Gelatin/hyaluronic acid nanofibrous scaffolds: biomimetics of extracellular matrix

Abstract

In living systems, extracellular matrix (ECM) plays a pivotal role in controlling cell behavior [1]. One of the most significant objectives in tissue engineering is to design and obtain scaffolds with the ability of biomimicking natural ECM in chemical compositions, physical structure, and biological functions [2,3]. Natural ECM is composed of a cross-linked porous network of multifibril collagens with diameters ranging from 50 to 500 nm and embedded in glycosaminoglycans [4,5]. Electrospinning technique has been recognized as an efficient processing method for the preparation of ECM analog scaffolds composed of nanoscale fibers, which possess high surface area to volume ratio and high porosity and thus can promote cell adhesion, migration, and proliferation [3]. Since gelatin (GE) is a protein biopolymer derived from partial hydrolysis of native collagens and hyaluronic acid (HA) is a kind of polysaccharide in natural ECM, electrospun nanofibrous scaffolds of GE and HA complex could biomimic both the composition and the nanofibrous structure of natural ECM for tissue engineering. Here, we report the preparation and preliminary characterization of GE/HA blended nanofibrous scaffolds. GE/HA nanofibrous scaffolds were prepared through an electrospinning method [6]. Pure GE (type A; SigmaAldrich, St Louis, USA) and HA (sodium salt, MW 1⁄4 200,000; Zhejiang Dali Technology, Hangzhou, China) were blended with different weight ratios (GE/HA 1⁄4 10 : 0, 9 : 1, 8 : 2, 7 : 3 and 6 : 4) and then dissolved in 2,2,2trifluoroethanol (TFE)/water (1 : 1; v/v) solvents and stirred at room temperature for 6 h. GE/HA ratios below 5 : 5 were not tested, because no fibers were formed due to high viscosity and surface tension of the blends solution. The concentration of solutions was set at 10% (w/v), because a small quantity of nanofibers with bead-on-strings occurred when concentrations were set ,8% (w/v) or .12% (w/v) (data not shown). The solutions were placed into a 2.5-ml plastic syringe with a blunt-ended needle with an inner diameter of 0.21 mm. The needle was located at a distance of 13–15 cm from the grounded collector. A syringe pump (789100C; Cole-Parmer, Vernon Hills, USA) was employed to feed solutions to the needle tip at a feed rate of 0.8 ml/h. A high electrospinning voltage (20 kV) was applied between the needle and the ground collector using a high voltage power supply (BGG6-358; BMEI, Beijing, China). The morphology of the electrospun fibers was observed with a scanning electronic microscope (SEM) (JSM-5600; JEOL, Tokyo, Japan). The diameter range of the fabricated ultrafine fibers was measured based on the SEM images using an image visualization software Image J 1.34s (National Institutes of Health, Bethesda, USA) and calculated by selecting 100 fibers randomly observed on the SEM images. Bead-on-strings occurs when pure GE is used. The nanofibers became uniform without bead-on-strings and the average diameters of nanofibers gradually increased with increasing HA content in the blends. The unusually high molecular weight of HA contributed to the distinctly increased viscosity of the mixed solution [7]. The SEM micrographs of GE/HA nanofibers with different weight ratios are shown in Fig. 1. The most appropriate GE/HA ratio was 7 : 3. The GE/HA nanofibrous scaffolds were finally cross-linked in 1.5 M ethanol solutions of N-(3-dimethylaminopropyl)N-ethylcarbodiimide hydrochloride at 48C for different times, and then dried in a vacuum at room temperature for 3 days. The SEM micrographs of GE/HA nanofibrous scaffolds with different cross-linking time are shown in Fig. 2. The optimal cross-linking time was 30 min. The surface wettability plays an important role in affecting cell attachment, proliferation, and migration [8]. To clarify surface wettability of electrospun GE/HA nanofibrous scaffolds, we measured the water contact angles of nanofibrous scaffolds before and after cross-linking (Table 1). Pure GE nanofibrous scaffolds were 88.38. With the increase of HA content, water contact angle on nanofibrous scaffolds Acta Biochim Biophys Sin 2013, 45: 700–703 | a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt032. Advance Access Publication 16 April 2013