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International Journal of Bioprinting 3D scaffold prevents tendon ossification

2.4. Mechanical testing of tissue-engineered 2.8. Transwell migration assay
Achilles tendon scaffolds To evaluate the migratory capacity of TSPCs in SF and SF–
Scaffolds were centrally positioned on a testing fixture and HPC scaffolds, a Transwell migration assay was conducted.
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subjected to uniaxial tensile loading at a constant rate of SF and SF–HPC scaffolds containing 1×10 cells were placed
1.0 mm/min using a universal mechanical testing machine in the upper chamber of Transwell inserts (Corning, USA)
(Instron, USA). Pressure was applied until scaffold rupture with serum-free medium. The lower chamber was filled
occurred, with mechanical parameters (stress, strain) with 500 μL complete medium containing 10% fetal bovine
automatically recorded. Elastic modulus was calculated serum. After 36 h incubation, scaffolds were removed, and
from the linear elastic region of the stress-strain curves. non-migrated cells on the upper membrane surface were
Additionally, cyclic tensile testing was performed on both carefully wiped off. Migrated cells on the lower membrane
SF and SF–HPC scaffolds. Data visualization and analysis were fixed with 4% paraformaldehyde and stained with
were performed using GraphPad Prism 9.0 (GraphPad, 0.5% crystal violet. Three random fields per insert were
USA) and Origin Pro 2023 software (Origin Lab, USA). imaged under an inverted microscope (Nikon, Japan), and
migrated cells were quantified using ImageJ with the Cell
2.5. Degradation assessment of scaffolds Counter plugin.
For in vitro degradation, scaffolds were incubated in PBS
containing 0.1 U/mL proteinase XIV or enzyme-free 2.9. Scratch wound healing assay
PBS at 37°C. Specimens were retrieved on days 0, 2, 4, TSPCs were harvested from SF and SF–HPC scaffolds and
8, and 16, followed by gravimetric analysis to calculate seeded into six-well plates until 80–90% confluency. A
degradation rates. For in vivo degradation, scaffolds were uniform scratch wound was created using a sterile 200 μL
subcutaneously implanted into nude mice (BALB/c-nu; pipette tip. Wound closure was monitored at 0, 12, 24, and
male’ 6 weeks old; obtained from Weitong Lihua Limited 36 h under phase-contrast microscopy (Leica, Germany).
Company (China); n = 24) under sodium pentobarbital Wound width was measured at three predefined positions
anesthesia (50 mg/kg, intramuscular). Animals were per well using ImageJ.
sacrificed at predetermined intervals (0, 2, 4, 8, and 16 2.10. Cell Counting Kit-8 proliferation assay
days) for scaffold retrieval and degradation rate calculation TSPCs (1×10 cells/well), harvested from scaffolds, were
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using the same gravimetric protocol. seeded into 96-well plates (Corning, US) in triplicate.
2.6. Scanning electron microscopy analysis of Cell proliferation was assessed at 12, 24, and 72 h using
scaffold microarchitecture a Cell Counting Kit-8 (CCK-8; Dojindo, Japan). After 4
Scaffolds were imaged using a scanning electron h of incubation with CCK-8 reagent at 37°C, absorbance
microscope (SEM; Hitachi, Japan). Prior to imaging, at 450 nm was measured using a microplate reader
scaffolds were pre-cooled at −20°C for 4 h, transferred to (Thermo, USA).
−80°C for 15 min, and lyophilized in a pre-chilled freeze 2.11. Quantitative reverse transcription polymerase
dryer for 12 h. Specimens were sectioned, mounted on chain reaction
SEM stubs, and sputter-coated with 10 nm gold/palladium TSPC-laden scaffolds were cultured for 2 and 4 weeks. Total
(Quorum, UK) to enhance conductivity. Microstructural RNA was extracted using TRIzol reagent (Thermo, USA)
images were acquired at an accelerating voltage of 5 kV. following scaffold homogenization. After centrifugation
Porosity and pore size distribution were quantified from (10,000 rpm; 10 min; 4°C), RNA was reverse-transcribed
SEM images using ImageJ (NIH, USA). into cDNA using the SuperScript IV First-Strand Synthesis
System (Thermo, USA). Quantitative reverse transcription
2.7. Live/dead cell staining polymerase chain reaction (PCR) was performed on a
TSPC-seeded scaffolds were cultured for 72 h in Dulbecco’s QuantStudio 5 System (Thermo, USA) using PowerUp
modified eagle medium/nutrient mixture F12 medium SYBR Green Master Mix. Gene expression of tenogenic
supplemented with 10% fetal bovine serum. Cells were (scleraxis [Scx]), chondrogenic (SRY-box transcription
stained using a viability/cytotoxicity kit (Thermo, USA) factor 9 [Sox9]), and osteogenic (bone morphogenetic
containing 4 μM calcein-acetoxymethyl (live cells; green) protein 2 [Bmp2]) markers was normalized to
and 2 μM ethidium homodimer-1 (EthD-1; dead cells; red) glyceraldehyde 3-phosphate dehydrogenase.
for 30 min at 37°C. Fluorescent images were captured with
a confocal microscope (Leica, Germany) at 488 nm/530 2.12. Achilles tendon transection repair and scaffold
nm (calcein) and 552 nm/617 nm (EthD-1) excitation/ implantation in rats
emission. Cell viability was calculated as (live cells/total Male Sprague–Dawley rats (aged 4–6 weeks; obtained
cells) × 100 using ImageJ with the Cell Counter plugin. from Weitong Lihua Limited Company (China); n = 36)

Volume 11 Issue 4 (2025) 300 doi: 10.36922/IJB025210203