To induce tenogenic differentiation, encapsulated PDLSCs and GMSCs as well as hBMMSCs (2×106 cells in 1 mL of TGF-β3-loaded alginate microspheres) were cultured in a tenogenic medium containing DMEM with 15% FBS, 2 mM L-glutamine, 100 nM Dex, 100 3M ascorbic acid, 2 mM sodium pyruvate (R&D Systems Inc), 100 U/mL penicillin, and 100 μg/mL streptomycin [26 (link)]. Cell-free RGD-coupled alginate microspheres without TGF-β3 were used as the negative control.
Four weeks after induction, the samples were fixed with 4% PFA, and paraffin sections were made. Sections were immunolabeled using antibodies against Tenomodulin (Tnmd), Eya 1, Eya2 (from Santa Cruz Biotechnology, Inc, Dallas, TX), and Scleraxis (Abcam) at 4°C overnight, detected using Alexa fluor conjugated secondary antibody (1:200 dilution; Invitrogen), and counterstained with DAPI.
Four weeks after induction, the samples were fixed with 4% PFA, and paraffin sections were made. Sections were immunolabeled using antibodies against Tenomodulin (Tnmd), Eya 1, Eya2 (from Santa Cruz Biotechnology, Inc, Dallas, TX), and Scleraxis (Abcam) at 4°C overnight, detected using Alexa fluor conjugated secondary antibody (1:200 dilution; Invitrogen), and counterstained with DAPI.