Fetal Cartilage Engineering from Amniotic Mesenchymal Progenitor Cells

Abstract

We determined whether cartilage could be engineered from mesenchymal progenitor cells (MPCs) normally found in amniotic fluid. Mesenchymal amniocytes were isolated from ovine amniotic fluid samples (n = 5) and had their identity confirmed by immunocytochemistry. Cells were expanded and then cultured as micromass pellets (n = 5) in a chondrogenic medium containing transforming growth factor-beta2 (TGF-beta2) and insulin growth factor-1 (IGF-1) for 6-12 weeks. Pellets derived from fetal dermal fibroblasts (n = 4) were cultured under identical conditions. Additionally, expanded mesenchymal amniocytes were seeded onto biodegradable polyglycolic acid scaffolds (n = 5) and maintained in the same chondrogenic medium within a rotating bioreactor for 10-15 weeks. Engineered specimens were analyzed quantitatively and compared with native fetal hyaline cartilage samples (n = 5). Statistical analysis was by the unpaired Student's t-test (p < 0.05). The isolated cells stained positively for vimentin and cytokeratins-8 and -18, but negatively for CD31. Micromass pellets derived from mesenchymal amniocytes exhibited chondrogenic differentiation by both standard and matrix-specific staining. In contrast, these findings could not be replicated in dermal fibroblast-based pellets. The engineered constructs derived from mesenchymal amniocytes similarly displayed histological evidence of chondrogenic differentiation and maintained their original size and three-dimensional architecture. Quantitative assays of the engineered constructs revealed lower concentrations of collagen type II, but similar amounts of glycosaminoglycans, elastin, and DNA, when compared to native fetal hyaline cartilage. We conclude that mesenchymal amniocytes can be used for the engineering of cartilaginous tissue in vitro. Cartilage engineering from the amniotic fluid may become a practical approach for the surgical treatment of select congenital anomalies.