Abstract
BackgroundSonic hedgehog (Shh) is an important signalling protein involved in the induction of early cartilaginous differentiation. Herein, we demonstrate that Shh markedly induces chondrogenesis of rabbit bone marrow stromal cells (BMSCs) under microgravity conditions, and promotes cartilage regeneration.ResultsIn the rotary cell culture system (RCCS), chondrogenic differentiation was revealed by stronger Toluidine Blue and collagen II immunohistochemical staining in the Shh transfection group, and chondroinductive activity of Shh was equivalent to that of TGF-β. Western blotting and qRT-PCR analysis results verified the stronger expression of Sox9, aggrecan (ACAN), and collagen II in rabbit BMSCs treated with Shh or TGF-β in a microgravity environment. Low levels of chondrogenic hypertrophy, osteogenesis, and adipogenesis-related factors were detected in all groups. After transplantation in vivo, histological analysis revealed a significant improvement in cartilage and subchondral repair in the Shh transfection group.ConclusionsThese results suggested that Shh signalling promoted chondrogenesis in rabbit BMSCs under microgravity conditions equivalent to TGF-β, and improved the early stages of the repair of cartilage and subchondral defects. Furthermore, RCCS provided a dynamic culture microenvironment conducive for cell proliferation, aggregation and differentiation.
Highlights
Sonic hedgehog (Shh) is an important signalling protein involved in the induction of early cartilaginous differentiation
Passage 2 rabbit bone marrow stromal cells (BMSCs) transfected with Shh adenovirus or green fluorescent protein (GFP) adenovirus plasmid were incubated in the rotary cell culture system (RCCS) environment
GFP-transfected BMSCs cultured with 10 ng/mL TGF-β3 served as a positive control
Summary
Sonic hedgehog (Shh) is an important signalling protein involved in the induction of early cartilaginous differentiation. We demonstrate that Shh markedly induces chondrogenesis of rabbit bone marrow stromal cells (BMSCs) under microgravity conditions, and promotes cartilage regeneration. Chondrocytes and extracellular matrix (ECM) are the main components of hyaline-type cartilage [1]. ECM is composed of collagen II, aggrecan (ACAN), chondroitin sulfate, and other glycosaminoglycans that are important for maintaining the biomechanical properties of articular cartilage [2, 3]. As physical activity is increasing among people of all ages, articular cartilage injuries are increasing. Most previous studies related to cartilage regeneration have focused on cell-based therapy using scaffolds or growth factors in a rabbit osteochondral defect model [6, 7]. Autologous chondrocyte implantation is not suitable for treating cartilage defects due to inconvenient sampling, limited sources and poor proliferation ability in vitro [7]. The mechanisms regulating their chondrogenic potential are not clearly understood
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