Alendronate

Programmed antibacterial and mineralization therapy for dental caries based on zinc-substituted hydroXyapatite/ alendronate-grafted polyacrylic acid hybrid material

Xiaoyang Xu, Nan Wang, Mingzhen Wu, Jie Wang, Dingqian Wang, ZhuoXin Chen,
Jing Xie, Chunmei Ding, Jianshu Li
a College of Polymer Science and Engineering, Sichuan University, Chengdu, China
b State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, China
c State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China

A B S T R A C T
The domination of cariogenic bacteria in dental plaque biofilms is the primary cause of dental caries. In view of this, for the purpose of an effective treatment of dental caries, it is of great importance to inhibit the activity of acidogenic bacteria and promote the remineralization of damaged teeth simultaneously. However, the expensive antibacterial agents and poor mineralization ability of materials limit the practical applications. Biomineralization regulated by non-collagenous proteins (NCPs) gives hints to combine the remineralization ability of NCPs with accessible antibacterial property effectively. In this work, we propose a programmed an- tibacterial and remineralization strategy for the therapy of dental caries based on zinc-substituted hydro- Xyapatite/ alendronate-grafted polyacrylic acid hybrid nanoneedles (ZHA@ALN-PAA). This hybrid material dissolves in the acidic caries environment and regulate the pH to nearly neutral (6.5). Abundant calcium/ phosphate ions are supplemented and the ALN-PAA embedded in it has also been released, which assists the biomineralization on tooth defect. It has been revealed that the inhibition ratio of ZHA@ALN-PAA against Streptococcus mutans is the highest (11.25 folds that of HA), which originates from the highest zinc ions released (132.9 mg/L). Besides, the interspace of etched enamel is fully filled with regenerated nanorods and the surface microhardness (SMH) is significantly improved (3.68 folds that of etched enamel) after only 3 days of miner- alization in vitro. This strategy developed here is simple and cost-effective, which can be referred to design the effective anti-caries materials applied for clinic treatment and daily oral care.

1. Introduction
As one of the most common diseases, dental caries is prevalent in both children and adults [1]. It has been well accepted that dental caries begins once demineralization dominates in the dynamic demi- neralization-remineralization balance in the mouth. Generally, the faster demineralization of teeth was closely related to the dental plaque biofilms [1,2]. That is to say, if the symbiotic steady-state microflora becomes a dominant group of acid-producing and acid-tolerant bacteria with frequent intake of fermentable carbohydrates or other stimula- tions, the produced organic acid results in the initiation of caries lesions[2–5]. The initial dental caries might lead to problems such as pulpitis,osteomyelitis and even worse complications if not treated in time [6], which could cause serious damages to the human body.
Oral hygiene helps the treatment of dental caries to certain extend, which removes the dental plaque and inhibits the erosion of acid pro- duced by cariogenic bacteria. However, teeth restoration is emergently demanded if serious damage happens. Metal, resin and ceramics are commonly used as restorative materials in clinic treatment. For the purpose of a well-fitting interface with defect area, materials possessing matched physicochemical properties with that of natural teeth are highly pursued. HydroXyapatites (HAs) are the main inorganic com- ponents of enamel with the weight ratio of 96 % [7]. Until now, many attempts have been undertaken to realize the restoration of enamel by HA, for example, crystalline paste [8], double-layered gel system [9], and precursor layer [10]. Among all, biomineralization gives hints to design a reasonable strategy for the repair of teeth.
The natural formation of teeth is regulated by non-collagenousproteins (NCPs) such as osteopontin, bone sialoprotein and dentin sia- lophosphoprotein [11,12]. It has been well-acknowledged that most NCPs related to biomineralization contain a large amount of acid groups, which play a considerable role in the nucleation and growth of HA crystals [13]. Inspired by this, in recent years, phosphorylated- terminated poly(amidoamine) [14], polyacrylic acid [15], and L-glu- tamic acid (L-Glu) [16] have been utilized as NCPs analogues to reg- ulate the biomineralization of enamel. Besides, it is worth noting that the localized microenvironment of oral caries is usually acidic accom- panied by the domination of cariogenic bacteria. Therefore, in order to achieve the effective treatment of dental caries, it is also very important to inhibit the activity of acidogenic bacteria [17]. For example, chicken immunoglobulin Y (IgY) was loaded on an amorphous calcium phos- phate nanocomposite, which shows significant antibacterial activity through the sustained release of IgY [18]. Silver [19,20], and chlor- hexidine [21] have been introduced into calcium phosphate composites for the treatment of dental lesion.
By virtue of the dissolution of calcium phosphate in acidic condi-tion, calcium ions, phosphates ions and antibacterial agents were re- leased, inducing antibacterial and remineralization effects simulta- neously. However, the effectiveness for the therapy of oral caries in the actual situation might not be so good. On one hand, the remineraliza- tion of calcium phosphate only depends on the locally increased ion concentrations, which lacks the regulation of external mediator such as NCPs and their analogues. On the other hand, aiming at the clinic ap- plications, the antibacterial agents should be more cost-effective and easily-prepared. In light of this, it seems a good strategy to combine the remineralization ability of NCPs with accessible antibacterial property effectively. Unfortunately, it is still a challenge until now.
Alendronate (ALN) is an effective bone resorption inhibitor, whichpossess great affinity with HA [22]. It has been found that this strong interaction is derived from the ligand exchange between the two phosphate groups of ALN and that of HA [23]. Taking advantage of this, ALN has been widely used for the treatment of osteoporosis and other hard-tissue targeted drug delivery systems [24,25].
Therefore, in this work, we designed a programmed antibacterial and remineralization strategy for the therapy of dental caries. First, alendronate-grafted polyacrylic acid (ALN-PAA) was prepared to si- mulate NCPs, then HAs were mineralized on its outer layer. After the substitution of zinc in HAs, ZHA@ALN-PAA hybrid materials were fi- nally obtained. Here zinc ions were utilized as antibacterial agents because of their cheapness, stability and environmental friendliness [26]. In the acidic caries environment, ZHA dissolves accompanied with the release of zinc ions for bactericidal treatment against cariogenic bacteria. Meanwhile, the pH can be restored and a large amount of calcium and phosphate ions are supplemented to increase the local supersaturation. In addition to that, the ALN-PAA embedded in it has also been released. On basis of the strong binding force between ALN and enamel [27], ALN-PAA can adsorb and promote the remineraliza- tion. The schematic illustration of this work is shown in Scheme 1. The effect of this hybrid materials for the therapy of dental caries was evaluated from two aspects: the antibacterial property against Strep- tococcus mutants (S. mutans, a model cariogenic bacteria) and the re- mineralization effect with the presence of bacterial filtrate. The results demonstrate the effectiveness of this strategy with the most minerals regenerated and highest hardness recovered after the treatment of ZHA@ALN-PAA compared with all other groups. This strategy devel- oped here is simple and cost-effective, which can be referred to design the effective anti-caries materials applied for clinic treatment and daily oral care.

2. Experiments
2.1. Materials
Brain Heart Infusion broth (BHI) was purchased from Thermo Fisher Technology Co. CaCl2, (NH4)2HPO4, ethyl(dimethylaminopropyl)car- bodiimide (EDC), N-HydroXy succinimide (NHS), alendronate (ALN), polyacrylic acid (PAA, average Mw = 2000) NaN3 and 4-(2-hydro- Xyethyl)-1-piperazineethanesulfonic acid (HEPES) were bought from Sigma-Aldrich. HCl was purchased from Haihong Chemical Co. Ltd. NaOH, H3PO4 (37 wt%), Zn(CH3COO) %2H2O, Na2HPO4 and KH2PO4were purchased from Aladdin, China. Phosphate buffer saline (PBS) was obtained from BaoXin Biotechology Company (Chengdu, China).

2.2. Synthesis of ALN-PAA
100 mg of PAA was dissolved in PBS (10 mg/mL). Subsequently, NHS (17.25 mg), EDC (28.75 mg) and ALN (48.75 mg) were added, and the pH was adjusted to 7.4 by NaOH. After stirring at room temperature for 24 h, the solution was evaporated under reduced pressure, and the sample obtained was dissolved in acetic acid for a concentration of 20 mg/mL. The miXture was then precipitated in ethyl acetate and ALN- PAA was obtained. The ALN-PAA was dissolved in D2O solution for 1H nuclear magnetic resonance (NMR) analysis by a Bruker Avance spec- trometer (Bruker AVII-400 MHZ; Germany). Fourier transform infrared (FTIR) spectra of the product was measured by Nicolet Spectrometer (Thermo Fisher, Nicolet iS50, USA).

2.3. Preparation of HA nanomaterials
To synthesize HA@PAA, 2 g of (NH4)2HPO4 and 70 mg of PAA were dissolved in deionized water (DIW, 50 mL) as solution A. 2.775 g of CaCl2 was dissolved in DIW (50 mL) as solution B. First, the solution A was placed in a round bottom flask, which was heated to the boiling point under refluX with continuous mechanical stirring. When the so- lution was cooled down to room temperature, solution B was added dropwise and the pH of the miXture was adjusted to 10 using NH4OH (0.8 M). The miXture was allowed to age at boiling temperature for one hour, and at room temperature for another 23 h. HA and HA@ALN-PAA were prepared following the same procedure except the composition of solution A, where PAA was not added (for HA) and PAA was replaced by ALN-PAA (for HA@ALN-PAA) respectively. The whole procedure was illustrated in Fig. S1. The precipitates were collected and washed by DIW through centrifugation at 6000 rpm. Finally, the vacuum-dried deposits were conducted for FTIR and X-ray diffraction (XRD, Rigaku, Ultima IV, Japan) measurements.

2.4. Substitution of zinc into HA
287 mg of as-prepared powder (HA, HA@PAA and HA@ALN-PAA) in Section 2.3 was added into ethanol solution (50 mL) containing 286 mg of Zn(CH3COO)2%2H2O. The obtained suspension was then magnetically stirred for 1 h. The precipitates were collected by cen- trifugation, washed with ethanol and dried under vacuum, which were named as ZHA, ZHA@PAA and ZHA@ALN-PAA respectively. Finally, they were analyzed with XRD and transmission electron microscopy (TEM, FEI, Tecnai G2 F20 S-TWIN, USA).

2.5. In vitro pH-dependent Ca2+ release of ZHA@ALN-PAA
In order to estimate the dissolution behavior of ZHA@ALN-PAA, the phosphate buffer (PB) with various pH values (4.0, 5.4 and 7.4) was utilized. In brief, 50 mg of ZHA@ALN-PAA was placed in a dialysis bag (molecular weight cut off: 1000 g/mol) which was filled with PB (2 mL). The dialysis membrane bag was placed in a centrifugal tube containing 5 mL of release medium (PB). The whole system was gently shaken at a shaking table (100 rpm, 37 ℃). At different time intervals, 2 mL of the release medium was withdrawn and 2 mL of fresh medium was added. Finally, the concentrations of the released calcium were analyzed with Calcium Assay Kit (Nanjing Jiancheng Bioengineering Institute, China), and the OD value at 600 nm was read by microplatereader (Molecular Devices, SpectraMax ABS plus, USA).

2.6. Preparation of enamel samples and enamel lesions
Sound bovine teeth were selected in this study. After removal of the root and organic contaminants, the teeth were cut into enamel slices using sintered diamond saw on a low-speed precision cutting machine. Then the enamel slices were then embedded into acrylic resin with the enamel exposed, followed by the polish of 400-, 800-, 1200- and 2400- grit carborundum discs in turn. After that, acid-resistant nail polish was painted on the enamel surfaces, leaving a working area of 4 × 4 mm2 exposed.
For the demineralization of enamel, all tooth slices were sonicated for 30 min first and then etched by 37 % phosphoric acid for 45 s. All the samples were stored in PBS (4 ℃) for further characterization.

2.7. The binding capability of ALN-PAA on tooth enamel of bovine teeth
ALN-PAA and PAA were dissolved separately in DIW for a con- centration of 25 mg/mL. The polymer solution (100 μL) was dropped on the working area of demineralized enamel, and dried at room tem- perature. The enamel slices were then rinsed with PBS and DIW each for three times in turn. Attenuated total reflectance infrared (ATR-IR)(NICOLET iS10, Thermo Scientific, USA) spectra of enamel surface were measured.

2.8. Biomineralization of polymer-coated enamel in artificial saliva
ALN-PAA or PAA aqueous solution (25 mg/mL, 20 μL) was dropped onto the working area of demineralized enamel. Enamel surfaces treated by PBS was used as control. The treated enamel slices were dried at room temperature and then soaked in artificial saliva at 37 ℃for biomineralization.
The artificial saliva contains CaCl2 (1.5 mM), KH2PO4 (0.9 mM), KCl (130 mM), HEPES (20 mM) and NaN3 (1 mM). The KOH (1 M) was used to adjust the pH of solution to 7.0. Each tooth slice was vertically placed in polystyrene tubes with the working area totally soaked in artificial saliva solution, which was refreshed every 24 h.
After the period of 11 days and 14 days, samples were removed from artificial saliva and rinsed with DIW for three times. The mor- phology of the samples was analyzed with scanning electron micro- scopy (SEM, Phenom world, Phenom, Netherlands).

2.9. Antibacterial test against S. mutans
S. mutans UA159 was chosen as a model for antibacterial test. Microbes were grown overnight in BHI at 37 °C. The concentration of bacterial suspension was made 103 per mL by determining the OD value of solution at 600 nm on the microplate reader (KHB ST-360, Shanghai Kehua, China). Then 1.2 mL of the bacterial suspension was co-cultured with 40 mg nanomaterials (HA, ZHA, HA@PAA, ZHA@PAA, HA@ALN- PAA and ZHA@ALN-PAA). The pH values of the culture media were measured by pH acidometer after cultivation of 6, 12, 18 and 24 h. Inaddition, for colony forming unit (CFU) measurement, 100 μL of bac- terial solution at the co-culture time of 24 h was taken from each groupand diluted in gradient (10°∼105). These diluted solutions were in- oculated on BHI solid agar plates for incubation of 12 h at 37 °C. Theinhibition ratio (IR) was calculated according to the number of CFU as Eq. (1):
IR = C0-C × 100%
C0 (1)
Here C stands for the CFU of nanomaterial treated groups and C0 re- presents that of the control group without any treatment.
The rest of the bacterial suspension was collected by filtering through 0.22 μm Millipore membranes (USA) and stored at 4 ℃ for the following remineralization experiment. The concentration of zinc ions in filtrate was estimated by inductively coupled plasma-optical emis- sion spectroscopy (ICP-OES) (Agilent, ICP-OES 5100 SVDV).
Similarly, enamel slices were co-cultured with bacteria and treated by different formulations for 24 h. After that, the slices were soaked in2.5 % glutaraldehyde at 4 ℃ for 4 h, dehydrated in gradient ethanol solution (30 %, 50 %, 70 %, 85 %, 95 % and 100 %), dried in super- critical condition and sputtered with gold for SEM observation.

2.10. Evaluation of mineralization ability by alternating immersion of simulated saliva and bacterial filtrate
The etched enamel slice was vertically placed in 24-well plates with the exposed working area totally immersed in artificial saliva for 20 h. Then the artificial saliva was replaced by bacterial filtrate (as-prepared in Section 2.9) for culture of 4 h. This process was repeated every 24 h. Enamel slices of different groups were washed with PBS and DIW for three times in sequence after mineralization of 3 days. The mechanical property of enamel slices was characterized by Knoop microhardnesstester (Duramin-1/-2; Struers, Copenhafen, Denmark).

2.11. Cytotoxicity test
The cytotoXicity of nanomaterials is evaluated by Cell Counting Kit- 8 (CCK-8 assay) using human oral keratinocyte (HOK) cells. In brief, HOK cells cultured in medium (10 % fetal bovine serum, 90 % α-MEMand 1 % penicillin/streptomycin) were incubated in a 96-well micro-plate for 24 h (3000 cells per well). Then the solution of different na- nomaterials was added for further incubation of 24 h. After that, the CCK-8 work solution was added in each well and incubated at 37 °C for 3 h. The OD of the solution in each well was measured at 450 nm. The cell viability of different groups was calculated as Eq. (2):
Cell viability C2 − C0
with the flow rate of 0.08∼1.40 mL/min [29], the binding capability of ALN-PAA to the enamel is crucial for further regeneration of minerals. In order to estimate the affinity of polymer with enamel, both PAA andALN-PAA aqueous solution was dropped onto the surfaces of tooth enamel samples, which were thoroughly washed with PBS to simulate the saliva flow process according to previous investigation [27,30]. Fig. 1B shows the ATR-IR spectra of enamel slices. The characteristic peak of PO 3− groups (P]O stretching vibration at 1025 cm-1) can beclearly observed on native enamel. After PAA/ALN-PAA coating, theintense peaks located at 1703 cm-1 newly appear, which are derived from C]O stretching vibration of abundant carbonyl groups in PAA. However, this peak attenuates sharply on PAA-treated enamel when the substrates were washed, suggesting few PAA retained on enamel sur- face. In contrast, the characteristic peak of carbonyl groups was still obviously detected on ALN-PAA treated sample, which implies that a lot of ALN-PAA molecules were still adsorbed on enamel surface. Based on above results, we can conclude that the binding affinity between ALN- PAA and enamel surface is tight enough to resist the sufficient rinse of PBS and DIW. Considering pure PAA can be easily removed from the enamel surface, this strong interaction is attributed to the robust ab- sorption of ALN moieties, which is consistent with the results reported in the literature [27].

2.12. Statistical analysis
The statistical analysis was performed using GraphPad Prism 6.01. The results are presented as mean value and standard deviation (SD) for at least three trials. A two-tail Student’s T-test was applied for com-parison between two groups. The p-value < 0.05 was regarded as sta-tistically significant. 3. Results and discussion 3.1. Characterization of ALN-PAA In consideration of the strong affinity between ALN and HA crystals [27], ALN was introduced into PAA polymer chain for a HA-specific binder. The 1H NMR spectra are shown in Fig S2. Characteristic che- mical shifts at 1.42–2.08 ppm (c, -CH2-CH-) and 2.23–2.61 ppm (d, -CH- CO-) are ascribed to the methylene and methine protons on the mainpolymer chain of PAA. After the graft of ALN to PAA, chemical shifts located at 2.00–2.09 (a,-CH2-CH2-,-CH2-CH2-NH-), 3.02–3.16 (b,-CH2-NH-) and 2.105 (e, HO-P = O) newly appear (cyan line in Fig S2),which belong to protons of ALN [28], demonstrating successful mod- ification of ALN on PAA. The FTIR spectrum of PAA (Fig. 1A) reveals the characteristic peak at 1709 cm−1, assignable to the C]O stretching vibration of carbonyl groups in PAA. After the modification of ALN, typical peaks of PO 3− moieties at 1025 cm−1 (P]O stretching vibration) and 577 cm−1 (P] O bending vibration) are clearly observed, representing the signal ofphosphate groups in ALN. Meanwhile, the characteristic absorption peak at 1648 cm−1 is assignable to the C]O stretching vibration of amide carbonyl, and the peak at 1546 cm−1 is ascribed to the NeH bending vibration of secondary amide. The NMR, together with FTIRresults prove the successful synthesis of ALN-PAA. 3.2. Binding capability of ALN-PAA to enamel Since the whole oral environment contains lots of flowing saliva Bovine incisors have the advantages of large flat surface and easy to obtain, thus they have been widely utilized as substitutes for human teeth [31–33]. Here enamel slices of bovine incisors were carefullypolished and then etched for further experiments. As shown in Fig. 2A,the tooth surface is relatively flat after polishing. Although the scaly- like prisms and interprisms are well-defined, the delicate structure of the crystals cannot be clearly distinguished. In contrast, after acid etching (Fig. 2B), the elaborate structures of prisms and interprisms becomes clearer. Crystals of interprisms exhibit a roof ridge structure with a width of about 1.5 μm, making a distinct separation betweenprisms. It is apparent that the roughness of tooth surface increasedsignificantly after acid etching. Subsequently, the etched-enamel slices were treated by PBS, PAA and ALN-PAA separately. Mineralization was carried out by soaking the treated samples in artificial saliva for 2 weeks. The morphology of all samples was characterized. Substrates in three groups exhibit distinct differences after 11 days, especially the coverage of regenerated mi- nerals (Fig. 2, C1-E1). For the PBS-treated enamel (Fig. 2, C1), part of the etched-prisms are covered by newly formed crystals, and the ridge structure becomes less obvious given the biomineralization-inducing ability of HA itself. Nanorod crystals are randomly distributed on PAA- treated enamel, which cover most of the prism area (Fig. 2, C1). For ALN-PAA treated group (Fig. 2, D1), it can be observed that the etched prisms are almost fully filled by newly-formed crystals, and some or- dered crystallites are perpendicular to the interprisms. These results reveal the promotion of polymer mediator to mineralization. Note that the interspace of the etched area is most covered by regenerated mi- nerals, ALN-PAA outperforms the other groups for mineralization during this period. After mineralization of 14 days, a large number of crystal mineralswere formed on the enamel surface of all three groups (Fig. 2, B2-D2). Even so, for PBS treated group, the roof-like structure is still obvious with part of the etched prisms exposed (Fig. 2, B2). It is noted that the surface of PAA (Fig. 2, C2) or ALN-PAA (Fig. 2, D2) treated enamel is completely covered by nanofibers, where the interspace and roof-like structure cannot be observed, in sharp contrast to the uneven surface on PBS-treated enamel. Based on above, it is shown that the adsorbed ALN- PAA on enamel surface is beneficial for the biomineralization behavior in artificial saliva, which induces the formation of a preferred relatively flat surface in the whole 2-week period. 3.3. Remineralization induced by PAA/ALN-PAA on acid-etched tooth = C1 − C0 × 100% Here C0 and C1 represent the OD value of culture media with the ab- sence/ presence of cells respectively. C2 stands for the OD value of groups with treatment of different nanomaterials. 3.4. Characterizations of synthesized nanoparticles Wet precipitation is used to prepare HA@ALN-PAA, meanwhile HA@PAA and HA were obtained for comparison following the same procedure. All three products were conducted for FTIR measurement. Characteristic peaks of HA are evident in all of the modified samples (Fig. S3), the main peaks at 1031 cm−1 are derived from the stretchingvibration of P]O groups and the distinct absorption peaks at 560cm−1and 600 cm−1 are assignable to the P]O bending vibration of PO 3− in HA [15,16,34]. Compared with that of HA, the FTIR spectrum of HA@PAA shows typical carbonyl band at 1560 cm−1 and 1720 cm−1, which are assignable to the stretching vibration of C]Ogroups [35,36]. This result demonstrates the existence of PAA in HA@ PAA. In addition to the signal of carbonyl moieties, the characteristic peak of amide (NeH bending vibration at 2921 cm−1) is detected onspectrum of HA@ALN-PAA. The results above demonstrate the suc-cessful preparation of HA@ALN-PAA. The products obtained above were extracted in ethanol for the loading of zinc, which are named as ZHA, ZHA@PAA and ZHA@ALN- PAA correspondingly after loading. The crystal structure of all samples before and after zinc substitution was analyzed by powder XRD. The XRD pattern of HA shows significant diffraction peaks at 2θ = 31.5°,32.2°, 32.7° (Fig. 3A), which are consistent with the diffraction of (211),(112) and (300) planes of HA (JCPDS File No. 86-0740) [37]. When the polymer (PAA or ALN-PAA) is introduced into hydroXyapatite, the diffraction peak of (300) plane becomes much stronger on spectra of HA@PAA and HA@ALN-PAA, which might be resulted from the pre- ferred plane for the attachment of PAA to apatite [38]. The XRD pat- terns show no significant differences after zinc extraction, and the general crystal forms remain constant. Note that the signal of the (111) and (300) planes vanished after zinc loading, which indicates that under our experimental conditions, zinc ions tend to exchange with calcium ions along (111) and (300) planes and destroy the corre- sponding crystal planes [39]. The TEM image (Fig. 3B) shows needle-like ZHA@ALN-PAA nano- particles were obtained with a diameter of 3∼10 nm and length of 70∼110 nm. The selected area electron diffraction (SAED) pattern(inset in Fig. 3B) exhibits the bright rings made up by small spots, in- dicating polynanocrystalline of nanoneedles. And the assignment of spots is in accordance with the diffraction planes detected in XRD. 3.5. pH-triggered dissolution of ZHA@ALN-PAA based on the release of Ca2+ Aiming at on-demand release of antibacterial agents and the sup- plement of calcium/ phosphate ions in cariogenic environment, the fabricated nanoparticles should maintain stable at physiological pH, and dissolve in acidic microenvironments. Therefore, the stability of nanoparticles was evaluated in solution of different pH values by measuring the content of calcium ions released. Generally, the demi- neralization of enamel occurs below the critical pH of 5.5 [40]. In consideration of this, the pH values of 7.4 (physiological condition), 5.4 (around the critical pH) and 4.0 (below the critical pH) were chosen for the experiment. The release profiles of Ca2+ from ZHA@ALN-PAA are shown in Fig. 4. It can be seen that under neutral condition, the amount of cumulatively released Ca2+ is 6.29 mg/L at 48 h. In contrast to that, the amount of released Ca2+ conducts sharp increase under critical pH (20.67 mg/L for pH 5.4), that is even much higher under more acidic condition (42.33 mg/L for pH 4.0). It is evident that the acidic pH conditions (pH 5.4 and 4.0) trigger the release of Ca2+ from nano- particles, implying the dissolution of ZHA@ALN-PAA. As a con- sequence, the zinc ions, phosphate ions and ALN-PAA were con- comitantly released in this process. 3.6. Antibacterial effect of synthesized nanoparticles against S. mutans S. mutans is regarded as the main cause of dental caries, which in- duces acidulation of the localized environment in the oral cavity andthe demineralization of teeth [41]. Thus S. mutans was chosen as model cariogenic bacteria in this study. Usually, the pH value of the bacterial suspension is as low as below 5.5 after culture of 24 h [42]. In this sense, the recovery of pH to a more neutral value is crucial for the remineralization of demineralized tooth. To evaluate the acid-inhibi- tion property of synthesized nanoparticles against S. mutans in vitro, which is also a reflection of bactericidal effect to some extent, first, the pH values of the co-culture medium containing bacteria and different samples were measured. As shown in Fig. 5A, the pH of all groups decreases in the first 6 h and then increases slowly as time prolongs. This phenomenon might be explained by catabolite repression [43]: glucose acts as preferred carbon source in the beginning with the production of lactic acid, so the solution becomes acidic. After glucose is exhausted, lactic acid is used as carbon source, and then the pH slightly increases. It is worth pointing out that after 24 h, the pH values of three zinc-unloaded groups are 6.12∼ 6.22, and that of three zinc-loaded groups are 6.55 ∼ 6.63. Thesubtle increase of pH for zinc-loaded samples (0.4 on average) indicates the positive role of zinc for the recovery of pH. In order to illustrate the antibacterial effect of the material, the IR of different samples against S. mutans was measured (Fig. 5B). It is shown that the IR of HA, HA@PAA and HA@ALN-PAA is low and more than 68 % of the microbes are alive. After the materials are loaded with zinc, the antibacterial ability is significantly improved, with the IR of 57.29%, 67.72 % and 75.05 % for ZHA, ZHA@PAA, ZHA@ALN-PAA correspondingly. It is noted that the antibacterial activity of ZHA@ALN-PAA outperforms that of ZHA@PAA and ZHA, and the IR exhibits 7.33 % and 17.76 % enhancement separately in comparison. We speculate that the antibacterial activity originates from the loaded zinc, given the excellent antibacterial ability of zinc ions [26,44]. To verify this as- sumption, the concentration of released Zn2+ was evaluated in bac- terial suspension after 24 h. As shown in Fig. 5C, nearly no zinc ions are detected for all three un-loaded groups. By contrast, the amount of zinc ions released from three loaded groups is much higher and ranks in the following order: ZHA@ALN-PAA (132.9 mg/L) > ZHA@PAA (90.0 mg/ L) > ZHA (45.9 mg/L). There is a positive correlation between IR and the amount of released Zn2+, that is to say, a higher amount of Zn2+ generates better antibacterial property. Based on above results, we infer that the antibacterial property of samples is attributed to the released Zn2+, and the highest concentration of Zn2+ in solution accounts for the outstanding antibacterial property of ZHA@ALN-PAA. As for the discrepancies of Zn2+ release, we suppose that there might exist a few PAA/ALN-PAA molecules exposed on the surface of HA, so in the fol- lowing extraction process, the strong chelation between zinc and car- boXyl/ alendronate groups facilitate the loading of zinc [45,46]. Be- cause of a stronger interaction of alendronate-zinc than carboXyl-zinc,more zinc ions can be loaded onto HA@ALN-PAA [47].
In addition, bacterial suspension was put on the surface of enamel slices with the treatment of different nanomaterials. The morphology of the enamel surfaces was then observed by SEM. As shown in Fig. S4, abundant chain-like microbes with smooth and intact appearance are located on enamel surface in the absence of nanomaterials (blank group). The treatment of HA, HA@PAA and HA@ALN-PAA generates no influence to microbial activity, considering massive normal cells are attached to the nanocrystals, which cover the whole surface of enamel. By contrast, the number of microbes on the surface treated by ZHA, ZHA@PAA and ZHA@ALN-PAA decreases sharply compared with that of the other four groups. Besides, it is noticeable that some sunken cells are clearly observed in these three groups (white arrow in Fig. S4), and the surface of microbes after ZHA@ALN-PAA treatment becomes rough (white circle in Fig. S4 G2), which might be derived from the released zinc ions. The SEM images further reveal the antibacterial property of zinc-loaded samples against S. mutans, in good accordance with the IR result.

3.7. Mineralization of acid etched teeth by alternate treatment of simulated saliva and bacterial filtrate
In order to simulate the actual state of the oral cavity, the re- mineralization of etched teeth was evaluated by alternate soaking in simulated saliva (20 h) and bacterial filtrate (4 h) [48]. Here the bac- terial filtrate is that of co-culture medium containing bacteria and dif- ferent samples after the cultivation of 24 h. The enamel treated with HA(Fig. 6A) exhibits obvious ladder-shaped structure with the step height of 20 ∼ 45 μm. The stair is composed by nanorod crystals about 200∼440 nm in length and 70∼90 nm in diameter (inset of Fig. 6A). Owing to the final treatment of bacterial filtrate for 4 h at the end of 3days, this ladder structure is supposed to be caused by the corrosion of lactic acid [49]. For group treated with HA@PAA, the ladder steps appear indistinctly, and short nanorods (60 ∼ 160 nm in length) are distributed on entire surface. By contrast, the surface of HA@ALN-PAA group is relatively smooth among three, which is also covered by na-norod crystals. The performance of HA@ALN-PAA is superior to the other two groups, demonstrating the promotion effect of ALN-PAA for the repair of teeth, in good accordance with the results of Fig. 2.
Besides, the enamel surface of zinc-loaded groups is relatively flat compared with that of corresponding unloaded groups, which is con- stituted by abundant nanorod crystallites. It is noteworthy that the length of newly generated crystals for ZHA, ZHA@PAA and ZHA@ALN- PAA groups is 200 ∼ 500 nm higher than that of HA, HA@PAA andHA@ALN-PAA groups on average. As mentioned in Section 3.6, the pHvalue of bacterial suspension treated by unloaded samples is slightly lower. As a consequence, more lactic acid exists in the bacterial filtrate, which hinders the growth of crystallites.
The hardness of all samples is characterized by Knoop microhard- ness test (Fig. 7). The etched enamel possesses the SMH as low as22.6 HV. After mineralization of 3 days, the SMH of all samples exhibits significant enhancement with the value higher than 60 HV. More im- portantly, the SMH of ZHA, ZHA@PAA and ZHA@ALN-PAA is 13.6 %,21.4 % and 29.9 % higher than that of HA, HA@PAA and HA@ALN- PAA, correspondingly. In addition, it is noted that the SMH of three zinc-loaded groups lies in the following order: ZHA@ALN-PAA > ZHA@PAA > ZHA. The above results reveal the crucial role of zinc and ALN-PAA for the repair of etched teeth, which agrees well with the results observed by SEM.
The cytotoXicity of nanomaterials was evaluated by HOK cell lines, which possess a cell viability higher than 80 % in all groups (Fig. S5) [50]. The acceptable cytocompatibility of nanomaterials guarantees their further applications.

4. Conclusions
In this work, ZHA@ALN-PAA nanoneedles were prepared for the therapy of dental caries which are featured by two important factors:
(1) Acid-triggered dissolution of nanomaterials for the antibacterial ability against cariogenic bacteria; (2) The supplement of ions and ALN- PAA mediator for biomineralization on defect teeth. The results reveal that the IR of ZHA@ALN-PAA against S. mutans is the highest among all groups (especially 11.25-fold enhancements compared with that of HA), which originates from the zinc ions released. In addition, the synthesized ALN-PAA can bind strongly with the tooth enamel and then induce in situ remineralization of HA on acid-etched enamel for a re- latively flat surface. The positive role of ALN-PAA, together with zinc ions might account for the results obtained under alternate soaking of simulated saliva and bacterial filtrate. It has been demonstrated that after 3 days of mineralization, the interspace of etched enamel is fully filled with regenerated nanorods for ZHA@ALN-PAA group, and the SMH of substrates is greatly boosted in comparison with other groups. This material presents a programmed antibacterial and remineraliza- tion strategy which will provide reference for the design and production of effective anti-caries materials for the therapy of dental caries.

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