Optimizing a novel method for low intensity ultrasound in chondrogenesis induction

Hyaline cartilage tissue of joints is susceptible to injuries due to avascularity. Mesenchymal stem cells (MSCs) are used for cartilage tissue engineering. Among MSCs, adipose stem cells (ASCs) are attractive because of accessibility, their large number, and rapid growth. Common in vitro protocols successfully induce chondrogenic differentiation by expression of multiple cartilage-specific molecules. However, transforming growth factor b (TGFb) promotes chondrogenesis to terminal stages. Despite much attention being given to the influences of biochemical factors on chondrogenesis of MSCs, few studies have examined the chondrogenic effect of mechanical factors such as ultrasound as a feasible tool.

In this study, we focused on inducing chondrogenesis in the early stages of differentiation by using low-intensity ultrasound (LIUS). Four groups of ASC pellets (control, ultrasound, TGFb, and ultrasound/TGF) were cultured under chondrogenic (10 ng/ml of TGFβ3) and ultrasound conditions (200 mW/cm2 , 10 min/day). After 2 weeks, differentiation was evaluated by histology, quantitative gene expression analysis, and immunohistochemistry.

Our data demonstrated that ultrasound differentiated pellets showed increased expression of early chondrogenesis marker, Col2A, than those in TGFb groups(P < 0.001), and Col2B and Col10 expression were more prominent in TGFb groups. Immunostaining of sections showed Col2 fibrils around lacuna in LIUS and TGFb treated groups.

Using LIUS resulted in early chondrogenesis in comparison with terminally differentiated chondrocytes by TGFb. Therefore, LIUS might provide an applicable, safe, efficient, and cheap tool for chondrogenic differentiation of ASCs in cartilage tissue engineering.