Liselotte F. Bulstra

Nerve Transfers to Restore Elbow Function

The purpose of this article is to provide an overview of the various nerve transfer options for restoration of elbow function. This article describes nerve transfer strategies for elbow flexion and extension including the indications, limitations, and expected outcomes based on current literature.

Recipient-derived angiogenesis with short term immunosuppression increases bone remodeling in bone vascularized composite allotransplantation: A pilot study in a swine tibial defect model.

Current vascularized composite allotransplantation (VCA) transplantation protocols rely upon life‐long immune modulation to maintain tissue perfusion. Alternatively, bone‐only VCA viability may be maintained in small animal models using surgical angiogenesis from implanted autogenous vessels to develop a neoangiogenic bone circulation that will not be rejected. This study tests the methods efficacy in a large animal model as a bridge to clinical practice, quantifying the remodeling and mechanical properties of porcine tibial VCAs. A segmental tibial defect was reconstructed in Yucatan miniature swine by transplantation of a matched tibia segment from an immunologically mismatched donor. Microsurgical repair of nutrient vessels was performed in all pigs, with simultaneous intramedullary placement of an autogenous arteriovenous (AV) bundle in Group 2. Group 1 served as a no‐angiogenesis control. All received 2 weeks of immunosuppression. After 16 weeks, micro‐CT and histomorphometric analyses were used to evaluate healing and remodeling. Axial compression and nanoindentation studies evaluated bone mechanical properties. Micro‐CT analysis demonstrated significantly more new bone formation and bone remodeling at the distal allotransplant/recipient junction and on the endosteal surfaces of Group 2 tibias (p = 0.03). Elastic modulus and hardness were not adversely affected by angiogenesis. The combination of 2 weeks of immunosuppression and autogenous AV‐bundle implantation within a microsurgically transplanted tibial allotransplant permitted long‐term allotransplant survival over the study period of 16 weeks in this large animal model. Angiogenesis increased bone formation and remodeling without adverse mechanical effects. The method may allow future composite‐tissue allotransplantation of bone without the risks associated with long‐term immunosuppression.

Noninvasive ultrasound of the tibial muscle for longitudinal analysis of nerve regeneration in rats

Background: Today’s criterion standards for measuring functional recovery after nerve trauma in experimental studies are the muscle mass ratio and the isometric tetanic force; both tests are invasive and require a sacrificial procedure. The authors propose ultrasound as a noninvasive method to determine muscle atrophy, and evaluate its validity and reliability by comparing it to muscle mass ratio, isometric tetanic force, and histology. Methods: Fifty rats sustained a 10-mm autograft sciatic nerve reconstruction. With a 2-week interval, five animals were tested with a total follow-up of 20 weeks. The functional recovery of the hind-limb muscles was measured with ultrasound, muscle mass ratio, and isometric tetanic force. In addition, neuromuscular junctions were analyzed histologically. The different evaluation techniques were compared and the reliability of the ultrasound was determined. Results: Four weeks after denervation, extensive muscle atrophy resulted in a decrease of muscle mass up to 30 percent. Ultrasound showed good correlations with muscle mass ratio for both tibial (r = 0.85) and gastrocnemius muscles (r = 0.89). Both intrarater reliability (r = 0.97) and interrater reliability (r = 0.88) of the ultrasound were high. The correlation with force was lower (0.62) but still statistically significant. Conclusions: Ultrasound measurement of muscle atrophy was highly correlated with the criterion standard muscle mass ratio and was also significantly correlated with isometric tetanic force. Histologic evaluation confirmed the regeneration pattern observed with ultrasound. The authors propose that ultrasound can be used as a valid alternative to muscle mass ratio to study muscle atrophy after nerve injury in a less-invasive and more animal-friendly manner.

Bone vascularized composite allotransplantation model in swine tibial defect: Evaluation of surgical angiogenesis and transplant viability

In 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.

Comparable functional motor outcomes after repair of peripheral nerve injury with an elastase-processed allograft in a rat sciatic nerve model

A 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.

Evaluate muscle tension using intramuscular pressure device in rabbit tibialis anterior model for improved tendon transfer surgery

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.

Motor Nerve Recovery in a Rabbit Model: Description and Validation of a Noninvasive Ultrasound Technique

PURPOSE To develop and validate a noninvasive ultrasound technique for the longitudinal analysis of functional recovery after segmental peroneal nerve reconstruction in a rabbit model. METHODS Twelve male New Zealand White rabbits underwent a 1-cm peroneal nerve autograft reconstruction. Ultrasound measurements were performed before surgery and at 1, 2, 4, 8, 12, and 16 weeks postoperatively. All rabbits were managed with manual restraint for the ultrasound procedure, avoiding the risks of anesthetics. At 12 and 16 weeks, we evaluated functional recovery using compound muscle action potential, isometric tetanic force measurements, wet muscle weight, and nerve histomorphometry. Data were compared with ultrasound measurements by calculating the Pearson correlation coefficient. We determined intra-rater and inter-rater reliability of the ultrasound measurements. RESULTS Ultrasound demonstrated good correlation with isometric tetanic force measurements and wet muscle weight, good correlation with nerve histomorphometry, and moderate correlation with compound muscle action potential. Both intra-rater and inter-rater reliability of the ultrasound technique was excellent. CONCLUSIONS Ultrasound analysis of the tibialis anterior muscle provided a reliable method for analysis of functional recovery in a rabbit peroneal nerve reconstruction model. The noninvasive nature allowed for longitudinal follow-up within the same animal and measurement of early recovery without the use of anesthesia. CLINICAL RELEVANCE Application of this noninvasive technique can reduce the variability and sample size necessary in peripheral nerve reconstruction studies and may provide an ideal tool for comparative studies in larger animal models.

Description and Validation of a Simple Histological Nerve Tissue Scoring System

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.

Functional Outcome After Reconstruction of a Long Nerve Gap in Rabbits Using Optimized Decellularized Nerve Allografts

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.