Substrate elasticity modulates TGF beta stimulated re-differentiation of expanded human articular chondrocytes

Substrate elasticity has emerged as important biomaterial design parameter. In particular, it has been reported that on soft substrates (~4 kPa) freshly isolated porcine chondrocytes better maintain their phenotype than on stiffer ones (>20 kPa). Thus, we investigated whether this also applies to re-differentiating, expanded/de-differentiated (EDD) human articular chondrocytes (HAC). EDD HAC were seeded onto Type I collagen functionalized poly acrylamide (PA) films with a Young's modulus of 0.26 ± 0.08 kPa (soft), 21.32 ± 0.79 kPa (intermediately stiff) and 74.88 ± 5.13 kPa (stiff), or type I collagen-coated plastic dishes (TCPS w/CI). Cells were cultured for 7 to 14 days in chondrogenic medium supplemented with transforming growth factor beta-1 (TGF-β1) and assessed for attachment, initial adhesion strength, proliferation, morphology as well as for expression of type I and II collagen at mRNA and type II collagen on protein level. Attachment and adhesion strength was similar on the different PA substrates and proliferation remained marginal (<1 doubling/week). On intermediately stiff to infinitely stiff substrates EDD HAC assumed a spindle shaped, fibroblastic morphology, whereas on the soft substrate they remained more spherical, as assessed by shape factor analysis, and had a reduced spreading area (up to 3.2-fold). F-actin organization on the soft substrate was restricted cortically, while on the stiffer substrates F-actin assembled into stress fibers. While type II collagen mRNA expression on the soft substrate was (similar to that in aggregate culture and) 18-fold higher than on TCPS w/CI, it was not detectable on protein level. On all substrates, in the absence of TGF-β1 type II collagen mRNA remained at levels expressed by EDD HAC. In summary, substrate elasticity modulated the TGF-β1 stimulated re-differentiation of EDD HAC. Mechanical compliance is thus an important parameter to be coupled with the delivery of appropriate morphogens in designing biomaterials for cartilage engineering and repair.