American Society for Peripheral Nerve

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Introduction of Adipose-Derived Stem Cells In Decellularized Nerve Allografts: An In-Vitro Analysis of the Gene Expression and Growth Factors
Nadia Rbia, MD, MSC1; Liselotte F. Bulstra, BsC2; Alexander Shin, MD2; AT Bishop, MD2; S.E.R. Hovius, MD, PhD1
1Department of Plastic, Reconstructive and Handsurgery, Erasmus Medical Center, Rotterdam, Netherlands, 2Mayo Clinic, Rochester, MN

Objective: Successful repair of segmental peripheral nerve defects remains a clinical challenge. The gold standard, an autologous nerve graft, creates donor site morbidity with loss of sensation and potential scarring. Processed nerve allografts have been used to bridge segmental nerve gaps. However, in large nerve defects, functional outcomes are not comparable to autograft reconstruction yet. Since all cellular material including Schwann cells and axons are removed after processing, the addition of supporting cells has been proposed to improve the 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 extracellular matrix (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 cDNA and qRT-PCR was performed in combination with gene specific assays for genes essential for nerve regeneration including: NGF, BDNF, PTN, VEGF and GAP43. Additional genes were analyzed to map AMSC characteristics. Growth factor production was evaluated and quantified using Enzyme-Linked Immunosorbent Assay (ELISA).

Results: Semi-quantitative RT-PCR analysis showed that the interaction of the allograft and AMSC enhanced the expression of neurotrophic factors NGF, BDNF and GAP43. The expression of the angiogenic molecule, VEGF-a was also increased and remained significantly elevated at 28 days post seeding. Analysis of ECM-related gene expression showed that LAMB2, COL1A1, COL3A1, FBLN1 were significantly elevated until 21 days. Angiogenic factor CD31 and neurotrophic factor PTN were down regulated in the seeded cells. ELISA analysis showed an upregulation of NGF and VEGF-a growth factor levels.

Conclusion: This study demonstrates that the remaining ECM of decellularized nerve allografts 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 and growth factors. The combination of patient's own easily accessible, abundant supply of AMSC's and the readily available processed nerve allograft is potentially a promising method for individualized peripheral nerve repair.

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