Human dental pulp stem cells transplantation combined with treadmill training in rats after traumatic spinal cord injury

Abstract

Spinal cord injury (SCI) is a disabling condition resulting in deficits of sensory and motor functions, and has no effective treatment. Considering that protocols with stem cell transplantation and treadmill training have shown promising results, the present study evaluated the effectiveness of stem cells from human exfoliated deciduous teeth (SHEDs) transplantation combined with treadmill training in rats with experimental spinal cord injury. Fifty-four Wistar rats were spinalized using NYU impactor. The rats were randomly distributed into 5 groups: Sham (laminectomy with no SCI, n=10); SCI (laminectomy followed by SCI, n=12); SHEDs (SCI treated with SHEDs, n=11); TT (SCI treated with treadmill training, n=11); SHEDs+TT (SCI treated with SHEDs and treadmill training; n=10). Treatment with SHEDs alone or in combination with treadmill training promoted functional recovery, reaching scores of 15 and 14, respectively, in the BBB scale, being different from the SCI group, which reached 11. SHEDs treatment was able to reduce the cystic cavity area and glial scar, increase neurofilament. Treadmill training alone had no functional effectiveness or tissue effects. In a second experiment, the SHEDs transplantation reduced the TNF-α levels in the cord tissue measured 6 h after the injury. Contrary to our hypothesis, treadmill training either alone or in combination, caused no functional improvement. However, SHEDs showed to be neuroprotective, by the reduction of TNF-α levels, the cystic cavity and the glial scar associated with the improvement of motor function after SCI. These results provide evidence that grafted SHEDs might be an effective therapy to spinal cord lesions, with possible anti-inflammatory action.

Figure 1. Experimental design (A). Photomicrographs of stem cells from human exfoliated deciduous teeth (SHEDs) labeled with Hoechst 33342 (B). SHEDs survived and migrated 1.190 µm along the spinal cord (arrows). BBB: Basso, Beatie, and Bresnahan scale.

Figure 2. Design of the second experiment (A). ELISA results for TNF-α (B). Naive: rats with no manipulation; SCI: rats with laminectomy followed by spinal cord injury; and SHEDs: rats with SCI treated with stem cells from human exfoliated deciduous teeth (SHEDs). Data reported as means±SE. ^#P<0.05, compared to Naive group; *P<0.05, compared to SCI group (one-way ANOVA followed by Tukey's post hoc test).

Figure 3. Progress of hind limbs functional recovery after incomplete spinal cord injury (SCI), as evaluated with the BBB scale, in sham operated (Sham), SCI, SCI treated with stem cells from human exfoliated deciduous teeth (SHEDs), SCI treated with treadmill training (TT), and SCI treated with SHEDs and treadmill training (SHEDs+TT) rats. Data are reported as means±SE. *Significantly different comparing the SHEDs group to the SCI group; **significantly different comparing the SHEDs and the SHEDs+TT groups to the SCI group. Statistical analysis was performed using repeated-measures ANOVA to detect treatment effects over time, and individual one-way ANOVA followed by Tukey's post hoc were performed to observe intraday differences among groups (P<0.05). BBB: Basso, Beatie, and Bresnahan scale.

Figure 4. Representative images of cavitation areas (A) and cavitation area analysis (B) of incomplete spinal cord injury (SCI), SCI treated with stem cells from human exfoliated deciduous teeth (SHEDs), SCI treated with treadmill training (TT), and SCI treated with SHEDs and treadmill training (SHEDs+TT) rats. Data are reported as means±SE. **P<0.05, SHEDs group compared to SCI group (one-way ANOVA followed by Tukey's post hoc test).

Figure 5. Representative immunostaining of GFAP expression in the epicenter of injured spinal cord, 6 weeks after spinal cord injury (SCI), SCI treated with stem cells from human exfoliated deciduous teeth (SHEDs), SCI treated with treadmill training (TT), and SCI treated with SHEDs and treadmill training (SHEDs+TT) rats (A). Densitometry of GFAP (B). Data are reported as means±SE. *P<0.05, SHEDs group compared to SCI group (one-way ANOVA followed by Tukey's post hoc test). Scale bar = 50 µm.

Figure 6. MBP staining in ventral white matter (VWM) (A), spinal regions of quantification (B), densitometry of MBP (C), spinal regions of NF-M staining in VWM (D), densitometry of NF-M (E) of spinal cord injury (SCI), SCI treated with stem cells from human exfoliated deciduous teeth (SHEDs), SCI treated with treadmill training (TT), and SCI treated with SHEDs and treadmill training (SHEDs+TT) rats. Data are reported as means±SE. *P<0.05, SHEDs group compared to SCI group. **P<0.05, TT group compared to SHEDs and SCI groups (one-way ANOVA followed by Tukey's post hoc test). Scale bar = 50 µm.