Nano-hydroxyapatite/collagen film as a favorable substrate to maintain the phenotype and promote the growth of chondrocytes cultured in vitro

摘要

Autologous chondrocyte implantation (ACI) has emerged as a novel approach to cartilage repair through the use of harvested chondrocytes. However, the expansion of the chondrocytes from the donor tissue in vitro is restricted by the limited cell numbers and the dedifferentiation of the chondrocytes. The present study investigated the effect of collagen-based films, including collagen, hydroxyapatite (HA)/collagen (HC) and in situ synthesis of nano-HC (nHC), on monolayer cultures of chondrocytes. As a substrate for the chondrocytes monolayer culture in vitro, nHC was able to restrain the dedifferentiation of chondrocytes and facilitate cell expansion, which was detected by methyl thiazolyl tetrazolium assay, scanning electron microscopy, calcein-acetoxymethyl/propidium iodide staining, hematoxylin and eosin staining, Safranin O staining, immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction. Furthermore, the nHC films significantly facilitated cell growth and enhanced the expression of cartilage-specific extracellular matrix (ECM) components, including aggrecan and type II collagen. In addition, nHC films markedly downregulated the expression of collagen type I, an indicator of dedifferentiation. The results indicated that nHC, a collagen-based substrate optimized by nanoparticles, was able to better support cell growth and preserve cell phenotype compared with collagen alone or HC. The nHC film, which favors cell growth and prevents the dedifferentiation of chondrocytes, may therefore serve as a useful cartilage-like ECM for chondrocytes. In conclusion, nHC film is a promising substrate for the culture of chondrocytes in cell-based therapy.