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Osteogenic Differentiation of Mc3t3-E1 Cells Regulated by Substrate Stiffness Requires Mapk Activation

Chirag B. Khatiwala1, Shelly R. Peyton1, and Andrew J. Putnam2. (1) Department of Chemical Engineering and Materials Science, University of California-Irvine, 3462, Natural Sciences II, Irvine, CA 92697, (2) Department of Chemical Engineering and Materials Science and Department of Biomedical Engineering, University of California-Irvine, 3462, Natural Sciences II, Irvine, CA 92697

The importance of mechanical forces in bone development and adaptation was first recognized nearly a century ago in the form of Wolff's Law. Increasing experimental evidence suggests that mechanical cues alone may be sufficient to dictate the fate of bone precursor cells. Furthermore, we recently showed that substrate stiffness influences focal adhesion formation, migration, proliferation, and, differentiation, of MC3T3-E1 cells, towards an osteoblastic phenotype as early as 4 days1. At the same time, there exist contrasting views as to the role of the MAPK pathway in the differentiation of these cells. While some studies support the fact that its activation is required for osteoblastic dfferentiation2 others show its inhibition promotes differentiation3. We hypothesize that structural-mechanical cues inherent in the extracellular matrix (ECM) can directly regulate the differentiation of these bone precursor cells over longer time scales via the MAPK pathway, and tested this hypothesis in vitro using a model substrate with tunable mechanical properties. Specifically, the pre-osteoblastic MC3T3-E1 cell line was cultured on the surface of poly(ethylene glycol) (PEG) hydrogels modified with the tri-peptide sequence RGD to support cellular adhesion. Gel compliance was manipulated by varying the total amount of PEG-diacrylate (PEGDA) and the ratio of PEG to PEGDA. Quantification of the macroscopic bulk elastic properties of the resulting hydrogels revealed Young's Moduli values of 13.7 and 423.9 kPa for hydrogels having 10% PEGDA (by weight) 50:50 PEGDA:PEG and 20% PEGDA 100:0 PEGDA:PEG respectively. Tissue-culture polystyrene (TCPS) was used as a rigid control. The osteoblastic differentiation of these cells was assessed over 14 days at the enzymatic level by quantifying alkaline phosphatase (ALP) activity, as well as at the genetic level by looking at expression levels of osteocalcin (OCN), bone sialo protein (BSP) and type-I collagen (α1 chain) via RT-PCR. Preliminary results show that phosphorylation of MAPK is dependent on substrate stiffness with significantly higher levels observed in cells cultured on the stiffer substrates (TCPS and 423.9 kPa gels) relative to those on the softest hydrogels (13.7 kPa) at both days 7 and 14. Furthermore, significantly higher ALP levels were seen at 7 and 14 days for cells on the stiffer substrates (TCPS and 423.9 kPa gels) over the softest hydrogels (13.7 kPa). At the genetic level significantly higher expression levels were seen for OCN and BSP at 7 and 14 days on stiff substrates versus soft substrates. However, uniform expression levels of type-I collagen were seen for all substrate stiffnesses at all timepoints (days 1, 7 and 14). To assess the role of the MAPK pathway on these stiffness-dependent differentiation events in MC3T3-E1 cells, studies were conducted using a well-known MAPK inhibitory drug, PD98059. Interestingly, we found that the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation at all timepoints for both soft (13.7 kPa) and stiff (423.9 kPa) hydrogels but no significant reduction in cells grown on TCPS. At the enzymatic level inhibiting MAPK activation resulted in significantly reduced levels of ALP for the 13.7 and 423.9 kPa hydrogel conditions at 7 and 14 days, but ALP levels on TCPS were same as that in the absence of the drug. Furthermore addition of the drug resulted in significantly reduced OCN gene expression levels for the 13.7 and 423.9 kPa hydrogel conditions at all timepoints while similar levels were seen on TCPS as in the absence of the drug. Expression levels of BSP gene were also reduced for all substrate stiffnesses at all timepoints. The presence of PD98059 did not influence the gene expression levels of type-I collagen. These data together indicate that substrate stiffness alone in the absence of other cues can regulate the activation of MAPK and osteogenic differentiation of these cells, and, inhibiting this activation results in retarding the rate of differentiation these MC3T3-E1 cells towards an osteogenic phenotype. The consequence of these findings will provide insight towards rational design of hydrogel biomaterials (e.g., polyethylene glycol, alginate, etc.) capable of supporting bony in-growth and tissue regeneration.

References 1. Khatiwala, C. B., Peyton, S. R. & Putnam, A. J. The Intrinsic Mechanical Properties of the Extracellular Matrix Affect the Behavior of Pre-Osteoblastic MC3T3-E1 Cells. Am J Physiol Cell Physiol (2006). 2. Hu, Y., Chan, E., Wang, S. X. &Li, B. Activation of p38 mitogen-activated protein kinase is required for osteoblast differentiation. Endocrinology 144, 2068-74. (2003). 3. Higuchi, C., Myoui, A., Hashimoto, N., Kuriyama, K., Yoshioka, K., Yoshikawa, H. & Itoh, K. Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix. J Bone Miner Res 17, 1785-94. (2002).