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injuries and aging associated diseases that affect joints. This study reports the development of a bilayered
scaffold, which consists of both bone and cartilage regions. On the other hand, amniotic fluid-derived
stem cells (AFSCs) could be differentiated into either osteogenic or chondrogenic cells, respectively. In
this study we have developed a bilayered scaffolding system, which includes a starch/polycaprolactone
(SPCL) scaffold for osteogenesis and an agarose hydrogel for chondrogenesis. AFSC-seeded scaffolds were
cultured for 1 or 2 weeks in an osteochondral-defined culture medium containing both osteogenic and
chondrogenic differentiation factors. Additionally, the effect of the presence or absence of insulin-like
growth factor-1 (IGF-1) in the culture medium was assessed. Cell viability and phenotypic expression
were assessed within the constructs in order to determine the influence of the osteochondral differentiation
medium. The results indicated that, after osteogenic differentiation, AFSCs that had been seeded
onto SPCL scaffolds did not require osteochondral medium to maintain their phenotype, and they produced
a protein-rich, mineralized extracellular matrix (ECM) for up to 2 weeks. However, AFSCs differentiated
into chondrocyte-like cells appeared to require osteochondral medium, but not IGF-1, to synthesize
ECM proteins and maintain the chondrogenic phenotype. Thus, although IGF-1 was not essential for creating
osteochondral constructs with AFSCs in this study, the osteochondral supplements used appear to
be important to generate cartilage in long-term tissue engineering approaches for osteochondral interfaces.
In addition, constructs generated from agarose–SPCL bilayered scaffolds containing pre-differentiated
AFSCs may be useful for potential applications in regeneration strategies for damaged or diseased
joints.