Objective:This study aimed to investigate the effect of differentiation osteogenic bone marrow mesenchymal stem cells (De-BMSCs) transplantation on the promotion of bone formation at the tendon-bone interface after anterior cruciate ligament reconstruction (ACLR), and further explored the molecular mechanism of the enhanced osteogenic effect of De-BMSCs.Methods:BMSCs from femur and tibia of New Zealand White rabbit were subjected to osteogenic induction and then cultured in no osteogenic factor medium; the obtained cell population was termed De-BMSCs. De-BMSCs were induced into osteo-, chondro-and adipo-differentiation in vitro to examine the characteristics of primitive stem cells. ACLR model with a semitendinosus tendon were performed in 48 adult rabbits, three groups were established: control group with alginate gel injectionat the tendon-bone interface, BMSCs group with the injection of alginate gel containing BMSCs, De-BMSCs group with the injection of alginate gel containing De-BMSCs. At 4 and 12 weeks after surgery, rabbits in each group were sacrificed to evaluate tendon-bone healing by histologic staining, micro-CT examination, and biomechanical test. During osteogenic differentiation of De-BMSCs, si-RNA of nuclear factor of activated T cells 2 (NFATc2) si-RNA of nuclear factor of activated T cells 1 (NFATc1) were used to verify the molecular mechanism of enhanced osteogenic effect of De-BMSCs.Results:De-BMSCs exhibited some properties similar to BMSCs including multiple differentiation potential and cell surface marker. At 4 weeks after surgery, the BV/TV value of the De-BMSCs group 0.36±0.01 was significantly higher than that of the control group 0.24±0.03 and BMSCs group 0.30±0.02 (all P<0.05), and the maximum load 40.34±1.19 N and stiffness 20.67±2.14 N/mm were significantly higher than those in the control group 14.88±2.74N, 8.67±2.19 N/mm and the BMSCs group 26.31±1.76 N, 13.81±2.14 N/mm (all P<0.05). At 12 weeks after surgery, the BV/TV value of the De-BMSCs transplantation group 0.47±0.02 was significantly higher than that of the control group 0.30±0.02 and the BMSCs group 0.35±0.03 (all P<0.05), and the maximum load 64.46±6.69 N and stiffness 25.18±3.11 N/mm were significantly higher than those in the control group 41.01±6.12 N, 11.59±2.54 N/mm and the BMSCs group 48.21±4.12 N, 15.89±2.94 N/mm (all P<0.05). During the osteogenic differentiation of De-BMSCs, the expressions of Nanog and NFATc1 were synergistically increased which promoted interaction of NFATc1 and Osterix ( P< 0.05), resulting in the increased expression of osteoblast marker genessuch as COL1A, OCN, OPN (all P< 0.05). Conclusion:De-BMSCs transplantation could promote bone formation at the tendon-bone interface after ACLR,Nanog/NFATc1/Osterix signaling pathway mediated the enhancement of the osteogenic differentiation effect of De-BMSCs.