Journal of Polymer Research

, 26:262 | Cite as

Factors affecting calcium phosphate mineralization within bulk alginate hydrogels

  • Vida Strasser
  • Nives Matijaković
  • Tea Mihelj Josipović
  • Jasminka Kontrec
  • Daniel M. Lyons
  • Damir Kralj
  • Maja Dutour SikirićEmail author


Alginate hydrogels are natural materials which can be used for biomedical purposes in order to trigger natural remineralization of damaged hard tissues. Mineralization of alginate hydrogels by using diverse protocols is extensively studied, with a goal of obtaining biocomposite materials having improved mechanical properties. The majority of the studies described in the literature are focused on the use of alginate in the form of beads, while alginate in bulk form has been more rarely studied. In this work, the influence of parameters, like phosphate concentration, type of media (water, HEPES and TRIS buffer) and pH (5.0, 7.4, 9.0), on gelation and mineralization of bulk alginate hydrogels (approx. 2 × 2 cm) was studied. Hydrogels were produced using semipermeable membranes. In all experiments, calcium chloride solution was added as a mineralizing solution to the alginate dissolved in medium containing disodium phosphate, which simultaneously triggered gelation and mineralization of hydrogels. Hydrogels were mineralized for 5 days with daily exchange of mineralizing solution. Such hydrogels were characterized using a combination of infrared spectroscopy, powder X-ray diffraction and electron microscopy. The obtained results demonstrated that the media and pH have a profound influence on gelation and the stability of the hydrogels. Octacalcium phosphate was formed in all hydrogels except in TRIS buffer at pH = 9.0 where amorphous calcium phosphate (ACP) was formed. ACP was stabilized during the experiments, as well as during the extraction from the hydrogel. The obtained results point to a protocol suitable for fine-tuning the properties of the mineralized hydrogel, by a simple change of experimental conditions.


alginate bulk hydrogel octacalcium phosphate amorphous calcium phosphate mineralization 



Authors are grateful to Krunoslav Užarević for auxiliary PXRD measurements.

Author Contributions

Conceptualization, Maja Dutour Sikirić; Funding acquisition, Damir Kralj; Investigation, Vida Strasser, Nives Matijaković, Daniel Mark Lyons, Tea Mihelj Josipović, Jasminka Kontrec and Damir Kralj; Methodology, Maja Dutour Sikirić; Writing – original draft, Maja Dutour Sikirić, Vida Strasser, Damir Kralj.

Funding Information

This research was funded by the Croatian Science Foundation under project HRZZ-IP-2013-11-5055.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10965_2019_1942_MOESM1_ESM.docx (2.7 mb)
ESM 1 (DOCX 2758 kb)


  1. 1.
    Crichton R (2019) Chapter 19 - Biomineralization. In: Crichton R (ed) Biological Inorganic Chemistry (Third Edition). Academic Press, pp 517–544CrossRefGoogle Scholar
  2. 2.
    Lowenstam HA, Weiner S (1989) On biomineralization. Oxford University Press, New YorkGoogle Scholar
  3. 3.
    Mann S (2001) Biomineralization: principles and concepts in bioinorganic materials chemistry. Oxford University Press, New YorkGoogle Scholar
  4. 4.
    Li X, Deng Y, Wang M et al (2018) Stabilization of Ca-deficient hydroxyapatite in biphasic calcium phosphate ceramics by adding alginate to enhance their biological performances. J Mater Chem B 6:84–97. CrossRefGoogle Scholar
  5. 5.
    Jung SW, Byun J-H, Oh SH et al (2018) Multivalent ion-based in situ gelling polysaccharide hydrogel as an injectable bone graft. Carbohydr Polym 180:216–225. CrossRefPubMedGoogle Scholar
  6. 6.
    Xie M, Olderøy MØ, Andreassen J-P et al (2010) Alginate-controlled formation of nanoscale calcium carbonate and hydroxyapatite mineral phase within hydrogel networks. Acta Biomater 6:3665–3675. CrossRefPubMedGoogle Scholar
  7. 7.
    Ma S, Yu B, Pei X, Zhou F (2016) Structural hydrogels. Polymer 98:516–535. CrossRefGoogle Scholar
  8. 8.
    Hasirci N, Kilic C, Kömez A, et al (2016) Hydrogels in Regenerative Medicine. In: Gels Handbook,Fundamentals, Properties and ApplicationsVolume 2: Applications of Hydrogels in Regenerative Medicine, Edited by: Mohammad Reza Abidian, Umut Atakan Gurkan, Faramarz Edalat. WORLD SCIENTIFIC, pp 1–52Google Scholar
  9. 9.
    Zhao W, Jin X, Cong Y et al (2013) Degradable natural polymer hydrogels for articular cartilage tissue engineering. J Chem Technol Biotechnol 88:327–339. CrossRefGoogle Scholar
  10. 10.
    Bai X, Gao M, Syed S et al (2018) Bioactive hydrogels for bone regeneration. Bioact Mater 3:401–417. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Nuttelman CR, Mortisen DJ, Henry SM, Anseth KS (2001) Attachment of fibronectin to poly(vinyl alcohol) hydrogels promotes NIH3T3 cell adhesion, proliferation, and migration. J Biomed Mater Res 57:217–223.<217::AID-JBM1161>3.0.CO;2-I CrossRefPubMedGoogle Scholar
  12. 12.
    Nair LS, Laurencin CT (2007) Biodegradable polymers as biomaterials. Prog Polym Sci 32:762–798. CrossRefGoogle Scholar
  13. 13.
    Mihaila SM, Reis RL, Marques AP, Gomes ME (2016) Hydrogels in Bone Tissue Engineering: A Multi-Parametric Approach. In: Gels Handbook. WORLD SCIENTIFIC, pp 165–197Google Scholar
  14. 14.
    Kosanović C, Fermani S, Falini G, Kralj D (2017) Crystallization of Calcium Carbonate in Alginate and Xanthan Hydrogels. Crystals 7:355. CrossRefGoogle Scholar
  15. 15.
    Ucar S, Bjørnøy SH, Bassett DC et al (2015) Nucleation and Growth of Brushite in the Presence of Alginate. Cryst Growth Des 15:5397–5405. CrossRefGoogle Scholar
  16. 16.
    Sancilio S, Gallorini M, Di Nisio C, et al (2018) Alginate/Hydroxyapatite-Based Nanocomposite Scaffolds for Bone Tissue Engineering Improve Dental Pulp Biomineralization and Differentiation. Stem Cells Int 2018, Article ID 9643721:13 pp. doi: CrossRefGoogle Scholar
  17. 17.
    Bjørnøy SH, Bassett DC, Ucar S et al (2016) Controlled mineralisation and recrystallisation of brushite within alginate hydrogels. Biomed Mater 11:015013. CrossRefPubMedGoogle Scholar
  18. 18.
    Dorozhkin SV (2012) Calcium Orthophosphates : Applications in Nature, Biology, and Medicine, 1st ed. Pan Stanford Publishing, New York, NYGoogle Scholar
  19. 19.
    Dorozhkin SV (2016) Multiphasic calcium orthophosphate (CaPO 4 ) bioceramics and their biomedical applications. Ceram Int 42:6529–6554. CrossRefGoogle Scholar
  20. 20.
    Yang N, Zhong Q, Zhou Y et al (2016) Controlled degradation pattern of hydroxyapatite/calcium carbonate composite microspheres: Controlled Degradation of Hydroxyapatite Microspheres. Microsc Res Tech 79:518–524. CrossRefPubMedGoogle Scholar
  21. 21.
    Spiller KL, Maher SA, Lowman AM (2011) Hydrogels for the Repair of Articular Cartilage Defects. Tissue Eng Part B Rev 17:281–299. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Cunniffe GM, Vinardell T, Thompson EM et al (2015) Chondrogenically primed mesenchymal stem cell-seeded alginate hydrogels promote early bone formation in critically-sized defects. Eur Polym J 72:464–472. CrossRefGoogle Scholar
  23. 23.
    Sawkins MJ, Bowen W, Dhadda P et al (2013) Hydrogels derived from demineralized and decellularized bone extracellular matrix. Acta Biomater 9:7865–7873. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Osmokrovic A, Jancic I, Vunduk J et al (2018) Achieving high antimicrobial activity: Composite alginate hydrogel beads releasing activated charcoal with an immobilized active agent. Carbohydr Polym 196:279–288. CrossRefPubMedGoogle Scholar
  25. 25.
    Abdalla K, Kamoun E, Maghraby G (2015) Optimization of the entrapment efficiency and release of ambroxol hydrochloride alginate beads. J Appl Pharm Sci 5:013–019. CrossRefGoogle Scholar
  26. 26.
    Leslie SK, Nicolini AM, Sundaresan G et al (2016) Development of a cell delivery system using alginate microbeads for tissue regeneration. J Mater Chem B 4:3515–3525. CrossRefGoogle Scholar
  27. 27.
    Xie M, Olderøy MØ, Zhang Z et al (2012) Biocomposites prepared by alkaline phosphatase mediated mineralization of alginate microbeads. RSC Adv 2:1457–1465. CrossRefGoogle Scholar
  28. 28.
    Cholewinski A, Yang FK, Zhao B (2017) Underwater Contact Behavior of Alginate and Catechol-Conjugated Alginate Hydrogel Beads. Langmuir 33:8353–8361. CrossRefPubMedGoogle Scholar
  29. 29.
    Bjørnøy SH, Bassett DC, Ucar S et al (2016) A correlative spatiotemporal microscale study of calcium phosphate formation and transformation within an alginate hydrogel matrix. Acta Biomater 44:254–266. CrossRefPubMedGoogle Scholar
  30. 30.
    Bjørnøy SH, Mandaric S, Bassett DC et al (2016) Gelling kinetics and in situ mineralization of alginate hydrogels: A correlative spatiotemporal characterization toolbox. Acta Biomater 44:243–253. CrossRefPubMedGoogle Scholar
  31. 31.
    Bajpai M, Shukla P, Bajpai SK (2017) Enhancement in the stability of alginate gels prepared with mixed solution of divalent ions using a diffusion through dialysis tube (DTDT) approach. J Macromol Sci Part A 54:301–310. CrossRefGoogle Scholar
  32. 32.
    LeGeros RZ, Kijkowska R, LeGeros JP (1984) Formation and transformation of octacalcium phosphate, OCP: a preliminary report. Scan Electron Microsc Pt 4:1771–1777Google Scholar
  33. 33.
    Boanini E, Torricelli P, Gazzano M et al (2012) The effect of alendronate doped calcium phosphates on bone cells activity. Bone 51:944–952. CrossRefPubMedGoogle Scholar
  34. 34.
    Fuji T, Anada T, Honda Y et al (2009) Octacalcium Phosphate–Precipitated Alginate Scaffold for Bone Regeneration. Tissue Eng Part A 15:3525–3535. CrossRefPubMedGoogle Scholar
  35. 35.
    Zhao K, Cheng G, Huang J, Ying X (2008) Rebinding and recognition properties of protein-macromolecularly imprinted calcium phosphate/alginate hybrid polymer microspheres. React Funct Polym 68:732–741. CrossRefGoogle Scholar
  36. 36.
    Karampas IA, Kontoyannis CG (2013) Characterization of calcium phosphates mixtures. Vib Spectrosc 64:126–133. CrossRefGoogle Scholar
  37. 37.
    Koutsopoulos S (2002) Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods. J Biomed Mater Res 62:600–612. CrossRefPubMedGoogle Scholar
  38. 38.
    Drouet C (2013) Apatite Formation: Why It May Not Work as Planned, and How to Conclusively Identify Apatite Compounds. BioMed Res Int 013, Article ID 490946:12 pp. doi: CrossRefGoogle Scholar
  39. 39.
    Markovic M, Fowler BO, Brown WE (1993) Octacalcium phosphate carboxylates. 1. Preparation and identification. Chem Mater 5:1401–1405. CrossRefGoogle Scholar
  40. 40.
    Ravichandran YD, Rajendiran R (2015) Development of a new carbon nanotube–alginate–hydroxyapatite tricomponent composite scaffold for application in bone tissue engineering. Int J Nanomedicine 10:7–15. CrossRefPubMedGoogle Scholar
  41. 41.
    Suzuki O (2010) Octacalcium phosphate: Osteoconductivity and crystal chemistry. Acta Biomater 6:3379–3387. CrossRefPubMedGoogle Scholar
  42. 42.
    Iijima M, Moriwaki Y, Wen HB et al (2002) Elongated Growth of Octacalcium Phosphate Crystals in Recombinant Amelogenin Gels under Controlled Ionic Flow. J Dent Res 81:69–73. CrossRefPubMedGoogle Scholar
  43. 43.
    Dabiri SMH, Lagazzo A, Barberis F et al (2016) Characterization of alginate-brushite in-situ hydrogel composites. Mater Sci Eng C 67:502–510. CrossRefGoogle Scholar
  44. 44.
    Amer W, Abdelouahdi K, Ramananarivo HR et al (2014) Smart designing of new hybrid materials based on brushite-alginate and monetite-alginate microspheres: Bio-inspired for sequential nucleation and growth. Mater Sci Eng C 35:341–346. CrossRefGoogle Scholar
  45. 45.
    Wang L, Nancollas GH (2009) Pathways to biomineralization and biodemineralization of calcium phosphates: the thermodynamic and kinetic controls. Dalton Trans 15:2665–2672. CrossRefGoogle Scholar
  46. 46.
    Li S, Wang L (2012) Phosphorylated osteopontin peptides inhibit crystallization by resisting the aggregation of calcium phosphate nanoparticles. CrystEngComm 14:8037. CrossRefGoogle Scholar
  47. 47.
    Bar-Yosef Ofir P, Govrin-Lippman R, Garti N, Füredi-Milhofer H (2004) The Influence of Polyelectrolytes on the Formation and Phase Transformation of Amorphous Calcium Phosphate. Cryst Growth Des 4:177–183. CrossRefGoogle Scholar
  48. 48.
    Selmani A, Coha I, Magdić K et al (2015) Multiscale study of the influence of cationic surfactants on amorphous calcium phosphate precipitation. CrystEngComm 17:8529–8548. CrossRefGoogle Scholar
  49. 49.
    LeRoux MA, Guilak F, Setton LA (1999) Compressive and shear properties of alginate gel: Effects of sodium ions and alginate concentration. J Biomed Mater Res 47:46–53.<46::AID-JBM6>3.0.CO;2-N CrossRefPubMedGoogle Scholar
  50. 50.
    Eanes ED, Gillessen IH, Posner AS (1965) Intermediate States in the Precipitation of Hydroxyapatite. Nature 208:365. CrossRefPubMedGoogle Scholar
  51. 51.
    Wang L, Nancollas GH (2008) Calcium Orthophosphates: Crystallization and Dissolution. Chem Rev 108:4628–4669. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Brečević Lj, Füredi-Milhofer H (1972) Precipitation of calcium phosphates from electrolyte solutions: II. The formation and transformation of the precipitates. Calcif Tissue Res 10:82–90. CrossRefPubMedGoogle Scholar
  53. 53.
    Pan H, Liu XY, Tang R, Xu HY (2010) Mystery of the transformation from amorphous calcium phosphate to hydroxyapatite. Chem Commun 46:7415. CrossRefGoogle Scholar
  54. 54.
    Jiang S, Jin W, Wang Y-N et al (2017) Effect of the aggregation state of amorphous calcium phosphate on hydroxyapatite nucleation kinetics. RSC Adv 7:25497–25503. CrossRefGoogle Scholar
  55. 55.
    Despotović R, Filipović-Vinceković N, Füredi-Milhofer H (1975) Precipitation of calcium phosphates from electrolyte solutions. Calcif Tissue Res 18:13–26. CrossRefPubMedGoogle Scholar
  56. 56.
    Christoffersen J, Christoffersen MR, Kibalczyc W, Andersen FA (1989) A contribution to the understanding of the formation of calcium phosphates. J Cryst Growth 94:767–777. CrossRefGoogle Scholar
  57. 57.
    Sikirić MD, Füredi-Milhofer H (2006) The influence of surface active molecules on the crystallization of biominerals in solution. Adv Colloid Interf Sci 128–130:135–158. CrossRefGoogle Scholar
  58. 58.
    Ding H, Pan H, Xu X, Tang R (2014) Toward a Detailed Understanding of Magnesium Ions on Hydroxyapatite Crystallization Inhibition. Cryst Growth Des 14:763–769. CrossRefGoogle Scholar
  59. 59.
    Chen Y, Gu W, Pan H et al (2014) Stabilizing amorphous calcium phosphate phase by citrate adsorption. CrystEngComm 16:1864–1867. CrossRefGoogle Scholar
  60. 60.
    Bleek K, Taubert A (2013) New developments in polymer-controlled, bioinspired calcium phosphate mineralization from aqueous solution. Acta Biomater 9:6283–6321. CrossRefPubMedGoogle Scholar
  61. 61.
    Frayssinet P, Trouillet JL, Rouquet N et al (1993) Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition. Biomaterials 14:423–429. CrossRefPubMedGoogle Scholar
  62. 62.
    Knaack D, Goad MEP, Aiolova M et al (1998) Resorbable calcium phosphate bone substitute. J Biomed Mater Res 43:399–409.<399::AID-JBM7>3.3.CO;2-A CrossRefPubMedGoogle Scholar
  63. 63.
    Kaunitz JD, Yamaguchi DT (2008) TNAP, TrAP, ecto-purinergic signaling, and bone remodeling. J Cell Biochem 105:655–662. CrossRefPubMedGoogle Scholar
  64. 64.
    Bushinsky DA (1996) Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts. Am J Phys 271:F216–F222. CrossRefGoogle Scholar
  65. 65.
    Liu W, Wang T, Yang C et al (2016) Alkaline biodegradable implants for osteoporotic bone defects--importance of microenvironment pH. Osteoporos Int J Establ Result Coop Eur Found Osteoporos Natl Osteoporos Found USA 27:93–104. CrossRefGoogle Scholar
  66. 66.
    Ito T, Saito M, Uchino T et al (2012) Preparation of injectable auto-forming alginate gel containing simvastatin with amorphous calcium phosphate as a controlled release medium and their therapeutic effect in osteoporosis model rat. J Mater Sci Mater Med 23:1291–1297. CrossRefPubMedGoogle Scholar

Copyright information

© The Polymer Society, Taipei 2019

Authors and Affiliations

  1. 1.Laboratory for Biocolloids and Surface Chemistry, Division of Physical ChemistryRuđer Bošković InstituteZagrebCroatia
  2. 2.Laboratory for Precipitation Processes, Division of Materials ChemistryRuđer Bošković InstituteZagrebCroatia
  3. 3.Laboratory for Marine Nanotechnology and Biotechnology, Center for Marine ResearchRuđer Bošković InstituteRovinjCroatia

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