Journal of Biomedical Materials Research Part A

Tissue engineered constructs are increasingly used for both modeling organs and disease in vitro as well as for therapeutic intervention. In addition to collagen, these constructs commonly include native extracellular matrix proteins (ECM), such as fibronectin and laminin. Given the critical role of inflammatory pathways in disease and in response to implanted materials, it is important to understand the role these proteins play in regulating the inflammatory environment. Fibronectin and laminin influence neutrophil function and endothelial activation in 2D, but their regulation of the inflammatory environment in 3D engineered constructs is not clear. For this study, we used an inflammation-on-a-chip device that includes a model blood vessel surrounded by a collagen I hydrogel with fibronectin and/or laminin. We investigated the additive effects of both proteins and a range of concentrations for each protein to determine concentration dependence. Both fibronectin and laminin have concertation dependent effects on neutrophils and the endothelium. High concentrations (50 {micro}g/mL) of fibronectin reduced neutrophil migration, while 20 {micro}g/mL laminin reduced neutrophil extravasation and migration, potentially due to lower ICAM-1 expression by the endothelium. Interestingly, 50 {micro}g/mL of laminin significantly disrupted endothelial vessel formation and reduced ICAM-1 and VE-cadherin expression, likely due to significant changes in the collagen architecture. The inclusion of fibronectin and laminin, even at physiological levels, results in significant effects on neutrophil behavior, endothelial vessel formation, and collagen architecture. These proteins impact the inflammatory environment and thus need to be considered when modeling diseases and designing therapeutics, especially when neutrophils or an endothelium are involved. Translational Impact StatementThis work uses an inflammation-on-a-chip device to study how fibronectin and laminin impact neutrophil behavior and vascular inflammation as these proteins are commonly used in engineered constructs. We found that fibronectin impairs neutrophil migration, while laminin decreases neutrophil extravasation and migration and at higher concentrations also prevents endothelial vessel formation. Therefore, researchers should be aware that these proteins will alter the inflammatory environment when including them in engineered constructs.

Biodegradable electrospun nanofiber (NF) scaffolds have emerged as promising materials for tissue engineering applications, including vascular grafts, because their mechanical properties and degradability can be tuned. However, their in vivo degradation behavior remains poorly understood. In this study, we characterized the in vivo degradation profiles of representative biodegradable NF materials widely used in small-caliber vascular graft research, namely polycaprolactone (PCL), poly(D,L-lactide) (PLA), polyglycolic acid (PGA), and a PCL/PLA blend, by monitoring molecular weight changes in subcutaneous and vascular environments. Electrospun NF sheets were implanted subcutaneously in mice, and tubular NF grafts were implanted into the abdominal aorta of rats. Samples were harvested for up to 48 weeks after implantation and analyzed primarily by size-exclusion chromatography (SEC) to assess time-dependent changes in molecular weight. Scanning electron microscopy (SEM) and solid-state 13C nuclear magnetic resonance (NMR) were additionally performed to evaluate ultrastructural and chemical changes associated with degradation. SEC analysis revealed distinct material-specific degradation patterns. PCL showed the slowest degradation and retained a relatively high weight-average molecular weight (Mw) in both environments. PLA exhibited marked environment dependence, with near-complete degradation in the subcutaneous environment by 48 weeks, whereas scaffold structure was maintained in the vascular environment. The PCL/PLA blend showed earlier reduction in the high-molecular-weight fraction than PCL, indicating faster scaffold breakdown. PGA degraded most rapidly and could not be evaluated beyond 2 weeks in the subcutaneous model or in the vascular model because of early graft rupture. SEM analysis further demonstrated that progressive loss of fibrous ultrastructure over time was a common feature across all materials. In addition, NF scaffolds became resistant to organic solvent after implantation in vivo, and solid-state 13C NMR analysis of the solvent-insoluble fractions detected polymer-derived signals together with additional signals consistent with biological constituents. These findings indicate that in vivo degradation of biodegradable NF scaffolds is material dependent, environment dependent, and more complex than simple hydrolytic chain cleavage alone. This study provides a quantitative framework for evaluating NF degradability and offers new insight into the design of biodegradable vascular grafts. HighlightsO_LISEC quantified long-term in vivo degradation of PCL, PLA, PGA, and PCL/PLA. C_LIO_LIDegradation was both material dependent and implantation environment dependent. C_LIO_LIIn vivo nanofiber degradation involved structural and chemical changes beyond hydrolysis. C_LI

PurposeFibrosis is the pathological remodeling of the extracellular matrix (ECM) that is largely orchestrated by activated fibroblasts. The mechanical properties of the ECM change drastically during fibrosis, and fibroblasts become increasingly activated by mechanical environments that mimic the properties of fibrotic tissues. While the effects of increased elastic modulus (stiffness) on fibroblast activation have been well-studied, the impact of changes in viscoelasticity are less clear. Here, we sought to determine how fibroblast activation is altered by changes in viscoelasticity in a three-dimensional, fibrillar microenvironment. MethodsWe employed 3D alginate collagen I hydrogels with independently tunable stiffness and stress relaxation rates. Dermal fibroblasts were encapsulated in hydrogels with four distinct mechanical profiles (soft: 3 kPa or stiff: 10 kPa, fast stress relaxing: {tau}1/2 {approx} 160 s or slow stress relaxing: {tau}1/2 {approx} 1600 s). We assessed fibroblast activation by changes in cell morphology, expression of key activation markers, and evidence of ECM remodeling. ResultsFibrillar alginate collagen networks enhanced fibroblast spreading, -smooth muscle actin stress fiber formation, and fibroblast activation protein- expression in matrices that were slow relaxing or stiff. The presence of the fibrillar network further enhanced fibroblast activation, independent of the changes driven by matrix viscoelasticity. ECM remodeling was also promoted by slow relaxing matrices, with increased fibronectin deposition and more remodeling of the local collagen fiber network. ConclusionsOur results demonstrate that fibroblast activation is highly responsive to matrix stress relaxation rate, and that models incorporating fibrillar, viscoelastic networks can provide new insights into the role of ECM mechanics driving fibroblast activation.

Hydrogels are widely used as three-dimensional cell culture systems to understand the impact of cellular mechanotransduction for tissue engineering applications. Photoinitiated thiol-ene click chemistry is a commonly utilized hydrogel crosslinking mechanism that provides spatial and temporal control over hydrogel network formation and resulting mesh size and compressive properties. Despite historically documented efficiency as step-growth reactions, these reactions do not always proceed as predicted. To understand the impact of cell confinement and microenvironmental mechanics on cellular function, thiol-ene network formation must be thoroughly characterized. To this end, the objective of this work was to investigate the crosslinking dynamics to determine hydrogel network formation as assessed via mesh size and mechanical properties using a pentenoate-functionalized hyaluronic acid thiol-ene reaction. Hydrogel parameters including polymer concentration and thiol:-ene crosslinker molar ratio were modulated (4, 6, or 8 polymer weight percent and 0.15:1, 0.5:1, or 1:1 molar ratio of thiol groups to reactive -ene groups) to tune network properties including shear storage modulus and relative mesh size. Molecular Dynamics (MD) simulations were used to simulate the thiol-ene crosslinking reaction and establish a method for predicting thiol-ene reaction efficiency. Lastly, the feasibility of this hydrogel system for in vitro modeling was confirmed via assessment of metabolic activity of encapsulated primary human meniscal cells.

Curcumin is a naturally occurring polyphenol that demonstrates considerable anti-cancer activity, however the aqueous insolubility, rapid metabolism and relatively low bioavailability are limiting to its clinical application. As such, a curcumin-magnesium (Cur-Mg) coordination complex was synthesized and subsequently encapsulated within DNA hydrogels (Cur-Mg-Hgel). The Cur-Mg complex was fully characterized using UV-Vis spectroscopy, FTIR and X-ray diffraction (XRD). UV-Vis, FTIR and XRD all support the formation of a coordination complex and suggest a decreased level of crystallinity compared to free curcumin. DNA hydrogels were formed and characterized using atomic force microscopy, rheology and swelling kinetic studies. In vitro cytotoxicity studies utilizing an MTT assay demonstrate dose dependent inhibition of HeLa cell proliferation and a slightly better retention of RPE-1 viability at low concentrations (suggesting some difference in sensitivity) though significant cell death is seen at higher concentrations and both cells. Intracellular production of ROS was measured using the DCFH-DA assay and is seen to increase when HeLa cells are treated with Cur-Mg-Hgel in comparison to un-treated controls. Annexin V/PI staining demonstrates primarily late or early apoptotic activity with minimal necrosis following treatment with Cur-Mg-Hgel. The evidence presented strongly supports the notion that Cur-Mg-Hgel is a ROS-modulating, pro-apoptotic Hydrogel suitable for cancer treatment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/724072v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@18727aeorg.highwire.dtl.DTLVardef@3e20adorg.highwire.dtl.DTLVardef@d3703eorg.highwire.dtl.DTLVardef@16e260e_HPS_FORMAT_FIGEXP M_FIG C_FIG

ObjectiveEvaluate the effects of bioabsorbable magnesium wires on dermal wound healing and tissue regeneration in a murine full-thickness wound model. Approach6 mm diameter stented dorsal skin wounds were created in C57BL/6J mice and treated with implanted WE43B magnesium alloy wires or PBS control. Wound healing was evaluated on days 7 and 28 by histology, immunohistochemistry, and micro-CT. Finite element analysis modeled mechanical strain distribution due to wire degradation during healing. ResultsAt day 7, magnesium wire-treated wounds showed 100% improved granulation tissue formation, reduced inflammation (37% fewer CD45+ leukocytes and 37% fewer F4/80+ macrophages), increased neovascularization (91% more CD31+ lumens), and 74% more nerve bundles. Improved wound closure (mean difference -1.48 mm) did not reach statistical significance (d = 1.06). By day 28, magnesium-treated wounds showed improved collagen organization and normalized epidermal thickness. The increase in dermal appendages (247%), and vascular density (41%) did not reach statistical significance. Micro-CT confirmed progressive wire degradation. Modeling revealed that degrading wires dynamically altered strain gradients in healing tissue, thereby modulating the spatial mechanical cues that govern fibroblast migration and extracellular matrix (ECM) remodeling. InnovationMagnesium is an essential trace element involved in cellular processes critical to wound repair, including angiogenesis, nerve growth, inflammation modulation, and ECM remodeling. Previous magnesium delivery systems incorporated polymers or other confounding materials that degrade rapidly. We directly applied bioabsorbable pure magnesium metal to provide sustained ion release and favorable mechanical properties to support regenerative healing. ConclusionBioabsorbable magnesium wires support regenerative wound healing by reducing inflammation, enhancing neovascularization, and promoting favorable ECM remodeling without adverse inflammatory reactions. These findings provide a strong rationale to harness magnesium metal use in wound healing applications.

BackgroundBone graft biomaterials play a critical role in bone regeneration by influencing osteoblast differentiation and mineralization. However, comparative data regarding the osteogenic potential of commonly used graft materials under standardized conditions remain limited. Method and materialIn this in vitro experimental study, osteoblast-like cells (MG-63) were cultured with four bone graft materials, including Bio-Oss, Cerasorb, Bio-Tiss Cerabone, and Pro Osteon. The relative mRNA expression of osteogenic markers (COL1 and OPN) was evaluated at 1, 7, 14, and 21 days using real-time PCR. Alkaline phosphatase (ALP) activity and mineralization capacity were also assessed using colorimetric assay and Alizarin Red staining. Data were analyzed using one-way ANOVA and Tukey post hoc test (P < 0.05). ResultsSignificant differences were observed among the tested materials across all evaluated parameters. Bio-Oss and Cerasorb demonstrated higher gene expression levels and ALP activity compared to Bio-Tiss Cerabone and Pro Osteon (P < 0.05). Mineralization analysis showed significantly greater calcium deposition in the Bio-Oss and Cerasorb groups, whereas Pro Osteon consistently exhibited the lowest osteogenic performance. ConclusionBone graft biomaterials significantly influence osteogenic activity in osteoblast-like cells. Bio-Oss and Cerasorb showed superior osteogenic potential, while Pro Osteon demonstrated weaker performance. These findings highlight the importance of material properties in optimizing bone regeneration.

We aimed to study the stability of a {beta}-SARS-CoV-2 virus-like particle (VLP) vaccine in a series of preliminary experiments using select stabilising excipients. {beta}-SARS-CoV-2 VLPs were produced and purified using established methodologies. The thermostability of VLPs was tested at 4{degrees}C and -30{degrees}C in the presence or absence of stabilizers polysorbate 80, sorbitol or L-histidine in the presence of a physiological NaCl concentration of 137mM. The integrity of VLPs was assessed using ELISA, Western immunoblot and dynamic light scatter (DLS). {beta}-SARS-CoV-2 VLPs were stable at 4{degrees}C for 14 days and the addition of stabilizing excipients improved stability compared to VLPs in PBS alone. Storage of VLPs at -80{degrees}C maintained particle integrity by DLS analysis for up to 2 years. Excipients helped to maintain the immunogenicity of the VLPs by ELISA and Western immunoblot and DLS analysis revealed that VLPs retained their particulate structure. ImportanceSARS-CoV-2 continues to circulate globally and cause significant illness. The problem of waning immunity to mRNA/LNPs has necessitated frequent boosters to keep pace of emerging variants. The development of alternative vaccines therefore remans a priority. Protein based vaccines, like VLPs, offer a safe alternative able to produce longer lasting immune responses. In this preliminary stability analysis, the {beta}-SARS-CoV-2 VLPs were found to be stable at 4{degrees}C and the addition of excipients improved VLP stability. Storage of VLPs at -30{degrees}C and -80{degrees}C also showed that the VLPs are stable for very long periods. Our findings will be of importance for the ongoing development of a SARS-CoV-2 VLP based vaccine.

Three-dimensional (3D) bioprinted liver scaffolds offer a promising platform for drug screening, disease modelling, and regenerative medicine, yet their broader adoption is limited by the absence of robust post-fabrication preservation strategies. This study aimed to evaluate the impact of -80{degrees}C (deep freezer) preservation and evaluate the structural integrity and hepatic functionality of GelMA-decellularized liver extra cellular matrix (dECM)-based 3D bioprinted liver scaffolds. Bioinks were formulated using synthesized GelMA and solubilized rat liver dECM, and 3D scaffolds were fabricated via extrusion bioprinting into rectilinear grid scaffolds. The 3D scaffold preservations was performed by immersion into two different medium (the culture DMEM media and the other FBS-DMSO cocktail) was evaluated using MTT viability assay, and albumin assay. Preserved 3D bioprinted scaffolds retained overall architecture and cell distribution in the FBS-DMSO cocktail demonstrated by the live dead assay. Together, the data demonstrate that -80{degrees}C storage can maintain the basic cell viability ([~]80%) and a substantial fraction of liver-specific functionality in 3D bioprinted scaffolds but also highlight sensitivity to preservation-induced injury. These findings underscore the need for further optimization of cryoprotectant formulations and freezing protocols tailored to 3D bioprinted liver scaffolds, and provide a foundational framework for developing ready-to-use, cryopreserved 3D liver models for translational applications.

Focal injuries to articular cartilage in load-bearing joints fail to heal and often progress to degeneration, underscoring the need for repair strategies that result in restored cartilage structure and function rather than fibrocartilage formation. Granular extracellular matrix (gECM) hydrogels, flowable grafts composed of densely-packed matrix particles, offer a promising approach but lack long-term functional validation in large-animal models. Here, we developed a flowable gECM hydrogel composed of decellularized cartilage microparticles incorporated within a thiol-functionalized hyaluronan matrix. Proteomic analysis confirmed enrichment of cartilage-specific gECM matrisome components. When implanted into critical-sized femoral condyle defects in a goat model and evaluated 12 months post-implantation, both gECM hydrogel and microdrilling (surgical controls) achieved >80% defect filling. However, in contrast to microdrilling, gECM repair tissue exhibited surface tribological (friction, adhesion) and compressive mechanical properties comparable to native cartilage, with a similar proteoglycan-to-collagen ratio, enrichment of type II collagen, minimal type I collagen (typical of a fibrous scar), improved quantitative MRI metrics, and evidence of lateral cartilage integration and subchondral bone remodeling. Together, these findings demonstrate that a flowable gECM hydrogel supports integrative, cartilage-like repair in a load-bearing joint, supporting advancement of this approach toward clinical translation. One Sentence SummaryA granular ECM hydrogel implanted in a goat condyle provided a robust repair, filling the defect tissue with integrated, hyaline-like cartilage at 12 months.

Intervertebral disc degeneration (IVDD) compromises disc structures and mechanics, yet systematic evaluations of the mechanical responses and their relationship to morphological changes in preclinical models remain limited. This systematic review and meta-analysis synthesized mechanical and morphological alterations following experimental disc injury in in vivo animal models. Searches of MEDLINE, EMBASE and Web of Science databases were conducted in accordance with PRISMA guidelines. Study quality and risk of bias were assessed using modified CAMARADES and SYRCLE tools. Twenty-eight studies were included. Pooled analyses showed significant reductions in stiffness, Youngs modulus, and disc height, and significant increases in range of motion and degeneration grade, indicating both mechanical and structural deterioration. Youngs modulus appeared to be the most sensitive marker of functional degeneration. By contrast, creep and other viscoelastic responses showed non-significant changes. High heterogeneity was evident across studies, reflecting variability in injury models, species, timepoints, and testing methods. Evidence of publication bias was detected in several domains, and moderate methodological quality was noted with overall insufficient blinding and lack of sample size calculations. In vivo animal models of IVDD demonstrate robust and consistent mechanical and morphological degeneration after injury. Youngs modulus is a sensitive mechanical indicator, supporting its use in future preclinical research. Standardization of outcome definitions, methodology, and reporting is essential to improve comparability and enhance translation of preclinical findings to clinical research.

Patellar osteochondral allograft (OCA) transplantation is widely used to treat large full-thickness cartilage defects, yet long-term failure and reoperation rates remain high. Although surface congruity and osseous integration are emphasized clinically, cartilage thickness and mechanical compatibility between donor and recipient are not considered. Our previous work suggests that cartilage thickness mismatch can amplify local deformation at the graft boundary, potentially compromising graft longevity. This study investigates how combined mismatches in cartilage thickness and mechanical properties influence the local strain environment at the patellar OCA interface. Simplified two-dimensional axisymmetric finite element models of patellar OCA repair were developed in ABAQUS. Donor-to-recipient cartilage thickness ratios ranging from 0.33 to 3.25 were evaluated together with donor-recipient Youngs modulus mismatches (2.5-7.0 MPa). Cartilage was modeled using homogeneous linear elastic and functionally graded material formulations to account for depth-dependent stiffness. A compressive pressure of 1.0 MPa was applied to represent patellofemoral joint loading, and peak compressive and shear strains were quantified at the graft boundary. Cartilage thickness mismatch produced localized high-strain regions (HSR) of compressive and shear strain at the donor-recipient interface that were absent in thickness-matched constructs. Strain amplification increased with both thickness and mechanical property mismatch. Compressive strain exhibited directional asymmetry, with donor-side-thicker configurations producing greater amplification than recipient-side-thicker configurations. Incorporating depth-dependent cartilage stiffness reduced peak strain magnitudes but did not eliminate mismatch-driven strain amplification. These findings demonstrate that cartilage thickness and mechanical disparity can create HSR at the patellar OCA graft boundary that may predispose grafts to impaired integration and long-term failure.

Critical-sized bone defects represent a challenge in bone tissue engineering, due to insufficient vascularization that results in implant failure. Scaffold pre-vascularization is a promising strategy to create a functional microvascular network that integrates with host vasculature. In this study, we present a hybrid 3D construct comprising a hyaluronic acid-based hydrogel and a 3D printed polycaprolactone/{beta}-tricalcium phosphate scaffold, to support vascular network formation and osteogenic differentiation. Peptide-functionalized (i.e. RGD, YIGSR, IKVAV, QK) hydrogels were obtained via thiol-ene chemistry, using two crosslinkers (PEG-diSH or MMP-diSH). Preliminary biological experiments assessed human mesenchymal stromal cells (hMSCs), endothelial cells (hUVECs), and their co-culture, on different gel formulations. All cell conditions displayed enhanced spreading and metabolic activity on gel formulations comprising RGD; thus these (i.e. RGD only and a combination of RGD/YIGSR) were selected for further studies. Cells were then mixed with the hydrogel precursor solutions, which were injected to embed the scaffolds and crosslinked using a UV lamp. After 7 days, tubule formation was observed only in co-culture conditions, highlighting the importance of cellular crosstalk for the formation of a vascular network. Significant differences were found across the tested formulations. In the RGD-PEG constructs, hUVECs formed tubule-like structures, surrounded by hMSCs, exhibiting pericyte-like behavior, supported by the upregulation of SMA gene. Conversely, in the RGD/YIGSR-MMP conditions, hMSCs were mostly located on the scaffold fibers, and showed the highest expression of early osteogenic markers (RUNX2 and ALP). Overall, we demonstrated that the hybrid system with tailored hydrogel chemistry can support simultaneous microvascular organization and osteogenic commitment, offering a promising platform for bone tissue engineering applications. However, further studies involving longer culture periods will aim at clarifying the complex interplay between material composition, cell crosstalk and spatial organization and their influence on the maturation and stability of the vascular network.

IntroductionCancer progression is driven not only by tumor cells but also by interactions between the extracellular matrix (ECM), stromal cells, and immune cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major drivers of ECM remodeling, assembling ECM with aberrant organization. Extra domain A fibronectin (EDA-FN), a cellular FN containing an extra type III domain, is upregulated in the TME. EDA-FN regulates cellular behavior and has been associated with poor patient prognosis. Macrophages are among the most abundant immune cells within the TME, where they contribute to TME remodeling and inflammation to promote cancer cell invasion and metastasis. However, how tumor-associated matrix-specific cues regulate macrophage behavior remains largely understudied. PurposeHere, we developed a fibroblast-derived matrix platform that captures the structural imprint of tumor-associated EDA-enriched matrices and investigated how matrix-specific cues regulate macrophage behavior in the absence of ongoing soluble factor cues. MethodHuman mammary fibroblasts (HMFs) preconditioned in incubated low-serum media (lNC, or control) and MDA-MB231 metastatic breast cancer cell-conditioned media (mTCM) were cultured on polyacrylamide gels of 2 kPa and 20 kPa, respectively, followed by decellularization. Matrix organization, including fiber alignment, width, and intrafibrillar spacing, was quantified from confocal images. Decellularized EDA-FN-enriched matrices were subsequently reseeded with macrophages to assess macrophage morphology, phenotype, and matrix interactions. ResultsThe combined effects of tumor-derived soluble factors and pathological stiffness induced a CAF-like phenotype in HMFs, accompanied by cytoskeletal reorganization and microarchitectural alterations of EDA-FN-enriched matrices. Tumor-associated matrices exhibited increased alignment, narrower fiber width, and enlarged intrafibrillar spacing compared to control matrices. These aberrant, tumor-associated matrix-derived features were associated with altered macrophage behavior, including heterogeneous morphology, enhanced localized EDA-FN matrix loss beneath the cell body, and a hybrid phenotype with a shift toward a CD206-dominant profile. ConclusionsThese findings demonstrate the feasibility of obtaining EDA-FN-enriched matrices to isolate matrix-specific cues for investigating macrophage-ECM interactions. Furthermore, this platform can be leveraged to identify matrix-targeting therapeutic approaches for modulating macrophage function within the TME.

Cultivated meat is an emerging biotechnology that aims to produce edible tissues in an ethical and sustainable manner. However, the recreation of skeletal muscle tissue that replicates the protein composition and sensory characteristics of traditional meat is a major challenge. Skeletal muscle tissue engineering requires non-animal-based scaffolds which are inexpensive and food-safe, while meeting specific mechanical requirements with respect to viscosity, stress-relaxation and stiffness. While many of these characteristics can be fulfilled by alginate-based biomaterials, a key limitation of alginate is its lack of intrinsic attachment sites for animal cells, preventing efficient adhesion, differentiation and tissue formation. Here, we established a screening platform to evaluate extracellular matrix (ECM)-mimicking peptides as functionalisations of alginate scaffolds in 2D. Our platform enables high-throughput assessment of cell/peptide interactions, serving as a predictive tool for 3D tissue constructs. Our screen identified two RGD-containing sequences (vitronectin- and fibronectin-mimicking peptides) as most effective in promoting attachment and myogenic fusion of bovine satellite cells. Notably, these peptides outperformed more complex mixtures containing up to seven different ECM-mimicking peptides. Our findings provide a streamlined approach for optimising biomaterial functionalisations for cultivated meat applications, and lay the groundwork for future advancements in scalable, sustainable skeletal muscle tissue engineering.

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

Inflammation is a fundamental immune response but, when dysregulated, contributes to the pathogenesis of numerous inflammatory disorders. Although there are several conventional anti-inflammatory drugs which are effective, their long term use is often associated with adverse side effects, which highlights the need for safer alternative therapeutic drugs. Probiotic derived membrane vesicles (MVs) have recently emerged as biologically active nanostructures capable of modulating host immune responses. In the present study, MVs isolated from Lactobacillus acidophilus MTCC 10307 were evaluated for their anti-inflammatory efficacy and safety profile using in vitro and in vivo models. In RAW 264.7 macrophages, L. acidophilus MVs significantly attenuated lipopolysaccharide induced expression of the pro-inflammatory mediators Il-1{beta}, Il-6, and iNOS, accompanied by reduced nitric oxide and reactive oxygen species production which was abolished in the proteinase K treated MVs. The protein levels of NF{kappa}B and IL1{beta} were also reduced in the treatment groups. Repeated dose oral toxicity studies revealed no adverse effects, as evidenced by body weight and histopathological evaluation of major organs. The anti-inflammatory properties of L. acidophilus MVs were further validated in an in vivo hind paw edema model, which shows inflammation resolution demonstrated by molecular and histological analysis. Proteomic analysis using LC-MS/MS identified the presence of surface-layer protein A (SlpA) which is a potential bioactive component which might contribute to the observed immunomodulatory effects. Collectively, these findings demonstrate that L. acidophilus MVs exert potent anti-inflammatory activity while maintaining an excellent safety profile using integrated in vitro and in vivo models.

Controlled delivery of growth factors and viable cells remains a significant challenge in bone tissue engineering. In this study, a 3D-printed hydrogel scaffold system was developed for the co-delivery of bone morphogenetic protein-9 (BMP-9) and preosteoblasts to enhance bone regeneration. The system consisted of a 3D-printed base scaffold containing BMP-9-coated calcium sulfate (CaS) microparticles and a photocurable hydrogel coating layer encapsulating viable cells. The scaffold design exploited electrostatic interactions between BMP-9 and gelatin matrices by incorporating gelatin type B in the base scaffold and gelatin type A in the coating layer. Differences in the isoelectric points of these gelatin types were utilized to regulate protein binding and release. Release studies demonstrated that CaS microparticles alone exhibited rapid burst release, with nearly 80% of BMP-9 released within 24 h. Encapsulation of BMP-9 coated CaS particles in the 3D-printed scaffolds reduced the release rate, while the addition of the coating layer significantly improved sustained release, limiting BMP-9 release to approximately 50-60% by day 5. Bioactivity studies showed enhanced cell attachment in BMP-9 containing scaffolds compared with controls. Live/Dead cytotoxicity assays demonstrated high cell viability (>80%) within the coating layer over the culture period, confirming that the encapsulation and photocuring processes did not adversely affect cell survival. Cell proliferation and differentiation were further evaluated using WST-1 and alkaline phosphatase assays. The results demonstrate that electrostatic interactions governed by gelatin type selection can regulate BMP-9 release while maintaining high cell viability, providing a promising platform for growth factors and cell delivery in bone tissue engineering.

Multidrug resistant (MDR) bacterial wound infections are an increasing clinical challenge and require alternatives to conventional antibiotics. Although antimicrobial proteins offer promise, their therapeutic use is limited by poor stability, proteolytic degradation, reduced activity under physiological conditions, and potential toxicity. This work reports pH-sensitive lipid nanocarriers composed of granulysin (GNLY) and oleic acid (OA) for antimicrobial delivery to infected tissues. At neutral pH, GNLY is retained within OA-based nanocarriers and protected from proteolytic degradation. At pH 5.0, such as in infected wounds, the carriers undergo structural reorganization and release GNLY, restoring antimicrobial activity. OAGNLY (32 {micro}g/mL) achieved >3-log reductions in Staphylococcus aureus and Escherichia coli within 1 hour, and up to 4-log reductions in Pseudomonas aeruginosa and Acinetobacter baumannii, at physiological salt concentrations where free GNLY was largely inactive. Minimum inhibitory concentrations were 16 {micro}g/mL for MRSA and 32 {micro}g/mL for colistin-resistant E. coli. Ultrastructural analysis using transmission electron microscopy revealed disruptions of bacterial membranes and intracellular structures following OAGNLY treatment. In a murine surgical wound infection model, topical application of OAGNLY for 4 hours reduced bacterial burden by >5 logs and significantly decreased inflammation, as confirmed by histological analysis. In parallel, OAGNLY demonstrated minimal cytotoxicity to mammalian cells at active concentrations. These findings identify OAGNLY nanocarriers as a promising platform for pH-responsive delivery of GNLY and highlight their potential application for treating MDR skin and soft tissue infections..

Injectable biomaterials with aligned microstructures play a critical role in tissue engineering and drug-delivery applications where control over the position and orientation of cells and nano/micron-scale architectures enhance intervention efficacy. Patients are often subject to MRI scans; for patient safety and treatment efficacy, we investigated the effects of MRI on a biomaterial treatment consisting of aligned magnetic microstructures being developed for guiding cell growth. Under MRI exposure, potential movement of aligned structures could be detrimental to nearby cells, and potential MRI-induced heating could adversely affect traumatized tissue. In this work, the alignment state and heat conduction of such a treatment were studied using a 9.4 T preclinical MRI. The treatment comprises short magnetic rod-shaped polycaprolactone fibers (rods) with embedded magnetic nanoparticles in a surrounding hydrogel (gelatin methacrylate), with rod alignment observed before and after a 45-minute MRI scan. No change in rod alignment state was observed, and no heat generation was measured. A theoretical framework was developed which supports the experimental observation that the biomaterial is stable under MRI. This work can be extended to other biomaterial systems with aligned architectures used in tissue engineering applications such as spinal cord, muscle and tendon.