Peripheral Nerve Regeneration by Secretomes of Stem Cells from Human Exfoliated Deciduous Teeth

Abstract

Peripheral nerve regeneration across nerve gaps is often suboptimal, with poor functional recovery. Stem cell transplantation-based regenerative therapy is a promising approach for axon regeneration and functional recovery of peripheral nerve injury; however, the mechanisms remain controversial and unclear. Recent studies suggest that transplanted stem cells promote tissue regeneration through a paracrine mechanism. We investigated the effects of conditioned media derived from stem cells from human exfoliated deciduous teeth (SHED-CM) on peripheral nerve regeneration. In vitro, SHED-CM-treated Schwann cells exhibited significantly increased proliferation, migration, and the expression of neuron-, extracellular matrix (ECM)-, and angiogenesis-related genes. SHED-CM stimulated neuritogenesis of dorsal root ganglia and increased cell viability. Similarly, SHED-CM enhanced tube formation in an angiogenesis assay. In vivo, a 10-mm rat sciatic nerve gap model was bridged by silicon conduits containing SHED-CM or serum-free Dulbecco's modified Eagle's medium. Light and electron microscopy confirmed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in the SHED-CM group at 12 weeks after nerve transection surgery. The sciatic functional index (SFI) and gastrocnemius (target muscle) wet weight ratio demonstrated functional recovery. Increased compound muscle action potentials and increased SFI in the SHED-CM group suggested sciatic nerve reinnervation of the target muscle and improved functional recovery. We also observed reduced muscle atrophy in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. SHED-CM may therefore provide a novel therapy that creates a more desirable extracellular microenvironment for peripheral nerve regeneration.

FIG. 1.

Effects of SHED-CM on SC migration and proliferation. (A) Transwell migration assay. The migration of SCs cultured in SHED-CM was enhanced relative to those cultured in DMEM (−). DMEM (−), SHED-CM, DMEM (15% FBS): n = 5 per group. Data are shown as mean ± SD. *P < 0.05. (B) Cell proliferation assay. SC proliferation is reported as the absorbance at 450 nm following MTT analysis. The proliferation of SCs cultured in SHED-CM was also enhanced relative to those cultured in DMEM (−). Cells cultured in DMEM (15% FBS) were used as a positive control. DMEM (−), SHED-CM, DMEM (15% FBS): n = 5 per group. Data are shown as mean ± SD. *P < 0.05. SHED, stem cells from human exfoliated deciduous teeth; SHED-CM, serum-free conditioned media from SHEDs; SC, Schwann cell; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.

FIG. 2.

Effect of SHED-CM on SC gene expression. The mRNA levels of (A) NGF, (B) BDNF, (C) NT-3, (D) CNTF, (E) GDNF, (F) VEGF, (G) laminin, (H) fibronectin, and (I) collagen IV were analyzed in SCs cultured with SHED-CM or DMEM (−) using real-time RT-PCR. The expression levels of NTF-, angiogenic molecule-, and ECM molecule-encoding genes were upregulated in SCs cultured with SHED-CM relative to those cultured with DMEM (−). DMEM (−), SHED-CM: n = 5 per group. Data are shown as mean ± SD. *P < 0.05. ECM, extracellular matrix; NTF, neurotrophic factor; RT-PCR, reverse transcriptase–polymerase chain reaction.

FIG. 3.

Effects of SHED-CM on DRG neuronal outgrowth and survival. (A) β-III-tubulin immunostaining of DRG neurons cultured with SHED-CM or DMEM (−) for 48 h. Scale bar = 100 μm. (B) Quantitative analysis of average neurite length. The average neurite outgrowth was significantly higher in neurons treated with SHED-CM than in those treated with DMEM (−). DMEM (−), SHED-CM: n = 5 per group. Data are shown as mean ± SD. *P < 0.05. (C) DRG neuron viability. Cell viability is reported as the absorbance at 450 nm following CCK-8 assay. Compared with DMEM (−), SHED-CM significantly increased the viability of DRG neurons. DMEM (−), SHED-CM: n = 5 per group. Data are shown as mean ± SD. *P < 0.05. DRG, dorsal root ganglion.

FIG. 4.

Effect of SHED-CM on capillary tube-like HUVEC formation. (A) HUVECs were cultured with EM, SHED-CM, VEGF (10 ng/mL), and HGF (10 ng/mL). After 11 days, capillary tube-like structures were observed. Scale bar = 100 μm. (B) Statistical analysis of the total blood vessel lengths. HUVECs cultured in SHED-CM exhibited significant increases in tube length when compared with cells cultured in DMEM (−). EM, SHED-CM, VEGF, HGF: n = 3 per group. Data are shown as mean ± SD. *P < 0.05. (C) Statistical analysis of the numbers of joints. HUVECs cultured in SHED-CM exhibited significantly higher numbers of joints when compared with cells cultured in DMEM (−). EM, SHED-CM, VEGF, HGF: n = 3 per group. Data are shown as mean ± SD. *P < 0.05. EM, endothelial cell medium; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor.

FIG. 5.

Regeneration in a sciatic nerve transection rat model. (A) The sciatic nerve was transected to leave a 10 mm length and was bridged with a silicon conduit. (B) Microscopic view of the regenerated nerves 12 weeks after surgery. Images are representatives of the following groups: Sham, Transection, DMEM (−), SHED-CM, and Autograft. The diameters of regenerated nerves were thicker in rats treated with SHED-CM relative to rats treated with DMEM (−).

FIG. 6.

Myelin sheath morphology in the regenerated nerves. (A) Toluidine blue staining of semi-thin cross sections from the distal portions of regenerated nerves at 12 weeks after surgery. Scale bar = 20 μm. (B) TEM images of ultrathin cross sections of the distal regenerated nerves at 12 weeks after surgery. Scale bars = 5.0 μm, 500 nm. (C) Densities of remyelinated axons in the distal nerve portions. The number of regenerated myelinated fibers was significantly higher in the SHED-CM and Autograft groups than in the DMEM (−) group. Sham, DMEM (−), SHED-CM, Autograft: n = 4 per group. Data are shown as mean ± SD. *P < 0.05. (D) Statistical analysis of the G-ratios. The G-ratio was significantly improved in the SHED-CM group relative to the DMEM (−) group. Sham, DMEM (−), SHED-CM, Autograft: n = 4 per group. Data are shown as mean ± SD. *P < 0.05. TEM, transmission electron microscope.

FIG. 7.

Motor function recovery after sciatic nerve transaction. (A) The SFI was analyzed to assess motor function at 3, 6, 9, and 12 weeks after surgery. The SFI at 12 weeks after repair was significantly higher in the SHED-CM and Autograft groups than in the DMEM (−) group. Sham, Transection, DMEM (−), SHED-CM, Autograft: n = 6 per group. Data are shown as mean ± SD. *P < 0.05. (B) Representative CMAP recordings of the injured side at 12 weeks after surgery. (C) Histograms show the peak CMAP amplitude, latency, and nerve conduction velocity. Sham, Transection, DMEM (−), SHED-CM, Autograft: n = 6 per group. Data are shown as mean ± SD. *P < 0.05. CMAP, compound muscle action potential; SFI, sciatic functional index.

FIG. 8.

Muscle wet weight and morphological analysis. (A) Macroscopic view of the gastrocnemius muscles of both hind limbs at 12 weeks after surgery. (B) Masson's trichrome staining of gastrocnemius muscle cross sections. (C) Statistical analysis of the gastrocnemius muscle wet weight ratios. The relative gastrocnemius muscle was greater in the SHED-CM and Autograft groups than in the DMEM (−) group. Sham, Transection, DMEM (−), SHED-CM, Autograft: n = 6 per group. Data are shown as mean ± SD. *P < 0.05. (D) Histograms show the average collagen fiber areas. The collagen fiber percentage was significantly lower in the SHED-CM and Autograft groups relative to the DMEM (−) group. Sham, Transection, DMEM (−), SHED-CM, Autograft: n = 6 per group. Data are shown as mean ± SD. *P < 0.05.