Abstract
Objective
The objective of this study was to develop a rapid and effective method to remineralize human carious-like enamel using chimaeric peptide-mediated nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP), mimicking the mineralizing pattern of the oriented assembly of ACP guided by amelogenin in the biomineralization of enamel.
Methods
CMC/ACP nanocomplex solution was first synthesized through the successive addition of carboxymethyl chitosan, calcium chloride, and dipotassium phosphate into distilled water. ACP nanoparticles were degraded by 1% NaClO from CMC/ACP nanocomplexes. The morphology of the particles at different periods was tested by transmission electron microscopy (TEM). The chimaeric peptides were added to guide the arrangement of ACP nanoparticles and to bind ACP nanoparticles to the demineralized enamel surface specifically. X-ray diffraction (XRD)/scanning electron microscope (SEM)/confocal laser scanning microscopy (CLSM)/nano-indentation tests were applied to check the remineralization effects.
Results
CMC/ACP nanocomplexes were obtained and could be kept without precipitation for a long time. After the degradation of NaClO and guidance of chimaeric peptides, ACP nanoparticles were arranged into oriented arrays before transforming into crystals, and the enamel-like crystals were tightly bound onto the demineralized surface. The newly formed enamel-like crystals were nearly well-organized and equipped with strong mechanical properties.
Significance The chimaeric peptide, which possesses guiding and binding motifs, was proven to be effective in demineralized enamel remineralization. This study provides a novel method of enamel remineralization, which is of great value in the prevention and therapy of enamel decay and tooth erosion, such as decay due to chalk caries after orthodontic therapy.
1
Introduction
Remineralization of dental hard tissue is an important curative technique in minimally invasive dentistry (MID) . Biomimetic remineralization plays an important role in the prevention and early treatment of enamel caries, dentin caries, and dentin hypersensitivity . It is well known that dental caries and tooth erosion begin at the outermost layer of teeth (enamel) with the damage of dental hard tissues. This process is called demineralization , which is attributed to the loss of mineral ions from the lattice of hydroxyapatite (HAP) through the action of organic acids produced by bacteria on the surface of enamel. Increasing numbers of researchers believe that dental caries formation is a dynamic disease process that is caused by the disturbance between the demineralization and remineralization processes. And tooth erosion is a disease that dental hard tissues were progressively etched by endogenous and exogenous acid . Therefore, the prophylaxis of preliminary enamel caries and enamel erosion can eliminate the progression of dental caries and further erosion, resulting in better preservation of natural dental tissue. One approach to preventing lesion progression is to treat the lesion with a remineralizing agent to tip the balance back toward remineralization, thus reversing the pathological process of caries and erosion formation .
Enamel formation is a typical biomineralization process that requires the syngeneic actions of both organic and inorganic components. With the assistance of complex development and degradation of organic components in the extracellular matrix, the inorganic elements are regulated and then begin to nucleate, grow, and assemble in an orderly manner in a certain time and space . Robinson et al. illustrated that spherical mineral particles with a diameter of 50 nm organize into chains in enamel formation . It was suggested that these nanoparticles are composed of amorphous calcium phosphate (ACP) and proteins associated with biomineralization and that the ACP can transform into HAP crystals. The small particles arrange in lines and then, with the help of functional proteins, the particles disappear and form crystals parallel to the c-axis of enamel. Among the natural proteins, amelogenin is now the most acknowledged, consisting of 173 amino acids with an N-terminus and C-terminus on each side. Some in vitro studies have shown that amelogenin can stabilize ACP to form Amel/ACP particles and guide the particles to arrange, fuse, and transform into HAP crystals . In addition, from the perspective of knowledge of material nanoparticles of ACP are much more active for enamel remineralization due to their small size and high surface area . Therefore, it is possible to utilize the nanoparticles of ACP stabilized by matrix to remineralize demineralized enamel.
Inspired by the stabilization ability of amelogenin, it is speculated that the existence of macromolecules can serve as a stabilizer to directly sequester supersaturated calcium and phosphate ions in solution from precipitates. Thus, some organic macromolecules, such as polyaspartic acid (PASP), polyacrylate acid (PAA), and casein phosphopeptide amorphous (CPP), have been applied to stabilize ACP nanoparticles in solution because of their chelating function resulting from rich carboxyl groups . However, the disadvantages of the aforementioned materials are also apparent, such as the low ability to stabilize high-ion supersaturation and the allergenic property of the materials to people who are sensitive to milk. Here, we used carboxymethyl chitosan (CMC), a derivative product of chitosan with biodegradable, biocompatible, nontoxic, and antibacterial properties , that is rich in carboxyl groups and is a good stabilizer of ACP nanoparticles as nanocomplexes of CMC/ACP to provide a remineralizing effect on dental tissues .
Regarding the remineralization materials, the economic operative time and effective binding to the enamel surface are basic demands of the dental clinical population. However, macromolecules such as PASP, PASA, CPP, and CMC, applied to stabilize amorphous calcium phosphate, cannot make the particles undergo crystal transformation quickly enough for them to have an enamel binding effect in clinical practice. In vivo , MMP-20 and KLK4 are the two key enamel proteases that can process amelogenin to generate the major cleavage products that accumulate during the secretory stage of amelogenesis . Thus, surfactant should be used in vitro to mimic the natural degradation protein-like proteases to digest amelogenin and release active ACP in a timely manner. NaClO is responsible for the oxidative degradation of carbohydrate polymers , which develop strong abilities to decompose CMC and release active ACP.
Regarding the binding effect and crystal promotion, we created a chimaeric peptide to arrange the amorphous calcium phosphate particles in order. The novelty of the peptides utilized in this study is derived from the HA promotion part of amelogenin because the entire synthesis of amelogenin is expensive and time-consuming. Leucine-rich amelogenin peptides (LRAPs) cannot transform ACP into HAP crystals with the removal of 16 amino acids of the C-terminus (LEAWPATDKTKREEVD) of amelogenin . In vivo research has also demonstrated that amelogenin-C-terminal gene knockout mice can exhibit enamel hypoplasia of the canines , illustrating the importance of the amelogenin-C-terminus in the formation of HAP crystals. However, some researchers have created inorganic binding peptides through phage surface display and cell surface display that can bind organic and inorganic surfaces together . HA6-1 is one of these products that can specifically bind to the enamel HAP surface with a sequence of SVSVGMKPSPRR . Therefore, we combined Ame-CT16 and HA6-1 into a single sequence with the flexible connecting peptide (GGGGS) as the guiding chimaeric peptide: SVSVGMKPSPRP -GGGGS- LEAWPATDKTKREEVD, which would guide the active ACP to form HAP crystals and tightly bind to the enamel surface.
The hypothesis of the study was that the chimaeric peptide would guide ACP nanoparticles degraded by NaClO from CMC/ACP nanocomplexes into ordered and oriented arrays and make them specifically bind to the demineralized enamel surface to accomplish rapid enamel-like crystal formation and biomimetic remineralization, which mimics natural mineralization in vitro and accomplishes enamel recovery through minimally invasive dentistry (MID).
2
Material and methods
The institutional review board of the Tianjin Medical University approved all of the procedures and methods conducted in this study (TMUh-MEC2012019).
2.1
Preparation of highly supersaturated CMC/ACP solutions
The CMC solution was first prepared by mixing 200 mg of CMC powder (95%, Qingdao Hong Hai Biological Technology Co., LTD, Qingdao, China) with 40 ml of double-distilled water and stirring (1000 rpm) until the powder was entirely dissolved. Next, 55.68 mg of K 2 HPO 4 was added to the CMC solution under slight stirring (500 rpm). A total of 94.08 mg of CaCl 2 ·2H 2 O was then added to 10 ml of double-distilled water, and this solution was instilled into the CMC solution gently under continuous stirring to form a highly supersaturated CMC/ACP solution. The final concentrations of calcium and phosphate ions were 8 and 16 mM, respectively. The CMC/ACP solution was stored in a refrigerator at 4 °C.
Before the application of CMC/ACP, 1 ml of 1% NaClO was added to 10 ml of CMC/ACP, followed by mixing at 500 rpm for 5 min.
The diameters of the CMC/ACP solution and NaClO degraded solution were tested through Mastersizer laser particle size analyzer (Mastersizer 2000, Malvern, Worcestershire, UK). The size of the particles can be calculated and analyzed.
2.2
Tooth sample preparation
Ten human unerupted third molars were obtained from patients aged between 18 and 30 years who needed preventive extraction before orthodontic therapy at the Hospital of Stomatology, Tianjin Medical University. All the patients agreed to the study and signed the written informed consent forms. The inclusion criteria were that the enamel of the teeth was mature and without caries, cracks, or other defects and that the integrity of the teeth was destroyed. With the removal of the root and pulp, 4 wafers were sawn from the buccal, lingual, mesial, and proximal surfaces of each tooth to obtain similar enamel slabs and were randomly distributed into groups A–D, respectively. Forty enamel blocks of approximately 3 × 3 × 0.5 mm 3 (length × width × height) in size in the 4 groups were prepared and sealed with water-resistant nail varnish, except for a small window area (2 × 2 mm 2 ) for demineralization and remineralization treatments. Before demineralizing treatment, all enamel samples were first cleaned through sonic oscillation for 30 min and were wiped with 75% ethyl alcohol repeatedly. Thereafter, 3 wafers from each tooth in groups A–C underwent demineralization treatment, prepared by coating a gel of 37% phosphoric acid (Gluma Gel, Heraeus Kulzer, Hanau, Germany) on the surface of the enamel for 30 s. One wafer from each tooth was left untreated as a natural tooth control in group D. Next, the samples were washed with sterile DI water for 60 s, sonicated again for 5 min, and stored at 4 °C in sterile DI water prior to use.
2.3
Synthesis and primary software test of guiding chimaeric peptides and assembly of nanoparticles
Two chimaeric peptides, Peptide A (SVSVGMKPSPRPGGGGSLEAWPATDKTKREEVD) and Peptide B (LEAWPATDKTKREEVD) as the control, were produced via solid-phase peptide synthesis at the Shanghai Top-peptide Co., Ltd. Peptide A was equipped with two functional motifs that control binding management and the transformation area together with the flexible peptide linker GGGGS. Peptide B was isolated from the binding domain, and both of the chimaeric peptides were labeled with FITC prior to fluorescence detection. The secondary structure of Peptide A was predicted using PSIPRED software ( http://bioinf.cs.ucl.ac.uk/psipred/ ).
A total of 10 mg of peptide was added to 10 ml of NaClO-degraded CMC/ACP with a stirring speed of 500 rpm to form the peptide-guiding ACP solution, which was placed at room temperature for at least 30 min for the assembly of nanoparticles before use.
2.4
TEM characterization of nanocomplexes of CMC/ACP and chimaeric-peptide-guided CMC/ACP
The size and morphology of the nanocomplexes of CMC/ACP nanoparticles, NaClO-degraded CMC/ACP, and chimaeric-peptide-guided ACP nanoparticles were characterized using transmission electron microscopy (TEM) (JEM-1230, JEOL, Tokyo, Japan) and selected area electron diffraction (SAED) at 110 kV. Fifteen milliliters of nanoparticle solution was dropped onto a 400-mesh copper TEM grid covered by a carbon support film at room temperature. Excess liquid was removed by the filter paper beneath the grid.
2.5
Remineralization of the demineralized enamel model
Samples in group A (n = 10), which served as the control group, were treated with demineralized enamel without a biomimetic by immersing the samples in simulated saliva (5 ml/sample) containing 1.5 mM Ca 2+ , 0.9 mM HPO 4 2+ , and HEPES. In group B, the remineralization treatments were carried out by coating NaClO-degraded CMC/ACP nanocomplex solutions (100 μl) onto the demineralized surface using disposable micro-applicators (TPC, Advanced Technology, USA) repeatedly for 10 min. Group C was treated with chimaeric-peptide-guided ACP nanoparticles on the surface of the window area of the samples. Both Group B and C samples were immersed in simulated saliva after treatment. Group D was the natural tooth control group and was directly immersed in simulated saliva. During the 7-day remineralization process, the remineralizing solution was changed every day, and the application of NaClO-degraded CMC/ACP nanocomplexes and chimaeric-peptide-guided ACP nanoparticles was performed daily.
2.6
Surface characterization
PBS buffer was used to wash the sample surface three times before every test. Samples of enamel with different treatments were prepared for X-ray diffraction (XRD: XRD-6000 X-ray diffractometer, SHIMADZU, Tokyo, Japan), field emission gun scanning electron microscopy (FE-SEM: JSM-5600LV, JEOL, Japan), and laser confocal scanning microscopy (LCSM: Leica SP8, Germany).
XRD patterns were recorded in a Rigaku diffractometer with Cu Ka radiation (k = 1.542 Å) operating at 70 kV and 50 mA in the 2-h range of 10–60°.
The surface morphology was detected by FE-SEM, with a beam voltage of 15 kV and a field emission gun scanning electron microscope (JSM-6701F, JEOL, Japan). The enamel samples were dehydrated using ethanol (at a gradient concentration of 70%, 80%, 90%, and 100% for 20 min) and were sputter-coated with gold before characterization.
The binding capability of chimaeric-peptide-guided CMC/ACP on human tooth enamel was observed using an FITE-labeled peptide. After remineralization treatment, the samples were dried and then observed by confocal laser scanning microscopy (CLSM).
2.7
Mechanical properties of remineralized tooth enamel
Before SEM characterization, all samples were tested using a commercial nanoindenter (Agilent nanoindenter G200; Agilent, USA) equipped with a Berkovich tip. A constant load of 100 mN was applied, and the loading times were kept at 15 s. The load process consisted of a loading approach to the surface (at 10 nm/s), a holding time at the peak load of 10 s, and an unloading period (at 10 nm/s). The nanoindenter machine calculations were performed using Agilent Nanosuite 6.1 Professional software. The nanohardness and elastic modulus were evaluated from the load versus depth curves. The mean value of the elastic modulus and hardness were determined and compared between the different groups after the nano-indentation test of every sample.
2.8
Statistical analysis
The data from the XRD of characteristic peak ratio, nano-particles of CMC/ACP diameters distribution and the hardness and elastic modulus of nano-indentation tests were evaluated using one-way ANOVA at the 95% confidence level. A multiple range test (Fisher’s least significant difference (LSD) procedure) at a 95% confidence level was performed to identify statistically homogeneous groups. For mechanical testing, Student’s t -test was applied to identify differences in the hardness and elastic modulus between the samples of etched, natural, and repaired enamel.
2
Material and methods
The institutional review board of the Tianjin Medical University approved all of the procedures and methods conducted in this study (TMUh-MEC2012019).
2.1
Preparation of highly supersaturated CMC/ACP solutions
The CMC solution was first prepared by mixing 200 mg of CMC powder (95%, Qingdao Hong Hai Biological Technology Co., LTD, Qingdao, China) with 40 ml of double-distilled water and stirring (1000 rpm) until the powder was entirely dissolved. Next, 55.68 mg of K 2 HPO 4 was added to the CMC solution under slight stirring (500 rpm). A total of 94.08 mg of CaCl 2 ·2H 2 O was then added to 10 ml of double-distilled water, and this solution was instilled into the CMC solution gently under continuous stirring to form a highly supersaturated CMC/ACP solution. The final concentrations of calcium and phosphate ions were 8 and 16 mM, respectively. The CMC/ACP solution was stored in a refrigerator at 4 °C.
Before the application of CMC/ACP, 1 ml of 1% NaClO was added to 10 ml of CMC/ACP, followed by mixing at 500 rpm for 5 min.
The diameters of the CMC/ACP solution and NaClO degraded solution were tested through Mastersizer laser particle size analyzer (Mastersizer 2000, Malvern, Worcestershire, UK). The size of the particles can be calculated and analyzed.
2.2
Tooth sample preparation
Ten human unerupted third molars were obtained from patients aged between 18 and 30 years who needed preventive extraction before orthodontic therapy at the Hospital of Stomatology, Tianjin Medical University. All the patients agreed to the study and signed the written informed consent forms. The inclusion criteria were that the enamel of the teeth was mature and without caries, cracks, or other defects and that the integrity of the teeth was destroyed. With the removal of the root and pulp, 4 wafers were sawn from the buccal, lingual, mesial, and proximal surfaces of each tooth to obtain similar enamel slabs and were randomly distributed into groups A–D, respectively. Forty enamel blocks of approximately 3 × 3 × 0.5 mm 3 (length × width × height) in size in the 4 groups were prepared and sealed with water-resistant nail varnish, except for a small window area (2 × 2 mm 2 ) for demineralization and remineralization treatments. Before demineralizing treatment, all enamel samples were first cleaned through sonic oscillation for 30 min and were wiped with 75% ethyl alcohol repeatedly. Thereafter, 3 wafers from each tooth in groups A–C underwent demineralization treatment, prepared by coating a gel of 37% phosphoric acid (Gluma Gel, Heraeus Kulzer, Hanau, Germany) on the surface of the enamel for 30 s. One wafer from each tooth was left untreated as a natural tooth control in group D. Next, the samples were washed with sterile DI water for 60 s, sonicated again for 5 min, and stored at 4 °C in sterile DI water prior to use.
2.3
Synthesis and primary software test of guiding chimaeric peptides and assembly of nanoparticles
Two chimaeric peptides, Peptide A (SVSVGMKPSPRPGGGGSLEAWPATDKTKREEVD) and Peptide B (LEAWPATDKTKREEVD) as the control, were produced via solid-phase peptide synthesis at the Shanghai Top-peptide Co., Ltd. Peptide A was equipped with two functional motifs that control binding management and the transformation area together with the flexible peptide linker GGGGS. Peptide B was isolated from the binding domain, and both of the chimaeric peptides were labeled with FITC prior to fluorescence detection. The secondary structure of Peptide A was predicted using PSIPRED software ( http://bioinf.cs.ucl.ac.uk/psipred/ ).
A total of 10 mg of peptide was added to 10 ml of NaClO-degraded CMC/ACP with a stirring speed of 500 rpm to form the peptide-guiding ACP solution, which was placed at room temperature for at least 30 min for the assembly of nanoparticles before use.
2.4
TEM characterization of nanocomplexes of CMC/ACP and chimaeric-peptide-guided CMC/ACP
The size and morphology of the nanocomplexes of CMC/ACP nanoparticles, NaClO-degraded CMC/ACP, and chimaeric-peptide-guided ACP nanoparticles were characterized using transmission electron microscopy (TEM) (JEM-1230, JEOL, Tokyo, Japan) and selected area electron diffraction (SAED) at 110 kV. Fifteen milliliters of nanoparticle solution was dropped onto a 400-mesh copper TEM grid covered by a carbon support film at room temperature. Excess liquid was removed by the filter paper beneath the grid.
2.5
Remineralization of the demineralized enamel model
Samples in group A (n = 10), which served as the control group, were treated with demineralized enamel without a biomimetic by immersing the samples in simulated saliva (5 ml/sample) containing 1.5 mM Ca 2+ , 0.9 mM HPO 4 2+ , and HEPES. In group B, the remineralization treatments were carried out by coating NaClO-degraded CMC/ACP nanocomplex solutions (100 μl) onto the demineralized surface using disposable micro-applicators (TPC, Advanced Technology, USA) repeatedly for 10 min. Group C was treated with chimaeric-peptide-guided ACP nanoparticles on the surface of the window area of the samples. Both Group B and C samples were immersed in simulated saliva after treatment. Group D was the natural tooth control group and was directly immersed in simulated saliva. During the 7-day remineralization process, the remineralizing solution was changed every day, and the application of NaClO-degraded CMC/ACP nanocomplexes and chimaeric-peptide-guided ACP nanoparticles was performed daily.
2.6
Surface characterization
PBS buffer was used to wash the sample surface three times before every test. Samples of enamel with different treatments were prepared for X-ray diffraction (XRD: XRD-6000 X-ray diffractometer, SHIMADZU, Tokyo, Japan), field emission gun scanning electron microscopy (FE-SEM: JSM-5600LV, JEOL, Japan), and laser confocal scanning microscopy (LCSM: Leica SP8, Germany).
XRD patterns were recorded in a Rigaku diffractometer with Cu Ka radiation (k = 1.542 Å) operating at 70 kV and 50 mA in the 2-h range of 10–60°.
The surface morphology was detected by FE-SEM, with a beam voltage of 15 kV and a field emission gun scanning electron microscope (JSM-6701F, JEOL, Japan). The enamel samples were dehydrated using ethanol (at a gradient concentration of 70%, 80%, 90%, and 100% for 20 min) and were sputter-coated with gold before characterization.
The binding capability of chimaeric-peptide-guided CMC/ACP on human tooth enamel was observed using an FITE-labeled peptide. After remineralization treatment, the samples were dried and then observed by confocal laser scanning microscopy (CLSM).
2.7
Mechanical properties of remineralized tooth enamel
Before SEM characterization, all samples were tested using a commercial nanoindenter (Agilent nanoindenter G200; Agilent, USA) equipped with a Berkovich tip. A constant load of 100 mN was applied, and the loading times were kept at 15 s. The load process consisted of a loading approach to the surface (at 10 nm/s), a holding time at the peak load of 10 s, and an unloading period (at 10 nm/s). The nanoindenter machine calculations were performed using Agilent Nanosuite 6.1 Professional software. The nanohardness and elastic modulus were evaluated from the load versus depth curves. The mean value of the elastic modulus and hardness were determined and compared between the different groups after the nano-indentation test of every sample.
2.8
Statistical analysis
The data from the XRD of characteristic peak ratio, nano-particles of CMC/ACP diameters distribution and the hardness and elastic modulus of nano-indentation tests were evaluated using one-way ANOVA at the 95% confidence level. A multiple range test (Fisher’s least significant difference (LSD) procedure) at a 95% confidence level was performed to identify statistically homogeneous groups. For mechanical testing, Student’s t -test was applied to identify differences in the hardness and elastic modulus between the samples of etched, natural, and repaired enamel.
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