Mesenchymal stem cells (MSC) from a number of sources have great potential for various tissue engineering (TE) approaches to repair connective tissues. To form the TE target tissue the differentiation pathways of the cells have to be stimulated. In vivo the cellular microenvironment and physical stimuli play an important role and thus the combination of biological and mechanical signals is likely to be critical. The goal of this work was to combine biochemical stimulation of cells with soluble factors or signals from substrates in defined microenvironments, plus mechanical stimulation via intermittent cyclic hydrostatic pressure (iHP) for osteogenic in vitro differentiation of human adult MSC. This hydrostatic pressure system allows for stimulation of all types and geometries of scaffold materials and cellular constructs. For these studies, a novel apparatus has been used that makes application of long term iHP to in vitro cell cultures [1]. Titanium discs and embroidered textile polycaprolactone substrates prepared were coated with collagen and/or chondroitin sulphate (CS), seeded with hMSC and subjected to iHP at various time points (1, 6, 13 days). Cell culture was performed either in DMEM (10 % FBS, 1 % P/S) or differentiation medium (50 μM ascorbic acid, 3.5 mM ß-glycerophosphate and 10 nM dexamethasone). It was shown that the extent of osteogenic differentiation as measured by ALP activity, mineral content, and expression of osteogenic markers is highly affected by the stimuli combinations applied. Thus, CS as a biochemical stimulus and iHP promoted stimulation with the effect of iHP being dependent on its timing within the time course of cell culture. Future work will study duration and details of HP application (duration, pressure level) on osteogenic differentiation together with more complex artificial extracellular matrices. [1] Myers K, Shrive NG, and Hart DA. J Biosci Bioeng 103:578-581 (2007)

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