Shin, Alexander Y.

AbstractIntroductionNerve regeneration involves multiple processes, which enhance blood supply that can be promoted by growth factors. Currently, tools are lacking to visualize the vascularization patterns in transplanted nerves in vivo. The purpose of this study was to describe three‐dimensional visualization of the vascular system in the rat sciatic nerve and to quantify angiogenesis of nerve reconstruction.Materials and MethodsIn 12 Lewis rats (weighing 250–300 g), 10 mm sciatic nerve gaps were repaired with ipsilateral reversed autologous nerve grafts. At 12 and 16 weeks of sacrifice, Microfil® contrast compound was injected in the aorta. Nerve autografts (N = 12) and contralateral untreated nerves (N = 12) were harvested and cleared while preserving the vasculature. The amount of vascularization was measured by quantifying the vascular surface area using conventional photography (two‐dimensional) and the vascular volume was calculated with microcomputed tomography (three‐dimensional). For each measurement, a vessel/nerve area ratio was calculated and expressed in percentages (vessel%).ResultsThe vascular volume measured 3.53 ± 0.43% in autografts and 4.83 ± 0.45% vessels in controls at 12 weeks and 4.95 ± 0.44% and 6.19 ± 0.29% vessels at 16 weeks, respectively. The vascular surface area measured 25.04 ± 2.77% in autografts and 26.87 ± 2.13% vessels in controls at 12 weeks, and 28.11 ± 3.47% and 33.71 ± 2.60% vessels at 16 weeks, respectively. The correlation between both methods was statistically significant (p = .049).ConclusionsBoth methods are considered to successfully reflect the degree of vascularization. Application of this technique could be used to visualize and objectively quantify angiogenesis of the transplanted nerve graft. Moreover, this simple method is easily reproducible and could be extrapolated to any other desired target organ ex vivo in small animals to investigate the vascular network.

ABSTRACTVascularized composite allotransplantation of bone is a possible alternative treatment for large osseous defects but requires life‐long immunosuppression. Surgical induction of autogenous neo‐angiogenic circulation maintains transplant viability without this requirement, providing encouraging results in small animal models [1–3]. A preliminary feasibility study in a swine tibia model demonstrated similar findings [4, 5]. This study in swine tibial allotransplantation tests its applicability in a pre‐clinical large animal model. Previously, we have demonstrated bone vascularized composite allotransplantation (VCA) survival was not the result of induction of tolerance nor an incompetent immune system [1]. Fourteen tibia vascularized bone allotransplants were microsurgically transplanted orthotopically to reconstruct size‐matched tibial defects in Yucatan miniature swine. Two weeks of immunosuppression was used to maintain allotransplant pedicle patency during angiogenesis from a simultaneously implanted autogenous arteriovenous bundle. The implanted arteriovenous bundle was patent in group 1 and ligated in group 2 (a neo‐angiogenesis control). At twenty weeks, we quantified the neo‐angiogenesis and correlated it with transplant viability, bone remodeling, and gene expression. All patent arteriovenous bundles maintained patency throughout the survival period. Micro‐angiographic, osteocyte cell count and bone remodeling parameters were significantly higher than controls due to the formation of a neo‐angiogenic autogenous circulation. Analysis of gene expression found maintained osteoblastic and osteoclastic activity as well as a significant increase in expression of endothelial growth factor‐like 6 (EGFL‐6) in the patent arteriovenous bundle group. Vascularized composite allotransplants of swine tibia maintained viability and actively remodeled over 20 weeks when short‐term immunosuppression is combined with simultaneous autogenous neo‐angiogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:288‐296, 2020

Background Bone vascularized composite allotransplantation (VCA) is a possible alternative for the treatment of large bone defects. Clinical application of VCAs is limited by the need for life-long immunosuppression (IS). We report an alternative method to maintain bone allotransplant viability in a large animal model without the need for life-long IS by using autogenous vessel implantation. Methods Fourteen bone only VCAs were transplanted in a porcine tibia defect model with short-term IS. Two groups were used to evaluate the effect of the implantation of an autogenous arteriovenous (AV)-bundle, therefore the only difference between the groups was the patency of the AV-bundle. We radiographically evaluated bone healing and allogenic pedicle patency. AV-bundle patency and union were evaluated with micro-CT. Bone remodeling was assessed with histomorphometry and material properties were evaluated with axial compression testing and cyclic reference point indentation. Results Two subjects did not reach the final time point. Twelve tibiae healed proximally, and nine at the distal transplant–bone interface. Bone allotransplants showed their viability in the first 4 to 6 weeks by significant periosteal bridging arising from the transplant and maintained pedicle patency. Bone material properties were not affected by the implantation of an AV-bundle when compared with ligated AV-bundle controls, but diminished compared with normal bone. Significantly higher bone formation rates resulted from the implantation of a patent AV-bundle. Conclusion New periosteal bone formation and subsequent bone healing result from blood flow through the microsurgically repaired nutrient blood supply, demonstrated by maintained allogenic pedicle patency. The implantation of a patent autogenous AV-bundle has no adverse effect on material properties, but a positive effect on bone remodeling of endosteal surfaces despite thrombosis of the allogenic pedicle. Bone material properties change after transplantation compared with normal bone, although 20-weeks survival time is relatively short for the final evaluation of bone material properties.

AbstractIntroductionThere is conflicting anatomic and innervation data regarding the rabbit brachial plexus injury model. This study aims to validate a rabbit brachial plexus injury model. We hypothesize the middle trunk (C6, C7) is the primary innervation of the biceps, and when cut and unrepaired, would demonstrate lack of recovery and when repaired would demonstrate evidence of recovery.Materials and MethodsTwenty two male New Zealand white rabbits (3–4 kg) underwent unilateral surgical division of the middle trunk. Five rabbits were randomly assigned to the “no‐repair” group while the remaining 17 rabbits underwent direct coaptation (“repair” group). Rabbits were followed for 12 weeks, with ultrasound measurement of biceps cross‐sectional area performed preoperatively, and at 4, 8, and 12 weeks postoperatively. At a euthanasia procedure, bilateral compound muscle action potential (CMAP) and isometric tetanic force (ITF) were measured. Bilateral biceps muscles were harvested and wet muscle weight was recorded. The operative side was expressed as a percentage of the non‐operated side, and differences between the no repair and repair rabbits were statistically compared.ResultsThe repair group demonstrated significantly higher CMA (23.3 vs. 0%, p < .05), ITF (25.6 vs. 0%, p < .05), and wet muscle weight (65.8 vs. 52.0%, p < .05) as compared to the unrepaired group. At 4 weeks postoperatively, ultrasound‐measured cross‐sectional area of the biceps demonstrated atrophy in both groups. At 12 weeks, the repair group had a significantly larger cross‐sectional area as compared to the no‐repair group (89.1 vs. 59.3%, p < .05).ConclusionsThis injury model demonstrated recovery with repair and lack of function without repair. Longer survival time is recommended for future investigations.

AbstractPurposeAnimal models can be helpful in evaluating new surgical strategies for brachial plexus reconstruction. While several groups have already used the rabbit brachial plexus to model injury, reports conflict in anatomic detail and do not identify a nerve‐muscle pair to measure motor function recovery after reconstruction. The purpose of the current study is to describe the innervations of the biceps and triceps muscles in rabbits, which are both amenable to study in brachial plexus injury models.Materials and MethodsThirteen rabbits weighing 2–2.5 kg were anesthetized. Six rabbits were sacrificed and dissected using loupe and microscope magnification to understand the overall morphology of the brachial plexus. Seven rabbits underwent electrophysiologic investigation. A bipolar nerve stimulator was used to systematically stimulate the roots, trunks and divisions, and nerve branches of the rabbit brachial plexus and compound muscle action potential was used to record muscle response. Nerve length and width measurements were not recorded.ResultsRoots contributing to the brachial plexus were C5, C6, C7, C8, and T1. In contrast to other anatomical studies, T2 did not contribute to the brachial plexus. The triceps was innervated by the radial nerve, which received contributions from C6 (1.6 mA), C7 (1.9 mA), C8, and T1 (12.2 mA).The biceps had dual innervation (proximally and distally). The proximal branch received contributions from C6 (3.5 mA) and C7 (5mA). The distal portion was innervated by a branch from the median nerve, which received innervation from C6, C7, C8, and T1.ConclusionsThe overall structure of rabbit brachial plexus is described and innervation of the biceps and triceps is described in detail. This anatomic investigation will form the basis of a future brachial plexus model of injury and repair.

We sought to identify predictors of failed ulnar nerve fascicle (to flexor carpi ulnaris) to biceps motor branch transfer. A retrospective review of adult brachial plexus patients treated with flexor carpi ulnaris to biceps transfer with a minimum 1-year follow-up was performed. Failure, defined as modified British Medical Research Council grade <3 elbow flexion was compared with randomly selected controls (M ≥ 4−). Ninety-one patients, of which 80% regained >M3 flexion met criteria. Eighteen failures and 18 controls, with similar follow-up (20 vs 23 months) were evaluated. Preoperative flexor carpi ulnaris weakness (M < 5) was significantly more common in failures (78% vs 33%). The rate of flexor carpi ulnaris recovery after operation was significantly higher in controls (86% vs 7%). Increased failure risk can be expected with impaired preoperative flexor carpi ulnaris function. The challenge is how to identify which patients will regain near normal flexor carpi ulnaris strength as excellent outcomes can be obtained. Level of evidence: III

ABSTRACTCryopreserved bone allografts (CBA) used to reconstruct segmental bone defects provide immediate structural stability, but are vulnerable to infection, non‐union and late stress fracture as the majority of the allograft remains largely avascular. We sought to improve the bone vascularity and bone formation of CBAs by surgical angiogenesis with an implanted arteriovenous (AV) bundle, using a porcine tibial defect model. Cryopreserved tibial bone allografts were transplanted in swine leukocyte antigen (SLA) mismatched Yucatan minipigs to reconstruct a 3.5 cm segmental tibial defect. A cranial tibial AV‐bundle was placed within its intramedullary canal to induce angiogenesis. The AV bundle was patent in eight pigs and ligated in a control group of eight pigs. At 20 weeks neo‐angiogenesis was evaluated by micro‐angiography. Bone formation was measured by quantitative histomorphometry and micro‐computed tomography. Seven of eight AV‐bundles in the revascularized group were patent. One had thrombosed due to allograft displacement. Total vascular volume was higher in the revascularized allografts compared to the ligated group (p = 0.015). Revascularized allografts had increased levels of bone formation on the allograft endosteal surface compared to the ligated control group (p = 0.05). Surgical angiogenesis of porcine tibial CBAs by intramedullary implantation of an AV‐bundle creates an enhanced autogenous neoangiogenic circulation and accelerates active bone formation on allograft endosteal surfaces. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1698–1708, 2019

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.

Background: There is controversy regarding the effectiveness of brachial plexus reconstruction for elbow function in older patients, as reported outcomes are generally poor. The purpose of this study was to evaluate elbow function outcomes in patients older than 50. Methods: Fifty-eight patients older than 50 years underwent nerve grafting, transfers, or free functioning muscle transfer to improve elbow function after traumatic brachial plexus injury. Patients were evaluated preoperatively and postoperatively for elbow flexion strength and range of motion; Disabilities of the Arm, Shoulder and Hand scores; pain; concomitant trauma; severity of trauma; and type of reconstruction. Results: The average age of the patients was 57.8 years, and the average follow-up was 24.0 months. The average modified British Medical Research Council elbow flexion grade improved significantly from 0.26 to 2.63. Thirty-three patients (60 percent) achieved functional flexion greater than or equal to M3 postoperatively, compared to zero patients preoperatively. There was no correlation between age and modified British Medical Research Council grade. Active elbow range of motion improved significantly postoperatively, with no effect of age on flexion motion. More patients achieved greater than or equal to M3 flexion with nerve transfers (69 percent) compared to free functioning muscle transfer (43 percent). Patients had worse outcomes with high-energy injuries. The mean Disabilities of the Arm, Shoulder and Hand score decreased from 51.5 to 49.6 postoperatively, and the average pain score decreased from 5.0 to 4.3. Conclusion: Brachial plexus reconstruction for elbow function in patients older than 50 can yield useful flexion. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.