Fibroblast Growth Factor Receptors in In Vitro and In Vivo Chondrogenesis: Relating Tissue Engineering Using Adult Mesenchymal Stem Cells to Embryonic Development Catharine A. Hellingman, M.D., 1 Wendy Koevoet, B.Sc., 1 Nicole Kops, B.Sc., 2 Eric Farrell, Ph.D., 1,2 Holger Jahr, Ph.D., 2 Wei Liu, B.S., 3 Robert J. Baatenburg de Jong, M.D., Ph.D., 1 Dorothy A. Frenz, Ph.D., 3 and Gerjo J.V.M. van Osch, Ph.D. 1,2 Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb devel- opment, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is fol- lowed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1–3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR ex- pression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation. Introduction T he potential of cell therapy to stimulate tissue re- generation is currently under investigation. Adult mes- enchymal stem cells (MSCs) are a promising candidate cell source for this purpose because of the ease with which they can be isolated and expanded as well as their capacity for multilineage differentiation. 1,2 The possibility of regenerating cartilage with the aid of MSCs is appealing because cartilage has a very limited spontaneous healing capacity. 3–5 As car- tilage consists of only one cell type and does not contain blood vessels or neural innervations, it seems an uncompli- cated tissue to engineer. However, to obtain optimal results in reconstruction of cartilage from different anatomical lo- cations, it will be crucial to generate cartilage of a specific subtype. Stable hyaline cartilage should be regenerated for articular surface regeneration, since (tissue-engineered) hy- pertrophic cartilage may mineralize and perhaps even ossify in vivo via the endochondral route. 6,7 The generation of hy- pertrophic cartilage, on the other hand, could be advanta- geous for bone repair. 7 High-cell-density culture systems are helpful in the differ- entiation of MSCs toward the chondrogenic lineage. For that reason, pellet culture is an accepted model to induce chon- drogenic differentiation. However, MSCs display signs of chondrocyte hypertrophy, such as production of collagen X and matrix mineralization, during an in vitro pellet culture period of several weeks. 6,7 Therefore, pellet culture of MSCs has been claimed to be an in vitro model for endochondral ossification. 6 For cartilage regeneration, the consistent terminal hypertrophic differentiation of in vitro–differentiated MSCs represents a lack of external control over their differentiation. 6 Departments of 1 Otorhinolaryngology and 2 Orthopaedics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Nether- lands. 3 Department of Otorhinolaryngology and Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, New York. TISSUE ENGINEERING: Part A Volume 16, Number 2, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2008.0551 545