W. Kafienah (Bristol, GB)
University of Bristol School of Cellular and Molecular MedicinePresenter Of 1 Presentation
10.4.1 - Over-Confluence of expanded mesenchymal stem cells ameliorates their chondrogenic capacity in 3D cartilage tissue engineering
Abstract
Purpose
Tissue engineering of high-quality articular cartilage constructs using bone marrow-derived mesenchymal stem cells (BM-MSCs) remains an elusive goal. During the limb bud mesenchyme stage of skeletal development, MSCs undergo a process of condensation prior to the initiation of chondrogenesis. We hypothesise that culturing BM-MSCs in vitro to over confluence can mimic this condensation phase, leading to robust chondrogenic cells for articular cartilage tissue engineering applications.
Methods and Materials
Eleven BM-MSC samples were cultured for 5 days (low confluence) and 24 days (over confluence) before being harvested, seeded onto polyglycolic scaffolds and stimulated with transforming growth factor-β3 (TGF-β3) for 35 days to drive chondrogenic differentiation. The mature constructs were digested and analysed by quantitative immunoassays and qPCR, to measure the biochemical composition of various ECM components and the expression of chondrogenic markers, respectively. Four samples were analysed by RNA Seq analysis to reveal major pathways implicated in the over-confluence process.
Results
Biochemical analysis of the generated cartilage constructs demonstrated a statistically significant increase in type II to type I collagen content ratio and a significant increase in the proteoglycan content, in over confluence constructs compared to paired low confluence constructs. Gene analysis showed a statistically significant increase in SOX9, type II collagen and aggrecan expression and a decrease in the expression of the hypertrophic marker, type X collagen, in over confluence constructs. KEGG enriched pathway analysis of RNA Seq data revealed significantly active pathways for focal adhesion and ECM-receptor interactions.
Conclusion
This study has demonstrated, for the first time, that BM-MSCs, permitted to proliferate past confluence in 2D culture, can produce biochemically superior 3D tissue-engineered cartilage. This simple adjustment to the common BM-MSC culture technique could impact most of cartilage tissue engineering modalities utilising these cells.