Confocal and Histological Features After Poly(Ethylene Glycol) Diacrylate Corneal Inlay Implantation

Purpose: To evaluate the in vivo biocompatibility of photopolymerized poly(ethylene glycol) diacrylate (PEGDA) intrastromal inlays in rabbit corneas. Methods: Sixty-three eyes of 42 New Zealand rabbits were included. Manual intrastromal pockets were dissected in 42 eyes. PEGDA inlays were obtained using a specifically designed photomask and were inserted in the intrastromal pocket of 21 eyes (inlay group); the remaining 21 right eyes did not receive any implant (pocket-only group). Twenty-one eyes with no intervention were used as controls. In vivo confocal microscopy (IVCM) was performed at every visit. After 2 months, rabbits were sacrificed and corneas removed for histological analysis. Results: Corneas remained clear in all but two animals, and five cases of corneal neovascularization were seen (P = 0.2). Inlays remained stable without evidence of lateral or anterior migration, and no other complications were observed. No changes in anterior and posterior keratocyte density (P = 0.3 and P = 0.1, respectively) or endothelial cell density (P = 0.23) were observed between groups during the study time by IVCM. On pathology samples, thinning of the epithelium over the inlay area and epithelial hyperplasia over the edges were observed. A polygonal empty space with no evidence of PEGDA hydrogel within the midstroma was seen in the inlay group. Keratocytes were normal in shape and number in the vicinity of the PEGDA implant area. Conclusions: Photopolymerized PEGDA intrastromal inlays have shown relatively good safety and stability in rabbit corneas. Inlays were biostable in the corneal environment and remained transparent during follow up. Translational Relevance: The investigated PEGDA is promising for the development of biocompatible intrastromal implants.Supported in part by the Spanish Ministry of Science and Education through Apply Research, grant MAT2006-13708-CO2-01. The authors alone are responsible for the content and writing of the paper. Disclosure: A. Bidaguren, None; J. Mendicute, None; I. Madarieta, Tecnalia (E); N. Garagorri, Tecnalia (E

3D Cell Cultures as Prospective Models to Study Extracellular Vesicles in Cancer

The improvement of culturing techniques to model the environment and physiological conditions surrounding tumors has also been applied to the study of extracellular vesicles (EVs) in cancer research. EVs role is not only limited to cell-to-cell communication in tumor physiology, they are also a promising source of biomarkers, and a tool to deliver drugs and induce antitumoral activity. In the present review, we have addressed the improvements achieved by using 3D culture models to evaluate the role of EVs in tumor progression and the potential applications of EVs in diagnostics and therapeutics. The most employed assays are gel-based spheroids, often utilized to examine the cell invasion rate and angiogenesis markers upon EVs treatment. To study EVs as drug carriers, a more complex multicellular cultures and organoids from cancer stem cell populations have been developed. Such strategies provide a closer response to in vivo physiology observed responses. They are also the best models to understand the complex interactions between different populations of cells and the extracellular matrix, in which tumor-derived EVs modify epithelial or mesenchymal cells to become protumor agents. Finally, the growth of cells in 3D bioreactor-like systems is appointed as the best approach to industrial EVs production, a necessary step toward clinical translation of EVs-based therapy.The review is supported by Spanish Ministry of Science and Innovation, within the national Plan RTI2018-094969-B-I00, and Excellence Severo Ochoa grant Innovative Research Grant (SEV-2016- 0644), and by the European Union’s Horizon 2020 research and innovation program, grant number 860303

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

Macrophages, cells effective in sensing, internalizing and killing Candida albicans, are intertwined with the extracellular matrix (ECM) through different signals, which include the release of specific cytokines. Due to the importance of these interactions, the employment of in vitro models mimicking a fungal infection scenario is essential to evaluate the ECM effects on the macrophage response. In this work, we have analyzed the effects of human and porcine decellularized adipose matrices (DAMs), obtained by either enzymatic or organic solvent treatment, on the macrophage/Candida albicans interface. The present study has allowed us to detect differences on the activation of macrophages cultured on either human- or porcine-derived DAMs, evidencing changes in the macrophage actin cytoskeleton, such as distinct F-actin-rich membrane structures to surround the pathogen. The macrophage morphological changes observed on these four DAMs are key to understand the defense capability of these cells against this fungal pathogen. This work has contributed to the knowledge of the influence that the extracellular matrix and its components can exert on macrophage metabolism, immunocompetence and capacity to respond to the microenvironment in a possible infection scenario.This work has been supported by the European Union’s Horizon 2020 Research and Innovation Programme (H2020-FETOPEN-2018-2020, NeuroStimSpinal Project, Grant AgreementNo. 829060). M.C. acknowledges the European Union0s Horizon 2020 Research and InnovationProgramme for her contract under the NeuroStimSpinal Project. LC is grateful to the Universidad Complutense de Madrid for a UCM fellowshi

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

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.This work has been supported by the European Union’s Horizon 2020 Research and Innovation Programme (H2020-FETOPEN-2018-2020, NeuroStimSpinal Project, Grant Agreement No. 829060). M.C. acknowledges the European Union0s Horizon 2020 Research and Innovation Programme for her contract under the NeuroStimSpinal Project. LC is grateful to the Universidad Complutense de Madrid for an UCM fellowship

Enhanced Adipogenic Differentiation of Human Dental Pulp Stem Cells in Enzymatically Decellularized Adipose Tissue Solid Foams

Engineered 3D human adipose tissue models and the development of physiological human 3D in vitro models to test new therapeutic compounds and advance in the study of pathophysiological mechanisms of disease is still technically challenging and expensive. To reduce costs and develop new technologies to study human adipogenesis and stem cell differentiation in a controlled in vitro system, here we report the design, characterization, and validation of extracellular matrix (ECM)-based materials of decellularized human adipose tissue (hDAT) or bovine collagen-I (bCOL-I) for 3D adipogenic stem cell culture. We aimed at recapitulating the dynamics, composition, and structure of the native ECM to optimize the adipogenic differentiation of human mesenchymal stem cells. hDAT was obtained by a two-enzymatic step decellularization protocol and post-processed by freeze-drying to produce 3D solid foams. These solid foams were employed either as pure hDAT, or combined with bCOL-I in a 3:1 proportion, to recreate a microenvironment compatible with stem cell survival and differentiation. We sought to investigate the effect of the adipogenic inductive extracellular 3D-microenvironment on human multipotent dental pulp stem cells (hDPSCs). We found that solid foams supported hDPSC viability and proliferation. Incubation of hDPSCs with adipogenic medium in hDAT-based solid foams increased the expression of mature adipocyte LPL and c/EBP gene markers as determined by RT-qPCR, with respect to bCOL-I solid foams. Moreover, hDPSC capability to differentiate towards adipocytes was assessed by PPAR-γ immunostaining and Oil-red lipid droplet staining. We found out that both hDAT and mixed 3:1 hDAT-COL-I solid foams could support adipogenesis in 3D-hDPSC stem cell cultures significantly more efficiently than solid foams of bCOL-I, opening the possibility to obtain hDAT-based solid foams with customized properties. The combination of human-derived ECM biomaterials with synthetic proteins can, thus, be envisaged to reduce fabrication costs, thus facilitating the widespread use of autologous stem cells and biomaterials for personalized medicine.This research was funded by the Basque Government (IT1751-22; to G.I.; ELKARTEK program 566 PLAKA KK-2019-00093; to N.B.), the Health Department of the Basque Government (grant No. 2021333012; to J.R.P.), and grant No. RYC-2013-13450 and grant No. PID2019-104766RB-C21 funded by MCIN/AEI/10.13039/501100011033 by the European Union (NextGenerationEU) “Plan de Recuperación Transformación y Resiliencia” (grants to J.R.P.)

Osteogenic differentiation of human dental pulp stem cells in decellularised adipose tissue solid foams

3D cell culture systems based on biological scaffold materials obtainable from both animal and human tissues constitute very interesting tools for cell therapy and personalised medicine applications. The white adipose tissue (AT) extracellular matrix (ECM) is a very promising biomaterial for tissue engineering due to its easy accessibility, malleability and proven biological activity. In the present study, human dental pulp stem cells (hDPSCs) were combined in vitro with ECM scaffolds from porcine and human decellularised adipose tissues (pDAT, hDAT) processed as 3D solid foams, to investigate their effects on the osteogenic differentiation capacity and bone matrix production of hDPSCs, compared to single-protein-based 3D solid foams of collagen type I and conventional 2D tissue-culture-treated polystyrene plates. pDAT solid foams supported the osteogenic differentiation of hDPSCs to similar levels to collagen type I, as assessed by alkaline phosphatase and alizarin red stainings, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and osteocalcin/bone gamma-carboxyglutamate protein (BGLAP) immunostaining. Interestingly, hDAT solid foams showed a markedly lower capacity to sustain hDPSC osteogenic differentiation and matrix calcification and a higher capacity to support adipogenesis, as assessed by RT-qPCR and oil red O staining. White ATs from both human and porcine origins are relatively abundant and available sources of raw material to obtain high quality ECM-derived biomedical products. These biomaterials could have promising applications in tissue engineering and personalised clinical therapy for the healing and regeneration of lesions involving not only a loss of calcified bone but also its associated soft non-calcified tissues.This research was supported by the Basque Government (ELKARTEK program PLAKA KK- 2019-00093; to NB), MICINN retos I+D+i (PID2019- 104766RB-C21, to JRP) and UPV/EHU (PPGA20/22; to FU, GI). The authors would like to thank the staff members of the SGIKER services of the UPV/EHU: Lipidomic service (Beatriz Abad) and Analytical Microscopy (Ricardo Andrade, Alejandro Díez-Torre and Irene Fernández) for their technical assistance

Wound-Microenvironment Engineering through Advanced-Dressing Bioprinting

In patients with comorbidities, a large number of wounds become chronic, representing an overwhelming economic burden for healthcare systems. Engineering the microenvironment is a paramount trend to activate cells and burst-healing mechanisms. The extrusion bioprinting of advanced dressings was performed with novel composite bioinks made by blending adipose decellularized extracellular matrix with plasma and human dermal fibroblasts. Rheological and microstructural assessments of the composite hydrogels supported post-printing cell viability and proliferation over time. Embedded fibroblasts expressed steady concentrations of extracellular matrix proteins, including type 1, 3 and 4 collagens and fibronectin. ELISA assessments, multiplex protein arrays and ensuing bioinformatic analyses revealed paracrine activities corresponding to wound-healing activation through the modulation of inflammation and angiogenesis. The two modalities of advanced dressings, differing in platelet number, showed differences in the release of inflammatory and angiogenic cytokines, including interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conditioned media stimulated human-dermal-cell proliferation over time. Our findings open the door to engineering the microenvironment as a strategy to enhance healing