In cardiac surgical interventions, the persistent shortage of human donor tissues highlights the urgent need for alternative therapeutic approaches. Advances in repairing, reconstructing, or regenerating damaged cardiac tissues could significantly expand treatment options, particularly when donor organs are scarce [1]. Biomaterials have emerged as promising tools in cardiovascular medicine, primarily due to their excellent biocompatibility. Among these, bovine pericardium (BP) has gained prominence, particularly for use in bioprosthetic heart valves (BHV) and transcatheter aortic valves, making it suitable for various cardiac and vascular repair procedures [2,3]. Unlike the commonly used porcine valves, BP exhibits superior hemodynamic characteristics, including lower pressure gradients and larger orifice areas, making it clinically preferable. Despite its widespread clinical use, commercial BP faces challenges such as degenerative processes, including stiffening and calcification. These issues are especially pronounced in pediatric and young adult patients, likely due to higher metabolic rates and more active immune responses [4]. Therefore, understanding the mechanisms of BP calcification in these patients is critical, as it directly impacts the durability and effectiveness of BHV in clinical settings.

The immune-inflammatory reaction plays a key role in BHV calcification. Studies have shown that implanted calcified BHV tissue exhibits significant immune cell infiltration and elevated cytokine concentrations [5,6]. Some researchers suggest that the mechanisms and risk factors underlying BP calcification may share similarities with atherosclerosis and native valve calcification [7,8]. These findings have sparked interest in potential therapeutic strategies, including pharmacological or dietary interventions, to mitigate BP calcification in BHV. Cellular pyroptosis, a programmed form of inflammatory cell death primarily mediated by caspase-1, is closely associated with calcification. Pyroptosis contributes to the development of atherosclerosis, and certain inhibitors or medications have been shown to slow disease progression by suppressing pyroptosis [9]. While BP calcification may share mechanistic similarities with vascular calcification and atherosclerosis, it remains unclear whether immune cell pyroptosis is directly involved in BP calcification and its progression.

Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, is closely linked to dietary intake, primarily originating from choline-rich foods such as red meat, fish, poultry, and eggs [10]. Previous studies have demonstrated its strong association with atherosclerosis and aortic valve calcification [11,12]. Recent findings suggest that TMAO promotes atrial structural remodeling by enhancing M1 macrophage infiltration and inducing cardiac pyroptosis [13]. Additionally, it has been shown to induce endothelial cell pyroptosis, accelerating atherosclerosis in mice [14]. However, the potential link between TMAO and BP calcification remains uncertain. Here, we test the hypothesis that TMAO promotes BP calcification by activating immune-inflammatory responses in both in vivo and in vitro models, using BP as the material in BHV.