Directed Differentiation of Human Mesenchymal Stem Cells into Chondrocytes Using Micropatterning Method

Abstract

The necessity of chondrocytes can be obtained by inducing Mesenchymal Stem Cells (MSCs) using internal and external stimuli. In this research, MSC was directed to be differentiated on modified substrate and L-ascorbic acid addition. The objective of this research is to direct human MSCs (hMSCs) to become chondrocytes on modified substrates by qualitative and quantitative evaluation using several spesific markers. Mesenchymal stem cells were obtained from adipose tissue (ADMSC). Cell stemness was evaluated using flow cytometry and multipotency analysis. Micropatterned substrate was developed using Parafilm™ by doing mechanical method and pith size was measured after getting Scanning Electron Microscope (SEM) image and analyzedusing imageJ. The cells were seeded in 2.4× 104 cells/mL and incubated for 12 days. The expression of Collagen II was observed by immunocytochemistry method and sulphated-GAG using Alcian Blue Staining on day 6 and day 12. Cell morphology was observed using SEM imaging. ADMSCs were successfully obtained from adipose tissue and complied the mesenchymal stem cellscriteria. The micropatterned substrate was well developed using Parafilm™ and could be used to modify the substrate because ADMSCs were only attached on pith (P) that was not covered by Parafilm™. ADMSCs expressed Collagen II and GAG on day 6 and 12 of observation.The cells morphology also confirmed that chondrogenicdifferentiation was affected by seeding density because ADMSCs were found in more rounded shape and also expressed more Collagen II in narrow pith than in widepith.