Injuries of tendons and ligaments give rise to significant morbidity. Tissue engineering offers promising treatment concepts such as seeding a scaffold with human bone marrow stem cells (hBMSCs) to create high-quality tendon replacement tissue. HBMSCs are usually isolated and cultured prior to seeding. Studies evaluating if previous isolation is superior to seeding with bone marrow aspirates have not been published yet. The aim of this study was to compare these two seeding methods in terms of cell viability, proliferation and differentiation. HBMSCs were harvested from the iliac crest during routine trauma surgery. In group A the scaffold (human achilles tendons) was seeded with bone marrow aspirates, while in group B hBMSCs were isolated, harvested and then seeded. Constructs were stimulated in perfusion bioreactors according to established protocols. Mean cell proliferation was significantly increased (p< 0.05) on tendons seeded with bone marrow aspirates. Cell viability, the concentration of alkaline phosphatase in the perfused media and the synthesis of procollagen - III - polypeptide (PIIIP) were not significantly different when comparing the two groups. The synthesis of procollagen-I-polypeptide (PIP) was significantly increased on tendons seeded with previously isolated hBMSCs p < 0.05). The results indicate that seeding a human tendon matrix scaffold with bone marrow aspirates may be equal to seeding with previously isolated hBMSCs. This new seeding method could facilitate and speed up the tissue engineering process.