Here we investigated 8 BHVs: BMVs (n = 4) and BTVs (n = 4) (KemCor or PeriCor devices, NeoCor, Kemerovo, Russia). KemCor and PeriCor are stented xenoaortic porcine BHVs designed for surgical implantation and manufactured from porcine aortic valves fixed with ethylene glycol diglycidyl ether instead of glutaraldehyde ( Figure 1 ) (16, 17). Both KemCor and PeriCor models have three leaflets. The primary difference between KemCor and PeriCor is that supporting polypropylene frame of KemCor is covered with synthetic fabric, in contrast to PeriCor which employs calf pericardium for better biocompatibility and higher resistance to infective endocarditis. Manufacturing of KemCor and PeriCor has been terminated in 2001 and 2009, respectively. However, these BHV models can be used for studying the pathophysiology of BHV failure to improve the design of next-generation BHVs.

During the primary heart valve replacement because of rheumatic heart disease, each of the 4 patients received identical BHVs (either KemCor or PeriCor) for the mitral and tricuspid positions, which were then pairwise excised during the repeated heart valve replacement because of BMV failure. The diagnosis [stage III SVD for BMVs and stage I for BTVs (8)] has been made after the echocardiographic examination. All BTVs retained their function by the time of the intervention, yet have also been replaced to prevent further development of SVD. Hence, BMVs and BTVs were implanted and excised concurrently in each patient, and the duration of functioning was identical for both valves. The same surgical incision was used to obtain an access to BMVs and BTVs for performing a multivalvular replacement. Specimen collection was approved by the Local Ethical Committee of the Research Institute for Complex Issues of Cardiovascular Diseases (ethical approval code 16/2024, approved on 16 August 2024), and a written informed consent was provided by all study participants after receiving a full explanation of the study. The investigation was carried out in accordance with the Good Clinical Practice and a latest revision of the Declaration of Helsinki (2013). Clinicopathological features of patients and BHVs are provided in Tables 1 , 2 , respectively.

Calcification of BHVs was evaluated by multislice computed tomography (MSCT, LightSpeed VCT 64, General Electric, Boston, MA, USA) using the following parameters: tube voltage 120 kV; tube current 160 mA; gantry rotation time 0.9 seconds; scanning time 6.8 seconds; table feed speed 39.37 mm per rotation. 3D reconstruction was conducted utilizing a slide thickness of 0.625 mm. Images were formed using a standard kernel.

Digital Imaging and Communications in Medicine (DICOM) files were analyzed employing a Pydicom package (Python 3.9, Python Software Foundation, Wilmington, DE, USA). All MSCT layers were united into a 3D array where XY plane contained the parameters of brightness for each pixel along the Z axis. The volume of each BHV and its calcium deposits were defined using a pixel brightness threshold (400 units for pericardium and 1700 units for calcium deposits). To quantify calcification volume, we reconstructed a 3D binary array using a calcification mask for each BHV. In such array, pixel brightness was quantified to 1 if calcification mask pixel was not 0. Next, the number of such pixels was multiplied by the volume of the image voxel to obtain the total volume of calcium deposits within the BHVs. The parameters of the image voxel were calculated utilizing the metadata of DICOM files. The verification of such values was performed in Materialize Mimics software (Materialize NV, Leuven, Belgium) with < 1% error. 2D images of calcium deposits were represented as an average of brightness amongst all layers of the 3D image.

For the histological analysis, we dissected the leaflet fragments in the central and commissural zones (from the base to the free margin) and in the fragments with pathological thickening, tears, or calcium deposits). Selected fragments were snap-frozen (Epredia Neg-50 Frozen Section Medium, 6502, Thermo Fisher Scientific, Waltham, MA, USA) and sectioned (6 µm thickness) using Microm HM525 cryostat (Thermo Fisher Scientific, Waltham, MA, USA). To conduct a general histological examination, sections were then stained with Gill’s hematoxylin (HK-G0-BL01, ErgoProduction, Saint Petersburg, Russia) for 10 minutes, rinsed in tap water for 5 minutes, immersed in distilled water, and incubated in alcoholic eosin Y solution (HK-ES-BL01, ErgoProduction, Saint Petersburg, Russia) for 1 minute. After the brief washing in distilled water and three changes in 95% ethanol (Kemerovo Pharmaceutical Plant, Kemerovo, Russia), sections were cleared with xylene (HP-XR-1L01, ErgoProduction, Saint Petersburg, Russia) for 3 minutes. Coverslips were mounted with Vitrogel (12-005, ErgoProduction, Saint Petersburg, Russia). Collagen content and lipid deposition were assessed by Russell-Movat’s pentachrome staining (ab245884, Abcam, Cambridge, UK) and Oil Red O staining (ab150678, Abcam, Cambridge, UK), respectively, according to the manufacturer’s protocols. For the Oil Red O staining, coverslips were mounted with 50% glycerol (8.06.00284, ChemExpress, Ufa, Russia). Whole slide images were acquired using (Vision Slide Assist, West Medica, Perm, Russia). Image post-processing and semi-quantitative image analysis (i.e., cell count and area measurements) were carried out in QuPath software v.0.5.1 (18). For the quantification of immune cell infiltration and lipid deposition, we selected 10 representative sections from each BHV (3 or 4 sections per each leaflet). Immune cell density was counted at both inflow and outflow sides (100 µm thickness) as well as in the depth of the leaflet. Lipid deposition was measured as a proportion of Oil Red O-positive area within the leaflet to the total leaflet area using Fiji software (National Institutes of Health, Bethesda, MD, USA) (19). As these BHVs were stored in 10% neutral phosphate-buffered formalin for ≥ 8 years, immunohistochemistry and Western blotting have not been applied in this study.

Ultrastructural analysis of excised BHVs was carried out by a backscattered scanning electron microscopy after epoxy resin embedding (EM-BSEM technique) (20). Leaflet fragments were fixed in 2 changes (12 hours per each) of 10% neutral phosphate-buffered formalin (B06-003, ErgoProduction, Saint Petersburg, Russia) at 4°C. Then, leaflets were washed in three changes (10 minutes per each) of phosphate-buffered saline (PBS, 0.1 mol/L, pH 7.4), postfixed in 1% phosphate-buffered osmium tetroxide (OsO₄, 19110, Electron Microscopy Sciences, Hatfield, PA, USA) for 16 hours and stained with 2% aqueous osmium tetroxide for 40 hours. Next, leaflets were washed in four changes (15 minutes per each) of PBS, dehydrated in ascending ethanol series (50%, 60%, 70%, 80%, and 95%, two changes per each concentration, 15 minutes per change), stained in 2% alcoholic uranyl acetate (22400-2, Electron Microscopy Sciences, Hatfield, PA, USA) for 16 hours, dehydrated in two changes (15 minutes per each) of 95% ethanol, isopropanol (06-002, ErgoProduction, Saint Petersburg, Russia) for 2 hours, and acetone (6-09-20-03-83, EKOS-1, Moscow, Russia) for 2 hours. After the dehydration, leaflets were impregnated with a blend of acetone and epoxy resin (1:1) for 16 hours and pure epoxy resin (Araldite 502, 13900, Electron Microscopy Sciences, Hatfield, PA, USA) for 24 hours, and embedded into the fresh epoxy resin at 60°C for another 24 hours. Following the epoxy resin polymerization, leaflets were grinded and polished (TegraPol-11, Struers, Copenhagen, Denmark) and counterstained with Reynolds’s lead citrate (17810, Electron Microscopy Sciences, Hatfield, PA, USA). After the washing in three changes (5 minutes per each) of distilled water, leaflets were sputter coated with carbon (10 nm thickness, Leica EM ACE200, Leica Microsystems, Wetzlar, Germany), and visualized by backscattered scanning electron microscopy (BSECOMP mode, S-3400N, Hitachi, Tokyo, Japan) at high vacuum and 10 or 15 kV accelerating voltage.

Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). For descriptive statistics, data were represented by the proportions, the median, and 25th and 75th percentiles. Groups were compared by the Mann-Whitney U-test. P values ≤ 0.05 were regarded as statistically significant.

All BMVs had perforations and tears localized primarily in the dome of the leaflets and around the commissures, and two BMVs (patients #3 and #4) had large intravalvular hemorrhages ( Figure 2 ). In contrast, only one BTV (patient #2) had minor perforation, whilst two BTVs (patients #1 and #3) had small intravalvular hemorrhages ( Figure 2 ). All BTVs and one BMV (patient #3) had a moderate pannus (i.e., a pathological outgrowth of the fibrotic tissue) at the base and commissural areas at the outflow side ( Figure 2 ). In two BTVs (patients #2 and #3), pannus restricted the valve opening, accounting for grade I stenosis revealed at echocardiography. All BMVs and BTVs suffered from minor calcification in the xenopericardial coating of the polypropylene frame that did not impact BHV functioning. In 3 out of 4 pairs (patient #1, patient #2, and patient #4), BMVs had higher amounts of calcium deposits than BTVs ( Figure 3 ). Hence, failing BMVs had typical signs of material fatigue because of higher hemodynamic load, whereas BTVs retained integrity.

Hematoxylin and eosin staining found sporadic cellular infiltrations in BMVs and BTVs ( Figure 4 ). Most of the cells were located at and beneath the inflow or outflow side at 50-100 µm depth, or within the areas of degraded ECM. In BMVs, cells were also detected along the edges of perforations and tears; in BTVs, dense cell layer was found between the xenopericardium and pannus ( Figure 4 ). Russell-Movat’s pentachrome staining identified abundant amounts of glycosaminoglycans within the pannus that confirmed its synthesis by the host cells, whereas bioprosthetic ECM was devoid of ground substance ( Figure 4 ). None of the BHVs contained fibrin at the inflow or outflow side. Oil Red O staining detected numerous lipid droplets in the spongiosa layer and beneath the free margin of BMVs and BTVs ( Figure 4 ). Notably, BMVs had multiple fatty spots and fatty streaks which were absent in BTVs.

Having performed an electron microscopy analysis of BHV leaflets, we recognized the most of the infiltrating host cells as macrophages, foam cells, and multinucleated giant cells ( Figures 5A, B ). Macrophages and multinucleated giant cells frequently contained electron-dense cytoplasmic inclusions suggestive of ECM degradation ( Figure 5C ). In addition to macrophages, BTVs also contained myofibroblasts in the pannus ( Figure 5C ).

Cell density at the outflow side was higher than at the inflow side in both BMVs ( Figure 6A ) and BTVs ( Figure 6B ). In all BHV pairs, total cell density was higher in BTVs than in BMVs ( Figure 6C ). In 3 out of 4 pairs, the area of lipid deposition was higher in BMVs as compared with BTVs, in keeping with the previous findings on increased ECM degradation and disintegration in BMVs ( Figure 6D ).