Background:

Stem cells are an effective method of biologic healing and can be used to enhance the natural enthesis of the tendon-to-bone junction in rotator cuff repair. The purpose of this study was to investigate if the application of engineered stem cell sheets using adipose-derived cells (ADSCs) was effective in regeneration of natural enthesis and if there was a difference in the result of repair depending on the applied location Methods A chronic rotator cuff tear model was induced for 2 weeks, and cell sheets made using ADSCs isolated from rats were transplanted into the tendon-to-bone junction during surgical repair. Depending on the transplant location of the cell sheet, the difference in rotator cuff healing level between the overlaid group and the interposition group was compared to the surgical repair only group. The samples were obtained based on the tendon-to-bone junction and analysis of gross morphology, histology staining, and biomechanical analysis were performed. Results The differentiation potentials of ADSCs as stem cells were confirmed, as was the potential for tenogenic differentiation by growth factors. ADSCs were prepared as a sheet form to maintain the shape at the target site and to be easily attached. GFP-expressing ADSCs were proliferated in vivo and observed at the transplantation site. The overall healing level was better in the cell sheet transplanted group than in the control group that surgical repair only. Additionally, differences in healing level were shown depending on the cell sheet location by morphological, histological, and biomechanical perspectives. Histological results showed that the interposition transplantation group (1.75 ± 0.43, P  = 0.004) showed better fibrocartilage formation and collagen orientation at the junction than the overlaid transplantation group (0.86 ± 0.83). Conclusion In the chronic rotator cuff repair model, the engineered stem cell sheets enhanced the regeneration of the tendon-to-bone junction. This regeneration was more effective when the stem cell sheet was interpositioned at the tendon-to-bone interface. Trial registration: Not applicable