Shane Browne

Diabetic foot ulcers (DFU) are debilitating chronic wounds which affect up to a third of diabetic patients. Due to the underlying diabetic pathophysiology, normal wound healing is perturbed. In particular, there is insufficient re‐vascularisation of the wound bed, ultimately leading to the development of chronic wounds. Current treatment protocols are insufficient to stimulate re‐vascularisation and wound closure, ultimately leading to ~20% of patients with a DFU requiring lower limb amputation. Extracellular matrix (ECM)‐derived biomaterials have demonstrated great promise to promote tissue vascularisation and wound repair. In the present study, the overarching goal was to develop a decellularised ECM from human induced pluripotent stem cell (hiPSC)‐derived vascular cells, and incorporate it into a hyaluronic acid‐based hydrogel for wound healing. Vascular differentiation of hiPSCs yielded a heterogenous population containing both CD31 expressing endothelial cells (iEC), and CD31‐negative stromal cells (iSC). ECM deposition by iSCs was stimulated by treatment with ascorbic acid and macromolecular crowder carrageenan. Post‐decellularisation, the ECM composition was analysed by immunofluorescent and histological staining, indicating the presence of ECM components such as fibronectin, fibrillar collagens, perlecan and glycosaminoglycans, all having implications in angiogenic processes. Decellularised ECM accelerated the capacity of iECs to form tube‐like structures when added to cell culture media in 2D. Decoration of hyaluronic acid (HyA)‐based hydrogels with this decellularised ECM enhanced metabolic activity and attachment of both HUVECs and human dermal fibroblasts seeded on these hydrogels. Taken together, this ECM‐decorated HyA‐based hydrogel has the potential to enhance angiogenesis and revascularisation in chronic wound applications such as DFU.

Disruption of the wound healing cascade can result in pathological outcomes, including fibrosis due to myofibroblast-mediated contraction and collagen deposition. Despite the clinical significance, effective treatments for fibrosis remain limited as current therapies often show inconsistent efficacy, adverse effects, and patient discomfort. Combinatorial therapeutic strategies integrating biomaterial scaffolds with gene delivery have shown promise in regenerative healing. MicroRNAs (miRNAs) are key regulators of fibrotic signalling in cells, including fibroblasts and myofibroblasts. Specifically, miRNA-29b is notable for downregulating pro-fibrotic genes, including collagen type I, reducing ECM accumulation, and limiting fibroblast/myofibroblast overactivation. In this context, the present work develops a collagen-GAG (CG) scaffold platform for delivery of miRNA-29b complexed GET nanoparticles to inhibit fibrosis. Initially, bioinformatic analysis of miRNA-29b validated its involvement in ECM-associated pathways and processes, followed by successful nanoparticle internationalisation in primary dermal fibroblasts. The anti-fibrotic efficacy of the optimised miRNA-29b nanoparticles was subsequently demonstrated by significant reductions in collagen deposition and -SMA expression, both key indicators of myofibroblast differentiation and fibrosis. The optimised miRNA-29b formulation was then incorporated into 3D porous collagen-GAG (CG) scaffolds, which modulated fibrotic gene expression while preserving scaffold structure conducive to fibroblast/myofibroblast infiltration and proliferation. Finally, functional outcomes of seeded TGF--stimulated fibroblasts, including reduced matrix contraction, -SMA expression, and ECM deposition, were comparable to those observed in non-fibrotic conditions, thereby confirming the therapeutic potential of scaffold-mediated miRNA-29b delivery. Together, these findings demonstrate that scaffold-mediated miRNA-29b delivery represents a promising anti-fibrotic strategy for wound healing by mitigating myofibroblast activation, limiting matrix contraction, and preventing pathological ECM accumulation.

Disruption of the wound healing cascade can result in pathological outcomes, including fibrosis due to myofibroblast-mediated contraction and collagen deposition. Despite the clinical significance, effective treatments for fibrosis remain limited as current therapies often show inconsistent efficacy, adverse effects, and patient discomfort. Combinatorial therapeutic strategies integrating biomaterial scaffolds with gene delivery have shown promise in regenerative healing. MicroRNAs (miRNAs) are key regulators of fibrotic signalling in cells, including fibroblasts and myofibroblasts. Specifically, miRNA-29b is notable for downregulating pro-fibrotic genes, including collagen type I, reducing ECM accumulation, and limiting fibroblast/myofibroblast overactivation. In this context, the present work develops a collagen-GAG (CG) scaffold platform for delivery of miRNA-29b complexed GET nanoparticles to inhibit fibrosis. Initially, bioinformatic analysis of miRNA-29b validated its involvement in ECM-associated pathways and processes, followed by successful nanoparticle internationalisation in primary dermal fibroblasts. The anti-fibrotic efficacy of the optimised miRNA-29b nanoparticles was subsequently demonstrated by significant reductions in collagen deposition and a-SMA expression, both key indicators of myofibroblast differentiation and fibrosis. The optimised miRNA-29b formulation was then incorporated into 3D porous collagen-GAG (CG) scaffolds, which modulated fibrotic gene expression while preserving scaffold structure conducive to fibroblast/myofibroblast infiltration and proliferation. Finally, functional outcomes of seeded TGF-b-stimulated fibroblasts, including reduced matrix contraction, a-SMA expression, and ECM deposition, were comparable to those observed in non-fibrotic conditions, thereby confirming the therapeutic potential of scaffold-mediated miRNA-29b delivery. Together, these findings demonstrate that scaffold-mediated miRNA-29b delivery represents a promising anti-fibrotic strategy for wound healing by mitigating myofibroblast activation, limiting matrix contraction, and preventing pathological ECM accumulation.

Disruption of the wound healing cascade can result in pathological outcomes, including fibrosis due to myofibroblast-mediated contraction and collagen deposition. Despite the clinical significance, effective treatments for fibrosis remain limited as current therapies often show inconsistent efficacy, adverse effects, and patient discomfort. Combinatorial therapeutic strategies integrating biomaterial scaffolds with gene delivery have shown promise in regenerative healing. MicroRNAs (miRNAs) are key regulators of fibrotic signalling in cells, including fibroblasts and myofibroblasts. Specifically, miRNA-29b is notable for downregulating pro-fibrotic genes, including collagen type I, reducing ECM accumulation, and limiting fibroblast/myofibroblast overactivation. In this context, the present work develops a collagen-GAG (CG) scaffold platform for delivery of miRNA-29b complexed GET nanoparticles to inhibit fibrosis. Initially, bioinformatic analysis of miRNA-29b validated its involvement in ECM-associated pathways and processes, followed by successful nanoparticle internationalisation in primary dermal fibroblasts. The anti-fibrotic efficacy of the optimised miRNA-29b nanoparticles was subsequently demonstrated by significant reductions in collagen deposition and α-SMA expression, both key indicators of myofibroblast differentiation and fibrosis. The optimised miRNA-29b formulation was then incorporated into 3D porous collagen-GAG (CG) scaffolds, which modulated fibrotic gene expression while preserving scaffold structure conducive to fibroblast/myofibroblast infiltration and proliferation. Finally, functional outcomes of seeded TGF-β-stimulated fibroblasts, including reduced matrix contraction, α-SMA expression, and ECM deposition, were comparable to those observed in non-fibrotic conditions, thereby confirming the therapeutic potential of scaffold-mediated miRNA-29b delivery. Together, these findings demonstrate that scaffold-mediated miRNA-29b delivery represents a promising anti-fibrotic strategy for wound healing by mitigating myofibroblast activation, limiting matrix contraction, and preventing pathological ECM accumulation.

Chronic wounds represent a significant clinical challenge due to persistent inflammation and impaired nerve regeneration that delay healing. Conventional treatments often yield inconsistent and limited success. Combinatorial strategies that integrate biomaterial scaffolds with gene delivery offer a promising approach to promote tissue repair. MicroRNAs (miRNAs), particularly miRNA-155, are key regulators of wound healing. miRNA-155 is highly expressed in inflammatory conditions and modulates macrophage activation, polarisation, and nerve regeneration. In this context, this study introduces a miRNA-155 inhibitor-activated scaffold designed to modulate the chronic wound environment by inhibiting miRNA-155. miRNA-155 inhibitor complexed GET nanoparticles were incorporated into collagen-glycosaminoglycan (CG) scaffolds. Scaffold-mediated miRNA-155 inhibition in both non-polarised (M0) and pro-inflammatory (M1) macrophages promoted anti-inflammatory (M2) polarisation, confirmed by molecular and protein analysis. The regenerative potential of this macrophage polarisation was validated through inflammatory and angiogenic functional assays with endothelial cells. In parallel, scaffold-mediated miRNA-155 inhibition in dorsal root ganglia (DRG) enhanced axonal regrowth, essential for the synergistic repair of chronic wounds across the skin-nerve axis. In vivo implantation in a chick model demonstrated successful scaffold integration without disrupting vascular development. Collectively, these findings establish the miRNA-155 inhibitor-activated scaffold as a multi-faceted regenerative platform with anti-inflammatory, angiogenic, and neurogenic outcomes for chronic wound healing applications.

When gut health is compromised by cancer or other disorders, an ostomy may be recommended. An ostomy or stoma is a surgically created opening from a hollow organ to the abdominal wall that facilitates expulsion of waste (dejecta), bypassing the pathological location in the gastrointestinal tract. However, ostomies are associated with complications, the most common being damage to skin surrounding the stoma—peristomal skin complications (PSCs). Leakage of dejecta is the greatest risk factor for PSCs, as it exposes skin to gut microbes, bile acids, and digestive enzymes. Among these constituents, microbes pose a unique risk as they can form biofilms, secrete proteolytic enzymes and release metabolic byproducts that alter the local microenvironment, leading to progressive skin barrier damage. This study identifies intestinal microbes commonly found in ileostomy dejecta and investigates the role of gut microbes in PSC formation using 2D and 3D in vitro human skin models. To investigate the role of gut microbes in PSC formation, dejecta with intact microbial communities was compared to dejecta with a substantially reduced microbial load using skin models. In 2D assays with keratinocytes and fibroblasts, cell migration and viability were compromised in the presence of intact/raw dejecta compared to microbe-reduced dejecta. In 3D skin constructs, intact/raw dejecta caused significant epidermal degradation, while microbe-reduced dejecta caused much less damage. These findings demonstrate the involvement of microbes in PSC-related skin injury. Analyses of dejecta revealed 38 distinct species with significant inter-donor variability. Facultative anaerobes were dominant (59.9 ± 23.8%), with Escherichia coli and Enterococcus faecium identified as the most frequent species. When applied to the 3D constructs, both species caused notable epidermal damage, supporting their role in PSC pathogenesis. This work establishes that microbes in dejecta can directly compromise skin epithelial integrity. By studying ex vivo dejecta, then Escherichia coli and Enterococcus faecium as contributors to epidermal breakdown, microbial factors as drivers of PSC development was demonstrated. These insights can help elucidate a mechanism by which gut microbes contribute to PSC formation and inform the development of preventative approaches aimed at reducing PSC incidence and improving quality of life for patients.

Chronic wounds represent a significant clinical challenge due to persistent inflammation and impaired nerve regeneration, both of which delay healing. Conventional treatments often yield limited success and inconsistent outcomes, especially in complex and recurring wounds. Combinatorial strategies that integrate biomaterial scaffolds with gene delivery offer a promising approach to promote tissue repair. MicroRNAs (miRNAs), particularly miRNA-155, have emerged as crucial regulators of wound healing. MiRNA-155 is highly expressed in inflammatory conditions and plays a key role in macrophage activation, polarisation, and nerve regeneration. In this context, this study introduces a miRNA-155 inhibitor-activated scaffold designed to modulate the chronic wound environment by inhibiting miRNA-155. MiRNA-155 inhibitor complexed GET nanoparticles were characterised and incorporated into collagen-glycosaminoglycan (CG) scaffolds. These supported dermal fibroblast and endothelial cell growth while enabling controlled inhibitor release. Scaffold-mediated miRNA-155 inhibition in both non-polarised (M0) and pro-inflammatory (M1) macrophages promoted anti-inflammatory (M2) polarisation. Macrophage secretome analysis showed reduced inflammatory cytokines and increased angiogenic growth factor secretion in both conditions. The regenerative potential of the miRNA-i-activated scaffold via macrophage polarisation was validated through inflammatory and angiogenic functional assays with endothelial cells. In parallel, scaffold-mediated miRNA-155 delivery to dorsal root ganglia (DRG) promoted neurogenic outcomes through enhanced axonal regrowth, essential for the synergistic repair of chronic wounds across the skin-nerve axis. In vivo implantation of miRNA-155 inhibitor-activated scaffolds in chicks demonstrated successful integration without disrupting vascular network formation. Collectively, these findings establish the miRNA-155 inhibitor-activated scaffold as a regenerative platform with anti-inflammatory, pro-angiogenic, and neurogenic outcomes, offering a multifaceted solution for chronic wound healing applications. Graphical

Cerebral organoids (COs) are multicellular, self‐organized, in vitro, 3D brain‐like tissues used for developmental biology, disease modelling, and drug screening. However, their lack of vascularity renders them less physiologically accurate. Vascularization of COs remains challenging due to the different requirements between COs and vascular cells, limited vascular network penetration within the organoid, and the absence of luminal perfusion. Here, an encapsulation approach is devised in which human brain microvascular endothelial cells (HBMVECs) are delivered to developing COs from progressively degrading extracellular matrix (ECM)‐based hydrogel droplets. By tuning this hydrogel concentration and media composition, an enhanced vascular‐like network formation is observed, expanding within the organoid tissue. Using pathway inhibitors, a subset of the endothelial cells (ECs) is shown to originate from the CO itself, promoting network integration. Endothelial networks displayed blood‐brain barrier (BBB) features, including astrocytic end‐foot‐like interactions, pericyte wrapping, and collagen‐laminin basal lamina. Vascularized COs exhibited greater media internalization and up to three‐fold lower apoptosis than non‐vascularized COs. This comprehensive 3D neurovascular model is a promising platform for cerebrovascular research and drug testing applications.

An ostomy, or stoma, is a surgically created percutaneous aperture from a hollow organ (e.g., small intestine) to the body's surface. Physicians may recommend an ostomy as a temporary or permanent solution to a range of disorders of the gastrointestinal tract, with up to 130 000 ostomies performed annually in the United States. An ostomy facilitates the expulsion of waste products, termed dejecta and circumvents the compromised organs. While an ostomy can be a lifesaving treatment, it is a disruption of regular digestive flow and has a number of associated complications including hernia, prolapse and necrosis. The most commonly observed complications are peristomal skin complications (PSCs), attributed to the leakage of dejecta onto the peristomal skin or the skin directly surrounding the stoma. Despite the prevalence of PSCs, little is known about the precise etiological factors that play a role in PSC formation. This review discusses the constituents of dejecta and their possible roles in PSC formation. Additionally, we identify a number of in vitro and in vivo skin models that could be used to study PSCs. Identification of the components of dejecta and understanding their interaction with skin models can facilitate the development of interventions to treat and prevent PSCs.