Use of fibrin sealant combined with mesenchymal stem cells in the repair of peripheral nerves through tubulization technique

Author(s)

Luciana Politti Cartarozzi

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2013-12-08

Examining board members

Alexandre Leite Rodrigues de Oliveira; Ana Maria Blanco Martinez; Cecília Amélia de Carvalho Zavaglia

• Advisor: Alexandre Leite Rodrigues de Oliveira

Abstract

Peripheral nerve regeneration is a complex process that is dependent on the rearrangement and activation of Schwann Cells (SC). Such stimulation of SCs may be achieved by the use of exogenous stem cells. In order to better understand the importance of mesenchymal stem cell (MSC) grafting in the peripheral regeneration process we have used the model of sciatic nerve tubulization. Tubular prostheses were prepared from polycaprolactone (PCL) membranes and filled with fibrin sealant (FS), which was used as a substrate for the MSC. The technique of tubulization was applied in adult Lewis female rats that were divided into four groups (n = 5 per group): normal, PCL (empty tube), FS (tube filled with fibrin sealant) and FS + MSC (tube filled with fibrin sealant and grafted with MSC). Sixty days after injury, the regenerated nerves were processed for imunohistochemistry and observed under fluorescence microscopy. The presence of GFP positive stem cells was detected in the nerves of the animals that received MSC grafts, indicating the long term survival of such cells. The axonal regeneration process was studied by immunohistochemistry and revealed the presence of the basic elements of the peripheral nerve, namely axons and basal lamina components that were equivalent in all experimental groups. The axonal organization was observed with anti-neurofilament immunostaining. The presence of SCs was analyzed with anti-S100 immunostaining and anti-type IV collagen was used to detect the basal lamina. Anti-p75NTR, the low affinity receptor for neurotrophins, was used to investigate the reactivity of the SCs. A basal positive labeling in uninjured nerves was detected, which was upregulated by the regenerative process, being statistically higher in FS + MSC group (77% relative to uninjured nerve; p<0.001). Moreover there was colocalization between GFP-positive MSC and anti-BNDF immunolabeling, showing a possible pathway that these cells induce the reactivity of SCs. From sciatic nerve semi-thin sections we were able to evaluate that the areas of regenerated nerves were statistically the same in the different experimental groups. When we quantified the number of myelinated axons in 50.000?m2, the FG+MSC group showed higher density of axons when compared with PCL group (25%, p<0,05). In the same way, the analysis of morphometric parameters showed that the FG+MSC group have a tendency to present higher caliber axons and ticker myelin sheath when compared with other groups, being that the myelin sheath thickness, in the interval between 1,46 to 2,25?m, was significantly higher in FG+MSC group when compared to PCL and FG (p<0,05). As the functional result of the findings above, the FG+MSC animals showed higher motor function recovery, analyzed by FFI, at seventh and eighth weeks after lesion. The findings herein show that MSC associated with the FS scaffold improve the regeneration process by positively modulating the reactivity of SCs (AU)

• FAPESP's process: 11/02889-0 - Use of fibrin sealant combined with mesenchymal stem cells in the repair of peripheral nerves through tubulization technique
• Grantee: Luciana Politti Cartarozzi
• Support Opportunities: Scholarships in Brazil - Master