Investigation of osteoblast-like cells cultured on nano-hydroxyapatite/chitosan based composite scaffold in the treatment of bone defects and limited mobility

Document Type : Research Paper


1 Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

2 Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Neuroscience Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

4 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Objective(s): Design and construction of biocompatible and biodegradable scaffolds are among the main goals of tissue engineering. Recently, use of nano-hydroxyapatite as a bioactive bioceramic agent with high similarity to the mineral phase of the human bone tissue, in combination with biodegradable polymers and implant coatings has attracted the attention of researchers in the field of biomaterial sciences. The present study aimed to assess the differentiation of bone marrow stromal cells (BMSCs) in osteoblast-like cells on the chitosan/polyethylene oxide (PEO)/nano-hydroxyapatite scaffold in mature rats.
Materials and Methods: Chitosan and PEO solution with the weight ratio of 80:20 and 70:30 were prepared, and 2% weight of nano-hydroxyapatite was added. Nanofibers were prepared using the electrospinning method, and the morphology was studied using scanning electron microscopy (SEM). Afterwards, the BMSCs of mature rats were cultured on nanofibers and differentiated by adding a differentiation medium. The survival of the differentiated cells was evaluated at the end of the first, second, and third week using acridine orange staining, and the morphology of the differentiated cells exposed to nanofibers was assessed using SEM.
Results: The mean diameter of the nanofibers with the ratio of 80:20 was 150±17 nanometers. The differentiation of BMSCs into the osteoblast-like cells on nanofibers was confirmed using Alizarin red staining. The results indicated a significant decrease in the survival of the differentiated cells in the nanofiber groups by the end of the third week of differentiation compared to the control samples.
Conclusion: According to the results, BMSCs could be differentiated into osteoblast-like cells in the presence of the chitosan/PEO nanofibers containing nano-hydroxyapatite.


1. Mohamed A, Xing M. Nanomaterials and nanotechnology for skin tissue engineering. Int J Burn Trauma. 2012; 2(1): 29-41.
2. Hashemi ZS, Soleimani M. Tissue Engineering Scaffolds: History, Types and Construction Methods. ASJ. 2011; 9(35): 146-168.
3. Curtis A, Riehle M. Tissue engineering: the biophysical background. Phys Med Biol. 2001; 46(4): 47-65.
4. Ma PX, Choi JW. Biodegradable Polymer Scaffolds with Well-Defined Interconnected Spherical Pore Network. Tissue Eng. 2001; 7(1); 23-33.
5. Bhattaraia N, Edmondson D, Veiseha O, Matsen FA, Zhang M. Electrospun chitosan-based nanofibers and their cellular compatibility. Biomaterials. 2005; 26(31): 6176–6184.
6. Fouda MFA, Nemat A, Gawish A, Baiuomy AR. Does the Coating of Titanium Implants by Hydroxyapatite affect the Elaboration of Free Radicals. An Experimental Study. AJBAS. 2009; 3(2): 1122-1129.
7. Nelea V, Pelletier H, Mille P, Muller D. High-energy ion beam implantation of hydroxyapatite thin films grown on TiN and ZrO inter-layers by pulsed laser deposition. Thin Solid Films. 2004; 453-454: 208-214.
8. Tohill M, Mantovani C, Wiberg M, Terenghi G. Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration. Neurosci Lett. 2004; 362(3): 200-203.
9. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiates in vitro according to a hierarchical model. J Cell Sci. 2000; 113(7); 1161-1166.
10. Emamgholi A, Rahimi M, Kaka G, Sadraie SH, Najafi S. Presentation of a novel model of chitosan-polyethylene oxide-nanohydroxyapatite nanofibers together with bone marrow stromal cells to repair and improve minor bone defects. IJBMS. 2015; 18: 887-893.
11. Yeu IS, Lee HY, Yi JS, Yang JH, Lee IW, Lee HK. The survival and migration pattern of the Bone marrow stromal cells after intracerebral transplantation in Rats. J Korean Neurosurg Soc. 2004; 36: 400-404.
12. Kulterer B, Friedl G, Jandrositz A, Sanchez-Cabo F, Prokesch A, Paar C, Scheideler M, Windhager R, Preisegger KH, Trajanoski Z. Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation. BMC Genomics. 2007; 8: 70.
13. Venkatesan J, Kim SK. Chitosan Composites for Bone Tissue Engineering-An overview. Mar Drugs. 2010; 8(8): 2252-2266.
14. Sheikh FA, Barakat NAM, Kanjwal MA, Park SJ, Park DK, Kim HY. Synthesis of Poly(vinyl alcohol) (PVA) nanofibers incorporating hydroxyapatite nanoparticles as future implant materials. 2010; 18(1): 59-66.
15. Shahrooz Z, Vahid H. A Nanofibrous Composite Scaffold of PCL/Hydroxyapatite-chitosan/PVA Prepared by Electrospinning. Iran Polym J. 2010; 19(6): 457-468.
16. Venugopal J, Low S, Choon AT, Kumar TSS, Ramakrishna S. Mineralization of osteoblasts with electrospun collagen/hydroxyapatite nanofibers. J Mater Sci: Mater Med. 2008; 19(5): 2039–2046.
17. Ramay HR, Zhang M. Biphasic calcium phosphate nanocomposite porous scaffolds for load-bearing bone tissue engineering. Biomaterials. 2004; 25(21); 5171-5180.
18. Liuyun J, Yubao L, Chengdong X. Preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering. J Biomed Sci. 2009; 16(1): 65.
19. Rahimi M, Emamgholi A, Seyyed Tabaei SJ, Khodadoust M, Taghipour H, Jafari A. Perspectives of chitosan nanofiber/film scaffolds with bone marrow stromal cells in tissue engineering and wound dressing. Nanomed J. 201 9; 6(1): 27-34.
20. Cao L, Liu G, Gan Y, Fan Q, Yang F, Zhang X, Tang T, Dai K. The use of autologous enriched bone marrow MSCs to enhance osteoporotic bone defect repair in long‐term estrogen deficient goats. Biomaterials 2012; 33(20): 5076‐5084.
21. Zhou H, Lee J. Nanoscale hydroxyapatite particles for bone tissue engineering. Acta Biomater. 2011; 7(7): 2769-2781.
22. Tripathi G, Basu B. A porous hydroxyapatite scaffold for bone tissue engineering: Physico-mechanical and biological evaluations. Ceram Int. 2012; 38(1): 341-349.
23. Leukers B, Gülkan H, Irsen SH, Milz S, Tille C, Schieker M, Seitz H. Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing. J Mater Sci Mater Med. 2005; 16(12): 1121-1124.
24. Guan J, Yang J, Dai J., Qin Y, Wang Y, Guo Y, Ke Q, Zhang C. Bioinspired nanostructured hydroxyapatite/collagen three-dimensional porous scaffolds for bone tissue engineering. RSC Adv. 2015; 5(46): 36175–36184.