Stem cells in nerve reconstruction: Hype, hope or reality?

The overarching goal of this thesis is to further improve outcomes after nerve reconstruction by individualizing nerve allograft repair with the addition of adipose-derived MSCs. The aim of the first part was to investigate the clinical problem. In chapter 2, an evidencebased overview of the effectiveness of nerve conduits and allografts in motor and mixed sensory/motor nerve reconstruction is provided. In chapter 3, the outcomes of digital nerve gap reconstruction with the NeuraGen type 1 collagen nerve conduit and the Avance Nerve Graft are reported in a retrospective observational study. The second part of this thesis focuses on the addition of adipose derived MSCs to decellularized nerve allografts and the in-vitro characteristics on human tissue, as well as the in-vivo characteristics in a rat-model. An adequate, reliable and validated cell seeding technique is an essential step for evaluating the translational utility of MSC-enhanced decellularized nerve grafts. Therefore in chapter 4, a new method to effectively seed decellularized nerve allografts with MSCs is described and validated. To understand how the functions of MSCs can be leveraged for peripheral nerve repair, in chapter 5, we investigated whether interactions of MSCs with decellularized nerve allografts can improve mRNA and protein expression of growth factors that may support nerve regeneration. After in-vitro testing, the MSC seeded nerve allograft was implemented in a rat model. As there is a paucity of information regarding the ultimate survivorship of implanted MSCs or if these cells remain where they are placed, in chapter 6, the in-vivo distribution and survival of MSCs seeded on a decellularized nerve allograft was tracked using luciferase based bioluminescent imaging (BLI). In chapter 7, the molecular mechanisms underlying nerve repair by a decelullarized nerve allograft preseeded with autologous, undifferentiated, adipose derived MSCs are studied and compared to the unseeded allograft and autograft nerve. In the general discussion (chapter 8) the results of this thesis are put into a broader perspective and compared to other recent publications. Furthermore, the implications of this research for future perspectives are discussed

Gene expression and growth factor analysis in early nerve regeneration following segmental nerve defect reconstruction with a mesenchymal stromal cell-enhanced decellularized nerve all

Background: The purpose of this study was to evaluate the molecular mechanisms underlying nerve repair by a decellularized nerve allograft seeded with adiposederived mesenchymal stromal cells (MSCs) and compare it to the unseeded allograft and autograft nerve. Methods: Undifferentiated MSCs were seeded onto decellularized nerve allografts and used to reconstruct a 10 mm gap in a rat sciatic nerve model. Gene expression profiles of genes essential for nerve regeneration and immunohistochemical staining (IHC) for PGP9.5, NGF, RECA-1, and S100 were obtained 2 weeks postoperatively. Results: Semi-quantitative RT-PCR analysis showed that the angiogenic molecule VEGFA was significantly increased in seeded allografts, and transcription factor SOX2 was downregulated in seeded allografts. Seeded grafts showed a significant increase in immunohistochemical markers NGF and RECA-1, when compared with unseeded allografts. Conclusions: MSCs contributed to the secretion of trophic factors. A beneficial effect of the MSCs on angiogenesis was found when compared with the unseeded nerve allograft, but implanted MSCs did not show evidence of differentiation into Schwann cell-like cells

Gene expression profiles of human adipose-derived mesenchymal stem cells dynamically seeded on clinically available processed nerve allografts and collagen nerve guides

It was hypothesized that mesenchymal stem cells (MSCs) could provide necessary trophic factors when seeded onto the surfaces of commonly used nerve graft substitutes. We aimed to determine the gene expression of MSCs when influenced by Avance® Nerve Grafts or NeuraGen® Nerve Guides. Human adipose-derived MSCs were cultured and dynamically seeded onto 30 Avance® Nerve Grafts and 30 NeuraGen® Nerve Guides for 12 hours. At six time points after seeding, quantitative polymerase chain reaction analyses were performed for five samples per group. Neurotrophic [nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), pleiotrophin (PTN), growth associated protein 43 (GAP43) and brain-derived neurotrophic factor (BDNF)], myelination [peripheral myelin protein 22 (PMP22) and myelin protein zero (MPZ)], angiogenic [platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) and vascular endothelial cell growth factor alpha (VEGFA)], extracellular matrix (ECM) [collagen type alpha I (COL1A1), collagen type alpha III (COL3A1), Fibulin 1 (FBLN1) and laminin subunit beta 2 (LAMB2)] and cell surface marker cluster of differentiation 96 (CD96) gene expression was quantified. Unseeded Avance® Nerve Grafts and NeuraGen® Nerve Guides were used to evaluate the baseline gene expression, and unseeded MSCs provided the baseline gene expression of MSCs. The interaction of MSCs with the Avance® Nerve Grafts led to a short-term upregulation of neurotrophic (NGF, GDNF and BDNF), myelination (PMP22 and MPZ) and angiogenic genes (CD31 and VEGFA) and a long-term upregulation of BDNF, VEGFA and COL1A1. The interaction between MSCs and the NeuraGen® Nerve Guide led to short term upregulation of neurotrophic (NGF, GDNF and BDNF) myelination (PMP22 and MPZ), angiogenic (CD31 and VEGFA), ECM (COL1A1) and cell surface (CD96) genes and long-term upregulation of neurotrophic (GDNF and BDNF), angiogenic (CD31 and VEGFA), ECM genes (COL1A1, COL3A1, and FBLN1) and cell surface (CD96) genes. Analysis demonstrated MSCs seeded onto NeuraGen® Nerve Guides expressed significantly higher levels of neurotrophic (PTN), angiogenic (VEGFA) and ECM (COL3A1, FBLN1) genes in the long term period compared to MSCs seeded onto Avance® Nerve Grafts. Overall, the interaction between human MSCs and both nerve graft substitutes resulted in a significant upregulation of the expression of numerous genes important for nerve regeneration over time. The in vitro interaction of MSCs with the NeuraGen® Nerve Guide was more pronounced, particularly in the long term period (> 14 days after seeding). These results suggest that MSC-seeding has potential to be applied in a clinical setting, which needs to be confirmed in future in vitro and in vivo research