Expanded hMSCs (2.0 × 106 cells per construct; passage 4) were thoroughly mixed with BMP-2–loaded MCM (1.6 μg/mg; 0.4 mg per construct) and TGF-β1–loaded GM (0.4 μg/mg; 1.5 mg per construct) in chemically defined medium [DMEM-HG (Sigma-Aldrich), 1% ITS+ Premix (Corning), 1 mM sodium pyruvate (HyClone), 100 μM nonessential amino acids (Lonza), 100 nM dexamethasone (MP Biomedicals, Solon, OH), 0.13 mM l-ascorbic acid-2-phosphate (Wako USA), and 1% P/S (Thermo Fisher Scientific)] (27, 39). Five hundred microliters of the suspension was seeded onto the prewetted membrane of transwell inserts (3-μm pore size and 12 mm in diameter; Corning) and allowed to self-assemble into hMSC sheets for 2 days. After 24 hours, medium in the lower compartment was replaced. Control constructs containing either unloaded MCM and/or GM were prepared and cultured in a similar fashion. After 48 hours, three microparticle-incorporated hMSC sheets per group were combined into a sterile perforated PCL mesh tube to form the mesenchymal condensations for implantation.

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