Thickness-Tunable Bilayer PBAT Nanofibrous Scaffolds for Enhancing r-AdMSCs’ Tenogenic Commitment in Supraspinatus Tendon Regeneration

Acute or chronic rotator cuff tears are major causes of shoulder dysfunction, motivating the development of scaffolds with tailored thickness and mechanics for supraspinatus tendon regeneration. This study aimed to investigate the effect of bilayer poly(butylene adipate-co-terephthalate) (PBAT) scaffold thickness on the tenogenic differentiation of rat adipose mesenchymal stem cells (r-AdMSCs) and supraspinatus tendon regeneration. Aligned fibers with a diameter of approximately 476 nm were deposited onto randomly oriented layers at different times (4 h; 4S, 6 h; 6S, 8 h; 8S), and scaffolds with increasing thicknesses from 441 µm (4S) to 1132 µm (8S) were produced. Mechanical testing showed comparable tensile strength for 4S and 6S (≈1.9–2.0 MPa) and modulus (5.5–7.3 MPa), while 8S exhibited markedly reduced stiffness (0.5 MPa) and hyper elastic deformation. Mechanical performance across degradation conditions remained strongly thickness-dependent: thinner scaffolds retained integrity and strengthened, with modulus increases during hydrolytic and enzymatic degradation, whereas thicker matrices showed limited remodeling and instability. Rat-AdMSCs’ were cultured on the scaffolds for 21 days. Cell-free and cell-laden mechanical responses further reflected thickness effects: cell-free samples stiffened due to media-induced passive matrix tightening, whereas cell-laden scaffolds showed extracellular matrix (ECM)-driven reinforcement, most prominently in 4S, which reached 2.1 MPa tensile strength with improved elasticity and balanced deformation. The 4S scaffold exhibited the highest tensile strength and significantly increased collagen-1 (col1), tenomodulin (tnmd) and scleraxis (scx) expression compared with the other groups. In conclusion, among all groups, 4S scaffolds demonstrated the most favorable mechanical and biological performance, suggesting that scaffold thickness plays a critical role in regulating tendon regeneration and will become even more suitable when matured in bioreactors.