Applications of AFM nanomechanical tests in studying cartilage aging and tissue engineering. (a) Comparison of the ultrastructure and compressive nanomechanics of native and engineered aggrecan. Top panel: compression resistance of end-attached newborn and adult (38 years old) human cartilage aggrecan monolayers measured via AFM force spectroscopy in 0.01 M NaCl solution (pH ≈5.6). Bottom panel: tapping mode AFM images of native human cartilage aggrecan and aggrecan synthesized by equine chondrocytes and bone marrow stromal cells (BMSCs) in self-assembled peptide hydrogel culture. The newborn human aggrecan shows core protein and CS-GAG side chains longer than that of the adult one. Meanwhile, engineered aggrecan by BMSCs shows longer core protein and longer CS-GAG side chains than that by age-matched chondrocytes. Adapted with permission from refs 16 and 17. Copyright 2012 Elsevier Inc. and Osteoarthritis Research Society International. (b) Impacts of growth factors on chondrocyte-engineered PCM. Top panel: schematic of AFM-nanoindentation on individual chondrons (chondrocytes-PCM composite) immobilized within silicon pyramidal wells. Bottom panel: representative indentation force versus depth loading curves (mean ± SEM, n ≥ 5 cells) on the individual composite after 21 and 28 day culture in DMEM with 10% FBS versus with insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1, also known as BMP-7). Adapted with permission from ref 119. Copyright 2016 Elsevier Ltd. (c) Impacts of predifferentiation on chondrogenetic activities of induced pluripotent stem cells (iPSCs). Top panel: Safranin-O/Fast-Green/hematoxylin stained section from pellets categorized by type II collagen (Col2)-driven green fluorescent protein (GFP): GFP+ versus GFP− iPSC cells after two passages. Bottom panel: indentation modulus Eind of the pellet bulk and cryo-sections, calculated by the Hertz model (mean ± SEM, *p < 0.05 by region, #p < 0.05 by cell type). Adapted with permission from ref 129. Copyright 2012 National Academy of Science.