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Multiscale Analysis Of Human Tissue Engineered Matrices For Heart Valve Tissue Engineering Applications
Nikolaos Poulis1, Pascal Breitenstein1, Simon Hofstede2, Maximilian Y. Emmert1, Simon P. Hoerstrup1, Emanuela Fioretta1.
1Institute for Regenerative Medicine, University of Zurich, Zurich, Switzerland, 2Laboratory for Orthopaedic Biomechanics, Institute for Biomechanics, ETH Zurich, Zurich, Switzerland.

OBJECTIVE: Tissue-engineered matrices (TEMs) based on in-vitro grown ECM and bioresorbable scaffolds have been proposed as a promising approach for in-situ tissue engineered heart valves (TEHVs). TEMs can provide crucial mechanical and biochemical cues to ensure in-vivo functionality and remodelling. However, TEM composition and ECM developmental processes during culture have not been fully characterized. We aim to perform an in-depth evaluation of the TEM, focusing on the relation between ECM proteome development and TEM mechanical properties. TEM-based TEHVs were tested in-vitro to assess their performance as aortic replacements.
METHODS: TEMs were produced by culturing human dermal fibroblasts on PGA-P4HB for 2, 4, and 6-weeks (n=4/time-point), and then decellularized. (Immuno-)histology, biochemical assays and mass spectrometry (LC-MS/MS) were used to characterize the ECM. The protein pathways involved in the ECM development were elucidated by geneset enrichment analysis (GSEA). Stiffness, stress, and strain at failure were obtained via uniaxial tensile test. TEM-based TEHVs after 6-weeks were tested under aortic-like pressure conditions using a pulse duplicator.
RESULTS: Immuno-histological and biochemical evaluations showed that ECM quantity and organization increased over tissue culture time. Mass spectrometry confirmed this time-dependent increase in ECM proteins and revealed the most abundant collagens (COL6,COL12), proteoglycans (HSPG2,VCAN), and glycoproteins (FN,TNC). GSEA identified the most represented protein pathways at 2-weeks (mRNA metabolic processes), 4-weeks (ECM production), and 6-weeks TEMs (ECM organization and maturation). Uniaxial mechanical testing showed increase in stiffness (+66.6%) and stress (+37.2%), and reduction in strain (-35.8%) in 6-weeks compared to 2-weeks TEMs. TEM-based TEHVs showed good performance under in-vitro simulated aortic conditions.
CONCLUSIONS: We observed the TEM proteome development and identified an increase in ECM proteins and organization over tissue culture time up to 6 weeks. These results suggest that longer tissue culture in TEMs induces improved mechanical properties to cope with high-pressure applications in-vitro.


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