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

Abstract

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.

Keywords: Avance®Nerve Grafts; NeuraGen®Nerve Guides; dynamic seeding; mesenchymal stem cell; peripheral nerve repair; qPCR.

Figure 1

Gene expression curves of human MSCs seeded onto Avance® Nerve Grafts and NeuraGen® Nerve Guides concerning neurotrophic genes NGF, GDNF, PTN, GAP43 and BDNF at six time points after seeding. The curves demonstrate the mRNA levels of the seeded MSCs on both nerve substitutes as a ratio of the average mRNA levels of unseeded MSCs (= reference group). n = 5 per group per timepoint. Comparisons between groups (MSCs seeded onto Avance® Nerve Grafts versus MSCs seeded onto NeuraGen® Nerve Guides) over time were obtained using two-way repeated measures analysis of variance with post hoc Bonferroni correction. Significant differences between groups are indicated by an *. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars = Standard error of the mean. BDNF: Brain-derived neurotrophic factor; GAP43: growth associated protein 43; GDNF: glial cell line-derived neurotrophic factor; MSC: mesenchymal stem cell; NGF: nerve growth factor; PTN: pleiotrophin.

Figure 2

Gene expression curves of MSCs seeded onto Avance® Nerve Grafts and NeuraGen® Nerve Guides concerning myelination genes PMP22 and MPZ at six different time points after seeding. The curves demonstrate the mRNA levels of the seeded MSCs on both nerve substitutes as a ratio of the average mRNA levels of unseeded MSCs (= reference group). n = 5 per group per timepoint. Comparisons between groups (MSCs seeded onto Avance® Nerve Grafts versus MSCs seeded onto NeuraGen® Nerve Guides) over time were obtained using two-way repeated measures analysis of variance with post hoc Bonferroni correction. Error bars = Standard error of the mean. MSC: Mesenchymal stem cell; MPZ: myelin protein zero; PMP22: peripheral myelin protein 22.

Figure 3

Gene expression curves of MSCs seeded onto Avance® Nerve Grafts and NeuraGen® Nerve Guides concerning angiogenic genes VEGFA and CD31 at six different time points after seeding. The curves demonstrate the mRNA levels of the seeded MSCs on both nerve substitutes as a ratio of the average mRNA levels of unseeded MSCs (= reference group). n = 5 per group per timepoint. Comparisons between groups (MSCs seeded onto Avance® Nerve Grafts versus MSCs seeded onto NeuraGen® Nerve Guides) over time were obtained using two-way repeated measures analysis of variance with post hoc Bonferroni correction. Significant differences between groups are indicated by an *. *P < 0.05. Error bars = Standard error of the mean. MSC: Mesenchymal stem cell; VEGFA: vascular endothelial cell growth factor alpha.

Figure 4

Gene expression curves of MSCs seeded onto Avance® Nerve Grafts and NeuraGen® Nerve Guides concerning extracellular matrix genes COL1A1, COL3A1, FBLN1 and LAMB2 at six different time points after seeding. The curves demonstrate the mRNA levels of the seeded MSCs on both nerve substitutes as a ratio of the average mRNA levels of unseeded MSCs (= reference group). n = 5 per group per timepoint. Comparisons between groups (MSCs seeded onto Avance® Nerve Grafts versus MSCs seeded onto NeuraGen® Nerve Guides) over time were obtained using two-way repeated measures analysis of variance with post hoc Bonferroni correction. Significant differences between groups are indicated by an *. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars = Standard error of the mean. COL1A1: Collagen type alpha I; COL3A1: collagen type alpha III; FBLN1: Fibulin 1; LAMB2: laminin subunit beta 2; MSC: mesenchymal stem cell.

Figure 5

Gene expression curve of MSCs seeded onto Avance® Nerve Grafts and NeuraGen® Nerve Guides concerning the immunoglobulin marker CD96 at six different time points after seeding. The curves demonstrate the mRNA levels of the seeded MSCs on both nerve substitutes as a ratio of the average mRNA levels of unseeded MSCs (= reference group). n = 5 per group per timepoint. Comparisons between groups (MSCs seeded onto Avance® Nerve Grafts versus MSCs seeded onto NeuraGen® Nerve Guides) over time were obtained using two-way repeated measures analysis of variance with post hoc Bonferroni correction. Significant differences between groups are indicated by an *. *P < 0.05. Error bars = Standard error of the mean. CD96: Cluster of differentiation 96; MSC: mesenchymal stem cell.

Figure 6

Proposed mechanism of MSC-seeding on nerve substitutes. The interaction between MSCs and the nerve substitute results in changes in gene expression profiles, leading to production of trophic factors that are involved in Wallerian degeneration and axon regeneration. BDNF: Brain-derived neurotrophic factor; CD96: cluster of differentiation 96; COL1A1: collagen type alpha I; COL3A1: collagen type alpha III; ECM: extracellular matrix; FBLN1: Fibulin 1; GAP43: growth associated protein 43; GDNF: glial cell line-derived neurotrophic factor; GF: growth factor; LAMB2: laminin subunit beta 2; MPZ: myelin protein zero; MSC: mesenchymal stem cell; NGF: nerve growth factor; PECAM1/CD31: platelet endothelial cell adhesion molecule 1; PMP22: peripheral myelin protein 22; PTN: pleiotrophin; VEGFA: vascular endothelial cell growth factor alpha.