Introduction: Electrospun nanofibers have shown great promise as a scaffold for regenerative medicine due to their biomimicry of the architecture of extracellular matrix (ECM) and the size of ECM collagen fibrils[1]. Traditional electrospinning typically produces uncontrolled and densely packed fibers, however, resulting in compact two-dimensional (2D) nanofiber mats/membranes and hindrance of both cell infiltration and growth throughout the nanofiber scaffolds[2]. Thus traditional 2D nanofiber mats are limited as an ideal substrate for their applications in regenerative medicine and engineered three-dimensional (3D) tissue models. There is an imperative need for the development of new methods for creating electrospun 3D nanofiber scaffolds. Herein, we present a new method for three-dimensionally expanding electrospun poly(ε-caprolactone) (PCL, an FDA-approved, biocompatible and biodegradable polymer) nanofiber mats utilizing a modified gas foaming technique followed by freeze-drying.
Materials and Methods: Nanofibers were produced utilizing a standard electrospinning setup, described previously[3]. Poly(ε-caprolactone) (PCL) (Mw=80 kDa, Sigma-Aldrich, St. Louis, MO) was dissolved in a solvent mixture consisting of dichloromethane (DCM) and N, N-dimethylformamide (DMF) (Fisher Chemical, Waltham, MA) with a ratio of 4:1 (v/v) (at a concentration 10% (w/v)). Rotating mandrels were employed to generate both random and aligned nanofiber mats. PCL nanofiber mats were immersed in 40 ml fresh prepared NaBH4 solutions and shaken at 50 rpm for varying lengths of time at 21oC.
Results and Discussion: Electrospun nanofiber mats were successfully expanded in the third dimension after treatment with the NaBH4 aqueous solutions (Fig. 1). The terminal thickness of treated nanofiber mats increased with increasing time in solution and with increasing concentration of NaBH4. Surprisingly, the thickness of nanofiber mats increased from 1 mm to 35.6 mm after only 24 h treatment with the 1 M NaBH4 aqueous solution. Similarly, the porosity of aligned PCL nanofiber scaffolds increased with increasing the reaction time and increasing concentration of NaBH4, which was in line with the trend of thickness. The scaffold porosity increased to 99.2% after treatment with a 1 M NaBH4 aqueous solution for 24 h from a baseline porosity of 83.6% for the starting nanofiber mat.
Fig. 1. Expanded nanofiber scaffolds. (A-C): photographs showing the nanofiber scaffold before and after expansion for 20 min and 24 h. (D-F): SEM images showing the cross-sectional morphologies of nanofiber scaffolds in (A-C).
In order to maintain the integrity of nanofiber scaffolds following expansion, the scaffolds were freeze-dried using a lyophilizer. Nanofiber scaffolds were then cut along two different planes (x-y, y-z) and examined via scanning electron microscopy (SEM) to reveal the detailed fiber architecture of scaffolds. Prior to expansion, aligned electrospun PCL nanofiber mats were composed of densely packed fibrillar structures. In contrast, nanofiber scaffolds expanded for 20 min and 24 h displayed layered structures with preserved nanotopographic cues rendered by aligned nanofibers. Gap distances were noted to increase with increasing reaction time. In contrast, layer thickness decreased from approximately 15 μm to 5 μm with increasing reaction time.
Cell culture results indicated that cells successfully infiltrated and proliferated throughout the bulk of expanded aligned nanofiber scaffolds. In comparison, cells did not penetrate the unexpanded nanofiber scaffolds and only proliferated on the surface of the material. Cellular morphology was consistent between expanded and unexpanded scaffolds, as cells displayed an elongated shape along the direction of fiber alignment.
Conclusion: In summary, we have demonstrated a controllable method for expanding electrospun nanofiber mats/membranes in the third dimension while preserving imparted anisotropic features and cues.
NIH NIGMS Grant 2P20GM103480-08 and UNMC Regenerative Medicine Program grant 37-1209-2004-007.
References:
[1] J. Xie, X. Li, and Y. Xia, Macromol. Rapid Commun., 2008, 29, 1775.
[2] B. A. Blakeney et al., Biomaterials 2011, 32, 1583.
[3] J. Jiang et al., ACS Biomater. Sci. & Eng., 2015, DOI: 10.1021/acsbiomaterials.5b00238.