Development and Optimization of the Novel Fabrication Method of Highly Macroporous Chitosan/Agarose/Nanohydroxyapatite Bone Scaffold for Potential Regenerative Medicine Applications
Abstract
:1. Introduction
2. Materials and Methods
2.1. Optimization of the Scaffold Fabrication Method
2.2. Porosity Determination
2.3. Compression Test
2.4. Bioactivity Test
2.5. Cell Culture Experiments
2.5.1. Cytotoxicity Tests
2.5.2. Cell Growth on the Surface of the Scaffold
2.6. Statistical Analysis
3. Results and Discussion
3.1. Porosity Determination
3.2. Compression Test
3.3. Bioactivity Test
3.4. Cell Culture Tests
4. Conclusions
5. Patents
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Rose, F.R.A.J.; Oreffo, R.O.C. Bone tissue engineering: Hope vs hype. Biochem. Biophys. Res. Commun. 2002, 292, 1–7. [Google Scholar] [CrossRef] [PubMed]
- Roseti, L.; Parisi, V.; Petretta, M.; Cavallo, C.; Desando, G.; Bartolotti, I.; Grigolo, B. Scaffolds for bone tissue engineering: State of the art and new perspectives. Mater. Sci. Eng. C 2017, 78, 1246–1262. [Google Scholar] [CrossRef] [PubMed]
- Przekora, A. The summary of the most important cell-biomaterial interactions that need to be considered during in vitro biocompatibility testing of bone scaffolds for tissue engineering applications. Mater. Sci. Eng. 2019, 97, 1036–1051. [Google Scholar] [CrossRef] [PubMed]
- Przekora, A.; Vandrovcova, M.; Travnickova, M.; Pajorova, J.; Molitor, M.; Ginalska, G.; Bacakova, L. Evaluation of the potential of chitosan/β-1,3-glucan/hydroxyapatite material as a scaffold for living bone graft production in vitro by comparison of ADSC and BMDSC behaviour on its surface. Biomed. Mater. 2017, 12. [Google Scholar] [CrossRef] [PubMed]
- Liao, H.-T.; Chen, C.-T. Osteogenic potential: Comparison between bone marrow and adipose-derived mesenchymal stem cells. World J. Stem Cells 2014, 6, 288–295. [Google Scholar] [CrossRef] [PubMed]
- Karageorgiou, V.; Kaplan, D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005, 26, 5474–5491. [Google Scholar] [CrossRef] [PubMed]
- Mour, M.; Das, D.; Winkler, T.; Hoenig, E.; Mielke, G.; Morlock, M.M.; Schilling, A.F. Advances in porous biomaterials for dental and orthopaedic applications. Materials 2010, 3, 2947–2974. [Google Scholar] [CrossRef]
- Sachot, N.; Engel, E.; Castano, O. Hybrid organic-inorganic scaffolding biomaterials for regenerative therapies. Curr. Org. Chem. 2014, 18, 2299–2314. [Google Scholar] [CrossRef]
- Chang, H.-I.; Wang, Y. Cell responses to surface and architecture of tissue engineering scaffolds. In Regenerative Medicine and Tissue Engineering Cells and Biomaterials; IntechOpen: London, UK, 2011. [Google Scholar] [CrossRef]
- Przekora, A.; Palka, K.; Ginalska, G. Biomedical potential of chitosan/HA and chitosan/β-1,3-glucan/HA biomaterials as scaffolds for bone regeneration—A comparative study. Mater. Sci. Eng. 2016, 58. [Google Scholar] [CrossRef]
- Chang, Y.-S.; Gu, H.-O.; Kobayashi, M.; Oka, M. Influence of various structure treatments on histological fixation of titanium implants. J. Arthroplasty 1998, 13, 816–825. [Google Scholar] [CrossRef]
- Subia, B.; Kundu, J.; Kundu, S.C. Biomaterial scaffold fabrication techniques for potential tissue engineering applications. In Tissue Engineering; Eberli, D., Ed.; IntechOpen: London, UK, 2010. [Google Scholar] [CrossRef]
- Liang, X.; Qi, Y.; Pan, Z.; He, Y.; Liu, X.; Cui, S.; Ding, J. Design and preparation of quasi-spherical salt particles as water-soluble porogens to fabricate hydrophobic porous scaffolds for tissue engineering and tissue regeneration. Mater. Chem. Front. 2018, 2, 1539–1553. [Google Scholar] [CrossRef]
- Felfel, R.M.; Gideon-adeniyi, M.J.; Zakir, K.M.; Roberts, G.A.F.; Grant, D.M. Structural, mechanical and swelling characteristics of 3D scaffolds from chitosan-agarose blends. Carbohydr. Polym. 2019, 204, 59–67. [Google Scholar] [CrossRef] [PubMed]
- Tang, Z.; Li, X.; Tan, Y.; Fan, H.; Zhang, X. The material and biological characteristics of osteoinductive calcium phosphate ceramics. Regen. Biomater. 2018, 5, 43–59. [Google Scholar] [CrossRef] [PubMed]
- Kar, S.; Kaur, T.; Thirugnanam, A. Microwave-assisted synthesis of porous chitosan-modified montmorillonite-hydroxyapatite composite scaffolds. Int. J. Biol. Macromol. 2016, 82, 628–636. [Google Scholar] [CrossRef] [PubMed]
- Li, S.H.; De Wijn, J.R.; Layrolle, P.; De Groot, K. Synthesis of macroporous hydroxyapatite scaffolds for bone tissue engineering. J. Biomed. Mater. Res. 2002, 61, 109–120. [Google Scholar] [CrossRef] [PubMed]
- Kim, H.L.; Jung, G.Y.; Yoon, J.H.; Han, J.S.; Park, Y.J.; Kim, D.G.; Zhang, M.; Kim, D.J. Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering. Mater. Sci. Eng. 2015, 54, 20–25. [Google Scholar] [CrossRef] [PubMed]
- Gómez, S.; Vlad, M.D.; López, J.; Fernández, E. Design and properties of 3D scaffolds for bone tissue engineering. Acta Biomater. 2016, 42, 341–350. [Google Scholar] [CrossRef] [PubMed]
- Turnbull, G.; Clarke, J.; Picard, F.; Riches, P.; Jia, L.; Han, F.; Li, B.; Shu, W. 3D bioactive composite scaffolds for bone tissue engineering. Bioact. Mater. 2018, 3, 278–314. [Google Scholar] [CrossRef] [PubMed]
- Zheng, Z.; Wu, J.; Liu, M.; Wang, H.; Li, C.; Rodriguez, M.J.; Li, G.; Wang, X.; Kaplan, D.L. 3D bioprinting of self-standing silk-based bioink. Adv. Healthc. Mater. 2018, 7, e1701026. [Google Scholar] [CrossRef]
- Yang, C.; Huan, Z.; Wang, X.; Wu, C.; Chang, J. 3D printed Fe scaffolds with HA nanocoating for bone regeneration. ACS Biomater. Sci. Eng. 2018, 4, 608–616. [Google Scholar] [CrossRef]
- Kim, S.H.; Yeon, Y.K.; Lee, J.M.; Chao, J.R.; Lee, Y.J.; Seo, Y.B.; Sultan, M.T.; Lee, O.J.; Lee, J.S.; Yoon, S.I.; et al. Precisely printable and biocompatible silk fibroin bioink for digital light processing 3D printing. Nat. Commun. 2018, 9, 1620. [Google Scholar] [CrossRef] [PubMed]
- Augustine, R.; Dan, P.; Sosnik, A.; Kalarikkal, N.; Tran, N.; Vincent, B.; Thomas, S.; Menu, P.; Rouxel, D. Electrospun poly(vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation. Nano Res. 2017, 10, 3358–3376. [Google Scholar] [CrossRef]
- Moradi, S.L.; Golchin, A.; Hajishafieeha, Z.; Khani, M.-M.; Ardeshirylajimi, A. Bone tissue engineering: Adult stem cells in combination with electrospun nanofibrous scaffolds. J. Cell. Physiol. 2018, 233, 6509–6522. [Google Scholar] [CrossRef] [PubMed]
- Xu, Z.; Zhao, R.; Huang, X.; Wang, X.; Tang, S. Fabrication and biocompatibility of agarose acetate nanofibrous membrane by electrospinning. Carbohydr. Polym. 2018, 197, 237–245. [Google Scholar] [CrossRef] [PubMed]
- Wang, J.; Chen, Y.; Zhu, X.; Yuan, T.; Tan, Y.; Fan, Y.; Zhang, X. Effect of phase composition on protein adsorption and osteoinduction of porous calcium phosphate ceramics in mice. J. Biomed. Mater. Res. 2014, 102, 4234–4243. [Google Scholar] [CrossRef]
- Hannink, G.; Arts, J.J.C. Bioresorbability, porosity and mechanical strength of bone substitutes: What is optimal for bone regeneration? Injury 2011, 42, S22–S25. [Google Scholar] [CrossRef] [Green Version]
- Wagoner Johnson, A.J.; Herschler, B.A. A review of the mechanical behavior of CaP and CaP/polymer composites for applications in bone replacement and repair. Acta. Biomater. 2011, 7, 16–30. [Google Scholar] [CrossRef]
- Huang, C.; Ogawa, R. Mechanotransduction in bone repair and regeneration. FASEB J. 2010, 24, 3625–3632. [Google Scholar] [CrossRef]
- Prasadh, S.; Wong, R.C.W. Unraveling the mechanical strength of biomaterials used as a bone scaffold in oral and maxillofacial defects. Oral Sci. Int. 2018, 15, 48–55. [Google Scholar] [CrossRef]
- Li, L.; Kommareddy, K.; Pilz, C.; Zhou, C.; Fratzl, P.; Manjubala, I. In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres. Acta Biomater. 2010, 6, 2525–2531. [Google Scholar] [CrossRef]
- El-Meliegy, E.; Abu-Elsaad, N.I.; El-Kady, A.M.; Ibrahim, M.A. Improvement of physico-chemical properties of dextran-chitosan composite scaffolds by addition of nano-hydroxyapatite. Sci. Rep. 2018, 8, 12180. [Google Scholar] [CrossRef] [PubMed]
- Zadpoor, A.A. Relationship between in vitro apatite-forming ability measured using simulated body fluid and in vivo bioactivity of biomaterials. Mater. Sci. Eng. 2014, 35, 134–143. [Google Scholar] [CrossRef] [PubMed]
- Kokubo, T.; Takadama, H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006, 27, 2907–2915. [Google Scholar] [CrossRef] [PubMed]
- Shibata, H.; Yokoi, T.; Goto, T.; Kim, I.Y.; Kawashita, M.; Kikuta, K.; Ohtsuki, C. Behavior of hydroxyapatite crystals in a simulated body fluid: Effects of crystal face. J. Ceram. Soc. Jpn. 2013, 121, 807–812. [Google Scholar] [CrossRef]
- Kim, H.M.; Himeno, T.; Kawashita, M.; Kokubo, T.; Nakamura, T. The mechanism of biomineralization of bone-like apatite on synthetic hydroxyapatite: An in vitro assessment. J. R. Soc. Interface 2004, 1, 17–22. [Google Scholar] [CrossRef]
- Weng, J.; Liu, Q.; Wolke, J.G.C.; Zhang, X.; De Groot, K. Formation and characteristics of the apatite layer on plasma-sprayed hydroxyapatite coatings in simulated body fluid. Biomaterials 1997, 18, 1027–1035. [Google Scholar] [CrossRef] [Green Version]
- Dorozhkin, S.V.; Epple, M. Biological and medical significance of calcilum phosphates. Angew. Chem. Int. Ed. 2002, 41, 3130–3146. [Google Scholar] [CrossRef]
- Przekora, A.; Czechowska, J.; Pijocha, D.; Ślósarczyk, A.; Ginalska, G. Do novel cement-type biomaterials reveal ion reactivity that affects cell viability in vitro? Cent. Eur. J. Biol. 2014, 9, 277–289. [Google Scholar] [CrossRef] [Green Version]
- Malafaya, P.B.; Reis, R.L. Bilayered chitosan-based scaffolds for osteochondral tissue engineering: Influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor. Acta Biomater. 2009, 5, 644–660. [Google Scholar] [CrossRef] [Green Version]
- Gustavsson, J.; Ginebra, M.P.; Engel, E.; Planell, J. Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media. Acta Biomater. 2011, 7, 4242–4252. [Google Scholar] [CrossRef]
- Humphries, J.D.; Wang, P.; Streuli, C.; Geiger, B.; Humphries, M.J.; Ballestrem, C. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 2007, 179, 1043–1057. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Sample Designation | CH3COOH (% v/v) | NaHCO3 (% w/v) |
---|---|---|
Mat_0.5_0.5 | 0.5 | 0.5 |
Mat_0.5_1 | 0.5 | 1 |
Mat_1_1 | 1 | 1 |
Mat_1_1.5 | 1 | 1.5 |
Mat_2_2 | 2 | 2 |
Control | 2 | none |
Porosity (%) | Control | Mat_0.5_0.5 | Mat_0.5_1 | Mat_1_1 | Mat_1_1.5 | Mat_2_2 |
---|---|---|---|---|---|---|
Total | 8.81 ± 1.61 | 57.46 ± 2.08 * | 47.88 ± 7.48 *# | 59.61 ± 3.00 * | 45.79 ± 9.96 *# | 70.32 ± 2.33 * |
Closed | 8.56 ± 1.56 | 33.22 ± 2.65 * | 32.48 ± 2.46 *#$ | 22.27 ± 2.20 * | 30.16 ± 4.85 *#$ | 19.99 ± 3.01 * |
Open | 0.24 ± 0.14 | 24.24 ± 1.16 * | 15.40 ± 8.14 #$ | 37.34 ± 3.78 * | 15.63 ± 12.30 #$ | 50.32 ± 4.28 * |
Mechanical Parameters (MPa) | Control | Mat_0.5_0.5 | Mat_0.5_1 | Mat_1_1 | Mat_1_1.5 | Mat_2_2 |
---|---|---|---|---|---|---|
Compressive Strength | 36.19 ± 8.77 | 1.32 ± 0.11 * | 2.35 ± 0.44 * | 0.74 ± 0.03 * | 0.77 ± 0.04 * | 0.86 ± 0.06 * |
Young’s Modulus | 179.14 ± 9.38 | 10.17 ± 1.37 * | 11.79 ± 3.45 * | 6.04 ± 0.30 * | 6.69 ± 0.81 * | 7.42 ± 0.16 * |
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Kazimierczak, P.; Palka, K.; Przekora, A. Development and Optimization of the Novel Fabrication Method of Highly Macroporous Chitosan/Agarose/Nanohydroxyapatite Bone Scaffold for Potential Regenerative Medicine Applications. Biomolecules 2019, 9, 434. https://doi.org/10.3390/biom9090434
Kazimierczak P, Palka K, Przekora A. Development and Optimization of the Novel Fabrication Method of Highly Macroporous Chitosan/Agarose/Nanohydroxyapatite Bone Scaffold for Potential Regenerative Medicine Applications. Biomolecules. 2019; 9(9):434. https://doi.org/10.3390/biom9090434
Chicago/Turabian StyleKazimierczak, Paulina, Krzysztof Palka, and Agata Przekora. 2019. "Development and Optimization of the Novel Fabrication Method of Highly Macroporous Chitosan/Agarose/Nanohydroxyapatite Bone Scaffold for Potential Regenerative Medicine Applications" Biomolecules 9, no. 9: 434. https://doi.org/10.3390/biom9090434