Skip to main content

Advertisement

SpringerLink
Log in

Effect of enzymatic degradation of chitosan in polyhydroxybutyrate/chitosan/calcium phosphate composites on in vitro osteoblast response

  • Tissue Engineering Constructs and Cell Substrates
  • Original Research
  • Published:
Journal of Materials Science: Materials in Medicine Aims and scope Submit manuscript

Abstract

Polyhydroxybutyrate/chitosan/calcium phosphate composites are interesting biomaterials for utilization in regenerative medicine and they may by applied in reconstruction of deeper subchondral defects. Insufficient informations were found in recent papers about the influence of lysozyme degradation of chitosan in calcium phosphate/chitosan based composites on in vitro cytotoxicity and proliferation activity of osteoblasts. The effect of enzymatic chitosan degradation on osteoblasts proliferation was studied on composite films in which the porosity of origin 3D scaffolds was eliminated and the surface texture was modified. The significantly enhanced proliferation activity with faster population growth of osteoblasts were found on enzymatically degraded biopolymer composite films with α-tricalcium phosphate and nanohydroxyapatite. No cytotoxicity of composite films prepared from lysozyme degraded scaffolds containing a large fraction of low molecular weight chitosans (LMWC), was revealed after 10 days of cultivation. Contrary to above in the higher cytotoxicity origin untreated nanohydroxyapatite films and porous composite scaffolds. The results showed that the synergistic effect of surface distribution, morphology of nanohydroxyapatite particles, microtopography and the presence of LMWC due to chitosan degradation in composite films were responsible for compensation of the cytotoxicity of nanohydroxyapatite composite films or porous composite scaffolds.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Muzzarelli R, Baldassarre V, Conti F, Ferrara P, Biagini G, Gazzanelli G, Vasi V. Biological activity of chitosan. Ultrastructural study. Biomaterials. 1988;9(3):247–52.

    Article  Google Scholar 

  2. Thein-Han WW, Misra RDK. Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering. Acta Biomater. 2009;5:1182–97.

    Article  Google Scholar 

  3. Kong L, Gao Y, Lu G, Gong Y, Zhao N, Zhang X. A study on the bioactivity of chitosan/nano-hydroxyapatite composite scaffolds for bone tissue engineering. Eur Polym J. 2006;42:3171–79.

    Article  Google Scholar 

  4. Siddiqui N, Pramanik K, Jabbari E. Osteogenic differentiation of human mesenchymal stem cells in freeze-gelled chitosan/nano β-tricalcium phosphate porous scaffolds crosslinked with genipin. Mater Sci Eng C. 2015;54:76–83.

    Article  Google Scholar 

  5. Seda Tıglı R, Karakecili A, Gümüsderelioglu M. In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree. J Mater Sci Mater Med. 2007;18:1665–74.

    Article  Google Scholar 

  6. Hesaraki S, Nezafati N. In vitro biocompatibility of chitosan/hyaluronic acid-containing calcium phosphate bone cements. Bioprocess Biosyst Eng. 2014;37:1507–16.

    Article  Google Scholar 

  7. Hockin XuHK, Simon CG. Fast setting calcium phosphate–chitosan scaffold:mechanical properties and biocompatibility. Biomaterials. 2005;26:1337–48.

    Article  Google Scholar 

  8. Lee JS, Baek SD, Venkatesan J, Bhatnagar I, Chang HK, Kim HT, Kim SK. In vivo study of chitosan-natural nano hydroxyapatite scaffolds for bone tissue regeneration. Int J Biol Macromol. 2014;67:360–66.

    Article  Google Scholar 

  9. Wu W, Hua X, He Z, Wang X, Yu X, Ren W. The bactericidal and biocompatible characteristics of reinforced calcium phosphate cements. Biomed Mater. 2012;7:045003. doi:10.1088/1748-6041/7/4/045003. (10pp)

    Article  Google Scholar 

  10. Mellegård H, Strand SP, Christensen BE, Granum PE, Hardy SP. Antibacterial activity of chemically defined chitosans: Influence of molecular weight, degree of acetylation and test organism. Int J Food Microbiol. 2011;148:48–54.

    Article  Google Scholar 

  11. Hou JW, Qian L, Kou JM, Zhang CW, Jia XJ, Tian W. Effect of water-soluble chitosan on the osteoblast function in MC3T3-E1 cells. Int J Biol Macromol. 2015;72:1041–43.

    Article  Google Scholar 

  12. Jack KS, Velayudhan S, Luckman P, Trau M, Grøndahl L, Cooper-White J. The fabrication and characterization of biodegradable HA/PHBV nanoparticle-polymer composite scaffolds. Acta Biomater. 2009;5(7):2657–67.

    Article  Google Scholar 

  13. Tai HY, Fu E, Cheng LP, Don TM. Fabrication of asymmetric membranes from polyhydroxybutyrate and biphasic calcium phosphate/chitosan for guided bone regeneration. J Polym Res. 2014;21:421. doi:10.1007/s10965-014-0421-8

    Article  Google Scholar 

  14. Sadat-Shojai M, Khorasani MT, Jamshidi A, Irani S. Nano-hydroxyapatite reinforced polyhydroxybutyrate composites: A comprehensive study on the structural and in vitro biological properties. Mater Sci Eng C. 2013;33:2776–87.

    Article  Google Scholar 

  15. Cool SM, Kenny B, Wu A, Nurcombe V, Trau M, Cassady AI, Grøndahl L. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite biomaterials for bone tissue regeneration: In vitro performance assessed by osteoblast proliferation, osteoclast adhesion and resorption, and macrophage proinflammatory response. J Biomed Mater Res. 2007;82A:599–610.

    Article  Google Scholar 

  16. Zheng Y, Wang Y, Yang H, Chen X, Chen Z. Characteristic comparison of bioactive scaffolds based on polyhydroxyalkoanate/bioceramic hybrids. J Biomed Mater Res Part B Appl Biomater. 2007;80B:236–43.

    Article  Google Scholar 

  17. Cheung MK, Wan KPY, Yu PH. Miscibility and morphology of chiral semicrystalline poly-(R)-(3-hydroxybutyrate)/chitosan and poly-(R)-(3-hydroxybutyrateco-3-hydroxyvalerate)/chitosan blends studied with DSC, 1H T1 and T1q CRAMPS. J Appl Polym Sci. 2002;86:1253–58.

    Article  Google Scholar 

  18. Ikejima T, Inoue Y. Crystallization behavior and environmental biodegradability of the blend films of poly(3- hydroxybutyric acid) with chitin and chitosan. Carbohydr Polym. 2000;41:351–56.

    Article  Google Scholar 

  19. Veleirinho B, Coelho DS, Dias PF, Maraschin M, Ribeiro-do-Valle RM, Lopes-da-Silva JA. Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/chitosan scaffolds for skin regeneration. Int J Biol Macromol. 2012;51:343–50.

    Article  Google Scholar 

  20. Ma G, Yang D, Wang K, Han J, Ding S, Song G, Nie J. Organic-soluble chitosan/polyhydroxybutyrate ultrafine fibers as skin regeneration prepared by electrospinning. J Appl Polym Sci. 2010;118:3619–24.

    Article  Google Scholar 

  21. Ni Y, Huang Ch, Kokot S. A kinetic spectrophotometric method for the determination of ternary mixtures of reducing sugars with the aid of artificial neural networks and multivariate calibration. Anal Chim Acta. 2003;480:53–65.

    Article  Google Scholar 

  22. Skrbic Z, Divjakovic V. Temperature influence on changes of parameters of the unit cell of biopolymer PHB. Polymer. 1996;37(3):505–07.

    Article  Google Scholar 

  23. Medvecky L, Giretova M, Stulajterova R. Properties and in vitro characterization of polyhydroxybutyrate–chitosan scaffolds prepared by modified precipitation method. J Mater Sci Mater Med. 2014; 25(3):777–89.

  24. Padermshoke A, Katsumoto Y, Sato H, Ekgasit S, Noda I, Ozaki Y. Melting behavior of poly(3-hydroxybutyrate) investigated by two-dimensional infrared correlation spectroscopy. Spectrochim Acta Part A. 2005;61:541–50.

    Article  Google Scholar 

  25. Ibrahim M, Osman O, Mahmoud AA. Spectroscopic analyses of cellulose and chitosan:FTIR and modeling approach. J Comput Theor Nanosci. 2011;8:117–23.

    Article  Google Scholar 

  26. Kumirska J, Czerwicka M, Kaczyński Z, Bychowska A, Brzozowski K, Thöming J, Stepnowski P. Application of spectroscopic methods for structural analysis of chitin and chitosan. Mar Drugs. 2012;8:1567–636. doi:10.3390/md8051567

    Article  Google Scholar 

  27. Nah JW, Jang MK. Spectroscopic characterization and preparation of low molecular, water-soluble chitosan with free-amine group by novel method. J Polym Sci Part A. 2002;40:3796–803.

    Article  Google Scholar 

  28. Thamaraiselvi TV, Prabakaran K, Rajeswari S. Synthesis of hydroxyapatite thatmimic bone mineralogy. Trends biomater. Artif Organs. 2006;19(2):81–3.

    Google Scholar 

  29. Slosarczyk A, Paszkiewicz Z. Paluszkiewizc C. FTIR and XRD evaluation of carbonated hydroxyapatite powders synthesized by wet methods. J Mol Struct. 2005;744–747:657–61.

    Article  Google Scholar 

  30. Tachaboonyakiat W, Netswasdi N, Srakaew V, Opaprakasit M. Elimination of inter- and intramolecular crosslinks of phosphorylated chitosan by sodium salt formation. Polym J. 2010;42:148–56.

    Article  Google Scholar 

  31. Takagi O, Kuramoto N, Ozawa M, Suzuki S. Adsorption/desorption of acidic and basic proteins on needlelike hydroxyapatite filter prepared by slip casting. Ceram Int. 2004;30:139–43.

    Article  Google Scholar 

  32. Chou YF, Huang W, Dunn JC, Miller TA, Wu BM. The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression. Biomaterials. 2005;26:285–95.

    Article  Google Scholar 

  33. Woo KM, Seo J, Zhang R, Ma PX. Suppression of apoptosis by enhanced protein adsorption on polymer / hydroxyapatite composite scaffolds. Biomaterials. 2007;28:2622–30.

    Article  Google Scholar 

  34. Shi Z, Huang X, Cai Y, Tang R, Yang D. Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells. Acta Biomater. 2009;5:338–45.

    Article  Google Scholar 

  35. Kizuki T, Ohgaki M, Katsura M, Nakamura S, Hashimoto S, Toda T, Udagawa S, Yamashita K. Effect of bone-like layer growth from culture medium on adherence of osteoblastlike cells. Biomaterials. 2003;24:941–47.

    Article  Google Scholar 

  36. Yuasa T, Miyamoto Y, Ishikawa K, Takechi M, Momota Y, Tatehara S, Nagayama M. Effects of apatite cements on proliferation and differentiation of human osteoblasts in vitro. Biomaterials. 2004;25:1159–66.

    Article  Google Scholar 

  37. Herten M, Rothamel D, Schwarz F, Friesen K, Koegler G, Becker J. Surface- and nonsurface-dependent in vitro effects of bone substitutes on cell viability. Clin Oral Invest. 2009;13:149–55.

    Article  Google Scholar 

  38. Okada S, Ito H, Nagai A, Komotori J, Imai H. Adhesion of osteoblast-like cells on nanostructured hydroxyapatite. Acta Biomater. 2010;6:591–97.

    Article  Google Scholar 

  39. Himno S, Tsuchida H, Nagao N. N-acetylation in chitosan and the rate of its enzymatic hydrolysis. Biomaterials. 1989;10:574–76.

    Article  Google Scholar 

  40. Pangburn SH, Trescony PV, Heller J. Lysozyme degradation of partially deacetylated chitin, its films and hydrogels. Biomaterials. 1982;3:105–08.

    Article  Google Scholar 

  41. Varum KM, Holme HK, Izume M, Stokke BT, Smidsrod O. Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans. Biochim Biophys Acta. 1996;1291:5–15.

    Article  Google Scholar 

  42. Davies RC, Neuberger A, Wilson BM. The dependence of lysozyme activity on pH and ionic strength. Biochim Biophys Acta. 1969;178:294–305.

    Article  Google Scholar 

  43. Deligianni DD, Katsala ND, Koutsoukos PG, Missirlis YF. Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength. Biomaterials. 2001;22:87–96.

    Article  Google Scholar 

  44. Rizzi SC, Heath DJ, Coombes AGA, Bock N, Textor M, Downes S. Biodegradable polymer/hydroxyapatite composites: Surface analysis and initial attachment of human osteoblasts. J Biomed Mater Res. 2001;55:475–86.

    Article  Google Scholar 

  45. Sadat-Shojai M, Khorasani MT, Jamshidi A, Irani S. Nano-hydroxyapatite reinforced polyhydroxybutyrate composites: A comprehensive study on the structural and in vitro biological properties. Mater Sci Eng C. 2013;33:2776–87.

    Article  Google Scholar 

  46. Wu CS, Hsu YC, Liao HT, Cai YX. Antibacterial activity and in vitro evaluation of the biocompatibility of chitosan-based polysaccharide/polyester membranes. Carbohydr Polym. 2015;134:438–47.

    Article  Google Scholar 

  47. Tanase CE, Sartoris A, Popa MI, Verestiuc L, Unger RE, Kirkpatrick CJ. In vitro evaluation of biomimetic chitosan–calcium phosphate scaffolds with potential application in bone tissue engineering. Biomed Mater. 2013;8:025002. doi:10.1088/1748-6041/8/2/025002

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the Slovak Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, Project No. 2/0047/14 and within the framework of the project “Advanced implants seeded with stem cells for hard tissues regeneration and reconstruction”, which is supported by the Operational Program “Research and Development” financed through the European Regional Development Fund.

Author information

Authors and Affiliations

  1. Institute of Materials Research of SAS, Watsonova 47, 04001, Kosice, Slovakia

    Maria Giretova, Lubomir Medvecky, Radoslava Stulajterova, Tibor Sopcak & Monika Tatarkova

  2. Institute of Geotechnics of SAS, Watsonova 47, 04001, Kosice, Slovakia

    Jaroslav Briancin

Authors
  1. Maria Giretova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lubomir Medvecky
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Radoslava Stulajterova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tibor Sopcak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jaroslav Briancin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Monika Tatarkova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Giretova.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Giretova, M., Medvecky, L., Stulajterova, R. et al. Effect of enzymatic degradation of chitosan in polyhydroxybutyrate/chitosan/calcium phosphate composites on in vitro osteoblast response. J Mater Sci: Mater Med 27, 181 (2016). https://doi.org/10.1007/s10856-016-5801-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10856-016-5801-7

Keywords

Search

Navigation