Copyright © 2012 American Scientific Publishers All rights reserved Printed in the United States of America Article Journal of Biomedical Nanotechnology Vol. 9, 1–10, 2012 www.aspbs.com/jbn In Vitro ALP and Osteocalcin Gene Expression Analysis and In Vivo Biocompatibility of N -Methylene Phosphonic Chitosan Nanofibers for Bone Regeneration Pallab Datta 1 , Paulomi Ghosh 1 , Kuntal Ghosh 2 , Pritiprasanna Maity 1 , Sintu Kumar Samanta 3 , Sudip Kumar Ghosh 3 , Pradeep Kumar Das Mahapatra 2 , Jyotirmoy Chatterjee 1 , and Santanu Dhara 1 ∗ 1 School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India 2 Department of Microbiology, Vidyasagar University, Midnapore, West Bengal 721102, India 3 Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India Most polymeric nanofibers used for bone tissue engineering lack adequate functional groups for bioactivity. This study explores the potential of nanofibers of phosphate functionalized derivative of chitosan-N-methylene phosphonic chi- tosan (NMPC) for bone tissue engineering. Nanofibers were fabricated by electrospinning of NMPC/PVA blend solutions. NMPC/PVA nanofibers exhibited 172% higher viability of MG-63 cells compared to pure PVA nanofibers. ALP and Col- lagen type I genes revealed higher expression in NMPC nanofibers on day 3 whereas osteocalcin gene was expressed on day 7. In rabbit tibial defects, NMPC based electrospun graft showed presence of no adverse tissue reaction by histo- logical examination while radiological examination suggested acceleration of bone healing by 300% compared to defects without any scaffold. Thus it is concluded NMPC based nanofibers may have potential for bone grafting applications. KEYWORDS: Osteoinduction, Bone Healing, Electrospinning, Osteocalcin, Tibial Defect. INTRODUCTION In the tryst to obtain successful bone grafts, deficien- cies associated with present bone grafts-autografts, allo- grafts, metals or ceramics—have put enhanced focus on investigations involving polymer and polymer composites to meet the unmet clinical requirements. 1 Advantages of polymers over other materials include their processability, degradability, viscoelasticity, and cost effective availability amongst others. Moreover, as tissue engineering approach gains momentum as an alternative treatment modality compared to conventional structural reinforcements pro- vided at injury site, polymers like chitosan, polylactic acid, polyethylether ketones, alginates, and polycaprolac- tone are increasingly receiving attention as ideal materi- als for bone regeneration applications. 2–4 Vital to success of tissue engineering approach, lies the creation of a ∗ Author to whom correspondence should be addressed. Email: sdhara@smst.iitkgp.ernet.in Received: 26 June 2012 Revised/Accepted: 18 November 2012 cell-scaffold construct as graft material which is required to possess three important characteristics of osteoconduc- tion, osteoinduction and osteogenicity for complete inte- gration at implantation site. 5 Amongst the various types of scaffolds investigated for bone tissue engineering or guided bone regeneration, electrospun nanofibrous scaf- folds have received considerable attention for their abil- ity to provide structures which mimic extracellular matrix architecture, help in cell attachment and improve osteo- conduction. The process is simple and amenable to many post-electrospinning modifications to incorporate bioac- tive agents and make matrix osteoinductive. 6 7 For exam- ple, Shim et al. observed favorable bone formation with electrospun mesh of polylactic acid in rabbit calvarial defects while Shin et al. employed chitosan nanofibers for guided bone regeneration. 8 9 Similarly, many other studies with nanofibers composed of silk fibroin, titania, polycaprolactone, polylactic acid, or collagen have shown promise in inducing in vitro osteogenesis or as selective membranes allowing guided bone regeneration in animal studies. 10–13 Notwithstanding above results, quest to fur- ther accelerate bone healing have witnessed incorporation J. Biomed. Nanotechnol. 2012, Vol. 9, No. 5 1550-7033/2012/9/001/010 doi:10.1166/jbn.2012.1592 1