Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INTRODUCTION: Numerous experimental efforts have been undertaken to induce the healing of lesions within articular cartilage by re-establishing competent repair tissue. Adult mesenchymal stem cells have attracted attention as a source of cells for cartilage tissue engineering. The purpose of this study was to investigate chondrogenesis employing periosteal mesenchymal cells. METHODS: Periosteum was harvested from patients who underwent orthopedic surgeries. Mesenchymal stem cells were characterized through flow cytometry using specific antibodies. The stem cells were divided into four groups. Two groups were stimulated with transforming growth factor beta 3 (TGF-beta 3), of which one group was cultivated in a monolayer culture and the other was cultured in a micromass culture. The remaining two groups were cultivated in monolayer or micromass cultures in the absence of TGF-beta 3. Cell differentiation was verified through quantitative reverse transcription-polymerase chain reaction (RT-PCR) and using western blot analysis. RESULT: In the groups cultured without TGF-beta 3, only the cells maintained in the micromass culture expressed type II collagen. Both the monolayer and the micromass groups that were stimulated with TGF-beta 3 expressed type II collagen, which was observed in both quantitative RT-PCR and western blot analysis. The expression of type II collagen was significantly greater in the micromass system than in the monolayer system. CONCLUSION: The results of this study demonstrate that the interactions between the cells in the micromass culture system can regulate the proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis and that this effect is enhanced by TGF-beta 3.663487492Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)