Various natural biological conduits have been investigated to bridge peripheral nerve injury especially in critical gap (greater than 3 cm in human). Autograft, the current gold standard, has several drawbacks including limited availability of donor graft, donor-site morbidity and mismatch in size in clinical practices. The aim of this study was to analyze the development of nerve conduit using decellularized human umbilical cord (HUC) artery seeded with neurodifferentiated human MSCs (ndMSCs) in bridging peripheral nerve gap. Artery conduits obtained from HUC were decellularized to remove native cells (n=3), then characterized by Hematoxylin and Eosin (H&E) staining and nuclei counterstaining with DAPI. The decellularized artery conduit was measured for every 2 weeks until 12 weeks. Next, mesenchymal stem cells (MSCs) were differentiated into neural lineage using 400 µg/mL of Centella asiatica. Then, 1.5×10 6 of MSCs or ndMSCs were seeded into decellularized artery conduit to study cell attachment. H&E staining and nuclei counterstaining with DAPI showed that all cellular components were removed from the HUC arteries. The decellularized artery conduit did not collapse and the lumen remained rigid for 12 weeks. Immunocytochemistry analysis with neural markers namely S100β, P75 NGFR, MBP and GFAP showed that MSCs had differentiated into neural lineage cells. H&E staining showed that the seeded MSCs and ndMSCs attached to the lumen of the conduits as early as 2 days. In conclusion, this study showed that nerve conduit using decellularized HUC artery seeded with neurodifferentiated human MSCs was successfully developed and have the potential to bridge critical nerve gap.

BACKGROUND: Centella asiatica (L.) is a plant with neuroprotective and neuroregenerative properties; however, its effects on the neurodifferentiation of mesenchymal stem cells (MSCs) and on peripheral nerve injury are poorly explored. This study aimed to investigate the effects of C. asiatica (L.)-neurodifferentiated MSCs on the regeneration of peripheral nerve in a critical-size defect animal model. 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.