Effects of local transplantation of autologous adipose-derived stromal vascular fraction on the hyperplastic scar formation in rabbit ears and the mechanism

Abstract

Objective: To explore the effects of local transplantation of autologous adipose-derived stromal vascular fraction (SVF) on the hyperplastic scar (HS) formation in rabbit ears and the mechanism. Methods: Twenty-four New Zealand white rabbits were used to reproduce HSs by making four full-thickness skin defect wounds with a diameter of 1 cm on the ventral surface of left ear of each rabbit. Wound epithelization and local-tissue proliferation were observed, and wound healing (complete epithelization) time and formation time of HS were recorded. The 24 rabbits were divided into SVF group, pure DMEM group, and pure HS group according to the random number table, with 8 rabbits and 32 wounds in each group. On post injury day (PID) 25 (after the complete epithelization of wounds), 0.2 mL of low glucose DMEM medium containing CM-Dil labeled autologous SVF was injected into HSs of rabbits in SVF group, while the same amount of low glucose DMEM medium was injected into HSs of rabbits in pure DMEM group. The frequency of injection was once every 5 days, totally for 3 times. HSs of rabbits in pure HS group did not receive any treatment. On PID 40, HSs of rabbits' ears in each group were harvested, then the histological form was observed by hematoxylin and eosin staining, the arrangement of collagen in HS was observed by Van Gieson staining, the distribution of CM-Dil-labeled SVF in the HS was observed with fluorescence microscope, and the mRNA expression and the protein expression of transforming growth factor β(1) (TGF-β(1)), Smad3, and Smad7 in HS were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. Data were processed with one-way analysis of variance and Tukey test. Results: (1) Complete epithelization time of wounds of rabbits' ears was (20.0±2.0) d post injury, and HSs were formed on PID 25. On PID 40, HSs of rabbits' ears in pure DMEM group and pure HS group were still in hyperplasia, while those in SVF group became smaller, flat, soft, and light colored. (2) On PID 40, compared with those in pure DMEM group and pure HS group, the number of epithelium foot like structures was more and the amount of inflammatory cells was less. The collagen of HSs of rabbits' ears in SVF group was arranged more regularly with broader gap between collagens. (3) On PID 40, CM-Dil-labeled SVF could still be observed in the HSs of rabbits' ears in SVF group. (4) On PID 40, compared with those in pure DMEM group and pure HS group, the mRNA expressions of TGF-β(1) and Smad3 in the HSs of rabbits' ears in SVF group were significantly down-regulated (P<0.05), while the mRNA expression of Smad7 was significantly up-regulated (P<0.05). There were no significant differences in the mRNA expressions of TGF-β(1), Smad3, and Smad7 in the HSs of rabbits' ears between pure DMEM group and pure HS group (P>0.05). (5) On PID 40, compared with those in pure DMEM group (0.74±0.03, 0.73±0.10, 0.54±0.09) and pure HS group (0.72±0.08, 0.71±0.12, 0.53±0.06), the protein expressions of TGF-β(1) and Smad3 in the HSs of rabbits' ears in SVF group (0.57±0.06, 0.42±0.09) were significantly down-regulated (P<0.05), while the protein expression of Smad7 (0.71±0.05) was significantly up-regulated (P<0.05). The protein expressions of TGF-β(1), Smad3, and Smad7 in the HSs of rabbits' ears in pure DMEM group and pure HS group were close (P>0.05). Conclusions: Autologous SVF transplantation can inhibit the formation of HS in the early stage of scar formation of rabbit, the mechanism may be related to the TGF-β(1)/Smad signaling pathway.