MT1-MMP controls hMSC osteogenesis. ALP staining (A) or activity (B) after osteogenesis induction in hMSCs embedded within 3D gels of type I collagen in the presence of dimethyl sulfoxide (DMSO) control, TIMP-1 (7.5 μg/mL), or TIMP-2 (2.5 μg/mL) for 9 days. ALP staining (C) or activity (D) after osteogenesis induction in hMSCs electroporated with Scr siRNA or MT1 siRNA and cultured within 3D type I collagen gels (2.2 mg/mL) or pepsin-extracted type I collagen gels (Vitrogen; 2.2 mg/mL) in osteogenesis differentiation medium for 7 days. In 3D collagen gels, phalloidin-stained hMSCs (green) display a dendritic phenotype after MT1-MMP silencing. (E) Real-time PCR quantification analysis (left panel) of ALP, Runx2, and BMP2 mRNA levels in Scr siRNA or MT1 siRNA-treated hMSCs induced for osteogenesis in collagen gels for 7 days. RT-PCR confirms MT1-MMP mRNA silencing and verifies down-regulation of ALP and Runx2 mRNA levels without affecting BMP2 expression (GAPDH used as a reference; right panel). (F) ALP staining and activity of hMSCs cotransfected with MT1-MMP siRNA and either a control expression vector (MT1siRNA + Vec) or mouse MT1-MMP expression vector (MT1siRNA + mMT1) cultured in 3D collagen with osteogenesis differentiation medium for 7 days. Pictures shown are representative of 3 or more experiments performed. Bars, 1 mm and 100 μm, respectively, as shown in each figure. Relative enzyme quantification was expressed as ng/mL ± SD of 3 experiments. *P < .05.