Neurotrophic effects of dental pulp stem cells in repair of peripheral nerve after crush injury

Figure 1 Isolation, culture, and multilineage differentiation of dental pulp stem cells. A: Morphology of dental pulp stem cells (DPSCs); B: Neural-like cells differentiated from DPSCs; C: Immunofluorescent staining for Nestin, S100, GFAP, p75, and NF200; D: Osteogenic induction; E: Neural induction assessed by real-time PCR; F: Flow cytometry for CD73, CD90, CD146, CD106, CD31, and CD34. Student’s t test was used to identify statistical significance. ^aP < 0.05, ^bP < 0.01.

Figure 2 Dental pulp stem cells can differentiate into neural-like cells, and neural-induced dental pulp stem cells are highly positive for neurotrophic factors. A: Morphology of neural-induced dental pulp stem cells; B: The expression of BDNF, GDNF, and NGF was assessed by Western blot; C: The expression of BDNF, GDNF, and NGF was assessed by immunofluorescent staining. ^bP < 0.01. DPSC: Dental pulp stem cells; hDPSCs: Human dental pulp stem cells.

Figure 3 The secretions of neural-induced dental pulp stem cells enhance the proliferation and migration of RSC96 cells. A: Migration ability assessed by transwell assays; B: Analysis of RSC96 cell migration; C: CCK-8 assays. Student’s t test was used to identify statistical significance. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. N-DPSCs: Neural-induced dental pulp stem cells; DPSC: Dental pulp stem cells; hDPSCs: Human dental pulp stem cells.

Figure 4 Neural-induced dental pulp stem cells enhance rat functional recovery after nerve crush injury. A: Nerve crush model of peripheral nerve injury and cell sheet implantation; B: Footprints of the four groups; C and D: Analysis of sciatic function index; E: Wet weight ratio of the gastrocnemius muscles; F: Appearance of the gastrocnemius muscle of four groups. Bar = 1 cm. All data represent the mean ± SD. n = 8 in each group. ^bP < 0.01. SFI: Sciatic function index; N-DPSCs: Neural-induced dental pulp stem cells; hDPSCs: Human dental pulp stem cells.

Figure 5 Hematoxylin & eosin staining and Masson staining of cross-sections of muscle fibers showed that neural-induced dental pulp stem cells could alleviate muscle atrophy. A: The degree of muscle atrophy in the neural-induced dental pulp stem cells (N-DPSC) group was significantly improved, as shown by the larger area ratio of well-organized muscle fibers compared with those of the control and DPSC groups; B: Histograms presenting the percentage of area of muscle fiber. ^bP < 0.01. H&E: Hematoxylin & eosin staining; DPSC: Dental pulp stem cells.

Figure 6 Histological and transmission electron microscopic evaluation showed that neural-induced dental pulp stem cells could help nerve reorganization and regeneration. A: Hematoxylin & eosin staining of longitudinal sections of crushed nerves; B: Transmission electron microscopy of the nerve myelin sheath. n = 8. Bar = 50 μm. hDPSC: Dental pulp stem cells; N-hDPSCs: Neural-induced human dental pulp stem cells.

Figure 7 Immunofluorescence staining of crushed nerves for Myelin basic protein and NF-200 implied that neural-induced dental pulp stem cells help nerve regeneration. A: Myelin basic protein (MBP) and NF200 staining; B and C: Percentage of positive area of MBP and NF200 staining. n = 8. Bar = 50 μm. ^bP < 0.01. MBP: Myelin basic protein; hDPSC: Dental pulp stem cells; N-hDPSCs: Neural-induced human dental pulp stem cells.