Evaluation of the Efficacy of Natural Biomaterial-Based Scaffolds in Promoting Optimal Regeneration of Peripheral Nerves

Peripheral nerve injuries (PNIs) less than 5 mm in length self-repair compared with those with wider gaps. Traditionally, PNIs of 20 mm or less have been repaired either by suturing nerves end-to-end or by grafting. However, these techniques present drawbacks for surgical and patient recovery, prompting the search for better technologies.

Nerve guidance conduits (NGCs) have shown promise to mitigate these limitations. However, the currently available commercial NGCs are still deficient in supporting satisfactory recovery of PNIs due to inadequate compatibility between the NGCs and the local environment. The ineffectiveness of current NGC materials can be attributed to their limited biocompatibility, inadequate bioresorbability, and unsuitable mechanical properties. Consequently, these material limitations underline the need for innovative bioresorbable materials. Such new materials should potentially enhance the outcomes of peripheral nerve injury treatments, particularly in cases involving longer gaps.
This study aims to enhance recovery of peripheral nerve injury by employing natural, biocompatible materials, specifically polyhydroxyalkanoates (PHAs) and bacterial cellulose. These materials result in nerve guidance conduits that provide superior physical and biochemical support for optimal regeneration of the damaged peripheral nerves.

Polyhydroxyalkanoates (PHAs), a family of biocompatible and biodegradable polymers, are synthesised by bacteria under nutrient-limited conditions. These polymers exhibit diverse mechanical characteristics, including both high elasticity and stiffness. This research produced medium-chain length PHA (MCL-PHA) and an SCL-PHA, P(3HB) through bacterial fermentation and extracted using solvent extraction. By combining them proportionally, various blends were produced. A comprehensive analysis of the material's chemical structure, thermal properties, mechanical properties, and cytocompatibility with NG108-15 neuronal and primary dissociated dorsal root ganglia of mice was carried out. These cytocompatibility studies demonstrated good attachment and neuronal extension.

The 50:50 blend of [P(3HB): MCL-PHA] was chosen, which demonstrated suitable characteristics for the production of nerve guidance conduits and good processability. The conduits demonstrated the capacity to support the recovery of longer nerve injury gaps in vivo. Similarly, large quantities of BC were produced under static conditions. The polymer was characterised with respect to its structural and thermal properties. Blends of SCL-PHAs, MCL-PHAs, and BC were produced, and their corresponding fibres, produced via electrospinning, underwent a thorough analysis to assess their thermal, morphological features, and cytocompatibility. The fibrous materials demonstrated good cell attachment, proliferation, and neurite outgrowth with NG108-15 neuronal cells.

Furthermore, the immiscibility of some of the blends led to the exploration of in-situ production of copolymers by co-culturing SCL-PHA and MCL-PHA-producing microorganisms under various media conditions, thus integrating the inherent properties of each polymer in the in-situ blend. This approach yielded a range of PHA-based blends with distinctive intermediary characteristics. To demonstrate the feasibility of incorporating nerve growth factors (NGF) into NGCs, an innovative, cost-effective single-step strategy was used to incorporate NGF into the polymer matrix of NGCs and solvent-cast films, confirmed by the ELISA release assay and s-SNOM (scattering-type Scanning Near-field Optical Microscopy) analysis.

In an in vivo study utilising a Thy-1-YFP-H mouse sciatic nerve injury model, the NGCs based on a 50:50 blend were implanted. The functional recovery of injured peripheral nerves and the bioresorbability of PHAs in physiological conditions were evaluated. The NGCs proved to be highly suturable, and after five weeks of implantation, they were functionally at par with nerve grafts. Notably, the repair conducted via nerve guidance conduits did not result in significantly higher astrocyte activation with respect to the activation by graft repair, a finding confirmed through immunostaining (Glial Fibrillary Acidic Protein - GFAP) and assessments of activated astrocytes in the injured regions, which theoretically suggests a lack of significant neuropathic pain markers.

In summary, this research confirmed the promise of PHAs and BC as platforms for nerve regeneration, providing valuable insights into the design of NGCs and the field of peripheral nerve tissue engineering.