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.

Applications of decellularized extracellular matrix in bone and cartilage tissue engineering

“…Zwitterionic detergents such as 3‐([3‐cholamidopropyl] dimethylammonio)‐1‐propanesulfonate (CHAPS) have a net zero charge and show characteristics of both ionic and nonionic detergents . Although zwitterionic detergents result in less denaturing of proteins compared to ionic detergents, they also tend to remove less cellular material than ionic detergents …”

“…15 Although zwitterionic detergents result in less denaturing of proteins compared to ionic detergents, they also tend to remove less cellular material than ionic detergents. 41,42…”

Water‐insoluble amorphous silk fibroin scaffolds from aqueous solutions

“…These changes suggested that further assembly occurred during the incubation procedure and freezing process at higher temperature, resulting in more nanofiber formation. Similar to acid-assisted RSF scaffold formation, the extracellular matrix-mimetic nanofibrous structure assembled in the TS scaffolds, which would provide favorable cues for cell growth and proliferation (Calle et al, 2016;Ding et al, 2017;Gu et al, 2014;Han et al, 2016). Flt-1 CGGAGAAATCTGCTC GCTATCTTGGAAGGGA CGACACG attributed to amorphous states (Bai et al, 2015;Sang et al, 2018;Wang et al, 2016).…”

Scarring vs. functional healing: Matrix-based strategies to regulate tissue repair

“…Quantitative proteomics provides a tool that will likely improve the characterization and quality control of tissue derived scaffolds. A recent study described the use of proteomic methods to enable refinement of processing methods to attain high fidelity ECM constructs from lung tissue [158]. Still, heterogeneity between donor tissues can result in heterogeneity in decellularization.…”