METHODS: The hMSCs derived from human Wharton's jelly umbilical cord (hWJMSCs; n = 6) were treated with RECA at different concentrations; 400, 800, 1200, 1600, 2000 and 2400 μg/ml. The cytotoxicity of RECA was evaluated via the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) and cell proliferation assays. The hWJMSCs were then induced to neural lineage for 9 days either with RECA alone or RECA in combination with neurotrophic factors (NF). Cell morphological changes were observed under an inverted microscope, while the expression of the neural markers S100β, p75 NGFR, MBP, GFAP and MOG was analyzed by quantitative polymerase chain reaction and immunocytochemistry. The cell cycle profile of differentiated and undifferentiated hWJMSCs was investigated through cell cycle analysis.
RESULTS: RECA exerted effects on both proliferation and neural differentiation of hWJMSCs in a dose-dependent manner. RECA reduced the proliferation of hWJMSCs and was cytotoxic to cells above 1600 μg/ml, with IC50 value, 1875 ± 55.67 μg/ml. In parallel with the reduction in cell viability, cell enlargement was also observed at the end of the induction. Cells treated with RECA alone had more obvious protein expression of the neural markers compared to the other groups. Meanwhile, gene expression of the aforementioned markers was detected at low levels across the experimental groups. The supplementation of hWJMSCs with RECA did not change the normal life cycle of the cells.
CONCLUSIONS: Although RECA reduced the proliferation of hWJMSCs, a low dose of RECA (400 μg/ml), alone or in combination of neurotrophic factors (NF + RECA 400 μg/ml), has the potential to differentiate hWJMSCs into Schwann cells and other neural lineage cells.
METHODS: Nerve conduit was developed using decellularised artery seeded with C. asiatica-neurodifferentiated MSCs (ndMSCs). A 1.5 cm sciatic nerve injury in Sprague-Dawley rat was bridged with reversed autograft (RA) (n = 3, the gold standard treatment), MSC-seeded conduit (MC) (n = 4) or ndMSC-seeded conduit (NC) (n = 4). Pinch test and nerve conduction study were performed every 2 weeks for a total of 12 weeks. At the 12th week, the conduits were examined by histology and transmission electron microscopy.
RESULTS: NC implantation improved the rats' sensory sensitivity in a similar manner to RA. At the 12th week, nerve conduction velocity was the highest in NC compared with that of RA and MC. Axonal regeneration was enhanced in NC and RA as shown by the expression of myelin basic protein (MBP). The average number of myelinated axons was significantly higher in NC than in MC but significantly lower than in RA. The myelin sheath thickness was higher in NC than in MC but lower than in RA.
CONCLUSION: NC showed promising effects on nerve regeneration and functional restoration similar to those of RA. These findings revealed the neuroregenerative properties of C. asiatica and its potential as an alternative strategy for the treatment of critical size nerve defect.