Fabrication and Characterization of Zirconium Oxide Grafted Chitosan/Hydroxyapatite Scaffolds for Bone Tissue Engineering

Abstract

In this study, zirconium oxide (ZrO₂)-containing chitosan/hydroxyapatite (CH/HAp) scaffolds were successfully fabricated using a vacuum freeze-drying technique. Subsequently, the impact of ZrO₂ incorporation on their structural, physicochemical, and biological performance was systematically investigated. XRD, FTIR, and SEM analyses confirmed that HAp and ZrO2 were distributed throughout the CH network, and indicated chemical interactions between CH and the incorporated components. Both CH/HAp and CH/HAp/ZrO₂ scaffolds presented a highly interconnected porous structure, together with a pore size range (9–88 µm) appropriate for tissue engineering. Enzymatic degradation studies revealed that after 35 days, the lysozyme induced degradation reached 63% for the CH/HAp and 30% for the CH/HAp/ZrO2 scaffolds, while scaffolds remained structurally stable in enzyme-free PBS. Flow cytometry analysis demonstrated that both CH/HAp and CH/HAp/ZrO₂ scaffolds maintained osteoblast viability (73.4%) without inducing significant apoptosis compared to control cells (p > 0.05). These results show that the degradation resistance of the scaffolds can be improved by the incorporation of ZrO2 without compromising the biological properties, making the CH/HAp/ZrO2 scaffold promising for bone regeneration.

Keywords

• Chitosan
• Hydroxyapatite
• Zirconium oxide
• Biocompatible
• Degradable

Ethical Statement

This study did not involve human participants or animals. The experiments were performed using commercially available osteoblast cell lines. Therefore, ethical approval was not required.

References

1. Chiarello, E., Cadossi, M., Tedesco, G., Capra, P., Calamelli, C., Shehu, A., et al., "Autograft, allograft and bone substitutes in reconstructive orthopedic surgery", Aging Clinical and Experimental Research, 25 (1), 101–103, 2013.
2. Betz, R. R., "Limitations of autograft and allograft: new synthetic solutions", Orthopedics, 25 (5), 561-570, 2002.
3. Sabouri, Z., Dequeecker, M., Anees, H., Adib, F. R., Jamous, R., Zheng, J., et al., "Recent advances in biomaterials for bone regeneration: Bridging innovation and clinical translation", Mater Today Bio, 36, 102685, 2026.
4. Gómez, S., Vlad, M. D., López, J. and Fernández, E., "Design and properties of 3D scaffolds for bone tissue engineering", Acta Biomater, 42, 341–350, 2016.
5. Turnbull, G., Clarke, J., Picard, F., Riches, P., Jia, L., Han, F., et al., "3D bioactive composite scaffolds for bone tissue engineering", Bioact Mater, 3 (3), 278–314, 2018.
6. Abdelaziz, A. G., Nageh, H., Abdo, S. M., Abdalla, S. S., Amer, A. A., Abdal-hay, A., et al., "A Review of 3D Polymeric Scaffolds for Bone Tissue Engineering: Principles, Fabrication Techniques, Immunomodulatory Roles, and Challenges", Bioengineering, 10 (2), 204, 2023.
7. Gönenmiş, D. E. ve Özcan, Y., "Preparation of Diatom-Doped Bio-Nanocomposite Materials for Bone Tissue Scaffolds", Materials Research, 25, e20220234, 2022.
8. Sökmen, B., Özcan, Y., Abdelsalam, A. H., Gök, C. ve Arslan, S., "Fabrication and Characterization of Novel Porous Bioactive Glass with Enhanced Properties and Biocompatibility for Bone Tissue Engineering", Arabian Journal for Science and Engineering, 51, 1769–1788, 2025.

9. Özcan, Y., Gönenmiş, D. E., Kızılhan, E. ve Gök, C., "Highly Porous Biocomposite Scaffolds Fabricated by Chitosan/Alginate/Diatom for Tissue Engineering", Polymer (Korea), 46 (4), 455–462, 2022.
10. Kocak, F. Z., Yar, M. ve Rehman, I. U., "In vitro degradation, swelling, and bioactivity performances of in situ forming injectable chitosan-matrixed hydrogels for bone regeneration and drug delivery", Biotechnol Bioeng, 121 (9), 2767–2779, 2024.
11. Daldiken, E., Koçak, F. Z. ve Küçükdeveci, N., "Synthesis and characterisations of clinoptilolite enriched hydroxyapatite nanoceramics by sol-gel route for bone regeneration", Ceram Int, 50 (11), 19650–19659, 2024.
12. Ji, C., Khademhosseini, A. and Dehghani, F., "Enhancing cell penetration and proliferation in chitosan hydrogels for tissue engineering applications", Biomaterials, 32 (36), 9719–9729, 2011.
13. Asghari, F., Samiei, M., Adibkia, K., Akbarzadeh, A. and Davaran, S., "Biodegradable and biocompatible polymers for tissue engineering application: a review", Artif Cells Nanomed Biotechnol, 45 (2), 185–192, 2017.
14. Eldeeb, A. E., Salah, S. and Elkasabgy, N. A., "Biomaterials for Tissue Engineering Applications and Current Updates in the Field: A Comprehensive Review", AAPS PharmSciTech, 23 (7), 267, 2022.
15. Ressler, A., "Chitosan-Based Biomaterials for Bone Tissue Engineering Applications: A Short Review", Polymers, 14 (16), 3430, 2022.
16. Ying, R., Wang, H., Sun, R. and Chen, K., "Preparation and properties of a highly dispersed nano-hydroxyapatite colloid used as a reinforcing filler for chitosan", Materials Science and Engineering: C, 110, 110689, 2020.
17. Mohamed, K. R. and Mostafa, A. A., "Preparation and bioactivity evaluation of hydroxyapatite-titania/chitosan-gelatin polymeric biocomposites", Materials Science and Engineering: C, 28 (7), 1087–1099, 2008.
18. Hammouda, H. F., Farag, M. M., El Deftar, M. M. F., Abdel-Gabbar, M. and Mohamed, B. B., "Effect of Ce-doped bioactive glass/collagen/chitosan nanocomposite scaffolds on the cell morphology and proliferation of rabbit’s bone marrow mesenchymal stem cells-derived osteogenic cells", Journal of Genetic Engineering and Biotechnology, 20(1), 33, 2022.
19. Okpe, P. C., Folorunso, O., Aigbodion, V. S. and Obayi, C., "Hydroxyapatite synthesis and characterization from waste animal bones and natural sources for biomedical applications", J Biomed Mater Res B Appl Biomater, 112 (7), e35440, 2024.
20. Kong, L., Gao, Y., Lu, G., Gong, Y., Zhao, N. and Zhang, X., "A study on the bioactivity of chitosan/nano-hydroxyapatite composite scaffolds for bone tissue engineering", Eur Polym J, 42 (12), 3171–3179, 2006.
21. Jahan, K., Manickam, G., Tabrizian, M. and Murshed, M., "In vitro and in vivo investigation of osteogenic properties of self-contained phosphate-releasing injectable purine-crosslinked chitosan-hydroxyapatite constructs", Scientific Reports, 10 (1), 11603, 2020.
22. Soriente, A., Fasolino, I., Gomez-Sánchez, A., Prokhorov, E., Buonocore, G. G., Luna-Barcenas, G., et al., "Chitosan/hydroxyapatite nanocomposite scaffolds to modulate osteogenic and inflammatory response", J Biomed Mater Res A, 110 (2), 266-277, 2022.
23. Jang, T. S., Jung, H. D., Pan, H. M., Han, W. T., Chen, S. and Song, J., "3D printing of hydrogel composite systems: Recent advances in technology for tissue engineering", Int J Bioprint, 4 (1), 126, 2018.
24. Liu, Y., Rath, B., Tingart, M. and Eschweiler, J., "Role of implants surface modification in osseointegration: A systematic review", J Biomed Mater Res A, 108 (3), 470–484, 2020.
25. Long, S., Zhu, J., Jing, Y., He, S., Cheng, L. and Shi, Z., "A Comprehensive Review of Surface Modification Techniques for Enhancing the Biocompatibility of 3D-Printed Titanium Implants", Coatings, 13 (11), 1917, 2023.
26. Tian, Y., Song, Y., Lan, S., Geng, R., Wang, M., Li, S., et al., "Ceria-Stabilized Zirconia/Alumina Nanocomposite (NANO-Zr) Surface Enhances Osteogenesis Through Regulation of Macrophage Polarization", Coatings, 14 (11), 1460, 2024.
27. Webster, T. J., Siegel, R. W. and Bizios, R., "Osteoblast adhesion on nanophase ceramics", Biomaterials, 20 (12), 1221–1227, 1999.
28. Jayakumar, R., Ramachandran, R., Sudheesh Kumar, P. T., Divyarani, V. V., Srinivasan, S., Chennazhi, K. P., et al., "Fabrication of chitin–chitosan/nano ZrO2 composite scaffolds for tissue engineering applications", Int J Biol Macromol, 49 (3), 274–280, 2011.
29. Girija, M. and Sampath Kumar, T., "Characterization of akermanite (AKT) and zirconia-infused PMMA bone cement composite with superior physicochemical, mechanical, and bioactive properties for enhanced orthopaedic performance", Compos Interfaces, 32 (6), 817–841, 2025.
30. Shao, R. X., Quan, R. F., Wang, T., Du, W. B., Jia, G. Y., Wang, D., et al., "Effects of a bone graft substitute consisting of porous gradient HA/ZrO2 and gelatin/chitosan slow-release hydrogel containing BMP-2 and BMSCs on lumbar vertebral defect repair in rhesus monkey", J Tissue Eng Regen Med, 12 (3), e1813–e1825, 2018.
31. Sollazzo, V., Pezzetti, F., Scarano, A., Piattelli, A., Bignozzi, C. A., Massari, L., et al., "Zirconium oxide coating improves implant osseointegration in vivo", Dental Materials, 24 (3), 357–361, 2008.
32. Al-Mohammedawi, N. A., Kashan, J. S. and Zaidan, S. A., "Biocompatibility Assessment of TiO2-CaO-ZrO2/HDPE Hybrid Bio-Nanocomposites Used in Bone-Tissue Engineering Scaffolds", International Journal of Nanoelectronics and Materials (IJNeaM), 18 (June), 33–46, 2025.
33. Thirugnanasambandam, A., Nallamuthu, R., Narayanaswamy, N., Rangappa, S. M., Siengchin, S. and Kechagias, J. D., "A comprehensive investigation of the 3D printed polylactic acid/yttria-stabilized zirconia nanocomposite scaffold for orthopedic applications", The International Journal of Advanced Manufacturing Technology, 139 (1), 929–944, 2025.
34. Ruiz-Aguilar, C., Alcántara-Quintana, L. E., Aguilar-Reyes, E. A. and Olivares-Pinto, U., "Fabrication, characterization, and in vitro evaluation of β-TCP/ZrO2-phosphate-based bioactive glass scaffolds for bone repair", Boletín de la Sociedad Española de Cerámica y Vidrio, 61 (3), 191–202, 2022.
35. Khoo, W., Nor, F. M., Ardhyananta, H. and Kurniawan, D., "Preparation of Natural Hydroxyapatite from Bovine Femur Bones Using Calcination at Various Temperatures", Procedia Manuf, 2, 196–201, 2015.
36. Kumar, P., "Nano-TiO2 Doped Chitosan Scaffold for the Bone Tissue Engineering Applications", Int J Biomater, 2018 (1), 6576157, 2018.
37. Venkatesan, J., Qian, Z. J., Ryu, B., Ashok Kumar, N. and Kim, S. K., "Preparation and characterization of carbon nanotube-grafted-chitosan – Natural hydroxyapatite composite for bone tissue engineering", Carbohydr Polym, 83 (2), 569–577, 2011.
38. Pinto, R. V., Gomes, P. S., Fernandes, M. H., Costa, M. E. V. and Almeida, M. M., "Glutaraldehyde-crosslinking chitosan scaffolds reinforced with calcium phosphate spray-dried granules for bone tissue applications", Materials Science and Engineering: C, 109, 110557, 2020.
39. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W., "NIH Image to ImageJ: 25 years of image analysis", Nature Methods, 9 (7), 671–675, 2012.
40. Ishrat, U. and Rafiuddin., "Synthesis, characterization and electrical properties of Titanium molybdate composite membrane", Desalination, 286, 8–15, 2012.
41. Thein-Han, W. W. and Misra, R. D. K., "Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering", Acta Biomater, 5 (4), 1182–1197, 2009.
42. Adekogbe, I. and Ghanem, A., "Fabrication and characterization of DTBP-crosslinked chitosan scaffolds for skin tissue engineering", Biomaterials, 26 (35), 7241–7250, 2005.
43. Swift, M. L., "GraphPad Prism, Data Analysis, and Scientific Graphing", J Chem Inf Comput Sci, 37 (2), 411–412, 1997.
44. Yang, C., Yang, P., Wang, W., Gai, S., Wang, J., Zhang, M., et al., "Synthesis and characterization of Eu-doped hydroxyapatite through a microwave assisted microemulsion process", Solid State Sci, 11 (11), 1923–1928, 2009.
45. Reddy, C. V., Babu, B., Reddy, I. N. and Shim, J., "Synthesis and characterization of pure tetragonal ZrO2 nanoparticles with enhanced photocatalytic activity", Ceram Int, 44 (6), 6940–6948, 2018.
46. Cui, L., Gao, S., Song, X., Huang, L., Dong, H., Liu, J., et al., "Preparation and characterization of chitosan membranes", RSC Adv, 8 (50), 28433–28439, 2018.
47. Sudatta, B. P., Sugumar, V., Varma, R. and Nigariga, P., "Extraction, characterization and antimicrobial activity of chitosan from pen shell, Pinna bicolor", Int J Biol Macromol, 163, 423–430, 2020.
48. Sahadat Hossain, M. and Ahmed, S., "FTIR spectrum analysis to predict the crystalline and amorphous phases of hydroxyapatite: a comparison of vibrational motion to reflection", RSC Adv, 13 (21), 14625–14630, 2023.
49. Khedr, R. F. and Elhady, M. A., "Development the Hydrophobic Property of Polyvinyl Alcohol/Silicon Dioxide/Titanium Dioxide Nanocomposites for Self-Cleaning and Soil Stabilization", Molecules, 30 (8), 1664, 2025.
50. Kumar, P., Brijnandan. and Dehiya, S. ve Sindhu, A., "Comparative study of chitosan and chitosan–gelatin scaffold for tissue engineering", International Nano Letters, 7 (4), 285–290, 2017.
51. Vladu, A. F., Motelica, L., Oprea, O. C., Doina Truşcă, R., Iordache, F., Ficai, D., et al., "Chitosan/Hydroxyapatite Composite Grafts for Bone Tissue Engineering", UPB Scientific Bulletin, Series B: Chemistry and Materials Science, 87 (3), 175-186, 2025.
52. Bhowmick, A., Jana, P., Pramanik, N., Mitra, T., Banerjee, L. S., Gnanamani, A., et al., "Multifunctional zirconium oxide doped chitosan based hybrid nanocomposites as bone tissue engineering materials", Carbohydr Polym, 151, 879–888, 2016.
53. Thommes, M., Kaneko, K., Neimark, A. V., Olivier, J. P., Rodriguez-Reinoso, F., Rouquerol, J., et al., "Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)", Pure and Applied Chemistry, 87 (9-10), 1051–1069, 2015.
54. Azadi, M., Teimouri, A. and Mehranzadeh, G., "Preparation, characterization and biocompatible properties of β-chitin/silk fibroin/nanohydroxyapatite composite scaffolds prepared using a freeze-drying method", RSC Adv, 6 (9), 7048–7060, 2016.
55. Xu, Y., Xia, D., Han, J., Yuan, S., Lin, H. and Zhao, C., "Design and fabrication of porous chitosan scaffolds with tunable structures and mechanical properties", Carbohydr Polym, 177, 210–216, 2017.
56. Reves, B. T., Jennings, J. A., Bumgardner, J. D. and Haggard, W. O., "Preparation and Functional Assessment of Composite Chitosan-Nano-Hydroxyapatite Scaffolds for Bone Regeneration", Journal of Functional Biomaterials, 3 (1), 114-130, 2012.
57. Attaeyan, A., Shahgholi, M. and Karimipour, A., "Enhancing Mechanical Properties of Chitosan–Silica Aerogels with Tricalcium Phosphate Nanoparticles: A Molecular Dynamics Study for Bone Tissue Engineering", Polymers, 17 (6), 755, 2025.
58. Felfel, R. M., Gideon-Adeniyi, M. J., Zakir Hossain, K. M., Roberts, G. A. F. and Grant, D. M., "Structural, mechanical and swelling characteristics of 3D scaffolds from chitosan-agarose blends", Carbohydr Polym, 204, 59–67, 2019.
59. Hu, Z., Hong, P., Liao, M., Kong, S., Huang, N., Ou, C., et al., "Preparation and Characterization of Chitosan—Agarose Composite Films", Materials, 9 (10), 816, 2016.
60. Martins, A. M., Santos, M. I., Azevedo, H. S., Malafaya, P. B. and Reis, R. L., "Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications", Acta Biomater, 4 (6), 1637–1645, 2008.
61. Hamidi, S., Maton, M., Hildebrand, F., Gaucher, V., Bossard, C., Cazaux, F., et al., "Design and Evaluation of a Crosslinked Chitosan-Based Scaffold Containing Hyaluronic Acid for Articular Cartilage Reconstruction", Molecules, 30 (10), 2202, 2025.
62. Jennings, J. A., "Controlling chitosan degradation properties in vitro and in vivo", Chitosan Based Biomaterials, 1, 159–182, 2017.
63. Sabido-Barahona, A. B., Vargas-Coronado, R. F., Hernández-Sánchez, F., Martínez-Richa, A., Gómez Ribelles, J. L., Cauich-Rodríguez, J. V., et al., "Segmented Polyurethanes Based on Adipate and Sebacate Biodegradable Polyesters for Use as Nerve Guide Conduits in Peripheral Nerve Regeneration", Polymers, 17 (12), 1692, 2025.
64. Gelati, L., Rabuffetti, M., Benaglia, M., Campisi, S., Gervasini, A., Speranza, G., et al., "Hydroxyapatite: An Eco-Friendly Material for Enzyme Immobilization", Chempluschem, 89 (8), e202400204, 2024.
65. Pramanik, S., Aggarwal, A., Kadi, A., Alhomrani, M., Alamri, A. S., Alsanie, W. F., et al., "Chitosan alchemy: transforming tissue engineering and wound healing", RSC Adv, 14 (27), 19219–19256, 2024.