Differentiation Capacity of Human Chondrocytes Embedded in Alginate Matrix

Abstract

Healing capacity of cartilage is low. Thus, cartilage defects do not regenerate as hyaline but mostly as fibrous cartilage which is a major drawback since this tissue is not well adapted to the mechanical loading within the joint. During in vitro cultivation in monolayers, chondrocytes proliferate and de-differentiate to fibroblasts. In three-dimensional cell cultures, de-differentiated chondrocytes could re-differentiate toward the chondrogenic lineage and re-express the chondrogenic phenotype. The objective of this study was to characterize the mesenchymal stem cell (MSC) potential of human chondrocytes isolated from articular cartilage. Furthermore, the differentiation capacity of human chondrocytes in three-dimensional cell cultures was analyzed to target differentiation direction into hyaline cartilage. After isolation and cultivation of chondrogenic cells, the expression of the MSC-associated markers: cluster of differentiation (CD)166, CD44, CD105, and CD29 was performed by flow cytometry. The differentiation capacity of human chondrocytes was analyzed in alginate matrix cultured in Dulbecco?s modified eagle medium with (chondrogenic stimulation) and without (control) chondrogenic growth factors. Additionally, the expression of collagen type II, aggrecan, and glycosaminoglycans was determined. Cultivated chondrocytes showed an enhanced expression of the MSC-associated markers with increasing passages. After chondrogenic stimulation in alginate matrix, the chondrocytes revealed a significant increase of cell number compared with unstimulated cells. Further, a higher synthesis rate of glycosaminoglycans and a positive collagen type II and aggrecan immunostaining was detected in stimulated alginate beads. Human chondrocytes showed plasticity whilst cells were encapsulated in alginate and stimulated by growth factors. Stimulated cells demonstrated characteristics of chondrogenic re-differentiation due to collagen type II and aggrecan synthesis.

3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads

“…Especially, cells embedded in sodium alginate display a suitable source for this investigations (Kumachev et al 2011;Pampaloni et al 2007;Xu et al 2013). The manual manufacturing is an established process in research (Jonitz et al 2011b;Fischbach et al 2009). The automated alginate bead production close to the manual manufacturing steps in combination with bioscreening is new and established in this paper.…”

“…The cells are likewise embedded in 1.2 % sodium alginate (2 9 10 6 cells/ml) and dropped in 100 mM CaCl 2 solution for bead formation. In the manual procedure 10-15 beads were pooled into a 48 well plate (Jonitz et al 2011b). Instead of this, one bead/well is produced in the adapted automated process for further individual tests by high throughput screening systems.…”

“…A suitable natural and biocompatible polysaccharide for immobilization of cells in a 3D construct is alginate. Popular 3D in vitro models are alginate beads, which are mainly used for environmental (Cassidy et al 1996) and basic research like drug development (Rimann and Graf-Hausner 2012), regenerative medicine (Jonitz et al 2011b;Fischbach et al 2009;Moshaverinia et al 2013a) including tissue engineering (Chen et al 2010) and investigation of cancer (Fischbach et al 2009). The alginate beads can also be used to encapsulate chondrocytes.…”

“…The alginate beads can also be used to encapsulate chondrocytes. The 3D cell culturing in the presence of several growth factors have lead to a differentiation process of the cells, which was shown by the accumulation of ECM components like collagen type II, aggrecan and glycosaminoglycans (Jonitz et al 2011b). Moreover, the influence of 3D cell cultures associated with growth factors on cancer cell angiogenic capability was investigated with oral squamous cell carcinoma cells (OSCC-3) encapsulated in alginate beads (Fischbach et al 2009).…”

Transient supplementation of growth factor TGF-β1 effectively initiates chondrogenic redifferentiation of human chondrocytes

“…The cartilage tissue was washed with phosphate buffered saline (PBS, PAA, Coelbe, Germany), minced and enzymatically digested using 1 % trypsin/EDTA (Gibco® Invitrogen, Darmstadt, Germany) for 20 minutes at 37 °C prior treatment with 0.2 % collagenase A (Roche, Mannheim, Germany) for three hours at 37 °C. The isolated chondrocytes were cultured in cell culture medium DMEM (Dulbecco's Modified Eagle Medium) containing 10 % foetal calf serum (FCS), 1 % amphotericin B and 1 % penicillin-streptomycin (all from Fisher Scientific, Darmstadt, Germany) supplemented with ascorbic acid (50 μg/mL, Sigma, Seelze, Germany) up to passage two [7].…”