ABSTRACT
Human adipose derived adult stem (hADAS) cells have the ability to differentiate into a chondrogenic phenotype in three-dimensional culture and media containing dexamethasone and TGF-beta. The current study examined the potential of a genetically engineered elastin-like polypeptide (ELP) to promote the chondrocytic differentiation of hADAS cells without exogenous chondrogenic supplements. hADAS cells were cultured in ELP hydrogels in either chondrogenic or standard medium at 5% O2 for up to 2 weeks. By day 14, constructs cultured in either medium exhibited significant increases in sulfated glycosaminoglycan (up to 100%) and collagen contents (up to 420%). Immunolabeling confirmed that the matrix formed consisted mainly of type II and not type I collagen. The composition of the constructs cultured in either medium did not differ significantly. To assess the effect of oxygen tension on the differentiation of the above constructs, samples were cultured in standard medium at either 5% or 20% O2 for 7 days and their gene expression profile was evaluated using real time RT-PCR. In both cases, the hADAS-ELP constructs upregulated SOX9 and type II collagen gene expression, while type I collagen was downregulated. However, constructs cultured in 20% O2 highly upregulated type X collagen, which was not detected in the 5% O2 cultures. The study suggests that ELP can promote chondrogenesis for hADAS cells in the absence of exogenous TGF-beta1 and dexamethasone, especially under low oxygen tension conditions.
Subject(s)
Adipose Tissue/cytology , Biocompatible Materials/chemistry , Cell Culture Techniques/methods , Chondrocytes/cytology , Elastin/chemistry , Peptides/chemistry , Cell Differentiation , Cells, Cultured , Chondrocytes/metabolism , Collagen/metabolism , Collagen Type I/metabolism , Collagen Type II/metabolism , Collagen Type X/biosynthesis , Cross-Linking Reagents/pharmacology , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate , Immunochemistry , Immunohistochemistry , Oxygen/chemistry , Oxygen/metabolism , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology , Time Factors , Tissue Engineering , Up-RegulationABSTRACT
Elastin-like polypeptides (ELPs) are a class of biocompatible, non-immunogenic and crosslinkable biomaterials that offer promise for use as an injectable scaffold for cartilage repair. In this study, an oligohistidine (His(6)) epitope tag was incorporated at the N-terminus of an ELP using recombinant DNA techniques to permit tracking without compromising on material biocompatibility. His(6)-tagged ELPs were successfully detected by Western blot analysis and quantified by ELISAs following digestion with trypsin. The mass of His(6) tagged ELP fragments freed from a crosslinked ELP hydrogel after digestion with trypsin correlated highly with hydrogel weight loss, providing evidence of the tag's capability to enable tracking of enzymatic degradation of the ELP hydrogel. The His(6) tag also facilitated recognition of crosslinked ELPs from background staining of articular cartilage. These results suggest that the His(6) epitope tag has the potential to track ELP scaffold loss independently of newly formed tissue mass for evaluating matrix remodeling in vivo.
