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Massive rotator cuff tears (MRCTs) represent a major clinical concern, especially when degeneration and chronicity are involved, which highly compromise healing capacity.To study the effect of the secretome of mesenchymal stem cells (MSCs) on tendon cells (TCs) followed by the combination of these activated TCs with an electrospun keratin-based scaffold to develop a tissue engineering strategy to improve tendon-bone interface (TBi) healing in a chronic MRCT rat model.Controlled laboratory study.Human TCs (hTCs) cultured with the human MSCs (hMSCs) secretome (as conditioned media [CM]) were combined with keratin electrospun scaffolds and further implanted in a chronic MRCT rat model. Wistar-Han rats (N = 15) were randomly assigned to 1 of 3 groups: untreated lesion (MRCT group, n = 5), lesion treated with a scaffold only (scaffold-only group, n = 5), and lesion treated with a scaffold seeded with hTCs preconditioned with hMSCs-CM (STC_hMSC_CM group, n = 5). After sacrifice, 16 weeks after surgery, the rotator cuff TBi was harvested for histological analysis and biomechanical testing.The hMSCs secretome increased hTCs viability and density in vitro. In vivo, a significant improvement of the tendon maturing score was observed in the STC_hMSC_CM group (mean ± standard error of the mean, 15.6 ± 1.08) compared with the MRCT group (11.0 ± 1.38; P < .05). Biomechanical tests revealed a significant increase in the total elongation to rupture (STC_hMSC_CM, 11.99 ± 3.30 mm; scaffold-only, 9.89 ± 3.47 mm; MRCT, 5.86 ± 3.16 mm; P < .05) as well as a lower stiffness (STC_hMSC_CM, 6.25 ± 1.74 N/mm; scaffold-only, 6.72 ± 1.28 N/mm; MRCT, 11.54 ± 2.99 N/mm; P < .01).The results demonstrated that hMSCs-CM increased hTCs viability and density in vitro. Clear benefits also were observed when these primed cells were integrated into a tissue engineering strategy with an electrospun keratin scaffold, as evidenced by improved histological and biomechanical properties for the STC_hMSC_CM group compared with the MRCT group.This work supports further investigation into the use of MSC secretome for priming TCs toward a more differentiated phenotype, and it promotes the tissue engineering strategy as a promising modality to help improve treatment outcomes for chronic MRCTs.