Abstract
In the research of bone tissue engineering and regeneration, nano-hydroxyapatite (nHAp), chitosan (CS), and gelatin (GeL)-based scaffold shows promising result because of their potentials of tailored properties by manipulating 3D networks. In this work, nHAp was synthesized by wet chemical precipitation method using calcium nitrate tetrahydrate [Ca(NO3)2∙4H2O] and di-ammonium hydrogen phosphate (NH4)2HPO4. Glutaraldehyde was used as the cross-linking agent to prepare 3D networks through a freeze-drying technique. X-ray diffraction (XRD) analysis confirmed the formation of single-phase nHAp. Fourier Transformation Infrared Spectroscopy (FTIR) spectra revealed the presence of hydroxyl (-OH) and phosphate \(\left( {{\text{PO}}_{4}^{3- } } \right)\) groups into the sample which confirmed the formation of hydroxyapatite. Raman Spectroscopy analysis elicited the conservancy of the nHAp and scaffold structure. Scanning Electron Microscopy (SEM) images revealed the formation of a 3D interconnected porous scaffold with a pore size in the range of 30–250 μm. Energy Dispersive X-ray Spectroscopy (EDS) of the scaffold affirmed that the prepared nHAp is calcium abundant nHAp. The cytotoxicity of the above scaffolds was studied by VERO cells, which revealed that the prepared samples were non-cytotoxic. Mechanical testing demonstrated that inclusion of higher nHAp concentration leads to increase the mechanical properties of the scaffolds.
Similar content being viewed by others
Availability of data and material
The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
Li, J., Chen, Y., Yin, Y., Yao, F., Yao, K.: Modulation of nano hydroxyapatite size via formation on chitosan-gelatin network film in situ. Biomaterials 28(5), 781–790 (2007). https://doi.org/10.1016/j.biomaterials.2006.09.042
Maji, K., Dasgupta, S., Pramanik, K., Bissoyi, A.: Preparation and characterization of gelatin-chitosan-nano-TCP based scaffold for orthopedic application. Mater. Sci. Eng. C 86(5), 83–94 (2018). https://doi.org/10.1016/j.msec.2018.02.001
Maji, K., Dasgupta, S., Pramanik, K., Bissoyi, A.: Development of gelatin-chitosan hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength. J. Biomater. Sci. Polym. Ed. 26(16), 1190–1209 (2015). https://doi.org/10.1080/09205063.2015.1082809
Lutz-Christian, G., Boccaccini, A.R.: Bioactive glass and glass-ceramic scaffolds for bone tissue engineering. Materials 3(7), 3867–3910 (2010). https://doi.org/10.3390/ma3073867
Jones, J.R.: New trends in bioactive scaffolds: the importance of nanostructure. J. Eur. Ceram. Soc. 29(7), 1275–1281 (2009). https://doi.org/10.1016/j.jeurceramsoc.2008.08.003
Amini, A.R., Laurencin, C.T., Nukavarapu, S.P.: Bone tissue engineering: recent advances and challenges. Crit. Rev. Biomed. Eng. 40(5), 363–408 (2012). https://doi.org/10.1615/CritRevBiomedEng.v40.i5.10
Yunos, D.M., Bretcanu, O., Boccaccini, A.R.: Polymer-bioceramic composites for tissue engineering scaffolds. J. Mater. Sci. 43(7), 4433–4442 (2008). https://doi.org/10.1007/s10853-008-2552-y
Mouriño, V., Cattalini, J.P., Roether, J.A., Dubey, P., Roy, I., et al.: Composite polymer-bioceramic scaffolds with drug delivery capability for bone tissue engineering. Expert Opin. Drug Deliv. 10(10), 1353–1365 (2013). https://doi.org/10.1517/17425247.2013.808183
Yeo, M.G., Kim, G.H.: Preparation and characterization of 3D composite scaffolds based on rapid-prototyped PCL/β-TCP struts and electrospun PCL coated with collagen and HA for bone Regeneration. Chem. Mater. 24(5), 903–913 (2011). https://doi.org/10.1021/cm201119q
Best, S., Porter, A., Thian, E., Huang, J.: Bioceramics: past, present and for the future. J. Eur. Ceram. Soc. 28(7), 1319–1327 (2008). https://doi.org/10.1016/j.jeurceramsoc.2007.12.001
Hoque, M.E., Sakinah, N., Chuan, Y.L., Ansari, M.N.M.: Synthesis and characterization of hydroxyapatite bioceramic. Int. J. Sci. Eng. Technol. 3(5), 458–462 (2014)
Wu, S., Ma, S., Zhang, C., Cao, G., Wu, D., Gao, C., Lakshmanan, S.: Cryogel biocomposite containing chitosan-gelatin/cerium–zinc doped hydroxyapatite for bone tissue engineering. Saudi J. Biol. Sci. 27(10), 2638–2644 (2020)
Cianferotti, L., Gomes, A., Fabbri, S., Tanini, A., Brandi, M.: The calcium-sensing receptor in bone metabolism: from bench to bedside and back. Osteoporos. Int. 26(6), 2055–2071 (2015). https://doi.org/10.1007/s00198-015-3203-1
Sowmya, S., Bumgardener, J.D., Chennazhi, K.P., Nair, S.V., Jayakumar, R.: Role of nanostructured biopolymers and bioceramics in enamel, dentin and periodontal tissue regeneration. Prog. Polym. Sci. 38(11), 1748–1772 (2013). https://doi.org/10.1016/j.progpolymsci.2013.05.005
Yaaguchi, I., Tokuchi, K., Fukuzaki, H., Koyama, Y., Takakada, K., et al.: Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites. J. Biomed. Mater. Res. 55(1), 20–28 (2001). https://doi.org/10.1002/1097-4636(200104)55:1%3c20::AID-JBM30%3e3.0.CO;2-F
Ahmed, O.E.: Gelatin-based nanoparticles as drug and gene delivery systems: reviewing three decades of research. J. Controll. Release 172(3), 1075–1091 (2013). https://doi.org/10.1016/j.jconrel.2013.09.019
Dan, Y., Liu, O., Liu, Y., Zhang, Y.Y., Li, S., et al.: Development of novel biocomposite scaffold of chitosan-gelatin/nano hydroxyapatite for potential bone tissue engineering applications. Nanoscale Res. Lett. 11(11), 487–493 (2016). https://doi.org/10.1186/s11671-016-1669-1
Sadat-Shojai, M., Taghi Khorasani, M., Dinpanah-Khoshdargi, E., Jamshidi, A.: Synthesis methods for nanosized hydroxyapatite in diverse structures. Acta Biomater. 9(8), 7591–7621 (2013). https://doi.org/10.1016/j.actbio.2013.04.012
Bouyer, E., Gitzhofer, F., Boulos, M.I.: Morphological study of hydroxyapatite nanocrystal suspension. J. Mater. Sci. Mater. Med. 11(7), 523–531 (2000). https://doi.org/10.1023/A:1008918110156
Maji, K., Dasgupta, S.: Comparative study on mechanical strength of macroporous hydroxyapatite-biopolymer based composite scaffold. In: International conference on advances in engineering and technology, 1 Singapore, pp. 474–480 (2014)
Wang, C.K., Ju, C.P., Chem Lin, J.H.: Effect of doped bioactive glass on structure and properties of sintered hydroxyapatite. Mater. Chem. Phys. 53(2), 138–149 (1998). https://doi.org/10.1016/S0254-0584(97)02074-9
Yin, Y.J., Zhao, F., Song, X.F., Yao, K.D., William, W.L., et al.: Preparation and characterization of hydroxyapatite/chitosan–gelatin network composite. J. Appl. Polym. Sci. 77(13), 2929–2938 (2000). https://doi.org/10.1002/1097-4628(20000923)77:13%3c2929::AID-APP16%3e3.0.CO;2-Q
Gardiner, D.J., Graves, P.R., Bowley, H.J.: Practical Raman Spectroscopy. Springer, Berlin, Heidelberg, New York (1989)
Valentyna, V.N., Anatoliy, M.Y., Volodymyr, M.D., Igor, P.V., Yuriy, A.R., et al.: Nature of some features in Raman spectra of hydroxyapatite containing materials. J. Raman Spectrosc. 47(6), 726–730 (2016). https://doi.org/10.1002/jrs.4883
Xiong, L., Hongping, Z., Yanan, G., Yingbo, W., Xiang, G., et al.: Hexagonal hydroxyapatite formation on TiO2 nanotubes under urea modulation. Cryst. Eng. Comm. 13(11), 3741–3749 (2011). https://doi.org/10.1039/C0CE00971G
Olsztyska-Janus, S., Gasior-Glogowska, M., Szymborska-Malek, K., Komorowska, M., Witkiewicz, W., et al.: Spectroscopic techniques in the study of human tissues and their components. Part II: Raman spectroscopy. Acta Bioeng. Biomech. 14(4), 121–133 (2012). https://doi.org/10.5277/abb120414
Zhang, L.J., Feng, X.S., Liu, H.G., Qian, D.J., Zhang, L., et al.: Hydroxyapatite/collagen composite materials formation in simulated body fluid environment. Mater. Lett. 58(5), 719–722 (2004). https://doi.org/10.1016/j.matlet.2003.07.009
van Apeldoorn, A.A., Aksenov, Y., Stigter, M., Hofland, I., de Bruijn, J.D., et al.: Parallel high-resolution confocal Raman SEM analysis of inorganic and organic bone matrix constituents. J. R. Soc. Interface 2(2), 39–45 (2005). https://doi.org/10.1098/rsif.2004.0018
Nabakumar, P., Debasish, M., Indranil, B., Tapas, K.M., Parag, B., et al.: Chemical synthesis, characterization, and biocompatibility study of hydroxyapatite/chitosan phosphate nanocomposite for bone tissue engineering applications. Int. J. Biomater. 2009(512417), 1–8 (2009). https://doi.org/10.1155/2009/512417
Karin, H.M., Michael, M., Alistair, J.P., Alexander, F.R., Catherine, M.S., et al.: The effect of particle agglomeration on the formation of a surface connected compartment induced by hydroxyaptite nanoparticles in human monocyte-derived macrophages. Biomaterials 35(3), 1074–1088 (2014). https://doi.org/10.1016/j.biomaterials.2013.10.041
Abidi, S.S.A., Murtaza, Q.: Synthesis and characterization of nanohydroxyapatite powder using wet chemical precipitation reaction. J. Mater. Sci. Technol. 30(4), 307–310 (2014). https://doi.org/10.1016/j.jmst.2013.10.011
Santos, M.H., de Oliveira, M., de Freitas Souza, L.P., Mansur, H.S., Vasconcelos, W.L.: Synthesis control and characterization of hydroxyapatite prepared by wet precipitation process. Mater. Res. 7(4), 625–630 (2004). https://doi.org/10.1590/S1516-14392004000400017
Dorozhkin, S.V., Dorozhkina, E.I., Epple, M.: Precipitation of carbonate apatite from a revised simulated body fluid in the presence of glucose. J. Appl. Biomater. Funct. Mater. 1(1), 200–208 (2003). https://doi.org/10.1177/228080000300100307
Sultana, N.: Biodegradable Polymer-Based Scaffolds for Bone Tissue Engineering. Springer, Berlin, Heidelberg (2013). https://doi.org/10.1007/978-3-642-34802-0
Bikiaris, D.: Can nanoparticles really enhance thermal stability of polymers? Part II: an overview on thermal decomposition of polycondensation polymers. Thermochim. Acta 523(1–2), 25–45 (2011). https://doi.org/10.1016/j.tca.2011.06.012
Cai, X., Tong, H., Shen, X.Y., Chen, W.X., Yan, J., et al.: Preparation and characterization of homogeneous chitosan–polylactic acid/hydroxyapatite nanocomposite for bone tissue engineering and evaluation of its mechanical properties. Acta Biomater. 5(7), 2693–2703 (2009). https://doi.org/10.1016/j.actbio.2009.03.005
Pang, Y.X., Bao, X.: Influence of temperature, ripening time and calcination on the morphology and crystallinity of hydroxyapatite nanoparticles. J. Eur. Ceram. Soc. 23(10), 1697–1704 (2003). https://doi.org/10.1016/S0955-2219(02)00413-2
Habiba, E., Felfel, R.M., Abd El-Hady, B.M., Reicha, F.M.: Effect of synthesis temperature on the crystallization and growth of in situ prepared nanohydroxyapatite in chitosan matrix. ISRN Biomater. 2014(897468), 1–8 (2014). https://doi.org/10.1155/2014/897468
Rahman, S., Maria, K.H., Ishtiaque, M.S., Nahar, A., Das, H., Hoque, S.M.: Evaluation of a novel nanocrystalline hydroxyapatite powder and a solid hydroxyapatite/chitosan-gelatin bioceramic for scaffold preparation using as bone substitute material. Turk. J. Chem. 44(4), 884–900 (2020). https://doi.org/10.3906/kim-1912-40
Chang, M.C., Ko, C.C., Douglas, W.H.: Preparation of hydroxyapatite-gelatin nanocomposite. Biomaterials 24(17), 2853–2862 (2003). https://doi.org/10.1016/S0142-9612(03)00115-7
Maji, K., Dasgupta, S.: Hydroxyapatite-chitosan and gelatin based scaffold for bone tissue engineering. Trans. Indian Ceram. Soc. 73(2), 110–114 (2014). https://doi.org/10.1080/0371750X.2014.922424
Thariga, S., Subashini, R., Pavithra, S., Meenachi, P., Kumar, P., Balashanmugam, P., Senthil, K.P.: In vitro evaluation of biodegradable nHAP-chitosan-gelatin-based scaffold for tissue engineering application. IET Nanobiotechnol. 13(3), 301–306 (2019). https://doi.org/10.1049/iet-nbt.2018.5204
Azhar, F.F., Olad, A., Salehi, R.: Fabrication and characterization of chitosan-gelatin/nanohydroxyapatite-polyaniline composite with potential application in tissue engineering scaffolds. Des. Monomers Polym. 17(7), 654–667 (2014). https://doi.org/10.1080/15685551.2014.907621
Tontowi, A.E., Anindyajati, A., Tangkudung, R.: Biocomposite of Hydroxyapatite/Gelatin/PVA for bone graft application. In: IEEEXplore, 1st international conference on bioinformatics, biotechnology, and biomedical engineering (BioMIC), pp. 1–6 (2018). https://doi.org/10.1109/BIOMIC.2018.8610571
Morgan, E.F., Unnikrisnan, G.U., Hussein, A.I.: Bone mechanical properties in healthy and diseased states. Annu. Rev. Biomed. Eng. 20, 119–143 (2018). https://doi.org/10.1146/annurev-bioeng-062117-121139
Acknowledgements
The authors acknowledge gratefully to Material Science Division, Bangladesh Atomic Energy commission, Dhaka for their laboratory support. We also thank Nano and advanced laboratory, Department of Physics and Centre for Advanced Research of Sciences, University of Dhaka, Dhaka, Bangladesh for technical support.
Funding
Authors did not receive any financial support to conduct this research.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical approval and consent to participate
This article does not contain any studies with human participants or animals performed by the author.
Competing interests and consent for publication
The authors declare no competing interest. Only the authors had the right to publish the results as they were involved in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Mohonta, S.K., Maria, K.H., Rahman, S. et al. Synthesis of hydroxyapatite nanoparticle and role of its size in hydroxyapatite/chitosan–gelatin biocomposite for bone grafting. Int Nano Lett 11, 381–393 (2021). https://doi.org/10.1007/s40089-021-00347-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40089-021-00347-9