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Surgical repairs of rotator cuff tears have high re-tear rates and many scaffolds have been developed to augment the repair. Understanding the interaction between patients’ cells and scaffolds is important for improving scaffold performance and tendon healing. In this in vitro study, we investigated the response of patient-derived tenocytes to eight different scaffolds. Tested scaffolds included X-Repair, Poly-Tape, LARS Ligament, BioFiber (synthetic scaffolds), BioFiber-CM (biosynthetic scaffold), GraftJacket, Permacol, and Conexa (biological scaffolds). Cell attachment, proliferation, gene expression, and morphology were assessed. After one day, more cells attached to synthetic scaffolds with dense, fine and aligned fibres (X-Repair and Poly-Tape). Despite low initial cell attachment, the human dermal scaffold (GraftJacket) promoted the greatest proliferation of cells over 13 days. Expression of collagen types I and III were upregulated in cells grown on non-cross-linked porcine dermis (Conexa). Interestingly, the ratio of collagen I to collagen III mRNA was lower on all dermal scaffolds compared to synthetic and biosynthetic scaffolds. These findings demonstrate significant differences in the response of patient-derived tendon cells to scaffolds that are routinely used for rotator cuff surgery. Synthetic scaffolds promoted increased cell adhesion and a tendon-like cellular phenotype, while biological scaffolds promoted cell proliferation and expression of collagen genes. However, no single scaffold was superior. Our results may help understand the way that patients’ cells interact with scaffolds and guide the development of new scaffolds in the future.