Freeze-drying and electrospun fiber hybrid 3D structure

Scanning electron micrographs of: a) porous structure of freeze-dried conduits, b) Aligned fibronectin nanofibers produced through electrospining process. [Mottaghitalab et al. PLoS ONE 2013; 8(9): e74417. doi:10.1371/journal.pone.0074417. This work is licensed under a Creative Commons Attribution 4.0 International.]

Freeze-drying or thermal induced phase separation is commonly used to construct three-dimensional (3D) scaffold. 3D porous structure made out of nanofibers may also be fabricated through careful control of the freeze-drying process. However, most freeze-dried scaffolds are composed of pores made from randomly distributed or ordered thin walls which lack the nanofibrous topography of most natural extracellular matrix (ECM). Salamian et al (2013) did a comparison of fibroblast cultured on electrospun and freeze-dried poly (lactic-co-glycolic acid) (PLGA) and showed that the cell proliferation and viability is better on electrospun scaffold probably due to its nanofibrous topography. A limitation of electrospinning is the difficulty in building up scaffold thickness. Therefore, a combination of electrospinnig and freeze drying has been explored to make use of the advantages offered by both techniques.

There are a few ways in which freeze-drying has been used in combination with electrospinning and electrospun membranes. Freeze dried membrane may be used as a base substrate in which electrospun fibers are deposited [Uzunalan et al 2013]. Alternate layers of freeze dried material and electrospun membranes are subsequently stacked to form a thick scaffold. Electrospun fibers and freeze dried substrate bilayer may be constructed depending on the applications. An important consideration is the adhesion between the two layers. Uzunalan et al (2013) created this bilayer to mimic skin structure. A macro-porous freeze-dried collagen layer was first created. Electrospinning is then carried out to directly deposit collagen onto the underlying collagen scaffold. To improve the stability of the bilayer, cross-linking was carried out after electrospun fibers have been deposited. An important advantage of electrospinning is its relative ease of constructing aligned fibrous membrane. This has been shown to facilitate cell contact guidance. To take advantage of this property, Mottaghitalab et al (2013) requires aligned nanofibers to form the inner lumen of their nerve conduit. For this construction, aligned fibers were first constructed using a rotating mandrel. The aligned fibrous membrane is subsequently transferred onto the freeze-dried substrate and rolled up to form a tube.

For a thick 3D block structure, the electrospun fibers may either be evenly distributed throughout the freeze-dried structure or fit in as distinct layers. Vaquette and Cooper-White (2013) demonstrated the fabrication of a multi-layered scaffold by stacking polycaprolactone (PCL) electrospun fibrous membrane into a holder filled with poly(lactic-co-glycolic acid) (PLGA) solution. They used quenching in liquid nitrogen followed by leaching in cold water to remove the solvent such that PLGA acts as binders. Based on the same concept freeze drying may also be carried out to form porous interlayers between the fibrous layers.

Electrospun fibers may also be mixed within the freeze drying solution prior to the freeze drying process such that the fibers are distributed within freeze dried composite scaffold. Kim et al fabricated bioactive glass nanofiber by electrospinning of bioactive glass sol-poly-vinyl-butyral mixture followed by sintering to remove the organic component. The resultant bioactive glass nanofiber membrane is mixed with collagen solution followed by freeze-drying [Kim et al 2006].