Improved preparation of acellular nerve scaffold and application of PKH26 fluorescent labeling combined with in vivo fluorescent imaging system in nerve tissue engineering

Abstract

Acellular nerve scaffold has been widely used for peripheral nerve defect treatment. However, the structure of traditional acellular nerve scaffold is dense; the interval porosity and void diameter are too small to meet the requirement of cell seeding, which limits the application. This study was designed to prepare a novel acellular nerve scaffold by the technique of hypotonic buffer combined with freeze-drying, and use PKH26 fluorescent labeling combined with in vivo fluorescent imaging system to evaluate the biological behavior of tissue-engineered nerve in vitro and in vivo. According to light and electron microscopy, the scaffold, which microarchitecture was similar to the fibrous framework of rabbit sciatic nerves, was cell-free and rich in laminin, collagen I and collagen III. In vitro experiment showed that the novel acellular nerve scaffold could provide a 3-D environment to support the attachment, proliferation and migration of adipose-derived stem cells (ADSCs). ADSCs labeled with fluorescent dye PKH26 were then seeded on scaffolds and implanted subcutaneously into nude mice. After 4 weeks, nerve-like tissue rounded by vessels formed. Cells in the tissue seemed to confirm that they originated from the labeled ADSCs, as confirmed by in vivo fluorescent imaging. In conclusion, the prepared novel acellular nerve scaffold can be used as a new kind of nerve scaffold material, which is more conducible for seeding cells; And PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo.

Keywords: ADSCs; Freeze-drying; In vivo fluorescent imaging; Nerve tissue engineering; PKH26; Scaffolds; adipose-derived stem cells.