Chronic diabetic wounds are often characterized by persistent hypoxia and poor healing outcomes, highlighting the need for regenerative grafts that not only promote tissue repair but also provide insights into the wound microenvironment. In this study, we introduce a novel strategy for diabetic ulcer treatment through the development of a structurally personalized skin graft. The graft is fabricated via 3D bioprinting of natural porcine skin extracellular matrix (psECM) and integrated with microsensors for oxygen monitoring. We established a porcine skin decellularization protocol that efficiently removed cellular components, while preserving the integrity of the ECM, as verified by DNA quantification and scanning electron microscopy. The resulting psECM bioink demonstrated rheological properties suitable for 3D printing, which depended on psECM concentration and exhibited temperature-responsive gelation behavior. Incorporation of LiNC-BuO oxygen microsensors into the bioink enabled real-time, non-invasive oxygen level monitoring within the printed constructs. Both in vitro and in vivo studies confirmed the cytocompatibility and low immunogenicity of the psECM-based grafts with embedded microsensors. Moreover, the 3D bioprinting technology enabled the manufacturing of grafts tailored to match individual wound geometries. The technological proof of concept presented herein for this multifunctional platform, which integrates the regenerative benefits of ECM scaffolds with advanced biosensing capabilities, represents a promising approach for enhancing future therapeutic outcomes in the management of diabetic ulcers.

One of the major challenges in developing cultured meat that presents a viable alternative to conventional meat is the design of scaffolds that are edible as well as sustainable, animal-free, and low-cost. In this work, we introduce the development of two technologies to process chickpea protein (CP) into edible microcarriers (CPMCs) and fibrous scaffolds (CP-FSs) to create minced and thick-cut cultured meat products, respectively. CP-MCs production is facilitated by the use of electrospray technology and coagulation through solvent exchange, resulting in stable spherical microcarriers with an average diameter of 577 mu m. The production of CP-FSs is enabled using electrospinning technology and solvent evaporation, which results in a mat of isotropic or aligned fibers, characterized by an average width of 4.5 mu m, and an elastic behavior. Chickpea protein demonstrates no cytotoxicity and supports the adhesion and proliferation of both mesenchymal stem cells and skeletal muscle cells. The produced scaffolds are validated to preserve the main proteins of the source CP, their molecular structures, and their thermal behavior. Furthermore, both types of scaffolds are shown to support cell culture and growth, leading to a complete coverage of CP-MC within 10 days of culture, and a cell expansion of 8-fold within a week of culture on the CP-FSs. Notably, the aligned orientation of CP-FSs fibers prompt the alignment of skeletal muscle cells. All in all, the chickpea-based scaffolds presented in this work exhibit impressive potential towards the future development of sustainable, edible, and affordable cultured meat products.

Bone defects resulting from trauma, tumors, or congenital conditions pose significant challenges for natural healing and often require grafting solutions. While autografts remain the gold standard, their limitations, such as restricted availability and donor site complications, underscore the need for alternative approaches. The present research investigates the potential of porcine-derived bone extracellular matrix (pbECM) hydrogel as a highly promising bioactive scaffold for bone regeneration, comparing it to the human-derived bECM (hbECM). Porcine and human cancellous bones were decellularized and characterized in terms of their composition and structure. Further, the ECMs were processed into hydrogels, and their rheological properties and cytocompatibility were studied in vitro while their biocompatibility was studied in vivo using a mouse model. The potential of the pbECM hydrogel as a bone graft was evaluated in vivo using a rat femoral defect model. Our results demonstrated the excellent preservation of essential ECM components in both the pbECM and hbECM with more than 90% collagen out of all proteins. Rheological analyses revealed the superior mechanical properties of the pbECM hydrogel compared to the hbECM, with an approximately 10-fold higher storage modulus and a significantly later deformation point. These stronger gel properties of the pbECM were attributed to the higher content of structural proteins and residual minerals. Both the pbECM and hbECM effectively supported mesenchymal stem cell adhesion, viability, and proliferation, achieving a 20-fold increase in cell number within 10 days and highlighting their strong bioactive potential. In vivo, pbECM hydrogels elicited a minimal immunogenic response. Most importantly, when implanted in a rat femoral defect model, pbECM hydrogel had significantly enhanced bone regeneration through graft integration, stem cell recruitment, and differentiation. New bone formation was observed at an average of 50% of the defect volume, outperforming the commercial demineralized bone matrix (DBM), in which the new bone filled only 35% of the defect volume. These results position pbECM hydrogel as a highly effective and biocompatible scaffold for bone tissue engineering, offering a promising alternative to traditional grafting methods and paving the way for future clinical applications in bone repair.

Type 1 diabetes (T1D) is caused by autoimmune-mediated destruction of pancreatic β-cells, resulting in insulin deficiency. While islet transplantation presents a potential therapeutic approach, its clinical application is impeded by limited donor availability and the risk of immune rejection. This study proposes an innovative islet encapsulation strategy that utilizes decellularized porcine pancreatic extracellular matrix (pECM) as the sole biomaterial to engineer bioactive, immunoprotective microcapsules. Rat islets were encapsulated within pECM-based microcapsules using the electrospray technology and were compared to conventional alginate-based microcapsules in terms of viability, function, and response to hypoxic stress. The pECM microcapsules maintained a spherical morphology, demonstrating mechanical robustness, and preserving essential ECM components (collagen I/IV, laminin, fibronectin). Encapsulated islets exhibited sustained viability and superior insulin secretion over a two-week period compared to alginate controls. The expression of key β-cell transcription factors (PDX1, MAFA) and structural integrity were preserved. Under hypoxic conditions, pECM microcapsules significantly reduced islet apoptosis, improved structural retention, and promoted functional recovery, likely due to antioxidant and ECM-derived cues inherent to the pECM. In vivo transplantation in immunocompetent mice confirmed the biocompatibility of pECM microcapsules, with minimal immune responses, stable insulin/glucagon expression, and no adverse systemic effects. These findings position pECM-based microencapsulation as a promising strategy for creating immunoprotective, bioactive niches for xenogeneic islet transplantation, with the potential to overcome current limitations in cell-based diabetes therapy.

Cardiac tissue engineering aims to efficiently replace or repair injured heart tissue using scaffolds, relevant cells, or their combination. While the combination of scaffolds and relevant cells holds the potential to rapidly remuscularize the heart, thereby avoiding the slow process of cell recruitment, the proper ex vivo cellularization of a scaffold poses a substantial challenge. First, proper diffusion of nutrients and oxygen should be provided to the cell-seeded scaffold. Second, to generate a functional tissue construct, cells can benefit from physiological-like conditions. To meet these challenges, we developed a modular bioreactor for the dynamic cellularization of full-thickness cardiac scaffolds under synchronized mechanical and electrical stimuli. In this unique bioreactor system, we designed a cyclic mechanical load that mimics the left ventricle volume inflation, thus achieving a steady stimulus, as well as an electrical stimulus with an action potential profile to mirror the cells’ microenvironment and electrical stimuli in the heart. These mechanical and electrical stimuli were synchronized according to cardiac physiology and regulated by constant feedback. When applied to a seeded thick porcine cardiac extracellular matrix (pcECM) scaffold, these stimuli improved the proliferation of mesenchymal stem/stromal cells (MSCs) and induced the formation of a dense tissue-like structure near the scaffold’s surface. Most importantly, after 35 d of cultivation, the MSCs presented the early cardiac progenitor markers Connexin-43 and α-actinin, which were absent in the control cells. Overall, this research developed a new bioreactor system for cellularizing cardiac scaffolds under cardiac-like conditions, aiming to restore a sustainable dynamic living tissue that can bear the essential cardiac excitation–contraction coupling.

A library of eight new fluoroquinolone-nuclease conjugates containing a guanidinoethyl or aminoethyl auxiliary pendant on the 1,4,7-triazacyclononane (TACN) moiety was designed and synthesized to investigate their potential as catalytic antibiotics. The Cu(ii) complexes of the designer structures showed significant in vitro hydrolytic and oxidative DNA cleavage activity and good antibacterial activity against both Gram-negative and Gram-positive bacteria. The observed activity of all the Cu(ii)-TACN-ciprofloxacin complexes was strongly inhibited in the presence of Cu(ii)-chelating agents, thereby demonstrating "vulnerability" under physiological conditions. However, selected TACN-ciprofloxacin conjugates in their metal-free form efficiently cleaved plasmid DNA under physiological conditions. The lead compound 1 showed good DNase activity which was retained in the presence of strong metal chelators and exhibited excellent antibacterial activity against both Gram-negative and Gram-positive bacteria. Density functional theory calculations combined with quantum mechanics/molecular mechanics simulations suggest a general base-general acid mechanism for the hydrolytic DNA cleavage mechanism by compound 1.

PURPOSE:Electron spin resonance (ESR) is used to measure oxygen partial pressure (pO2) in biological media with many clinical applications. Traditional clinical ESR involves large magnets that encompass the subject of measurement. However, certain applications might benefit from a scanner operating within local static magnetic fields. Our group recently developed such a compact scanner for transcutaneous (surface) pO2 measurements of skin tissue. Here we extend this capability to subsurface (subcutaneous) pO2 measurements and verify it using an artificial tissue emulating (ATE) phantom. METHODS:We introduce a new scanner, tailored for subcutaneous measurements up to 2 mm beneath the skin's surface. This scanner captures pulsed ESR signals from embedded approximate 1-mm oxygen-sensing solid paramagnetic implant, OxyChip. The scanner features a static magnetic field source, producing a uniform region outside its surface, and a compact microwave resonator, for exciting and receiving ESR signals. RESULTS:ESR readings derived from an OxyChip, positioned approximately 1.5 mm from the scanner's surface, embedded in ATE phantom, exhibited a linear relation of 1/T2 versus pO2 for pO2 levels at 0, 7.6, 30, and 160 mmHg, with relative reading accuracy of about 10%. CONCLUSION:The compact ESR scanner can report pO2 data in ATE phantom from an external position relative to the scanner. Implementing this scanner in preclinical and clinical applications for subcutaneous pO2 measurements is a feasible next phase for this development. This innovative design also has the potential to operate in conjunction with artificial skin graft for wound healing, combining therapeutic and pO2 diagnostic features.

Scaffold design is a key step for cultivated meat development. In addition to providing nutritional values and texture, scaffolds are crucial for cell adherence and proliferation. The technologies applied for the manufacturing of cultivated meat scaffolds are often adapted from the field of tissue engineering, where structures are designed to support mammalian cell culture. Nevertheless, the application of tissue engineering methodologies for cultivated meat scaffolds presents several challenges. Safety and edibility should be primarily considered, in addition to the nutritional values and textural properties. The process and material sustainability should not be compromised, nor the low cost of the final product, to achieve commercial viability. Technologies adapted from the food industry, on the other hand, impose challenges such as proper support of cell adherence and proliferation. Here, we detail the main technologies and materials suggested for cultivated meat scaffolds and discuss their pros and cons considering the unique attributes essential for cultivated meat.

The pancreatic extracellular matrix (ECM) is an enormously complex construct. Previous studies underline the challenges to identify the optimal combinations and ratios of individual ECM proteins for promoting survival and function of isolated and transplanted islets. This study aimed on assessing the efficiency of solubilized natural ECM extracted from juvenile pigs, an unlimited donor source. Isolated human islets were cultured under a hypoxic atmosphere (2% oxygen) in media supplemented with either solubilized porcine pancreatic ECM (ppECM) or a mixture of human ECM proteins composed of collagen-IV, laminin-521, and nidogen-1 (hEPM). Control islets were cultured under identical conditions without ECM-compounds. Reactive oxygen species production increased three-fold in controls but was reduced by hEPM or ppECM. Early apoptosis remained on preculture levels when islets were treated with hEPM or ppECM. Preculture viability was preserved when hEPM or ppECM was administered. Whilst controls failed to respond to glucose challenge, treatment with hEPM or ppECM preserved the physiological insulin response. In summary, overall survival was significantly highest in ppECM-treated islets. This study presents a new approach to protect human islets from hypoxia-induced damage by supplementing media with ppECM extracted from an unlimited donor source. The findings may also serve as starting point for a novel encapsulation technique to protect isolated human islets.

With the increasing global demand for meat, cultured meat technologies are emerging, offering more sustainable solutions that aim to evade a future shortage of meat. Here, we demonstrate a cultured meat platform composed of edible microcarriers and an oleogel-based fat substitute. Scalable expansion of bovine mesenchymal stem cells on edible chitosan-collagen microcarriers is optimized to generate cellularized microtissues. In parallel, an oleogel system incorporated with plant protein is developed as a fat substitute, which is comparable to beef fat in appearance and texture. Combining the cellularized microtissues with the developed fat substitute, two types of cultured meat prototypes are introduced: layered cultured meat and burger-like cultured meat. While the layered prototype benefits enhanced stiffness, the burger-like prototype has a marbling meat-like appearance and a softer texture. Overall, this platform and the established technological basis may contribute to the development of different cultured meat products and promote their commercial production.

Microencapsulation is a promising strategy to prolong the survival and function of transplanted pancreatic islets for diabetes therapy, albeit its translation has been impeded by incoherent graft performance. The use of decellularized ECM has lately gained substantial research momentum due to its innate capacity to augment the function of cells originating from the same tissue type. In the present study, the advantages of both these approaches are leveraged in a porcine pancreatic ECM (pECM)-based microencapsulation platform, thus significantly enhancing murine pancreatic islet performance. pECM-encapsulated islets sustain high insulin secretion levels in vitro, surpassing those of islets encapsulated in conventional alginate microcapsules. Moreover, pECM-encapsulated islet cells proliferate and produce an enriched intra-islet ECM framework, displaying a distinctive structural rearrangement. The beneficial effect of pECM encapsulation is further reinforced by the temporary protection against cytokine-induced cytotoxicity. In-vivo, this platform significantly improves glucose tolerance and achieves glycemic correction in 100% of immunocompetent diabetic mice without any immunosuppression, compared to only 50% mice achieved glycemic correction by alginate encapsulation. Altogether, the results presented herein reveal that pECM-based microencapsulation offers a natural pancreatic niche that can restore the function of isolated pancreatic islets and deliver them safely, avoiding the need for immunosuppression. STATEMENT OF SIGNIFICANCE: Aiming to improve pancreatic islet transplantation outcomes in diabetic patients, we developed a microencapsulation platform based on pancreatic extracellular matrix (pECM). In these microcapsules the islets are entrapped within a pECM hydrogel that mimics the natural pancreatic microenvironment. We show that pECM encapsulation supports the islets' viability and function in culture, and provides temporal protection against cytokine-induced stress. In a diabetic mouse model, pECM encapsulation significantly improved glucose tolerance and achieved glycemic correction without any immunosuppression. These results reveal the potential of pECM encapsulation as a viable treatment for diabetes, providing a solid scientific basis for more advanced preclinical studies.

Offering conventional meat-like nutritional values and eating experience without harming animals or the environment, cultured meat from livestock animal cells is considered one of the leading solutions to the global food crisis. Cell microcarriers (MCs) have been widely used for the scalable expansion of anchorage-dependent cells for biotechnology applications as well as cultured meat. In the current work, however, we suggest a different concept in which edible MCs can be incorporated into the final product, thus avoiding costly harvesting steps, and contributing to its nutritional values, textures, and appearance. Through addressing the different technological and biological aspects, we present the development of edible hydrogel MCs from chitosan and collagen, edible materials with a wide range of applications in tissue engineering. The obtained composite MCs have a uniform spherical shape of 571 mu m diameter, a smooth surface, and suitable mechanical properties. These MCs support the attachment and rapid proliferation of mouse skeletal C2C12 myoblasts, rabbit smooth muscle cells, sheep fibroblasts, and bovine umbilical cord mesenchymal stem cells, achieving complete coverage of the carrier surface within only a few days in culture. These findings indicate the high potential of the edible microcarriers as a platform for the development of prospective cultured meat products.

Background: Cultured meat aims to solve the current sustainability and environmental issues of conventional livestock husbandry by converging tissue engineering practices with food innovation science to recapitulate the components and structure of animal-derived meat. To this end, various scaffolding technologies were adopted and developed to support the cultivation, expansion, and differentiation of cells using edible, low-cost, and sustainable materials and methods. The cell-scaffold constructs can benefit different processing strategies that include structuring approaches and additives to generate final cultured meat products. Scope and approach: In this paper, we elaborate on the main considerations for the design of scaffolds for cultured meat applications, review the leading scaffolding technologies, and discuss their current or potential application for the engineering of cultured meat. Key findings and conclusions: The extensive research efforts in recent years attest to the boundless potential of novel cultured meat scaffolds. Moreover, different scaffolding technologies originally developed for tissue en-gineering can be adapted to cultured meat by using edible materials and avoiding toxic crosslinkers and reagents throughout all of the development steps, thus ensuring compliance with food and safety regulations. Altogether, the combination of tissue engineering and food science technologies holds the promise of technologically and commercially viable scaffolds towards the realization of the cultured meat vision.

Human-induced pluripotent stem cells (hiPSCs) hold great promise in the fields of regenerative therapy and personalized in vitro tissue models as they provide an almost endless autologous source of virtually any cell type. Their potential benefits can be highly enhanced through polymeric cell microencapsulation, which offers the cells structural support and immune protection from the host tissue. However, in contrast to biologically-inert polymers, decellularized extracellular matrix (dECM)-based microencapsulation also provides the cells a physiologically mimetic bioactive microenvironment while providing reproducibility and scalability. Here we describe the entrapment of hiPSCs in a porcine pancreatic ECM (pECM)-based microencapsulation platform and address its key aspects. To promote high levels of cell survival and growth, several encapsulation parameters were optimized, including cell dissociation level, the process layout, and Rho-associated kinase (ROCK) inhibitor addition. This has allowed extensive proliferation of the cells for at least 21 days in vitro. Furthermore, the encapsulation of embryoid bodies (EBs) was assessed to address the entrapment of hiPSCs at various stages of differentiation, and the culture conditions that promote lasting encapsulated EB survival were investigated. Finally, to study the in vivo safety of dECM-encapsulated hiPSC-derived products, completely undifferentiated pECM-encapsulated hiPSCs were implanted in mice. The cells displayed a slower proliferation rate in vivo and showed no signs of cell escape from the microcapsules. Altogether, the results of this study point to the potential of dECM-based microencapsulation to provide an efficient polymeric platform for transplantation of hiPSC-derived products and the development of iPSC-based 3D tissue models.

The current unmet clinical need for post-myocardial infarction (MI) treatments has driven the development of diverse scaffolds for regenerating the infarcted area, based on natural and synthetic polymers. Decellularized porcine cardiac extracellular matrix (pcECM) has emerged as a promising biomaterial for cardiac regeneration, due to its unique bioactivity and microstructure that mimic the natural tissue. We have previously reported the development of an electrospun pcECM cardiac scaffold that was shown to preserve the mechanical, structural, and biological properties of cardiac ECM while allowing a controllable reproducible production. In the present work, however, we reveal the potential of this unique scaffold as a possible treatment post-MI. Two types of electrospun pcECM scaffolds, varying in thickness, were applied to rat hearts, 4 weeks following MI induction, thus allowing cardiac deterioration and a scar tissue formation before treatment (chronic model). Our results show moderated remodeling, decreased scar, and reduced wall thinning accompanied by a partial functional recovery in the hearts of both treatment groups when compared to the control. When examining the different structural and functional parameters of the heart, however, the advantage of the thicker electrospun pcECM scaffold is revealed. Hence, significant improvement was obtained in important structural parameters such as wall thickness and functional parameters such as ejection fraction. Altogether, our results indicate the potential applicability of electrospun pcECM scaffolds for cardiac regeneration, establishing the basis for advanced preclinical research using large animal models.

Porcine extracellular matrix (pECM)-derived hydrogels were introduced, in recent years, aiming to benefit the pECM’s microstructure and bioactivity, while controlling the biomaterial’s physical and mechanical properties. The use of pECM from different tissues, however, offers tissue-specific features that can better serve different applications. In this study, pECM hydrogels derived from cardiac, artery, pancreas, and adipose tissues were compared in terms of composition, structure, and mechanical properties. While major similarities were demonstrated between all the pECM hydrogels, their distinctive attributes were also identified, and their substantial effects on cell-ECM interactions were revealed. Furthermore, through comprehensive protein and gene expression analyses, we show, for the first time, that each pECM hydrogel supports the spontaneous differentiation of induced pluripotent stem cells towards the resident cells of its origin tissue. These findings imply that the origin of ECM should be carefully considered when designing a biomedical platform, to achieve a maximal bioactive impact.

Aiming to restore the normal function of diseased or injured tissues, regenerative therapy approaches are generally based on the engineering of complex tissue-mimicking grafts, encompassing biomaterial scaffolds, stem cells, or their combinations [1-4]. Due to the major role of stem cells in physiological regenerative mechanisms, regenerative therapies normally rely on either stem cells transplantation or stem cell recruitment from the neighboring tissue into the implanted scaffold

Aim: Islet isolation essentially requires the dissociation of the islet basement membrane by collagenolytic enzymes. Basement membrane loss is associated with reduced islet function and increased cell death. Previous ex-vivo and in-vivo studies demonstrated that individual extracellular matrix (ECM)-proteins can increase islet survival. As the natural ECM is a tissue-defined complex network we propose a novel concept for creating a specific islet matrix by using the whole pancreatic ECM (WPM). Methods: Islets, isolated from 6 human pancreases, were cultured for 4–5 days in 2% oxygen. Islets were suspended in CMRL 1066 (2% FCS) and WPM-gel (200 µg/mL) extracted and purified from porcine pancreatic tissue. The WPM-gel was compared with a pretested combination of 80 µg/mL human Collagen-IV, 10 µg/mL Laminin-521, and 10 µg/mL Nidogen-1 (CLN). Sham-treated islets cultured without any ECM components served as controls. Post-culture characterisation included yield of islet equivalents (IEQ) or islet particle number (IN), viability (FDA-PI staining), early and late apoptosis (Annexin V-PI), glucose stimulation index (SI: 2 vs 20 vs 2 mM) and production of reactive oxygen species (ROS). All parameters were normalised to IEQ, related to pre-culture data if appropriate and presented as mean ± SEM. Results: Post-culture yield was significantly highest when hypoxic human islets were cultured in WPM-gel and compared to controls (65±10% vs 38±10%, p<0.01). Although fragmentation, determined as IN/IEQ ratio, increased after all treatments, this increase was lowest in the presence of WPM-gel (0.62±0.05 vs 0.80±0.14 vs 0.93±0.27, NS). FDA-PI staining revealed that pre-culture viability was preserved by nearly 100% when CLN (99±10% vs 79±10%, p<0.01) or WPM-gel (92±8%, p<0.05) were administered. ROS production in controls increased nearly 3-fold (127±15 AU/IEQ) but was significantly halved in the presence of CLM (61±14 AU/IEQ, p<0.01) or WPM-gel (65±18 AU/IEQ, p<0.05). Whilst initial early apoptosis remained stable when human islets were treated with CLN (90±13% vs 136±14%, p<0.01) or WPM-gel (84±10%, p<0.01), apo-necrosis increased substantially in the presence of CLN (172±33%, NS) or WPM-gel (154±195 vs 214±24%, p<0.05). Control islets did not respond with adequate insulin secretion after glucose stimulation (SI 0.85±0.14). In contrast, supplementation with CLN (1.29±0.09, p<0.05) or WPM-gel (1.34±0.09, p<0.01) preserved the physiological insulin response during hypoxia. Overall survival, considering the recovery of viable cells only, was significantly increased using CLN (56±8% vs 34±8%, p<0.01) or WPM-gel (58±8%, p<0.01). Conclusion: This initial study presents a novel and efficient approach to protect human islets from hypoxia-induced damage by supplementing culture media with selected ECM-proteins or with whole pancreatic ECM. The promising findings may serve as starting point for a new encapsulation technique to protect transplanted islets. European Union’s Horizon 2020 (645991). Juvenile Diabetes Research Foundation (JDRF) award (31-2008-617).