Therapy with human dental pulp stem cells spheroids after avulsion of spinal motor roots in adult rats

Author(s)

Sabrina Moreira Paes

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2021-10-22

Examining board members

Alexandre Leite Rodrigues de Oliveira; Bruno de Siqueira Mietto; Marimélia Aparecida Porcionatto

• Advisor: Alexandre Leite Rodrigues de Oliveira

Abstract

Traumatic spinal cord injuries result in severe neurological deficits and high neuronal loss, with consequent difficulty in repair and functional recovery. In this sense, mesenchymal stem cells have shown promising results in the treatment of this type of injury, in addition to leading to significant nerve regeneration, not achieved with conventional treatment methods. In view of its multiple applications in the field of regenerative medicine and the promising results obtained with cell therapy, the aim of this work was to compare the neuroprotective and inducer potential of axonal regeneration of human dental pulp stem cells (hDPSCs) cultivated in monolayer (2D) or in spheroids (3D), after avulsion and reimplantation of spinal motor roots in adult rats. For this purpose, 72 adult female Lewis rats were divided into 4 groups: Avulsion (AV); Avulsion associated with reimplantation with fibrin sealant (AV + R); Avulsion associated with reimplantation and 2D cell therapy (AV + 2D) and Avulsion associated with reimplantation and 3D cell therapy (AV + 3D), under the approval of the Committee on Ethics in the Use of Animals (CEUA/UNICAMP; protocols: 5355-1/2019 and 5713-1/2021). The application of cells in 2D and 3D was performed by microsurgery, in a spinal motor root avulsion model, with subsequent functional assessment by means of a gait test (Catwalk system), until the twelfth week of survival. The expression of GFAP (astrocyte marker), Iba-1 (microglial marker) and synaptophysin (synaptic marker) was evaluated by immunohistochemistry, as well as neuronal survival at 4 and 12 weeks post-surgery by means of Nissl staining. There was also analysis of gene expression, with one week of survival, of iNOS, Arginase 1, IL-6, VEGF?, TGF?, TNF?, IL-4 and GDNF transcripts through qRT-PCR. As for neuronal survival, the animals in the AV + 3D and AV + 2D groups had the highest rates, being 70 and 65% respectively (Reimplantation + 3D: 0.71 ± 0.06; Reimplantation + 2D: 0. 65 ± 0.03; ipsi/contralateral ratio, mean ± SEM). In the AV + 3D and AV + 2D groups, immunofluorescence revealed a reduction in the expression of GFAP (Reimplantation + 3D: 1.52 ± 0.05; Reimplantation + 2D: 1.50 ± 0.17) and Iba-1 (Reimplantation + 3D: 1.84 ± 0.14; Reimplantation + 2D: 1.60 ± 0.13) and synaptophysin preservation (Reimplantation + 3D: 0.8073 ± 0.01; Re-implantation + 2D: 0.75 ± 0.01 ), indicating a reduction in the reactivity of astrocytes and microglia, in addition to the maintenance of synaptic inputs in these groups. As for gene expression, there was a statistically significant increase in transcripts of anti-inflammatory factors (IL-4, TGF?) and a reduction in pro-inflammatory factors (IL-6, TNF?) in animals treated with reimplantation and hDPSCs. The iNOS and Arginase 1 factors showed a statistically significant increased gene expression in the reimplantation + 2D group, indicating favored migration and cell action before the injury. As for functional recovery, in all parameters analyzed, the AV + 2D group was the one with a statistically significant difference compared to the AV group (p=0.0019). The results allow us to infer that cell therapy associating reimplantation with fibrin sealant and hDPSCs cultivated in monolayer is superior to isolated reimplantation and to reimplantation with hDPSCs in 3D. Future analyzes will seek to understand the mechanisms involved in the difference in regenerative potential of hDPSCs when in 2D and 3D cultivation (AU)

• FAPESP's process: 19/17402-0 - Human dental pulp stem cell spheroid therapy after spinal motor root avulsion in adult rats
• Grantee: Sabrina Moreira Paes
• Support Opportunities: Scholarships in Brazil - Master