Journal of Materials Science

, Volume 54, Issue 4, pp 3403–3420 | Cite as

Fabrication and in vitro biocompatibility of sodium tripolyphosphate-crosslinked chitosan–hydroxyapatite scaffolds for bone regeneration

  • Chin Yee Goh
  • Siew Shee LimEmail author
  • Kim Yeow Tshai
  • Ahmed Wael Zaki Zaki El Azab
  • Hwei-San LohEmail author
Materials for life sciences


The aims of this study were to fabricate a novel chitosan–hydroxyapatite (CHA) scaffold crosslinked with sodium tripolyphosphate (TPP) and evaluate its in vitro biocompatibility. CHA scaffolds were fabricated via direct blending and lyophilization and further crosslinked with different concentrations (0.1 M, 0.2 M and 0.4 M) of TPP. Microstructure of TPP-CHA scaffolds was examined by using field emission scanning electron microscope. Porosity and compressive modulus of composite scaffolds were analyzed. The biodegradability of TPP-crosslinked CHA scaffolds was studied for 30 days and in vitro biocompatibility and functionality were evaluated using osteoblast-like cells, MG63, in terms of cell viability, adhesion, proliferation and early differentiation. All scaffolds showed an interconnected honeycomb-like microstructure except 0.4 M TPP-CHA scaffolds, which demonstrated the most compact and least porous structure with pore sizes of 62–185 µm. In contrast, 0.1 M TPP-CHA scaffolds exhibited the highest porosity, measured as 58.6% and pore sizes of 74–207 µm. Besides, 0.1 M TPP-CHA scaffolds also showed the lowest compressive modulus of 2.54 kPa. All TPP-crosslinked CHA scaffolds degraded to a similar extent (1.93–5.03%). Current findings revealed that 0.1 M TPP-CHA scaffolds are the most biocompatible one by promoting good cell viability with the highest adhesion, proliferation and early differentiation activities on MG63 cells among the other scaffolds. In conclusion, 0.1 M TPP-CHA scaffolds exhibited the most promising physiochemical and biocompatible properties which can be used as an alternative regenerative material for bone tissue engineering.



This study was conducted under the collaboration between Faculty of Science and Faculty of Engineering and funded by the University of Nottingham Malaysia Campus.

Compliance with ethical standards

Conflict of interest

The authors declare that they do not have any conflict of interest.


  1. 1.
    Barati D (2016) Biodegradable hybrid tissue engineering scaffolds for reconstruction of large bone defects. Doctoral dissertation, University of South CarolinaGoogle Scholar
  2. 2.
    Office of the Surgeon General (US) (2014) Bone health and osteoporosis: a report of the surgeon general. Office of the Surgeon General (US), RockvilleGoogle Scholar
  3. 3.
    Bose S, Roy M, Bandyopadhyay A (2012) Recent advances in bone tissue engineering scaffolds. Trends Biotechnol 30(10):546–554Google Scholar
  4. 4.
    Rosetia L, Parisia V, Petrettaa M, Cavalloa C, Desandoa G, Bartolottia I, Grigolo B (2017) Scaffolds for bone tissue engineering: state of the art and new perspectives. Mater Sci Eng, C 78:1246–1262Google Scholar
  5. 5.
    Amini AR, Laurencin CT, Nukavarapu SP (2012) Bone tissue engineering: recent advances and challenges. Crit Rev Biomed Eng 40(5):363–408Google Scholar
  6. 6.
    Tatkare D (2016) Bone grafts and substitutes market by product (allografts, bone grafts substitutes, and cell-based matrices), by application (spinal fusion, long bone, foot & ankle, craniomaxillofacial, joint reconstruction, and dental bone grafting)—Global opportunity analysis and industry forecast 2014–2022, Allied Market Research databaseGoogle Scholar
  7. 7.
    Luo T, Zhang W, Shi B, Cheng X, Zhang Y (2012) Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery. Clin Oral Implant Res 23:467–474Google Scholar
  8. 8.
    Chan WD, Perinpanayagam H, Goldberg HA, Hunter GK, Dixon SJ, Santos GC Jr, Rizkalla AS (2009) Tissue engineering scaffolds for the regeneration of craniofacial bone. J Can Dental Assoc 75(5):373–377Google Scholar
  9. 9.
    Falisi G, Galli M, Vittorini-Velasquez P, Gallegos-Rivera JC, Minasi R, De Biase A, Di Paolo C (2013) Use of 3D cartilage scaffolds for the stabilization of implants and bone regeneration with the fit-lock technique. Int J Appl Basic Dental Res 26(3):167–172Google Scholar
  10. 10.
    Roberts TT, Rosenbaum AJ (2012) Bone grafts, bone substitutes and orthobiologics: the bridge between basic science and clinical advancements in fracture healing. Organogenesis 8(4):114–124Google Scholar
  11. 11.
    Alvarez K, Nakajima H (2009) Metallic scaffolds for bone regeneration. Materials 2(3):790–832Google Scholar
  12. 12.
    Arsad SMM, Lee PM, Lee KH (2011) Synthesis and characterization of hydroxyapatite nanoparticles and β-TCP particles. Second Int Conf Biotechnol Food Sci 7:184–188Google Scholar
  13. 13.
    Yang J, Kang Y, Browne C, Jiang T, Yang Y (2015) Graded porous β-tricalcium phosphate scaffolds enhance bone regeneration in mandible augmentation. J Craniofac Surg 26(2):148–153Google Scholar
  14. 14.
    Piétua G, Ehlinger M (2017) Minimally invasive internal fixation of distal femur fractures. Orthop Traumatol Surg Res 103(1):161–169Google Scholar
  15. 15.
    Mohamed KR, Beherei HH, El-Rashidy ZM (2014) in vitro study of nano-hydroxyapatite/chitosan–gelatin composites for bio-applications. J Adv Res 5(2):201–208Google Scholar
  16. 16.
    Thein-Han WW, Misra RDK (2009) Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering. Acta Biomater 5(4):1182–1197Google Scholar
  17. 17.
    Dreifke MB, Ebraheim NA, Jayasuriya AC (2013) Investigation of potential injectable polymeric biomaterials for bone regeneration. J Biomed Mater Res, Part A 101(8):2436–2447Google Scholar
  18. 18.
    Zhou T, Wu J, Liu J, Luo Y, Wan Y (2015) Fabrication and characterization of layered chitosan/silk fibroin/nano-hydroxyapatite scaffolds with designed composition and mechanical properties. Biomed Mater 10(4):045013. Google Scholar
  19. 19.
    Li B, Huang L, Wang X, Ma J, Xie F, Xia L (2013) Effect of micropores and citric acid on the bioactivity of phosphorylated chitosan/chitosan/hydroxyapatite composites. Ceram Int 39(3):3423–3427Google Scholar
  20. 20.
    Scalera F, Gervaso F, Sanosh KP, Sannino A, Licciulli A (2013) Influence of the calcination temperature on morphological and mechanical properties of highly porous hydroxyapatite scaffolds. Ceram Int 39(5):4839–4846Google Scholar
  21. 21.
    Wu Q, Zhang X, Wu B, Huang W (2013) Effects of microwave sintering on the properties of porous hydroxyapatite scaffolds. Ceram Int 39(3):2389–2395Google Scholar
  22. 22.
    Sionkowska A, Kaczmarek B (2017) Preparation and characterization of composites based on the blends of collagen, chitosan and hyaluronic acid with nano-hydroxyapatite. Int J Biol Macromol 102:658–666Google Scholar
  23. 23.
    Murugan R, Ramakrishna S (2004) Nano-structured biomaterials. In: Nalwa HS (ed) Encyclopedia of nanoscience and nanotechnology. American Scientific Publishers, California, p 595Google Scholar
  24. 24.
    Arsad MSM, Lee PM, Lee KH (2010) Morphology and particle size analysis of hydroxyapatite micro- and nano-particles. In: International conference on science and social research (CSSR 2010), pp 1030–1034Google Scholar
  25. 25.
    Rishna R, Mayer J, Winter E, Kam M, Leong W (2001) Biomedical applications of polymer-composite materials a review. Compos Sci Technol 61(9):1189–1224Google Scholar
  26. 26.
    Kikuclin M, Itoh S, Ichinose S, Shinomiya K, Tanaka J (2001) Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo. Biomaterials 22(13):1705–1711Google Scholar
  27. 27.
    Toworfe GK, Composto RJ, Shapiro IM, Ducheyne P (2006) Nucleation and growth of calcium phosphate on amine-, carboxyl- and hydroxyl-silane self-assembled monolayers. Biomaterials 27(4):631–642Google Scholar
  28. 28.
    Family R, Solati-Hashjin M, Namjoy Nik S, Nemati A (2012) Surface modification for titanium implants by hydroxyapatite nanocomposite. Casp J Intern Med 3(3):460–465Google Scholar
  29. 29.
    Yamaguchi I, Tokuchi K, Fukuzaki H, Koyama Y, Takakuda K, Monma H, Tanaka J (2001) Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites. J Biomed Mater Res, Part A 55(1):20–27Google Scholar
  30. 30.
    Li Z, Yubao L, Aiping Y, Xuelin P, Xuejiang W, Xiang Z (2005) Preparation and in vitro investigation of chitosan/nano-hydroxyapatite composite used as bone substitute materials. J Mater Sci Mater Med 16(3):213–219Google Scholar
  31. 31.
    Ibrahim HM, El-Bisi MK, Taha GM, El-Alfy EA (2015) Chitosan nanoparticles loaded antibiotics as drug delivery biomaterial. J Appl Pharm Sci 5(10):85–90Google Scholar
  32. 32.
    Huang Y, Caia Y, Lapitsky Y (2015) Factors affecting the stability of chitosan/tripolyphosphate micro- and nanogels: resolving the opposing findings. J Mater Chem B 3(29):5957–5970Google Scholar
  33. 33.
    Dash M, Chiellini F, Ottenbrite RM, Chiellini E (2011) Chitosan—a versatile semi-synthetic polymer in biomedical applications. Prog Polym Sci 36(8):981–1014Google Scholar
  34. 34.
    Levengood SKL, Zhang MQ (2014) Chitosan-based scaffolds for bone tissue engineering. J Mater Chem B 2(21):3161–3184Google Scholar
  35. 35.
    Martinez-Alvarez C, Gonzalez-Meli B, Berenguer-Froehner B et al (2013) Injection and adhesion palatoplasty: a preliminary study in a canine model. J Surg Res 183(2):654–662Google Scholar
  36. 36.
    Mogosanu GD, Grumezescu AM (2014) Natural and synthetic polymers for wounds and burns dressing. Int J Pharm 463(2):127–136Google Scholar
  37. 37.
    Uswatta SP, Okeke IU, Jayasuriya AC (2016) Injectable porous nano-hydroxyapatite/chitosan/tripolyphosphate scaffolds with improved compressive strength for bone regeneration. Mater Sci Eng C Mater Biol Appl 69:505–512Google Scholar
  38. 38.
    Venkatesan J, Kim SK (2010) Chitosan composites for bone tissue engineering—an overview. Mar Drugs 8(8):2252–2266Google Scholar
  39. 39.
    Silva SS, Luna SM, Gomes ME, Benesch J, Pashkuleva I, Mano JF, Reis RL (2008) Plasma surface modification of chitosan membranes: characterization and preliminary cell response studies. Macromol Biosci 8(6):568–576Google Scholar
  40. 40.
    Bose S, Tarafder S (2012) Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review. Acta Biomater 8(4):1401–1421Google Scholar
  41. 41.
    Jayakumar R, Prabaharan M, Nair SV, Tamura H (2010) Novel chitin and chitosan nanofibers in biomedical applications. Biotechnol Adv 28(1):142–150Google Scholar
  42. 42.
    Zhang Y, Zhang MQ (2001) Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering. J Biomed Mater Res, Part A 55(3):304–312Google Scholar
  43. 43.
    Jarquín-Yáñez K, Arenas-Alatorre JA, Piñón-Zárate G, Arellano-Olivares RM (2016) Structural effect of different EDC crosslinker concentration in gelatin- hyaluronic acid scaffolds. J Bioeng Biomed Sci 6(2):182. Google Scholar
  44. 44.
    Pati F, Adhikari B, Dhara S (2012) Development of chitosan-tripolyphosphate non-woven fibrous scaffolds for tissue engineering application. J Mater Sci Mater Med 23(4):1085–1096Google Scholar
  45. 45.
    Gabriel Paulraj M, Ignacimuthu S, Gandhi MR, Shajahan A, Ganesan P, Packiam SM, Al-Dhabi NA (2017) Comparative studies of tripolyphosphate and glutaraldehyde cross-linked chitosan-botanical pesticide nanoparticles and their agricultural applications. Int J Biol Macromol 104(Pt B):1813–1819Google Scholar
  46. 46.
    Siddiqui N, Pramanik K, Jabbari E (2015) Osteogenic differentiation of human mesenchymal stem cells in freeze-gelled chitosan/nano β-tricalcium phosphate porous scaffolds crosslinked with genipin. Mater Sci Eng C Mater Biol Appl 54:76–83Google Scholar
  47. 47.
    Pinto CA, Saripella KK, Loka NC, Neau SH (2018) Development and characterization of chitosan cross-linked with tripolyphosphate as a sustained release agent in tablets, part I: design of experiments and optimization. J Pharm Sci 107(4):1063–1075Google Scholar
  48. 48.
    Kyzioł A, Mazgała A, Michna J, Regiel-Futyra A, Sebastian V (2017) Preparation and characterization of alginate/chitosan formulations for ciprofloxacin-controlled delivery. J Biomater Appl 32(2):162–174Google Scholar
  49. 49.
    Machul A, Mikołajczyk D, Regiel-Futyra A et al (2015) Study on inhibitory activity of chitosan-based materials against biofilm producing Pseudomonas aeruginosa strains. J Biomater Appl 30(3):269–278Google Scholar
  50. 50.
    Servat-Medina L, González-Gómez A, Reyes-Ortega F et al (2015) Chitosan–tripolyphosphate nanoparticles as Arrabidaea chica standardized extract carrier: synthesis, characterization, biocompatibility, and antiulcerogenic activity. Int J Nanomed 10:3897–3909Google Scholar
  51. 51.
    Karimi M, Avci P, Ahi M, Gazori T, Hamblin MR, Naderi-Manesh H (2013) Evaluation of chitosan-tripolyphosphate nanoparticles as a p-shRNA delivery vector: formulation, optimization and cellular uptake study. J Nanopharm Drug Deliv 1(3):266–278Google Scholar
  52. 52.
    Fisher JP, Reddi AH (2003) Functional tissue engineering of bone: signals and scaffolds. Top Tissue Eng 1:1–29Google Scholar
  53. 53.
    Bhushan B (2010) Scanning probe microscopy in nanoscience and nanotechnology. Springer, Berlin, p 471Google Scholar
  54. 54.
    Unosson J, Persson C, Engqvist H (2015) An evaluation of methods to determine the porosity of calcium phosphate cements. J Biomed Mater Res B Appl Biomater 103B:62–71Google Scholar
  55. 55.
    Shimojo AAM, Galdames SEM, Perez AGM et al (2016) In vitro performance of injectable chitosan-tripolyphosphate scaffolds combined with platelet-rich plasma. Tissue Eng Regen Med 13(1):21–30Google Scholar
  56. 56.
    Fan J (2016) Hemocytometer cell count and trypan blue cell viability. McMaster UniversityGoogle Scholar
  57. 57.
    Kanade S, Nataraj G, Ubale M, Mehta P (2016) Fluorescein diacetate vital staining for detecting viability of acid-fast bacilli in patients on antituberculosis treatment. Int J Mycobacteriol 5(3):294–298Google Scholar
  58. 58.
    Jo HY, Kim Y, Park HW et al (2015) The unreliability of MTT assay in the cytotoxic test of primary cultured glioblastoma cells. Exp Neurobiol 24(3):235–245Google Scholar
  59. 59.
    Aboudzadeh N, Imani M, Shokrgozar MA, Khavandi A, Javadpour J, Shafieyan Y, Farokhi M (2010) Fabrication and characterization of poly(d, l-lactide-co-glycolide)/hydroxyapatite nanocomposite scaffolds for bone tissue regeneration. J Biomed Mater Res, Part A 94(1):137–145Google Scholar
  60. 60.
    Zhang Y, Ni M, Zhang M, Ratner B (2003) Calcium phosphate-chitosan composite scaffolds for bone tissue engineering. Tissue Eng 9(2):337–345Google Scholar
  61. 61.
    Zhang L, Morsi Y, Wang Y, Li Y, Ramakrishna S (2013) Review scaffold design and stem cells for tooth regeneration. Jpn Dental Sci Rev 49(1):14–26Google Scholar
  62. 62.
    O’Brien F (2011) Biomaterials and scaffolds for tissue engineering. Materials 14(3):88–95Google Scholar
  63. 63.
    Dhandayuthapani B, Yoshida Y, Maekawa T, Kumar DS (2011) Polymeric scaffolds in tissue engineering application: a review. Int J Polym Sci 2011:19. Google Scholar
  64. 64.
    Lu T, Li Y, Chen T (2013) Techniques for fabrication and construction of three-dimensional scaffolds for tissue engineering. Int J Nanomed 8:337–350Google Scholar
  65. 65.
    Shavandi A, Ael-D B, Ali MA, Sun Z, Gould M (2015) Development and characterization of hydroxyapatite/β-TCP/chitosan composites for tissue engineering applications. Mater Sci Eng, C 56:481–493Google Scholar
  66. 66.
    Loh QL, Choong C (2013) Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. Tissue Eng Part B Rev 19(6):485–502Google Scholar
  67. 67.
    Roosa SMM, Kemppainen JM, Moffitt EN, Krebsbach PH, Hollister SJ (2009) The pore size of polycaprolactone scaffolds has limited influence on bone regeneration in an in vivo model. J Biomed Mater Res, Part A 92(1):359–368Google Scholar
  68. 68.
    Murphy CM, O’Brien FJ (2010) Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds. Cell Adhes Migr 4(3):377–381Google Scholar
  69. 69.
    Rogina A, Rico P, Ferrer G, Ivanković M, Ivanković H (2016) In situ hydroxyapatite content affects the cell differentiation on porous chitosan/hydroxyapatite scaffolds. Ann Biomed Eng 44(4):1107–1119Google Scholar
  70. 70.
    Bružauskaitė I, Bironaitė D, Bagdonas E, Bernotienė E (2016) Scaffolds and cells for tissue regeneration: different scaffold pore sizes—different cell effects. Cytotechnology 68(3):355–369Google Scholar
  71. 71.
    Olad A, Azhar FF (2014) The synergetic effect of bioactive ceramic and nanoclay on the properties of chitosan–gelatin/nanohydroxyapatite–montmorillonite scaffold for bone tissue engineering. Ceram Int 40(7):10061–10072Google Scholar
  72. 72.
    Karageorgiou V, Kaplan D (2005) Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26(27):5474–5491Google Scholar
  73. 73.
    Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21(24):2529–2543Google Scholar
  74. 74.
    Yook SW, Jung HD, Park CH, Shin KH, Koh YH, Estrin Y, Kim HE (2012) Reverse freeze casting: a new method for fabricating highly porous titanium scaffolds with aligned large pores. Acta Biomater 8(6):2401–2410Google Scholar
  75. 75.
    Hadjicharalambous C, Prymak O, Loza K, Buyakov A, Kulkov S, Chatzinikolaidou M (2015) Effect of porosity of alumina and zirconia ceramics toward pre-osteoblast response. Front Bioeng Biotechnol 3:175. Google Scholar
  76. 76.
    Pilia M, Guda T, Appleford M (2013) Development of composite scaffolds for load-bearing segmental bone defects. Biomed Res Int 2013:458253. Google Scholar
  77. 77.
    Pati F, Kalita H, Adhikari B, Dhara S (2013) Osteoblastic cellular responses on ionically crosslinked chitosan-tripolyphosphate fibrous 3-D mesh scaffolds. J Biomed Mater Res, Part A 101(9):2526–2537Google Scholar
  78. 78.
    Hanninka G, Arts JJC (2011) Bioresorbability, porosity and mechanical strength of bone substitutes: What is optimal for bone regeneration? Injury 42(2):22–25Google Scholar
  79. 79.
    Wong FSY, Chan BP, Lo ACY (2014) Carriers in cell-based therapies for neurological disorders. Int J Mol Sci 15(6):10669–10723Google Scholar
  80. 80.
    Gibon E, Lu LY, Nathan K, Goodman SB (2017) Inflammation, ageing, and bone regeneration. J Orthop Transl 10:28–35Google Scholar
  81. 81.
    Murphy CM, O’Brien FJ, Little DG, Schindeler A (2013) Cell-scaffold interactions in the bone tissue engineering triad. Eur Cells Mater 26:120–132Google Scholar
  82. 82.
    Trichet L, Le Digabel J, Hawkins RJ et al (2012) Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness. Proc Natl Acad Sci USA 109(18):6933–6938Google Scholar
  83. 83.
    Zhou D, Qi C, Chen Y-X et al (2017) Comparative study of porous hydroxyapatite/chitosan and whitlockite/chitosan scaffolds for bone regeneration in calvarial defects. Int J Nanomed 12:2673–2687Google Scholar
  84. 84.
    Kong LJ, Qiang A, Jing X, Zhang L, Gong YD, Zhao NM, Zhang XF (2007) Proliferation and differentiation of MC 3T3-E1 cells cultured on nanohydroxyapatite/chitosan composite scaffolds. Chin J Biotechnol 23(2):262–267Google Scholar
  85. 85.
    Yeo MG, Jung WK, Kim GH (2012) Fabrication, characterisation and biological activity of phlorotannin-conjugated PCL/β-TCP composite scaffolds for bone tissue regeneration. J Mater Chem 22:3568–3577Google Scholar
  86. 86.
    Lim SS, Chai CY, Loh H-S (2017) in vitro evaluation of osteoblast adhesion, proliferation and differentiation on chitosan-TiO2 nanotubes scaffolds with Ca2+ ions. Mater Sci Eng, C 76:144–152Google Scholar
  87. 87.
    Chen Y, Huang Z, Li X et al (2012) In vitro biocompatibility and osteoblast differentiation of an injectable chitosan/nano-hydroxyapatite/collagen scaffold. J Nanomater 3:95. Google Scholar
  88. 88.
    Golub EE, Boesze-Battaglia K (2007) The role of alkaline phosphatase in mineralization. Curr Opin Orthop 18(5):444–448Google Scholar
  89. 89.
    Tanahashi M, Matsuda T (1997) Surface functional group dependence on apatite formation on self-assembled monolayers in a simulated body fluid. J Biomed Mater Res, Part A 34(3):305–315Google Scholar
  90. 90.
    Varma HK, Yokogawa Y, Espinosa FF, Kawamoto Y, Nishizawa K, Nagata F, Kameyama T (1999) Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method. Biomaterials 20(9):879–884Google Scholar
  91. 91.
    Bastiaansen-Jenniskens YM, de Bart ACW, Koevoet W et al (2010) Elevated levels of cartilage oligomeric matrix protein during in vitro cartilage matrix generation decrease collagen fibril diameter. Cartilage 1(3):200–210Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Biosciences, Faculty of ScienceUniversity of Nottingham Malaysia CampusSemenyihMalaysia
  2. 2.Department of Chemical with Environmental Engineering, Faculty of EngineeringUniversity of Nottingham Malaysia CampusSemenyihMalaysia
  3. 3.Department of Mechanical, Materials and Manufacturing Engineering, Faculty of EngineeringUniversity of Nottingham Malaysia CampusSemenyihMalaysia

Personalised recommendations