Bulstra, Liselotte F.

AbstractBackgroundIn nerve allograft development, currently used subjective histological scoring systems to evaluate nerve structure in experimental studies are not uniform and have not been validated.The aim of this study was to describe and validate a simple, fast and inexpensive method to compare structural properties of nerve allografts on a histological level.Materials and methodsA total of 113 histological sections of rat (sciatic nerves) and human peripheral nerve segments (thoracodorsal and long thoracic nerve for motor and sural for sensory nerve) treated with various decellularization protocols were analyzed. Slides were stained with Hematoxylin & Eosin (H&E), Toluidine Blue and Laminin. A subjective scoring system using a 5‐point scale was used to score each nerve section by four independent researchers. For validation of this score both inter‐ and intra‐rater reliability were calculated using the Intraclass Correlation Coefficient (ICC).ResultsOverall, an excellent correlation between the different observers was found for the Toluidine Blue (ICC = 0.919, 95% CI: 0.891–0.941), H&E (ICC = 0.9, 95% CI: 0.865–0.927) and Laminin staining (ICC = 0.897, 95% CI: 0.860–0.926). Intra‐rater reliability was good for all three staining techniques (ICC >0.8).ConclusionThis study demonstrates the validity of this simple scoring system to evaluate nerve structure after different processing protocols. All three evaluated staining techniques can reliably be used for both rat and human nerve tissue. We believe this method is suitable to compare different processing techniques to create processed nerve allografts, especially when the average scores of multiple raters are used.

Background: Processed nerve allografts are a promising alternative to nerve autografts, providing an unlimited, readily available supply and avoiding donor-site morbidity and the need for immunosuppression. Currently, clinically available nerve allografts do not provide satisfactory results for motor reconstruction. This study evaluated motor recovery after reconstruction of a long nerve gap using a processed nerve allograft and the influence of storage techniques. Methods: Nerve allografts were decellularized using elastase and detergents and stored at either 4° or −80°C. In 36 New Zealand White rabbits, a 3-cm peroneal nerve gap was repaired with either an autograft (group 1, control) or a cold-stored (group 2) or frozen-stored (group 3) processed nerve allograft. Nerve recovery was evaluated using longitudinal ultrasound measurements, electrophysiology (compound muscle action potentials), isometric tetanic force, wet muscle weight, and histomorphometry after 24 weeks. Results: Longitudinal ultrasound measurements showed that the cold-stored allograft provided earlier regeneration than the frozen-stored allograft. Furthermore, ultrasound showed significantly inferior recovery in group 3 than in both other groups (p < 0.05). Muscle weight and isometric tetanic force showed similar outcomes in the autograft and cold-stored allograft groups [p = 0.096 (muscle weight) and p = 0.286 (isometric tetanic force)], and confirmed the inferiority of the frozen-stored allograft to the autograft [p < 0.01 (muscle weight) and p = 0.02 (isometric tetanic force)]. Conclusions: Frozen storage of the nerve allograft significantly impairs functional recovery and should be avoided. The cold-stored optimized nerve allograft yields functional recovery similar to the gold standard autograft in the reconstruction of a 3-cm motor nerve defect. Future studies should focus on further improvement of the nerve allograft.

Background: The purpose of this study was to evaluate the molecular mechanisms underlying nerve repair by a decellularized nerve allograft seeded with adipose-derived mesenchymal stromal cells (MSCs) and compare it to the unseeded allograft and autograft nerve. Methods: Undifferentiated MSCs were seeded onto decellularized nerve allografts and used to reconstruct a 10 mm gap in a rat sciatic nerve model. Gene expression profiles of genes essential for nerve regeneration and immunohistochemical staining (IHC) for PGP9.5, NGF, RECA-1, and S100 were obtained 2 weeks postoperatively. Results: Semi-quantitative RT-PCR analysis showed that the angiogenic molecule VEGFA was significantly increased in seeded allografts, and transcription factor SOX2 was downregulated in seeded allografts. Seeded grafts showed a significant increase in immunohistochemical markers NGF and RECA-1, when compared with unseeded allografts. Conclusions: MSCs contributed to the secretion of trophic factors. A beneficial effect of the MSCs on angiogenesis was found when compared with the unseeded nerve allograft, but implanted MSCs did not show evidence of differentiation into Schwann cell-like cells.

Background: Elbow flexion after upper brachial plexus injury may be restored by a nerve transfer from the ulnar nerve to the biceps motor branch with an optional nerve transfer from the median nerve to the brachialis motor branch (single and double fascicular nerve transfer). This meta-analysis assesses the effectiveness of both techniques and the added value of additional reinnervation of the brachialis muscle. Methods: Comprehensive searches were performed identifying studies concerning restoration of elbow flexion through single and double fascicular nerve transfers. Only C5 to C6 lesion patients were included in quantitative analysis to prevent confounding by indication. Primary outcome was the proportion of patients reaching British Medical Research Council elbow flexion grade 3 or greater. Meta-analysis was performed with random effects models. Results: Thirty-five studies were included (n = 688). In quantitative analysis, 29 studies were included (n = 341). After single fascicular nerve transfer, 190 of 207 patients reached Medical Research Council grade 3 or higher (random effects model, 95.6 percent; 95 percent CI, 92.9 to 98.2 percent); and after double fascicular nerve transfer, 128 of 134 patients reached grade 3 or higher (random effects model, 97.5 percent; 95 percent CI, 95.0 to 100 percent; p = 0.301). Significantly more double nerve transfer patients reached grade 4 or greater if preoperative delay was 6 months or less (84 of 101 versus 49 of 51; p = 0.035). Conclusions: Additional reinnervation of the brachialis muscle did not result in significantly more patients reaching Medical Research Council grade 3 or higher for elbow flexion. Double fascicular nerve transfer may result in more patients reaching grade 4 or higher in patients with a preoperative delay less than 6 months. The median nerve may be preserved or used for another nerve transfer without substantially impairing elbow flexion restoration.

AbstractBackgroundA bridging nerve autograft is the gold standard for the repair of segmental nerve injury that cannot be repaired directly. However, limited availability and donor site morbidity remain major disadvantages of autografts. Here, a nerve allograft decellularized with elastase was compared with an autograft regarding functional motor outcome in a rat sciatic segmental nerve defect model. Furthermore, the effect of storage on this allograft was studied.MethodsSixty‐six Lewis rats (250–300 g) underwent a 10‐mm sciatic nerve reconstruction using either a cold‐ (n = 22) or frozen‐stored (n = 22) decellularized nerve allograft or an autograft (n = 22). Sprague–Dawley rats (300–350 g) served as full major histocompatibility complex‐mismatched donors. Functional motor outcome was evaluated after 12 and 16 weeks. Ankle angle, compound muscle action potential (CMAP), isometric tetanic force, wet muscle weight, and histomorphometry were tested bilaterally.ResultsFor CMAP and isometric tetanic force, no significant differences were observed between groups. In contrast, for ankle angle, histomorphometry and muscle weight, the cold‐stored allograft performed comparable to the autograft, while the frozen‐stored allograft performed significantly inferior to the autograft. At week 16, ankle angle was 88.0 ± 3.1% in the cold‐stored group, 77.4 ± 3.6% in the frozen‐stored group, and 74.1 ± 3.1% in the autograft group (P < .001); At week 16, the muscle weight showed a recovery up to 71.1 ± 4.8% in the autograft group, 67.0 ± 6.6% in the cold‐stored group, and 64.7 ± 3.7% in the frozen‐stored group (P < .05).ConclusionsA nerve allograft decellularized with elastase, if stored under the right conditions, results in comparable functional motor outcomes as the gold standard, the autograft.

Background: The addition of adipose-derived mesenchymal stromal cells to decellularized nerve allografts may improve outcomes of nerve reconstruction. Prior techniques used for cell seeding are traumatic to both the mesenchymal stromal cells and nerve graft. An adequate, reliable, and validated cell seeding technique is an essential step for evaluating the translational utility of mesenchymal stromal cell–enhanced decellularized nerve grafts. The purpose of this study was to develop a simple seeding strategy with an optimal seeding duration. Methods: A dynamic bioreactor was used to seed rat and human mesenchymal stromal cells separately onto rat and human decellularized nerve allografts. Cell viability was evaluated by MTS assays and cellular topology after seeding was determined by scanning electron microscopy. Cell density and distribution were determined by Live/Dead assays and Hoechst staining at four different time points (6, 12, 24, and 72 hours). The validity and reliability of the seeding method were calculated. Results: Cells remained viable at all time points, and mesenchymal stromal cells exhibited exponential growth in the first 12 hours of seeding. Seeding efficiency increased significantly from 79.5 percent at 6 hours to 89.2 percent after 12 hours of seeding (p = 0.004). Both intrarater reliability (r = 0.97) and interrater reliability (r = 0.92) of the technique were high. Conclusions: This study describes and validates a new method of effectively seeding decellularized nerve allografts with mesenchymal stromal cells. This method is reproducible, distributes cells homogenously over the graft, and does not traumatize the intraneural architecture of the allograft. Use of this validated seeding technique will permit critical comparison of graft outcomes.

IntroductionIn prior small animal studies, we maintained vascularized bone allotransplant viability without long‐term immunotherapy. Instead, an autogenous neoangiogenic circulation is created from implanted vessels, sufficient to maintain bone viability with only 2 weeks immunosupression. Blood flow is maintained despite rejection of the allogeneic vascular pedicle thereafter. We have previously described a large animal (swine) pre‐clinical model, reconstructing tibial defects with vascularized tibial allotransplants. In this manuscript, autologous angiogenesis is evaluated in this model and correlated with bone viability.Materials and methodsAllogeneic tibial segments were transplanted across a major swine leukocyte antigen mismatch. Microvascular repair of the bone VCA pedicle was combined with intraosseous implantation of an autogenous arteriovenous (AV) bundle. The bundle was ligated in group 1 (n = 4), and allowed to perfuse in group 2 (n = 4). Three‐drug immunotherapy was given for 2 weeks. At 16 weeks micro‐CT angiography quantified neoangiogenic vessel volume. Bone viability, rejection grade, and bone healing were analyzed.ResultsA substantial neoangiogenic circulation developed from the implanted AV‐bundle in group 2, with vessel density superior to ligated AV‐bundle controls (0.11 ± 0.05 vs. 0.01 ± 0.01, P = .029). Bone allotransplant viability was also significantly enhanced by neoangiogenesis (78.7 ± 4.4% vs. 27.7 ± 5.8%, P = .028) with higher bone healing scores (21.4 ± 2.9 vs. 12.5 ± 3.7, P = .029). Ligated control tibias demonstrated disorganized bone morphology and higher local inflammation (P = .143).ConclusionImplantation of autogenous AV bundles into vascularized bone allotransplants resulted in the rapid formation of a neoangiogenic autogenous blood supply in a swine tibia model that maintained bone viability, improved bone healing, and minimized rejection.

OBJECTIVEReconstructive options for brachial plexus lesions continue to expand and improve. The purpose of this study was to evaluate the prevalence and quality of restored elbow extension in patients with brachial plexus injuries who underwent transfer of the spinal accessory nerve to the motor branch of the radial nerve to the long head of the triceps muscle with an intervening autologous nerve graft and to identify patient and injury factors that influence functional triceps outcome.METHODSA total of 42 patients were included in this retrospective review. All patients underwent transfer of the spinal accessory nerve to the motor branch of the radial nerve to the long head of the triceps muscle as part of their reconstruction plan after brachial plexus injury. The primary outcome was elbow extension strength according to the modified Medical Research Council muscle grading scale, and signs of triceps muscle recovery were recorded using electromyography.RESULTSWhen evaluating the entire study population (follow-up range 12–45 months, mean 24.3 months), 52.4% of patients achieved meaningful recovery. More specifically, 45.2% reached Grade 0 or 1 recovery, 19.1% obtained Grade 2, and 35.7% improved to Grade 3 or better. The presence of a vascular injury impaired functional outcome. In the subgroup with a minimum follow-up of 20 months (n = 26), meaningful recovery was obtained by 69.5%. In this subgroup, 7.7% had no recovery (Grade 0), 19.2% had recovery to Grade 1, and 23.1% had recovery to Grade 2. Grade 3 or better was reached by 50% of patients, of whom 34.5% obtained Grade 4 elbow extension.CONCLUSIONSTransfer of the spinal accessory nerve to the radial nerve branch to the long head of the triceps muscle with an interposition nerve graft is an adequate option for restoration of elbow extension, despite the relatively long time required for reinnervation. The presence of vascular injury impairs functional recovery of the triceps muscle, and the use of shorter nerve grafts is recommended when and if possible.

Abstract Quantitative evaluation of passive tension in a muscle is important in tendon transfer surgeries, however, currently appropriate intraoperative measurement techniques are lacking. Objective : Intramuscular pressure (IMP) is explored as an application to access force. Approach : The tibialis anterior (TA) in New Zealand white rabbits ( n = 9) was used to test the hypothesis of a strong correlation between the IMP, muscle force, and length. This study also helped to develop intraoperative techniques for future human studies evaluating various insertion techniques (parallel versus perpendicular). Main results : The Pearson correlation between IMP and force for all trials was 0.74 ± 0.30. Separating out the parallel insertion from the perpendicular insertion revealed a significantly higher correlation for parallel, 0.91 ± 0.13 versus 0.56 ± 0.32. Significance : These data indicate IMP sensors can be used to assess force in a single muscle and the parallel insertion method should be used. New findings • What is the central question of this study? Successful outcomes of tendon and muscle transfers depend on proper muscle tension. A near linear relationship has been seen between muscle force and intramuscular pressure. This study aims to develop an intraoperative technique for assessing passive muscle tension using intramuscular pressure. • What is the main finding and its importance? The findings from this study reveal a high correlation between pressure and passive tension in a single muscle. The techniques developed in this study will allow the translation to a human model. The work will help to improve surgical outcomes and aim to retain muscle strength in the patient following procedures such as tendon and muscle transfers.

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.