VEGFA-modified DPSCs combined with LC-YE-PLGA NGCs promote facial nerve injury repair in rats

Abstract

Objective: The aim of this research was to investigate the effect of vascular endothelial growth factor A (VEGFA)-overexpressing rat dental pulp stem cells (rDPSCs) combined with laminin-coated and yarn-encapsulated poly(_l-lactide-co-glycolide) (PLGA) nerve guidance conduit (LC-YE-PLGA NGC) in repairing 10 mm facial nerve injury in rats.

Study design: rDPSCs isolated from rat mandibular central incisor were cultured and identified in vitro and further transfected with the lentiviral vectors (Lv-VEGFA). To investigate the role and mechanisms of VEGFA in neurogenic differentiation in vitro, semaxanib (SU5416), Cell Counting Kit-8 (CCK-8), real-time quantitative polymerase chain reaction (qPCR) and Western blotting were performed. Ten-millimeter facial nerve defect models in rats were established and bridged by LC-YE-PLGA NGCs. The repair effects were detected by transmission electron microscopy (TEM), compound muscle action potential (CMAP), immunohistochemistry and immunofluorescence.

Results: Extracted cells exhibited spindle-shaped morphology, presented typical markers (CD44⁺CD90⁺CD34^-CD45^-), and presented multidirectional differentiation potential. The DPSCs with VEGFA overexpression were constructed successfully. VEGFA enhanced the proliferation and neural differentiation ability of rDPSCs, and the expression of neuron-specific enolase (NSE) and βIII-tubulin was increased. However, these trends were reversed with the addition of SU5416. This suggests that VEGFA mediates the above effects mainly through vascular endothelial growth factor receptor 2 (VEGFR2) binding. The LC-YE-NGC basically meet the requirements of facial nerve repair. For the in vivo experiment, the CMAP latency period was shorter in DPSCS-VEGFA-NGC group in comparison with other experimental groups, while the amplitude was increased. Such functional recovery correlated well with an increase in histological improvement. Further study suggested that VEGFA-modified DPSCs could increase the myelin number, thickness and axon diameter of facial nerve. NSE, βIII-tubulin and S100 fluorescence intensity and immunohistochemical staining intensity were significantly enhanced.

Conclusion: VEGFA-modified rDPSCs combined with LC-YE-PLGA NGCs have certain advantages in the growth and functional recovery of facial nerves in rats.

Keywords: Facial nerve defects; Neural differentiation; Neural tissue engineering; SU5416; VEGFA; rDPSCs.

Graphical abstract

Fig. 1

Biological characteristics of DPSCs. (A) Primary DPSCs cultured for 48 h. (B) First generation of DPSCs. (C) Third generation of DPSCs. (A–C) Scale bars: 200 μm. (D) FACS results of rDPSCs in vitro. CD90 and CD44 were positive, and CD34 and CD45 were negative. (E) Osteogenic differentiation of DPSCs. (F) Adipogenic differentiation of DPSCs. (E, F) Scale bars: 100 μm.

Fig. 2

Validation of the transfection effect of lentivirus carrying VEGFA. (A) Lentivirus carrying VEGFA-infected DPSCs (MOI = 50). Scale bars: 50 μm. (B) qPCR showed the expression of VEGFA. ***P < 0.001. (C,D) Western blot images and analysis of VEGFA. GAPDH was used as a reference protein. ***P < 0.001. Data are expressed as the mean ± SD (n = 3 per group, one-way analysis of variance followed by Tukey's multiple comparisons test). DPSCs, unmodified rDPSCs; DPSCs-vector, empty vector-infected rDPSCs; DPSCs-VEGFA, VEGFA-overexpressing rDPSCs.

Fig. 3

Effects of VEGFA on neural differentiation of rDPSCs in vitro. (A) Cells following induction in neural-induced medium exhibit neuronal-like morphological changes. a: After 24 h of induction, cells formed clusters; b: After 7 days of induction, cells formed clusters. Scale bars: 100 μm. (B) qPCR showed the expression of βIII-tubulin and NSE. **P < 0.01, ***P < 0.001. (C) Western blot images and analysis of βIII-tubulin and NSE in the four groups. GAPDH was used as a reference protein.*P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SD (n = 3 per group, one-way analysis of variance followed by Tukey's multiple comparisons test). DPSCs, unmodified rDPSCs; DPSCs-vector, empty vector-infected rDPSCs; DPSCs-VEGFA, VEGFA-overexpressing rDPSCs; SU5416, VEGFA-overexpressing rDPSCs + SU5416.

Fig. 4

Effects of VEGFA on the viability of rDPSCs in vitro. CCK8 showed the effects of VEGFA on rDPSC viability. **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SD (n = 3 per group, one-way analysis of variance followed by Tukey's multiple comparisons test). DPSCs, unmodified rDPSCs; DPSCs-vector, empty vector-infected rDPSCs; DPSCs-VEGFA, VEGFA-overexpressing rDPSCs; SU5416, VEGFA-overexpressing rDPSCs + SU5416; OD, optical density.

Fig. 5

Morphology and use of LC-YE-PLGA NGCs. (A) Morphology of LC-YE-PLGA NGC. (B) The length of LC-YE-PLGA NGC. (C) The inner diameter of LC-YE-PLGA NGC. (D) LC-YE-PLGA NGC was used to bridge facial nerve defects in rat.

Fig. 6

The Incubation Period and The Maximum Amplitude of CMAP. (A) The incubation period of CMAP. (B) The maximum amplitude of CMAP. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SD (n = 3 per group, one-way analysis of variance followed by Tukey's multiple comparisons test). Autograft, facial nerve autograft reconstruction; NGC, nerve guidance conduit; DPSCs-NGC, nerve guidance conduit contains rDPSCs; DPSCs-VEGFA-NGC, nerve guidance conduit contains VEGFA-overexpressing rDPSCs.

Fig. 7

TEM features of regenerated nerve. (A–D) Axonal and myelin staining in the Autograft group, NGC group, DPSCs-NGC group and DPSCs-VEGFA-NGC group shown under TEM. Scale bars: 5 μm. (E) Analysis of myelin sheath thickness in each group. (F) Analysis of axon diameter in each group. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SD (n = 3 per group, one-way analysis of variance followed by Tukey's multiple comparisons test). Autograft, facial nerve autograft reconstruction; NGC, nerve guidance conduit; DPSCs-NGC, nerve guidance conduit contains rDPSCs; DPSCs-VEGFA-NGC, nerve guidance conduit contains VEGFA-overexpressing rDPSCs.

Fig. 8

Immunofluorescence of NSE and βIII-tubulin at 12 weeks postoperation. (A) Immunofluorescent images of NSE-positive cells and βIII-tubulin-positive cells captured using an inverted fluorescence microscope. Scale bars: 50 μm. (B) Image J software was used to measure the percentage of NSE and βIII-tubulin positive cells. *P < 0.05, **P < 0.01, ***P < 0.001. Autograft, facial nerve autograft reconstruction; NGC, nerve guidance conduit; DPSCs-NGC, nerve guidance conduit contains rDPSCs; DPSCs-VEGFA-NGC, nerve guidance conduit contains VEGFA-overexpressing rDPSCs.

Fig. 9

Immunohistochemical analysis of NSE, βIII-tubulin and S100 at 12 weeks postoperatively. (A) Images of immunohistochemistry sections obtained using a light microscope. Scale bars: 50 μm. (Blue: nuclei; Brown: NSE, βIII-tubulin and S100; The circular highlighted area is material). (B) Image J software was used to measure the mean optical density.*P < 0.05, **P < 0.01, ***P < 0.001. Autograft, facial nerve autograft reconstruction; NGC, nerve guidance conduit; DPSCs-NGC, nerve guidance conduit contains rDPSCs; DPSCs-VEGFA-NGC, nerve guidance conduit contains VEGFA-overexpressing rDPSCs; Mod, mean optical density. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)