SCIENCE CHINA Materials, Volume 60, Issue 6: 516-528(2017) https://doi.org/10.1007/s40843-017-9038-5

Symmetry-breaking assembled porous calcite microspheres and their multiple dental applications

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  • ReceivedMar 10, 2017
  • AcceptedApr 19, 2017
  • PublishedMay 16, 2017


Biomedical applications of porous calcium carbonate (CaCO3) microspheres have been mainly restricted by their aqueous instability and low remineralization rate. To overcome these obstacles, a novel symmetry-breaking assembled porous calcite microsphere (PCMS) was constructed in an ethanol/water mixed system using a two-step vapor-diffusion/aging crystallization strategy. In contrast to the conventional additive-induced crystallization method, the present strategy was performed under mild conditions and was free from any foreign additives, thus avoiding the potential contamination of the final product. Meanwhile, the prepared PCMSs were characterized by their highly uniform spherical morphology and large open pores, which are favorable for large protein delivery. An antimicrobial study of immunoglobulin Y (IgY)-loaded PCMSs revealed excellent antimicrobial activity against Streptococcus mutans. More importantly, they showed surprisingly rapid transformation to bone minerals in physiological medium. Evaluation of the in vitro efficacy of PCMSs in dentinal tubule occlusion demonstrated their powerful potential to serve as a catalyst in the repair of dental hard tissue. Therefore, the developed PCMSs show great promise as multifunctional biomaterials for dental treatment applications.

Funded by

National Natural Science Foundation of China(51402329,81500806)

Science Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfine Microstructures(SKL201404)

Shanghai Excellent Academic Leaders Program(14XD1403800)


This work was supported by the National Natural Science Foundation of China (51402329 and 81500806), the Science Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfine Microstructures (SKL201404) and Shanghai Excellent Academic Leaders Program (14XD1403800).

Interest statement

The authors declare that they have no conflict of interest.

Contributions statement

Ma M and Yan Y contributed to this work equally. Ma M designed and synthesized the samples; Ma M, Yan Y, Chern S, Qi S and Shang G performed the experiments; Ma M wrote the paper with support from Qi C, Chen H and Wang R. All authors contributed to the general discussion.

Author information

Ming Ma received his PhD degree from Shanghai Institute of Ceramics, Chinese Academy of Sciences (SICCAS) in 2013. He is now an associate professor of SICCAS. His research focuses on the design of mesoporous materials for biomedical applications including anticancer drug delivery, medical imaging and dental restoration.

Yanhong Yan obtained her PhD degree from Wuhan University under the supervision of Prof. Mingwen Fan. She is now a dentist at the Department of Pediatric Dentistry, School of Stomatology, Tongji University. Her research focuses on the structure and properties of anti-dental caries biomaterials.

Raorao Wang obtained his PhD degree from Matsumoto Dental University in Japan under the supervision of Prof. Hiroo Miyazawa. He is now the director of Stomatological Department, Shanghai Tenth People’s Hospital of Tongji University. His research interest focuses on the stem cell-based biological tooth repair and regeneration.

Hangrong Chen received her PhD degree from SICCAS in 2001. She is now a professor of SICCAS, and deputy director of the State Key Laboratory of High Performance Ceramics and Superfine Microstructure. Her research areas include the synthesis of mesoporous materials, multifunctional inorganic biomedical nanomaterials and environmental catalytic materials.


Supplementary information

Supporting data are available in the online version of the paper.


[1] Trushina DB, Bukreeva TV, Kovalchuk MV, et al. CaCO3 vaterite microparticles for biomedical and personal care applications. Mater Sci Eng-C, 2014, 45: 644-658 CrossRef PubMed Google Scholar

[2] Cury JA, Simões GS, Del Bel Cury AA, et al. Effect of a calcium carbonate-based dentifrice on in situ enamel remineralization. Caries Res, 2005, 39: 255-257 CrossRef PubMed Google Scholar

[3] Elabbadi A, Jeckelmann N, Haefliger O, et al. Selective coprecipitation of polyphenols in bioactive/inorganic complexes. ACS Appl Mater Interfaces, 2011, 3: 2764-2771 CrossRef PubMed Google Scholar

[4] Lu P, Arai K, Kuboyama N. Possibility of application of calcium carbonate in pulpotomy of rat molars. Pediatric Dental J, 2010, 20: 45-56 CrossRef Google Scholar

[5] Pickles MJ, Evans M, Philpotts CJ, et al. In vitro efficacy of a whitening toothpaste containing calcium carbonate and perlite. Int Dental J, 2005, 55: 197-202 CrossRef Google Scholar

[6] Ueno Y, Futagawa H, Takagi Y, et al. Drug-incorporating calcium carbonate nanoparticles for a new delivery system. J Control Release, 2005, 103: 93-98 CrossRef PubMed Google Scholar

[7] Wei W, Ma GH, Hu G, et al. Preparation of hierarchical hollow CaCO3 particles and the application as anticancer drug carrier. J Am Chem Soc, 2008, 130: 15808-15810 CrossRef PubMed Google Scholar

[8] Kim YY, Schenk AS, Ihli J, et al. A critical analysis of calcium carbonate mesocrystals. Nat Commun, 2014, 5: 4341 CrossRef PubMed ADS Google Scholar

[9] Yu JG, Guo H, Davis SA, et al. Fabrication of hollow inorganic microspheres by chemically induced self-transformation. Adv Funct Mater, 2006, 16: 2035-2041 CrossRef Google Scholar

[10] Lee K, Wagermaier W, Masic A, et al. Self-assembly of amorphous calcium carbonate microlens arrays. Nat Commun, 2012, 3: 725 CrossRef PubMed ADS Google Scholar

[11] Wolf SE, Leiterer J, Pipich V, et al. Strong stabilization of amorphous calcium carbonate emulsion by ovalbumin: gaining insight into the mechanism of ‘polymer-induced liquid precursor’ processes. J Am Chem Soc, 2011, 133: 12642-12649 CrossRef PubMed Google Scholar

[12] Qi C, Zhu YJ, Lu BQ, et al. ATP-stabilized amorphous calcium carbonate nanospheres and their application in protein adsorption. Small, 2014, 10: 2047-2056 CrossRef PubMed Google Scholar

[13] Hetherington NBJ, Kulak AN, Kim YY, et al. Porous single crystals of calcite from colloidal crystal templates: ACC is not required for nanoscale templating. Adv Funct Mater, 2011, 21: 948-954 CrossRef Google Scholar

[14] Won YH, Jang HS, Chung DW, et al. Multifunctional calcium carbonate microparticles: synthesis and biological applications. J Mater Chem, 2010, 20: 7728-7733 CrossRef Google Scholar

[15] Zhang J, Li Y, Xie H, et al. Calcium carbonate nanoplate assemblies with directed high-energy facets: additive-free synthesis, high drug loading, and sustainable releasing. ACS Appl Mater Interfaces, 2015, 7: 15686-15691 CrossRef Google Scholar

[16] Volodkin DV, Petrov AI, Prevot M, et al. Matrix polyelectrolyte microcapsules:  new system for macromolecule encapsulation. Langmuir, 2004, 20: 3398-3406 CrossRef Google Scholar

[17] Wang X, Kong R, Pan X, et al. Role of ovalbumin in the stabilization of metastable vaterite in calcium carbonate biomineralization. J Phys Chem B, 2009, 113: 8975-8982 CrossRef PubMed Google Scholar

[18] Zhang Z, Gao D, Zhao H, et al. Biomimetic assembly of polypeptide-stabilized CaCO3 nanoparticles. J Phys Chem B, 2006, 110: 8613-8618 CrossRef PubMed Google Scholar

[19] Cölfen H, Antonietti M. Crystal design of calcium carbonate microparticles using double-hydrophilic block copolymers. Langmuir, 1998, 14: 582-589 CrossRef Google Scholar

[20] Chen SF, Yu SH, Jiang J, et al. Polymorph discrimination of CaCO3 mineral in an ethanol/water solution:  formation of complex vaterite superstructures and aragonite rods. Chem Mater, 2006, 18: 115-122 CrossRef Google Scholar

[21] de Leeuw NH, Parker SC. Surface structure and morphology of calcium carbonate polymorphs calcite, aragonite, and vaterite:  an atomistic approach. J Phys Chem B, 1998, 102: 2914-2922 CrossRef Google Scholar

[22] Montes-Hernandez G, Renard F, Findling N, et al. Formation of porous calcite mesocrystals from CO2–H2O–Ca(OH)2 slurry in the presence of common domestic drinks. CrystEngComm, 2015, 17: 5725-5733 CrossRef Google Scholar

[23] Wang J, Li D, Li T, et al. Gelatin tight-coated poly(lactide-co-glycolide) scaffold incorporating rhBMP-2 for bone tissue engineering. Materials, 2015, 8: 1009-1026 CrossRef ADS Google Scholar

[24] Zhang N, Wang Y, Xu W, et al. Poly(lactide-co-glycolide)/hydroxyapatite porous scaffold with microchannels for bone regeneration. Polymers, 2016, 8: 218 CrossRef Google Scholar

[25] Domingo C, Loste E, Gómez-Morales J, et al. Calcite precipitation by a high-pressure CO2 carbonation route. J Supercritical Fluids, 2006, 36: 202-215 CrossRef Google Scholar

[26] Lee HS, Ha TH, Kim K. Fabrication of unusually stable amorphous calcium carbonate in an ethanol medium. Mater Chem Phys, 2005, 93: 376-382 CrossRef Google Scholar

[27] Saleem IY, Vordermeier M, Barralet JE, et al. Improving peptide-based assays to differentiate between vaccination and mycobacterium bovis infection in cattle using nanoparticle carriers for adsorbed antigens. J Control Release, 2005, 102: 551-561 CrossRef PubMed Google Scholar

[28] Ikoma T, Tonegawa T, Watanaba H, et al. Drug-supported microparticles of calcium carbonate nanocrystals and its covering with hydroxyapatite. J Nanosci Nanotech, 2007, 7: 822-827 CrossRef Google Scholar

[29] Cai WY, Feng LD, Liu SH, et al. Hemoglobin-CdTe-CaCO3 @polyelectrolytes 3D architecture: fabrication, characterization, and application in biosensing. Adv Funct Mater, 2008, 18: 3127-3136 CrossRef Google Scholar

[30] Sand KK, Rodriguez-Blanco JD, Makovicky E, et al. Crystallization of CaCO3 in water–alcohol mixtures: spherulitic growth, polymorph stabilization, and morphology change. Cryst Growth Des, 2012, 12: 842-853 CrossRef Google Scholar

[31] Wang T, Cölfen H, Antonietti M. Nonclassical crystallization:  mesocrystals and morphology change of CaCO3 crystals in the presence of a polyelectrolyte additive. J Am Chem Soc, 2005, 127: 3246-3247 CrossRef PubMed Google Scholar

[32] Smith DJ, King WF, Godiska R. Passive transfer of immunoglobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries. Infection Immun, 2001, 69: 3135-3142 CrossRef PubMed Google Scholar

[33] Nilsson E, Kollberg H, Johannesson M, et al. More than 10 years' continuous oral treatment with specific immunoglobulin Y for the prevention of Pseudomonas aeruginosa infections: a case report. J Medicinal Food, 2007, 10: 375-378 CrossRef PubMed Google Scholar

[34] Klein MI, Hwang G, Santos PHS, et al. Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms. Front Cell Infect Microbiol, 2015, 5: 10 CrossRef Google Scholar

[35] Kim S, Park CB. Mussel-inspired transformation of CaCO3 to bone minerals. Biomaterials, 2010, 31: 6628-6634 CrossRef PubMed Google Scholar

[36] de La Pierre M, Carteret C, Maschio L, et al. The Raman spectrum of CaCO3 polymorphs calcite and aragonite: a combined experimental and computational study. J Chem Phys, 2014, 140: 164509 CrossRef PubMed ADS Google Scholar

[37] Yamini D, Devanand Venkatasubbu G, Kumar J, et al. Raman scattering studies on PEG functionalized hydroxyapatite nanoparticles. Spectrochimica Acta Part A-Mol Biomolecular Spectroscopy, 2014, 117: 299-303 CrossRef PubMed ADS Google Scholar

[38] Chiang YC, Lin HP, Chang HH, et al. A mesoporous silica biomaterial for dental biomimetic crystallization. ACS Nano, 2014, 8: 12502-12513 CrossRef PubMed Google Scholar

[39] Absi EG, Addy M, Adams D. Dentine hypersensitivity. A study of the patency of dentinal tubules in sensitive and non-sensitive cervical dentine. J Clin Periodontol, 1987, 14: 280-284 CrossRef Google Scholar

[40] Oh DX, Prajatelistia E, Ju SW, et al. A rapid, efficient, and facile solution for dental hypersensitivity: the tannin–iron complex. Sci Rep, 2015, 5: 10884 CrossRef PubMed ADS Google Scholar

[41] Tsai WS, Placa SJ, Panagakos PS. Clinical evaluation of an in-office desensitizing paste containing 8% arginine and calcium carbonate for relief of dentin hypersensitivity prior to dental prophylaxis. Am J Dent, 2012, 25: 165-170. Google Scholar

[42] Suge T, Ishikawa K, Kawasaki A, et al. Duration of dentinal tubule occlusion formed by calcium phosphate precipitation method: in vitro evaluation using synthetic saliva. J Dental Res, 1995, 74: 1709-1714 CrossRef PubMed Google Scholar

  • Figure 1

    (a) Schematic illustration of the two-step synthetic procedure for PCMSs. Step I: vapor-diffusion process. Step 2: aging crystallization process via soaking PCMS in a mixed water/ethanol solution (R=1/50) at 30°C for 7 days. (b–e) Morphological and XRD characterizations of PCMSs: (b) TEM image of PCMSs. (c, d) SEM images of PCMSs. Fig. 1d shows the enlargement of the selected square region in (c). The arrows indicate the large-pore on the MPs surface. (e) TEM size distribution of PCMSs. Over 200 particles in different fields of views were analyzed for the size distribution. (f) The XRD pattern of PCMSs. The inset high-resolution TEM image shows the lattice fringe of PCMSs.

  • Figure 2

    SEM images of samples obtained by two-step vapor-diffusion/aging crystallization strategy. (a, c, e) SEM images of the intermediate products synthesized in the first step: (a) R=0; (c) R=1/160; (e) R=1/40. (b, d, f) SEM images of the final products after two-step synthetic process: (b) R=0; (d) R=1/160; (f) R=1/40.

  • Figure 3

    Characterizations of IgY protein loading capacity for PCMSs. (a) FTIR spectra of free IgY, empty PCMSs and PCMSs-IgY. The arrow indicates the specific absorption peak at 1648 cm−1. (b) UV-vis spectra of supernatant solution after loading PCMSs and CMCs with the same initial concentration of IgY (1 mg mL−1), separately. The inside table shows the calculated encapsulation efficiency and loading capacity of each sample.

  • Figure 4

    (a) Schematic demonstration of the in vitro antibacterial activity evaluation of the PCMSs-IgY against S. mutans. (b, c) The S. mutans colonies (b) and the corresponding surviving S. mutans counts (c) after different treatments for 12 h.

  • Figure 5

    SEM images and Raman spectra showing the conversion of samples to hydroxyapatite in PBS solution. (a, c) SEM images of PCMSs after soaking in PBS for (a) 2 and (c) 24 h. Lath-like surface morphology formed after soaking for 2h. (b) Raman spectra of PCMSs after soaking in PBS for 2 h. H indicate the characteristic peaks of hydroxyapatite. (d, f) SEM images of counterpart samples CMCs after soaking in PBS for (a) 2 and (c) 24 h. Nanometer sized lath-like agglomerates are shown on the surface of rhombohedral calcite after soaking CMCs in PBS for 2 h. The arrow in Fig. 5f indicates the unreacted rhombohedral calcite. (e) Raman spectrum of CMCs after soaking in PBS for 2 h. C indicates the characteristic peaks of calcite. Only a minority of calcite converts to hydroxyapatite structure for CMCs within 2 h.

  • Figure 6

    SEM images of dentinal tubules before (a) and after treatment with the PCMSs (b–e) and MCCPs group (f). (b, c) The PCMSs-treated dentin surface was incubated in PBS solution for 15 min. The arrows in Fig. 6c indicate the broken porous particles within tubules. (d–f) SEM images of the groups containing PCMSs (d, e) and MCCPs (f) after tooth-brushing for 7 days: (d, f) magnified ×1000, (e) magnified ×3000. (g) Schematic illustration of the hydroxyapatite formation mechanism of PCMSs within dentin tubules.

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