Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture

Abstract

Without using biochemical agents, in this study, we sought to investigate the potential of controlling the differentiation of mesenchymal stem cells (MSCs) into a specific cell type through the use of 3D co-culturing and mechanical stimuli. MSCs and primary cultured chondrocytes were separately encapsulated into alginate beads, and the two types of beads were separated by a membrane. For the investigation a computer-controllable bioreactor was designed and used to engage intermittent hydrostatic pressure (IHP). Five different magnitudes (0.20, 0.10, 0.05, 0.02 MPa and no stimulation) of IHP were applied. The stimulation pattern was the same for all groups: 2 h/day for 7 days starting at 24 h after seeding; 2 and 15 min cycles of stimulating and resting, respectively. Biochemical (DNA and GAG contents), histological (Alcian blue), and RT-PCR (Col II, SOX9, AGC) analyses were performed on days 1, 5, 10, and 20. The results from these analyses showed that stimulation with higher magnitudes of IHP (≥0.10 MPa) were more effective on the proliferation and differentiation of co-cultured MSCs. Together, these data demonstrate the potential of using mechanical stimulation and co-culturing for the proliferation and differentiation of MSCs, even without biochemical agents.

Adipose Stromal Cells Contain Phenotypically Distinct Adipogenic Progenitors Derived from Neural Crest

“…4C ). To further verify chondrogenesis, RT-qPCR was performed to compare the expression of chondrogenic markers Aggrecan , collagen type2a1 (Col2a1) , and Sox9 [26], [27]. The results showed that the expression of all three markers in the GFP+ cells were markedly lower than that in the GFP- cells after chondrogenic induction ( Fig.…”

Development of a new co-culture system, the “separable-close co-culture system,” to enhance stem-cell-to-chondrocyte differentiation

“…The increase was more than threefold compared with the indirect co-culture system and the monoculture (Figs. 3a, b) although Jeong et al (2012) concluded that even an indirect co-culture system between MSCs and chondrocytes was able to increase the expression levels of SOX9 mRNA. Additionally, the expression levels of SOX9 mRNA in the separable-close co-culture system were considerably higher than those in the direct co-culture system (Fig.…”

“…Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control), aggrecan, and SOX9 were analyzed using pre-designed minor groove binder probes (Applied Biosystems), TaqMan PCR Master Mix (Applied Biosystems), and a Light Cycler apparatus (ABI 7300; Applied Biosystems). Aggrecan, a typical cartilage-specific proteoglycan core protein, and SOX9, a transcription factor, are commonly used to evaluate chondrogenic differentiation in cells (Jeong et al 2012;Lin et al 2006;Maxson and Burg 2010;Safshekan et al 2014). Gene expression levels were calculated using the standard curve method and normalized relative to the levels of GAPDH gene expression.…”

Mesenchymal stromal cells for cartilage repair in osteoarthritis

“…Co-culture of MSCs with juvenile articular chondrocytes (ACs) resulted in efficient chondrogenic differentiation 30 . It has been observed that coculture with primary chondrocytes and mechanical stimulation helped to achieve MSC chondrogenic differentiation without biochemical agents 31 . Co-cultures of MSCs and ACs with reduced concentration and duration of TGF-b3 showed effective chondrogenic potential 32,33 .…”