Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells in Self-Gelling Alginate Discs Reveals Novel Chondrogenic Signature Gene Clusters

Abstract

We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/ pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants.

Application of Sodium Alginate Hydrogel

“…beside the interaction of cells that can be obtained with the involving to hydrogel through peptide-coupled alginates and that was discussed in Section 2.3.1,In order to get a proper control on the gels elasticity,alginate concentration optimization showed be highly controlled, the alginate kind and the crosslinkage strategy that will be chosen ( whether ionic or covalent) beside the density of the cross-linkage, hydrogels of the alginate could be fabricated to contrast the elastic function of most tissue kinds. (Figure 5 www.iosrjournals.org 26 | Page been highly correlated with the density of the existent peptides (7.5-150 μM RGD concentration) and the rigidity of the matrix (2.5-110 kPa) therefore, delivering the major roles of the mentioned two parameters [69].…”

“…The RGD peptides amount for each chain of alginate , and the vacancy with the RGD peptides clusters, specifically of the total RGD ligands density, highly influence the cells rebound to the gels of alginate with RGD-modification [66,67], probably these factors can impact the integrin receptors clusters [68]. However, the availability of the RGD ligands identically improves the differentiation and the adhesion of cells, chondrogenic gene expression in addition to the BMSCs accumulated matrix can undergo encapsulation in RGD-gels of alginate (3-D) that prevented with an high density of the RGD in vitro [69], Nowadays, gels of alginate have been expressed in a microfluidic apparatus via the release of the light-triggered caged calcium within the usage of DM-nitrophen™ complexes, and can be also utilized as a three-dimensional substrate of the cell culture. Prosteoblasts (MC3T3-E1) and the umbilical vein of the human endothelial cells were gonr through culture in the microfluidic apparatus by the manipulation of the photo-patterning of hydrogels derived from alginate, and this way lead to a valuable way for three-dimensional culture microenvironments integration with the microfluidic processes [70].…”

Bone and Cartilage from Stem Cells: Growth Optimalization and Stabilization of Cell Phenotypes

“…Hence, it is necessary to identify a transcriptome, which is reflecting the "true" osteoblast and chondrocyte phenotypes pertaining to the function and localization of such cells in the skeleton. Many excellent articles have addressed this task over the past 10 years, of which only some are mentioned here (Kulterer, Friedl et al 2007;Grundberg, Brandstrom et al 2008;Duggal, Fronsdal et al 2009;Morsczeck, Schmalz et al 2009;Sundelacruz and Kaplan 2009;Bernstein, Sticht et al 2010;Granchi, Ochoa et al 2010;Piek, Sleumer et al 2010;Sun, Mauerhan et al 2010;van der Zande, Walboomers et al 2010;Herlofsen, Kuchler et al 2011). Two transcriptomes featuring putative gene markers of osteoblasts and chondrocytes (two each), respectively, will be described in detail.…”

“…Bone and cartilage engineering revisited). As for the chondrocytic differentiation, it is referred to the papers of Bernstein et al (Bernstein, Sticht et al 2010) and Herlofsen et al (Herlofsen, Kuchler et al 2011). Bernstein and co-workers used chondrocytes from articular cartilage and MSCs in an intricate array of manipulations.…”

“…The features listed above have been extensively described and confirmed in the literature, however, a joint approach to produce well adapting engineered osteoblasts and chondrocytes has hitherto not been the subject of review articles or book chapters. The present outline encompasses a combined literature review of phenomena to take into consideration when engineering such cells from stem cells (SCs): sources of SCs to use (Logeart-Avramoglou, Anagnostou et al 2005;van Osch, Brittberg et al 2009), genes or microRNAs to manipulate (Goldring, Tsuchimochi et al 2006;Betz 2008;Grundberg, Brandstrom et al 2008;Duggal, Fronsdal et al 2009;Gordeladze, Djouad et al 2009;Lin 2009;Granchi, Ochoa et al 2010;Herlofsen, Kuchler et al 2011), selection of gene and microRNA delivery systems (Saraf and Mikos 2006;Phillips, Gersbach et al 2007), choice of humoral growth factors to facilitate SC differentiation (Shahdadfar, Fronsdal et al 2005;Boeuf and Richter 2010;van der Kraan, Davidson et al 2010), selection of appropriate scaffolds to support "asymmetric" SC differentiation (Vinatier, Bouffi et al 2009;Oh, Kim et al 2010;Seidi, Ramalingam et al 2011), combination of stem cell niches and/or co-cultures to ensure organ mimicry reflecting proper cell-cell interactions (Grad and Salzmann 2009;Grellier, Bordenave et al 2009;Boeuf and Richter 2010;Tare, Kanczler et al 2010), mechano-stimulation of cells (Kelly and Jacobs 2010;Nowlan, Sharpe et al 2010), and three-dimensional (3D) organ printing (Williams 2009;Visconti, Kasyanov et al 2010). Furthermore, this review also discusses how to stabilize osteoblasts and chondrocytes obtained by differentiation of SCs, i.e.…”