The cell metabolism was examined using an alamarBlue assay (Thermo Fisher Scientific, USA), as reported previously (43). Briefly, human tenocytes (10,000 cells in 200 μl) were seeded onto the as-fabricated tendon scaffold (sample size, 1 cm in length and 4 mm in diameter) in low-adhesion 24-well plates (BD Biosciences, USA). Cells were incubated for 6 hours to allow initial attachment and cultured with the addition of another 800 μl of growth medium for proliferation. For the analysis of cell metabolic levels, the culture medium was replaced by 10% alamarBlue medium (1 ml per well) and incubated for 4 hours. The fluorescence of supernatant medium was read at 560/590 nm (excitation/emission wavelength) using a Microplate Reader (Synergy H1, BioTek, Singapore) at 1, 3, 7, and 14 days of culturing. The metabolic levels of cells cultured on the disassembled shell (10,000 cells cm−1) and core (10,000 cells cm−1) portions of the tendon scaffold, raw PCL film (as the control of scaffold shell portion; 10,000 cells cm−1) and fiber mesh (as the control of scaffold core portion; 10,000 cells cm−1), and tissue culture plate (10,000 cells cm−1; Thermo Fisher Scientific, USA) were also investigated. Six distinct replicates were tested for each group at all time points.

The viability of human tenocytes seeded in the as-fabricated tendon scaffold was examined using a confocal laser microscopy (FV1000, Olympus, Japan) (44). Briefly, after 14 days of culture, cells (seeded with 10,000 cells per sample) were incubated with fluorescein diacetate (FDA; 5 μg μl−1 in PBS, 10 min) and, subsequently, with propidium iodide (PI; 4 μg μl−1 in PBS, 5 min) at room temperature. After washing thrice with PBS, the scaffold samples were disassembled into separated shell and core portions for imaging immediately. The live and dead cells were identified as FDA-labeled green and PI-labeled red colors, respectively.

To characterize the organization and morphology of live cells, human tenocytes (10,000 cells cm−1; sample size, 1 × 1 cm2) were seeded on the disassembled shell and core portions of the tendon scaffold and cultured for predetermined periods (1, 3, 7, and 14 days). Cells were fluorescence-labeled with FDA (5 μg μl−1 in PBS, 10 min; Sigma-Aldrich), and after washing thrice with PBS, they were imaged immediately using a confocal microscopy. The images of subconfluent cells were analyzed using the built-in function of ImageJ software (NIH, USA). All cells in contact with other cells and image edges were manually removed from the datasets toward single-cell analysis. Cellular angle was determined as the direction of the major elliptic axis of individual cell, as reported previously (44). A preferential cell orientation was determined and set as 0°, and cell angles were then normalized to the preferential cell orientation. The number of cells within each degree from −90° to +89° was calculated and normalized such that the total sum was unity. An isotropic sample would be expected to have an even distribution of cell angles in each degree (the percentage of cell angles = 1 × 180−1 × 100%). Cells with angles to fall in ±15° were considered to be aligned. Cell elongation was determined by a CSI (circularity = 4 × π × area × perimeter−2), with a value of 1 representing the circle. Cells cultured on the raw PCL film (as the control of shell portion; 10,000 cells cm−1) and fiber mesh (as the control of core portion; 10,000 cells cm−1) were also studied. Images were taken at three to four random regions of each sample for data analysis. Three distinct replicates were tested for each group at all time points.

To analyze cell cytoskeleton and nucleus, human tenocytes cultured at the predetermined periods were fixed with paraformaldehyde [3.7 weight % (wt %) in PBS, 15 min; Sigma-Aldrich], permeabilized using Triton X-100 (0.1 wt % in PBS, 5 min; Sigma-Aldrich), and blocked with bovine serum albumin (2 wt % in PBS, 30 min; Sigma-Aldrich). Cells were then incubated with tetramethyl rhodamine isothiocyanate–conjugated phalloidin (1:200 dilution in PBS; Sigma-Aldrich) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000 dilution in PBS; Sigma-Aldrich) for the labeling of F-actin filaments and nucleus DNA, respectively. Confocal images were taken and analyzed using the built-in function of ImageJ software (NIH, USA). Cell nuclei with angles falling in the range of ±15° were considered to be aligned. Nucleus elongation was described using an NSI, with a value of 1 representing the circle. Images were taken at three to four random regions of each sample for data analysis. Three distinct replicates were tested for the different groups at each time point.

The major matrix protein (COL-I) and regulating proteins (TNC and DCN) of human tendon were examined using immunofluorescence. Cells were seeded (10,000 cells cm−1) on the disassembled shell and core portions (1 × 1 cm2) of the tendon scaffold. The raw PCL film and fiber mesh (1 × 1 cm2) were set as the controls of the shell and core portions, respectively. After 7 days of culturing, the cells were treated for fixation, permeabilization, and blocking purposes. Cells were then incubated with the mouse-derived antihuman primary monoclonal antibodies [SC-59772 (COL-I), SC-25328 (TNC), and SC-73896 (DCN), all from Axil Scientific; table S2] as testing groups and related normal mouse IgG isotypes as negative controls (SC-3877, Axil Scientific; table S2) at room temperature for 60 min. The cells were then washed thrice with PBS and incubated with the fluorescence-labeled second goat anti-mouse IgG (H + L) antibody (A-11005, Thermo Fisher Scientific; table S2) at room temperature for 60 min. After washing thrice with PBS, the cells were further incubated with DAPI for nucleus visualization. The cells were finally washed thrice with PBS and imaged using a confocal microscopy with the identical parameters for all the groups of each testing marker.

The major protein of human tendon matrix was determined quantitatively on the basis of the measurement of COL-I C-terminal propeptide (CICP) using the MicroVue EIA Kit (mouse-derived antihuman antibodies: MK101, Quidel). Briefly, tenocytes (10,000 cells cm−1) were cultured on the disassembled shell and core portions (1 × 1 cm2) of the tendon scaffolds for 3, 7, and 14 days. The raw PCL film and fiber mesh were set as the control. For the culturing period of days 0 to 7, the cell medium was changed at days 3 and 7 and kept at a total volume of 1 ml. For the culturing period of days 7 to 14, the addition of 1 ml of new medium was performed at day 10 with a final volume of 2 ml. To measure CICP expression, the collected cultured medium was diluted with an assay buffer (1:12, v/v) and added (100 μl per well) to 96-well stripwells (murine monoclonal anti-CICP antibody coating) for 2 hours of incubation at room temperature. After washing thrice with a wash buffer, the stripwells were added with polyclonal rabbit anti-CICP antibody (100 μl per well) and incubated at room temperature for 45 min. The stripwells, after washing thrice, were then incubated with goat anti-rabbit IgG antibody conjugated to alkaline phosphatase (100 μl per well; room temperature) for 45 min. Last, after washing thrice, the stripwells were incubated with substrate solution containing p-nitrophenyl phosphate (100 μl well−1; 30 min), followed by the addition of stop solution (50 μl per well). A series of known concentrations (CICP: 0 to 74.3 ng ml−1) were used for the preparation of a standard curve. Immunofluorescence detection was performed at 405 nm using the microplate reader, and the concentration of CICP in culture medium was obtained according to the predetermined standard curve. Cumulative CICP production (CCP) was calculated using an equation as followsEmbedded Image(1)where Ci and Vi were the measured CICP concentration and the volume of culture medium after i days of culturing (i = 3, 7, and 14). The production of COL-I on testing samples was expressed by dividing CCP by sample area, while the level of cells to produce COL-I was expressed by dividing CCP by total cell number. Four distinct replicates were tested for each sample group at all time points.

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