Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e., bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species’ scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species’ scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use.

Notwithstanding their wide exploitation, biological prosthetic heart valves are characterized by limited durability (10–15 years). The treatment of biological tissues with chemical crosslinking agents such as glutaraldehyde accounts for the enhanced risk of structural deterioration associated with the early failure of bioprosthetic valves. To overcome the shortcomings of the currently available solutions, adoption of decellularized biological tissues of animal origin has emerged as a promising approach. The present study aims to assess in vitro cardiovascular scaffolds composed of bovine pericardium decellularized with the novel TRITDOC (TRIton-X100 and TauroDeOxyCholic acid) procedure. The effects of the treatment have been assessed by means of histological, biomolecular, cellular, biochemical and biomechanical analyses. The TRITDOC procedure grants the complete decellularization of bovine pericardial scaffolds while preserving the extracellular matrix architecture and the biomechanical properties. With a dedicated ELISA test, the TRITDOC procedure has been proven to ensure the complete removal of the alphaGal antigen, responsible for hyperacute rejection and for long-term deterioration of xenogenic biomaterials. Static seeding of the acellular pericardial patches with human adipose-derived stem cells resulted in an evenly repopulated scaffold without signs of calcification. The in vitro cyto-/immuno-compatibility response of the TRITDOC-bovine pericardium was compared with glutaraldehyde-treated xenogenic pericardium collected from two bioprosthetic devices currently used in clinical practice: PERIMOUNT MAGNA and TRIFECTA ^TM . TRITDOC-bovine pericardium exhibited lower complement activation, lower cytotoxicity and a lower tendency to secrete pro-inflammatory cytokines compared to the tested commercial bioprostheses. Therefore, TRITDOC-decellularized pericardium could be considered as possible candidate material for the production of prosthetic heart valves.

Functional kleptoplasty is a photosymbiotic relationship, in which photosynthetically active chloroplasts serve as an intracellular symbiont for a heterotrophic host. Among Metazoa, functional kleptoplasty is only found in marine sea slugs belonging to the Sacoglossa and recently described in Rhabdocoela worms. Although functional kleptoplasty has been intensively studied in Sacoglossa, the fundamentals of the specific recognition of the chloroplasts and their subsequent incorporation are unknown. The key to ensure the initiation of any symbiosis is the ability to specifically recognize the symbiont and to differentiate a symbiont from a pathogen. For instance, in photosymbiotic cnidarians, several studies have shown that the host innate immune system, in particular scavenger receptors (SRs) and thrombospondin‐type‐1 repeat (TSR) protein superfamily, is playing a major role in the process of recognizing and differentiating symbionts from pathogens. In the present study, SRs and TSRs of three Sacoglossa sea slugs, Elysia cornigera, Elysia timida, and Elysia chlorotica, were identified by translating available transcriptomes into potential proteins and searching for receptor specific protein and/or transmembrane domains. Both receptors classes are highly diverse in the slugs, and many new domain arrangements for each receptor class were found. The analyses of the gene expression of these three species provided a set of species‐specific candidate genes, that is, SR‐Bs, SR‐Es, C‐type lectins, and TSRs, that are potentially relevant for the recognition of kleptoplasts. The results set the base for future experimental studies to understand if and how these candidate receptors are indeed involved in chloroplast recognition.

Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. Herein, we propose the use of supercritical carbon dioxide (scCO₂) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cell’s own ECM, to be used in the preparation of bioinks. The ECM extraction protocol best fitted for cell sheets was defined by considering efficient DNA removal with a minor effect on the ECM. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs) were processed using a customised supercritical system by varying the pressure of the reactor, presence, exposure time, and type of co-solvent. A quantification of the amount of DNA, protein, and sulfated glycosaminoglycans (sGAGs) was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. The bioinks containing the extracted ECM were fabricated by combining them with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amounts on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cell viability was assessed. The optimised scCO₂ protocol allowed efficient removal of DNA while preserving a higher number of proteins and sGAGs than the standard methodologies. The characterization of extract’s composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) is distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source. Overall, this work highlights the benefits of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs.