Periosteum as a source of mesenchymal stem cells: the effects of TGF-β3 on chondrogenesis

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 β3 (TGF-β3), 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-β3. 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-β3, only the cells maintained in the micromass culture expressed type II collagen. Both the monolayer and the micromass groups that were stimulated with TGF-β3 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-β3.

Participação do HIF-1'alfa' na expressão de colageno tipo II e agrecano na cartilagem articular mediada pela IL-1'beta' e TNF'alfa' / The participation of HIF-1'alpha' in collagen type II and aggrecan expression on articular cartilage mediated by IL-1'beta' and TNF'alpha' cytokines

O Fator Induzido por Hipóxia-1 (Hypoxia Inducible Factor-1 - HIF-1) é um fator de transcrição responsável por transcrever genes relacionados às alterações nas concentrações de oxigênio e sobrevivência celular. A cartilagem articular é um tecido avascular e o ambiente dos condrócitos é caracterizado por condições de hipóxia dentro da matriz. Nestas condições a proteína HIF-1α do Fator de transcrição Induzido por Hipóxia-1α (HIF-1α) é necessário para controlar o metabolismo e a integridade funcional da cartilagem. Além da hipóxia algumas citocinas como IL-1β e TNFα são também capazes de estabilizar HIF-1α, além de serem consideradas as principais mediadoras da osteoartrite (OA). Objetivo: Verificar a participação da IL–1β na regulação do HIF-1α em condições normais de oxigênio; Verificar a utilização da via da Fosfatidil-Inositol-3-Kinase (PI-3K) pelo HIF-1α e analisar a participação HIF-1α na expressão de colágeno tipo II e agrecano e a sua regulação pelas citocinas TNF-α e da IL–1β. Material e Métodos: Condrócitos humanos provenientes de pacientes em OA, submetidos à artroplastia...

Introduction: Hypoxia Inducible Factor -1 (HIF-1) is a transcription factor that regulates the expression of genes related with oxygen concentration e cellular survive. Articular cartilage is a non-vascular tissue and the condrocytes microenviroment are caracterized by hypoxic conditions inside the extracelluar matrix. In this condition, the protein HIF-1α, from HIF-1 is necessary to the metabolism control and cartilage functional intengrity. Some citokines like IL-1β and TNFα, as well as hypoxia, are capable to stabilize HIF-1α and are essential in oateoarthritis (OA) progression disease. Objective: To verify the participation of IL–1β in the HIF-1α regulation under normal conditions of oxygen; To verify if the pathway of phosphatidilynositol-3-Kinase (PI-3K) is used in this modulation; To analyse HIF-1α participation in the collagen type II and aggrecan gene expression and your regulation by TNF-α and IL–1β. Material and Methods: Human OA chondrocytes were obtained from patients with OA that underwent total knee joint replacement surgery...

Comparação do processo de reparo ósseo em tíbias de ratas normais e osteopênicas / Bone repair process in normal and osteopenic female rats' tibiae: a comparative study

O objetivo foi comparar a consolidação óssea em tíbias de ratas normais e osteopênicas. 49 ratas albinas fêmeas, linhagem Wistar, peso médio de 160 (± 20g) e 100 dias foram distribuídas em 2 grupos: Ooforectomizado (OOF) e Pseudo-ooforectomizado (Grupo controle - SHAM). 30 dias após a ooforectomia e/ou cirurgia simulada, todas foram submetidas à produção de lesão óssea cortical. Foram sacrificadas na 2ª, 4ª, 6ª e 8ª semanas. Os osteoblastos foram contados. O peso aumentou progressivamente, porém as OOF apresentaram maior peso (p<0,05) quando comparadas as SHAM, à época da segunda cirurgia. 15 dias pós-lesão óssea, as OOF apresentaram maior número de osteoblastos (p<0,05) quando comparados as SHAM. 30 dias pós-lesão óssea houve diminuição no número de osteoblastos, porém os valores foram equivalentes entre os dois grupos OOF e SHAM. 45 dias pós-lesão, apesar da diminuição constante de osteoblastos, o grupo OOF permaneceu elevado quando comparado ao grupo controle (p<0,05). Aos 60 dias o grupo SHAM apresentou menos osteoblastos, sugerindo processo avançado de reparo ósseo. Os animais osteopênicos apresentaram resposta inicial acelerada à lesão óssea, possibilitando a equivalência entre os grupos 30 dias pós-lesão. Mas, após este período apresentaram retardo na mineralização do osteóide, sugerindo atraso tardio no processo de reparo ósseo.

The purpose was to compare tibial bone union in normal and osteopenic female rats. Forty-nine Wistar albino female rats weighing 160 g (±20g) and 100 days were distributed into 2 groups: Oophorectomized (OOF) and Pseudo-oophorectomized (SHAM). Thirty days later, a cortical injury was produced in all the animals. They were sacrificed in the 2nd, 4th, 6th and 8th weeks. Osteoblasts count was performed. Progressive weight increase was observed, but the OOF group was shown to have gained more weight (p£0.05) than the SHAM group, at the time of the second surgery. After 15 days post-injury, the animals in the OOF group presented a higher number of osteoblasts (p£0.05) compared to the SHAM group. Thirty days after injury, the number of osteoblasts was reduced, but both groups showed similar amounts. Forty-five days after injury, despite a constant reduction, the number of osteoblasts in the OOF group remained high when compared to SHAM (p£0.05) group. After 60 days, we found less osteoblasts in the SHAM group, suggesting an advanced bone repair process. The osteopenic animals showed an early accelerated response, which became equivalent between both groups 30 days after injury. However, after that period, they showed a delayed osteoid mineralization, suggesting delayed late bone repair process.