Preparation of NET samples and decellularized book-shaped enthesis scaffolds
The experiment design is illustrated as in Fig. 1. In total, 32 female New Zealand rabbits weighing 3.1 ± 0.3 kg (supplied by the Experimental Animal Center of Central South University, Changsha, China) were euthanized (intraperitoneal injection of sodium pentobarbital, 100 mg/kg) in this study and NET harvested from the supraspinatus were sectioned into cuboid (about 30 mm × 1.2 mm × 10 mm) for decalcified and then vertically sliced at the boundary between fibrocartilage and tendon. The specimens were sectioned into book shape from the tendinous end to bony end along myotility direction with layer thicknesses of 250 μm. Layers 250 μm in thickness were selected for preparing book-shaped scaffold and the DEM was conducted by Su M et al’ protocol.
Histology and scanning electron microscopy (SEM) analysis
Hematoxylin and eosin (H&E), toluidine blue fast green and 4’,6-diamidino-2-Phenyl
indole (DAPI) for histological observation was used to evaluate decellularized efficacy (N=3). The decellularized book-shaped enthesis scaffolds from rabbit rotator cuff were embedded in paraffin and sectioned into 7 μm slices, then staining with HE, toluidine blue fast green and DAPI for observing the retention of nuclear materials. The samples (N=3) used for SEM scanning were fixed with 2.5% glutaraldehyde for 24 h and then washed with PBS, dehydrated by gradient ethanol, and soaked with isoamyl acetate. The microstructure of the decellularized book-shaped enthesis scaffolds surface was observed and the decellularized components was detected.
In this study, we innovatively applied the synchrotron radiation-Fourier transform infrared spectroscopy (SR-FTIR) to comparatively evaluate the preservation of collagen and PGs in the NET and DEM (N=3). The result of decellularization on the extracellular matrix components were evaluated using synchrotron radiation-Fourier transform infrared spectroscopy (SR-FTIR) at the BL01B beamline of National Facility for Protein Science Shanghai and Shanghai Synchrotron Radiation Facility, where synchrotron radiation from a bending magnet was collected, collimated and transported to a commercial FTIR interferometer bench. The peak area of amide I (1720-1590 cm-1) and carbohydrate (1140-985 cm-1) in the infrared spectrum were respectively calculated to characterize the distribution and content of collagen and PGs of the NET or DEM. The specifical procedures are as previously described according to the published literature .
DNA content analysis and mechanical tests
The quantifications of DNA in the decellularized book-shaped enthesis scaffolds were performed using DNeasy Blood & Tissue protocol according to the manufacturer’s instructions . Specifically, the decellularized book-shaped enthesis scaffolds (N=3) were weighed and minced after freeze-dried for 24 h using a lyophilizer (SIM International Group, USA). Then the decellularized bone-tendon scaffold (10 mg) was digested with proteinase K at 56℃ for 3 h. Finally, the DNA content in the decellularized book-shaped enthesis scaffolds was quantified by DNeasy Blood&Tissue Kit (Qiagen, USA) together with PicoGreen DNA assay kit (Invitrogen, USA).
The mechanical properties of the NET and DEM were comparatively evaluated with mechanical testing system (preloaded: 1 N, tension rate: 20 mm min−1, 23 MTS Insight, MTS, USA) (N=3). The mechanical index should be included as follows: failure load and stiffness.
Attachment and Viability Assay
After washing for three times with PBS (3 × 30 min), the decellularized book-shaped enthesis scaffolds were sterilized and immersed in complete medium overnight, then 104 BMSCs were respectively seeded onto the decellularized book-shaped enthesis scaffolds. To evaluate the cytotoxicity of the decellularized book-shaped enthesis scaffolds on BMSCs, cell viability was evaluated with a Live/Dead Assay kit (Invitrogen) at day 3 after seeding, Live/Dead assay showed that the green- and red-stained cells were captured by fluorescence with excitation wavelength of 488/594 nm to quantify cell viability (N=3).
All statistical analyses were performed using SPSS software (Version 23.0, Chicago, USA). Data was expressed as mean ± standard deviation. Statistical significance of the experimental variables was then evaluated using Student’s t-test (P < 0.05 was considered statistically significant).