To investigate the ability of muscle-derived stem cells (MDSCs) supplemented with growth and differentiation factor-5 (GDF-5) to improve tendon healing compared with bone marrow stromal cells (BMSCs) in an in vitro tendon culture model.Eighty canine flexor digitorum profundus tendons were assigned into 5 groups: repaired tendon (1) without gel patch interposition (no cell group), (2) with BMSC-seeded gel patch interposition (BMSC group), (3) with MDSC-seeded gel patch interposition (MDSC group), (4) with GDF-5–treated BMSC-seeded gel patch interposition (BMSC+GDF-5 group), and (5) with GDF-5–treated MDSC-seeded gel patch interposition (MDSC+GDF-5 group). After culturing for 2 or 4 weeks, the failure strength of the healing tendons was measured. The tendons were also evaluated histologically.The failure strength of the repaired tendon in the MDSC+GDF-5 group was significantly higher than that of the non-cell and BMSC groups. The stiffness of the repaired tendons in the MDSC+GDF-5 group was significantly higher than that of the non-cell group. Histologically, the implanted cells became incorporated into the original tendon in all 4 cell-seeded groups.Interposition of a multilayered GDF-5 and MDSC-seeded collagen gel patch at the repair site enhanced tendon healing compared with a similar patch using BMSC. However, this increase in vitro was relatively small. In the clinical setting, differences between MDSC and BMSC may not be substantially different, and it remains to be shown that such methods might enhance the results of an uncomplicated tendon repair clinically.Muscle-derived stem cell implantation and administration of GDF-5 may improve the outcome of tendon repair.