Abstract
Remodelling of the extracellular matrix is accomplished by altering the balance between matrix macromolecule production and degradation. However, it is not well understood how cells balance production of new matrix molecules and degradation of existing ones during tissue remodelling and regeneration. In this study, we used decellularized lung scaffolds repopulated with allogenic lung fibroblasts cultured with stable isotope labelled amino acids to quantify the balance between matrix production and degradation at a proteome-wide scale. Specific temporal dynamics of different matrisome proteins were found to correspond to the proliferative activity of the repopulating cells and the degree of extracellular deposition. The remodeling of the scaffold was characterized by an initial phase with cell proliferation and high production of cell adhesion proteins such as emilin-1 and fibronectin. Extended culture time resulted in increased levels of core matrisome proteins. In a comparison with monolayer cultures on plastic, culture in lung scaffolds lead to a pronounced accumulation of proteoglycans, such as versican and decorin, resulting in regeneration of an extracellular matrix with greater resemblance to native lung tissue compared to standard monolayer cultures. Collectively, the study presents a promising technique for increasing the understanding of cell- extracellular matrix interactions under healthy and diseased conditions.
Highlights
The human lung is continuously challenged with damaging agents such as microbes, allergens, polluted air and smoke
SILAC-mass spectrometry (MS) is a promising alternative for quantitative analysis of extracellular matrix (ECM) turnover, provided that ECM integrity is maintained after decellularization while cell remnants are sufficiently removed to allow for proper repopulation[36]
We used a recently described SILAC-MS strategy to quantify for the first time the ECM turnover in acellular lung scaffolds repopulated with primary lung fibroblasts, grown in media supplemented with stable heavy isotope labelled amino acids
Summary
The human lung is continuously challenged with damaging agents such as microbes, allergens, polluted air and smoke. Using decellularized vocal folds it was shown that immortalized vocal fold fibroblasts cultured with isotopically labelled amino acids produced heavy labelled versions of all newly synthesized proteins These heavy labelled, newly synthesized proteins were distinguishable from the resident proteins found in the acellular scaffolds using mass spectrometry (MS)[25]. Quantitative comparison of the heavy and light proteins provides a direct measurement of protein turnover in these repopulated scaffolds and represents a promising and emerging technology in tissue engineering In this communication, we have adapted the SILAC mass spectrometry strategy to investigate matrisome turnover by primary human lung fibroblasts in acellular scaffolds obtained from healthy human lung tissues. The outlined results reveal missing and important knowledge about the turnover of ECM in repopulated biological scaffolds and the complex interplay between ECM and tissue resident cells
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