Proteomic analysis of naturally-sourced biological scaffolds

Abstract

A key challenge to the clinical implementation of decellularized scaffold-based tissue engineering lies in understanding the process of removing cells and immunogenic material from a donor tissue/organ while maintaining the biochemical and biophysical properties of the scaffold that will promote growth of newly seeded cells. Current criteria for evaluating whole organ decellularization are primarily based on nucleic acids, as they are easy to quantify and have been directly correlated to adverse host responses. However, numerous proteins cause immunogenic responses and thus should be measured directly to further understand and quantify the efficacy of decellularization. In addition, there has been increasing appreciation for the role of the various protein components of the extracellular matrix (ECM) in directing cell growth and regulating organ function. We performed in-depth proteomic analysis on four types of biological scaffolds and identified a large number of both remnant cellular and ECM proteins. Measurements of individual protein abundances during the decellularization process revealed significant removal of numerous cellular proteins, but preservation of most structural matrix proteins. The observation that decellularized scaffolds still contain many cellular proteins, although at decreased abundance, indicates that elimination of DNA does not assure adequate removal of all cellular material. Thus, proteomic analysis provides crucial characterization of the decellularization process to create biological scaffolds for future tissue/organ replacement therapies.

Keywords: Decellularized human lung; Decellularized rat liver; Mass spectrometry; Matrigel; Matrisome; Proteomics.

Figure 1

Proteomic results for GFR-Matrigel samples. A) Numbers of proteins and unique peptides identified, along with the percentage of iBAQ intensity (iBAQ%) arising from matrisome proteins. B) Under the condition of 1.7 μm column and 6 peptide fractions, the number of proteins and the percentage of iBAQ intensity for each matrisome subcategory. n=3 biological replicates.

Figure 2

Proteomic results and DNA content for the naïve, partially-decellularized (part-decell), and decellularized (decell) rat livers.

Figure 3

Quantitative proteomic results for rat livers. (A) Images of naïve, part-decell, and decell livers (scale bar: 10 mm), along with a heatmap showing changes in protein abundances for these three conditions. Numbers in the color code are log-transformed-corrected LFQ intensity. (B) Volcano plot showing quantitative comparison of the naïve versus the decell rat livers. The purple rectangle denotes cutoff criteria for significant removal of a protein in the decell sample compared to the naïve sample (fold change < 1/8; Benjamini Hochberg-adjusted p-value < 0.01). The orange rectangle encompasses the proteins that are considered retained in the decell sample (fold change > 1/8). Four proteins that were chosen for further validation are highlighted in the volcano plot. n=3 biological replicates.

Figure 4

Western blots of four proteins – FN1, SERPINA3K, FLNA, GAPDH – in the naïve, partially-decellularized (part-decell), and decellularized (decell) rat livers. n=3 biological replicates.

Figure 5

Proteomic results for naïve and decellularized (decell) human lung. Both samples were subjected to peptide pre-fractionation (6 fractions).

Figure 6

Venn diagram with the number of matrisome protein identifications for rat-tail type I collagen, GFR-Matrigel, and decellularized rat liver. Note that the GFR-Matrigel samples were subjected to peptide fractionation (6 fractions), while the collagen gel and rat liver samples were not, which could account for the relatively large number of proteins only detected in the GFR-Matrigel sample.

Figure 7

Matrisome compositions by sub-category for decellularized rat liver and decellularized human lung.