Regenerative medicine: the red planet for clinicians

Regenerative medicine represents the forefront of health sciences and holds promises for the treatment and, possibly, the cure of a number of challenging conditions. It relies on the use of stem cells, tissue engineering, and gene therapy alone or in different combinations. The goal is to deliver cells, tissues, or organs to repair, regenerate, or replace the damaged ones. Among stem-cell populations, both haematopoietic and mesenchymal stem cells have been employed in the treatment of refractory chronic inflammatory diseases with promising results. However, only mesenchymal stem cells seem advantageous as both systemic and local injections may be performed without the need for immune ablation. Recently, also induced pluripotent stem cells have been exploited for therapeutic purposes given their tremendous potential to be an unlimited source of any tissue-specific cells. Moreover, through the development of technologies that make organ fabrication possible using cells and supporting scaffolding materials, regenerative medicine promises to enable organ-on-demand, whereby patients will receive organs in a timely fashion without the risk of rejection. Finally, gene therapy is emerging as a successful strategy not only in monogenic diseases, but also in multifactorial conditions. Several of these approaches have recently received approval for commercialization, thus opening a new therapeutic era. This is why both General Practitioners and Internists should be aware of these great advancements

Resealable, optically accessible, PDMS-free fluidic platform for ex vivo interrogation of pancreatic islets

We report the design and fabrication of a robust fluidic platform built out of inert plastic materials and micromachined features that promote optimized convective fluid transport. The platform is tested for perfusion interrogation of rodent and human pancreatic islets, dynamic secretion of hormones, concomitant live-cell imaging, and optogenetic stimulation of genetically engineered islets. A coupled quantitative fluid dynamics computational model of glucose stimulated insulin secretion and fluid dynamics was first utilized to design device geometries that are optimal for complete perfusion of three-dimensional islets, effective collection of secreted insulin, and minimization of system volumes and associated delays. Fluidic devices were then fabricated through rapid prototyping techniques, such as micromilling and laser engraving, as two interlocking parts from materials that are non-absorbent and inert. Finally, the assembly was tested for performance using both rodent and human islets with multiple assays conducted in parallel, such as dynamic perfusion, staining and optogenetics on standard microscopes, as well as for integration with commercial perfusion machines. The optimized design of convective fluid flows, use of bio-inert and non-absorbent materials, reversible assembly, manual access for loading and unloading of islets, and straightforward integration with commercial imaging and fluid handling systems proved to be critical for perfusion assay, and particularly suited for time-resolved optogenetics studies

Comprehensive characterization of the human pancreatic proteome for bioengineering applications

Interactions between the pancreatic extracellular matrix (ECM) and islet cells are known to regulate multiple aspects of islet physiology, including survival, proliferation, and glucose-stimulated insulin secretion. Recognizing the essential role of ECM in islet survival and function, various engineering approaches have been developed that aim to utilize ECM-based materials to recreate a native-like microenvironment. However, a major impediment to the success of these approaches has been the lack of a robust and comprehensive characterization of the human pancreatic proteome. Herein, by combining mass spectrometry (MS) and multiplex ELISA, we have provided an improved workflow for the in-depth profiling of the proteome, including minor constituents that are generally underrepresented. Moreover, we have further validated the effectiveness of our detergent-free decellularization protocol in the removal of cellular proteins and retention of the matrisome. It has also been established that the decellularized ECM and its derivatives can provide more tissue-specific cues than traditionally used biological scaffolds and are therefore more physiologically relevant for the development of hydrogels, bioinks and medium additives, in order to create a pancreatic niche. The data generated in this study would contribute significantly to the efforts of comprehensively defining the ECM atlas and also serve as a standard for the human pancreatic proteome to provide further guidance for design and engineering strategies for improved tissue engineering scaffolds. [Display omitted

Extracellular matrix-based hydrogels obtained from human tissues: a work still in progress

Purpose of reviewThe current review summarizes contemporary decellularization and hydrogel manufacturing strategies in the field of tissue engineering and regenerative medicine.Recent findingsDecellularized extracellular matrix (ECM) bioscaffolds are a valuable biomaterial that can be purposed into various forms of synthetic tissues such as hydrogels. ECM-based hydrogels can be of animal or human origin. The use of human tissues as a source for ECM hydrogels in the clinical setting is still in its infancy and current literature is scant and anecdotal, resulting in inconclusive results.SummaryThus far the methods used to obtain hydrogels from human tissues remains a work in progress. Gelation, the most complex technique in obtaining hydrogels, is challenging due to remarkable heterogeneity of the tissues secondary to interindividual variability. Age, sex, ethnicity, and preexisting conditions are factors that dramatically undermine the technical feasibility of the gelation process. This is contrasted with animals whose well defined anatomical and histological characteristics have been selectively bred for the goal of manufacturing hydrogels

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Abstract Type 2 diabetic cardiomyopathy features Ca 2+ signaling abnormalities, notably an altered mitochondrial Ca 2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca 2+ homeostasis, the reticulum–mitochondrial Ca 2+ coupling, and/or the mitochondrial Ca 2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca 2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca 2+ sensors were performed to measure Ca 2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca 2+ transfer to mitochondria, with no changes in reticular Ca 2+ level, cytosolic Ca 2+ transients, and mitochondrial Ca 2+ uniporter function. Disruption of organelle Ca 2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca 2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca 2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca 2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Abstract Type 2 diabetic cardiomyopathy features Ca 2+ signaling abnormalities, notably an altered mitochondrial Ca 2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca 2+ homeostasis, the reticulum–mitochondrial Ca 2+ coupling, and/or the mitochondrial Ca 2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca 2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca 2+ sensors were performed to measure Ca 2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca 2+ transfer to mitochondria, with no changes in reticular Ca 2+ level, cytosolic Ca 2+ transients, and mitochondrial Ca 2+ uniporter function. Disruption of organelle Ca 2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca 2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca 2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca 2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy

Decellularized human pancreatic extracellular matrix-based physiomimetic microenvironment for human islet culture

A strategy that seeks to combine the biophysical properties of inert encapsulation materials like alginate with the biochemical niche provided by pancreatic extracellular matrix (ECM)-derived biomaterials, could provide a physiomimetic pancreatic microenvironment for maintaining long-term islet viability and func-tion in culture. Herein, we have demonstrated that incorporating human pancreatic decellularized ECM within alginate microcapsules results in a significant increase in Glucose Stimulation Index (GSI) and to-tal insulin secreted by encapsulated human islets, compared to free islets and islets encapsulated in only alginate. ECM supplementation also resulted in long-term (58 days) maintenance of GSI levels, similar to that observed in free islets at the first time point (day 5). At early time points in culture, ECM promoted gene expression changes through ECM-and cell adhesion-mediated pathways, while it demonstrated a mitochondria-protective effect in the long-term.Statement of significanceThe islet isolation process can damage the islet extracellular matrix, resulting in loss of viability and func-tion. We have recently developed a detergent-free, DI-water based method for decellularization of human pancreas to produce a potent solubilized ECM. This ECM was added to alginate for microencapsulation of human islets, which resulted in significantly higher stimulation index and total insulin production, com-pared to only alginate capsules and free islets, over long-term culture. Using ECM to preserve islet health and function can improve transplantation outcomes, as well as provide novel materials and platforms for studying islet biology in microfluidic, organ-on-a-chip, bioreactor and 3D bioprinted systems.(c) 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved