title: Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads.
abstract: Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.
authors.affiliation: Tissue Engineering Group, Department of Orthopaedic Surgery (NOCERAL), Faculty of Medicine, University of Malaya , Kuala Lumpur , Malaysia.
authors.forename: Havva
authors.initials: H
authors.lastname: Dashtdar
authors.affiliation: Tissue Engineering Group, Department of Orthopaedic Surgery (NOCERAL), Faculty of Medicine, University of Malaya , Kuala Lumpur , Malaysia.
authors.forename: Malliga Raman
authors.initials: MR
authors.lastname: Murali
authors.affiliation: School of Medicine and Health Sciences, Monash University Malaysia , Sunway Campus Selangor , Malaysia.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Selvaratnam
authors.affiliation: Tissue Engineering Group, Department of Orthopaedic Surgery (NOCERAL), Faculty of Medicine, University of Malaya , Kuala Lumpur , Malaysia.
authors.forename: Hanumantharao
authors.initials: H
authors.lastname: Balaji Raghavendran
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya , Kuala Lumpur , Malaysia.
authors.forename: Abdulrazzaq Mahmod
authors.initials: AM
authors.lastname: Suhaeb
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya , Kuala Lumpur , Malaysia.
authors.forename: Tunku Sara
authors.initials: TS
authors.lastname: Ahmad
authors.affiliation: Tissue Engineering Group, Department of Orthopaedic Surgery (NOCERAL), Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Clinical Investigative Centre (CIC), University Malaya Medical Centre, Kuala Lumpur, Malaysia.
authors.forename: Tunku
authors.initials: T
authors.lastname: Kamarul
keywords: Alginate; Chondrogenesis; Hypertrophic condensation; Mesenchymal stromal cells; Pellet culture; Tissue engineering