Micro-aggregates vs. dispersed cells: what is best for chondrogenic differentiation of BMSCs?
Citation for published version (APA):
Potier, E., Rivron, N., Blitterswijk, C. A., & Ito, K. (2011). Micro-aggregates vs. dispersed cells: what is best for chondrogenic differentiation of BMSCs?. Poster session presented at Mate Poster Award 2011 : 16th Annual Poster Contest.
Document status and date: Published: 01/01/2011 Document Version:
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Cell phenotype – Chondrogenic differentiation (fig.3)
• All markers increased in time for all groups.
• Higher up-regulation for dispersed cells than for micro-aggregates.
• Higher up-regulation for 7 days of TGFβ treatment.
Matrix production (fig.4)
• GAG/DNA content increased in time for all groups.
• Higher up-regulation for 21 days of treatment and dispersed conditions.
• Matrix deposition increased with days of TGFβ treatment.
Fig.4. (A) GAG/DNA content per alginate bead.Values are mean +/- St Dev; n = 3. (B) Alcian blue staining (dark blue color indicates chondrogenic
matrix deposition). Scale: 50 µm.
Conclusion
• In all conditions, bovine BMSCs differentiated toward the chondrogenic phenotype.
• Cell-cell contact (micro-aggregates) has a negative effect on chondrogenic marker expression but it is not reflected at the matrix level.
• The up-regulation of chondrogenic marker expression was
higher for 7 days of TGFβ treatment but the matrix production was higher for 21 days of treatment.
• Overall, chondrogenic differentiation of bovine BMSCs is better for dispersed cells and 21 days of TGFβ treatment
References
[1] Chamberlain et al. 2007. Stem cells (25) [2] Guo et al. 1989. Connect
Tissue Res (19) [3] Mauck et al. 2006. OsteoArthritis and Cartilage (14).
Introduction
Bone marrow-derived stromal cells (BMSCs) are envisioned as regenerative cells for numerous tissues[1], including cartilage. Success of BMSC-based therapies, however, relies on a number of methodological improvements, among which a better understanding/control of their differentiation pathways. We investigated here if different paracrine signaling and cell-cell contact conditions (micro-aggregates vs dispersed cells) can affect their chondrogenic potential and if different stimulation patterns can modify these effects.
Materials and Methods
Cells and 3D scaffolds: bovine BMSCs (n=3 donors) were
encapsulated in alginate beads (1.2%)[1] as dispersed cells and as micro-aggregates at 7 million cells/ml; thus creating different paracrine signaling and cell-cell contact conditions.
Culture conditions: BMSCs were cultured for 21 days at 2%O2 in high glucose DMEM containing dexamethasone, ITS-1+, BSA,
ascorbic-2-phosphate, L-Proline and sodium pyruvate[3]. Medium was
supplemented with TGFβ3 (10ng/ml) for 0, 7, and 21 days (Fig. 1).
Fig.1. Seeding and culture conditions.
Data analysis: cell phenotype was characterized by RT-qPCR (type II
collagen, sox9, aggrecan); produced matrix by histology (Alcian blue staining) and biochemical assays (glycosaminoglycan (GAG) and DNA content); and cell viability by calcein/propidium iodide staining.
Results
Cell viability (fig.2) and proliferation
• BMSCs stayed viable in all conditions and DNA content increased slightly after 21 days for all dispersed conditions.
• BMSCs formed aggregates after 21 days, with bigger
structures under micro-aggregate conditions and 21 days of TGFβ treatment.
Fig.2. Cell viability after 21 days of culture
Calcein (alive) and propidium iodide (dead) staining.
Orthopaedic Biomechanics
/ Department of Biomedical Engineering
Micro-aggregates vs. dispersed
cells: what is best for chondrogenic
differentiation of BMSCs?
Potier E, Rivron N*, van Blitterswijk C*, Ito K
*Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands
Dispersed 21 0 7 21 Micro-aggregates 0 7 D isper sed 0d 7d 21d 0d 7d 21d Mic ro -aggr egat es Days of TGFβ treatment Type II collagen Sox9 Aggrecan Dispersed Micro-aggregates
Fig.3. Gene expression of chondrogenic markers.
Values are mean +/- St Dev; n = 3.
A. Dispersed B.
