Effects of combined transplantation of rat Schwann cells and fibroblasts on nerve regeneration of denervated perforator flaps in rats and the mechanism

Objective To explore the effects of combined transplantation of the rat Schwann cells and fibroblasts (Fbs) on the nerve regeneration of denervated perforator flaps in rats and the mechanism. Methods (1) Fbs were isolated from the trunk of 2 Sprague-Dawley (SD) rats embryos of 14-16 days′ pregnancy and cultured, and the morphology of the cells was observed. The third passage of cells were used for subsequent experiments. The protein expressions of fibronectin and Ephrin-B2 were observed by immunohistochemical method. The mRNA expression of Ephrin-B2 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (n=3). (2) Schwann cells were isolated from the bilateral sciatic nerves and brachial plexus nerves of 45 SD rats born for 1-3 days and cultured, and the morphology of the cells was observed. The third passage of cells were used for subsequent experiments. The rate of S100 positive cells was detected by immunofluorescence method and flow cytometer, with sample numbers of 9 and 3 respectively. (3) In Dulbecco′s modified Eagle medium (DMEM) high glucose medium, 1 mL Fbs and 1 mL Schwann cells both in the concentration of 1×10⁵ cells/mL were co-cultured as Schwann cells+ Fbs co-culture group, and 2 mL Schwann cells in the concentration of 1×10⁵ cells/mL were cultured alone as Schwann cells alone culture group, with 5 wells in each group. The clusters of Schwann cells in the two groups were observed and counted under inverted phase contrast microscope at post culture hour (PCH) 6 and 24 respectively. The clusters of Schwann cells in Schwann cells+ Fbs co-culture group were observed by immunofluorescence method at PCH 24 too. The protein expressions of EphB2, Sox2, and N-cadherin in Schwann cells of two groups at PCH 24 were detected by Western blotting (n=20). (4) Totally 100 8-week-old male SD rats were selected, and an in situ replanted peritoneal denervated perforator flap was made in each rat. According to the random number table, the rats were divided into simple flap group, Fbs alone transplantation group, Schwann cells alone transplantation group, Schwann cells+ Fbs co-transplantation group, with 25 rats in each group. Flaps of rats in Fbs alone transplantation group and Schwann cells alone transplantation group were injected with 0.4 mL Fb and 0.4 mL Schwann cells respectively (2×10⁶ cells each). Flaps of rats in Schwann cells+ Fbs co-transplantation group were injected with 0.4 mL Fbs and Schwann cells mixed cells (totally 2×10⁶ cells, cell number ratio: 1∶1), and flaps of rats of simple flap group were injected with the same volume of DMEM high glucose medium. On post injection day (PID) 2, 5, 7, 9, and 14, 5 rats in each group were selected respectively according to the random number table. The flap tissue was collected, and the number, diameter, and arrangement of regenerated nerves were observed by immunofluorescence method. Data were processed with completely random designed t test, analysis of variance for repeated measurement, t test, and Bonferroni correction. Results (1) The third passage of cells isolated and cultured from the rat embryo trunks were uniform in size and shape, long spindle-shaped, with a large proportion of nuclei. Strong positive expressions of fibronectin and Ephrin-B2 protein in cells were observed, and the mRNA expression of Ephrin-B2 was 0.004 1±0.000 8. The cells were identified as Fbs. (2) After 5 days of culture, the primary cells isolated from the sciatic nerves and brachial plexus nerves of neonatal rats were elongated in cell bodies and grew in nest, fence, or vortex-like shape. The third passage of cells were detected by immunofluorescence method and flow cytometer, and the corresponding S100 positive cell rates were (95.9±1.0)% and (95.8±1.1)% respectively. The cells were identified as Schwann cells. (3) At PCH 6 and 24, the cluster numbers of Schwann cells in Schwann cells+ Fbs co-culture group were significantly higher than those of Schwann cells alone culture group (t=6.500, 10.614, P<0.01). At PCH 24, the Schwann cells in Schwann cells+ Fbs co-culture group aggregated into clusters, Fbs dispersed around the Schwann cell clusters, and the protein expressions of EphB2, N-cadherin, and Sox2 in Schwann cells were significantly higher than those in Schwann cells alone culture group (t=2.975, 19.717, 11.159, P<0.05 or P<0.01). (4) On PID 2, a small number of scattered, disordered, short, and thin nerve fibers were observed in the flap tissue of rats in the four groups. From PID 5 to 14, the number of nerve fibers in the flap tissue of rats of Schwann cells+ Fbs co-transplantation group increased gradually, and the nerve fibers were with long diameter and arranged orderly. The number of nerve fibers in the flap tissue of rats of Schwann cells alone transplantation group increased, but the nerve fibers were with short diameter and arranged disorderly, and the number was smaller than that of Schwann cells+ Fbs co-transplantation group. In simple flap group and Fbs alone transplantation group, the nerve fibers in the flap tissue of rats gradually degenerated with gradually decreased number or even disappeared. Conclusions The combined transplantation of Fbs and Schwann cells in rats can regulate Schwann cells migration and clustering by activating Ephrin/Eph-Sox2-N-cadherin signaling pathway, thus promoting the orderly nerve regeneration of denervated perforator flaps in rats.