Introduction of Adipose-Derived Stem Cells in Decellularized Nerve Allografts

Abstract

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.