Bishop, A. T.

Vascularization is an important factor in nerve graft survival and function. The specific molecular regulations and patterns of angiogenesis following peripheral nerve injury are in a broad complex of pathways. This review aims to summarize current knowledge on the role of vascularization in nerve regeneration, including the key regulation molecules, and mechanisms and patterns of revascularization after nerve injury. Angiogenesis, the maturation of pre-existing vessels into new areas, is stimulated through angiogenic factors such as vascular endothelial growth factor and precedes the repair of damaged nerves. Vascular endothelial growth factor administration to nerves has demonstrated to increase revascularization after injury in basic science research. In the clinical setting, vascularized nerve grafts could be used in the reconstruction of large segmental peripheral nerve injuries. Vascularized nerve grafts are postulated to accelerate revascularization and enhance nerve regeneration by providing an optimal nutritional environment, especially in scarred beds, and decrease fibroblast infiltration. This could improve functional recovery after nerve grafting, however, conclusive evidence of the superiority of vascularized nerve grafts is lacking in human studies. A well-designed randomized controlled trial comparing vascularized nerve grafts to non-vascularized nerve grafts involving patients with similar injuries, nerve graft repair and follow-up times is necessary to demonstrate the efficacy of vascularized nerve grafts. Due to technical challenges, composite transfer of a nerve graft along with its adipose tissue has been proposed to provide a healthy tissue bed. Basic science research has shown that a vascularized fascial flap containing adipose tissue and a vascular bundle improves revascularization through excreted angiogenic factors, provided by the stem cells in the adipose tissue as well as by the blood supply and environmental support. While it was previously believed that revascularization occurred from both nerve ends, recent studies propose that revascularization occurs primarily from the proximal nerve coaptation. Fascial flaps or vascularized nerve grafts have limited applicability and future directions could lead towards off-the-shelf alternatives to autografting, such as biodegradable nerve scaffolds which include capillary-like networks to enable vascularization and avoid graft necrosis and ischemia.

Objective: The current gold standard treatment for peripheral nerve gaps remains the use of nerve autografts. Decellularized nerve allografts provide a potential ideal alternative, avoiding important disadvantages of the autograft such as donor site morbidity and the limited supply without the need for immunosuppressive therapy. In the ideal decellularized nerve allograft, all cellular debris is removed while maintaining the nerves’ ultrastructure. For the optimization of processing techniques for nerve allografts, it is essential to compare the effect of different processing techniques on a histological level. Although several studies describe some form of (subjective) histological scoring system, evaluation techniques are not uniform and nothing has been validated yet. A validated method that is simple and could easily be adopted by other groups would allow for a more standardized comparison of nerve decellularization techniques. Therefore, the aim of this study was to describe and validate an easy, fast, and inexpensive method to compare different nerve decellularization strategies on a histological level. Materials and Methods: Fifty rat sciatic nerve and 70 human nerve segments (35 sensory and 35 motor) were used for this study. Nerve segments were processed using 5 different processing techniques and compared with unprocessed nerve segments (control). Three different stainings were used to visualize general morphology and different components of the nerve tissue. Toluidine blue was used for visualization of myelin sheaths, hematoxylin-eosin (H&E) for general morphology, and laminin y-l for visualization of the basal lamina. All slides were scored by 4 independent observers who were blinded for the different processing techniques used. Each slide was given a score from 1 to 5 where 5 resembles a “perfect” structure similar to the native nerve and 1 resembles severe structural damage or absence of the component. To validate this scoring system, the agreement between the observers was calculated using the intraclass correlation coefficient (ICC). Results: The 4 different observers similarly scored the processed nerve segments. Significant differences between the processing groups could be identified. We found a good correlation between the different observers for the Toluidine Blue (ICC = .73, 95% confidence interval [CI], 0.66-0.79), H&E (ICC = .72, 95% CI, 0.64-0.78), and Laminin staining (ICC = .74, 95% CI, 0.67-0.80). Conclusion: This study demonstrates the validity of the scoring system to evaluate nerve tissue after different processing protocols. A good correlation was found for the scoring of the structural integrity (Toluidine Blue, H&E, and Laminin). This method is suitable to compare different processing techniques in vitro on the route to engineering the ideal nerve allograft. Use of this method by other groups will make it possible to compare results of different nerve processing studies. Using valid outcome measurements is essential when striving to create new innovative options for nerve reconstruction.

Objective/Hypothesis: Processed nerve allografts provide a promising alternative to nerve autografts with the advantages of a readily available unlimited supply, no donor site morbidity, and without the need for immunosuppression. The nerve allografts currently available for clinical use do not provide satisfactory results. The processing technique was optimized to reduce the allografts immunogenicity and cellular debris while maintaining the ultrastructural properties. The aim of this study was to evaluate the functional recovery after reconstruction of a long nerve gap using the optimized nerve allograft in a rabbit model. To study the influence of storage techniques on functional recovery, processed nerve allografts were either cold or frozen stored. The nerve allografts were challenged to the gold standard, the nerve autograft. Materials and Methods: Nerve allografts were decellularized using our optimized protocol and stored at either 4°C or −80°C. In 36 New Zealand White rabbits, a 3-cm peroneal nerve gap was repaired with either a cold or frozen stored processed nerve allograft or a nerve autograft (control). Nerve recovery was longitudinally evaluated with ultrasound measurements performed prior to surgery and at 10, 16, and 24 weeks follow-up. Functional motor recovery was determined 24 weeks postoperatively using electrophysiology (compound muscle action potentials [CMAP]), isometric tetanic force (ITF), wet muscle weight (MW), and histomorphometry. Results: The longitudinal ultrasound measurements showed that the cold stored nerve allograft provides faster and earlier regeneration than the frozen stored nerve allograft. At 24 weeks, the cold stored allograft showed equal results compared with the autograft based on ultrasound measurements. Ultrasound furthermore showed significantly impaired recovery in the frozen stored allograft group compared with both the cold stored allograft and autograft ( P < .05). The functional outcome measurements, MV and ITF, both showed that the cold stored allograft proved to have a similar result compared with the autograft ( P > .05 for both MW and ITF, respectively). The frozen stored allograft, however, had a significantly inferior result to the autograft ( P < .05 for both MW and ITF). The CMAP and histomorphometry results showed no significant differences between all 3 groups. Conclusions: Nerve reconstruction with the cold stored optimized nerve allograft results in successful recovery of a long motor nerve defect in a rabbit model, statistically equivalent to the gold standard nerve autograft. Freeze storage of the processed allograft impairs the functional outcome.