Evaluation of diastolic function is a pivotal challenge due to limitations of the conventional echocardiography, especially when the heart rate is rapid as in rats. Currently, by using color M-mode echocardiography (CMME), intraventricular pressure difference (IVPD) and intraventricular pressure gradient (IVPG) in early diastole can be generated and are available as echocardiographic indices. These indices are expected to be useful for the early diagnosis of heart failure (HF), especially diastolic dysfunction. There have not been any studies demonstrating changes in IVPD and IVPG in response to changes in loading conditions in rats. Therefore, the present study aims to evaluate CMME-derived IVPD and IVPG changes in rats under various loading conditions. Twenty rats were included, divided into two groups for two different experiments, and underwent jugular vein catheterization under inhalational anesthetics. Conventional echocardiography, CMME, and 2D speckle tracking echocardiography were measured at the baseline (BL), after intravenous infusion of milrinone (MIL, n = 10), and after the infusion of hydroxyethyl starch (HES, n = 10). Left ventricular IVPD and IVPG were calculated from color M-mode images and categorized into total, basal, mid-to-apical, mid, and apical parts, and the percentage of the corresponding part was calculated. In comparison to the BL, the ejection fraction, mid-to-apical IVPG, mid IVPG, and apical IVPD were significantly increased after MIL administration (p < 0.05); meanwhile, the end-diastolic volume, E-wave velocity, total IVPD, and basal IVPD were significantly increased with the administration of HES (p < 0.05). The increase in mid-to-apical IVPD, mid IVPD, and apical IVPD indicated increased relaxation. A significant increase in basal IVPD reflected volume overloading by HES. CMME-derived IVPD and IVPG are useful tools for the evaluation of various loading conditions in rats. The approach used in this study provides a model for continuous data acquisition in chronic cardiac disease models without drug testing.

Silk fibroin (SF) is a suitable material for vascular prostheses for small arteries. SF is useful not only as a base material for artificial vascular grafts but also as a coating material. This study prepared three types of transgenic SF (vascular endothelial growth factor (VEGF), Arg-Glu-Asp-Val (REDV), and Tyr-Ile-Gly-Ser-Arg (YIGSR)) incorporating expression factors that are thought to be effective for endothelialization as coating materials. We compared the contribution of these materials to early endothelialization in vivo when using them as a porous transgenic SF coating. A porous coating of transgenic SF containing VEGF, REDV, and YIGSR was applied to a silk small-diameter artificial vascular graft base with a diameter of 1.5 mm and a length of 3 cm. Two and four weeks after implantation of these artificial grafts into the abdominal aorta of rats, they were removed and evaluated by histologic examination. Transgenic SF coating incorporating VEGF and REDV demonstrated higher tissue infiltration and continuous endothelialization in the center of the graft compared to YIGSR at 4 weeks after implantation. VEGF and REDV are capable of endothelialization as early as 4 weeks after implantation, confirming the usefulness of transgenic SF when used as a porous coating.

Vascular replacement is one treatment for cardiovascular disease. However, in blood vessels with a diameter less than 6 mm, the existing artificial vascular grafts may be occluded by thrombus formation or intimal hyperplasia. Thus, new artificial vascular grafts need to be developed. We have developed a small-diameter artificial vascular graft made of silk fibroin. The implantation of such graft has been evaluated mainly in rats. However, only a few reports describe long-term implantation in large animal models. Therefore, modified silk fibroin artificial vascular grafts were implanted in the femoral arteries of dogs, and their patency and remodeling ability were investigated. Six beagles weighing 10 to 12 kg were used for the in vivo study. Grafts (4 cm length × 3.5 mm inner diameter) were implanted in the femoral artery of 6 dogs. The patency of the graft was monitored using vascular ultrasound apparatus. At 3 months', 5 months', and 1-year postimplantation, the graft was retrieved and conducted histopathologic examination. No side effects, such as ischemia, paralysis, and edema of the hind legs, were observed postimplantation. Five of the 6 grafts exhibited a high patency rate, and the lumen was covered with vascular endothelial cells in the central part of the graft 3 months’ postimplantation. Based on these results, artificial silk fibroin vascular grafts implanted in the femoral arteries of dogs exhibit high patency and remodeling ability. Silk fibroin grafts may be clinically applicable as an artificial vascular graft in small-diameter <6 mm.

In recent years, the demand for functional small-diameter (< 6 mm) artificial vascular grafts has greatly increased due to an increase in the number of patients with vascular heart disease. However, currently, there are no available commercial small-diameter grafts. The objective of this research was to develop a porous silk fibroin (SF)-coated poly(ethylene terephthalate) (PET) graft with a diameter < 6 mm. The graft was compared with a gelatin-coated PET graft because the latter PET graft with a diameter ~ 6 mm was widely used as a commercial vascular graft. Initially, porous SF was prepared using Glyc as the porogen [termed SF(Glyc)] and the PET grafts were prepared through the double-Raschel knitting method. Subsequently, the degradation of the SF coating was monitored using protease XIV in vitro and was compared with that observed in gelatin-coated PET grafts. Finally, these grafts were also implanted into rats for an in vivo comparison. In degradation experiments, after 7 days, the SF was clearly digested by protease XIV, but the gelatin on the graft was still remained at the outer surface. In implantation experiments in rats, the SF(Glyc)-coated PET graft was rapidly degraded in vivo and remodeling to self-tissues was promoted compared with the gelatin-coated PET graft. Thrombus formation and intimal hyperplasia were observed in the gelatin-coated PET graft; however, such side reactions were not observed in the SF(Glyc)-coated PET graft. Thus, the porous SF(Glyc)-coated PET graft with a small diameter < 6 mm may be useful as a commercial vascular graft.

Mesenchymal stem cells (MSCs) derived from adipose tissue are evolved into various cell-based regenerative approaches. Adipose-derived stem cells (ASCs) isolated from rats are commonly used in tissue engineering studies. Still, there is a gap in knowledge about how the harvest locations influence and guide cell differentiation. This study aims to investigate how the harvesting site affects stem-cell-specific surface markers expression, pluripotency, and differentiation potential of ASCs in female Sprague Dawley rats. ASCs were extracted from the adipose tissue of the peri-ovarian, peri-renal, and mesenteric depots and were compared in terms of cell morphology. MSCs phenotype was validated by cell surfaces markers using flow cytometry. Moreover, pluripotent gene expression of Oct4, Nanog, Sox2, Rex-1, and Tert was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). ASCs multipotency was evaluated by specific histological stains, and the results were confirmed by quantitative polymerase chain reaction (RT-qPCR) expression analysis of specific genes. There was a non-significant difference detected in the cell morphology and immunophenotype between different harvesting sites. ASCs from multiple locations were significantly varied in their capacity to differentiate into adipocytes, osteoblastic cells, and chondrocytes. To conclude, depot selection is a critical element that should be considered when using ASCs in tissue-specific cell-based regenerative therapies research.

Recently, Bombyx mori silk fibroin (SF) has been shown to be a suitable material for vascular prostheses for small arteries. In this study, we developed a softer SF graft by coating water-dispersed biodegradable polyurethane (PU) based on polycaprolactone and an SF composite sponge on the knitted SF vascular graft. Three kinds of ¹³C solid-state nuclear magnetic resonance (NMR), namely carbon-13 (¹³C) cross-polarization/magic angle spinning (MAS), ¹³C dipolar decoupled MAS, and ¹³C refocused insensitive nuclei enhanced by polarization transfer (r-INEPT) NMR, were used to characterize the PU-SF coating sponge. Especially the ¹³C r-INEPT NMR spectrum of water-dispersed biodegradable PU showed that both main components of the non-crystalline domain of PU and amorphous domain of SF were highly mobile in the hydrated state. Then, the small-diameter SF artificial vascular grafts coated with this sponge were evaluated through implantation experiments with rats. The implanted PU-SF-coated SF grafts showed a high patency rate. It was confirmed that the inside of the SF grafts was covered with vascular endothelial cells 4 weeks after implantation. These results showed that the water-dispersed biodegradable PU-SF-coated SF graft created in this study could be a strong candidate for small-diameter artificial vascular graft.