The increasing prevalence of cardiovascular disease (CVD) has significantly heightened the clinical demand for vascular grafts [[1], [2], [3]]. Clinicians typically utilize autografts derived from the great saphenous vein, radial artery, and internal mammary artery, which are typically regarded as the gold standard choice for vascular replacement [4,5]. However, the availability of autologous blood vessels can be limited [6]. Therefore, artificial vascular graft transplantation has garnered increasing attention. Large-diameter vascular grafts have been employed in clinical applications [7], whereas the majority of small-diameter vascular grafts (SDVGs < 6 mm) have remained within the research and preclinical phases of development due to complications associated with their long-term implantation [8,9]. One such complication that serves as a critical determinant of the failure of long-term implanted SDVGs is calcification, characterized by the calcium hydroxyapatite deposits on the graft wall [10,11]. Calcification is a widespread and serious problem in cardiovascular implants, biomaterials, and grafts, determining the effect of long-term implantation and posing the biggest challenge to achieving clinical transformation [[12], [13], [14], [15]]. Therefore, it is necessary to develop SDVGs with anti-calcification properties.

Currently, efforts are primarily directed toward achieving rapid endothelialization and preventing intimal hyperplasia (IH) and thrombosis in SDVGs [16,17], with less emphasis on addressing calcification. Moreover, in studies of SDVGs, calcification is considered as one smaller aspect of vascular graft evaluation, and in-depth evaluation of SDVG calcification remains scarce in the literature. Vascular graft calcification is a complex process influenced by the material degradation rate, by-products of degradation, material physicochemical properties, host metabolism, and inflammatory responses [12,18,19]. In previous works, we fabricated a tri-layered vascular graft using native porcine thoracic aorta dECM powder and PLCL [20]. The study found that the developed vascular graft significantly promoted tissue regeneration but encountered calcification after implantation. The possible causes of calcification were speculated to be as follows: 1) the addition of dECM powder during the construction of the vascular graft, which was rich in collagen and elastin, provided favorable sites for the deposition of calcium ions [21]; and 2) inflammation was the probable trigger of calcification.

Porcine aortic elastin contains 18.4% polar amino acids (including glutamic acid, proline, hydroxyproline, and cystine, etc.), and their anionic groups (such as COO-, COOH, and OH), can attract calcium ions through electrostatic forces to act as nucleation sites [22,23]. Crosslinking of collagen and elastin with natural compounds is one effective way to reduce calcification. Procyanidins are natural polyphenolic crosslinking agents that interact with proteins in a specific and selective manner [22]. Procyanidins possess phenolic hydroxyl groups, which together with protein amide carbonyl groups form hydrogen bonds. These compounds are recognized for their exceptionally high affinity for proline-rich proteins, as the proline residue serves as an effective hydrogen bond acceptor [22,24]. Han et al. [24] reported that collagen materials treated with procyanidins showed no signs of calcification in rats. Additionally, Wang et al. [23] employed procyanidins to crosslink aortic elastin and found that it effectively inhibited elastin-initiated calcification.

Baicalin and its aglycone, baicalein, are the predominant glycosyloxyflavone ingredients found in Scutellaria baicalensis Georgi (SBG) [[25], [26], [27]], which possesses a range of pharmacological activities, such as antioxidant, anticancer, anti-inflammatory, and antithrombotic properties [[28], [29], [30], [31]]. In general, most protein-flavonoid complexes are stabilized by non-covalent interactions (hydrophobic interactions, hydrogen bonding, van der Waals forces, and electrostatic interactions) [32,33]. Baicalin contains electron-rich carbonyl, carboxyl groups, and hydroxyl groups, which may interact with chemical groups in dECM powder. This process may in turn block nucleation sites in elastin and collagen fiber, resulting in reduced calcification. Therefore, employing baicalin as a crosslinking agent with similar functionality to proanthocyanidins may also effectively reduce the deposition of calcium ions. In addition, scutellarin, was shown to inhibit osteoclastogenesis by suppressing MAPK and NF-κB signaling pathways [34]. Other research demonstrated that pretreatment of vascular smooth muscle cells (VSMCs) with baicalein significantly reduced calcium content in calcifying VSMCs, downregulated the expression of Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2) and increased typical VSMC markers smooth muscle protein 22α (SM22-α) and α-smooth muscle actin (α-SMA) to mitigate vascular calcification (VC) [35]. In pathological conditions such as vessel injury and atherosclerosis, VSMCs exhibit a synthetic phenotype, and its markers (e.g., SM22-α, α-SMA) are reduced [36,37]. VC is also a pathological condition that has been associated with phenotypic transformations in VSMCs.

The aggregation of macrophages in vascular grafts causes inflammation, which may eventually lead to calcification [18,38]. Cathepsin S (Cat S) is a potent cysteine protease, and it was found that in chronic kidney disease (CKD) and apolipoprotein-deficient mouse models, Cat S induced inflammation played a role in the calcification of atherosclerotic lesions [39,40]. It was suggested that Cat S accelerated calcification by inducing the transformation of mesenchymal cells into osteochondrogenic phenotypes [40]. Figueiredo et al. [41] found that the Cat S inhibitor slowed down the progress of atherosclerotic lesions by simultaneously reducing the immunoreactivity of Cat S, macrophage accumulation, elastin degradation, alkaline phosphatase activity, and plaque size [39], which suggests that Cat S inhibitors may represent a promising intervention to reduce calcification.

Therefore, this study intends to evaluate the feasibility of utilizing the anti-calcification properties of baicalin and Cat S inhibitor to improve dECM/PLCL SDVGs. Firstly, we designed dECM/PLCL SDVGs combined with baicalin and Cat S inhibitor. Then, we evaluated the scaffold of calcium deposition, osteogenic differentiation, and polarization of macrophages in vitro. Finally, we assessed the grafts in subcutaneous implantation and abdominal aortic replacement in rats. The data showed that vascular grafts combined with baicalin and Cat S inhibitor effectively reduced the osteogenic transition of VSMCs, and inflammation occurrence, and exhibited anti-calcification effects, providing a promising strategy for the design and construction of anti-calcification SDVGs (Scheme 1).