%, demonstrating a statistically significant difference when compared to the Control-Primer (P < 0.05) (Fig. 7A).

Fig. 7.

The degree of conversion (DC) and dispersive capacity of BRPs-Primer. A presents the DC of different primer groups, with * indicating the statistical difference (P< 0.05). B shows the state of the 5% BRPs-Primer and the 5% 45S5-Primer after standing for 1 h, with the red arrow indicating apparent stratification for the 5% 45S5-Primer. C, D demonstrate the SEM morphology of the 5% BRPs-Primer bonded with dentin, and the white arrows indicate that the BRPs entered the dentin tubules

Following a 1-h incubation period, no noticeable delamination was observed in the 5% BRPs-Primer group, whereas significant delamination occurred in the 45S5-Primer group. This indicates excellent stability of BRPs within the bonding primer, owing to their amphiphilic characteristics (Fig. 7B). After the application of 5% BRPs-Primer to the demineralized dentin surface, SEM analysis revealed that BRPs effectively penetrated the exposed dentinal tubules and adhered to the demineralized collagen fibers with the monomers of the primer (Fig. 7C and D).

Discussion

The aging of dentin bonding interfaces significantly impacts the long-term effectiveness of direct resin bonding restoration. Various factors contribute to the degradation and aging of the resin bonding interface, such as residual water, endogenous and exogenous proteolytic enzymes, temperature fluctuations in the oral environment, and masticatory forces [4, 22, 23]. During the bonding process, the presence of free water at the bonding interface is inevitable, leading to the formation of water trees and water droplets within the bonding hybrid layer [4, 24]. Meanwhile, the acidic environment during the bonding process triggers the activation of various hydrolases, including matrix metalloproteinases (MMPs), which effectively facilitate subsequent hydrolysis of demineralized collagen and resin monomers within dentin [25, 26]. These ultimately lead to microleakage or nanoleakage, facilitating bacterial infiltration and secondary caries formation, thereby resulting in the failure of resin bonding restoration [27].

The process of biomineralization involves the removal of free water. Researchers have aimed to utilize biomineralization to gradually displace non-bound water with forming apatite crystals within the bonding hybrid layer, thereby reducing free water at the dentin bonding interface [10, 28]. The incorporation of nano bioactive mineralization fillers into the dentin bonding system can help maintain its mineralization performance and enhance interface stability to a certain extent [29–31]. In this study, obvious mineralization was also observed for both 5% BRPs-Primer and 10% BRPs-Primer groups through SEM and XRD analyses. These findings were consistent with the results of previous studies [30, 31]. Therefore, the null hypothesis that BRPs-Primer could not exert a mineralization effect was rejected.

The BRPs possess hydrophilic bioactive domains that exert biomineralizing effects. In this study, SEM revealed the formation of distinct flower-like clusters of new minerals on BRPs after SBF immersion. XRD results indicated the presence of characteristic peaks associated with HAP, suggesting excellent mineralization performance of BRPs. BRPs possess a similar structure to BG [19]. Specifically, a large amount of Si-OH on the surface of BRPs, which can combine with Ca²⁺. Therefore, it is postulated that the mineralization mechanism of BRPs resembles that of BG. Upon contacting with SBF, Ca²⁺ ions exchange with H⁺ ions in the SBF, leading to the formation of Si-OH groups on the surface of BRPs. These Si-OH groups subsequently condense to form a silica gel layer. Subsequently, PO₄³⁻ and Ca²⁺ ions from the SBF migrate toward this silica gel layer, resulting in the formation of an amorphous calcium-phosphate (Ca-P) layer, which gradually crystallizes into HAP [32].

However, the physical and chemical properties of the fillers can influence the fundamental characteristics of the dentin bonding system. Liang et al. reported that the incorporation of amorphous fluorinated calcium phosphate nanoparticles (AFCP) into Clearfil S3 Bond resulted in a significant decrease in dentin bond strength, and the application of an MDP-containing primer was necessary to enhance bonding performance [17]. Abuna G et al. observed comparable outcomes for nano-bioactive glass (nBG) [33]. This detrimental effect could be primarily attributed to the incompatibility between inorganic fillers and organic monomers. Previous studies indicated that achieving a more uniform dispersion of nanomaterials within the organic monomers resulted in stronger interaction with the resin matrix, thereby significantly enhancing its overall performance [34]. The BRPs used in this study were synthesized through the Pickering emulsion method, resulting in particles with a size of about 100 nm and dendritic-like surfaces with a high specific surface area. Moreover, the amphiphilic nature of BRPs was attributed to the hydrophobic polymer domain formed by TPM, enabling them to exhibit good compatibility with resin monomers and facilitating their excellent dispersion as fillers in resin monomers [19, 20].

Research has shown that microleakage within the hybrid layer progressively worsens over time, primarily due to hydrolysis originating at the base of the dentin bonding hybrid [35, 36]. Consequently, achieving remineralization at the base of the hybrid layer is critical to ensuring the longevity and stability of the bonding interface. BRPs were added to the bonding primer in this study, and SEM showed that BRPs could enter the dentin tubules with the primer matrix, which can promote the mineralization effect at the base of the dentin bonding hybrid layer. SEM analysis demonstrated that immersion of the 5% BRPs-Primer in SBF for 1 month led to the deposition of newly formed minerals, which was further characterized by XRD analysis with peaks corresponding to HAP. These findings suggested that the 5 wt% BRPs in bonding primer could facilitate mineralization and hydroxyapatite formation. Previous investigations conducted by our research team have also demonstrated the capacity of BRPs to induce mineralization within a specific time frame when incorporated into organic polymers [19]. The ability of BRPs to promote mineralization in the bonding primer may be linked to water channels formed at the bonding interface. As mentioned above, capillary action facilitates water infiltration into the hybrid layer during the dentin bonding process. Moreover, the presence of hydrophilic monomers and the leaching of unreacted monomers further promote the formation of water channels within the dentin-bonding hybrid layer [4, 24, 37]. These water channels serve as pathways for ion exchange between mineralized fillers and body fluids within the primer, thereby achieving mineralization effects.

The BRPs content in the bonding primer impacted the dentin bond strength. The results of this study revealed that the µTBS of the 5% BRPs-Primer group was statistically similar to that of the Control-Primer group. However, the 10% BRPs-Primer group exhibited a significant decline in the µTBS. Many previous studies have also demonstrated that the dentin bond strength decreases as the fillers content increases [38–40]. This could be attributed to that the increased fillers impact the bonding primer mobility and subsequent impaired micromechanical interlocking [38]. Furthermore, higher filler content may affect dispersibility and lead to agglomeration, potentially creating stress concentrations and resulting in weak points that compromise the adhesive strength itself [41]. Although the 5% BRPs-Primer showed good dispersive capacity, agglomeration may still occur as the BRPs content increases to 10wt%. Therefore, 5 wt% BRPs was an appropriate concentration in this study that could not only maintain the dentin bond strength, but also confer mineralization properties. Meanwhile, the DC values of the 5% BRPs-Primer group did not differ significantly from the Control-Primer group. Previous studies have indicated that an appropriate content of fillers can scatter light to activate photoinitiators (such as CQ) [42], thereby promoting a more efficient polymerization reaction without affecting the degree of monomer conversion.

In this study, after 6-month aging, the 5% BRPs-Primer group exhibited the highest bond strength, with no statistically significant reduction compared to the 24-h measurement. Thus, the null hypothesis that BRPs had no effect on maintaining the durability of the dentin bond strength could not be accepted. Previous studies have suggested that resin containing mineral fillers can reduce endogenous hydrolase activity through the formation of amorphous calcium phosphate (ACP) during the mineralization process, which interacts with hydrolases like MMPs to form Ca/P-MMP macromolecular complexes [14, 43, 44]. We speculate that BRPs may form Ca/P minerals to bind with MMPs, thereby inhibiting the activity of MMPs and protecting hybrid collagen to maintain bond strength. Additionally, BRPs have the potential to release mineral ions, which interact with the demineralized dentin collagen and lead to the formation of new mineral deposits [45]. This process may help compensate for micro-gaps caused by aging at the bonding interface, thereby reducing microleakage and enhancing interface integrity [30]. Consequently, 5% BRPs-Primer has the potential to remineralize the dentin bonding interface and enhance the long-term durability of dentin bond through biomineralization.

The present study has several limitations. Further experiments are necessary to characterize the fracture patterns at the bonding interface and to confirm whether BRPs have an inhibitory effect on MMPs. Moreover, we only evaluated the effect of BRPs in an etch-and-rinse bonding system; therefore, further research is needed to explore their impact in self-etching dentin bonding systems. Additionally, since this study was conducted in vitro, it may not fully reflect the complex biological conditions present in vivo.

Conclusions

The BRPs exhibited superior ions release performance and effective mineralization capabilities. A novel biomineralizing primer was developed by incorporating 5 wt% BRPs into the dentin bonding primer, which demonstrated notable mineralization effects and dispersion stability. Furthermore, the 5% BRPs-Primer not only achieved high initial dentin bond strength but also maintained bond strength after 6 months of aging.

Abbreviations

BRPs

Bioactive amphiphilic raspberry-like nanoparticles

HAP

Hydroxyapatite

BG

Bioactive glass

NACP

Nano-amorphous calcium phosphate

TPM

3-(trimethoxysilyl) propyl methacrylate

KPS

Potassium persulfate

SEM

Scanning electron microscopy

EDS

Energy-dispersive spectrometer

EtOH

Ethanol

SBF

Simulated body fluid

ICP-MS

Inductively coupled plasma mass spectrometer

XRD

X-ray diffraction

UDMA

Urethane dimethacrylate

Bis-GMA

Bisphenol A diglycidylmethacrylate

TEGDMA

Trimethylene glycol dimethacrylate

HEMA

Hydroxyethyl methacrylate

EDMAB

Ethyl dimethylamino benzoate

CQ

Camphoroquinone

μTBS

Micro-tensile bond strength

ATR-FTIR

Attenuated total internal reflection Fourier transform infrared spectroscopy

DC

Degree of conversion

MMPs

Matrix metalloproteinases

ACP

Amorphous calcium phosphate

Authors’ contributions

Xaiofeng Huang and Ailing Li designed the method of the study. Qiuju Li and Guibin Huang conducted the research, collected the data and contributed to analyzing data. Qiuju Li, Wei An, and Xiaofeng Huang interpreted the results and drafted the manuscript. All authors read and approved the final manuscript.

Funding

Self-funded.

Data availability

The datasets generated and analyzed during this study are available from the corresponding author upon reasonable request. All essential data supporting the findings are included within the manuscript.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Ethics Committee of Beijing Friendship Hospital, Capital Medical University (Approval No. BFHHZS20240030).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.