Fabrication a novel cartilage ECM-derived 3-D porous acellular matrix scaffold and in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells.

Abstract

Objective The objective of this study is non-destructive evaluation of tissue engineering constructs in vivo using PKH26 and molecular light imaging system, and exploring the feasibility for tissue engineering cartilage using canine chondrocytes and porous cartilage acellular matrix scaffold. Methods After induced by chondrogenic medium, BMSCs were seeded into the CEDPS scaffolds, cell attachment was confirmed by SEM and the viability of attached cells on the scaffold was confirmed by a live/dead assess-ment. Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds in vitro and implanted subcutaneously into nude mice. Then in vivo fluorescent imaging system was used for e-valuating the cell-scaffold constructs. After 4 weeks, the constructs was analyzed by histology, immunohisto-chemistry and immunofluorescence examnation. Results SEM showed a large mount of extraceilular matrix around the cells as time grown. Dead/Live staining in the confocal microscopy of cell-scaffold constructs re-vealed cells with green fluorescence (live cell). Four weeks later, cartilage-like tissue formed in nude mice, with positive staining for Safranin O, tuoluidine blue and collagen Ⅱ. Cells in the samples seemed to confirm that they originated from the labeled BMSCs, as confirmed by in vivo fluorescent imaging and immunofluo-rescence examination. Conclusion Cartilage ECM-derived scaffolds can be used for effective cartilage tis-sue engineering both in vitro and in vivo. As well, PKH26 fluorescent labeling and in vivo fluorescent imag-ing can be useful for cell tracking and analyzing cell-scaffold constructs in vivo. Key words: Cartilage; Tissue engineering; Mesenchymal stem cells; Bone marrow; Fluorescence