Mathot, Femke

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.

AbstractPurposeAdipose derived mesenchymal stem cells (MSCs) are hypothesized to supplement tissues with growth factors essential for regeneration and neovascularization. The purpose of this study was to determine the effect of MSCs with respect to neoangiogenesis when seeded onto a decellularized nerve allograft in a rat sciatic nerve defect model.MethodsAllograft nerves were harvested from Sprague–Dawley rats and decellularized. MSCs were obtained from Lewis rats. 10 mm sciatic nerve defects in Lewis rats were reconstructed with reversed autograft nerves, decellularized allografts, decellularized allografts seeded with undifferentiated MSC or decellularized allografts seeded with differentiated MSCs. At 16 weeks, the vascular surface area and volume were evaluated.ResultsThe vascular surface area in normal nerves (34.9 ± 5.7%), autografts (29.5 ± 8.7%), allografts seeded with differentiated (38.9 ± 7.0%) and undifferentiated MSCs (29.2 ± 3.4%) did not significantly differ from each other. Unseeded allografts (21.2 ± 6.2%) had a significantly lower vascular surface area percentage than normal nonoperated nerves (13.7%, p = .001) and allografts seeded with differentiated MSCs (17.8%, p = .001). Although the vascular surface area was significantly correlated to the vascular volume (r = .416; p = .008), no significant differences were found between groups concerning vascular volumes. The vascularization pattern in allografts seeded with MSCs consisted of an extensive nonaligned network of microvessels with a centripetal pattern, while the vessels in autografts and normal nerves were more longitudinally aligned with longitudinal inosculation patterns.ConclusionsNeoangiogenesis of decellularized allograft nerves was enhanced by stem cell seeding, in particular by differentiated MSCs. The pattern of vascularization was different between decellularized allograft nerves seeded with MSCs compared to autograft nerves.

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.