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Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.


ABSTRACT: Extracellular matrix is a key component of many products in regenerative medicine. Multiple regenerative medicine products currently in the clinic are comprised of human or xenogeneic extracellular matrix. In addition, whole-organ regeneration exploits decellularized native organs as scaffolds for organotypic cell culture. However, precise understanding of the constituents of such extracellular matrix-based implants and scaffolds has sorely lagged behind their use. We present here an advanced protein extraction method using known quantities of proteotypic 13C-labeled peptides to quantify matrix proteins in native and decellularized lung tissues. Using quantitative proteomics that produce picomole-level measurements of a large number of matrix proteins, we show that a mild decellularization technique ("Triton/SDC") results in near-native retention of laminins, proteoglycans, and other basement membrane and ECM-associated proteins. Retention of these biologically important glycoproteins and proteoglycans is quantified to be up to 27-fold higher in gently-decellularized lung scaffolds compared to scaffolds generated using a previously published decellularization regimen. Cells seeded onto this new decellularized matrix also proliferate robustly, showing positive staining for proliferating cell nuclear antigen (PCNA). The high fidelity of the gently decellularized scaffold as compared to the original lung extracellular matrix represents an important step forward in the ultimate recapitulation of whole organs using tissue-engineering techniques. This method of ECM and scaffold protein analysis allows for better understanding, and ultimately quality control, of matrices that are used for tissue engineering and human implantation. These results should advance regenerative medicine in general, and whole organ regeneration in particular. STATEMENT OF SIGNIFICANCE:The extracellular matrix (ECM) in large part defines the biochemical and mechanical properties of tissues and organs; these inherent cues make acellular ECM scaffolds potent substrates for tissue regeneration. As such, they are increasingly prevalent in the clinic and the laboratory. However, the exact composition of these scaffolds has been difficult to ascertain. This paper uses targeted proteomics to definitively quantify 71 proteins present in acellular lung ECM scaffolds. We use this technique to compare two decellularization methods and demonstrate superior retention of ECM proteins important for cell adhesion, migration, proliferation, and differentiation in scaffolds treated with low-concentration detergent solutions. In the long term, the ability to acquire quantitative biochemical data about biological substrates will facilitate the rational design of engineered tissues and organs based on precise cell-matrix interactions.

SUBMITTER: Calle EA 

PROVIDER: S-EPMC5451113 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.

Calle Elizabeth A EA   Hill Ryan C RC   Leiby Katherine L KL   Le Andrew V AV   Gard Ashley L AL   Madri Joseph A JA   Hansen Kirk C KC   Niklason Laura E LE  

Acta biomaterialia 20160929


Extracellular matrix is a key component of many products in regenerative medicine. Multiple regenerative medicine products currently in the clinic are comprised of human or xenogeneic extracellular matrix. In addition, whole-organ regeneration exploits decellularized native organs as scaffolds for organotypic cell culture. However, precise understanding of the constituents of such extracellular matrix-based implants and scaffolds has sorely lagged behind their use. We present here an advanced pr  ...[more]

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