Biomaterials

The lung is a common site of metastasis and is continuously deformed by breathing. Whether physiological breathing mechanics influence tumor cell extravasation is unknown. To address this, we developed a mechanically actuated microphysiological system combining perfusable lung-specific microvascular networks with breathing-like cyclic deformation. Under static conditions, lung-specific networks exhibited more lung-like vascular features and higher tumor cell extravasation rates than generic networks. The mechanically actuated device reproduced physiological tissue and vascular deformation, while 24 h of cyclic actuation did not alter vessel viability, permeability, or morphology compared with unactuated controls. Pre-actuation for 24 h, followed by tumor cell perfusion under static conditions, did not alter subsequent extravasation, whereas actuation initiated immediately after perfusion significantly increased extravasation compared with unactuated controls. This biomimetic platform enables direct investigation of how breathing-like vascular deformation influences metastasis and provides a versatile tool for studying vascular mechanobiology in the lung and other mechanically active organs.

The tumor microenvironment plays a pivotal role in tumor development, harboring elements such as endothelial cells, immune cells, fibroblasts, and soluble factors such as transforming growth factor-{beta} (TGF-{beta}) family. TGF-{beta} family regulates cell development and promotes tumor invasion, metastasis, angiogenesis, and endothelial-to-mesenchymal transition (EndoMT). Here, we investigate the effects of TGF-{beta} signaling on vascular remodeling using a three-dimensional (3D) vascular network in a microfluidic device. Using both a co-culture (3D-Co) and simplified endothelial monoculture (3D-CM), we demonstrate that TGF-{beta} signaling reduces the quality and functionality of the vasculature by regressing them. In addition, we observed the upregulation of EndoMT-related markers in mRNA and protein expressions, suggesting the induction of EndoMT in 3D vascular networks. The increased vascular permeability stimulated by TGF-{beta}2 also supports the loss of endothelial identity in the 3D-Co. Transcriptomic analysis revealed the coordinated activation of pathways associated with cell migration and EndoMT, along with the suppression of cell cycle progression. A comparative analysis of two-dimensional (2D) and 3D cultures revealed a fundamentally distinct endothelial response to TGF-{beta}2 in the 3D context, including metabolic reprogramming. These findings demonstrate that the 3D microenvironment critically modulates endothelial responses to TGF-{beta} and enables the emergence of vascular phenotypes not captured in 2D systems. This study provides a more physiologically relevant platform to investigate endothelial dysfunction and vascular remodeling.

Collagen, a major component of the extracellular matrix, regulates cellular behaviors, such as adhesion, differentiation, and angiogenesis. These functions are mediated by interactions between specific amino acid motifs within the collagen triple-helical structure and collagen-binding biomolecules. These include cell-surface receptors, such as integrins, discoidin domain receptors (DDRs), and syndecans, a family of transmembrane heparan sulfate proteoglycans (HSPGs). Signals mediated by these receptors are integrated to regulate cell fate. However, native collagen simultaneously presents multiple receptor-binding motifs, making it difficult to isolate receptor-specific functions and to evaluate receptor crosstalk. Here, we introduce a composition-controlled artificial collagen matrix platform that enables independent tuning of multiple receptor-binding motifs within a constant triple-helical scaffold. This material was produced by disulfide crosslinking of chemically synthesized collagen-like triple-helical peptides, each bearing a single defined receptor-binding sequence. By varying the mixing ratios of these peptides before crosslinking, we systematically controlled the composition of receptor-binding motifs within the matrices. We applied this platform to nerve growth factor-dependent neuronal differentiation of PC12 cells, a process supported by collagen. Matrices containing only integrin-binding sequences were sufficient to support this differentiation. Incorporation of an HSPG-binding sequence had little additional effect, whereas incorporation of a DDR-binding sequence suppressed integrin-mediated differentiation and coincided with DDR phosphorylation. These results reveal opposing roles of collagen-binding integrins and DDRs in regulating PC12 cell differentiation. Composition-controlled artificial collagen provides a versatile matrix platform for dissecting functional crosstalk among collagen receptors.

The mammary gland relies on reciprocal interactions between epithelial cells and their surrounding extracellular matrix (ECM) to form and maintain milk-producing tissue structures. Yet these processes remain difficult to study in human model systems. Mammary epithelial cells (MECs) can be isolated noninvasively from breast milk, but whether they generate three-dimensional organoids and respond to matrix cues has been unclear. Here, human milk-derived MECs (milk MECs) spontaneously form complex organoids, including polarized acinar and terminal duct lobular unit-like structures after isolation. To investigate how matrix composition shapes these organoids, milk MECs were cultured in decellularized mammary ECM (dECM), Matrigel, and collagen I. In dECM, milk MECs formed polarized branched networks with aligned actin organization along collagen fibrils, whereas in Matrigel they adopted a more lactation-associated state, marked by {beta}-casein expression and milk fat globules. Together, these findings establish breast milk-derived MEC organoids as a human model to study how ECM context regulates mammary morphogenesis and lactation biology.

Chronic inflammation drives persistent systemic cytokine signaling that contributes to vascular dysfunction and secondary vasculitis, yet mechanistic studies are limited by models that fail to capture the multicellular architecture and dynamics of human arteries. In contrast, perfusing intact vessels ex vivo has limited tractability because of material availability and difficulty of genetic or biochemical manipulation. We developed a modular, perfused artery-on-a-chip platform by tri-axially bioprinting primary human vascular cells to recapitulate the concentric organization of the intimal, medial, and adventitial layers. The engineered vessels are viable longer than 21 days, with functional endothelial barriers, contractile smooth muscle behavior, and actively remodeled extracellular matrices bearing hallmarks of native vascular tissue. Addition of tumor necrosis factor alpha (TNF) induces altered transcript levels of proinflammatory mediators and secretion of cytokines and matrix-remodeling enzymes without compromising vessel viability. Importantly, this secretory response is effectively attenuated by both a small-molecule JAK1 inhibitor (ABT-317) and anti-TNF antibody (Infliximab), demonstrating the models utility for therapeutic evaluation.

Musculoskeletal symptoms are frequently reported following silicone breast implantation. However, the biological mechanisms linking implant-derived silicone exposure to skeletal muscle alterations remain poorly understood, partly because the biological effects of silicone have long been debated in the context of its biocompatibility. Here, we chemically characterized the low-molecular-weight fraction of the breast implant silicone exposome, readily released from implant gel through gel bleed, and investigated its potential biological consequences using an integrated approach combining analytical chemistry, clinical transcriptomics and histology, and controlled in vitro muscle experiments. Transcriptomic analyses of periprosthetic tissues associated with silicone implant rupture revealed unexpected myogenic and neuromuscular signatures in tissue conventionally regarded as predominantly fibrous, together with alterations in lipid metabolism and transport. These findings were supported by histological evidence of close interactions between periprosthetic tissue and skeletal muscle. Chemical analysis of the implant-gel extract detected linear siloxane L2 and cyclic siloxanes D3-D8, with tentative assignment of D9. In vitro, C2C12 cells exposed to the implant-gel extract showed up to 30% reduced viability and decreased expression of key neuromyogenic genes. Together, these findings provide convergent chemical, clinical, and experimental evidence that low-molecular-weight constituents of the breast implant silicone exposome may constitute a biologically active exposure capable of affecting skeletal muscle. The associated alterations in lipid metabolism and transport further provide a mechanistic framework for investigating the cellular handling and potential tissue distribution of hydrophobic silicone-derived species. These findings position silicone gel bleed as a biologically relevant source of chemical exposure rather than solely a material-integrity phenomenon.

Matrix stiffness is a hallmark of aggressive solid tumors and strongly associated with therapeutic resistance and poor prognosis. This mechanical barrier represents a major unmet challenge in oncology, as current strategies rarely address how matrix mechanics themselves drive treatment resistance. Here, we identified ferroptosis, an iron-dependent form of cell death, as a potential long-sought vulnerability that can be exploited to eliminate mechanically resistant tumors. Using genetic and chemical approaches, we investigated how matrix stiffness sensitizes cancer cells to ferroptosis and whether this vulnerability can be therapeutically targeted. We found that cancer cells cultured on stiff matrices were more sensitive to ferroptosis than those on soft matrices, as evidenced by reduced cell viability and elevated lipid peroxidation. Furthermore, we found that matrix stiffness induced YAP activation, which upregulated its target gene SKP2, a key mediator required for the heightened ferroptosis sensitivity observed under stiff conditions. Finally, using an injectable hydrogel system, we demonstrated that stiffened tumors were resistant to chemotherapy but remained highly susceptible to ferroptosis induction, resulting in markedly reduced tumor volume and weight. Our findings established ferroptosis as a stiffness-dependent vulnerability mediated by a YAP-SKP2 signaling axis, revealing a mechanotransductive pathway that may be leveraged to target treatment-refractory tumors.

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

Most licensed vaccines against infectious diseases mediate protection by priming B cells and humoral immune responses, where efficacy largely relies on activation of germinal centers (GCs) in lymphoid tissues that serve as the training ground for a high-affinity antibody response. While subunit vaccines offer safety advantages, they tend to be poorly immunogenic with weak and short-lived antibody responses, presenting a need for engineering strategies that enhance immunogenicity. Amphiphile-protein vaccines, consisting of protein antigens modified with an albumin-binding lipid tail via a polyethylene glycol (PEG) linker, hitchhike on albumin following subcutaneous injection to enhance lymphatic trafficking and antigen-specific immune activation. Amph-vaccines also demonstrate an ability to insert their lipid tail into cell membranes, effectively 'painting' cells with multivalent antigen. We hypothesized that cell painting may play a role in driving humoral immunity by sustaining antigen persistence in lymph nodes and generating multivalent antigen presentation for B cell activation. Here, we investigated how amph-vaccine molecular properties (antigen MW and PEG linker length) influenced conformational behaviors (micelle formation, albumin hitchhiking, and membrane insertion) along with the resulting humoral immune response. Using HIV env proteins as model antigens (eOD monomer and MD39 trimer), we investigated cell membrane insertion and B cell activation via calcium flux assay in vitro, followed by lymphatic trafficking and immunogenicity studies in vivo. Our results demonstrate that amphiphile conjugation is an effective strategy for enhancing humoral immunogenicity of both monomer and trimer protein antigens, where cell painting plays a significant role in driving B cell, GC, and humoral immune activation.

Encapsulins are self-assembling prokaryotic protein nanocages with growing potential as systemic drug delivery systems, but their pharmacokinetic behaviour remains poorly characterised, and rapid immune recognition and clearance may limit delivery to target tissues. Here, we show that modular PEGylation of a SpyCatcher-decorated encapsulin Alkaliphilus metalliredigens (Am-S) markedly reduces macrophage uptake and extends systemic circulation. Site-directed surface PEGylation using SpyTagged PEG achieved 82% conjugation efficiency, corresponding to an estimated average of 49 PEG chains per 60 subunit Am-S nanocage, without compromising nanocage assembly, morphology, or colloidal stability. PEGylated Am-S also retained solubility and protein integrity following freeze-thaw cycling and six months of storage. When interacted with RAW 264.7 macrophages in vitro, PEGylation substantially reduced nanocage association and internalisation relative to non-PEGylated nanocages. Following intravenous administration in BALB/c mice, PEGylated nanocages exhibited markedly prolonged circulation, with >50% of the injected dose remaining after 1 h compared with 2.6% for non-PEGylated Am-S, and a circulatory half-life of 1 h 43 min. To our knowledge, this represents the first pharmacokinetic characterisation of an encapsulin nanocage. Together, these findings demonstrate that controlled PEGylation can substantially reduce macrophage interactions and prolong encapsulin circulation in vivo.

Engineering a transplantable artificial liver requires reconstituting the hepatic lobule, whose densely cellular parenchyma is organized around a hierarchically branched vascular network. Achieving high cellular density and hierarchical vascularization within a single fabrication step remains a major challenge. Here, we present an ultrasonic standing wave (USW)-based strategy that concurrently organizes vascular and parenchymal compartments during a single extrusion. HepG2-endothelial cell aggregates were fabricated as building blocks and suspended in a liver-derived dECM-alginate hydrogel for rapid, ultraviolet-free crosslinking. They were then co-extruded with single endothelial cells through a glass capillary under USW actuation. Due to their size difference, the single cells were focused at the pressure nodes, forming patterns that transitioned from a single supplying line to four microvascular lines. The aggregates instead accumulated at the pressure antinodes, reconstituting the basic microvascular-parenchymal unit of the lobule. Aggregate size increased with seeding density while maintaining uniformity and over 90% viability. The patterned endothelial cells formed perfusable lumina through which 5 um microparticles flowed freely. Compared with hepatocyte-only cultures, the vascularized constructs showed a 1.7-fold increase in urea production, and 2.7- and 6.2-fold increases in CYP1A2 and CYP3A4 expression, respectively. Following implantation into the mouse liver, the scaffolds showed favorable biocompatibility, sustained proliferation over 21 days, and progressive host integration. The smallest blood-containing lumina matured from 26.1 um to 7.9 um in diameter, accompanied by increasing collagen deposition at the graft-host interface. This approach provides a template-free route to hierarchically vascularized hepatic units and a promising step towards implantable liver tissue.

Osteochondral repair requires restoration of zonally organised articular cartilage and subchondral bone, yet translatable implants rarely reproduce this spatial complexity. Here, we developed an off-the-shelf, cell-free multilayer scaffold comprising a superficial 2% (w/v) articular cartilage extracellular matrix (AC-ECM) phase, an intermediate 5% AC-ECM phase and a basal 6% bone ECM (BN-ECM) phase. The scaffold formed continuous interfaces, displayed regionally distinct pore sizes and resisted permanent deformation during cyclic compression. In vitro, constructs seeded with caprine mesenchymal stromal and articular cartilage progenitor cells supported cell expansion and the accumulation of sulfated glycosaminoglycan- and collagen-rich matrix, with regional differences in collagen I, II and X deposition. Following eight weeks of subcutaneous implantation, cell-seeded scaffolds contained more collagenous matrix than unseeded controls, while vascularisation preferentially localised to the BN-ECM phase. The scaffold was then evaluated against empty defects in a caprine osteochondral model for six months. Scaffold treatment significantly improved macroscopic and histological repair, increased chondral tissue fill (~60% versus ~40%), limited cartilage-like tissue extension into the subchondral region and generated a more native-like superficial collagen organisation. Repair tissue further exhibited greater collagen II immunoreactivity, increased ACAN and COL2A1 expression and reduced COL1A2 expression relative to empty defects, although deeper bone repair was not significantly improved. These findings demonstrate that tissue-specific ECM layering can spatially guide endogenous repair and substantially improve cartilage restoration without exogenous cells or growth factors in a clinically relevant large-animal model, while identifying subchondral bone regeneration as the remaining design challenge for complete osteochondral repair.

Neonatal mammals exhibit a remarkable capacity for cardiac repair that declines rapidly after birth and continues to diminish with age. This loss of regenerative potential is accompanied by age-dependent changes in macrophage behavior, shifting from pro-regenerative to pro-fibrotic responses following injury. We investigated whether extracellular matrix (ECM)-derived matrikines from different developmental ages regulate macrophage polarization. Cardiac ECM was isolated from neonatal (P2), adolescent (6-week), and adult (16-20-week) rat hearts through decellularization and pepsin digestion. ECM peptides were adsorbed onto tissue culture surfaces, and murine RAW 264.7 macrophages were cultured on ECM-coated or uncoated controls. After 24 hours, macrophage phenotype and gene expression were assessed using marker analysis and RNA sequencing. Macrophages cultured on neonatal cardiac ECM displayed reduced inflammatory activation and maintained expression of pro-reparative markers, even in the presence of inflammatory stimuli. Transcriptomic analysis revealed a distinct ECM-driven phenotype that differed from canonical M1 and M2 polarization states. Gene ontology analysis demonstrated significant downregulation of pathways associated with antiviral responses, immune activation, and interferon signaling in macrophages exposed to neonatal ECM. These effects were not observed with adolescent or adult ECM. Collectively, these findings suggest that neonatal ECM matrikines suppress interferon-mediated inflammatory signaling, potentially contributing to the reduced inflammation and enhanced repair observed following myocardial injury in neonatal hearts.

Objective: The maturation of an autologous arteriovenous fistula (AVF) depends on the coordinated hemodynamic adaptation of the inflow artery and the outflow vein. While venous remodeling has been extensively studied, the mechanisms of arterial adaptation are poorly understood. We addressed this knowledge gap with the first temporal single-cell atlas of the porcine AVF model. Methods: Six Yorkshire pigs underwent bilateral femoro-femoral AVF creation for tissue harvest at 2 and 21 days postoperatively or sham operation to collect native vessels after 2 days. Arteries and veins were analyzed by droplet-based single-cell RNA sequencing, histology, and immunohistochemistry. Results: All fistulas were patent, with flows reaching 1185.0 +/- 170.6 mL/min despite mild to moderate neointima formation in the veins. Arteries and veins demonstrated >60% endothelial denudation and increased neovascularization. Arterial remodeling was characterized by a progressive increase in venous-like transcriptional features. Arteries presented a venous-like endothelial cell (EC) composition at 21 days postop and a temporal increase in venous-associated phenotypes of myofibroblasts and fibroblasts. In both vessels, the latter populations were the main bearers of mechanosensitive signatures at 2 days. Despite the temporal convergence in phenotypes, arteries and veins retained vessel-selective characteristics. Arterial ECs expressed high levels of nitric oxide synthase, whereas venous ECs were largely inflammatory. Arterial myofibroblasts and fibroblasts had higher expression of Notch signaling transducers (HEY2, HEYL) and cellular communication network factors (CCN3, CCN5), while Wnt signaling regulators (DKK2, SFRP4) and complement genes (C3, C7, CFD) were upregulated in veins. Anti-inflammatory macrophages increased in both vessels during remodeling, likely contributing to the maturation of the fistulas. Conclusion: These analyses challenge the traditional paradigm of exclusive vein arterialization and support a complementary process of arterial venification after anastomosis. Recognition of this underappreciated phenomenon may reshape our understanding of AVF biology and guide the discovery of both vascular-wide and vessel-selective anti-stenotic therapies.

Recurrent urinary tract infection (rUTI) is a significant public health problem. The most common cause of rUTI is uropathogenic Escherichia coli (UPEC). Antimicrobial prophylactic and therapeutic regimens for rUTI disrupts the microbiome and leads to infection with antimicrobial resistant organisms. Therefore, there is an urgent need to develop microbiome-sparing alternative approaches for preventing rUTI. Asymptomatic bacteriuria (ABU) E. coli strain 83972 (ABEC) outcompetes UPEC in the urinary tract without causing UTI symptoms, but its limited persistence in the bladder restricts its efficacy. Here, we investigate a device made from an engineered living material (ELM) that releases ABEC in a sustained manner as an antibiotic-free platform against UTIs and rUTIs. Using physiologically relevant in vitro models incorporating human urothelial cells, human urine, and periodic urine exchange, we show that ABEC-releasing ELMs suppress UPEC proliferation and inhibit UPEC attachment to and invasion into urothelial cells, particularly when ABEC is maintained at equal or higher levels than UPEC. Because ELMs continuously release ABEC, they sustain competitive pressure even as planktonic bacteria are cleared during voiding, outperforming a single dose of free-floating ABEC. In a rUTI model, sustained ABEC release from ELMs reduces the proliferation and re-invasion of UPEC expelled from infected urothelial cells, while UPEC infiltration into fractured ELMs remains negligible. Finally, we design a first-generation ELM device that can be transurethrally delivered and retained within the mouse urinary bladder, achieving sustained ABEC release in vivo, with ABEC persisting in the bladder, kidneys, and urine for at least 4 days. In summary, we report the development of an ELM device that continuously releases ABEC to suppress UPEC proliferation and urothelial cell invasion in in vitro models of rUTI, and demonstrate sustained ABEC release in vivo in a mouse bladder.

During tumor growth and progression, cancer cells are exposed to sustained physical confinement and volumetric compression that can alter cell volume, cytoskeletal organization, mechanotransduction, and invasive behavior. However, whether breast cancer cells retain a compression-induced mechanical memory after release from sustained volumetric compression, and how this memory influences subsequent migration and invasion, remains poorly understood. Here, by controlling cell volume using PEG - mediated volumetric compression, we investigated the compression and post-compression recovery responses of MCF-7 breast cancer cells. Cells were compressed for four days, followed by four days of recovery after PEG removal, and analyzed using daily morphological tracking, single-cell time-lapse imaging, F-actin and YAP staining, wound healing assays, and 3D spheroid invasion assays. We show that sustained volumetric compression shifts MCF-7 cells into a compact, jammed-like, low-motility state characterized by reduced morphodynamic remodeling, suppressed collective migration, and limited spheroid invasion. In contrast, post-compression recovery induces a distinct mechanobiological state marked by increased cell area and perimeter, altered single-cell trajectories, heterogeneous F-actin remodeling, enhanced YAP nuclear localization in enlarged recovered cells, accelerated wound closure, and increased spheroid invasion and cell dissemination. These findings suggest that prior volumetric compression can prime breast cancer cells for enhanced migration and invasion after stress release, supporting post-compression recovery as a form of mechanical memory that may contribute to tumor dissemination.

Macrophages are highly dynamic cells that maintain tissue homeostasis by regulating both initiation and resolution of inflammation. During efferocytosis, macrophages recognize the eat me signal, phosphatidylserine (PS), exposed at the surface of apoptotic cells, leading to the resolution of inflammation and acquisition of a pro-resolving phenotype. Inspired by this endogenous mechanism, PS-based biomaterials have demonstrated immunomodulatory potential. However, the molecular mechanisms underlying PS-mediated macrophage reprogramming remain poorly understood. Here, submicron PS-exposing polymeric particles (PSPs; [~]300 nm) were developed to improve the suitability of PSP formulations for future systemic administration while preserving their immunomodulatory activity. PSPs were efficiently internalized by macrophages through both actin- and dynamin-dependent pathways. PSP treatment significantly reduced IL-6 and IL-12p70 production in LPS-stimulated macrophages, whereas induction of the classical anti-inflammatory M2 marker CD206 was limited. Transcriptomic analysis revealed coordinated attenuation of inflammatory signaling pathways, including downregulation of Myd88, Nfkb1, Rel, and Irf8, together with activation of NRF2-associated antioxidant pathways characterized by increased expression of Nfe2l2, Hmox1, Prdx1, Gclm, and Gclc. Activation of antioxidant-associated genes together with reduced Irf8 expression suggests that PSP promotes inflammatory resolution through coordinated redox adaptation and selective attenuation of inflammatory signaling. Collectively, these findings provide mechanistic insight into PS-mediated macrophage reprogramming and support the future development of systemically administered therapies for chronic inflammatory diseases, including vascular inflammatory disorders. HighlightsO_LISubmicron PSPs retain immunomodulatory activity of apoptotic cell-mimicking biomaterials. C_LIO_LIPSPs are rapidly internalized through actin- and dynamin-dependent pathways. C_LIO_LIPSPs attenuate inflammatory signaling and selectively suppress IL-6 and IL-12p70 production. C_LIO_LIPSPs induce NRF2-associated antioxidant and glutathione responses. C_LIO_LITranscriptomics reveals an early redox-adaptive macrophage program. C_LI

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-{beta}-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.

Myocardial infarction highlights an urgent need for strategies to regenerate functional cardiac tissue. Cardiac tissue engineering offers a promising approach; however, fabricating scaffolds that simultaneously integrate precise architectural anisotropy, mechanical compliance, and electrical conductivity remains an open challenge. In this work, we utilized melt electrowriting (MEW) to construct well-defined, 20-layer anisotropic rhomboidal polycaprolactone (PCL) scaffolds. We characterised them by tensile testing and by micro- and nanoscale microscopy. While introducing electrical conductivity via bulk blending with fillers (polypyrrole (PPy), polyaniline, or graphene oxide) compromised MEW print fidelity and failed to achieve physiological conductivity, surface coating strategies effectively combined conductivity from structural mechanics. Electrical and mechanical testing revealed that gold sputter coating and in situ PPy polymerization both imparted robust electrical conductivity while preserving the microfibrous architecture. However, when seeded with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in fibrin hydrogels, only the gold-coated scaffolds supported synchronized, robust, and sustained contractile activity. PPy-coating resulted in functionally restricted constructs, suggesting that excessive structural rigidity limited tissue deformability. Gene expression analysis further revealed that elevated electrical conductivity alone does not drive hiPSC-CM maturation. Our data indicates that successful cardiac patch design relies on the integrated optimization of mechanics and architecture rather than treating conductivity as an isolated parameter, offering foundational guidelines for developing translational bioengineered heart patches.

Living myocardial slices (LMS) are a highly relevant ex vivo model for investigating cardiac physiology and disease, as they preserve the native three-dimensional architecture, cellular diversity, and extracellular matrix of the heart. In addition, LMS enable longitudinal functional and molecular analyses. In this study, we established and compared two LMS culture approaches: an air-liquid interface system and a biomimetic culture system. We further examined how different slicing techniques affect tissue quality and longevity within the biomimetic setup. To develop a fibrosis model, LMS were stimulated with transforming growth factor-beta1 (TGF-beta1) and/or exposed to increased mechanical load. Tissue viability was assessed using LIVE/DEAD staining and the MTT assay, while cytotoxicity was evaluated with the LDH-Glo-TM Cytotoxicity assay. Contractile function was measured, and fibrotic remodelling was analysed using RT-qPCR, ELISA, and immunohistochemistry. Our results demonstrate that LMS cultured in the biomimetic system exhibit superior viability, structural integrity, and functional performance compared with those maintained at the air-liquid interface. Mouse LMS could be stably cultured for up to one week in the biomimetic system. Importantly, sample preparation, particularly the slicing method, had a significant impact on tissue quality and culture duration. While TGF-beta1 stimulation alone did not consistently induce fibrosis, combining TGF-beta1 treatment with increased mechanical load led to more pronounced fibrotic remodelling in LMS. These findings highlight the importance of biomechanical cues in modelling cardiac fibrosis ex vivo and support the biomimetic system as a robust platform for functional and disease-relevant studies.