Speaker
Description
Introduction:
The in vitro differentiation of bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to chondrocytes is mainly driven by the exogeneous administration of transforming growth factor β (TGF-β)[1]. Multiaxial load-induced activation of TGF-β1 (TGF-β1), which is secreted by the cells in a latent form has been shown to drive chondrogenesis[2]. In the present work, we apply a factorial design of experiment (DOE) and planned contrasts to establish the main and interactive effects between different loading parameters/factors in order to find a loading protocol that maximizes TGF-β1 activation in a biomaterial. Furthermore, we investigate whether the different loading protocols lead to changes in the secretion of biomarkers that affect chondrogenesis, such as bone morphogenetic protein 2 (BMP2).
Methodology:
BM-MSCs were obtained from three donors after acquiring written consent from the patients (Freiburg, EK-326/08) and encapsulated in fibrin–poly(ester-urethane) scaffolds (5 x 106 cells per scaffold). Samples were cultured in TGF-β1-free chondropermissive medium either in an unloaded state or subjected to joint-mimicking multiaxial load (combination of shear and compression) within a multiaxial bioreactor. The loading protocol consisted of 1 h of loading per day during a period of 12 days. Different combinations of three factors, namely type of indenter, shear frequency and compressive strain were used to investigate the effect of the factors' interaction on biomarker secretion. Two types of counterfaces were used: a cylinder and a ball. Two different settings were used for the shear frequency (0.2 and 1 Hz) and for the compressive strain (5% and 20%). The setting at 0.6 Hz shear frequency and 10% compressive strain represents the center point that demonstrates whether the relationship between factors is linear and to ensure process stability. Culture medium was collected and replaced every 2/3 days. ELISAs were performed to quantify BMP2 and TGF-β1. Nitrite was detected using the Griess assay and sulphated glycosaminoglycans using DMMB. The different outputs were all normalized to the DNA content (Hoechst dye).
Results:
Among the different donors, both active TGF-β1 and BMP2 showed very similar trends (r = 0.88). A protocol could be selected that resulted in the highest values for both biomarkers. Significant interaction could be found between the different factors using contrast analysis and a linear mixed model. DNA content remained consistent across all groups.
Conclusion:
We show that varying multiple parameters does have an effect on biomarker secretion. Active TGF-β1 and BMP2 were highest with a very specific loading regime (cylinder, shear frequency: 1 Hz, compressive strain: 20%). Certain biomarkers seem to be differentially regulated by mechanical stress. We conclude that a DOE is a useful tool to quickly screen complex relationships between multiple different factors in a biomaterial, which would take much longer in a one-factor-at-a-time approach.
[1] Johnstone, B. et al., Exp Cell Res, 238(1), 265-272 (1998)
[2] Li, Z. et al. J Cell Mol Med, 14(6A), 1338-1346 (2010)
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