Multiscale analysis of human tissue engineered matrices for next generation heart valve applications

Human tissue-engineered matrices (hTEMs) have been proposed as a promising approach for in situ tissue engineered heart valves (TEHVs). However, there is still a limited understanding on how ECM composition in hTEMs develops over tissue culture time. Therefore, we performed a longitudinal hTEM assessment by 1) multiscale evaluation of hTEM composition during culture time (2, 4, 6-weeks), using (immuno)histology, biochemical assays, and mass spectrometry (LC-MS/MS); 2) analysis of protein pathways involved in ECM development using gene set enrichment analysis (GSEA); and 3) assessment of hTEM mechanical characterization using uniaxial tensile testing. Finally, as a proof-of-concept, TEHVs manufactured using 6-weeks hTEM samples were tested in a pulse duplicator. LC-MS/MS confirmed the tissue culture time-dependent increase in ECM proteins observed in histology and biochemical assays, revealing the most abundant collagens (COL6, COL12), proteoglycans (HSPG2, VCAN), and glycoproteins (FN, TNC). GSEA identified the most represented protein pathways in the hTEM at 2-weeks (mRNA metabolic processes), 4-weeks (ECM production), and 6-weeks (ECM organization and maturation). Uniaxial mechanical testing showed increased stiffness and stress at failure, and reduction in strain over tissue culture time. hTEM-based TEHVs demonstrated promising in vitro performance at both pulmonary and aortic pressure conditions, with symmetric leaflet coaptation and no stenosis. In conclusion, ECM protein abundance and maturation increased over tissue culture time, with consequent improvement of hTEM mechanical characteristics. These findings suggest that longer tissue culture impacts tissue organization, leading to an hTEM that may be suitable for high-pressure applications. STATEMENT OF SIGNIFICANCE: It is believed that the composition of the extracellular matrix (ECM) in the human tissue engineered matrices (hTEM) may favor tissue engineered heart valve (TEHV) remodeling upon implantation. However, the exact protein composition of the hTEM, and how this impacts tissue mechanical properties, remains unclear. Hence, we developed a reproducible rotation-based tissue culture method to produce hTEM samples. We performed a longitudinal assessment using different analytical techniques and mass spectrometry. Our data provided an in-depth characterization of the hTEM proteome with focus on ECM components, their development, and how they may impact the mechanical properties. Based on these results, we manufactured functional hTEM-based TEHVs at aortic-like condition in vitro. These outcomes pose an important step in translating hTEM-based TEHVs into clinics and in predicting their remodeling potential upon implantation.