In this experimental study, 24 male New Zealand white rabbits, weighing 3-4 kg, were sourced from Shanghai SLAC Laboratory Animal Co., Ltd. Besides, the rabbits were housed in a room maintained on a 12/12 h light/dark cycle and continuously supplied with food and water. Also, in our study, the use of animals was approved by the Animal Studies Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University.
Immersion/Agitation decellularization protocol
Briefly, the New Zealand white rabbits were anesthetized using intraperitoneal injection with 6% sodium pentobarbital (0.5 ml/kg). Thereafter, heparinization of the rabbits was performed by injecting heparin（3 mg/kg） through the marginal ear vein. The anterior neck muscles were opened to exposed, separated, and removed the envelope surrounding the thyroid gland. The thyroid gland was excised and placed in a phosphate buffer saline (PBS) glass container. Samples collected from the thyroid gland were placed in the Petri dish and mechanically agitated by the shaker before their complete immersion in 1% (v/v) SDS. Hence, decellularization in 1%SDS was administered for 72 h, at a frequency of 100 rpm following its replenished after every 4 hs. After the decellularization, thyroid gland scaffolds were washed in deionized water for another 24 h. Replacement of the deionized water was done every 4 hs. However, the thyroid gland scaffolds were stored at 40C in PBS with 1% (v/v) 100 U/ml penicillin and 100μg / ml streptomycin (P/S) for subsequent applications.
Histological and immunostaining analyses
Fresh native and decellularized thyroid gland (DTG) scaffolds were fixed in 4% paraformaldehyde and sliced to 5μm-thick sections. For tissue structure analysis, hematoxylin and eosin (H&E) were performed on deparaffinized sections, where images were captured by a light microscope.
Besides, the sections were operated according to to the manufacturer's procedures for immunostaining analysis. Here,the collected samples were incubated with primary antibodies against collagen I (1:100, Invitrogen), collagen IV (1:100, Invitrogen), laminin (LN) (1:100, Invitrogen) and fibronectin (FN) (1:100, Invitrogen) at 4oC overnight. Afterwards, the samples were incubated with secondary antibodies (DyLight 488- or 594- ) (1:200, Santa Cruz). Besides, the cell nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI) (1:10,000, Beyotime) after being rinsed with PBS. Consequently, immunostaining images were taken by a fluorescence microscope (Nikon, Japan).
Scanning electron microscopy (SEM)
Fresh native and DTG scaffolds were fixed in 10% formalin buffer at 4oC overnight after extensively washed in deionized water. The fixed tissues were dehydrated in graded ethanol solutions after rinsing. Thereafter, dehydrated tissues were soaked in liquid carbon dioxide for critical point drying, sliced into 1mm thickness sections, and analyzed by SEM (S3000-N, Hitachi).
The extraction of of total DNA was done using the TRIzol reagent (TaKaRa Inc., Kyoto, Japan) from fresh native and DTG scaffolds. The DNA was quantified using ultraviolet spectrophotometry (OnedropTM OD-1000, PerkinElme).
Mechanical properties were carried out using a mechanical analyzer (Zwick/Roell, BZ2.5/TN1S, Germany). The tissue specimens were subjected to uniaxial tension until failure. The fresh native and DTG scaffolds were cut into 5*5 mm2 pieces before rehydrated with PBS for 1 h. The specimens were mounted on the Instron, a preloading of 0.015 N was imposed and the specimens length were reported, then a preload of 0.003 N was set.. The elastic modulus and stiffness were calculated to evaluate the mechanical properties.
The enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, USA) was used to analyze cytokines in DTG scaffolds. The concentrations of various cytokines including vascular endothelial growth factor (VEGF), transforming growth factor-β(TGF-β), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), connective tissue growth factor (CTGF) and platelet-derived growth factor (PDGF) were assayed with a microplate reader at 450 nm.
Human thyroid follicular cells line (HTFCs) were purchased from the Chinese Academy of Sciences Kunming Cell Bank (Kunming, Yunnan, China). The HTFCs were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 1% (v/v) penicillin and streptomycin under cell incubation conditions of 37ºCwith 5% CO2 air atmosphere.
The Cytotoxicity of DTG scaffolds was conducted through the Cell Counting Kit-8 (CCK-8) assay. Herein, samples of decellularized were sliced into 1mm thickness sections before sterilized adequately with P/S. After placing the sections at the bottom of the 96-well plate, they were seeded with HTFCs (1x105 cells per well) for 24, 48, 72 h at 37ºC with 5% CO2 complete medium. Experimentally, HTFCs without exposure to any samples were used as control. Moreover, 6 wells per sample were prepared and the tests were triplicated.
Recellularization of decellularized scaffolds
Accordingly, the DTG scaffolds were sliced into sections of 0.5 mm thickness and 5 mm in diameter (3D scaffolds) following sterilizing with P/S before seeding. Thereafter, the sections were placed at the bottom of the 6-well plates, followed by seeding with HTFCs at a density of 1x107 cells per well. Then, the seeded scaffolds were cultured in a cell incubator (37ºC, 5% CO2) with a complete medium for 7 days. The proliferation of HTFCs on DTG scaffolds was examined by CCK-8. Besides, Immunostaining analysis was conducted to detect the thyroid peroxidase (TPO) expression of HTFCs in DTG scaffolds.
Data were represented as the mean ± standard deviation (SD). Student's t-test was adopted in two groups, whereas a one-way analysis of variance was used for the multi-groups (SPSS statistics software v.20, IBM). The level of significance was set at p = 0.05.