Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

Alteração olfatória na esclerose sistêmica: frequência e fatores associados / Olfactory abnormalities in systemic sclerosis: frequency and associated factors

Resumo: O sistema olfativo medeia a interação entre o sistema nervoso central (SNC), humor, cognição e alterações inflamatórias. Anormalidades olfativas são frequentes em doenças autoimunes, mas em pacientes com esclerose sistêmica (SSc) são pouco exploradas. A SSc é uma doença complexa caracterizada por inflamação crônica, vasculopatia e fibrose de pele e órgãos internos. Trinta e cinco pacientes consecutivos com SSc e 40 controles pareados por idade e sexo foram acompanhados durante um ano. A função olfatória avaliada pelos três estágios do Sniffin 'Sticks test (SST), que inclui limiar de odor (TH), discriminação de odor (DIS) e identificação de odor (ID). O TDI é a soma deles, e foi calculado separadamente para sexo e grupos etários. As pontuações destes estágios foram analisadas de acordo com o inventário de ansiedade (BAI) e depressão de Beck (BDI), capacidade cognitiva pela avaliação cognitiva de Montreal (MOCA), atividade e gravidade da doença bem como volume de hipocampo e amígdala com ressonância magnética (RM). Todas as análises foram realizadas duas vezes sendo a 1º no início do curso e a 2º um ano depois. A capacidade olfatória piorou com a idade (p?0,05). A alteração olfatória foi associada com idade, tempo de doença, atividade da doença e volumes do hipocampo e amigdala da primeira análise. A análise longitudinal com regressão linear mostrou que a pontuação dos três estágios e a total obtidas na segunda análise foram correlacionadas com variáveis da primeira análise. O TDI foi correlacionado com a pontuação de BAI (IC 95% = 0,042-0,430, p = 0,019), volume do hipocampo direito (IC 95% = 0,007-0,025, p = 0,001), volume do hipocampo esquerdo (IC de 95% = (-0,021) - (- 0,004), p = 0,006) e volume da amígdala direita (IC 95% = -0,030-0,000, p = 0,050), sendo o R2=0,588. O DIS foi correlacionado com o volume do hipocampo direito (IC 95% = 0,004-0,013, p = 0,001) e esquerdo (IC 95% = (-0,009) - (-0,001), p = 0,019), sendo o R2=0,471. O ID foi correlacionado com o volume do hipocampo direito (IC 95% = 0,000-0,006, p = 0,05) e esquerdo (IC 95% = -0,006-0,000, p = 0,05), sendo o R2=0,457. O TH foi correlacionado com a pontuação do MOCA (IC 95% = (-0,574) - (- 0,030), p = 0,031), pontuação do BAI (IC 95% = 0,020-0,271, p = 0,024), pontuação do BDI (95% CI = 0,004-0,013, p = 0,001) e volume da amígdala esquerda (IC 95% = (-0,020) -0,000), p = 0,046), sendo o R2=0,492. A análise olfativa forneceu informações sobre o SNC, envolvimentos clínicos específicos sendo útil para monitorar a evolução da doença e o TH seria um marcador precoce do comprometimento olfatório na SSc(AU)

Abstract: The olfactory system mediated an interaction among the central nervous system (CNS), mood, cognition and inflammatory changes. Olfactory abnormalities are frequent in autoimmune diseases, but in patients with systemic sclerosis (SSc) it is little explored. SSc is a complex disease characterized by chronic inflammation, vasculopathy and fibrosis of the skin and internal organs. Thirty-five consecutives patients with SSc following for one years and 40 age and sex matched controls were assessed olfactory function using the three stages of the Sniffin 'Sticks test (SST), which include odor threshold (TH), odor discrimination (DIS), and odor identification (ID). The TDI is the sum of them, and it was calculate separately for gender and age groups. All these scores were analysed according with Beck's Depression (BDI) and Beck's Anxiety Inventories (BAI), cognitive capacity by the Montreal Cognitive Assessment (MOCA), activity and severity disease and the hippocampus and amygdala volumes with magnetic resonance (RM). All analyzes were performed twice, first at the study entry and second one years later. At all SST stages we observed that the olfactory capacity worsen with age (p?0.05). Olfactory abnormalities was significantly associated with age, disease duration, disease activity, and hippocampal and amygdala volumes. The linear regression showed that the scores obtained from the of the second analysis were correlate with the variables of the first analysis. The BAI score (95%CI=0.042-0.430, p=0.019), right hippocampus volume (95%CI= 0.007-0.025, p=0.001), left hippocampus volume (95%CI= (-0.021)-(-0.004), p=0.006) and right amygdala volume (95%CI= -0.030-0.000, p=0.050), R2=0.588. The DIS score was correlated with right hippocampus (95%CI= 0.004-0.013, p=0.001) and left hippocampus volume (95%CI= (-0.009)-(-0.001), p=0.019), R2=0.471. The ID score was correlated with right hippocampi (95%CI= 0.000-0.006, p=0.05) and left hippocampus volume (95%CI= -0.006-0.000, p=0.05), R2=0.457. The TH score was correlated with MOCA score (95%CI= (-0.574)-(-0.030), p=0.031), BAI score (95%CI= 0.020-0.271, p=0.024), BDI score (95%CI= 0.004-0.013, p=0.001) and left amygdala volume (95%CI= (-0.020)-0.000), p=0.046), R2=0.492. The olfactory analyses can provide information about CNS, specific clinical involvement being useful for monitoring disease progression and TH would be an early marker of olfactory abnormality in SSc(AU)