- Email: [email protected]
Regeneration of biomimetic hydroxyapatite on etched human enamel by anionic PAMAM template in vitro
archives of oral biology 58 (2013) 975–980
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Regeneration of biomimetic hydroxyapatite on etched human enamel by anionic PAMAM template in vitro Liang Chen a,b, Kunneng Liang a,b, Jianshu Li c, Duo Wu c, Xuedong Zhou a,b, Jiyao Li a,b,* a
State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China Department of Endodontics and Operative Dentistry, West China School of Stomatology, Sichuan University, Chengdu, China c Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu, China b
article info
abstract
Article history:
Objective: To repair the demineralized enamel by biomimetic method, and the effect of Poly
Accepted 17 March 2013
(amido amine) (PAMAM) dendrimers on the crystallization of hydroxyapatite on etched
Keywords:
Design: PAMAM dendrimers were synthesized step by step following the classical method
Dental caries
and modified with the carboxylic acid groups (COOH) on the surface. Demineralized human
Biomimetic mineralization
enamel samples were immersed in 10,000 ppm PAMAM-COOH solution for 30 min and then
enamel surface is investigated.
Human enamel
in calcium phosphorous solution with or without fluoride under near-clinical conditions for
PAMAM
20 h. Other samples without PAMAM-COOH were immersed in calcium phosphorous solu-
Remineralization
tion as the control group. After the immersion, the micro structure, morphology and composition of the regrown crystals on the longitudinal and transversal enamel surfaces were investigated by SEM, XRD and FTIR, and the results were compared with etched enamel and intact enamel. Results: With the PAMAM-COOH templates, well-arranged rod-like crystals were formed and they were parallel to the long axis of enamel crystals, which was more obvious on the longitudinal enamel surface. Otherwise, irregular flake-like crystals were obtained without PAMAM-COOH. Fluorapatite was not influenced by the PAMAM-COOH but its specific distribution also shown the patterns of the PAMAM-COOH temples XRD spectra showed that the main phase of the obtained crystals with PAMAM-COOH was hydroxyapatite and their morphology and structure were close to the intact enamel. Amide I band and two bands of methylene groups of PAMAM-COOH detected by FTIR demonstrated the presence of PAMAM-COOH within the biomimetic coating. Conclusions: It was concluded that PAMAM-COOH can play as the organic template on the demineralized enamel surface to induce the formation of HAP crystals with the same structure, orientation and mineral phase of the intact enamel in relatively short time. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental enamel is the hardest mineralized tissue in the human body.1,2 This hard tissue comprises 96% inorganic materials
and 4% organic materials and water by weight. The inorganic content is nanorod-like hydroxyapatite crystals arranged into highly organized hierarchical microstructures. These special structures play an important role in determining the unique physicochemical properties of dental enamel. Recent studies
* Corresponding author at: Department of Operative Dentistry and Endodontics, West China Hospital of Stomatology, No. 14, Unit 3, Renmin Nan Road, Chengdu City 610041, Sichuan Province, China. Tel.: +86 28 85501439; fax: +86 28 85582167. E-mail addresses: [email protected], [email protected] (J. Li). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.03.008
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archives of oral biology 58 (2013) 975–980
have reported that extracellular matrix proteins, such as amelogenin, are essential for the control and modulation of these special structures during the biomineralization of enamel.3–10 However, these proteins that induce and control the crystallization of apatite are almost completely degraded or removed during enamel maturation.4,6 Therefore, unlike dentine and bone, mature enamel has no ability to reform highly organized crystals. Conventional treatments replace the defected enamel with substitute materials, such as resin and amalgam. These substitute materials are quite different from the normal enamel in chemical composition and crystal structure. Moreover, these materials require the sacrifice of healthy tooth tissue. Thus, the materials are not ideal for repairing defective enamel. Presently, the biomimetic synthesis of enamel-like hydroxyapatite (HAP) has attracted much interest from research groups. Many methods have been used in vitro to form enamel-like crystals, including amelogenin, calcium phosphate solution and nano-hydroxyapatite. Some have used amelogenin to mimic the biomineralization process of enamel.11–18 However, the structure of amelogenin and mechanisms of amelogenin-mediated mineralization have not yet been fully elucidated. Additionally, amelogenin is difficult to extract and store, features that make it relatively unsuitable for further clinical use. Poly(amido amine) (PAMAM) dendrimers are highly branched polymers. Unlike conventional linear polymers, they are characterized by the presence of internal cavities, a large number of reactive end groups and well-defined size and shape. These structures allow it to be used as biomimetic macromolecules for proteins. Zhou et al.19 and Yan et al.20 demonstrated that PAMAM dendrimers with carboxylic groups have an effective influence on the size and shape of hydroxyapatite nanostructures. Zhang et al.21 reported that PAMAM dendrimers appeared to be an inhibitor for crystal formation and affected crystal morphology and particle size during mineralization in vitro. Chen et al.22,23 demonstrated that PAMAM dendrimers capped with carboxylic acid can be absorbed to enamel crystals. In addition, Sheng Yang et al.24 reported an amphiphilic PAMAM dendrimer that mimics the self-assembly behaviour of amelogenins in vitro. They observed that SaPAMAM-Asp self-assembled into nanospheres initially and further translated into linear chains either by increasing the concentration or by adding calcium ion at pH 7.4 and 37 8C in aqueous solution. The critical aggregation concentration for this translation was 5.5 106 M, and linear chains of 100 nm to 1.5 mm in length were finally observed. Therefore, the effect of PAMAM on HAP synthesis has casted some light on the biomimetic mineralization of enamel crystals. Based on previous studies, the aim of the present study was to investigate the effect of G3.0 PAMAM dendrimers with carboxylic acid (PAMAM-COOH) on crystal growth on etched enamel at 37 8C and pH 7.0. The morphology and mineral phase of the regrown crystal are analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), and a possible mechanism is explored explaining how the enamel-like structures are formed. The hypothesis is that the PAMAM dendrimers could act as an organic template to control the growth of crystals on the enamel surface.
2.
Materials and methods
2.1.
PAMAM synthesis
The divergent synthesis of PAMAM dendrimers included a two-step interactive sequence to produce amine-terminated structures. Iterative sequencing involved alkylation with methyl acrylate (MA) followed by amidation with excess 1,2ethylenediamine (EDA). The alkylation step produced esterterminated intermediates that were referred to as ‘‘halfgenerations’’. The second step involved amidation of the ester-terminated intermediates with a large excess of EDA to produce amine terminated intermediates, which are referred to as ‘‘full-generations’’. G3.0 PAMAM dendrimers were synthesized for further carboxyl modification. 2 g of dry G3.0 PAMAM was dissolved in 25 mL dimethyl sulfoxide (DMSO) in a round-bottom flask. To this dendrimer solution, 15 mL of the DMSO solution containing 5.15 g succinic anhydride (molar ratio of SAH/–NH2 = 5:1) was added under vigorous stirring and was reacted for 24 h without oxygen. The DMSO solution was dialyzed against water to remove the excess succinic anhydride as well as the organic solvent. The aqueous retentate was filtered using a 0.45 mm diameter membrane, and then was lyophilized.
2.2.
Tooth sample preparation
Human third molars were obtained from subjects aged between 18 and 30 years at the West China Hospital of Stomatology, Sichuan University, on the criterion that the enamel of the teeth had matured without caries, cracks or other defects. The study was approved by the hospital’s Institutional Review Board. The roots were removed, and the enamels were cut longitudinally or transversely using a lowspeed diamond saw (Minitom, Struers, Denmark) with water to obtain enamel samples that are approximately 3 3 1 mm, and then the test surfaces were polished using 600-, 1000-, 2000-, 4000-grit SiC wet paper in sequence with the machine wheel set to 250 rpm. All the enamel samples were stored in 0.1% thymol solution at 4 8C before use.
2.3.
PAMAM coating preparation
The enamel samples were etched by 3% HNO3 solution for 50 s, ultrasonically cleaned for 10 min, and then rinsed with sufficient de-ionized water. A 10,000-ppm PAMAM solution was prepared and statically placed for 18 h to ensure the selfassembly process was completed. The PAMAM coating was obtained by immersing the etched enamel samples in the PAMAM solution. After 30 min, the enamel samples were removed from the solution, rinsed with running de-ionized water for 50 s and air-dried.
2.4.
Mineralization solution
The samples were immersed in 3 mL of calcification solution containing 2.58 mM CaCl2 and 1.55 mM KH2PO4 at 37 8C, buffered by 20 mM HEPES and 180 mM NaCl. The concentration of the mineralization solution was referred from previous
archives of oral biology 58 (2013) 975–980
studies, in which amelogenin was used as the template to form the biomimetic HAP on the etched enamel.15,18 The pH of the mineralization solution was adjusted to 7.00 using 1 M NaOH. NaF was added to obtain 1 mg/L fluoride, immediately before immersing tooth samples. The mineralization solution was sealed air-tight in a 5-mL microcentrifuge tube and incubated at 37 8C statically for 20 h. After the desired time, the tooth samples were removed from the solution, rinsed with running de-ionized water for 50 s and air-dried.
2.5.
Characterization
The enamel surfaces were analyzed by SEM (Inspect F50; FEI, USA). The surfaces were sputter coated with Au before observation. The crystal sizes in the digital SEM images were measured using Photoshop software (n > 15). ATR-IR (NICOLET iS10; Thermo Scientific, USA) spectra were used for compositional analysis of the regrown crystals. X-ray diffraction analysis (X’Pert PRO MPD; PANalytical, Netherlands) was performed to detect the crystal orientation and mineral phase of the new crystals. The samples for XRD analysis were freshly prepared to prevent hydrolysis of octacalcium phosphate. The average crystallite sizes along the c and a axes were calculated
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from the (0 0 2) and (2 1 1) indexed XRD peaks, respectively, using the Scherrer equation as follows: Kl D ðnmÞ ¼ n cos u where D is the crystallite size in nm, K is the Scherrer constant (here, K = 0.9), l is the X-ray wavelength in nm (here, l = 0.154), b is the half-maximum intensity of the diffraction peaks, and u is the diffraction angle for the diffraction peaks.
3.
Results
The SEM results of the longitudinal and transversal enamel surfaces showed that, after the etching, peripheral regions of enamel prisms were more demineralized than central regions, so the outline of the prisms became more distinct. After soaking in the calcium phosphate solution, irregularly arranged flake-like crystals with a thickness of 2 mm formed on the enamel surface. There were large gaps between the flakes. With 1 ppm NaF in the remineralization solutions, the morphology of the crystals changed to a needle-like morphology with lengths of 1–2 mm. In the presence of PAMAM-COOH,
Fig. 1 – SEM images of the enamel surface after acidic etching (A). SEM images of crystals grown on acid-etched enamel for 20 h at 37 8C in HEPES-buffered calcium phosphate solution without the PAMAM-COOH template and fluoride (B), with 1 ppm sodium fluoride (C), with the PAMAM-COOH template (D), and with both 1 ppm sodium fluoride and the PAMAMCOOH template (E) (Group 1 for transversal surface and Group 2 for longitudinal surface).
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archives of oral biology 58 (2013) 975–980
Fig. 2 – SEM images of crystals grown on longitudinal enamel with 1 ppm sodium fluoride and the PAMAMCOOH template. The distribution of F-HAP was significantly influenced by the PAMAM-COOH template.
the gaps between enamel prisms were restored by rod-like crystals on the transversal section, and the new crystals preferred to grow in the peripheral regions than in the central regions of enamel prisms. On the longitudinal section, short rod-like crystals were formed on the enamel surface, interprismatic gaps disappeared, and the direction of the new crystals was parallel to the long axis of the enamel crystals Figs. 1–4. The results of ATR FT-IR indicated that the amide I band at 1650 cm1 appeared on the biomimetic coating. On the etched
Fig. 4 – The ATR FT-IR spectrum of the biomimetic coating on enamel prepared after 20 h compared with intact enamel and etched enamel. The presence of an amide I band at 1650 cmS1, and two bands at 2926 cmS1 and 2854 cmS1 resulting from the methylene groups in PAMAM-COOH were significant.
enamel surface, there were no characteristic peaks detected for organics. The XRD results showed that the length of the particles was 37.05 nm for intact enamel, 36.40 nm for etched enamel and 38.27 nm for the biomimetic coating; the width was 24.01 nm for intact enamel, 26.65 nm for etched enamel and 21.67 nm for the biomimetic coating. The aspect ratio was 1.54, 1.36 and 1.76, respectively. The results revealed that the average crystal sizes and preferential orientation of the biomimetic coating were almost the same with the intact enamel.25
4.
Fig. 3 – The XRD spectrum of the biomimetic coating on enamel with the PAMAM-COOH template. The presence of a hydroxyapatite diffraction band (0 0 2) at 25.9, (2 1 1) at 31.8, (1 1 2) at 32.2, and (3 0 0) at 32.9 was clearly detected. The PAMAM-COOH template did not alter the mineral phase of the calcium phosphate coating significantly, compared with the intact enamel.
Discussion
Chen et al. have demonstrated that PAMAM-COOH can be bound to single crystals from enamel through the interaction between anionic branches of the dendrimers and cationic calcium on the crystal surface.22,23 In the present study, FTIR showed an amide I band at 1650 cm1 at the surface of intact enamel, which may be induced by residual proteins15; after the etching and rinsing, all the proteins were supposed to be removed. However, the amide I band at 1650 cm1 reappeared at the remineralized surface due to the PAMAM template. Additionally, two characteristic bands of methylene groups in PAMAM-COOH at 2926 and 2754 cm1 were also detected. Both results demonstrated the presence of PAMAM-COOH within the biomimetic coating.26 The interaction between proteins and hydroxyapatite indicate that acidic proteins are preferentially adsorbed on the crystal face along the c axis because the crystal face is more positively charged than other faces.27–29 PAMAM-COOH demonstrated the same characteristic through AFM observation.22 In the present study, due to demineralization, peripheral regions of enamel prisms were suggested to be more exposed areas along with the c axis than at central regions,
archives of oral biology 58 (2013) 975–980
which can absorb more PAMAM self-assemblies to increase nucleation and growth of new crystals around enamel prisms. Therefore, Fig. 1 shows processes of crystals surrounding the prisms on the transversal surface. This finding also explains why the phenomenon was more obvious in SEM of the longitudinal section. In recent studies, many methods in vitro have been used to mimic the biomineralization of human enamel, including amelogenin, calcium phosphate solution and nano-hydroxyapatite. These results show the successful formation of enamel-like crystals. However, the experimental conditions are too harsh for clinics, and the retained crystals are perpendicular to the enamel surface without consideration of the direction of the original enamel prisms. The original crystals are very important because we believe that the principle of caries treatment, which require complete removal of infected enamel, must also be suitable for the future application of enamel biomineralization in clinics. Cavities surrounded by healthy enamel with prisms of different directions must be prepared, and if the regrown enamel layers are perpendicular to the cavity walls – particularly the axial walls – the micro structure of the final repaired enamel will be totally different from the original enamel. In the present study, we demonstrated that with the effect of the PAMAM-COOH template, the biomimetic crystals can be formed in a relatively short time and under near-clinical conditions, and the direction of the regrown crystals can be consistent with the enamel prisms on both longitudinal and transversal surfaces. This result has not been reported in any previous study. Conclusively, we proposed that PAMAM self-assemblies play as an organic template between the regrown crystals and etched enamel regulating both mineral nucleation and crystal growth. First, the chain-like PAMAM self-assemblies were adsorbed on the surface of the enamel crystals by electrostatic interaction between carboxylic terminates and enamel crystals. When in the calcium phosphate solution, the template served as a nucleation site due to calcium binding on the carboxylic acid terminals. The interaction led to a high local concentration of calcium ions, and the deposition of HAP occurs when the phosphate ions arrived. As the deposition continued, HAP crystals started growing at specific sites along the long axis of enamel crystals and finally formed a rod-like morphology, parallel to the enamel prisms. Although the FHAP crystals were not parallel with the enamel prism, and the underlying cause warrants further study, Fig. 2 shows that the distribution of F-HAP was significantly influenced by the PAMAM-COOH template. We can obviously see the patterns of enamel prisms through the F-Hap layer. It is assumed that the nucleation of F-HAP was controlled by the PAMAM-COOH template. However, subsequently, F-HAP crystals grew too fast in length to be controlled by the PAMAM-COOH template. Therefore, needle-like crystals are still observed even with the template.
5.
Conclusions
Previous studies of biomimetic remineralization of enamel only focused on the morphology and structure of the
979
newborn crystals, but few studies were concerned with their relationship with the original enamel crystals. This biomimetic structure is very important for the regeneration of teeth in reparative and restorative dentistry. Initially, in the present study, we successfully formed new rod-like crystals with the same structure, orientation and mineral phase of intact enamel, and the hydroxyapatite nanorods were closely paralleled to the original prisms. Therefore, we conclude that G3-PAMAM-COOH can be absorbed on the exposed enamel prisms and can function as the organic template for biomimetic remineralization on etched enamel.
Funding National Natural Science Foundation of China (Grant No. 81170958 and 51073102). Specialized Research Fund for the Doctoral Program of Higher Education (20100181110056).
Competing interest The authors do not have any possible conflicts of interest.
Ethical approval statement None.
Acknowledgments This research was supported by the National Natural Science Foundation of China (Grant Nos. 81170958 and 51073102) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20100181110056).
references
1. Palmer LC, Newcomb CJ, Kaltz SR, Spoerke ED, Stupp SI, Bone I. Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel. Chemical Reviews 2008;108(11):4754–83. 2. Simmer JP, Fincham AG. Molecular mechanisms of dental enamel formation. Critical Reviews in Oral Biology and Medicine 1995;6(2):84–108. 3. Moradian-Oldak J. Amelogenins: assembly, processing and control of crystal morphology. Matrix Biology 2001;20(5/ 6):293–305. 4. Ye L, Le TQ, Zhu L, Butcher K, Schneider RA, Li W, et al. Amelogenins in human developing and mature dental pulp. Journal of Dental Research 2006;85(9):814–8. 5. Robinson C, Brookes SJ, Kirkham J, Bonass WA, Shore RC. Crystal growth in dental enamel: the role of amelogenins and albumin. Advances in Dental Research 1996;10(2):173–9. 6. Le T, Denbesten P, Huang Y, Zhu L, Uskokovic V, Habelitz S, et al. Altered self-assembly and apatite binding of amelogenin induced by N-terminal proline mutation. Archives of Oral Biology 2011;56(4):331–6.
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7. Robinson C, Connell S, Brookes SJ, Kirkham J, Shore RC, Smith DA. Surface chemistry of enamel apatite during maturation in relation to pH: implications for protein removal and crystal growth. Archives of Oral Biology 2005;50(2):267–70. 8. Moradian-Oldak J, Iijima M, Bouropoulos N, Wen HB. Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology. Connective Tissue Research 2003;44(Suppl. (1)):58–64. 9. Du C, Falini G, Fermani S, Abbott C, Moradian-Oldak J. Supramolecular assembly of amelogenin nanospheres into birefringent microribbons. Science 2005;307(5714):1450–4. 10. Moradian-Oldak J, Du C, Falini G. On the formation of amelogenin microribbons. European Journal of Oral Sciences 2006;114(Suppl. (22)):289–96. 11. No¨r JE. Buonocore memorial lecture. Operative Dentistry 2006;31(6):633–42. 12. Iijima M, Du C, Abbott C, Doi Y, Moradian-Oldak J. Control of apatite crystal growth by the co-operative effect of a recombinant porcine amelogenin and fluoride. European Journal of Oral Sciences 2006;114(Suppl. (1)):304–7. 13. Hunter GK, Curtis HA, Grynpas MD, Simmer JP, Fincham AG. Effects of recombinant amelogenin on hydroxyapatite formation in vitro. Calcified Tissue International 1999;65(3):226–31. 14. Uskokovic´ V, Li W, Habelitz S. Amelogenin as a promoter of nucleation and crystal growth of apatite. Journal of Crystal Growth 2011;316(1):106–17. 15. Fan Y, Sun Z, Moradian-Oldak J. Controlled remineralization of enamel in the presence of amelogenin and fluoride. Biomaterials 2009;30(4):478–83. 16. Iijima M, Moriwaki Y, Wen HB, Fincham AG, MoradianOldak J. Elongated growth of octacalcium phosphate crystals in recombinant amelogenin gels under controlled ionic flow. Journal of Dental Research 2002;81(1):69–73. 17. Bouropoulos N, Moradian-Oldak J. Induction of apatite by the cooperative effect of amelogenin and the 32-kDa enamelin. Journal of Dental Research 2004;83(4):278–82. 18. Fan Y, Nelson JR, Alvarez JR, Hagan J, Berrier A, Xu X. Amelogenin-assisted ex vivo remineralization of human enamel: effects of supersaturation degree and fluoride concentration. Acta Biomaterialia 2011;7(5):2293–302.
19. Zhou Z-H, Zhou P-L, Yang S-P, Yu X-B, Yang L-Z. Controllable synthesis of hydroxyapatite nanocrystals via a dendrimer-assisted hydrothermal process. Materials Research Bulletin 2007;42(9):1611–8. 20. Yan S, Zhou Z-H, Zhang F, Yang S-P, Yang L, Yu X-B. Effect of anionic PAMAM with amido groups starburst dendrimers on the crystallization of Ca10(PO4)6(OH)2 by hydrothermal method. Materials Chemistry and Physics 2006;99(1):164–9. 21. Zhang F, Zhou Z, Yang S-P, Mao L, Chen H, Yu X. Hydrothermal synthesis of hydroxyapatite nanorods in the presence of anionic starburst dendrimer. Materials Letters 2005;59(11):1422–5. 22. Chen H, Banaszak Holl M, Orr BG, Majoros I, Clarkson BH. Interaction of dendrimers (artificial proteins) with biological hydroxyapatite crystals. Journal of Dental Research 2003;82(6):443–8. 23. Chen H, Chen Y, Orr BG, Holl MMB, Majoros I, Clarkson BH. Nanoscale probing of the enamel nanorod surface using polyamidoamine dendrimers. Langmuir 2004;20(10):4168–71. 24. Sheng Yang. Hai He. Lei Wang. Xinru Jia. Hailan Feng. Oriented crystallization of hydroxyapatite by the biomimetic amelogenin nanospheres from self-assemblies of amphiphilic dendrons. Chemical Communications 2011;47(36):10100–2. 25. Xue J, Zhang L, Zou L, Liao Y, Li J, Xiao L, et al. Highresolution X-ray microdiffraction analysis of natural teeth. Journal of Synchrotron Radiation 2008;15(Pt 3):235–8. 26. Suk J, Lee J, Kwak J. Electrochemistry on alternate structures of gold nanoparticles and ferrocene-tethered polyamidoamine dendrimers. Bulletin of the Korean Chemical Society 2004;25(11):1681–6. 27. Fujisawa R, Kuboki Y. Preferential adsorption of dentin and bone acidic proteins on the (1 0 0) face of hydroxyapatite crystals. Biochimica et Biophysica Acta 1991;1075(1):56–60. 28. Okazaki M, Yoshida Y, Yamaguchi S, Kaneno M, Elliott JC. Affinity binding phenomena of DNA onto apatite crystals. Biomaterials 2001;22(18):2459–64. 29. Matsumoto T, Okazaki M, Inoue M, Hamada Y, Taira M, Takahashi J. Crystallinity and solubility characteristics of hydroxyapatite adsorbed amino acid. Biomaterials 2002;23(10):2241–7.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Regeneration of biomimetic hydroxyapatite on etched human enamel by anionic PAMAM template in vitro Liang Chen a,b, Kunneng Liang a,b, Jianshu Li c, Duo Wu c, Xuedong Zhou a,b, Jiyao Li a,b,* a
State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China Department of Endodontics and Operative Dentistry, West China School of Stomatology, Sichuan University, Chengdu, China c Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu, China b
article info
abstract
Article history:
Objective: To repair the demineralized enamel by biomimetic method, and the effect of Poly
Accepted 17 March 2013
(amido amine) (PAMAM) dendrimers on the crystallization of hydroxyapatite on etched
Keywords:
Design: PAMAM dendrimers were synthesized step by step following the classical method
Dental caries
and modified with the carboxylic acid groups (COOH) on the surface. Demineralized human
Biomimetic mineralization
enamel samples were immersed in 10,000 ppm PAMAM-COOH solution for 30 min and then
enamel surface is investigated.
Human enamel
in calcium phosphorous solution with or without fluoride under near-clinical conditions for
PAMAM
20 h. Other samples without PAMAM-COOH were immersed in calcium phosphorous solu-
Remineralization
tion as the control group. After the immersion, the micro structure, morphology and composition of the regrown crystals on the longitudinal and transversal enamel surfaces were investigated by SEM, XRD and FTIR, and the results were compared with etched enamel and intact enamel. Results: With the PAMAM-COOH templates, well-arranged rod-like crystals were formed and they were parallel to the long axis of enamel crystals, which was more obvious on the longitudinal enamel surface. Otherwise, irregular flake-like crystals were obtained without PAMAM-COOH. Fluorapatite was not influenced by the PAMAM-COOH but its specific distribution also shown the patterns of the PAMAM-COOH temples XRD spectra showed that the main phase of the obtained crystals with PAMAM-COOH was hydroxyapatite and their morphology and structure were close to the intact enamel. Amide I band and two bands of methylene groups of PAMAM-COOH detected by FTIR demonstrated the presence of PAMAM-COOH within the biomimetic coating. Conclusions: It was concluded that PAMAM-COOH can play as the organic template on the demineralized enamel surface to induce the formation of HAP crystals with the same structure, orientation and mineral phase of the intact enamel in relatively short time. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental enamel is the hardest mineralized tissue in the human body.1,2 This hard tissue comprises 96% inorganic materials
and 4% organic materials and water by weight. The inorganic content is nanorod-like hydroxyapatite crystals arranged into highly organized hierarchical microstructures. These special structures play an important role in determining the unique physicochemical properties of dental enamel. Recent studies
* Corresponding author at: Department of Operative Dentistry and Endodontics, West China Hospital of Stomatology, No. 14, Unit 3, Renmin Nan Road, Chengdu City 610041, Sichuan Province, China. Tel.: +86 28 85501439; fax: +86 28 85582167. E-mail addresses: [email protected], [email protected] (J. Li). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.03.008
976
archives of oral biology 58 (2013) 975–980
have reported that extracellular matrix proteins, such as amelogenin, are essential for the control and modulation of these special structures during the biomineralization of enamel.3–10 However, these proteins that induce and control the crystallization of apatite are almost completely degraded or removed during enamel maturation.4,6 Therefore, unlike dentine and bone, mature enamel has no ability to reform highly organized crystals. Conventional treatments replace the defected enamel with substitute materials, such as resin and amalgam. These substitute materials are quite different from the normal enamel in chemical composition and crystal structure. Moreover, these materials require the sacrifice of healthy tooth tissue. Thus, the materials are not ideal for repairing defective enamel. Presently, the biomimetic synthesis of enamel-like hydroxyapatite (HAP) has attracted much interest from research groups. Many methods have been used in vitro to form enamel-like crystals, including amelogenin, calcium phosphate solution and nano-hydroxyapatite. Some have used amelogenin to mimic the biomineralization process of enamel.11–18 However, the structure of amelogenin and mechanisms of amelogenin-mediated mineralization have not yet been fully elucidated. Additionally, amelogenin is difficult to extract and store, features that make it relatively unsuitable for further clinical use. Poly(amido amine) (PAMAM) dendrimers are highly branched polymers. Unlike conventional linear polymers, they are characterized by the presence of internal cavities, a large number of reactive end groups and well-defined size and shape. These structures allow it to be used as biomimetic macromolecules for proteins. Zhou et al.19 and Yan et al.20 demonstrated that PAMAM dendrimers with carboxylic groups have an effective influence on the size and shape of hydroxyapatite nanostructures. Zhang et al.21 reported that PAMAM dendrimers appeared to be an inhibitor for crystal formation and affected crystal morphology and particle size during mineralization in vitro. Chen et al.22,23 demonstrated that PAMAM dendrimers capped with carboxylic acid can be absorbed to enamel crystals. In addition, Sheng Yang et al.24 reported an amphiphilic PAMAM dendrimer that mimics the self-assembly behaviour of amelogenins in vitro. They observed that SaPAMAM-Asp self-assembled into nanospheres initially and further translated into linear chains either by increasing the concentration or by adding calcium ion at pH 7.4 and 37 8C in aqueous solution. The critical aggregation concentration for this translation was 5.5 106 M, and linear chains of 100 nm to 1.5 mm in length were finally observed. Therefore, the effect of PAMAM on HAP synthesis has casted some light on the biomimetic mineralization of enamel crystals. Based on previous studies, the aim of the present study was to investigate the effect of G3.0 PAMAM dendrimers with carboxylic acid (PAMAM-COOH) on crystal growth on etched enamel at 37 8C and pH 7.0. The morphology and mineral phase of the regrown crystal are analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), and a possible mechanism is explored explaining how the enamel-like structures are formed. The hypothesis is that the PAMAM dendrimers could act as an organic template to control the growth of crystals on the enamel surface.
2.
Materials and methods
2.1.
PAMAM synthesis
The divergent synthesis of PAMAM dendrimers included a two-step interactive sequence to produce amine-terminated structures. Iterative sequencing involved alkylation with methyl acrylate (MA) followed by amidation with excess 1,2ethylenediamine (EDA). The alkylation step produced esterterminated intermediates that were referred to as ‘‘halfgenerations’’. The second step involved amidation of the ester-terminated intermediates with a large excess of EDA to produce amine terminated intermediates, which are referred to as ‘‘full-generations’’. G3.0 PAMAM dendrimers were synthesized for further carboxyl modification. 2 g of dry G3.0 PAMAM was dissolved in 25 mL dimethyl sulfoxide (DMSO) in a round-bottom flask. To this dendrimer solution, 15 mL of the DMSO solution containing 5.15 g succinic anhydride (molar ratio of SAH/–NH2 = 5:1) was added under vigorous stirring and was reacted for 24 h without oxygen. The DMSO solution was dialyzed against water to remove the excess succinic anhydride as well as the organic solvent. The aqueous retentate was filtered using a 0.45 mm diameter membrane, and then was lyophilized.
2.2.
Tooth sample preparation
Human third molars were obtained from subjects aged between 18 and 30 years at the West China Hospital of Stomatology, Sichuan University, on the criterion that the enamel of the teeth had matured without caries, cracks or other defects. The study was approved by the hospital’s Institutional Review Board. The roots were removed, and the enamels were cut longitudinally or transversely using a lowspeed diamond saw (Minitom, Struers, Denmark) with water to obtain enamel samples that are approximately 3 3 1 mm, and then the test surfaces were polished using 600-, 1000-, 2000-, 4000-grit SiC wet paper in sequence with the machine wheel set to 250 rpm. All the enamel samples were stored in 0.1% thymol solution at 4 8C before use.
2.3.
PAMAM coating preparation
The enamel samples were etched by 3% HNO3 solution for 50 s, ultrasonically cleaned for 10 min, and then rinsed with sufficient de-ionized water. A 10,000-ppm PAMAM solution was prepared and statically placed for 18 h to ensure the selfassembly process was completed. The PAMAM coating was obtained by immersing the etched enamel samples in the PAMAM solution. After 30 min, the enamel samples were removed from the solution, rinsed with running de-ionized water for 50 s and air-dried.
2.4.
Mineralization solution
The samples were immersed in 3 mL of calcification solution containing 2.58 mM CaCl2 and 1.55 mM KH2PO4 at 37 8C, buffered by 20 mM HEPES and 180 mM NaCl. The concentration of the mineralization solution was referred from previous
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studies, in which amelogenin was used as the template to form the biomimetic HAP on the etched enamel.15,18 The pH of the mineralization solution was adjusted to 7.00 using 1 M NaOH. NaF was added to obtain 1 mg/L fluoride, immediately before immersing tooth samples. The mineralization solution was sealed air-tight in a 5-mL microcentrifuge tube and incubated at 37 8C statically for 20 h. After the desired time, the tooth samples were removed from the solution, rinsed with running de-ionized water for 50 s and air-dried.
2.5.
Characterization
The enamel surfaces were analyzed by SEM (Inspect F50; FEI, USA). The surfaces were sputter coated with Au before observation. The crystal sizes in the digital SEM images were measured using Photoshop software (n > 15). ATR-IR (NICOLET iS10; Thermo Scientific, USA) spectra were used for compositional analysis of the regrown crystals. X-ray diffraction analysis (X’Pert PRO MPD; PANalytical, Netherlands) was performed to detect the crystal orientation and mineral phase of the new crystals. The samples for XRD analysis were freshly prepared to prevent hydrolysis of octacalcium phosphate. The average crystallite sizes along the c and a axes were calculated
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from the (0 0 2) and (2 1 1) indexed XRD peaks, respectively, using the Scherrer equation as follows: Kl D ðnmÞ ¼ n cos u where D is the crystallite size in nm, K is the Scherrer constant (here, K = 0.9), l is the X-ray wavelength in nm (here, l = 0.154), b is the half-maximum intensity of the diffraction peaks, and u is the diffraction angle for the diffraction peaks.
3.
Results
The SEM results of the longitudinal and transversal enamel surfaces showed that, after the etching, peripheral regions of enamel prisms were more demineralized than central regions, so the outline of the prisms became more distinct. After soaking in the calcium phosphate solution, irregularly arranged flake-like crystals with a thickness of 2 mm formed on the enamel surface. There were large gaps between the flakes. With 1 ppm NaF in the remineralization solutions, the morphology of the crystals changed to a needle-like morphology with lengths of 1–2 mm. In the presence of PAMAM-COOH,
Fig. 1 – SEM images of the enamel surface after acidic etching (A). SEM images of crystals grown on acid-etched enamel for 20 h at 37 8C in HEPES-buffered calcium phosphate solution without the PAMAM-COOH template and fluoride (B), with 1 ppm sodium fluoride (C), with the PAMAM-COOH template (D), and with both 1 ppm sodium fluoride and the PAMAMCOOH template (E) (Group 1 for transversal surface and Group 2 for longitudinal surface).
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Fig. 2 – SEM images of crystals grown on longitudinal enamel with 1 ppm sodium fluoride and the PAMAMCOOH template. The distribution of F-HAP was significantly influenced by the PAMAM-COOH template.
the gaps between enamel prisms were restored by rod-like crystals on the transversal section, and the new crystals preferred to grow in the peripheral regions than in the central regions of enamel prisms. On the longitudinal section, short rod-like crystals were formed on the enamel surface, interprismatic gaps disappeared, and the direction of the new crystals was parallel to the long axis of the enamel crystals Figs. 1–4. The results of ATR FT-IR indicated that the amide I band at 1650 cm1 appeared on the biomimetic coating. On the etched
Fig. 4 – The ATR FT-IR spectrum of the biomimetic coating on enamel prepared after 20 h compared with intact enamel and etched enamel. The presence of an amide I band at 1650 cmS1, and two bands at 2926 cmS1 and 2854 cmS1 resulting from the methylene groups in PAMAM-COOH were significant.
enamel surface, there were no characteristic peaks detected for organics. The XRD results showed that the length of the particles was 37.05 nm for intact enamel, 36.40 nm for etched enamel and 38.27 nm for the biomimetic coating; the width was 24.01 nm for intact enamel, 26.65 nm for etched enamel and 21.67 nm for the biomimetic coating. The aspect ratio was 1.54, 1.36 and 1.76, respectively. The results revealed that the average crystal sizes and preferential orientation of the biomimetic coating were almost the same with the intact enamel.25
4.
Fig. 3 – The XRD spectrum of the biomimetic coating on enamel with the PAMAM-COOH template. The presence of a hydroxyapatite diffraction band (0 0 2) at 25.9, (2 1 1) at 31.8, (1 1 2) at 32.2, and (3 0 0) at 32.9 was clearly detected. The PAMAM-COOH template did not alter the mineral phase of the calcium phosphate coating significantly, compared with the intact enamel.
Discussion
Chen et al. have demonstrated that PAMAM-COOH can be bound to single crystals from enamel through the interaction between anionic branches of the dendrimers and cationic calcium on the crystal surface.22,23 In the present study, FTIR showed an amide I band at 1650 cm1 at the surface of intact enamel, which may be induced by residual proteins15; after the etching and rinsing, all the proteins were supposed to be removed. However, the amide I band at 1650 cm1 reappeared at the remineralized surface due to the PAMAM template. Additionally, two characteristic bands of methylene groups in PAMAM-COOH at 2926 and 2754 cm1 were also detected. Both results demonstrated the presence of PAMAM-COOH within the biomimetic coating.26 The interaction between proteins and hydroxyapatite indicate that acidic proteins are preferentially adsorbed on the crystal face along the c axis because the crystal face is more positively charged than other faces.27–29 PAMAM-COOH demonstrated the same characteristic through AFM observation.22 In the present study, due to demineralization, peripheral regions of enamel prisms were suggested to be more exposed areas along with the c axis than at central regions,
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which can absorb more PAMAM self-assemblies to increase nucleation and growth of new crystals around enamel prisms. Therefore, Fig. 1 shows processes of crystals surrounding the prisms on the transversal surface. This finding also explains why the phenomenon was more obvious in SEM of the longitudinal section. In recent studies, many methods in vitro have been used to mimic the biomineralization of human enamel, including amelogenin, calcium phosphate solution and nano-hydroxyapatite. These results show the successful formation of enamel-like crystals. However, the experimental conditions are too harsh for clinics, and the retained crystals are perpendicular to the enamel surface without consideration of the direction of the original enamel prisms. The original crystals are very important because we believe that the principle of caries treatment, which require complete removal of infected enamel, must also be suitable for the future application of enamel biomineralization in clinics. Cavities surrounded by healthy enamel with prisms of different directions must be prepared, and if the regrown enamel layers are perpendicular to the cavity walls – particularly the axial walls – the micro structure of the final repaired enamel will be totally different from the original enamel. In the present study, we demonstrated that with the effect of the PAMAM-COOH template, the biomimetic crystals can be formed in a relatively short time and under near-clinical conditions, and the direction of the regrown crystals can be consistent with the enamel prisms on both longitudinal and transversal surfaces. This result has not been reported in any previous study. Conclusively, we proposed that PAMAM self-assemblies play as an organic template between the regrown crystals and etched enamel regulating both mineral nucleation and crystal growth. First, the chain-like PAMAM self-assemblies were adsorbed on the surface of the enamel crystals by electrostatic interaction between carboxylic terminates and enamel crystals. When in the calcium phosphate solution, the template served as a nucleation site due to calcium binding on the carboxylic acid terminals. The interaction led to a high local concentration of calcium ions, and the deposition of HAP occurs when the phosphate ions arrived. As the deposition continued, HAP crystals started growing at specific sites along the long axis of enamel crystals and finally formed a rod-like morphology, parallel to the enamel prisms. Although the FHAP crystals were not parallel with the enamel prism, and the underlying cause warrants further study, Fig. 2 shows that the distribution of F-HAP was significantly influenced by the PAMAM-COOH template. We can obviously see the patterns of enamel prisms through the F-Hap layer. It is assumed that the nucleation of F-HAP was controlled by the PAMAM-COOH template. However, subsequently, F-HAP crystals grew too fast in length to be controlled by the PAMAM-COOH template. Therefore, needle-like crystals are still observed even with the template.
5.
Conclusions
Previous studies of biomimetic remineralization of enamel only focused on the morphology and structure of the
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newborn crystals, but few studies were concerned with their relationship with the original enamel crystals. This biomimetic structure is very important for the regeneration of teeth in reparative and restorative dentistry. Initially, in the present study, we successfully formed new rod-like crystals with the same structure, orientation and mineral phase of intact enamel, and the hydroxyapatite nanorods were closely paralleled to the original prisms. Therefore, we conclude that G3-PAMAM-COOH can be absorbed on the exposed enamel prisms and can function as the organic template for biomimetic remineralization on etched enamel.
Funding National Natural Science Foundation of China (Grant No. 81170958 and 51073102). Specialized Research Fund for the Doctoral Program of Higher Education (20100181110056).
Competing interest The authors do not have any possible conflicts of interest.
Ethical approval statement None.
Acknowledgments This research was supported by the National Natural Science Foundation of China (Grant Nos. 81170958 and 51073102) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20100181110056).
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