A Psychrophilic GelMA: Breaking Technical and Immunological Barriers for Multimaterial High-Resolution 3D Bioprinting.

The increasing demand for tissue replacement has encouraged scientists worldwide to focus on developing new biofabrication technologies. Multimaterials/cells printed with stringent resolutions are necessary to address the high complexity of tissues. Advanced inkjet 3D printing can use multimaterials and attain high resolution and complexity of printed structures. However, a decisive yet limiting aspect of translational 3D bioprinting is selecting the befitting material to be used as bioink; there is a complete lack of cytoactive bioinks with adequate rheological, mechanical, and reactive properties. This work strives to achieve the right balance between resolution and cell support through methacrylamide functionalization of a psychrophilic gelatin and new fluorosurfactants used to engineer a photo-cross-linkable and immunoevasive bioink. The syntonized parameters following optimal formulation conditions allow proficient printability in a PolyJet 3D printer comparable in resolution to a commercial synthetic ink (∼150 µm). The bioink formulation achieved the desired viability (∼80%) and proliferation of co-printed cells while demonstrating in vivo immune tolerance of printed structures. The practical usage of existing high-resolution 3D printing systems using a novel bioink is shown here, allowing 3D bioprinted structures with potentially unprecedented complexity.

Lactate metabolism coordinates macrophage response and regeneration in zebrafish.

Rationale: Macrophages are multifunctional cells with a pivotal role on tissue development, homeostasis and regeneration. Indeed, in response to tissue injury and the ensuing regeneration process, macrophages are challenged and undergo massive metabolic adaptations and changes. However, the control of this metabolic reprogramming by macrophage microenvironment has never been deciphered in vivo. Methods: In this study, we used zebrafish model and caudal fin resection as a robust regeneration system. We explored specific changes in gene expression after tissue amputation via single-cell RNA sequencing analysis and whole-tissue transcriptomic analysis. Based on the identification of key modifications, we confirmed the role of the lactate pathway in macrophage response and fin regeneration, through the combination of chemical and genetic inhibitors of this pathway. Results: Single cell RNA sequencing revealed the upregulation of different genes associated with glycolysis and lactate metabolism in macrophages, upon fin regeneration. Hence, using chemical inhibitors of the LDH enzyme, we confirmed the role of lactate in macrophage recruitment and polarization, to promote a pro-inflammatory phenotype and enhance fin regeneration. The genetic modulation of monocarboxylate transporters illustrated a complex regulation of lactate levels, based on both intracellular and extracellular supplies. Commonly, the different sources of lactate resulted in macrophage activation with an increased expression level of inflammatory cytokines such as TNFa during the first 24 hours of regeneration. Transcriptomic analyses confirmed that lactate induced a global modification of gene expression in macrophages. Conclusion: Altogether, our findings highlight the crucial role of lactate at the onset of macrophage differentiation toward a pro-inflammatory phenotype. The deep modifications of macrophage phenotype mediated by lactate and downstream effectors play a key role to coordinate inflammatory response and tissue regeneration.

PPARß/δ priming enhances the anti-apoptotic and therapeutic properties of mesenchymal stromal cells in myocardial ischemia-reperfusion injury.

BACKGROUND: Mesenchymal Stromal Cells (MSC) have been widely used for their therapeutic properties in many clinical applications including myocardial infarction. Despite promising preclinical results and evidences of safety and efficacy in phases I/ II, inconsistencies in phase III trials have been reported. In a previous study, we have shown using MSC derived from the bone marrow of PPARß/δ (Peroxisome proliferator-activated receptors ß/δ) knockout mice that the acute cardioprotective properties of MSC during the first hour of reperfusion are PPARß/δ-dependent but not related to the anti-inflammatory effect of MSC. However, the role of the modulation of PPARß/δ expression on MSC cardioprotective and anti-apoptotic properties has never been investigated. OBJECTIVES: The aim of this study was to investigate the role of PPARß/δ modulation (inhibition or activation) in MSC therapeutic properties in vitro and ex vivo in an experimental model of myocardial infarction. METHODS AND RESULTS: Naïve MSC and MSC pharmacologically activated or inhibited for PPARß/δ were challenged with H2O2. Through specific DNA fragmentation quantification and qRT-PCR experiments, we evidenced in vitro an increased resistance to oxidative stress in MSC pre-treated by the PPARß/δ agonist GW0742 versus naïve MSC. In addition, PPARß/δ-priming allowed to reveal the anti-apoptotic effect of MSC on cardiomyocytes and endothelial cells in vitro. When injected during reperfusion, in an ex vivo heart model of myocardial infarction, 3.75 × 105 PPARß/δ-primed MSC/heart provided the same cardioprotective efficiency than 7.5 × 105 naïve MSC, identified as the optimal dose in our experimental model. This enhanced short-term cardioprotective effect was associated with an increase in both anti-apoptotic effects and the number of MSC detected in the left ventricular wall at 1 h of reperfusion. By contrast, PPARß/δ inhibition in MSC before their administration in post-ischemic hearts during reperfusion decreased their cardioprotective effects. CONCLUSION: Altogether these results revealed that PPARß/δ-primed MSC exhibit an increased resistance to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARß/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the therapeutic injected doses. These results could be of major interest to improve MSC efficacy for the cardioprotection of injured myocardium in AMI patients.

Author Correction: Rapid fabrication of reinforced and cell-laden vascular grafts structurally inspired by human coronary arteries.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rapid fabrication of reinforced and cell-laden vascular grafts structurally inspired by human coronary arteries.

Design strategies for small diameter vascular grafts are converging toward native-inspired tissue engineered grafts. A new automated technology is presented that combines a dip-spinning methodology for depositioning concentric cell-laden hydrogel layers, with an adapted solution blow spinning (SBS) device for intercalated placement of aligned reinforcement nanofibres. This additive manufacture approach allows the assembly of bio-inspired structural configurations of concentric cell patterns with fibres at specific angles and wavy arrangements. The middle and outer layers were tuned to structurally mimic the media and adventitia layers of native arteries, enabling the fabrication of small bore grafts that exhibit the J-shape mechanical response and compliance of human coronary arteries. This scalable automated system can fabricate cellularized multilayer grafts within 30 min. Grafts were evaluated by hemocompatibility studies and a preliminary in vivo carotid rabbit model. The dip-spinning-SBS technology generates constructs with native mechanical properties and cell-derived biological activities, critical for clinical bypass applications.

Cold-adaptation of a methacrylamide gelatin towards the expansion of the biomaterial toolbox for specialized functionalities in tissue engineering.

Tissue regeneration is witnessing a significant surge in advanced medicine. It requires the interaction of scaffolds with different cell types for efficient tissue formation post-implantation. The presence of tissue subtypes in more complex organs demands the co-existence of different biomaterials showing different hydrolysis rate for specialized cell-dependent remodeling. To expand the available toolbox of biomaterials with sufficient mechanical strength and variable rate of enzymatic degradation, a cold-adapted methacrylamide gelatin was developed from salmon skin. Compared with mammalian methacrylamide gelatin (GelMA), hydrogels derived from salmon GelMA displayed similar mechanical properties than the former. Nevertheless, salmon gelatin and salmon GelMA-derived hydrogels presented characteristics common of cold-adaptation, such as reduced activation energy for collagenase, increased enzymatic hydrolysis turnover of hydrogels, increased interconnected polypeptides molecular mobility and lower physical gelation capability. These properties resulted in increased cell-remodeling rate in vitro and in vivo, proving the potential and biological tolerance of this mechanically adequate cold-adapted biomaterial as alternative scaffold subtypes with improved cell invasion and tissue fusion capacity.

IFN-γ and IL-33 modulate mesenchymal stem cells function targeting Th1/Th17 axis in a murine skin transplantation model.

The immune regulatory properties of IL-33 have indicated that this cytokine has the capacity to target several immune cells under a variety of immunological responses, including overt inflammation and tolerance. Due to its versatile mechanistics, we sought to investigate the role of IL-33 on mesenchymal stem cells (MSC), a population of cells with recognizable modulatory functions. Our data indicates that IL-33 does not affect the expression of classical MSC markers such as CD29, CD44 and CD73, or the lack of CD45, CD11b and CD117. Also, we found that IL-33 greatly induces iNOS expression and stimulates the secretion of TGF-ß and IL-6. Next, we decided to test IFN-γ/IL-33-treated MSC using a skin transplantation model. Our data indicate that allogeneic skin-grafted animals treated with IFN-γ/IL-33-modulated MSC reject as controls. Complementing this finding, we observed that ex vivo re-stimulated draining lymph nodes (dLN) cells from these mice secrete lower amounts of IFN-γ and a slightly higher amount of IL-17. Beside a reduction in CD4+ and CD8+ T cells number, we preliminarily found an increment in the frequencies of CD4+Foxp3+IL-17+ T cells. Altogether, our data propose that IL-33 and IFN-γ modulate MSC phenotype and function, most likely targeting Th1/Th17 axis.

Mesenchymal stem cells and their immunosuppressive role in transplantation tolerance.

Since they were first described, mesenchymal stem cells (MSCs) have been shown to have important effector mechanisms and the potential for use in cell therapy. A great deal of research has been focused on unveiling how MSCs contribute to anti-inflammatory responses, including describing several cell populations involved and identifying soluble and other effector molecules. In this review, we discuss some of the contemporary evidence for use of MSCs in the field of immune tolerance, with a special emphasis on transplantation. Although considerable effort has been devoted to understanding the biological function of MSCs, additional resources are required to clarify the mechanisms of their induction of immune tolerance, which will undoubtedly lead to improved clinical outcomes for MSC-based therapies.

IL-33 enhances retinoic acid signaling on CD4+ T cells.

Several molecules have been described as CD4+ T cells differentiation modulators and among them retinoic acid (RA) and more recently, IL-33, have been studied. Due to the similarities in T helper cell skewing properties between RA and IL-33, we asked whether IL-33 intersects, directly or indirectly, the RA signaling pathway. Total CD4+ T cells from DR5-luciferase mice were activated in the presence of RA with or without IL-33, and RA signaling was visualized using ex vivo imaging. Our results demonstrate that IL-33 itself is able to trigger RA signaling on CD4+ T cells, which is highly increased when IL-33 is added in conjunction with RA. This study presents IL-33 as a potential player that may synergize with RA in controlling T cell differentiation, and suggests that IL-33 may be an attractive target in controlling T cell differentiation in vivo.

Alarmin' Immunologists: IL-33 as a Putative Target for Modulating T Cell-Dependent Responses.

IL-33 is a known member of the IL-1 cytokine superfamily classically named "atypical" due to its diverse functions. The receptor for this cytokine is the ST2 chain (or IL-1RL1), part of the IL-1R family, and the accessory chain IL-1R. ST2 can be found as both soluble and membrane-bound forms, property that explains, at least in part, its wide range of functions. IL-33 has increasingly gained our attention as a potential target to modulate immune responses. At the beginning, it was known as one of the participants during the development of allergic states and other Th2-mediated responses and it is now accepted that IL-33 contributes to Th1-driven pathologies as demonstrated in animal models of experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis, and trinitrobenzene sulfonic acid-induced experimental colitis, among others. Interestingly, current data are placing IL-33 as a novel regulator of immune tolerance by affecting regulatory T cells (Tregs); although the mechanism is not fully understood, it seems that dendritic cells and myeloid suppressor-derived cells may be cooperating in the generation and/or establishment of IL-33-mediated tolerance. Here, we review the most updated literature on IL-33, its role on T cell biology, and its impact in immune tolerance.