Calcification in Pulmonary Heart Valve Tissue Engineering: A Systematic Review and Meta-Analysis of Large-Animal Studies

Abstract

Tissue-engineered heart valves (TEHVs) are emerging alternatives to current valve prostheses and prospectively a lifelong replacement. Calcification, a pathological complication for biological protheses, has been reported in preclinical TEHV studies. Systematic analysis of its occurrence is missing. This review aims to: 1) systematically review reported calcification of pulmonary TEHVs in large-animal studies; and 2) analyze the influence of engineering methodology (choice of scaffold material, cell preseeding) and animal model (animal species and age) on calcification. Baseline analysis included 80 studies, of which 41 studies containing 108 experimental groups were included in meta-analysis. Inclusion was low because only 55% of studies reported on calcification. Meta-analysis showed an overall average calcification event rate of 35% (95% CI: 28%-43%). Calcification was more prominent (P = 0.023) in the arterial conduit region (34%; 95% CI: 26%-43%) than in the valve leaflets (21%; 95% CI: 17%-27%), and was mostly (42% in leaflets, 60% in conduits) present in a mild form. Time-analysis showed an initial surge within 1 month after implantation, decreased calcification between 1 and 3 months, and then progression over time. There were no significant differences in degree of calcification between TEHV strategy nor animal models. Much variability between individual studies was observed in degree of calcification as well as quality of analysis and reporting thereof, hampering adequate comparisons between studies. These findings underline the need for improved analysis and better reporting standards of calcification in TEHVs. It also necessitates control-based research to further enlighten the risk of calcification for tissue-engineered transplants compared to current options. This can bring the field of heart valve tissue engineering forward toward safe clinical use.

Keywords: biomaterials; cardiac valve prosthesis; mineralization; preclinical; regeneration; scaffold; tissue-engineered heart valve.

Graphical abstract

Figure 1

Tissue Engineering Strategies Valves were categorized as natural or synthetic, based on the scaffold base material. Within the natural and synthetic groups, valves were subdivided into decellularized (Decell), preseeded (Seeded), or unseeded’ subgroups, based on their cellularization state at the time of implantation. ∗On the fly preseeded valves were included in the preseeded groups. ECM = extracellular matrix. Bottom figures were reprinted with permission from (left to right) Reimer et al, Wilhelmi et al, Iop et al, Driessen-Mol et al, Dijkman et al, and Kluin et al.

Central Illustration

Systematic Review of Calcification in Pulmonary Heart Valve Tissue Engineering Meta-analysis of leaflet and conduit calcification in natural and synthetic implants showed calcification in 35% of preclinical animal pulmonary valves, with more prone calcification in the valvular conduit (34%) compared with in the leaflet (21%) region.

Figure 2

Qualitative Analysis of Location and Severity of Calcification (A to E) Examples of leaflet calcification severity as shown by von Kossa–stained histological samples. (A) Example of no calcification. Reprinted with permission from Goecke et al.(B) Example of microcalcification (score 1). Reprinted with permission from Fioretta et al.(C) Example of mild calcification (score 2). Reprinted with permission from Emmert et al.(D) Example of moderate calcification (score 3). Reprinted with permission from Fioretta et al.(E) Example of severe calcification (score 4). Note: calcification shown here was described in the retrieved study to be distributed throughout the whole leaflet. Reprinted with permission from Leyh et al.(F) Schematic Representation of localization of calcification within the valve showing different conduit and leaflet regions with more (red) or less (yellow) calcification. p = calcification associated with polymeric remnants.

Figure 3

Analyses of the Quality of Model Description and Reporting on Outcome Parameters (A) Key points describing analysis-specific risk of bias. (B) Analysis of reporting of histological features shows a need for better reporting of calcification. (C) Analysis of study method of calcification. Calcification was not studied in 34% of the cases; 6% of which were not applicable (NA) because of the short follow-up time (<1 week). Calcification was studied in 11% of the cases only through macroscopic evaluation, and in 55% of the cases through other methods, mainly containing histology (75%). ∗Assessed for qualitatively, compared outcomes. ∗∗Except for animals excluded during operation and 1-week follow-up. Angio = angiography; CMR = cardiac magnetic resonance; Echo = echocardiography; His = histology; MA = macroscopic assessment only; NR = not reported; Spect = (atomic force) spectroscopy; TEM = transmission electron microscopy.

Figure 4

Meta-Analysis of the Effect of Scaffold Base Material and Cell-Seeding on Average Overall Calcification (A, B) Forest plot of all experimental groups within natural (A) and synthetic (B) base materials. Overall analysis is shown by red line. Dots represent estimate event rate per experimental group and dot size the relative weight. Lines represent 95% CIs, diamonds subgroup mixed effect analysis, and dotted lines subgroup average. (C) Average event rate and 95% CI of natural (orange) and synthetic (blue) base materials showed no significant difference between base materials. (D) Average event rate and 95% CI of preseeded (Seeded) versus unseeded natural scaffolds showed no significant difference between seeding method within the natural base material group. (E) Average event rate and 95% CI of decellularized (Decell), preseeded, and unseeded synthetic scaffolds showed no significant difference between seeding method within the synthetic base material group. Shown as subgroup event rate (bar height) with 95% CI (whiskers). Horizontal line represents grouped event rate, and dashed lines are 95% CI. Horizontal red dotted line is overall event rate at 35%.

Figure 5

Subgroup Analyses Animal Model (A to C) Subgroup analyses of animal species (A), sex (B), and age (C) showed no significant difference between subgroups. (D) Subgroup analysis of anticoagulation use showed no significant difference between anticoagulation use. (E) Subgroup analysis of follow-up time showed trends of higher calcification before 1 month and increasing calcification after longer follow-up times after 1 month, though not significantly. Shown as subgroup event rate (bar height) with 95% CI (whiskers). Horizontal line represents grouped event rate and dashed lines are 95% CI. Horizontal red dotted line is overall event rate at 35%.

Figure 6

Analysis of Location-specific Calcification (A to C) Leaflet calcification. (D to F) Conduit calcification. (A, D) Natural versus synthetic scaffolds. (B, E) Natural scaffold with compared to cell-seeding methods. (C, F) Synthetic scaffolds compared to cell-seeding methods. Shown as subgroup event rate (bar height) with 95% CI (whiskers). Horizontal line represents grouped event rate and dashed lines are 95% CI. Horizontal red dotted line is overall event rate at 35%.