Hovius, Steven E. R.

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.

Objective: Successful repair of segmental peripheral nerve defects remains a clinical challenge. The current clinical gold standard for such injuries is to use an autologous nerve graft, but this creates donor site morbidity with loss of sensation and potential scarring. Processed nerve allografts have been used to bridge segmental nerve gaps experimentally and clinically. However, in large nerve defects, functional outcomes are not comparable with autograft reconstruction yet; the longer the nerve gap, the poorer the outcome. Nerve architecture and the extracellular matrix (ECM) are preserved after processing; however, all cellular material including Schwann cells and axons are removed. The addition of supporting cells has been proposed to improve the decellularized allograft. Adipose-Derived Mesenchymal Stem Cells (AMSC) can potentially provide the necessary support for nerve regeneration due to local production of essential growth factors. While the mechanisms underlying the neurotrophic potential of AMSC remains unknown, it is postulated that the remaining ECM still has biological activity that influences the AMSC and their differentiation. Therefore, the purpose of this study was to quantitate the changes in gene expression profiles of the cells and quantify the actual produced growth factors after seeding the allograft with AMSC in vitro. Method: A total of 35 human nerve allografts were decellularized and seeded with human AMSC. At each time point (1, 3, 14, 21, and 28 days), total RNA was extracted, reverse transcribed into complementary DNA (cDNA), and quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed in combination with gene specific assays for genes essential for nerve regeneration including nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), pleiotrophin vascular endothelial growth factor (PTN VEGF), and growth associated protein 43 (GAP43).Additional genes were analyzed to map AMSC characteristics including proliferation, apoptosis, myelinization, and ECM molecules. Production of growth factors was evaluated and quantified using enzyme-linked immunosorbent assay (ELISA). All samples were analyzed in triplicates and data were compared with AMSC cultured in media only. Results: Semiquantitative RT-PCR analysis showed that the interaction of the processed allograft and AMSC enhanced the expression of the neurotrophic factors NGF, BDNF, and GAP43. The expression of the angiogenic molecules, vascular endothelial growth factor A (VEGF-A) was also increased and remained significantly elevated at 28 days post seeding. Analysis of ECM-related gene expression showed that laminin subunit beta-2 (LAMB2), collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), fibulin 1 (FBLN1), were significantly elevated until 21 days post seeding while after 28 days, these levels were normalized. Angiogenic factor cluster of differentiation 31 (CD31) and neurotrophic factor PTN expression were down regulated in the seeded cells. Quantitative results of the ELISA analysis are pending. We anticipate differences in growth factor levels released by the seeded allograft compared with the control expression levels. Conclusion: This study demonstrates that the remaining ECM of the decellularized nerve allograft has a stimulating effect on AMSC. Upon the seeding, secretion of neurotrophic and angiogenic factors were triggered; the cells cultured on the allograft showed enhanced levels of neurotrophic genes. This is hypothesized to be a consequence of specific cell-ECM interactions within the allograft. The combination of patient’s own easily accessible, abundant supply of stem cells harvested from adipose tissue and the readily available processed nerve allograft is potentially a promising method for individualized peripheral nerve repair.

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.