Comparative proteomic analyses of human adipose extracellular matrices decellularized using alternative procedures

Abstract

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting, and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for deoxyribose nucleic acid depletion, extracellular matrix composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy. Mass spectrometry based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP⁺ transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2481-2493, 2018.

Keywords: adipose tissue; bioscaffold; decellularization; extracellular matrix; mass spectrometry proteomics; regenerative medicine.

Figure 1

Microscopic Analysis of Untreated and Decellularized Adipose Tissue Samples. Untreated control tissue and the products of the M1, M2, and M3 decellularization procedures were evaluated by in H&E staining (top), Masson’s Trichrome stain (middle), and Scanning Electron Microscopy (SEM) (shown at 1500 X magnification). Individual images are representative of at least n = 3.

Figure 2

Immunofluorescent staining for collagen VI, actin, and vitronectin. Paraffin fixed slides prepared with tissue from untreated controls or scaffolds decellularized using methods M1 and M2 were stained with fluorochrome labeled antibodies to collagen VI, actin, and vitronectin. Images are representative of n =3.

Figure 3

Subcellular Localization of the Top Twenty Peptides (based on number of hits) in Each Decellularized Scaffold Relative to Untreated Tissue Control.

Figure 4

Immunohistochemical Detection of Adipogenic Biomarker (Perilipin) and Host Cells (GFP) in M1 and M2 Tissue Scaffold In Vivo Implants. Scaffolds were implanted into C57BL/6 mice transgenic for ubiquitous expression of the green fluorescent protein (GFP) for periods of 3, 6, or 9 weeks (top, middle, or bottom panels). A native adipose tissue served as positive controls for both antibodies.

Figure 5

Immunofluorescent Detection of CD 31. demonstrated the vascularization of the implanted scaffolds denoting their integration. Scaffolds were implanted into C57BL/6 mice transgenic for ubiquitous expression of the green fluorescent protein (GFP) for periods of 3 or 6 weeks. A silk scaffold implant and native adipose tissue served as positive controls. Sections were stained with DAPI for detection of nuclei (blue) or anti-CD31 fluorochrome labeled antibodies (red).