In Vitro Calcification Evaluation of Polycarbonate Urethane—Impact of Production Processes

TL;DR

This study finds that the production method of a heart valve material affects its tendency to calcify, with hot pressing increasing calcification risk.

Contribution

The study reveals that hot pressing alters the chemical structure of polycarbonate urethane, increasing calcification propensity.

Findings

Hot-pressed polycarbonate urethane patches showed calcification, while solution-cast patches did not.

Hot pressing caused chemical and structural changes in the material, likely promoting calcification.

Cytocompatibility was similar between hot-pressed and solution-cast polycarbonate urethane.

Abstract

Heart valve diseases remain a leading cause of death in industrialized nations. Polycarbonate urethane (PCU) is a promising material for heart valve prostheses due to its biocompatibility and low calcification tendency. However, the impact of processing methods on calcification remains unclear.

PCU patches were fabricated via hot pressing or solution casting. Both groups (n = 3 each), along with bovine pericardium patches as positive controls (n = 3), were incubated for 10 weeks in a custom in vitro calcification fluid. Calcification, cytocompatibility, and material properties were assessed using light and electron microscopy, infrared spectroscopy, and gel permeation chromatography (GPC).

Calcification was observed in hot‐pressed PCU and control patches but not in solution‐cast PCU. Both PCU types showed comparable cytocompatibility. Spectroscopy and GPC revealed chemical and structural changes in hot‐pressed PCU, likely promoting calcification.

Hot pressing alters the chemical structure of PCU and increases its calcification propensity without affecting cytocompatibility. These findings highlight the importance of process control and in vitro screening during heart valve material development.

The study shows that hot pressing induces chemical changes in PCU, promoting calcification, while solution‐cast patches remain unaffected.

Figures

Exemplary structure of a polyurethane carbonate. Highlighted in green are the urethane groups while the carbonate group is highlighted in blue. The residuals R 1 und R 2 can be equal but don't have to be. R 3 is the residual group of the employed isocyanate during synthesis. R 1–R 3 can be varied depending on the application of the final product. [Color figure can be viewed at wileyonlinelibrary.com]

Overview of the employed patches before testing. Top row, the PCU‐patches made from solution. Middle row, the patches made by hot‐pressing. Bottom row, the pericardium patches used as control. [Color figure can be viewed at wileyonlinelibrary.com]

Comparison of the patches' back sides after the test. Patches 1, 4, 5, and 6 show calcifications. Only Patch 2 does not show calcification inside the region of interest. [Color figure can be viewed at wileyonlinelibrary.com]

Calcification of the pericardium patches. Pericardium Patch 2 is shown with calcification in 10× (top left). The calcification is referenced via arrows in the close‐up images in 50× magnification. [Color figure can be viewed at wileyonlinelibrary.com]

Overview of the residual material at 50× magnification. Top row patch material from solution before testing (left) and after testing (right). Bottom row hot‐pressed patch material before testing (left) and after testing (right).

Calcification on Patch 4 after 10 weeks of testing—Light microscopy (20×) linked to the SEM image (100×). [Color figure can be viewed at wileyonlinelibrary.com]

Mean transmission spectra of the residual material from the hot‐pressed patches and the ones made from solution. [Color figure can be viewed at wileyonlinelibrary.com]

Microscopy results of live/dead staining after 3 days of cytocompatibility testing of solution‐casted and hot‐pressed PCU patches as well as untreated cells (positive control) and Triton X‐100–treated lysed cells (negative control) (100× magnification). [Color figure can be viewed at wileyonlinelibrary.com]

Tables

TABLE 1. Fluid composition used in the experiment.

Component	Concentration in mmol/L
CaCl2	1.8
CaT	1.8
KH2PO4	1.0
PT	1.0
NaCl	115
KCl	4.0

TABLE 2. Results of the patches' GPC measurement.

Sample	Mn in g/mol	Mean Mn in g/mol	Mw in g/mol	Mean Mw in g/mol	PDI	Mean PDI
Patch 1	101 000	101 467	220 100	216 833	2.180	2.137
Patch 2	103 000		214 800		2.086
Patch 3	100 400		215 600		2.146
Patch 4	87 070	103 490	200 200	232 933	2.299	2.251
Patch 5	120 400		274 700		2.282
Patch 6	103 000		223 900		2.172

Full text

Introduction

1

Cardiovascular diseases are still one of the main causes of death in developed countries [1]. Among therapeutic measures, heart valve‐related interventions are on the rise [2]. Bioprosthetic or mechanical valves are eligible for replacement therapy. The choice of valve type is patient‐specific and depends on several factors [3]. In recent years, the trend in research has been toward tissue‐engineered heart valves, which suggest better hemocompatibility and anticalcific behavior than mechanical or bioprosthetic ones. Although bioprosthetic valves, typically fabricated from glutaraldehyde‐treated bovine or porcine pericardium, offer good hemocompatibility, they are prone to structural valve deterioration over time. Particularly in younger patients, early degeneration due to calcification, enzymatic breakdown, or immune‐mediated damage remains a major limitation. Moreover, variability in donor tissue and complex processing introduce challenges in reproducibility and durability. These drawbacks have motivated the search for synthetic alternatives that combine long‐term stability with consistent quality [4].

Among synthetic materials, polymer‐based prosthetic heart valves represent an interesting research approach. This group is dominated by polyurethanes, while not being exclusively used [5]. Polyurethanes are a widely used class of materials in biomedical engineering. A broad range of polyurethane blends is available with outstanding biomedical performance regarding blood and tissue compatibility [6]. In a cardiovascular environment, the ability to withstand calcification is crucial. Polyurethane blends, especially polycarbonate urethanes (PCU), generally show better anticalcific behavior than other widely applied materials such as bovine or porcine pericardium, from which most bioprosthetic valves are made. Due to their structure (Figure 1) with the absence of ester groups and the resistance to hydrolysis and oxidation, they exhibit intrinsic resistance to calcification and degradation by endogenous processes. In addition, they show high mechanical integrity and flexibility [7, 8]. Furthermore, PCUs have been shown to provoke a reduced immune response compared to bioprosthetic materials [9].

Exemplary structure of a polyurethane carbonate. Highlighted in green are the urethane groups while the carbonate group is highlighted in blue. The residuals R 1 und R 2 can be equal but don't have to be. R 3 is the residual group of the employed isocyanate during synthesis. R 1–R 3 can be varied depending on the application of the final product. [Color figure can be viewed at wileyonlinelibrary.com]

The clinical potential of PCUs has been demonstrated in early prototype designs. For instance, Daebritz et al. introduced a flexible polymeric heart valve with a PCU leaflet design optimized for the mitral position, showing promising results in a pulsatile flow environment [10]. More recently, Todesco et al. reviewed current developments in polymeric heart valves and highlighted PCUs as strong candidates for durable, anticalcific prostheses with customizable design parameters [11]. Therefore, polyurethanes offer the potential for future artificial heart valves with fewer limitations [12].

Research on polyurethane and other prosthetic heart valves shows that leaflet calcification occurs more frequently near the belly, where high mechanical stress during valve motion promotes material fatigue, environmental stress cracking, and subsequent calcification. Surface roughness also contributes, as seen in polymer vascular grafts. Calcification is further influenced by proteins, particularly collagen in pericardium, which offers nucleation sites via exposed functional groups and its helical structure, making it a target for anti‐calcification treatments. The chemical composition of polymers significantly affects calcification, but surface structure and interactions with surrounding fluids are also critical. One hypothesis suggests plasma proteins or phospholipids become trapped in surface micro‐gaps, initiating calcification. Notably, polyurethanes may calcify even without mechanical load, and smoother surfaces exhibit lower calcification potential than cracked or rough ones [13, 14, 15, 16, 17, 18, 19, 20]. Several of these aspects can already be addressed during the fabrication process and the process parameters.

To evaluate calcification, researchers use various animal models that replicate human physiological and pathological mechanisms [21]. In parallel, several attempts have been made to facilitate this kind of testing in vitro. Available tests differ in test setup as well as in the choice of calcification medium [22, 23, 24, 25].

This study aimed to assess whether processing differences in identical PCU blends—and the resulting subtle surface variations—affect calcification behavior, a critical factor in polyurethane heart valve manufacturing. Identifying any correlation between processing parameters and calcification resistance is essential. Additionally, we examined calcification linked to material fatigue by comparing the in vitro calcification of two differently manufactured PCU patch groups (solution‐cast and hot‐pressed) with that of three bovine pericardium patches.

Materials and Methods

2

At our institute, we use a modified heart valve durability tester with a specially created calcification fluid to investigate the calcification of materials and heart valves without the need for animal studies [26, 27, 28].

Patches

2.1

Patches were divided into three groups based on material and manufacturing methods. Patches 1–6 used thermoplastic PCU (Carbothane TPU PU3575, Lubrizol) and were produced by two methods. Patches 1–3 were made by evaporating a 5% w/w PCU solution in chloroform at room temperature in petri dishes, covered with inverted 1 L glass beakers to slow evaporation and achieve a smooth surface. As chloroform fully evaporates from PCU within 24 h [29], the patches were flipped after 24 h and cut into 38 mm discs after another 24 h. Residual material was retained for further analysis.

Patches 4–6 were produced by hot pressing PCU pellets at ~3.5 bar and 150°C for 15 min using a Carver Laboratory Press (Model 4122, Carver Inc., USA). PTFE‐coated glass fabric (0.15 mm, Hightechflon) prevented sticking, while 0.5 mm steel shims ensured uniform thickness. After cooling to room temperature, samples were stored in a desiccator and later cut into 38 mm diameter patches. Residual material was retained and used as ‘before testing’ reference in SEM.

The last three patches (No. 7–9) were made of bovine pericardium by St. Jude Medical (SJM Pericardial Patch with EnCap Technology), an approved medical grade material with specific anti‐calcification treatment, and were therefore included as a positive control. They were punched to form round patches (of 38 mm diameter) like the PCU ones.

Test System and Settings

2.2

Calcification testing was conducted using a modified in‐house durability tester (CVE‐FT2) designed for accelerated patches and heart valve evaluation according to DIN EN ISO 5840 [26]. The tester consists of 12 compartments designed for testing either patches or full‐size heart valves. A shaker plate beneath the compartments compresses stainless steel bellows to simulate the heartbeat, thereby generating a pulsatile flow within the chambers.

Patches were clamped between holders (38 mm outer, 16 mm inner diameter) and placed in separate compartments. Each was subjected to normotensive pressure conditions per ISO 5840, targeting a 100 mmHg peak differential pressure for at least 5% of the cycle, at 300 bpm to accelerate testing. Compartments were filled with the same batch of calcification fluid developed by our group [26, 27] to ensure consistent ionic conditions. A static control fluid sample in a PE bottle was included to monitor fluid stability. Tests ran at 37°C for 10 weeks, which corresponds to 30 million heart cycles, representing a relevant time frame for early‐stage in vitro calcification screening.

The fluid was changed on a weekly basis to maintain stable physiological conditions, according to the previously developed test method [27]. Prior to the fluid exchange, fluid samples were taken for the analysis of the ionic concentrations, and the pH value was measured. Pressure conditions and temperature were controlled at least twice a week as well. Additionally, images of the patches (top and bottom side) were taken to continuously monitor the progress of calcification. The tester was inspected visually on a daily basis.

Calcification Solution

2.3

For this study, a previously developed calcification fluid [26] was modified to prevent spontaneous precipitation, making it more suitable for long‐term use and selective biomaterial calcification [27]. The fluid's calcium and phosphate levels were adjusted and stabilized by modifying the ionic strength with sodium and potassium salts. Weekly analysis of the freshly prepared fluid ensured consistent conditions.

The fluid concentrations are based on fluid L from Kiesendahl et al. and listed in Table 1 [27].

Ion concentrations (sodium, potassium, calcium, phosphate) were measured using the Vitros Chemistry System 350 (Ortho‐Clinical Diagnostics) at the Institute of Laboratory Animal Science, Uniklinik RWTH Aachen, via a colorimetric method.

Digital Light Microscopy

2.4

The digital light microscopy was performed using a Keyence VHX‐950F.

Prior to the actual testing procedure, all patches were screened by light microscopy (top and bottom side) to detect possible damages or surface structure anomalies. Images were taken at 5× and 10× magnification. After 10 weeks of experiment, the patches were analyzed by using light microscopy again to screen for calcified spots and damages on the surface, which may have resulted from the constant pressure, induced stretching during each cycle.

Scanning Electron Microscopy

2.5

The aim of the scanning electron microscopy (SEM) imaging was to detect surface irregularities, such as grooves or microstructural defects, potentially resulting from processing. The SEM took place at the Electron Microscopic Facility of the Uniklinik RWTH Aachen on an ESEM XL 30 FEG by FEI. The measurements were conducted using an acceleration voltage of 10 kV with a backscatter detector in a high vacuum. Before the actual imaging, the patches were coated with a 10 nm gold palladium layer in the sputter coater EN SCD500 by Leica. We used the SEM to receive higher resolution images of the patches' surface to determine the reasons for the calcification.

ATR‐IR Spectroscopy

2.6

Residual materials from Patches 1–3 and 4–6 were analyzed via ATR‐IR spectroscopy to assess chemical changes from hot pressing. Pericardium patches were excluded, as they underwent no thermal or pressure treatment. Measurements were performed at the Fraunhofer Institute of Lasertechnology in Aachen using a PerkinElmer Frontier MIR FTIR spectrometer with a diamond ATR unit and Spectrum v10.03.07 software. Each series began with a background scan, which was automatically subtracted. Solid PCU samples were pressed onto the ATR crystal, and spectra were recorded over 4000–600 cm^−1^ at 4 cm^−1^ resolution, averaged across five scans.

Gel Permeation Chromatography

2.7

Gel permeation chromatography (GPC) was used to assess molecular changes in PCU due to hot pressing, such as heat‐induced chain degradation. Analyses were conducted at the Institute of Technical and Macromolecular Chemistry, RWTH Aachen, using size‐exclusion chromatography (SEC) to determine M_n_ (number‐average molecular weight), M_w_ (weight‐average molecular weight), and M_w_/M_n_. Number‐average molecular weight (M_n_) reflects the average size of polymer chains, giving equal weight to each molecule. Weight‐average molecular weight (M_w_) gives more emphasis to larger chains. A bigger difference between M_w_ and M_n_ indicates a broader molecular weight distribution, affecting material properties like strength and stability [30].

DMF (≥ 99.9%, VWR) with 1 g/L LiBr (≥ 99%, Sigma‐Aldrich) served as the eluent. Samples contained 2 μL/mL toluene (≥ 99%, Sigma‐Aldrich) as an internal standard, measured at 260 nm. The system included an HPLC pump (1260 Infinity), refractive index detector, viscosity detector (DVD 1260), and UV detector (VWD 1290 Infinity II, all Agilent). One 8 × 50 mm pre‐column and three 8 × 300 mm GRAM gel columns (10 μm particles, 30/1000/1000 Å pores, Polymer Standards Service) were used at 60°C and 1.0 mL/min flow rate. Calibration employed narrow PMMA standards, and data were evaluated with PSS WinGPC UniChrom (v8.3.2).

Cytocompatibility Assay

2.8

A cell‐based assay was used to evaluate potential cytotoxic effects of both hot‐pressed and solution‐cast PCU samples prior to calcification testing, following DIN EN ISO 10993‐5 [31]. Indirect cytotoxicity and cell viability were assessed after 24 h and 3 days using live/dead staining of L929 murine fibroblasts. Cells were seeded in 48‐well plates at 180000 cells/cm^2^. Two patches per manufacturing method were incubated in medium for 24 h and 3 days, and the conditioned medium was then applied to the cells. Viability was analyzed using a Leica DMI 6000 B fluorescence microscope and LAS X software (v3.7.4.23463). Fluorescence microscopy data were further evaluated using ImageJ (v1.53i) to determine the relative proportion of live cells. Controls included untreated cells (positive) and Triton X‐100–treated lysed cells (negative).

Results

3

Digital Light Microscopy

3.1

Before calcification testing, polyurethane patches 1–3 had a smooth reflective surface. Polyurethane patches 4–6 were not as reflective as the ones made from solution because of a groovy surface (Figure 2).

Overview of the employed patches before testing. Top row, the PCU‐patches made from solution. Middle row, the patches made by hot‐pressing. Bottom row, the pericardium patches used as control. [Color figure can be viewed at wileyonlinelibrary.com]

The pericardium patches showed no damage before the test. The distinct surface structure of the pericardium's fibrous layer (used as top side) is clearly visible (Figure 2).

After testing, the polyurethane patches made from the chloroform solution were visibly stretched in the z‐direction in the region of interest while not showing any signs of calcification on their bottom side. The top side of patch 1 showed strong calcification, which was of a grainy structure. Patches 2 and 3 had no signs of calcification in the region of interest on their top side. However, the bottom sides showed no calcification on Patches 2 and 3, while Patch 1 showed slight calcification near the ring bearing (Figure 3).

Comparison of the patches' back sides after the test. Patches 1, 4, 5, and 6 show calcifications. Only Patch 2 does not show calcification inside the region of interest. [Color figure can be viewed at wileyonlinelibrary.com]

The patches prepared by hot pressing were similarly stretched in the z‐direction as the patches made from solution. All patches [4, 5, 6] had small calcific deposits on the bottom side. Patches 4 and 6 additionally showed strong calcification on the top side.

The pericardium patches were stretched in the region of interest as well. All pericardium patches used as a positive control had calcifications on their top and bottom sides. The calcification was more intense than the ones found on the hot‐pressed PCU patches. The calcification on Patch 2 is exemplarily shown in Figure 4. The additional images are available in the Figure S1.

Calcification of the pericardium patches. Pericardium Patch 2 is shown with calcification in 10× (top left). The calcification is referenced via arrows in the close‐up images in 50× magnification. [Color figure can be viewed at wileyonlinelibrary.com]

Scanning Electron Microscopy

3.2

Images of Patches 1–3 (from chloroform solution) showed smooth surfaces without cracks or scratches (Figure 5). Especially, there was no difference between the patches from the tester after 10 weeks and the residual materials prior to the experiment. The additional images are available in the Figures S3 and S4.

Overview of the residual material at 50× magnification. Top row patch material from solution before testing (left) and after testing (right). Bottom row hot‐pressed patch material before testing (left) and after testing (right).

Patches 4–6 (from the hot press) all showed grooves along their surface (Patch 5 shown exemplary in Figure 5). No differences regarding the grooves were found between the patches from the tester after 10 weeks and the residual pieces. Most significantly, no calcification formed along or anywhere specific to the grooves. It appeared to be randomly distributed on the patches' surface.

Figure 6 shows the exemplary calcification detected on Patch 4 after 10 weeks of testing. The leftmost image and the image in the middle were both taken by light microscopy at a 20× magnification. The middle image was cropped and manually magnified to better highlight the calcification. The image on the right is the corresponding calcification taken by SEM at 100× magnification.

Calcification on Patch 4 after 10 weeks of testing—Light microscopy (20×) linked to the SEM image (100×). [Color figure can be viewed at wileyonlinelibrary.com]

ATR‐IR‐Spectroscopy

3.3

The spectra of each patch group were nearly identical, allowing us to average the three spectra per group and present them in Figure 7 (black: solution‐cast, red: hot‐pressed). Peaks were divided into 11 regions. Both showed a peak at 3301 cm^−1^, sharper in the hot‐pressed group, corresponding to N–H stretching in urethane linkages. In the 3000–2750 cm^−1^ region, both spectra exhibited C–H stretching from aliphatic chains, more pronounced in hot‐pressed patches. Peaks at 1739/1740 cm^−1^ reflect C=O stretching in ester or carbonate groups, while 1640 cm^−1^ corresponds to C=O stretching in urethane. The 1524/1526 cm^−1^ peaks indicate N–H bending and C–N stretching. Peaks at 1464/1465 cm^−1^ and 1403 cm^−1^ represent C–H bending. C–O stretching appears at 1241/1242 cm^−1^. Unique to the hot‐pressed samples are peaks at 1150, 1086, 1062, and 1046 cm^−1^, attributed to C–O–C stretching in carbonate groups. Both groups shared peaks at 958, 901, 847, 791, and 731 cm^−1^ within the fingerprint region [32].

Mean transmission spectra of the residual material from the hot‐pressed patches and the ones made from solution. [Color figure can be viewed at wileyonlinelibrary.com]

Gel Permeation Chromatography

3.4

The results from the GPC display the differences between the two groups of PCUs (Table 1). The number averaged molar masses are almost identical for the first group. In contrast, they vary greatly in the second group. The same is obvious for the mass averaged molar masses for each group, respectively. The averaged molar masses were higher in the second group of patches. The mean polydispersity index (PDI) of the second group is as well slightly higher than of the first group's (Table 2).

Cytocompatibility Assay

3.5

The live/dead‐staining revealed only a negligible fraction of dead cells (Figure 8). The cell viability was higher than 95%; therefore, the materials are still considered cytocompatible according to DIN EN ISO 10993‐5. There was no quantitative difference between the two types of patches.

Microscopy results of live/dead staining after 3 days of cytocompatibility testing of solution‐casted and hot‐pressed PCU patches as well as untreated cells (positive control) and Triton X‐100–treated lysed cells (negative control) (100× magnification). [Color figure can be viewed at wileyonlinelibrary.com]

Discussion

4

The in vitro tests of differently processed PCU patches revealed varying calcification behavior. The hot‐pressed patches exhibited calcification, while patches produced from a solution showed no signs of calcification. The material surface plays a crucial role in the calcification of biomaterials [16]. Although grooves were visible on the hot‐pressed patches to the naked eye, it could not be determined whether they were related to calcification. Therefore, the patches were subjected to further investigation.

Patches 1, 4, and 6 showed little calcification debris at some parts in the upper section in week 6. Therefore, only the bottom sides of the patches were included in the analysis to avoid any false‐positive calcification at the patches that may be due to minor contamination.

Digital light microscopy of the patches' bottom sides revealed no visible signs explaining localized calcification. Scanning electron microscopy showed surface grooves in hot‐pressed patches, yet calcification did not follow these patterns. In the center region of the patches, they appeared to be randomly distributed, suggesting chemical composition rather than surface features as the trigger. Infrared spectroscopy supported this, showing changes in PCU macromolecular chains likely caused by hot pressing. Notable were elevated amino group peaks and reduced C–O stretching, similar to changes observed by Cipriani et al. upon thermal cycling of polyurethane. These changes stem from altered polymer morphology, as PCUs—composed of hard (polyurethane) and soft (polycarbonate) segments—lose microdomain organization above 100°C due to hydrogen bond breakdown, with incomplete recovery upon cooling. GPC analysis showed molecular weight shifts in hot‐pressed samples, indicating chain scission and rearrangement. Some patches showed chain shortening, while others showed apparent cross‐linking. Similar behavior has been reported in polyurethane recycling studies. These molecular changes likely created new nucleation sites for calcification. Despite these alterations, both PCU variants maintained excellent cytocompatibility, with cell viability exceeding 95%, confirming that hot pressing does not compromise biocompatibility [32, 33, 34, 35].

While these findings suggest a material‐driven mechanism of calcification initiation, the spatial distribution of deposits requires further consideration. Notably, both hot‐pressed and pericardium patches exhibited central calcification, which supports a chemical/material‐based initiation process independent of external stress. However, the highest density of calcific deposits was consistently found near the patch holder, an area associated with elevated mechanical stress. This pattern was observed across all patch types. Therefore, we propose a synergistic mechanism: chemical alterations caused by processing define the material's intrinsic susceptibility to calcification, while mechanical stress influences the localization, extent, and progression of deposit formation. In other words, stress may not initiate calcification alone, but it likely accelerates it in materials already predisposed due to their chemical structure.

Concluding from the experiment's results, the manufacturing processes for polyurethanes used as leaflet material for heart valve prostheses are limited. The widely used hot‐pressing procedure in other fields does not appear to be useful for producing leaflets due to the implicated rise in calcification propensity. Instead, heart valve leaflets can be formed by a variety of other methods. A prominent alternative is dipping a mold into a solution of the desired polymer. However, this process has other limitations [36]. Reproducibility is probably the most important one, while still being a costly procedure.

The results from this study once again show the importance of patch/material testing before manufacturing and subsequently testing an actual heart valve. With the pretesting, many unnecessary steps can be avoided if the material, like in this case, tends to calcify during the first line of testing.

Some limitations must be considered before extrapolating these results. First, only patches—not full‐size heart valve prostheses—were tested. Since patches experience different loading, with high stress concentrated near the holder, calcification was expected and observed in this area. However, both hot‐pressed and pericardium patches also showed central calcification, suggesting a material‐driven rather than stress‐induced mechanism. Despite this, further testing of full prostheses may be unnecessary due to the material's already poor calcification resistance. Second, the composition of the calcific deposits could not be analyzed due to their minimal quantity. The test fluid, designed to mimic in vivo conditions, likely produced a mix of octacalcium phosphate and hydroxyapatite, as shown in previous studies. However, the fluid's simplified composition omits factors such as non‐collagenous proteins, as discussed by Kiesendahl et al. [27].

Our findings yield three key insights. First, the manufacturing process significantly affects PCU calcification behavior. Despite identical starting material, differences in processing led to distinct calcification patterns. This is likely due to heat‐induced supramolecular rearrangements and chain scission, both of which create nucleation sites for calcific deposits. Second, surface grooves were not identified as primary calcification sites. Electron microscopy showed that surface irregularities alone do not trigger calcification, though the precise quantitative relationship remains unclear. Third, most calcific deposits formed in high‐stress regions, regardless of material type. This suggests that mechanical stress has a greater influence on calcification than chemical composition or surface structure. These results highlight the need to test full polymer heart valve prostheses to better understand stress‐related calcification during valve function. Overall, the study emphasizes the value of in vitro calcification testing in guiding the design of improved prosthetic heart valves.

Conclusion

5

To address the persistent issue of calcification of polymeric heart valves, we investigated the impact of processing parameters on the calcification propensity of polyurethane carbonate (PCU) patches. After 10 weeks of accelerated testing in a durability tester with a specifically designed calcification fluid, calcification of hot‐pressed and solution‐cast PCU patches was evaluated and compared to bovine pericardium patches. The study revealed that only the hot‐pressed PCU patches exhibited calcification, while solution‐cast patches remained unaffected. Scanning electron microscopy revealed surface grooves on the hot‐pressed samples, but these did not correlate with the location of calcific deposits. Instead, infrared spectroscopy and gel permeation chromatography (GPC) indicated chemical changes—such as increased amino group content and chain scission—likely caused by thermal processing, which may have promoted nucleation and calcification. Importantly, both PCU types maintained high cytocompatibility, suggesting that biocompatibility was not compromised by the processing method. These findings indicate that hot pressing increases the risk of calcification and should be avoided in the fabrication of polymeric heart valve leaflets. The study highlights the critical role of in vitro screening in guiding material and process selection for future valve designs.

Author Contributions

Jan Ritter drafted the manuscript. Study concept and design was developed by Jan Ritter, Thomas Schmitz‐Rode, Johanna C. Clauser and Cécile Oury. Acquisition, analysis, or interpretation of data was performed by Jan Ritter, Christoph Schmitz, Stephan Rütten, Abdelhafid Aqil and Johanna C. Clauser. Critical revision of the manuscript was performed by Jan Ritter, Johanna C. Clauser, Cécile Oury, Willi Jahnen‐Dechent, Thomas Schmitz‐Rode and Ulrich Steinseifer. Funding was obtained by Johanna C. Clauser, Cécile Oury, Thomas Schmitz‐Rode, Willi Jahnen‐Dechent and Ulrich Steinseifer.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Pericardium Patches 1–3 before testing (5× magnification). Figure S2: Pericardium patches with calcification after 10 weeks testing (5× magnification). Figure S3: Patches from solution before and after testing. Figure S4: Hot‐pressed patches before and after testing. PU1 from the tester is displayed in another magnification 25× instead of 50×.