Biomaterials Science

We report on the elaboration of a biocompatible therapeutic model system (TMS) capable of detecting the state of chronicity of a wound and responding to the latter by releasing the appropriate healing agent (antibiotic but not only). Different formulations of PVA-borate hydrogel systems containing methylene blue (MB) and levofloxacin (L) drug antibiotic were prepared. We were able to demonstrate sensitivity to microenvironmental factors such as pH, and high levels of ROS - a major biomarker of a chronic inflammation state. Furthermore, these reversible gels "open" and enact an ROS triggered drug delivery response. These results are promising in that they provide a potent wound dressing layer capable of sensing the chronic state of a wound and responding conditionally to the latter by delivering the appropriate amount of drug only when required. Furthermore, our system may be optimized for chronic wound dressing healing and promote better wound care by limiting unnecessary overexposure of the patient to the antibiotic. Our system provides a potent novel tool to the arsenal of existing strategies aiming at combatting bacterial resistance to antibiotics - a global societal challenge. Our results additionally confirmed good biocompatibility for skin tissue regeneration.

Nanoplastics (NPs) are increasingly recognized as pervasive environmental toxicants, however, their interactions with gut and renal barriers, and the resulting systemic consequences remain poorly understood. Here, we studied the uptake of 50 nm polystyrene (PS) nanoparticles using a multi-scale approach integrating zebrafish models, isolated perfused mouse kidneys, and in vitro assays to delineate uptake and barrier-dependent organ distribution. In zebrafish larvae, PS-NPs were efficiently absorbed via the intestinal tract, as visualized by confocal and label-free stimulated Raman scattering (SRS) microscopy, leading to gut microbiota dysbiosis and systemic inflammatory responses. Despite widespread systemic dissemination, renal accumulation was minimal under physiological conditions, whereas both zebrafish and isolated perfused mouse kidneys exhibited substantial PS-NPs retention only when the glomerular filtration barrier was disrupted. In vitro glomerular endothelial cells and podocytes readily internalized PS-NPs without altering key glomerular identity markers, highlighting their intrinsic uptake capacity that is normally restricted in vivo by barrier integrity. Our findings establish the glomerular filtration barrier as a crucial gatekeeper that prevents renal nanoplastic deposition. Furthermore, we revealed a microbiota-mediated axis that may prime the kidney for the environmentally induced stressing in long term.

Cultivated meat, a sustainable alternative to traditional livestock farming, has gained attention for its potential environmental and health benefits. However, concerns about microplastic contamination pose challenges, especially when sourcing cells from marine organisms prone to microplastic bioaccumulation. Additionally, the pervasive presence of microplastics in laboratory settings, ingredients, and during the production, increases the risk of unintentional contamination. This study focused on Atlantic mackerel (Scomber scombrus) skeletal muscle cell lines to examine the effects of microplastic exposure, represented by fluorescent polyethylene microspheres (10-45 {micro}m) on cell performance including cell proliferation, cell viability, gene expression, and differentiation processes critical for cultivated meat production. The results revealed significant impacts on cell attachment and proliferation at microplastic concentrations of 1 {micro}g/mL, 10 {micro}g/mL, and 50 {micro}g/mL. Notably, the 10 {micro}g/mL concentration exerted the most pronounced effects on cell viability during both attachment and proliferation phases. While the results indicated that both microplastic concentration and size influence cell viability, cell differentiation remained unaffected, and additional contributing factors require further investigation. These findings underscore the necessity of thoroughly exploring microplastic-cell interactions to ensure food safety and safeguard health within the burgeoning cultivated meat industry.

Extracellular vesicles (EVs) within the extracellular matrix (ECM) are often studied as passive elements whose diffusion and behaviour are subject to the composition and structure of the ECM. While EV diffusion and distribution in tissues are indeed governed by matrix interactions, accumulating evidence suggests that EVs contain much of the cellular machinery required for actively remodeling ECM. Using rheology and confocal reflectance microscopy, we investigate the gelation of collagen I hydrogels formed in the presence of EVs, and show that EVs can play an active role in ECM formation. EVs appear to nucleate new fibrils, recruiting collagen molecules from solution and accelerating their polymerization. Trypsinization of EVs shows that collagen-EV interactions are primarily mediated by surface proteins. The use of extruded plasma membrane vesicles shows that membrane composition determines final fibril length and matrix structure. EVs also become integrated into the fibril structures that they help form, reminiscent of matrix vesicles found in situ within tissues. This represents a plausible way by which EVs are deposited into the ECM, becoming signaling cues for resident cells. Our data show that EV-matrix interactions are dynamic and can contribute to the remodeling of tissue microenvironments. SignificanceExtracellular vesicles (EVs) are nanoscale membrane structures known for their role in facilitating cellto-cell trafficking of proteins, lipids, RNA, and other signaling molecules. In this report, we show that EVs derived from breast cancer cells are not merely passive messengers, but also direct active effectors of extracellular matrix (ECM) remodeling processes. Bulk rheology and confocal microscopy show that these EVs have the ability to nucleate new collagen fibrils and accelerate the formation of dense fibrillar collagenous networks. This has important implications in cancer pathology, where matrix density is often associated with worse disease outcomes, but could also potentially be exploitable in future tissue engineering applications.

Dysregulated macrophage function is implicated in a wide range of disorders. In vitro hydrogel culture systems are often used as matrices to model and explore the effect of various external stimuli on macrophage polarization and behaviour. Here, we show that 3D alginate hydrogels are not "macrophage inert" and instead help to direct the maturation of primary human macrophages towards specific phenotypes. We compared polarization of M1-like and M2-like cells activated on planar substrates or in 3D alginate hydrogels (with or without adhesion motifs (RGD)). We show that culture in 3D alginate systems selectively alters M2 polarisation following activation; cells show a 2.6-fold increase in CD86 expression compared to cells matured on planar controls, and increase IL1{beta} cytokine secretion even in response to an M2-like stimulus (LPS alone in the absence of IFN{gamma}). Our results suggest that alginate materials may intrinsically stimulate M2 macrophages to acquire a unique polarization state (resembling M2b), characterized by enhanced expression of CD86 and IL1{beta} secretion while retaining low IL12 and high IL10 secretion typical for M2 macrophages. This has important implications for researchers using alginate hydrogels to study macrophage behavior in culture and co-culture systems, as alginate itself may induce direct phenotypic changes independently or in conjunction with other stimuli.

Implantation of medical devices and biomaterials can help restore form and function of missing or damaged tissue. It is known that the immune system plays a critical role in both positive and negative outcomes of these implanted materials. The foreign body response is characterized by protein deposition and clotting followed by macrophage inflammation, frustrated phagocytosis, giant cell formation, and ultimately fibrosis. This can inhibit the function of implanted devices (e.g. Insulin pumps) as well as cosmesis (e.g. capsular contracture in breast implants) and persistent inflammation has been associated with more severe outcomes in some patients, including emergence of autoimmune-like pathologies. On the other hand, the immune system plays a constructive role in tissue remodeling and regeneration and is needed for the positive effects of some biomaterials, such as extracellular matrix-based scaffolds in muscle repair. Given these factors, we sought to understand potential variations in post-operative complications in individuals with primary and secondary immune disorders - both autoimmune and immunodeficiencies. This preliminary observational study using electronic health record mining showed increased complication odds for individuals with both autoimmune conditions and immunodeficiencies, with variations dependent upon the individuals sex and age as well as the type of material implanted. Future prospective studies could yield improved insight into both mechanisms of immune response to materials in humans and identify potential risk factors for individual patients undergoing plastics and reconstructive surgeries.

In vitro cell models have undergone a shift from 2D models on glass slides to 3D models that better reflect the native 3D microenvironment. 3D bioprinting promises to progress the field by allowing the high throughput production of reproducible cell-laden structures with high fidelity. As this technology is relatively new, the current stiffness range of printable matrices surrounding the cells that mimics the extracellular matrix environment remains limited. The work presented here aims to expand the range of stiffnesses by utilising a 4-armed polyethylene glycol with maleimide functionalised arms. The complementary crosslinkers comprised a matrix metalloprotease (MMP)-degradable peptide and a 4-armed thiolated polymer which were adjusted in ratio to tune the stiffness. The modularity of this system allows for a simple method of controlling stiffness and the addition of biological motifs. The application of this system in drop-on-demand printing is validated in this work using MCF-7 cells which were monitored for viability and proliferation. This study shows the potential of this system for the high-throughput investigation of the effects of stiffness and biological motif compositions in relation to cell behaviours.

Extracellular matrix (ECM) stiffness is fundamental in cell division, movement and differentiation. The stiffness that cells sense is determined not only by the elastic modulus of the ECM material, but also by ECM geometry and cell density. We hypothesised that these factors would influence cell-traction-induced matrix deformations and cellular differentiation in bone marrow stromal cells (BMSCs). To achieve this, we cultivated BMSCs on polyacrylamide hydrogels that varied in elastic modulus and geometry and measured cell spreading, cell-imparted matrix-deformations and differentiation. At low cell density BMSCs spread to a greater extent on stiff compared to soft hydrogels, or on thin compared to thick hydrogels. Cell-imparted matrix deformations were greater on soft compared to stiff hydrogels or thick compared to thin hydrogels. There were no significant differences in osteogenic differentiation relative to hydrogel elastic modulus and thickness. However, increased cell density and/or prolonged culture significantly reduced matrix deformations on soft hydrogels to levels similar to those on stiff substrates. This suggests that at high cell densities cell traction-induced matrix displacements are reduced by both neighbouring cells and the constraint imposed by an underlying stiff support. This may explain observations of the lack of difference in osteogenic differentiation as a function of stiffness.

Self-assembling peptides are promising candidates as scaffolds for 3D cell cultures. These hydrogels offer favourable biocompatibility, nanofibrillar structures that mimic native tissues, and the convenient integration of bioactive peptide sequences, such as arginine-glycine-aspartic acid (RGD), which can enable the development of therapeutically valuable cell types. In the treatment of osteoarthritis (OA) attempts have been made to combine hydrogel scaffolds with mesenchymal stem cells (MSCs) to harness their regenerative potential. This involves the deposition of extracellular matrix (ECM) components like collagen and proteoglycans. Here, we employ the hydrogel-forming peptide Fmoc-diphenylalanine (Fmoc-FF) and incorporate stoichiometric amounts of Fmoc-RGD. We investigate the impact of RGD on nanofibrillar morphologies, hydrogel stability, MSC viability, and the deposition of collagen, proteoglycans, and glycosaminoglycans. Elevating RGD content enhances cell viability and collagen deposition. However, at higher RGD concentrations, the stability of the hydrogels is compromised. To characterise collagen deposition, we introduce a non-destructive and label-free method using a plasmon-enhanced colorimetric histology technique. This innovation provides a practical means to image collagen without resorting to intricate and destructive sample processing and complex immunohistological staining procedures. This simple approach holds broad potential for routine and label-free quantification of collagen-rich biomaterials, promising widespread applications across various research and clinical settings.

BackgroundSepsis-induced mortality is frequently driven by the systemic dissemination of pore-forming toxins (PFTs), such as Staphylococcus aureus alpha-hemolysin. Biomimetic "nanosponges" which are nanoparticles coated in red blood cell (RBC) membranes have emerged as a promising detoxification strategy. However, current methods rely largely on empirical iteration, often failing to optimize the competitive binding kinetics required to outcompete native RBCs in a high-flow hemodynamic environment. MethodsWe developed a deterministic ordinary differential equation (ODE) kinetic model based on the law of mass action to simulate the competitive inhibition of alpha-toxin by decoy nanoparticles. Unlike prior geometric models, this study explicitly tracked molar receptor concentrations to enforce saturation kinetics and mass conservation. We performed a multi-parametric sweep of nanoparticle radius (r_{NP}: 50-200 nm) and receptor surface density (d_{rec: 200-10,000 sites {micro}m{square}2) to identify the design window that maximizes toxin sequestration efficiency within a clinically relevant timeframe (60 minutes). ResultsBaseline simulations established a native RBC receptor concentration of 3.34 x 10^{-7} M. The optimization landscape revealed a non-linear dependence on receptor density rather than particle size. The optimal design window was identified at a receptor density of >8,000 sites {micro}m{square}2 on an 80 nm vector, achieving a theoretical toxin neutralization efficiency of 91.79%. Notably, complete (100%) neutralization was not observed even under optimized conditions, suggesting a theoretical upper bound imposed by physiological competition. In contrast, standard biomimetic formulations (low-density, 100 nm) achieved suboptimal capture, failing to prevent significant toxin-RBC interaction. ConclusionWe demonstrate that "decoy" efficacy is governed primarily by receptor surface density rather than geometric surface area. Our model suggests that current manufacturing protocols, which prioritize particle stability over receptor enrichment, may be kinetically insufficient for human application. These findings provide a rational design framework for next-generation nanotoxoid therapeutics.

BackgroundProtein fibrillation represents a critical challenge in therapeutic insulin delivery, yet the structural determinants and immunological consequences of insulin-derived fibrils (IDFs) formed in the presence of phenolic preservatives remain poorly characterized. ObjectiveThis study investigated the structural characteristics of IDFs formed with (IDF (+)) and without (IDF (-)) phenolic preservatives and elucidated their differential immunomodulatory mechanisms in bone marrow-derived macrophages (BMDMs). MethodsIDF structural properties were characterized using Thioflavin T fluorescence and nanoparticle tracking analysis (Spectradyne nCS1). BMDMs were treated with serial dilutions of IDF (+), IDF (-), or m-cresol. Cytotoxicity, reactive oxygen species (ROS) production, MIP-1 levels, and expression of signaling pathways were quantified. ResultsStructural analysis revealed similar aggregation states between IDF (+) and IDF (-). However, IDF (+) induced greater cytotoxicity and ROS production than IDF (-), which produced minimal ROS. Both fibrils increased MIP-1 chemokine levels. Additionally, IDF (-) upregulated NRF2 whereas m-cresol downregulated STAT6 compared to control. Together, these results support the existence of distinct mechanisms of macrophage activation and suggest that protein aggregates can directly induce macrophage responses independent of ROS production. ConclusionsInsulin fibrils activate macrophage inflammatory pathways through ROS-independent mechanisms. Phenolic preservatives enhance fibril cytotoxicity and likely ROS production while differentially modulating inflammatory signaling. These findings suggest that strategies to remove or reduce the effects of IDFs in insulin infusion therapy may increase longevity and biocompatibility of these devices.

We have developed hydrogel systems with tunable porosity through dialysis casting by varying alginate and poloxamer compositions from 0% to 10%. These gels feature diverse porosity topologies, yielding loss factors between 16% and 29% in the 50 Hz to 300 Hz frequency range. The dynamic modulus shows a remarkable increase of over an order of magnitude, reaching approximately 3 MPa compared to the static modulus. Vibration transmissibility tests and dynamic mechanical analysis reveal that the poroelastic and pneumatic-like effects from the tunable porous structures contribute significantly to this damping effect. Furthermore, these hydrogels are biosourced and biodegradable, providing a sustainable alternative to conventional fossil-based damping materials.

APOSECTM, a complex mixture of secreted proteins, lipids, and extracellular vesicles from stressed peripheral blood monocytes, is currently in clinical trials for the treatment of chronic, poorly healing wounds. When applied to open wounds, 1 mL reconstituted APOSECTM lyophilisate is syringe-mixed with 3 g sterile hydrogel prior to administration. This study investigates the pharmaceutical performance of this novel administration system. A gel formulation (APOgel) was developed for terminal sterilisation in pre-filled syringes with post-sterilisation viscosity ([~]325-350{square}Pa*s at 1{square}s-1) comparable to a commercial benchmark gel. Syringe mixing of APOgel with a liquid APOSECTM surrogate (3:1) reduced viscosity by [~]67% but was highly reproducible across different operators (CV < 6%). Administration of three sequential dose units of the mixture from the syringe revealed an [~]20% higher content of active ingredients in the first and final dispensed compared to the middle unit, indicating non-uniform mixing in the closed syringe system. In vitro release studies over 72{square}h showed a 32% and 48% higher release of a small molecule marker and total proteins from the sterile APOgel compared to the benchmark gel as well as more pronounced gel swelling. However, efficacy studies in a murine wound healing model showed no significant difference between APOgel and the benchmark. These findings indicate that terminal sterilisation of gels for topical applications may provide benefits for more rapid release of active agents but syringe mixing of gels and a liquid requires optimisation to ensure uniform drug distribution. HighlightsO_LIAn autoclavable hydrogel for APOSECTM delivery was developed C_LIO_LIA novel syringe-mixing system for combining a gel with a liquid with subsequent dispensing of different volume units showed non-homogenous active ingredient distribution C_LIO_LIFinal optimised APOSECTM-APOgel formulation maintains functional wound-healing efficacy C_LI

BackgroundAbnormal scarring and fibrotic skin disorders arise from dysregulated wound healing processes. While Aloe vera is widely recognized for its therapeutic properties, the potential of its extracellular vesicles (Av-EVs) remains underexplored. ObjectiveThis study aimed to isolate and characterize Av-EVs and evaluate their antioxidant, anti-inflammatory, and antifibrotic properties in vitro, focusing on the impact of extraction method and plant maturity. MethodsAv-EVs were isolated from mature and young Aloe vera leaves using manual (NB) or blender-based (B) homogenization. Vesicles were characterized by nanoparticle tracking analysis, transmission electron microscopy, and protein quantification. Antioxidant and cytotoxicity assays (DPPH, alamarBlue) were followed by functional anti-inflammatory and antifibrotic analyses respectively in LPS-stimulated THP-1 macrophages and TGF-{beta}1/Vitamin C-activated human dermal fibroblasts (RT-qPCR, immunofluorescence, proteomics). ResultsNB-derived EVs from mature leaves exhibited the most potent activity across all assays, showing superior antioxidant capacity, greater suppression of pro-inflammatory cytokines, enhanced M2 macrophage polarization, and significant downregulation of COL1A1 and -SMA. In contrast, B-derived and young leaf-derived EVs showed reduced bioactivity, with young EVs failing to inhibit fibrotic markers. ConclusionManually extracted Av-EVs from mature leaves demonstrate superior multifunctional bioactivity, highlighting their potential as plant-derived nanotherapeutics for fibrotic scar modulation.

We assessed cell mechanical properties in both 2D and 3D environments employing compliant type I collagen matrices. Firstly, collagen gels of varying stiffness were prepared using a photocrosslinker to increase gel stiffness. Using methacrylic anhydride and UV light, a 10-fold increase in apparent Youngs modulus with respect to the soft collagen gel was achieved (0.2 kPa to 2 kPa). In addition, cells were plated onto the different collagen gels and hard Petri dishes (as a super stiff substrate) and their mechanical properties were evaluated. An increase in apparent Youngs modulus was observed in Dupuytren fibroblasts behavior when increasing substrate stiffness, supporting its myofibroblast phenotype (3.8 kPa to 5.2 kPa from soft collagen gels to hard Petri dishes). Secondly, gels mechanics, in which fibroblasts were embedded, were evaluated over time to assess cells contraction properties. Gels apparent Youngs modulus increased over time regardless of fibroblasts type and cells presented dendritic protrusions. Rheological properties of both cells and gels were extracted using AFM sweep frequency scheme and power law structural damping model for data analysis. As a summary, we have found that fibroblasts contractile properties, related to myofibroblast differentiation and development are highly influenced on the mechanical properties of the surrounding environment, being stiffer environments those that favor the increase in fibroblast mechanical tension.

Injectable hydrogels with shear-thinning and self-healing properties are critical for biomedical applications including 3D bioprinting and regenerative medicine. While granular hydrogels inherently exhibit these properties, they often lack post-injection stability. Here, we developed an electrostatically stabilized granular hydrogel system composed of norbornene-modified hyaluronic acid (NorHA) microgels and cationic gelatin ((+) Gel). NorHA microgels (9.91 {+/-} 4.85 m diameter) were synthesized via batch emulsification, while (+) Gel was prepared by modifying gelatin with ethylene diamine to increase zeta potential from 2.08 {+/-} 0.97 mV to 13.76 {+/-} 1.11 mV. The negatively charged NorHA microgels formed stable materials when combined with (+) Gel through electrostatic interactions, confirmed by gel inversion tests and salt sensitivity studies. Rheological characterization revealed that (+) Gel addition produced poroelastic behavior and strain-stiffening properties, with storage modulus and yield onset increasing under compression. Large amplitude oscillatory shear analysis showed strain-stiffening behavior (e3 > 0) that enhanced with both (+) Gel concentration and compression. Confocal microscopy demonstrated tunable porosity through gelatin fraction control, with (+) Gel forming aggregate-like clusters. Extrusion testing showed formulations required low injection pressures (0.47-0.91 kPa) comparable to PBS and significantly lower than Pluronic, while forming robust filaments up to 23 mm in length. The materials exhibited rapid self-healing behavior and maintained structural integrity post-extrusion. This electrostatically stabilized granular hydrogel system offers a promising platform for injectable biomaterials that combine ease of delivery with post-injection stability for wound healing and 3D bioprinting applications.

Inflammation serves as a critical defense mechanism against pathogens and tissue damage but can lead to chronic diseases, such as cardiovascular disease and diabetes, when dysregulated. Macrophages play a pivotal role in orchestrating inflammatory responses, transitioning from pro-inflammatory M1 to anti-inflammatory M2 phenotypes to resolve inflammation and promote tissue repair. Current approaches to modulate macrophage phenotype predominantly rely on biochemical cues, which may induce systemic side effects. Given the mechanosensitivity of macrophages, this study investigates biophysical cues, specifically substrate curvature, as a localized strategy to regulate macrophage phenotype and minimize systemic repercussions. We hypothesized that substrate curvature influences macrophage immunophenotype by modulating F-actin polymerization. To test this hypothesis, we fabricated spherical microgels with tunable curvatures and characterized their biophysical properties. Our findings indicate that macrophages adhere to microgel surfaces irrespective of curvature, but the curvature significantly alters F-actin dynamics. Furthermore, manipulating cytoskeletal dynamics via selective actin inhibition partially reversed curvature-induced changes in macrophage phenotype. These results underscore the pivotal role of substrate curvature in modulating macrophage behavior and immunophenotype. Overall, our study demonstrates that substrate curvature significantly influences macrophage cytoskeletal dynamics and resulting immunophenotype. This simple approach can be utilized as a localized immunomodulatory treatment for inflammatory diseases.

Rheumatoid arthritis is an autoimmune disease that affects about 250,000 Colombians, 82% of whom are women. Current treatments include disease-modifying antirheumatic drugs (DMARDs), such as methotrexate (MTX), analgesics and physiotherapy. The most common DMARD is MTX, which binds to plasma proteins with low efficiency (50%) and has a half-life of 6 hours. Due to its limitations when administered orally, nanoparticles (NPs) have been proposed to overcome these limitations. NPs support the release of therapeutic molecules, minimizing side effects and increasing the bioavailability of the drug in a controlled administration. NPs synthesized from biodegradable polymers, such as polyglycolic lactic acid (PLGA), are convenient for drug delivery due to their high biocompatibility and ability to bind DMARDs such as MTX. PLGA NPs loaded with MTX (MTX-PLGA-NPs) have reduced the presence of proinflammatory factors such as IL-10 and TGF-{beta}, suggesting their potential as anti-inflammatory therapies for arthritis. Therefore, this study aims to develop MTX-PLGA-NPs in bioactive and biocompatible hyaluronic acid-eucalyptus (GelHA-E) hydrogels to preserve their stability and delay their degradation. PLGA-NPs were synthesized with an average hydrodynamic diameter of 200 nm, the 1237 cm-1 band found in FITR indicated the successful covalent conjugation with MTX; the mass loss of only 1% in GelHA-E indicated the thermogravimetric stability of the biomaterial and the low hemolytic and platelet aggregation percentage confirmed the biocompatibility of the biomaterial as a potential localized, anti-inflammatory, and injectable treatment for rheumatoid arthritis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive fibrosis and tissue stiffness, in which monocytes and macrophages are increasingly recognized as key contributors to pro-fibrotic myofibroblast formation and activation, although the underlying mechanisms remain incompletely understood. Here, we used a three-dimensional (3D) skin model to study how CD14+ monocytes, M1 and M2-like macrophages induce (myo)fibroblasts activation/contraction in collagen type I hydrogels. We identified that co-culture of fibroblasts with monocytes displayed strong spontaneous hydrogel contraction, coupled with an upregulation of myofibroblasts activation-associated markers, such as -SMA and fibroblast activation protein. Using transcription-factor reporter constructs and small molecules inhibitors, we demonstrated that monocyte-fibroblast communication was mediated by JAK/STAT3 and TGF-{beta}/Smad2/3 signaling pathways. Flow cytometry analyses revealed that monocytes, after interacting with fibroblasts, differentiated into a mixed M1/M2 polarization phenotype, characterized by CD163, CD206, CD86, and HLA-DR expression. Both M1 and M2-like macrophages promoted significant myofibroblast contraction, which could be mimicked by supernatant transfer. TGF-{beta} neutralization but not IL-6 blocking abolished this effect. This study demonstrates that monocytes/macrophages can strongly induce (myo)fibroblasts activation/contraction. Together, our work contributes to elucidating pathways and mechanisms associated with skin fibrosis in SSc and paves the way for developing new platforms for targeted therapy testing.

BackgroundHydrogel-based 3D culture systems are emerging as a valuable tool for preclinical screening of cell-based immunotherapies against solid and hematological malignancies, such as chimeric antigen receptor T (CAR-T) cells. Hydrogels can influence T cell function in a non-desired manner due to their mechanical properties and chemical composition, potentially skewing results in preclinical testing of novel immunotherapeutic compounds. MethodsIn this study, we assess CD4+ T and CAR-T cell activation and proliferation in chemically-undefined matrices (Matrigel and basement membrane extract, BME) and compare them to a synthetic nanofibrillar cellulose (NFC) hydrogel. ResultsRheometric analyses show that NFC is more rigid than Matrigel and BME. Murine CD4+ T cells acquire a regulatory T cell (Treg) phenotype in Matrigel and BME, while this is not observed in NFC. Proliferation and activation of human T cells are higher in NFC than in Matrigel or BME. Similarly, we show that CAR-T cell activation and proliferation is significantly impaired in Matrigel and BME, in contrast to NFC. ConclusionsOur findings highlight the impact of hydrogel choice on (CAR-)T cell behavior, with direct implications for preclinical immunotherapy testing. In contrast to Matrigel and BME, NFC offers a chemically-defined 3D environment where T cell function is preserved. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSIn 3D (preclinical) tumor-killing assays for evaluating engineered T cell cytotoxicity, the surrounding matrix can influence immune cell phenotype and function, potentially skewing T cell activity. Basement membrane hydrogels such as Matrigel and basement membrane extract (BME), widely used as scaffolds for 3D culture, are inherently heterogeneous and contain extracellular matrix components that can influence lymphocyte function. What this study addsHere, we show that (CAR-)T cell function is significantly reduced in Matrigel and BME as compared to standard (2D) culture conditions. In contrast, (CAR-)T cell activity is preserved in synthetic nanofibrillar cellulose (NFC) gels. Importantly, murine T cells spontaneously acquire a Treg phenotype in Matrigel and BME. T cell proliferation and cytokine secretion are >10-fold lower in Matrigel than in NFC. Similarly, CAR-T cell survival and expansion are 10-fold higher in NFC than in Matrigel or BME. How this study might affect research, practice or policyWe report that the intrinsic cytotoxic and proliferative potential of (CAR-)T cells can be underestimated when performing assays in 3D cultures based on Matrigel or BME. As an alternative, we suggest the use of chemically defined synthetic gels, and we show that nanofibrillar cellulose hydrogels are suitable 3D matrices for preserving T cell phenotype and activation.