Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads.

Havva Dashtdar,Malliga Raman Murali,Abdulrazzaq Mahmod Suhaeb,Tunku Kamarul,Tunku Sara Ahmad,Lakshmi Selvaratnam,Hanumantharao Balaji Raghavendran

doi:10.7717/peerj.1650

Havva Dashtdar, Malliga Raman Murali + Show 5 more

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https://doi.org/10.7717/peerj.1650

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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.

Highlights

Chondrogenic differentiation is a unique process that starts with cell–cell interactions and is influenced by the presence of specific growth and differentiation factors
Pellet, and alginate bead cultures showed positive for Collagen type II staining on 21 days of culture (Figs. 2A–2C), compared with the negative controls (Figs. 2G–2I)
Immunochemistry of aggrecan showed positive for pellet and alginate bead cultures (Figs. 2D–2E); no positive staining was observed in monolayer (Fig. 2F) compared with the negative control (Figs. 2J–2L)

Summary

Introduction

Chondrogenic differentiation is a unique process that starts with cell–cell interactions and is influenced by the presence of specific growth and differentiation factors. In addition to inducing chondrogenesis, most of these culture conditions lead towards a hypertrophic differentiation after the initial chondrogenic induction, similar to that observed in the terminal differentiation of hypertrophic chondrocytes during endochondral ossification (Ma et al, 2003; Steinert et al, 2003; Ichinose et al, 2005; Xu et al, 2008; Bian et al, 2011). This unstable chondrogenic phenotypic expression after the initial induction was considered to be the major hurdle to in vitro chondrogenesis for application in cartilage tissue engineering

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