Effects of Human and Porcine Adipose Extracellular Matrices Decellularized by Enzymatic or Chemical Methods on Macrophage Polarization and Immunocompetence

Abstract

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage’s functional role.

Candida albicans/Macrophage Biointerface on Human and Porcine Decellularized Adipose Matrices

“…Our group has previously published the effective decellularization and delipidation of porcine and human AT by two different methods, enzymatic (method 1) and organic solvent (method 2). [ 34 ]. All the resultant DAMs (hDAM1, pDAM1, hDAM2 and pDAM2) meet the decellularization criteria stablished by the research community (less than 50 ng DNA/mg dry weight) and the delipidation was also effective.…”

“…Tissues were decellularized following the methodology previously published by our group [ 34 ]. Briefly, protein pellets were obtained by homogenization (Polytron PT3100) at 12,000 rpm for 5 min, centrifugation (5000 rpm for 5 min) with ultrapure water and manual discarding of lipids conserving the protein pellets.…”

“…DAMs were characterized for remnant DNA and lipids as well as for protein composition following the methodology previously described by our group [ 34 ]. Briefly, DNA was extracted from the DAMs (QiAmp kit, Qiagen, Madrid, Spain) and absolute quantification was done by quantitative real-time polymerase chain reaction (qRT-PCR) and based on standard curves of known human and porcine DNA samples.…”

“…Milled DAMs were used to prepare coatings following the methodology previously published by our group [ 34 ]. Briefly, milled DAMs were dissolved in 1M acetic acid (Panreac Quimica SLU, Barcelona, Spain); the solutions were added to polystyrene plates and glass coverslips (5 min room temperature), washed (3 × 5 min) with ultrapure Milli-Q water (Millipore) and dried (room temperature, 2 h).…”

“…In this work, we have prepared coatings of four DAMs obtained by two different treatments from human and porcine AT, following a methodology previously published by our group [ 34 ], and analyzed their effect on the macrophage/ Candida albicans interface. Thus, the objective of this study is to know how macrophages exposed to biomaterials prepared with these DAMs would respond in a fungal infection scenario.…”