RESUMO
The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P < 0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P < 0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P < 0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P < 0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P < 0.05). The deposition of calcium was decreased on day 21 (P < 0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.
Assuntos
Condrócitos/efeitos dos fármacos , Meios de Cultura/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Fenolsulfonaftaleína/farmacologia , Agrecanas/antagonistas & inibidores , Agrecanas/genética , Agrecanas/metabolismo , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/antagonistas & inibidores , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/antagonistas & inibidores , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Meios de Cultura/química , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Proteoglicanas/biossíntese , Fatores de Transcrição SOX9/antagonistas & inibidores , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Adulto JovemRESUMO
PURPOSE: The purpose of the study was to investigate the effect of dermatan sulphate (DS) addition to biodegradable methoxy polyethylene glycol (MPEG) substituted polylactide-co-glycolic acid (PLGA) scaffolds for cartilage repair in vitro and in vivo. METHODS: Human chondrocytes from eight patients undergoing anterior cruciate ligament reconstruction were isolated and cultured in 5% oxygen on MPEG-PLGA scaffolds±DS for one, three, seven and 14 days. Analyses were performed using quantitative gene expression analysis for chondrogenic and cell attachment markers. An osteochondral drill hole defect was created in the intertrochlear groove of the distal femur in 20 New Zealand white rabbits (defects n=20). When bleeding was observed, the defects were treated with MPEG-PLGA scaffolds±DS. Twelve weeks after surgery the rabbits were sacrificed and the defects were analysed using histological grading with O'Driscoll scoring. RESULTS: DS addition to MPEG-PLGA scaffolds resulted in a significant upregulation of fibronectin gene expression on day 1. No differences were observed in chondrogenic gene expression. There were no differences between the two groups in histological grading (+DS 10.3 and -DS 9.6). CONCLUSIONS: Upregulation of fibronectin in vitro indicating early cell-scaffold interaction and attachment did not result in improved cartilage repair in an osteochondral defect model in rabbits.
