Biomaterials Advances

Introduction: Advanced biomaterials for regenerative medicine are increasingly expected to combine multifunctionality and compatibility with tissue-specific cellular processes. In this context, hydroxyapatite-based platforms modified through ionic substitution represent promising candidates, as they may integrate structural similarity to bone mineral with additional biological functionality and luminescent properties, enabling diagnostic applications and real-time monitoring. In this study, we evaluated whether silicate-phosphate substituted calcium hydroxyapatite Ca10(PO4)6-x(SiO4)x(OH)2 (where x = 1.5) co-doped with lithium(I), europium(III), and gadolinium(III) ions (Si-HAp-LEG) affects the osteogenic, chondrogenic, and adipogenic differentiation potential of human bone marrow stromal/stem cells (BMSCs). Methods: Human BMSCs were cultured under lineage-specific differentiation conditions in the presence of undoped silicate-substituted phosphate hydroxyapatite (abbr. as Si-HAp), which served as a control, and two distinct Si-HAp-LEG formulations differing in gadolinium(III) (Gd3+) as well as lithium (Li+) and europium(III) (Eu3+) ion concentrations: Si-HAp-LEG-221 (1 mol% Gd3+ ion) and Si-HAp-LEG-222 (2 mol% Gd3+ ion). Differentiation-associated phenotypic outcomes, including extracellular matrix formation and lipid accumulation, were evaluated using Safranin O, Alizarin Red, and Oil Red O staining. In parallel, biomaterial-induced molecular responses were characterized at the transcriptomic and protein levels using RT-qPCR for selected coding and non-coding RNAs and Western blot analysis for representative lineage-associated proteins. Results: Histochemical evaluation confirmed that, across all tested biomaterial groups, BMSCs retained the ability to form mineralized calcium deposits, proteoglycan-rich extracellular matrix, and intracellular lipid accumulation under osteogenic, chondrogenic, and adipogenic conditions, respectively. Quantitative staining analysis revealed no significant Si-HAp-LEG-dependent enhancement of terminal differentiation outcomes compared with undoped Si-HAp. In turn, the molecular response differed between biomaterials modifications. Si-HAp-LEG-222 induced the most prominent changes in transcriptional and post-transcriptional regulators, particularly within BMP/SMAD-associated pathways under osteogenic and chondrogenic conditions, underlying a potential link between gadolinium concentration and osteogenic lineage commitment. However, these transcriptomic responses were not mirrored by consistent changes at the protein level. The results suggest that silicate-phosphate substituted hydroxyapatite co-doped with Li+, Eu3+, and Gd3+ ions primarily affects the early regulatory pathways associated with BMSCs differentiation rather than enhancing their terminal maturation. Discussion: In conclusion, the collective data indicate that Li+, Eu3+, and Gd3+ ions LEG co-doping broadens the multifunctional potential of Si-HAp by introducing imaging-related properties while preserving its underlying pro-regenerative character. Li+, Eu3+, and Gd3+ ions co-doped LEG-substituted Si-HAp may therefore be considered a compatible biomaterial platform that maintains BMSC cellular plasticity and supports balanced, differentiation-dependent modulation of lineage-associated molecular responses.

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

Sensory and motor neurons differ significantly in their morphology, structural organization, functional properties, and mode of action. However, despite this heterogeneity, many in vitro studies focus only on a single neuronal subtype, mainly being sensory neurons, limiting the translational potential and relevance of these studies for the evaluation of therapeutic options for spinal cord injury. In this study, we investigate the differentiation, maturation, and neuronal outgrowth of human induced pluripotent stem cell (iPSC)-derived motor and sensory neurospheres using polyethylene glycol (PEG)-microgels with various bioactive coatings. Our results show subtype-specific responses to the PEG-microgel scaffolds, with respect to motor and sensory neurosphere morphology and size. Furthermore, we compare the formation of the PEG-microgel/scaffolds when starting from iPSCs-derived precursor neuron spheres versus undifferentiated iPSCs. We observe notable differences in structural organization, maturity, and neuronal outgrowth between the two approaches, as well as between motor and sensory neurospheres. Together, these results underline the importance of studying motor and sensory neurons separately and highlight the need for a controlled, tunable culture platform to assess the impact of the microenvironment and to improve the physiological relevance of in vitro platforms for neuron-based research.

Most antimicrobial drug candidates currently in development are derivatives of established antibiotic classes. In contrast, antimicrobial heteroatom-doped carbon quantum dot (CQD) nanoparticles vastly differ from their chemical antibiotic counterparts and exhibit potent antibacterial activity and favourable biocompatibility, representing a promising alternative strategy, particularly for topical applications. Here, we report the incorporation of cobalt-doped carbon quantum dots (Co-CQDs) into injectable, biocompatible hydrogels capable of both sensing pH and eliminating bacteria. Ultrasmall Co-CQDs demonstrated broad-spectrum activity against gram-positive Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Pseudomonas aeruginosa (PAO1), mediated by membrane hyperpolarisation and reactive oxygen species (ROS) induced membrane damage. The particles showed negligible effect on primary fibroblast and endothelial cell viability at concentrations that were bactericidal to MRSA. Polymeric hydrogels were fabricated via electrospinning of chitosan, polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA) polymer blends incorporating Co-CQD and pH-responsive HPTS particles. This approach provided accurate measurement of environmental pH within the physiological range observed across healthy and chronic wounds. In vivo, the injectable hydrogels exhibited robust antimicrobial efficacy against MRSA without impairing wound closure relative to untreated controls, while also reducing inflammatory immune responses in infected tissues. Collectively, these findings demonstrate the potential of ultrasmall metal-doped CQDs for infection control and their integration into 3D matrices as multifunctional theragnostic platforms.

Mesenchymal stem/stromal cells (MSCs) are key players in regenerative medicine due to their immunomodulatory properties and ability to promote tissue repair. However, their therapeutic efficacy is often limited by rapid clearance following transplantation. MSC spheroids have shown enhanced functional properties, and we hypothesize that encapsulating them within hydrogel microbeads could offer additional protection and improve their viability. In this study, we developed a novel droplet-based microfluidic protocol for human MSCs derived from the apical papilla (SCAP) encapsulation and In-Bead spheroid formation within alginate microbeads. Optimization of the protocol allowed the formation of MSC spheroids in alginate droplets overnight (In-Bead), before alginate cross-linking and retrieval of alginate beads loaded with MSC spheroids. SCAP were successfully encapsulated within 275 {micro}m alginate microbeads, forming spheroids of approximately 80 {micro}m in diameter. Encapsulated SCAP spheroids retained their immunomodulatory properties. The process was further optimized by incorporating nanomedicines into the alginate solution before the formation of droplets and then spheroids, forming thus hybrid beads (Sph.Beads/NP). Nanomedicines were loaded with NecroX-5, a necrosis inhibitor, to improve SCAP viability further. Live/Dead assays indicated a protective effect of the nanomedicines, supporting the potential of this system for advanced cell delivery in regenerative applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/732109v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@278324org.highwire.dtl.DTLVardef@12bad2org.highwire.dtl.DTLVardef@1a7489forg.highwire.dtl.DTLVardef@190e793_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractA combination strategy enhancing MSC viability through spheroid formation, microencapsulation, and nanomedicine association achieved by microfluidic encapsulation with In-Bead spheroid formation. Created with BioRender

One crucial step during early osteogenesis is the embedding of osteoblasts within a collagen-rich extracellular matrix (osteoid), where they subsequently differentiate into a functional network of osteocytes. However, reconstructing 3D osteocyte networks in vitro remains a major challenge. We recently developed a synthetic microporous hydrogel to support the in vitro culture of 3D bone cell networks. Although matrix biodegradability facilitates cell-material interactions, the influence of micropores on bone tissue morphogenesis and differentiation remains poorly understood. Here, we investigate the effect of cell-scale micropores on bone cell morphogenesis and osteogenic differentiation in vitro. By exploiting polymerization-induced phase separation (PIPS) between 4-arm polyethylene glycol vinyl sulfones and dextran in the presence of hyaluronan, we generated matrix metalloproteinase-sensitive hydrogels with cell-scale micropores. Increasing the dextran concentration enlarged the average pore size from 4 m to 8 m, accompanied by a slight decrease in mechanical stiffness. Following encapsulation within these hydrogels, primary human osteoblasts remained highly viable. Hydrogels with larger pores supported extensive 3D cell network formation, whereas hydrogels with smaller pores exhibited enhanced osteogenic differentiation following 21 days of osteogenic culture. Together, these findings highlight that bone cells are sensitive to microporous physical cues and even minor changes over pore sizes can make an impact on osteocyte-like morphogenesis and differentiation in vitro.

Tissue engineered skin models are important tools for the in vitro study of physiological and pathophysiological processes as well as the valuation of therapeutic strategies and the efficacy of pharmaceutical and cosmetic compounds. Replicating the functional anatomy of cutaneous tissue is a crucial aspect in ensuring that observations made using these models are translatable to the actual situation in native skin. However, most contemporary full-thickness skin models neglect the reconstruction of the undulated microtopography of the dermal-epidermal junction (DEJ), which not only contributes to the biological functionality of the skin (e.g. stem cell niches), but also affects tissue mechanics and drug diffusion. Herein, we fabricated bilayer skin models with DEJ-like microtopographies introduced by interfacial wrinkling between a hydrogel and a nanofibrous membrane through a controllable swelling-deswelling approach. The interfacial wrinkles contributed to the structural integrity of the bilayer models. Their formation could be induced in the presence of living cells through mechanical stress-driven buckling instabilities, thus differentiating the process from commonly used pre-patterning techniques. Bilayer models supported the co-culture of human dermal fibroblasts and human epidermal keratinocytes, and the formation of stratified epithelia. Our findings provide a potential alternative method to introduce DEJ-like anatomical features into full-thickness skin tissue models.

Articular cartilage damage often progresses to osteoarthritis (OA), a degenerative joint disease characterized by chronic pain, limited mobility, and reduced quality of life. Tissue engineering approaches using poly (ethylene glycol) (PEG)-based hydrogels offer tunable mechanical properties and bioactive functionalization, yet the influence of surface charge on cartilage regeneration remains underexplored. Moreover, recombinant fibroblast growth factor 18 (FGF18) has successfully improved cartilage tissue thickness in clinical trials, but required high dosages and recurring injections may limit compliance. Here, we developed a granular microgel-based platform with bioorthogonally tethered FGF18 to evaluate the interplay of microgel surface charge and growth factor presentation on chondrogenesis. Azide groups were incorporated onto the microgel surfaces to enable site specific FGF18 conjugation across microgel scaffolds with distinct surface charges. When seeded with mesenchymal stromal cells, microgel scaffolds functionalized with FGF18 outperformed their unmodified counterparts, evidenced by higher GAG content, collagen content, and compressive modulus. In a murine microfracture model, anionic and zwitterionic microgel scaffolds tethered with FGF18 increased cartilage regeneration compared to nonionic microgels. We detected increased collagen II content within defects treated with tethered FGF18 microgel scaffolds. This work demonstrates the role of surface charge and growth factor presentation in directing cell behavior and tissue repair, advancing the design of biomaterials for cartilage regeneration. HIGHLIGHTSO_LICovalent tethering of FGF18 to microgel surface enables localized bioactivity C_LIO_LIFGF18 tethered microgel scaffolds enhance extracellular matrix deposition and chondrogenic differentiation of murine mesenchymal stromal cells in vitro C_LIO_LILocalized FGF18 presentation improves cartilage tissue formation and mechanical properties in vivo, with anionic and zwitterionic microgel scaffolds outperforming nonionic scaffolds C_LIO_LIFGF18 presentation is a more potent stimulus than microgel surface charge for cartilage regeneration C_LI

Tissue regeneration using bioactive biomaterials has made great progress in the field of wound healing. Biopolymers play a cardinal role in regenerative medicine by providing safe, biocompatible and bioresorbable support. The electrospinning fabrication technique has been used in creating suitable wound care materials. PHBV and PLLA are FDA approved polymers having important applications in biomedical field. In this study, to increase the wound healing potential, PHBV was functionalized with -COOH group and electrospun nano-fibrous mat was produced using PHBV-COOH and PLLA blended solution. Antibiofilm peptide (IDR-1018) with immunomodulatory activity was incorporated into the blended solution to improve infected wound treatment by actively fighting against bacterial infections. Furthermore, in-vitro experiments including cell cytotoxicity assay and scratch wound healing assay were done to evaluate the potential of the synthesized bioactive nanofibrous mat as a potential wound management aid.

Scaling engineered living materials to clinically relevant dimensions is limited by diffusion-dependent depletion of oxygen and nutrients, which rapidly induces metabolic failure. We introduce glycogen as nutritional nanoparticle that provides cell-mediated, autonomous nutrient release to support long-term survival under extreme metabolic stress. We demonstrate that human mesenchymal stromal cells (hMSCs) survive for weeks in anoxia and serum deprivation when provided extracellular glycogen. Contrary to long-held assumptions, hMSCs secrete glycogen-degrading enzymes, enabling cell-density controlled extracellular glycogenolysis and sustained release of glucose and metabolic intermediates, positioning glycogen as the first-of-its-kind metabolic battery. This cell-responsive process maintains metabolic activity, limits glycolytic acidosis, and enhances pro-angiogenic signaling. To translate this mechanism into a versatile materials platform, we engineered core-shell dextran-tyramine microcapsules that stably encapsulate glycogen while permitting diffusion of enzymes and degradation products. Integrated into centimeter-scale GelMA constructs, these microcapsules maintained hMSC viability and function for at least one month under anoxia. In vivo, glycogen-loaded implants promote deep cellular infiltration, enhanced matrix remodeling, increased M2 macrophage polarization, and orchestrated accelerated vascularization. This work establishes the novel concept of glycogen-based nutritional nanoparticles as metabolic batteries to endow engineered tissues with autonomous self-feeding capacity, enabling scalable and functional living materials for regenerative medicine and related technologies.

Chronic diabetic wounds remain a major clinical challenge because current therapies address infection or supplement growth factors without correcting the cellular dysfunction that prevents regeneration. We identify pathological glycocalyx thickening in diabetic dermal fibroblasts (DDFs) as a driver of elevated caspase 3, 8, and 9 expression and heightened apoptosis -- deficits that stall wound repair. Cleavage of sialic acid residues by neuraminidase (NMase) reverses this dysfunction, restoring fibroblast migration, proliferation, and contractility. We engineer a photo-crosslinked hybrid hydrogel combining methacrylated gelatin (GelMA) with high-molecular-weight methacrylated chitosan (HMW ChMA). ChMA increases storage modulus 8-12-fold, reduces pore size, and confers antibacterial activity against gram-positive and gram-negative bacteria, addressing infection susceptibility. The fortified HMW hybrid (HMWH) network enables sustained, localized NMase delivery that outperforms GelMA alone in resisting degradation and controlling release kinetics. NMase-loaded HMWH (N-HMWH) gels enhance DDF proliferation and migration in vitro, correlating with reduced focal adhesion size and increased turnover. In a diabetic rat model, N-HMWH patches achieve superior wound closure, outperforming EGF therapy, with robust epidermal regeneration, neovascularization, and collagen deposition. This work establishes glycocalyx-targeting hydrogels as a new class of wound therapeutics addressing the root cause of diabetic fibroblast failure, not just compensating with growth factors.

Osteosarcoma (OS) is one of the top ranking and deadliest primary malignant bone tumor of youngsters and adolescents. OS has poor prognostic features due to its immunosuppressive "cold" tumor microenvironment, obstructing the anti-tumor effector functions of immune cells (including cytotoxic CD8 T lymphocytes). Recent developments have focused on the role of Gut Microbiota in modulating effector immune responses in various cancers. However, the immunomodulatory role of gut microbiota and its derived gut-associated metabolites (GAMs) in OS still remains unclear. Here we report that peripheral CD8 T lymphocytes in osteosarcoma patients are less frequent in circulation, hypo-producers of effector cytokines (IFN-{gamma} and TNF-), and have compromised metabolic fitness. We characterized and investigated the effect of a panel of microbiota-derived GAMs on CD8 T lymphocytes, confirming their immunomodulatory role with respect to CD8 T lymphocytes activation, cellular metabolism and effector functions. Indole-3-lactic acid (ILA; product of tryptophan metabolism), was observed to be the strongest immunostimulant among all the GAMs studied. ILA enhanced the anti-tumor effector functions of CD8 T lymphocytes through increased glucose uptake, a higher mitochondrial bio-mass and increased production of IFN-{gamma} and TNF-. Moreover, ILA-primed cytotoxic T lymphocytes exhibited considerably higher cytotoxicity and increased apoptosis of osteosarcoma cells (U2OS). Altogether, our data demonstrates that ILA acts as a microbial-derived immune modulator to metabolically reprogram and restore dysfunctional CD8 T lymphocytes against osteosarcoma. This study for the first time demonstrates the therapeutic potential of exploiting the nexus between "Gut-Immune-Bone Tumor" ternary as a safe and cost-effective combinatorial immunotherapy against osteosarcoma. Graphical AbstractIndole-3-Lactic Acid (ILA) Reinvigorates CD8+ T-Cell Immunity in Osteosarcoma Circulating CD8+ T cells from osteosarcoma patients exhibit impaired metabolic fitness, reduced effector cytokine production, and diminished tumoricidal activity. Screening of gut-associated metabolites identified the microbial tryptophan metabolite indole-3-lactic acid (ILA) as the most potent immunomodulator. ILA restores glucose uptake and mitochondrial biomass, enhances IFN-{gamma} and TNF- production, promotes polyfunctional CD8+ T-cell responses, and significantly improves CTL-mediated killing of osteosarcoma cells, highlighting its potential as a microbiota-derived immunometabolic therapeutic for osteosarcoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/741694v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10416a4org.highwire.dtl.DTLVardef@16a99b8org.highwire.dtl.DTLVardef@19201cforg.highwire.dtl.DTLVardef@b4ca26_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hydrogels have emerged as attractive vaccine delivery platforms because they enable controlled modulation of antigen availability. However, how different hydrogel environments affect the release and functionality of mRNA-loaded lipid nanoparticles (mRNA-LNPs) remains poorly understood. Here, we investigated the release, stability, cellular uptake, and transfection capability of LNPs released from four hydrogel systems representing distinct crosslinking mechanisms: covalently crosslinked poly(ethylene glycol) (PEG), ionically crosslinked alginate, thermoresponsive Poloxamer 407 (P407), and protein-based Matrigel/collagen hydrogels. All hydrogels enabled release of LNPs over days, with kinetics strongly depending on hydrogel composition and polymer concentration. LNPs were quantitatively recovered from all hydrogel types, except from Matrigel/collagen where incomplete matrix dissolution was the limiting step. Lower polymer concentrations generally accelerated nanoparticle release. PEG offered greatest tunability of release kinetics; at the same time the recovery of the LNP-incorporated fluorescent dye DiI was reduced to about 80 %, indicating partial dye leakage. Alginate hydrogels exhibited recovery of DiI below 50 % and broader particle size distributions after release, while P407 hydrogels largely preserved LNP characteristics. Although quantitative recovery from Matrigel/collagen hydrogels was limited, released LNPs remained readily available for cellular uptake. Notably, LNPs released from low- and intermediate-concentration Matrigel/collagen hydrogels achieved approximately 80-90 % of the eGFP expression compared to mRNA-LNP that were not embedded into a hydrogel. Importantly, cellular uptake and transfection experiments demonstrated that all investigated hydrogels released biologically active mRNA-LNPs capable of mediating protein expression. Moreover, our findings show that hydrogel composition is a critical determinant of mRNA-LNP release, stability, and functional delivery. This work provides design principles for the development of hydrogel-based mRNA delivery systems aimed at sustained antigen availability and prolonged vaccine responses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/741169v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@138d9eforg.highwire.dtl.DTLVardef@16c0edaorg.highwire.dtl.DTLVardef@1432dd1org.highwire.dtl.DTLVardef@17511b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell therapies involving human adipose-derived stromal cells (hASCs) have shown promise for a range of clinical applications due to their ability to stimulate angiogenesis and dampen inflammation via paracrine mechanisms. However, a major barrier to the successful clinical translation of hASC-based therapies is that standard culture methods for expansion on rigid 2D tissue-culture polystyrene under static conditions diminish the pro-regenerative functionality of the cells. To address these limitations, the current project focused on the development of an in vitro bioreactor system for preconditioning hASCs to augment their capacity to stimulate regeneration through paracrine mechanisms. Specifically, the combined effects of decellularized adipose tissue (DAT) coatings, shear-stress stimulation, and varying oxygen tensions on hASC expansion and paracrine factor secretion were assessed. Additional studies were performed to characterize the effects of stimulating hASCs within the rocking bioreactor system using the pro-inflammatory cytokines IFN-{gamma} and TNF-. Expansion in the bioreactor under all conditions supported hASC growth with no observable morphological differences. However, dynamic culture on DAT coatings enhanced intracellular indoleamine 2,3-dioxygenase (IDO) expression in hASCs cultured under 20% O2. Moreover, culturing under dynamic conditions and/or on DAT coatings significantly increased secretion of the pro-angiogenic factors VEGF, HGF, and angiogenin. When pro-inflammatory cytokine priming was introduced, the expression of all tested paracrine factors was enhanced, particularly the immunomodulatory factors IL-6, IL-8 and MCP-1. Overall, a novel bioreactor system was developed for hASC expansion and preconditioning, demonstrating that the cell microenvironment can be tuned to modulate hASC paracrine factor secretion.

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.

Three-dimensional (3D) spheroid culture provides a physiologically relevant alternative to conventional two-dimensional culture, but reliable spheroid formation in microwells depends on limiting cell-substrate adhesion. Pluronic F127 is an amphiphilic triblock copolymer that forms a hydrated surface layer, reducing protein adsorption. Here, we evaluated whether this intrinsic anti-fouling property could restore an anti-adhesive surface in used microwell plates to promote spheroid formation. Using chondrogenic ATDC5 and pancreatic {beta}-cell INS-1 cells, we characterized spheroid assembly kinetics, F127 cytotoxicity, surface hydrophilicity, protein adsorption, and spheroid morphology including size and shape factor. Both cell types formed compact spheroids within 24 hours on commercial anti-adhesive microwells. F127 coating markedly reduced water contact angle and protein adsorption, confirming increased surface hydrophilicity and reduced protein fouling. In microwells stripped of their original surface coating, F127 coating amounts of approximately 0.011-0.045 mg/cm2 consistently promoted spheroid formation across both cell types. Soluble F127 concentrations were confirmed to be non-cytotoxic up to 0.625% (w/v), while even complete dissolution of the highest tested coating amount would correspond to only 0.025% (w/v) F127. This simple, reproducible, and low-cost surface-modification strategy may provide an accessible approach for re-functionalizing microwell platforms for 3D cell culture.

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.

Vocal fold (VF) fibrosis is a major cause of persistent dysphonia due to excessive extracellular matrix deposition and tissue stiffening that disrupt normal vocal fold vibration. Current treatment approaches are limited by the need for repeated local injections and inadequate long-term therapeutic control. Pirfenidone (PFD), an FDA-approved antifibrotic agent, has demonstrated potential for reducing fibrosis; however, its short half-life and systemic adverse effects limit conventional administration strategies. In this study, we developed a sustained and near-infrared (NIR)-responsive local delivery platform by integrating PFD-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles into biodegradable PLGA implants for dose-controllable antifibrotic delivery. PFD-loaded PLGA nanoparticles were fabricated using an oil-in-water emulsion solvent evaporation method and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Nanoparticles with small, medium, and large hydrodynamic diameters were generated to evaluate the effect of particle size on release behavior. Gold nanorods (AuNRs) were incorporated to enable photothermal NIR-triggered release enhancement. The nanoparticles were subsequently loaded into non-porous PLGA (90:10) implants and evaluated for long-term in vitro release under physiological conditions with and without pulsed 1064 nm laser irradiation. The nanoparticle-loaded implants demonstrated sustained PFD release for over 190 days with minimal initial burst release (<2.5%). NIR irradiation enhanced PFD release compared with non-irradiated controls across all nanoparticle sizes. Smaller nanoparticles produced greater cumulative release than medium and large nanoparticles due to shorter diffusion pathways and larger surface-area-to-volume ratios. Prior to implant fracture, cumulative PFD release reached approximately 20.2%, 14.8%, and 12.3% of total loading for small, medium, and large nanoparticle groups under 2-min irradiation conditions, respectively. Dialysis membrane studies further demonstrated that the PLGA capsule acted as an additional diffusion barrier that substantially prolonged release compared with nanoparticles alone. Overall, this study demonstrates a hybrid nanoparticle-in-implant strategy capable of providing sustained and irradiation-enhanced local PFD delivery with tunable release characteristics. These findings support the potential of biodegradable, dose-controllable implant systems for long-term management of vocal fold fibrosis while reducing the need for repeated interventions.

Bacterial biofilms on indwelling medical devices is a major driver of healthcare-associated infection despite significant advances in antifouling surface engineering, suggesting that laboratory antibacterial performance does not fully capture the determinants of clinical colonization. Here we show that adherent immune cells constitute a previously underappreciated conditioning layer that promotes biofilm formation on otherwise antifouling biomaterials. Pre-exposure of clinically used substrates to macrophages, monocytes, neutrophils or human peripheral blood cells markedly increased Staphylococcus aureus and Escherichia coli adhesion and aggregation. These studies also reveal that immune cells promote biofilms even after cell death with cellular debris acting as a conditioning agent. We demonstrate that reactive-oxygen-species amplification by incorporating bismuth telluride into a silicone composite converts adherent immune cells from passive conditioning agents into active bactericidal effectors. We note that this antimicrobial composite confers durable antibacterial protection across early, delayed and late infection time points in a murine implantation model. Together, these findings introduce a class of immune-coupled antibacterial materials as an alternative to the current antifouling paradigm.

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.