Thou shall not heal: overcoming the non-healing behaviour of diabetic foot ulcers by engineering the inflammatory microenvironment

Diabetic foot ulcers (DFUs) are a devastating complication for diabetic patients that have debilitating effects and can ultimately lead to limb amputation. Healthy wounds progress through the phases of healing leading to tissue regeneration and restoration of the barrier function of the skin. In contrast, in diabetic patients dysregulation of these phases leads to chronic, non-healing wounds. In particular, unresolved inflammation in the DFU microenvironment has been identified as a key facet of chronic wounds in hyperglyceamic patients, as DFUs fail to progress beyond the inflammatory phase and towards resolution. Thus, control over and modulation of the inflammatory response is a promising therapeutic avenue for DFU treatment. This review discusses the current state-of-the art regarding control of the inflammatory response in the DFU microenvironment, with a specific focus on the development of biomaterials-based delivery strategies and their cargos to direct tissue regeneration in the DFU microenvironment. </p

Thou shall not heal: overcoming the non-healing behaviour of diabetic foot ulcers by engineering the inflammatory microenvironment

Diabetic foot ulcers (DFUs) are a devastating complication for diabetic patients that have debilitating effects and can ultimately lead to limb amputation. Healthy wounds progress through the phases of healing leading to tissue regeneration and restoration of the barrier function of the skin. In contrast, in diabetic patients dysregulation of these phases leads to chronic, non-healing wounds. In particular, unresolved inflammation in the DFU microenvironment has been identified as a key facet of chronic wounds in hyperglyceamic patients, as DFUs fail to progress beyond the inflammatory phase and towards resolution. Thus, control over and modulation of the inflammatory response is a promising therapeutic avenue for DFU treatment. This review discusses the current state-of-the art regarding control of the inflammatory response in the DFU microenvironment, with a specific focus on the development of biomaterials-based delivery strategies and their cargos to direct tissue regeneration in the DFU microenvironment. </p

The rationale and emergence of electroconductive biomaterial scaffolds in cardiac tissue engineering

The human heart possesses minimal regenerative potential, which can often lead to chronic heart failure following myocardial infarction. Despite the successes of assistive support devices and pharmacological therapies, only a whole heart transplantation can sufficiently address heart failure. Engineered scaffolds, implantable patches, and injectable hydrogels are among the most promising solutions to restore cardiac function and coax regeneration; however, current biomaterials have yet to achieve ideal tissue regeneration and adequate integration due a mismatch of material physicochemical properties. Conductive fillers such as graphene, carbon nanotubes, metallic nanoparticles, and MXenes and conjugated polymers such as polyaniline, polypyrrole, and poly(3,4-ethylendioxythiophene) can possibly achieve optimal electrical conductivities for cardiac applications with appropriate suitability for tissue engineering approaches. Many studies have focused on the use of these materials in multiple fields, with promising effects on the regeneration of electrically active biological tissues such as orthopedic, neural, and cardiac tissue. In this review, we critically discuss the role of heart electrophysiology and the rationale toward the use of electroconductive biomaterials for cardiac tissue engineering. We present the emerging applications of these smart materials to create supportive platforms and discuss the crucial role that electrical stimulation has been shown to exert in maturation of cardiac progenitor cells

Time-dependent anabolic response of hMSC-derived cartilage grafts to hydrostatic pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions. </p

The rationale and emergence of electroconductive biomaterial scaffolds in cardiac tissue engineering

The human heart possesses minimal regenerative potential, which can often lead to chronic heart failure following myocardial infarction. Despite the successes of assistive support devices and pharmacological therapies, only a whole heart transplantation can sufficiently address heart failure. Engineered scaffolds, implantable patches, and injectable hydrogels are among the most promising solutions to restore cardiac function and coax regeneration; however, current biomaterials have yet to achieve ideal tissue regeneration and adequate integration due a mismatch of material physicochemical properties. Conductive fillers such as graphene, carbon nanotubes, metallic nanoparticles, and MXenes and conjugated polymers such as polyaniline, polypyrrole, and poly(3,4-ethylendioxythiophene) can possibly achieve optimal electrical conductivities for cardiac applications with appropriate suitability for tissue engineering approaches. Many studies have focused on the use of these materials in multiple fields, with promising effects on the regeneration of electrically active biological tissues such as orthopedic, neural, and cardiac tissue. In this review, we critically discuss the role of heart electrophysiology and the rationale toward the use of electroconductive biomaterials for cardiac tissue engineering. We present the emerging applications of these smart materials to create supportive platforms and discuss the crucial role that electrical stimulation has been shown to exert in maturation of cardiac progenitor cells

Time-dependent anabolic response of hMSC-derived cartilage grafts to hydrostatic pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions. </p

Impact of the reduction time-dependent electrical conductivity of graphene nanoplatelet-coated aligned <i>Bombyx mori</i> silk scaffolds on electrically stimulated axonal growth

Graphene-based nanomaterials, renowned for their outstanding electrical conductivity, have been extensively studied as electroconductive biomaterials (ECBs) for electrically stimulated tissue regeneration. However, using eco-friendly reducing agents like l-ascorbic acid (l-Aa) can result in lower conductive properties in these ECBs, limiting their full potential for smooth charge transfer in living tissues. Moreover, creating a flexible biomaterial scaffold using these materials that accurately mimics a specific tissue microarchitecture, such as nerves, poses additional challenges. To address these issues, this study developed a microfibrous scaffold of Bombyx mori (Bm) silk fibroin uniformly coated with graphene nanoplatelets (GNPs) through a vacuum coating method. The scaffold's electrical conductivity was optimized by varying the reduction period using l-Aa. The research systematically investigated how different reduction periods impact scaffold properties, focusing on electrical conductivity and its significance on electrically stimulated axonal growth in PC12 cells. Results showed that a 48 h reduction significantly increased surface electrical conductivity by 100-1000 times compared to a shorter or no reduction process. l-Aa contributed to stabilizing the reduced GNPs, demonstrated by a slow degradation profile and sustained conductivity even after 60 days in a proteolytic environment. β (III) tubulin immunostaining of PC12 cells on varied silk:GNP scaffolds under pulsed electrical stimulation (ES, 50 Hz frequency, 1 ms pulse width, and amplitudes of 100 and 300 mV/cm) demonstrates accelerated axonal growth on scaffolds exhibiting higher conductivity. This is supported by upregulated intracellular Ca2+ dynamics immediately after ES on the scaffolds with higher conductivity, subjected to a prolonged reduction period. The study showcases a sustainable reduction approach using l-Aa in combination with natural Bm silk fibroin to create a highly conductive, mechanically robust, and stable silk:GNP-based aligned fibrous scaffold. These scaffolds hold promise for functional regeneration in electrically excitable tissues such as nerves, cardiac tissue, and muscles. </p

From innovation to clinic: emerging strategies harnessing electrically conductive polymers to enhance electrically stimulated peripheral nerve repair

Peripheral nerve repair (PNR) is a major healthcare challenge due to the limited regenerative capacity of the nervous system, often leading to severe functional impairments. While nerve autografts are the gold standard, their implications are constrained by issues such as donor site morbidity and limited availability, necessitating innovative alternatives like nerve guidance conduits (NGCs). However, the inherently slow nerve growth rate (∼1 mm/day) and prolonged neuroinflammation, delay recovery even with the use of passive (no-conductive) NGCs, resulting in muscle atrophy and loss of locomotor function. Electrical stimulation (ES) has the ability to enhance nerve regeneration rate by modulating the innate bioelectrical microenvironment of nerve tissue while simultaneously fostering a reparative environment through immunoregulation. In this context, electrically conductive polymer (ECP)-based biomaterials offer unique advantages for nerve repair combining their flexibility, akin to traditional plastics, and mixed ionic-electronic conductivity, similar to ionically conductive nerve tissue, as well as their biocompatibility and ease of fabrication. This review focuses on the progress, challenges, and emerging techniques for integrating ECP based NGCs with ES for functional nerve regeneration. It critically evaluates the various approaches using ECP based scaffolds, identifying gaps that have hindered clinical translation. Key challenges discussed include designing effective 3D NGCs with high electroactivity, optimizing ES modules, and better understanding of immunoregulation during nerve repair. The review also explores innovative strategies in material development and wireless, self-powered ES methods. Furthermore, it emphasizes the need for non-invasive ES delivery methods combined with hybrid ECP based neural scaffolds, highlighting future directions for advancing preclinical and clinical translation. Together, ECP based NGCs combined with ES represent a promising avenue for advancing PNR and improving patient outcomes.</p

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

Background: Human mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source. Methods: In this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality-two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred. Results: During osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism. Conclusion: In summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.</p

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

Background: Human mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source. Methods: In this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality-two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred. Results: During osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism. Conclusion: In summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.</p