PKH26 dye for tracing the seed cells of tissue-engineered peripheral nerve in vivo

Abstract

Objective To assess the validity of PKH26 dye for labeling the bone marrow mesenchymal stem cells (BMSCs) and the feasibility of this method for tracing the seed cells of tissue engineering nerve in vivo. Methods BMSCs isolated from Wistar rat's bone marrow were labeled with the PKH26 dye, the marked validity was observed by the fluorescence microscopy and flow cytometer, the cellular vitality was de-tected by MTT, the differentiated abilities were tested by in vitro osteablastic and lipoblastic differentiation. After that, the labelld cells were micro-injected into the acellular nerve grafts(ANG) to build compound grafts and cultured for 3,5,7,14 days in vitro, then frozen sections were made respectively to observe the survival and migration of the implanted cells in ANG. Simultaneously, these compound grafts were used to bridge the 1.5 cm sciatic nerve defects of the Wistar rats, 1,4,6,8 weeks postoperation, the grafts were taken out and frozen section were made in order to observe the survival of the implanted stem cells in vivo inside the ANG. Results PKH26 dye was good for labelling the BMSCs, marked validity was higher than 95%. The cellular vitality and differentiated abilities were not affected compared with the unlabelled cells. Further more, BMSCs could adhere to the ANG, and suvive,magrate in vitro. In vivo, BMSCs was able to survive at least 8 weeks in the nerve regenerative microenvironment. Conclusion PKH26 dye could label the BMSCs effectively, and it's suitable for in vivo seed cells tracing in tissue engineering nerve model. Key words: Tissue engineering; Peripheral nerve; Bone marrow mesenchymal stem cells