The use of electrospun nanofibers for guided bone regeneration or bone scaffolds are becoming increasingly important in tissue engineering for next-generation healthcare. However, understanding the interaction between the nanofiber scaffolds that organize into complex 3D organizations and cells for optimized growth is a recurrent problem yet to be overcome.

In this study we exploit high resolution 3D imaging using scanning electron microscopy (SEM) and focussed ion beam (FIB) microscopy to evaluate cryo-prepared electrospun nanofiber scaffolds. Specifically, a biodegradable electrospun nanofiber membrane fabricated from poly(D,L-lactide-co-glycolide) (PLGA) is used for guided bone regeneration for orthopaedic applications. The electrospun scaffolds are produced with randomly oriented and aligned nanofibers, creating a range of void sizes, which are crucial for controlling cells penetration into electrospun scaffolds. The secondary electron imaging in Figure 1 and 2 demonstrates osteoblasts spreading over the random and aligned nanofiber mat surface respectively and within the fibrous membrane after 4 days in culture. Osteoblasts tend to growth both along the principal fiber axis and migrate in all directions towards neighboring fibrous filopodia, localized at the edges of osteoblasts, help the sheets of cells to align in the nanofiber direction and participate in cell-cell adhesion. It is presumed that these filopodia–like–protrusions are responsible for osteoblast elongation and migration into the 3D network of the electrospun nanofiber mat.

The interaction between osteoblasts and osteoblast-derived mineralized nodule formation on the nanofiber membrane is visualized using 3D FIB-SEM imaging based on a ‘slice-and-view’ approach. The 3D imaging highlights a coherent interface at small length scales between osteoblasts and nanofiber surfaces through connections between osteoblast, their filopodia and the nanofibers that promote cell growth. These imaging results are supported by biochemical approaches that indicate the formation of proteins responsible for focal adhesion in osteoblasts.¹ The presented 3D tomography therefore presents a new approach in high resolution visualization the cell growth on electrospun nanofibers, and potentially other biomaterials, that will develop and design new biomaterials for a range of clinically important applications including orthopaedics of this work.

Acknowledgments

The study was conducted within the funding from SONATA 8 project granted by National Science Centre in Poland, No 2014/15/D/ST6/02598 

References

1.   U. Stachewicz, T. Qiao, S.C.F. Rawlinson, F.V. Almeida, W.-Q Li, M. Cattell, A.H. Barber,  “3D Imaging of Cell Interactions with Electrospun PLGA Nanofiber Membranes for Bone Regeneration”,  Acta Biomater, 27 (2015), 88-100.

Figures:

Figure 1. Scanning electron micrograph of electrospun PLGA fibers with osteoblasts showing cells spreading over the aligned fibers in electrospun mat.

Figure 2. Scanning electron micrograph of electrospun PLGA fibers with osteoblasts showing cells spreading over the random fibers in electrospun mat.

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EMC Abstracts - https://emc-proceedings.com/abstract/3d-imaging-via-fib-sem-tomography-at-nanoscale-for-tissue-engineering-applications/