Table 1.
The sequences below were identified in NCBI’s Primer-BLAST.
Fig 1.
Schematic illustrations of the cell sheets for enhancing regeneration after rotator cuff tear repair.
(A) The concept of producing a cell sheet using adipose-derived stem cells isolated from rats. The sheets were harvested by changing the hydrophilicity of the dish surface and decreasing the temperature. (B) The process of creating a chronic rotator cuff tear model. The tendons were separated entirely and maintained for two weeks using a plastic drain in two places to block spontaneous healing. T: tendon, B: bone, Blue arrow: plastic drain.
Fig 2.
Study design for experiments using rats.
(A) The experiment was divided into three groups, with eight rats in each group. (B) The completion of surgical repair and stem cell sheet transplantation. Control group: a surgical repair-only, experimental group: Cell sheet transplantation on top of the repair site (overlay group) or between the tendon and bone (interposition group). The cell sheet is highlighted with a yellow circle in the illustration, and the suturing thread is shown as a blue triangle in the images.
Table 2.
Histological scoring system at the tendon-to-bone location.
Fig 3.
Characterization of rat adipose-derived stem cells (ASCs).
(A) The morphology of the cell sheet with plastic adhesion (left) and the shape of the shrunken cell sheet (right). The size bar represents 400 μm (magnification 10X). (B) The expression of MSC markers in rat ASCs (n = 3). (C) The adipogenic, osteogenic, and chondrogenic differentiation potential of rat ASCs (n = 3). The differentiation sites are shown in red or blue. All size bars represent 100 μm (magnification 40X). (D) The tenogenic differentiation potential of rat ASCs with 100 ng/ml GDF-7 treatment for two weeks. The side that has not treated the GDF-7 was used as a control. The fluorescence images of tendon markers in rat ASCs (upper). All size bars represent 100 μm (magnification 40X). The gene expression level of representative tendon markers in cells. Error bars represent standard deviation. *P < 0.05 (n = 3) (lower). (E) The tenogenic differentiation potential of rat ASC-derived cell sheet with GDF-7 treatment for two weeks. Green: differentiated cells, blue: nuclei acid. All size bars represent 100 μm (magnification 40X).
Fig 4.
The confirmation of GFP expression depending on the location of the transplanted cell sheets.
The cell sheets were transplanted above (left) or between (right) the tendon-to-bone junction. The GFP-tagging cells were used for the transplanted sheets, and it was confirmed two weeks after repair. GFP expression is located at the black box and shown as brown dots.
Fig 5.
Histological analysis according to the application of the cell sheets after repair.
(A) The different gross morphology depends on the location of the transplanted cell sheets and histological representation of the tendon-to-bone junction. T: tendon, J: junction, B: bone. The size bars represent 200 μm (magnification 20X). (B) The confirmation of the tendon marker expression for each group. The expression level of the markers was confirmed by green fluorescence. Blue dot is a nucleic acid. The size bars represent 200 μm (magnification 20X). All figures were selected as representative samples of each group.
Fig 6.
(A) The three groups were evaluated on four criteria. The above subscript indicates statistically significant relationships between groups. (B) Graph quantifying (left) the relative fibrocartilage formation and (right) percentages of the produced tidemark length relative to the fibrocartilage width at the tendon-to-bone junction. Error bars represent standard deviation. *P < 0.05, ***P < 0.001 (n = 4).
Fig 7.
Biomechanical analysis of the application of cell sheets.
(A) The image shows a tensile test device (left) and a mode of failure by tendon tearing at the enthesis (right). The 2 mm above from the junction was fixed, and a gradient tension of 1 mm/min was applied. (B) Three graphs indicate the maximal load, stiffness, and Young’s modulus of the tendon-to-bone interface. Error bars represent standard deviation. *P < 0.05, **P < 0.01 (n = 4).