Processed Human Extracellular Matrix Coating of Human Amniotic Epicardial Patch Material Modulates Its Immunomodulatory Capacity

Objectives: The epicardium is a novel target of cardiac regenerative strategies. We previously demonstrated in vivo that the epicardial application of cell-free human amniotic membrane (DecellAM) supports the recovery of the myocardium following infarction and also developed a hydrogel based on decellularized human cardiac extracellular matrix (hECM) that exerts specific cytoprotective effects. Here, we created a DecellAM/cECM composite patch material and tested its biologic activity in vitro

Methods: Amniotic membrane was isolated from human placenta, decellularized via Tris/EDTA and SDS incubation, and lyophilized. Human myocardium from explanted hearts was decellularized using a specific three-step protocol, characterized, and processed to a hydrogel via lyophilization, milling and pepsin digestion. DecellAM samples were then reconstituted with hECM hydrogel and incubated at 37°C for dry coating (dcAM) or hcECM hydrogel formation (gelAM). Adhesion and cell death of cardiac fibroblasts (CF) were determined via Calcein staining and LDH release. Cytokine secretion and surface marker expression of stimulated and naive monocytes incubated with drAM or gelAM were analyzed, as was macrophage polarization via ELISA and flow cytometry.

Results: hcECM processing preserved its cytoprotective and differentiation-guiding effects on iPSC and HL-1 cells. Whereas “naked” DecellAM patch material only allowed for limited adhesion of CF, both dcAM and gelAM displayed significantly enhanced adhesion-promoting properties. LDH release was not influenced by the presence hcECM. Macrophage polarization assays (CD80/CD163/CD206) indicated no polarization toward the pro-inflammatory M1 state with either dcAM or gelAM. Monocyte secretion of pro-inflammatory cytokines (IL-6, TNFα) was significantly reduced in presence of hcECM, and most prominently so when cultured on dcAM. This effect was even more pronounced when lipopolysaccharide-activated monocytes were used. Similarly, T cell secretion of IL-6 and TNFα was significantly reduced by dcAM.

Conclusion: The immunomodulatory properties of decellularized amniotic membrane are further improved by dry coating with processed hcECM. This novel composite material holds promise for epicardial applications to prevent remodelling and induce tissue regeneration.