Growth factors, hormones, and other regulatory molecules are traditionally required in tissue engineering studies to direct the differentiation of progenitor cells along specific lineages. We demonstrate that mechanical stimulation in vitro, without ligament-selective exogenous growth and differentiation factors, induces the differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages). A bioreactor was designed to permit the controlled application of ligament-like multidimensional mechanical strains (translational and rotational strain) to the undifferentiated cells embedded in a collagen gel. The application of mechanical stress over a period of 21 days up-regulated ligament fibroblast markers, including collagen types I and III and tenascin-C, fostered statistically significant cell alignment and density and resulted in the formation of oriented collagen fibers, all features characteristic of ligament cells. At the same time, no up-regulation of bone or cartilage-specific cell markers was observed.

We have investigated the effects of different growth factors on the proliferation and osteogenic potential of primary cultures of human bone marrow stromal cells (BMSC). Fibroblast growth factor (FGF)-2 was the most effective in promoting growth of these cells in vitro. The size of colonies formed in clonal conditions was approximately 2.5 times larger in presence of FGF-2. Also the morphology of BMSC was affected: cells cultured in 10% FCS alone became flattened, whereas FGF-2 expanded cells maintained a fibroblast-like elongated phenotype. Levels of alkaline phosphatase activity in BMSC expanded with FGF-2 were significantly lower (56%) than control and, after stimulation with ascorbic acid, betaGlycerophosphate and dexamethasone, FGF-2 expanded BMSC deposited approximately 3-fold more mineralized matrix than control cells. We have assessed osteogenicity of BMSC on hydroxyapatite porous scaffolds (bioceramics) by an ectopic bone formation assay. FGF-2 expanded BMSC yielded a higher bone formation (>20-fold) than control cells. We conclude that FGF-2, promoting BMSC proliferation, maintains cells in a more immature state allowing in vitro expansion of human osteo-progenitors which, associated with bioceramics, can differentiate in vivo and form bone tissue.

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The role of electromagnetism in the physiology of the human body and in the healing process is well accepted. Electroactive polymers, such as electrically conducting polymers, by virtue of their susceptibility to either electrical, mechanical, optical or thermal phenomena, could be utilized as an interface between the external world and the physiological environment1. In this study we have evaluated the utility of polypyrrole (PPy)-a conductive polymer, as an interactive substrate for inducing differentiation in bovine bone marrow stromal cells (BMSC). It was observed, the PPy is an excellent substrate for BMSC attachment, proliferation and differentiation. Furthermore, an application of an electrical stimulus (ES) to BMSC via PPy substrate was found to enhance the osteogenic commitment of these cells.