Fabrication of IP-Dip nanogrid using two-photon lithography. (A) The 3D nanogrid was designed using computer aided design software. Next, photolithography was used to print the IP-Dip scaffold. Finally, the nanogrid was chemically developed. (B) SEM image of IP-Dip 3D scaffold. (Bi) Magnified view of the aligned fiber mesh. (Bii) Ultrastructure of the surface morphology of single fibers, where the feature of longitudinal grooves is highlighted. Scale bars: 100 µm (B), 20 µm (Bi), 1 µm (Bii).
Fabrication of IP-Dip nanogrid using two-photon lithography. (A) The 3D nanogrid was designed using computer aided design software. Next, photolithography was used to print the IP-Dip scaffold. Finally, the nanogrid was chemically developed. (B) SEM image of IP-Dip 3D scaffold. (Bi) Magnified view of the aligned fiber mesh. (Bii) Ultrastructure of the surface morphology of single fibers, where the feature of longitudinal grooves is highlighted. Scale bars: 100 µm (B), 20 µm (Bi), 1 µm (Bii).

Personalized three-dimensional polymer scaffolds for regrowing nerve cells could help the millions of people every year suffering spinal cord and peripheral nerve injuries. Now a team from Okinawa Institute of Science and Technology Graduate University in Japan have used a new technique called 2-photon lithography to fabricate highly ordered 3D structures that enhance the growth of neurons [Agrawal et al., Materials Science & Engineering C 131 (2021) 112502, https://doi.org/10.1016/j.msec.2021.11.112502 ].

“We wanted to develop a 3D personalized scaffold with precise texture, shape, and size that would encourage aligned growth of axons, a parameter we think is critical to facilitating the reconnection of traumatic nerve lesions,” explains Marco Terenzio, who let the effort.

Neural tissue engineering with synthetic scaffolds aims to boost the regeneration of damaged nerves and restore function. Scaffolds designed to mimic the extracellular matrix (ECM) provide mechanical support and the right chemical cues to encourage the migration, differentiation, attachment, and development of regrowing cells. Natural polymers, which provide cell signaling cues and bionic properties but little structural support, or synthetic polymers, which offer excellent control of the architecture but less biocompatibility, can be used. Their structures can be random, or feature aligned fibrous architectures, which support neural stem cell attachment and growth more effectively. But scaffolds for larger tissue repairs with highly aligned fibers are challenging to fabricate.

The researchers used two-photon polymerization (2-PP) laser lithography to print 3D scaffolds from a commercially available resin, IP-Dip photoresist, with nanoscale patterns and features, controlled porosity, and mechanical properties that mimic those of the ECM. The 1 mm x 1 mm scaffolds feature 1- μm fibers with 200 nm grooves and inter-fiber spacings of 5-μm that can accommodate neuron cells.

“This level of precision and control is very difficult, if not impossible, to achieve with conventional fabrication methods such as electrospinning,” points out Terenzio.

The scaffolds proved compatible with two cell types, dorsal ganglia (DRG) and motor (MN) neurons, used to treat spinal cord and peripheral nerve damage. In fact, DRG neurons populate the entire scaffold, rather than just the surface, creating a truly 3D network of axons, the nerve fibers that transmit signals between neurons.

The researchers believe that the combination of the texture of the fibrous scaffolds, alignment, strength, and a laminin coating improves the attachment and survival of neurons. This proof-of-concept study indicates that nanofibrous scaffolds could form the basis of complex implants for encouraging and supporting the 3D growth of axons and boosting nerve regeneration after damage or injury to nerves or the spinal column.

“Our approach [is] feasible [for] future in vivo applications [but] the road to practical in vivo implants is still long, if not exciting,” says Terenzio.