The effects of chondrocytes derived conditioned medium supplemented with TGFβ-3/ IGF--1 on chondrogenesis in amniotic fluid derived cells

Cartilage is known for its paracrine activity and humoral factors released by chondrocytes especially TGF-β3 and IGF-1 are considered to play vital role in chondrogenic differentiation. The chondrogenesis can be characterized by expression of certain markers like SOX9, COL2A1, TGF-β1 and ACAN. Although, various protocols for chondrogenic differentiation of stem cells exist, yet these methods are unable to mimic intrinsic mechanisms of cartilage regeneration successfully. To better imitate the natural mechanism of chondrogenesis, conditioned media was opted for chondrogenic differentiation either with supplementation of TGF-β3 and/or IGF-1 or alone. It was also aimed to demonstrate whether external supplementation of conditioned media with standard chondrogenic induction media affects its chondrogenesis potential or not. Fot this purpose, rat amniotic fluid derived stem cells and chondrocytes were isolated and were characterized by Real-Time PCR and immunofluorescence studies. Following the characterization of chondrocytes, conditioned media was collected and rat amniotic fluid derived stem cells were differentiated into chondrocytes in alginate beads using conditioned media with TGF-β3 and/or IGF-1 supplementation or alone or with standard chondrogenic induction media. The results of differentiation studies were supported with Real-Time PCR, immunofluorescence and immunohistochemistry assays. The results showed that conditioned media have potential to induce chondrogenic differentiation successfully both alone and with the supplementation of TGF-β3 and/or IGF-1. Nonetheless, joint use of conditioned media and standard chondrogenic induction media provided the best results for COL2A1, ACAN and SOX9 expressions. In conclusion, it is believed that the constituents of induction media facilitate better chondrogenic differentiation when combine with the humoral factors from conditioned media. Yet, it is important to validate the components of conditioned media and their exact amounts for better understanding mechanisms of conditioned media based chondrogenic differentiation. Besides, the results of the conducted study should be tested with further in vivo experiments.