Three-dimensional Reconstruction of the Microstructure of Human Acellular Nerve Allograft

Abstract

The exact inner 3D microstructure of the human peripheral nerve has been a mystery for decades. Therefore, it has been difficult to solve several problems regarding peripheral nerve injury and repair. We used high-resolution X-ray computed microtomography (microCT) to scan a freeze-dried human acellular nerve allograft (hANA). The microCT images were then used to reconstruct a 3D digital model, which was used to print a 3D resin model of the nerve graft. The 3D digital model of the hANA allowed visualization of all planes. The magnified 3D resin model clearly showed the nerve bundles and basement membrane tubes of the hANA. Scanning electron microscopy (SEM) was used to analyse the microstructure of the hANA. Compared to the SEM images, the microCT image clearly demonstrated the microstructure of the hANA cross section at a resolution of up to 1.2 μm. The 3D digital model of the hANA facilitates a clear and easy understanding of peripheral nerve microstructure. Furthermore, the enlarged 3D resin model duplicates the unique inner structure of each individual hANA. This is a crucial step towards achieving 3D printing of a hANA or nerve that can be used as a nerve graft.

MicroCT optimisation for imaging fascicular anatomy in peripheral nerves

“…In addition, the size of the data obtained, and thus memory required, from this more accessible Nikon scanner is significantly lower (2 to <5 GB per scan of 1 cm of nerve for rat sciatic and pig vagus nerves respectively) than from the synchrotron scanners. This obviates the requirement for supercomputers whilst still maintaining the advantage of microCT over the predominant soft-tissue imaging technique of magnetic resonance imaging (MRI) by obtaining high voxel resolution in 3D (Zhu et al, 2016). Additionally, microCT exceeds the imaging penetration depth of another prevailing imaging technique, optical coherence tomography (Islam et al, 2012).…”

“…However, the imaging of biological soft tissue, limited by the low intrinsic X-ray contrast of non-mineralised tissues, has increased in the recent years with methods incorporating the use of contrast enhancement agents such as osmium , reduced silver (Mizutani et al, 2007), resin perfusion (Wirkner and Prendini, 2007;Wirkner and Richter, 2004), and iodine (de Crespigny et al, 2008;Degenhardt et al, 2010;Gignac and Kley, 2014;Heimel et al, 2019;Jeffery et al, 2011;Metscher, 2009;Yan et al, 2017). It has been successfully applied to tendons (Kalson et al, 2012), ligaments (Shearer et al, 2014) and nerves (Yan et al, 2017;Zhu et al, 2016). However, there are some shortcomings of the method used hitherto.…”

The neurochemistry of peripheral nerve regeneration

“…[98] A new technology, 3D printing technology, has been combined with micro-computed tomography to both demonstrate and replicate the internal structure of acellular nerve allografts. [99] Cellular therapies in nerve regeneration may be performed with stem cells or SCs. Stem cells can differentiate into glial fibrillary acidic protein-positive SCs and can support myelination and the repair process.…”

Advances and Future Applications of Augmented Peripheral Nerve Regeneration

“…The addition of 3D printing technology is promising as it is possible to create “smart” scaffolding with internal cues and spatial gradients of growth factors to direct nerve regeneration in mixed nerves and across bifurcations [ 68 ]. 3D printing technology has been combined with computed microtomography (microCT) to both demonstrate and replicate the internal structure of acellular nerve allografts [ 139 ]. It is quite foreseeable to have a peripheral nerve replacement that spans across a bifurcation with a native internal structure for directed regeneration.…”