Histologic Comparison of the Fascicular Area of Processed Nerve Allograft Versus Cabled Sural Nerve Autograft.

Background: The use of multiple cables of sural nerve autograft is common for peripheral nerve reconstruction when injured nerve caliber exceeds the nerve graft caliber. Although the optimal matching of neural to nonneural elements and its association with functional outcomes are unknown, it is reasonable to consider maximizing the neural tissue structure available for nerve regeneration. No prior studies have compared directly the cross-sectional fascicular area between cabled nerve autografts and size-selected nerve allografts. This study evaluated the cross-sectional fascicular area between native nerve stumps and two reconstructive nerve grafting methods: cabled sural nerve autograft (CSNA) and processed nerve allograft (PNA). Methods: CSNA from matched cadaveric specimens and PNA were used to reconstruct nerve defects in the median and ulnar nerves of six pairs of cadaveric specimens. Nerve reconstructions were done by fellowship-trained hand surgeons. The total nerve area, fascicular area, and nonfascicular area were measured histologically. Results: The CSNA grafts had significantly less fascicular area than PNA and caliber-matched native nerve. The PNA grafts had a significantly higher percent fascicular area compared with the intercalary CNSA graft. Conclusions: Fascicular area was significantly greater in PNA versus CSNA. The PNA consistently demonstrated a match in fascicular area closer to the native nerve stumps than CSNA, where CSNA had significantly smaller fascicular area compared with native nerve stumps.

Local FK506 drug delivery enhances nerve regeneration through fresh, unprocessed peripheral nerve allografts.

OBJECTIVE: Nerve allografts offer many advantages in the reconstruction of peripheral nerve gaps: they retain their native microstructure, contain pro-regenerative Schwann cells, are widely available, and avoid donor site morbidity. Unfortunately, clinical use of nerve allografts is limited by the need for systemic immunosuppression and its adverse effects. To eliminate the toxicity of the systemic immunosuppressant FK506, we developed a local FK506 drug delivery system (DDS) to provide drug release over 28 days. The study objective was to investigate if the local FK506 DDS enhances nerve regeneration in a rodent model of nerve gap defect reconstruction with immunologically-disparate nerve allografts. METHODS: In male Lewis rats, a common peroneal nerve gap defect was reconstructed with either a 20 mm nerve isograft from a donor Lewis rat or a 20 mm fresh, unprocessed nerve allograft from an immunologically incompatible donor ACI rat. After 4 weeks of survival, nerve regeneration was evaluated using retrograde neuronal labelling, quantitative histomorphometry, and serum cytokine profile. RESULTS: Treatment with both systemic FK506 and the local FK506 DDS significantly improved motor and sensory neuronal regeneration, as well as histomorphometric indices including myelinated axon number. Rats with nerve allografts treated with either systemic or local FK506 had significantly reduced serum concentrations of the pro-inflammatory cytokine IL-12 compared to untreated vehicle control rats with nerve allografts. Serum FK506 levels were undetectable in rats with local FK506 DDS. INTERPRETATION: The local FK506 DDS improved motor and sensory nerve regeneration through fresh nerve allografts to a level equal to that of either systemic FK506 or nerve isografting. This treatment may be clinically translatable in peripheral nerve reconstruction or vascularized composite allotransplantation.

Local delivery of FK506 to injured peripheral nerve enhances axon regeneration after surgical nerve repair in rats.

Administration of FK506, an FDA approved immunosuppressant, has been shown to enhance nerve regeneration following peripheral nerve injuries. However, the severe side effects of the systemically delivered FK506 has prevented clinicians from the routine use of the drug. In this study, we analyzed the effectiveness of our fibrin gel-based FK506 delivery system to promote axon regeneration in a rat peripheral nerve transection and immediate surgical repair model. In addition, biodistribution of FK506 from the local delivery system to the surrounding tissues was analyzed in vivo. Rats in the negative control groups either did not receive any delivery system treatment or received fibrin gel with empty microspheres. The experimental groups included rats treated with fibrin gel loaded with solubilized, particulate, and poly(lactic-co-glycolic) acid microspheres-encapsulated FK506. Rats in experimental groups receiving FK506 microspheres and the particulate FK506 regenerated the highest number of motor and sensory neurons. Histomorphometric analysis also demonstrated greater numbers of myelinated axons following particulate FK506 and FK506 microspheres treatment compared to the negative control groups. In biodistribution studies, FK506 was found at the nerve repair site, the sciatic nerve, and spinal cord, with little to no drug detection in other vital organs. Hence, the local application of FK506 via our delivery systems enhanced axon regeneration whilst avoiding the toxicity of systemic FK506. This local delivery strategy represents a new opportunity for clinicians to use for cases of peripheral nerve injuries. STATEMENT OF SIGNIFICANCE: This work for the first time investigated the influence of locally administered FK506 to the site of nerve injury and immediate repair directly on the number of motor and sensory neurons that regenerated their axons. Furthermore, using the immediate nerve repair model, we obtained valuable information about the biodistribution of FK506 within the nervous system following its release from the delivery system implanted at the site of nerve injury and repair. The strategy of local FK506 delivery holds a great promise in the clinical translation, as the localized delivery circumvents the main limitation of the systemic delivery of FK506, that of immunosuppression and toxicity.

Local FK506 dose-dependent study using a novel three-dimensional organotypic assay.

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG-ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.

Matrices, scaffolds, and carriers for protein and molecule delivery in peripheral nerve regeneration.

Local application of exogenous agents with neurotrophic properties enhances the regenerative capacity of injured neurons, especially following reconstructions of long nerve gaps and delayed nerve repairs. Recent advances in biomaterials and biomedical engineering have provided options for the sustained and controlled release of macromolecules to the peripheral nerve. Here, we review five methods for delivering macromolecules to the peripheral nerve including mini-osmotic pumps, hydrogel-based delivery systems, nerve guidance conduits, electrospun fibers, and nerve wraps. In addition to controlling the release of bioactive macromolecules, the ease of clinical use and versatility in implantation at a variety of "real-world" anatomical locations are key factors in designing an ideal delivery system. The incorporation of both mechanical and biological cues into such devices also helps optimize these systems.

A glial cell line-derived neurotrophic factor delivery system enhances nerve regeneration across acellular nerve allografts.

Acellular nerve allografts (ANAs) are used clinically to bridge nerve gaps but these grafts, lacking Schwann cells and therapeutic levels of neurotrophic factors, do not support regeneration to the same extent as autografts. Here we investigated a local drug delivery system (DDS) for glial cell line-derived neurotrophic factor (GDNF) controlled release to implanted ANAs in rats using drug-loaded polymeric microspheres (MSs) embedded in a fibrin gel. In a rat hindlimb nerve gap model, a 10mm ANA was used to bridge a 5mm common peroneal (CP) nerve gap. Experimental groups received DDS treatment at both suture sites of the allografts releasing GDNF for either 2 weeks or 4 weeks. In negative control groups, rats received no DDS treatment or empty DDS. Rats receiving nerve isografts served as the positive control group. The numbers of motor and sensory neurons that regenerated their axons in all the groups with GDNF MS and isograft treatment were indistinguishable and significantly higher as compared to the negative control groups. Nerve histology distal to the nerve graft demonstrated increased axon counts and a shift to larger fiber diameters due to GDNF MS treatment. The sustained delivery of GDNF to the implanted ANA achieved in this study demonstrates the promise of this DDS for the management of severe nerve injuries in which allografts are placed. STATEMENT OF SIGNIFICANCE: This work addresses the common clinical situation in which a nerve gap is bridged using acellular nerve allografts. However, these allografts are not as effective in supporting nerve regeneration as the gold standard method of autografting. The novel local drug delivery system used in this study provides sustained and controlled release of glial cell line-derived neurotrophic factor (GDNF), one of the most potent neurotrophic factors, which significantly improves nerve regeneration following severe nerve injuries. Results from this research will provide a mean of improving nerve allografts with locally delivered GDNF. This strategy may lead to a novel "off the shelf" alternative to the current management of severe nerve injuries.

An engineered biocompatible drug delivery system enhances nerve regeneration after delayed repair.

Localized drug delivery strategies could greatly benefit patients with peripheral nerve injury and could be easy for surgeons to implement. We developed a local drug delivery system (DDS) using drug-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) embedded in a fibrin gel. In an in vitro study, we investigated the biocompatibility of this DDS by performing a toxicity assay in which we incubated PC-12 cells with the medium released from the DDS in vitro. In an in vivo study, this DDS was applied at the rat common peroneal (CP) nerve injury site to deliver exogenous glial cell line-derived neurotrophic factor (GDNF) to the regenerating axons after delayed nerve repair. In vitro, PC-12 cells incubated with released media samples from the DDS had similar viability to control cells cultured with normal media, demonstrating that the DDS was not toxic. In vivo, the numbers of motor and sensory neurons that regenerated their axons with empty MS treatment were the same as when there was no MS treatment. The DDS increased the numbers of regenerating motor- and sensory neurons to levels indistinguishable from those observed with immediate nerve repair. The DDS increased neuron regeneration to levels double those observed with negative control groups. This biocompatible, nontoxic, fibrin gel-based DDS enhances outcomes following severe peripheral nerve injuries.

A novel polymeric drug delivery system for localized and sustained release of tacrolimus (FK506).

Despite substantial improvement in microsurgical techniques for nerve repair, recovery after peripheral nerve injury is usually incomplete. FK506, an FDA approved immunosuppressant, improves functional recovery and reinnervation following peripheral nerve injury in animal models. However, systemically delivered FK506 causes undesirable global immunosuppression. We have, therefore, engineered a biodegradable local delivery system for FK506 using fibrin gel as a drug reservoir that could be placed at a site of nerve injury. FK506 was incorporated into fibrin gel in solubilized, particulated, and poly(lactic-co-glycolic) acid (PLGA) microspheres-encapsulated forms. A tunable release of FK506 in the fibrin gel from days to weeks was observed with the rate of release being most rapid for the solubilized form and then the particulate form. The most prolonged period of release was seen with the PLGA microsphere-encapsulated form. As analyzed by in vitro dorsal root ganglion (DRG) neurite extension assay, PLGA microsphere encapsulation of FK506 did not alter the drug's properties and the released FK506 maintained its bioactivity over the entire period of release. This study suggests that local delivery of FK506 with fibrin hydrogel could be used to enhance peripheral nerve regeneration.