Saffari, Tiam M.

Purpose: Application of mesenchymal stem cells (MSCs) or tacrolimus (FK506), an FDA approved immunosuppressant, to nerve grafts has been a topic of interest to enhance peripheral nerve regeneration. The aim of this study was to investigate the combined effect of MSCs and local delivery of FK506 on nerve regeneration when applied to nerve autografts and decellularized nerve allografts. Methods: A three-dimensional (3D) in vitro compartmented cell culture system, validated by Tajdaran et al (2019), consisted of rat neonatal dorsal root ganglion (DRG) adjacent to rat nerve autograft or decellularized allograft. This model was used to evaluate regenerating neurites from the DRG into the peripheral nerve scaffold. Nerve autografts and decellularized allografts were augmented with (i) dynamic undifferentiated MSC seeding, (ii) local application of FK506 (100 ng/mL) or (iii) both (N=9/group). Local application was ensured by isolating the central system (i.e. DRG side) from the peripheral system (i.e. nerve graft side), where treatment was applied. After 48-hours of incubation, DRG-nerve graft constructs were collected, fixed, sectioned and stained against neurofilament-160 to measure neurite extension. CD90 staining was used to confirm stem cell characterization. Results: All grafts treated with MSCs confirmed CD90 expression. Compared to untreated autografts, neurite extension in autografts treated with FK506 and autografts treated with MSCs and FK506 combined were found superior (P<0.001 and P=0.0001, respectively), and comparable to autografts treated with MSCs (P=0.12). Compared to untreated allografts, allografts treated with FK506, and allografts treated with MSCs and FK506 were found superior (P<0.001 and P=0.0001, respectively), and allografts treated with MSCs were found comparable (P=0.09). All autograft groups were found superior compared to their respective allograft treatment group (P<0.05). Solely allografts receiving combined treatment were found superior to untreated autografts (P<0.05). Conclusion: MSCs or FK506 treatment improved neurite outgrowth and when combined, this resulted in significant synergistic neurite extension in both autografts and allografts in comparable patterns. Schematic overview of 3D compartmented cell culture system for isolated evaluation of treatment with MSCs and local FK506 in vitro. A 3.5 mm autograft or allograft with or without undifferentiated MSC seeding is attached to a DRG. DRG-nerve graft constructs are placed through a silicone isolator in the middle of a 24-wells plate to isolate the DRG from the nerve graft. FK506 containing media was added to the nerve graft side.

Background: Surgical angiogenesis applied to nerve grafts has been suggested to enhance nerve regeneration after nerve injury. The authors hypothesized that surgical angiogenesis to decellularized nerve allografts would improve functional recovery in a rat sciatic nerve defect model. Methods: Sixty Lewis rats were divided in three groups of 20 animals each. Unilateral sciatic nerve defects were repaired with (1) autografts, (2) decellularized allografts, and (3) decellularized allografts wrapped with a superficial inferior epigastric artery fascial flap to add surgical angiogenesis. Twelve and 16 weeks after surgery, nerve regeneration was assessed using functional, electrophysiologic, histologic, and immunofluorescence analyses. Ultrasonography was used during the survival period to noninvasively evaluate muscle atrophy and reinnervation by measuring cross-sectional muscle area. Results: Surgical angiogenesis of allografts demonstrated significantly improved isometric tetanic force recovery at 12 weeks, compared to allograft alone, which normalized between groups at 16 weeks. Cross-sectional muscle areas showed no differences between groups. Electrophysiology showed superiority of autografts at both time points. No differences were found in histologic analysis, besides a significantly inferior N ratio in allografts at 12 weeks. Immunofluorescent expression of CD34, indicating vascularity, was significantly enhanced in the superficial inferior epigastric artery fascial group compared to allografts at 12 weeks, with highest expression at 16 weeks compared to all groups. Conclusion: Surgical angiogenesis with an adipofascial flap to the nerve allograft increases vascularity in the nerve graft, with subsequent improvement of early muscle force recovery, comparable to autografts.

Background: Mesenchymal stem cells have the potential to produce neurotrophic growth factors and establish a supportive microenvironment for neural regeneration. The purpose of this study was to determine the effect of undifferentiated and differentiated mesenchymal stem cells dynamically seeded onto decellularized nerve allografts on functional outcomes when used in peripheral nerve repair. Methods: In 80 Lewis rats, a 10-mm sciatic nerve defect was reconstructed with (1) autograft, (2) decellularized allograft, (3) decellularized allograft seeded with undifferentiated mesenchymal stem cells, or (4) decellularized allograft seeded with mesenchymal stem cells differentiated into Schwann cell–like cells. Nerve regeneration was evaluated over time by cross-sectional tibial muscle ultrasound measurements, and at 12 and 16 weeks by isometric tetanic force measurements, compound muscle action potentials, muscle mass, histology, and immunofluorescence analyses. Results: At 12 weeks, undifferentiated mesenchymal stem cells significantly improved isometric tetanic force measurement and compound muscle action potential outcomes compared to decellularized allograft alone, whereas differentiated mesenchymal stem cells significantly improved compound muscle action potential outcomes. The autografts outperformed both stem cell groups histologically at 12 weeks. At 16 weeks, functional outcomes normalized between groups. At both time points, the effect of undifferentiated versus differentiated mesenchymal stem cells was not significantly different. Conclusions: Undifferentiated and differentiated mesenchymal stem cells significantly improved functional outcomes of decellularized allografts at 12 weeks and were similar to autograft results in the majority of measurements. At 16 weeks, outcomes normalized as expected. Although differences between both cell types were not statistically significant, undifferentiated mesenchymal stem cells improved functional outcomes of decellularized nerve allografts to a greater extent and had practical benefits for clinical translation by limiting preparation time and costs.

AbstractThe application of scaffold‐based stem cell transplantation to enhance peripheral nerve regeneration has great potential. Recently, the neuroregenerative potential of tacrolimus (a U.S. Food and Drug Administration‐approved immunosuppressant) has been explored. In this study, a fibrin gel‐based drug delivery system for sustained and localized tacrolimus release was combined with rat adipose‐derived mesenchymal stem cells (MSC) to investigate cell viability in vitro. Tacrolimus was encapsulated in poly(lactic‐co‐glycolic) acid (PLGA) microspheres and suspended in fibrin hydrogel, using concentrations of 0.01 and 100 ng/ml. Drug release over time was measured. MSCs were cultured in drug‐released media collected at various days to mimic systemic exposure. MSCs were combined with (i) hydrogel only, (ii) empty PLGA microspheres in the hydrogel, (iii) 0.01, and (iv) 100 ng/ml of tacrolimus PLGA microspheres in the hydrogel. Stem cell presence and viability were evaluated. A sustained release of 100 ng/ml tacrolimus microspheres was observed for up to 35 days. Stem cell presence was confirmed and cell viability was observed up to 7 days, with no significant differences between groups. This study suggests that combined delivery of 100 ng/ml tacrolimus and MSCs in fibrin hydrogel does not result in cytotoxic effects and could be used to enhance peripheral nerve regeneration.

We report a patient who has been on tacrolimus for bilateral lung transplantation and presented with a brachial plexus injury (BPI), with unusual improvement of lower trunk innervated hand function. The lower trunk injury with resultant left hand paralysis had developed after his sternotomy 18 months ago. He has been treated with tacrolimus as part of his immunosuppression protocol since the surgery, without severe side effects. Physical examination at 18 months demonstrated unusual excellent grip pattern and full opposition of his thumb with slight claw deformity of his ulnar two digits. While the neurotoxic effects of tacrolimus are more emphasised, the neuroregenerative properties have been recently explored. The recovery in this patient is unique and unusual after BPI and is most likely as a result of the low dose tacrolimus treatment.

Abstract Background An adipofascial flap in the rat may provide new options for adding vascularization to scarred or nonvascularized beds for a variety of research studies. Current literature lacks sufficient description for a simple reproducible flap model for a vascularized pedicled flap in rats, in particular for neovascularization of allograft nerves for the reconstruction of sciatic nerve defects. The purpose of this study was to describe a surgical technique and determine long-term survivability for the pedicled superficial inferior epigastric artery fascial (SIEF) flap in the rat to meet requirement for a tunneled adipofascial flap to add vascularization to the sciatic nerve area. Methods The technique and use of a 4 × 3-cm SIEF flap are described. Twenty Lewis rats underwent the technique to determine feasibility. The flap was wrapped around processed allograft nerve reconstructions and viability of the flap was evaluated after 12 and 16 weeks. To visualize vessels, nerve grafts were harvested at 12 weeks and stained with hematoxylin-eosin and an antibody against microvessels (CD-34). Results All flaps remained viable after survival of 12 and 16 weeks. Complications included one hematoma formation and two lymphocele formations that did not have any impact on the flap. Immunohistochemistry confirmed an increase in microvessels and Schwann cell nuclei in the SIEF group compared with nerve samples from the unoperated, contralateral side. Conclusion A pedicled adipofascial flap model in the rat to provide a vascular bed for sciatic nerve reconstruction is detailed with long-term survivability evaluation of the flap. This flap is technically simple to be harvested and is suitable for revascularization procedures of various tissues in the lower abdomen, genital area, thigh, or upper limb of the rat.

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.