Combined Treatment With Platelet-rich Plasma And BDNF-overexpressing Bone Marrow Stromal Cells Promotes Neural Regeneration After Spinal Cord Injury

[Backgrounds and Objectives] Spinal cord injury (SCI) can lead to devastating loss of neurological function, affecting all levels below the site of injury. Unfortunately, injured axons in the mammalian spinal cord fail to regenerate across the lesion site and do not re-establish synaptic contacts with their destinations, in large part due to a "natural barrier" and a diminished intrinsic regenerative capacity. This barrier consists of a cystic cavity and a non-permissive environment which includes inhibitory extracellular matrices. Transplanting embryonic stem cells, neural stem cells, and bone marrow stromal cells (BMSCs) are experimental treatments that have provided promising results for improving neurological function in animal models of SCI. BMSCs may be the best source for their unique advantage, making them ideal cellular targets for genetic engineering and neurotrophic factor production. Platelet-rich plasma is a biological product of concentrated platelets, which is collected from autologous blood. PRP secretes a large quantity of growth factors (PRGFs), including platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1and-β2, insulin-like growth factor (IGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF).Brain-derived neutrophic factor (BDNF), which has been shown to promote sprouting of corticospinal tract neurons after SCI Zurita et al. demonstrated that PRP scaffolds with BDNF allowed most BMSCs to survive and differentiate into neural phenotype cells compared to fibrin scaffolds. Previous studies have shown that the combined use of PRP and MSCs has a synergistic effect on enhancing peripheral nerve regeneration. We hypothesized that a combined treatment with BDNF-overexpressing BMSCs and a PRP scaffold in animals with SCI would have more beneficial effects for protecting neurons and promoting axonal regeneration.To observe the effect of combined treatment of Platelet-rich plasma and BDNF-overexpressing bone marrow stromal cells on axonal regeneration after spinal cord injury, both in vitro and in vivo experiments.[Methods] In vitro:3×105BMSC or BDNF-MSC were co-cultured with10μl PRP for14days, the control group were cultured for the same time without any treatment. In vivo:1×106BMSC or BDNF-MSC was mixed with10μl PRP and then was transplanted to the hemisectioned spinal cord of the Sprague-Dawley rats, the control group was transplanted with PRP alone, MSC or BDNF-MSC alone. BBB scores was used to evaluate the locomotor improvement between the control and experimental group at the1st,7th,14th,21st,28th,56th day after transplantation. The expression of BDNF was measured in the injured spinal cord at1,4and8weeks after surgery. The expression of neurofilament-200(NF-200), glia fibrillary acidic protein (GFAP) at4and8weeks after surgery were evaluated by realtime-PCR and western bot. The immunohistochemisty observation of GFAP and5-histamine (5-HT) in the injured spinal cord was performed. [Results] MSC and BDNF-MSCs cultured with PRP could proliferated and differentiated into neurons with the increased expression of NF-200, GFAP and microtubule-associated protein2(MAP2) in vitro. The expression of BDNF in the injured spinal cord of PRP/BDNF-MSC and BDNF-MSC transplanted group were increased significantly at1week post surgery. More astrocytes were observed in the epicenter of lesion area and less in the graft-host interface in PRP/BDNF-MSC group, with the hightest BBB scores at8weeks. The astrocyte migrated throughout the epicenter of the hemi-section area and connected the rostral and caudal spinal cord. Most grafted cells in the lesion area were linear alignment and exhibited5-HT immunoreactivity.[Conclusion]1. Adenovirus vector containing BDNF was constructed by DNA recombinant techniques and BDNF-overexpressing bone marrow stromal cells were harvested in vitro.2. PRP and MSC or BDNF-MSC loaded Scaffolds were prepared and PRP was demonstrated to be able to promote MSC differentiation into a neural phenotype cell or astrocytes.3. The combined treatment of PRP and BDNF-MSC promotes the astrocyte migration and improves loco-motor recovery in a rat hemi-section model which may be related to the regenerated serotonergic fibers.