Electrospun gelatin nanofibers: Optimization of genipin cross-linking to preserve fiber morphology after exposure to water Silvia Panzavolta a , Michela Gioffrè a , Maria Letizia Focarete a , Chiara Gualandi a , Laura Foroni b , Adriana Bigi a,⇑ a Department of Chemistry ‘‘G. Ciamician’’ and National Consortium of Materials Science and Technology (INSTM, Bologna RU), University of Bologna, Via Selmi 2, 40126 Bologna, Italy b Department of Specialistic Surgical and Anaesthesiological Sciences, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy article info Article history: Received 25 August 2010 Received in revised form 8 November 2010 Accepted 17 November 2010 Available online 21 November 2010 Keywords: Scaffold Gelatin Electrospinning Genipin Cross-linking abstract The development of suitable biomimetic three-dimensional scaffolds is a fundamental requirement of tis- sue engineering. This paper presents the first successful attempt to obtain electrospun gelatin nanofibers cross-linked with a low toxicity agent, genipin, and able to retain the original nanofiber morphology after water exposure. The optimized procedure involves an electrospinning solution containing 30 wt.% gelatin in 60/40 acetic acid/water (v/v) and a small amount of genipin, followed by further cross-linking of the as-electrospun mats in 5% genipin solution for 7 days, rinsing in phosphate-buffered saline and then air drying at 37 °C. The results of scanning electron microscopy investigations indicated that the cross-linked nanofibers were defect free and very regular and they also maintained the original morphology after exposure to water. Genipin addition to the electrospinning solution dramatically reduced the extensibil- ity of the as-electrospun mats, which displayed further remarkable improvements in elastic modulus and stress at break after successive cross-linking up to values of about 990 and 21 MPa, respectively. The results of the preliminary in vitro tests carried out using vascular wall mesenchymal stem cells indicated good cell viability and adhesion to the gelatin scaffolds. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction A suitable three-dimensional porous bioresorbable scaffold is considered mandatory for the successful development of an engi- neered tissue as a biological substitute. To this end, the scaffold should display structural and chemical properties matching as far as possible those of the extracellular matrix (ECM) [1–3]. Electrospinning is a simple and cost-effective technique that al- lows the fabrication of scaffolds, from both synthetic and natural polymers, mimicking the three-dimensional nano-scaled features of the ECM [4]. Synthetic bioresorbable polymers provide struc- tural functionalities to the scaffold. On the other hand, natural polymers display unique bioactive properties and excellent cellular affinity. Collagen type I, the most abundant structural protein in the human body, has frequently been used to produce electrospun scaffolds [5–8]. Compared with collagen, gelatin, which can be ob- tained through collagen thermal denaturation or physical and chemical degradation, is cheaper and does not show antigenicity under physiological conditions [9,10]. Moreover, gelatin is biode- gradable, biocompatible and displays many integrin binding sites for cell adhesion and differentiation. For these reasons gelatin is widely used in the pharmaceutical and medical fields in a variety of applications, including tissue engineering, wound dressing, drug delivery and gene therapy [11]. Although gelatin has been successfully electrospun into ultra- fine fibers, the preparation of gelatin scaffolds by means of elec- trospinning raises some critical issues. First, highly toxic solvents, among which are 1,1,1,3,3,3-hexaflouro-2-propanol [12] and 2,2,2-trifluoroethanol [9,13], are typically used to dissolve the pro- tein. Acetic acid [14], as well as water mixtures with acetic acid and ethyl acetate [15], have been proposed as solvents to circum- vent this problem. The solubility of the as-spun gelatin fibers is a further drawback for long-term medical applications. Cross-link- ing, which is necessary to improve the water resistance of the nanofibers, introduces an additional major issue when gelatin is used to create tissue engineered scaffolds. Indeed, cross-linking treatment of these materials must not only prevent gelatin dissolu- tion in water but also preserve the peculiar biomimetic nanofi- brous morphology of the electrospun scaffolds. Collagenous materials can be cross-linked using either physical methods, such as dehydrothermal treatment and ultraviolet and gamma irradiation, or chemical agents. Chemical cross-linking ex- ploits the large number of functional gelatin side groups and usu- ally involves bifunctional reagents such as glutaraldehyde and diisocyanates, as well as genipin, carbodiimides, acyl azide and polyepoxy compounds [10,16–18]. Cross-linking with glutaralde- 1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.11.021 ⇑ Corresponding author. Tel.: +39 051 2099551; fax: +39 051 2099456. E-mail address: adriana.bigi@unibo.it (A. Bigi). Acta Biomaterialia 7 (2011) 1702–1709 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat