SpringerLink
Log in

In-vitro Studies on Copper Nanoparticles and Nano-hydroxyapatite Infused Biopolymeric Composite Scaffolds for Bone Bioengineering Applications

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

This research study deals with the development of copper nanoparticles (CN) and nano-hydroxyapatite (nHAP) infused chitosan (C) and gelatin (G) based nanocomposite scaffolds for bone tissue engineering applications. Human-origin osteoblast cells (MG-63) were seeded over the scaffolds to investigate the novel biomimetic extracellular matrix system. The scanning electron microscopy (SEM) showed an average pore size between 100–146 µm for all the C-G-nHAP-CN based scaffolds. The in-vitro degradation study showed 74–83% degradation for CN-based scaffolds. For 0.03% CN based scaffold degradation rate (84%) was very close to the control scaffold. Swelling ratio was highest for the chitosan scaffold and it was in the range between 5.25–5.93 mg/mL for other scaffolds. Compressive moduli were highest for 0.03% CN scaffold (3.32 MPa) which was relatively very high in comparison to C-G-nHAP scaffold with 2.4 MPa strength in a wet state. Stress-strain graphs also show the maximum displacement by 0.03% CN scaffold. The functional and structural analysis for the scaffolds showed the presence of nHAP in the scaffold and CN peaks within the composite structure. Differential scanning colorimetry testing showed reduced crystallinity in CN-based scaffolds with a melting temperature of 320°C. Their 2D cell behaviour in the Electrical Cell Impedance System (ECIS) study showed maximum cell spreading and growth in 0.02% CN-based scaffold. The cell-seeded composite was tested for 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), 4,6-diamidino-2-phenylindole (DAPI), and acridine orange and propidium iodide (AOPI) assay for testing its cytocompatibility for MG-63 cell line. Cell proliferation and cell spreading was observed by SEM in all the CN-based scaffolds. Alkaline phosphatase (ALP) activity was highest in 0.03% CN scaffold with 2.0 optical density (OD) value. Alizarin Red Stain (ARS) staining was performed to support this study. It can be statistically depicted that nHAP and 0.03% CN-based scaffold could be potential biomaterial for minor to severe bone-related tissue regeneration applications.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Fernandez de Grado, G., L. Keller, Y. Idoux-Gillet, Q. Wagner, A.-M. Musset, N. Benkirane-Jessel, F. Bornert, and D. Offner (2018) Bone substitutes: a review of their characteristics, clinical use, and perspectives for large bone defects management. J. Tissue Eng. 9: 2041731418776819.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Amini, A. R., C. T. Laurencin, and S. P. Nukavarapu (2012) Bone tissue engineering: recent advances and challenges. Crit. Rev. Biomed. Eng. 40: 363–408.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Oryan, A., S. Alidadi, A. Moshiri, and N. Maffulli (2014) Bone regenerative medicine: classic options, novel strategies, and future directions. J. Orthop. Surg. Res. 9: 18.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Sheikh, Z., S. Najeeb, Z. Khurshid, V. Verma, H. Rashid, and M. Glogauer (2015) Biodegradable materials for bone repair and tissue engineering applications. Materials (Basel) 8: 5744–5794.

    Article  CAS  PubMed  Google Scholar 

  5. Feng, X. (2009) Chemical and biochemical basis of cell-bone matrix interaction in health and disease. Curr. Chem. Biol. 3: 189–196.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Nikolova, M. P. and M. S. Chavali (2019) Recent advances in biomaterials for 3D scaffolds: a review. Bioact. Mater. 4: 271–292.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Grass, G., C. Rensing, and M. Solioz (2011) Metallic copper as an antimicrobial surface. Appl. Environ. Microbiol. 77: 1541–1547.

    Article  CAS  PubMed  Google Scholar 

  8. **e, H. and Y. J. Kang (2009) Role of copper in angiogenesis and its medicinal implications. Curr. Med. Chem. 16: 1304–1314.

    Article  CAS  PubMed  Google Scholar 

  9. D’Andrea, L. D., A. Romanelli, R. Di Stasi, and C. Pedone (2010) Bioinorganic aspects of angiogenesis. Dalton Trans. 39: 7625–7636.

    Article  PubMed  Google Scholar 

  10. Turski, M. L. and D. J. Thiele (2009) New roles for copper metabolism in cell proliferation, signaling, and disease. J. Biol. Chem. 284: 717–721.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kumar, N. and L. S. B. Upadhyay (2020) Enzyme immobilization over polystyrene surface using cysteine functionalized copper nanoparticle as a linker molecule. Appl. Biochem. Biotechnol. 191: 1247–1257.

    Article  CAS  PubMed  Google Scholar 

  12. Zhou, M., M. Tian, and C. Li (2016) Copper-based nanomaterials for cancer imaging and therapy. Bioconjug. Chem. 27: 1188–1199.

    Article  CAS  PubMed  Google Scholar 

  13. Halevas, E. G. and A. A. Pantazaki (2018) Copper nanoparticles as therapeutic anticancer agents. Nanomed. Nanotechnol. J. 2: 119–139.

    Google Scholar 

  14. **, R., C. Hiemstra, Z. Zhong, and J. Feijen (2007) Enzymemediated fast in situ formation of hydrogels from dextran-tyramine conjugates. Biomaterials 28: 2791–2800.

    Article  CAS  PubMed  Google Scholar 

  15. Kudr, J., Y. Haddad, L. Richtera, Z. Heger, M. Cernak, V. Adam, and O. Zitka (2017) Magnetic nanoparticles: from design and synthesis to real world applications. Nanomaterials (Basel) 7: 243.

    Article  PubMed  Google Scholar 

  16. Kumari, S., B. N. Singh, and P. Srivastava (2019) Effect of copper nanoparticles on physico-chemical properties of chitosan and gelatin-based scaffold developed for skin tissue engineering application. 3 Biotech 9: 102.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Maji, K. and S. Dasgupta (2014) Hydroxyapatite-chitosan and gelatin based scaffold for bone tissue engineering. Trans. Indian Ceram. Soc. 73: 110–114.

    Article  CAS  Google Scholar 

  18. Echave, M. C., L. Saenz del Burgo, J. L. Pedraz, and G. Orive (2017) Gelatin as biomaterial for tissue engineering. Curr. Pharm. Des. 23: 3567–3584.

    Article  CAS  PubMed  Google Scholar 

  19. Raucci, M. G., U. D’Amora, A. Ronca, C. Demitri, and L. Ambrosio (2019) Bioactivation routes of gelatin-based scaffolds to enhance at nanoscale level bone tissue regeneration. Front. Bioeng. Biotechnol. 7: 27.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Elieh-Ali-Komi, D. and M. R. Hamblin (2016) Chitin and chitosan: production and application of versatile biomedical nanomaterials. Int. J. Adv. Res. (Indore) 4: 411–427.

    CAS  PubMed  Google Scholar 

  21. Zang, S., L. Zhu, K. Luo, R. Mu, F. Chen, X. Wei, X. Yan, B. Han, X. Shi, Q. Wang, and L. ** (2017) Chitosan composite scaffold combined with bone marrow-derived mesenchymal stem cells for bone regeneration: in vitro and in vivo evaluation. Oncotarget 8: 110890–110903.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Tang, T., G. Zhang, C. P. Y. Lau, L. Z. Zheng, X. H. **e, X. L. Wang, X. H. Wang, K. He, Y. Patrick, L. Qin, and S. M. Kumta (2011) Effect of water-soluble P-chitosan and S-chitosan on human primary osteoblasts and giant cell tumor of bone stromal cells. Biomed. Mater. 6: 015004.

    Article  CAS  PubMed  Google Scholar 

  23. Gadgey, K. K. and G. S. Sharma (2020) Investigation of mechanical properties of chitosan based films prepared from Narmada riverside crab shells. Int. J. Mech. Eng. Technol. 11: 21–28.

    Google Scholar 

  24. Maji, K., S. Dasgupta, B. Kundu, and A. Bissoyi (2015) Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength. J. Biomater. Sci. Polym. Ed. 26: 1190–1209.

    Article  CAS  PubMed  Google Scholar 

  25. Wang, Y., W. Zhang, and Q. Yao (2021) Copper-based biomaterials for bone and cartilage tissue engineering. J. Orthop. Translat. 29: 60–71.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Maia, F. R., V. M. Correlo, J. M. Oliveira, and R. L. Reis (2019) Natural origin materials for bone tissue engineering: properties, processing, and performance. pp. 535–558. In: A. Atala, R. Lanza, A. G. Mikos, and R. Nerem (eds.). Principles of Regenerative Medicine. 3rd ed. Academic Press, London, UK.

    Chapter  Google Scholar 

  27. Polo-Corrales, L., M. Latorre-Esteves, and J. E. Ramirez-Vick (2014) Scaffold design for bone regeneration. J. Nanosci. Nanotechnol. 14: 15–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kattimani, V. S., S. Kondaka, and K. P. Lingamaneni (2016) Hydroxyapatite-past, present, and future in bone regeneration. Bone Tissue Regen. Insights 7: BTRI.S36138.

    Article  Google Scholar 

  29. Tampieri, A., M. Iafisco, S. Sprio, A. Ruffini, S. Panseri, M. Montesi, A. Adamiano, and M. Sandri (2016) Hydroxyapatite: from nanocrystals to hybrid nanocomposites for regenerative medicine. pp. 119–144. In: I. V. Antoniac (ed.). Handbook of Bioceramics and Biocomposites. Springer, Cham, Switzerland.

    Chapter  Google Scholar 

  30. Turnbull, G., J. Clarke, F. Picard, P. Riches, L. Jia, F. Han, B. Li, and W. Shu (2018) 3D bioactive composite scaffolds for bone tissue engineering. Bioact. Mater. 3: 278–314.

    Article  PubMed  Google Scholar 

  31. Dan, Y., O. Liu, Y. Liu, Y.-Y. Zhang, S. Li, X. Feng, Z. Shao, C. Yang, S.-H. Yang, and J. Hong (2016) Development of novel biocomposite scaffold of chitosan-gelatin/nanohydroxyapatite for potential bone tissue engineering applications. Nanoscale Res. Lett. 11: 487.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Guan, J., K. L. Fujimoto, M. S. Sacks, and W. R. Wagner (2005) Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications. Biomaterials 26: 3961–3971.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Basu, P., N. Saha, and P. Saha (2020) Swelling and rheological study of calcium phosphate filled bacterial cellulose-based hydrogel scaffold. J. Appl. Polym. Sci. 137: 48522.

    Article  CAS  Google Scholar 

  34. Wang, P.-Y. and W.-B. Tsai (2013) Modulation of the proliferation and matrix synthesis of chondrocytes by dynamic compression on genipin-crosslinked chitosan/collagen scaffolds. J. Biomater. Sci. Polym. Ed. 24: 507–519.

    Article  CAS  PubMed  Google Scholar 

  35. Singh, B. N., V. Veeresh, S. P. Mallick, S. Sinha, A. Rastogi, and P. Srivastava (2020) Generation of scaffold incorporated with nanobioglass encapsulated in chitosan/chondroitin sulfate complex for bone tissue engineering. Int. J. Biol. Macromol. 153: 1–16.

    Article  CAS  PubMed  Google Scholar 

  36. Sahi, A. K., N. Varshney, S. Poddar, S. Gundu, and S. K. Mahto (2021) Fabrication and characterization of silk fibroin-based nanofibrous scaffolds supplemented with gelatin for corneal tissue engineering. Cells Tissues Organs 210: 173–194.

    Article  CAS  PubMed  Google Scholar 

  37. Jain, M., R. G. Vaze, S. C. Ugrani, and K. P. Sharma (2018) Mechanoresponsive and recyclable biocatalytic sponges from enzyme-polymer surfactant conjugates and nanoparticles. RSC Adv. 8: 39029–39038.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zernik, J., K. Twarog, and W. B. Upholt (1990) Regulation of alkaline phosphatase and alpha 2(I) procollagen synthesis during early intramembranous bone formation in the rat mandible. Differentiation 44: 207–215.

    Article  CAS  PubMed  Google Scholar 

  39. Chandrasekar, A., S. Sagadevan, and A. Dakshnamoorthy (2013) Synthesis and characterization of nano-hydroxyapatite (n-HAP) using the wet chemical technique. Int. J. Phys. Sci. 8: 1639–1645.

    Google Scholar 

  40. Mokhtari, A., H. Belhouchet, and A. Guermat (2019) In situ high-temperature X-ray diffraction, FT-IR and thermal analysis studies of the reaction between natural hydroxyapatite and aluminum powder. J. Therm. Anal. Calorim. 136: 1515–1526.

    Article  CAS  Google Scholar 

  41. Abbasi, N., S. Hamlet, R. M. Love, and N.-T. Nguyen (2020) Porous scaffolds for bone regeneration. J. Sci. Adv. Mater. Devices 5: 1–9.

    Article  Google Scholar 

  42. Pradini, D., H. Juwono, K. A. Madurani, and F. Kurniawan (2018) A preliminary study of identification halal gelatin using quartz crystal microbalance (QCM) sensor. Malays. J. Fundam. Appl. Sci. 14: 325–330.

    Article  Google Scholar 

  43. Lewandowska, K., A. Sionkowska, S. Grabska, and M. Michalska (2017) Characterisation of chitosan/hyaluronic acid blend films modified by collagen. Prog. Chem. Appl. Chitin Deriv. 22: 125–134.

    CAS  Google Scholar 

  44. Konovalova, I., V. Novikov, Y. Kuchina, and N. Dolgopiatova (2020) Technology and properties of chondroitin sulfate from marine hydrobionts. KnE Life Sci. 5: 305–314.

    Google Scholar 

  45. Isikli, C., V. Hasirci, and N. Hasirci (2012) Development of porous chitosan-gelatin/hydroxyapatite composite scaffolds for hard tissue-engineering applications. J. Tissue Eng. Regen. Med. 6: 135–143.

    Article  CAS  PubMed  Google Scholar 

  46. Sheikh, L., S. Sinha, Y. N. Singhababu, V. Verma, S. Tripathy, and S. Nayar (2018) Traversing the profile of biomimetically nanoengineered iron substituted hydroxyapatite: synthesis, characterization, property evaluation, and drug release modeling. RSC Adv. 8: 19389–19401.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kumar, P., B. S. Dehiya, and A. Sindhu (2017) Comparative study of chitosan and chitosan-gelatin scaffold for tissue engineering. Int. Nano Lett. 7: 285–290.

    Article  CAS  Google Scholar 

  48. Lim, J. Y., J. C. Hansen, C. A. Siedlecki, J. Runt, and H. J. Donahue (2005) Human foetal osteoblastic cell response to polymer-demixed nanotopographic interfaces. J. R. Soc. Interface 2: 97–108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lee, S. J., J. S. Choi, K. S. Park, G. Khang, Y. M. Lee, and H. B. Lee (2004) Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes. Biomaterials 25: 4699–4707.

    Article  CAS  PubMed  Google Scholar 

  50. Qu, F., J. L. Holloway, J. L. Esterhai, J. A. Burdick, and R. L. Mauck (2017) Programmed biomolecule delivery to enable and direct cell migration for connective tissue repair. Nat. Commun. 8: 1780.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Singh, B. N., V. Veeresh, S. P. Mallick, Y. Jain, S. Sinha, A. Rastogi, and P. Srivastava (2019) Design and evaluation of chitosan/chondroitin sulfate/nano-bioglass based composite scaffold for bone tissue engineering. Int. J. Biol. Macromol. 133: 817–830.

    Article  CAS  PubMed  Google Scholar 

  52. Peter, M., N. Ganesh, N. Selvamurugan, S. V. Nair, T. Furuike, H. Tamura, and R. Jayakumar (2010) Preparation and characterization of chitosan-gelatin/nanohydroxyapatite composite scaffolds for tissue engineering applications. Carbohydr. Polym. 80: 687–694.

    Article  CAS  Google Scholar 

  53. Shuai, C., W. Yang, C. He, S. Peng, C. Gao, Y. Yang, F. Qi, and P. Feng (2020) A magnetic micro-environment in scaffolds for stimulating bone regeneration. Mater. Des. 185: 108275.

    Article  CAS  Google Scholar 

  54. Alsberg, E., H. J. Kong, Y. Hirano, M. K. Smith, A. Albeiruti, and D. J. Mooney (2003) Regulating bone formation via controlled scaffold degradation. J. Dent. Res. 82: 903–908.

    Article  CAS  PubMed  Google Scholar 

  55. Fu, Q., M. N. Rahaman, H. Fu, and X. Liu (2010) Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. I. Preparation and in vitro degradation. J. Biomed. Mater. Res. A 95: 164–171.

    Article  PubMed  Google Scholar 

  56. Siddiquei, H. R., A. N. Nordin, M. I. Ibrahimy, M. A. Arifin, N. H. Sulong, M. Mel, and I. Voiculescu (2010) Electrical cellsubstrate impedance sensing (ECIS) based biosensor for characterization of DF-1 cells. Proceedings of International Conference on Computer and Communication Engineering (ICCCE’10). May 11–12. Kuala Lumpur, Malaysia.

  57. Kashiwazaki, H., Y. Kishiya, A. Matsuda, K. Yamaguchi, T. Iizuka, J. Tanaka, and N. Inoue (2009) Fabrication of porous chitosan/hydroxyapatite nanocomposites: their mechanical and biological properties. Biomed. Mater. Eng. 19: 133–140.

    PubMed  Google Scholar 

  58. Pepla, E., L. K. Besharat, G. Palaia, G. Tenore, and G. Migliau (2014) Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature. Ann. Stomatol. (Roma) 5: 108–114.

    PubMed  Google Scholar 

Download references

Acknowledgements

Central Instrument Facility Center, IIT (BHU) Varanasi supported this research study by providing several instruments for it. It was funded by SPARC, IUSSTF also. We are also thankful to Dr. Li for providing his lab to perform ECIS experiments. The authors are grateful to the Ministry of Human Resource Development for granting the fellowship for completing our research work. Finally IIT (BHU) for all the lab facilities provided for carrying out the research work to the first author.

Author information

Authors and Affiliations

  1. School of Biochemical Engineering, IIT BHU (Varanasi), Varanasi, U.P., 221005, India

    Shikha Kumari, Abha Mishra, Divakar Singh & Pradeep Srivastava

  2. Department of Biochemistry and Molecular Biology, Tulane University, New Orleans, LA, 70118, USA

    Chenzhong Li

Authors
  1. Shikha Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abha Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Divakar Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chenzhong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pradeep Srivastava
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pradeep Srivastava.

Ethics declarations

The authors declare there is no conflict of interest. Neither ethical approval nor informed consent was required for this study.

Additional information

Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kumari, S., Mishra, A., Singh, D. et al. In-vitro Studies on Copper Nanoparticles and Nano-hydroxyapatite Infused Biopolymeric Composite Scaffolds for Bone Bioengineering Applications. Biotechnol Bioproc E 28, 162–180 (2023). https://doi.org/10.1007/s12257-022-0236-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-022-0236-0

Keywords

Search

Navigation