Expression of N-Cadherins on Chondrogenically Differentiating Human Adipose Derived Stem Cells Using Single-Molecule Force Spectroscopy

Abstract

N-cadherins are important in the initial steps of mesenchymal stem cell chondrogenic differentiation referred to as pre-cartilage condensation. Because of that, the expression of N-cadherinson the surfaces of human adipose-derived stem cells (hASCs) differentiating toward chondrogenesis was investigated using single molecule force spectroscopy (SMFS). To engineer articular cartilage (AC), hASCs were grown in a unique centrifugal bioreactor (CBR) with cyclic oscillating hydrostatic pressure (OHP) and/or transforming growth factor (TGF-?3) conditions mimicking in ivo environments. Static AC tissues grown using micromass or pellet cultures were used as controls. To perform SMFS, anti-N-cadherin monoclonal antibodies were attached to atomic force microscopy (AFM) probes and forces were measured as these probes approached cells. Specific adhesion forces between antibody-functionalized AFM probes and cell surface N-cadherins were then identified, quantified and used to map the distribution of N-cadherins on cells. Our results indicate that a single antibody-antigen interaction has an adhesion force of 79 pN. Multiple antibody-antigen bindings are found to occupy multiples of 79 pN, independent of the culturing method. For tissues grown in the CBR, TGF-?3 elicited an increase in N-cadherin count whereSOX9 expression was directly proportional to the increase in N-cadherin. Tissue Young’s modulus also increased with the increase in N-cadherin. When tissues grown in static cultures were compared, they had higher N-cadherin counts and their Young’s moduli were significantly lower than those of the CBR tissues. Our results suggest that the addition of TGF-?3 in the CBR improves chondrogenic differentiation through a path dependent on N-cadherin expression.