The donor rats were euthanized via CO2 inhalation. The thoracic aorta was excised from the donor rats and divided into five groups according to the method of tissue preparation. For decalcification, 0.625% GA in phosphate-buffered saline (PBS; 2% glutaraldehyde fixative [Muto Pure Chemicals Co., Ltd, Tokyo, Japan] in 100 mL of PBS [pH 7.4; Gibco, NY, USA]) and 80% ethanol (400 mL of 99.5% ethanol [FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan] in 100 mL of distilled water) were used (7, 9). Group 1 contained untreated tissues as the control; group 2 contained tissues fixed with GA for 3 min; group 3 contained tissues fixed with GA for 3 min and post-treated with ethanol for 10 min; group 4 contained tissues fixed with GA for 3 min and post-treated with ethanol for 20 min; and group 5 contained tissues fixed with GA for 3 min and post-treated with ethanol for 30 min. The samples were fixed at room temperature and rinsed in normal saline to remove residual GA before implantation.
Rat model of subdermal embedding
Eleven 3-week-old male Wistar rats (Oriental Yeast Co., Ltd.) were used as recipients. The rats were anesthetized via the inhalation of isoflurane maintained at 2%–3%, after which the hairs were shaved and dorsal subdermal pouches were created in each rat. The treated tissue samples were implanted, and the wounds were closed with 5-0 polypropylene suture (Ethicon Inc., Somerville, NJ, USA). This subdermal implantation model has been proven to be a rapid and reliable tool for evaluating the effects of anticalcification treatment on tissues (10). Each rat was maintained and observed in its own cage after surgery. The rats were provided dry food and water ad libitum through an automatic watering system. The housing rooms of the rats were maintained at 24°C ± 1°C and a humidity between 50% and 60% under a 12-:12-h photoperiod. Ceftriaxone sodium hydrate (0.3 g) was subcutaneously injected into the rats post-operation. After 28 d, the rats were euthanized via CO2 inhalation, and the implanted donor tissues were harvested and rinsed with normal saline. All tissue samples were immediately stored in 10% buffered formalin. All rats survived the implantation and experimental period.
Histological processing of the explants
Formalin-fixed explants were embedded in paraffin, cut into 5-µm thick sections, and stained with hematoxylin and eosin (H&E) and Victoria blue (VB) for histological analysis. The slides were examined by light microscopy and evaluated blinded by pathologists for calcification, granulation, and host inflammatory response.
Tissue calcification and granulation analysis
The stained slides were scanned and digitally converted into virtual slides using a Hamamatsu NDP slide scanner (Hamamatsu NanoZoomer 2.0-HT; Hamamatsu Photonics, Hamamatsu, Japan). The virtual slides were viewed and analyzed using NDP.View2 (Hamamatsu Photonics). The calcification and granulation levels in each sample were semiquantitatively evaluated by annotating and measuring the visual area (Fig. 1).
Data were analyzed using EZR for Windows (Saitama Medical Center, Jichi Medical University, Saitama, Japan). Analysis of variance was used for multiple comparisons among different time points. For paired comparisons, the t-test and Kruskal–Wallis test were used to detect differences between the experimental groups. Statistical significance was set at P < 0.05. All data are expressed as mean ± standard deviation.