Journal of Biomedical Materials Research Part A

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.

Treating extensive full-thickness burn wounds remains difficult in clinical practice because available donor skin is often limited, the risk of infection is high, and many standard dressings do not perform well when defects are large or structurally complex. These limitations have shifted attention to decellularized extracellular matrix (dECM) scaffolds, which can provide physical coverage while preserving biochemical cues that may support tissue repair. Based on this rationale, we designed a decellularization method that improves reagent penetration to produce a full-thickness porcine decellularized small intestine (dSI) scaffold for use in burn wound coverage. The protocol removed most cellular material while leaving low levels of detergent residue, and it maintained the native three-layer structure of the intestinal wall. Most key ECM components, such as collagen and glycosaminoglycans, were also retained. In this study, the dSI showed several properties relevant to burn care, capacity to absorb large amounts of fluid, water vapor transmission rates similar to those reported for skin, and resisted microbial penetration in vitro. From a mechanical standpoint, the scaffold retained anisotropic behaviour and remained stable under cyclic loading. This pattern indicates that it could withstand repeated deformation instead of acting like a fragile membrane. Degradation tests under enzymatic and oxidative conditions indicate that the material breaks down in a controlled way over a period that appears consistent with typical wound-healing timelines. In vitro assays indicated that the scaffold was cytocompatible, as human dermal fibroblasts and keratinocytes both attached to its surface and continued to proliferate. Cell responses differed depending on surface orientation, suggesting that preserved intestinal layers may shape cell behaviour in ways that are often missing in thinner or more uniform matrices. Overall, full-thickness dSI appears to act as a biologically active scaffold and shows mechanical properties that exceed those of many currently used burn dressings.

Cardiac fibrosis is a pathological process in which the myocardium stiffens due to the overproduction of extracellular matrix (ECM) proteins. Cardiac fibroblasts activate to myofibroblasts in response to the inflammatory cytokine transforming growth factor beta1 (TGF-{beta}1) to promote fibrotic scarring. Biological sex also influences cardiac fibrosis progression and patient outcomes, where males exhibit increased fibrotic scarring after acute inflammation relative to females. At the cellular level, sex differences in TGF-{beta}1-mediated cardiac myofibroblast activation processes have not been clearly defined. We hypothesized that TGF-{beta}1 would cause sex-specific cardiac myofibroblast activation levels and alter the secretion of bioactive molecules to modulate sex differences in cardiac fibrosis. Primary left ventricle cardiac fibroblasts were isolated from male and female C57BL/6J mice and cultured on hydrogel biomaterials mimicking native myocardial ECM stiffness and treated with TGF-{beta}1 and/or the TGF-{beta}1 receptor inhibitor SD208. Male myofibroblasts exhibited increased -SMA stress fiber formation, increased SMAD2/3 localization, and greater resistance to SD208 inhibition compared to female myofibroblasts on hydrogels at various time points tested. Sex differences in relative secreted cytokine abundance were also determined, with male CFs secreting increased vascular endothelial growth factor (VEGF) and female CFs producing increased periostin and fibroblast growth factor 21 in response to TGF-{beta}1. Our findings establish that TGF-{beta}1 mediates sex differences in cardiac myofibroblast activation on hydrogels and secreted factors that may modulate the myocardial microenvironment. Our work underscores the importance of using hydrogels as cell culture platforms to recapitulate sex-specific cardiac fibrosis phenotypes as a steppingstone towards identifying sex-dependent therapeutic interventions for cardiac fibrosis.

Alginate hydrogels are widely used for biocompatible encapsulation due to their low cost, mild gelation conditions, and scalability; however, their limited mechanical strength and poor chemical stability under physiological conditions restrict their utility for oral delivery applications. In particular, the development of robust alginate formulations capable of surviving gastrointestinal salt and pH exposures is critical for advancing encapsulated microbial therapeutics for chronic kidney disease (CKD). In this study, we investigated the incorporation of ferric iron into calcium alginate networks as a strategy to enhance gel stability while maintaining biocompatibility. Using a three-ion competition approach, we achieved controlled introduction of ferric ions into calcium alginate gels without significantly altering bulk mechanical properties relative to standard calcium alginate. Although the initial ferric-containing gels displayed comparable modulus and structure, post-treatment with chitosan under mildly acidic conditions produced a dramatic increase in gel stability in physiological salt concentrations across both acidic and neutral pH environments. Ferric-containing gels formed at pH 4.6 absorbed negligible chitosan, in contrast to iron-free alginate gels, which incorporated substantial chitosan under identical conditions. These results support the formation of a thin, dense interfacial complex between chitosan, ferric ions, and alginate at the gel surface, which reinforces the matrix and inhibits dissolution. The resulting hybrid ferric-calcium alginate formulation enabled the production of sub-millimeter beads capable of encapsulating live Thauera aminoaromatica while preserving anaerobic p-cresol degradation activity at 37 {degrees}C using nitrate as an electron acceptor. Collectively, these findings establish ferric-modified alginate hydrogels as a promising, scalable platform for stable oral delivery of encapsulated microbial therapeutics.

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.

Articular cartilage repair is limited by the poor regenerative capacity of chondrocytes and their rapid dedifferentiation during in vitro expansion. This study investigated whether a decellularized and lyophilized cell-secreted matrix (CSM) could function as a bioactive material to regulate cell behavior, promote chondrogenic differentiation, and attenuate or reverse chondrocyte dedifferentiation without exogenous growth factor supplementation. CSM was generated from rabbit auricular perichondrial cells, decellularized, lyophilized, and characterized by histology, biochemical assays, and proteomic analysis. The resulting matrix was enriched in structurally and functionally relevant extracellular matrix proteins, including collagens, fibronectin, fibrillin, proteoglycans, and matricellular regulators, with minimal intracellular contamination and good batch-to-batch reproducibility. Functionally, CSM supported robust adhesion and proliferation of allogeneic and xenogeneic cells. Human articular chondrocytes cultured on CSM exhibited enhanced proliferation, sustained expression of cartilage-specific markers, and preserved type II collagen production over serial passages compared with standard plastic culture. CSM also promoted chondrogenic differentiation of human progenitor cells and partially reversed established chondrocyte dedifferentiation, as evidenced by increased expression of COL2A1, ACAN, SOX9, and COMP, with reduced COL1 expression and no induction of hypertrophic markers. These findings demonstrate that lyophilized CSM is a stable, off-the-shelf biomaterial capable of directing chondrocyte fate through intrinsic matrix-derived cues, highlighting its potential for cartilage tissue engineering and cell manufacturing applications.

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.

Tissue engineering scaffolds such as collagen-based biomaterials have long been used to mimic native extracellular matrix in a wide range of regenerative applications. Their high porosity, tunable degradation and mechanics, and cell adhesion sites provide a structure upon which cells can grow and differentiate, while they also have the potential to act as carriers for loading and release of biomolecules to aid in healing. Here we describe the inclusion of a second lyophilization step in the fabrication process to enable improved loading efficiency of bone morphogenic protein 2 as well as increased ease of end-user handling. We report mineralized collagen scaffolds demonstrate maintained microarchitecture and mechanical properties post-relyophilization with reduced variability in biomolecule loading. Relyophilization allows consistent loading and release profiles and suggests the potential to improve the translational potential of collagen scaffold biomaterials for regenerative medicine applications.

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.

The present study evaluated the immunogenicity and protective efficacy of the chitosan (CS)-modified poly-lactide-co-glycolic acid (PLGA) nanoparticles (NPs) delivering Brucella abortus L7/L12 DNA in a mouse model. The NPs were prepared by solvent displacement method and characterized for size, charge, morphology, cellular uptake and cytotoxicity. The cationic CS-PLGA NPs were spherical with a mean size of [~]165 nm with a positive zeta potential (+20 mV). DNA loading efficiency of 1.2% and DNA adsorption shifted zeta potential to -45 mV. In vitro studies in RAW 264.7 cell line demonstrated efficient uptake of the DNA loaded cationic NPs and expression of L7/L12 protein. Intramuscular immunization of the L7/L12 DNA vaccine loaded CS-PLGA NPs elicited both humoral and cell-mediated immunity with upregulation of Th1 and Th2 cytokines along with induction of IgG antibodies in mice. IFN-{gamma}, IL-2, and IL-4 levels were significantly (P < 0.001) higher than control group. The protective efficacy of the DNA loaded NPs against virulent B. abortus 544 infection (105 CFU) was significantly higher than that of the naked DNA (P<0.001). These findings suggest that the CS-PLGA NPs were efficiently delivered L7/L12 DNA and exhibited adjuvant potential, conferring protection against experimental murine brucellosis.

Stretch is an important biomechanical stimulus facilitating tissue development in the respiratory system by programming the epithelium, endothelium, and extracellular matrix (ECM). Lung tissue undergoes stretch induced lung differentiation under normal prenatal and postnatal development. Furthermore, supraphysiological and aberrant stretch responses are known mechanisms of acute lung injury and ECM disruption. Current in vitro human tissue cyclic mechanical stretch (CMS) models suffer from significant, well-recognized disadvantages and are poorly validated in vivo for longer-term study. In vitro precision-cut lung slice (PCLS) models are commonly used to study the complex structural arrangement and cellular interactions of human tissue, as well as various lung diseases, including BPD.3 PCLS maintain lung tissue architecture and the variety of cell types present in the lung, allowing for a more realistic imitation of the lung microenvironment.3 Existing agarose-inflated PCLS models are hindered by retention of agarose media in the tissue, affecting material properties and complicating stretch studies. Our novel PCLS approach utilizes several technical innovations including a removable hydrogel for inflation and uses supportive poly(ethylene glycol) (PEG) hydrogels enable improved viability and phenotype retention during cyclic mechanical stretch (CMS). This platform will induce PCLS CMS for biochemical assays (e.g. transcriptomics, proteomics) after exposure.

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.

ObjectivesThis study examined methylglyoxal (MGO) as a collagen crosslinker to reinforce demineralized dentin, enhance its enzymatic resistance, and improve long-term durability of the resin-dentin bond. MethodsDemineralized dentinal collagen films were treated with 0.5, 1, or 3 M MGO and subsequently exposed to collagenase to assess their resistance to enzymatic degradation. MGO-induced crosslink formation was monitored using Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) spectroscopy by tracking the carbohydrate-associated band at 1180 cm-{superscript 1}. The apparent elastic modulus of the treated specimens was measured using a three-point bending test. For bond strength evaluation, resin-dentin beams were prepared and tested using microtensile bond strength (TBS) to assess the influence of MGO pretreatment on interfacial adhesion. ResultsThe ATR-FTIR spectra demonstrated increased intensity in the carbohydrate double bands (1000-1180 cm-{superscript 1}) in glycated samples compared to the control. Glycation with 3 M MGO exhibited the highest resistance to enzymatic degradation, persisting for up to 60 hours with a 78-fold increase in resistance factor compared to the control group (p < 0.05). Furthermore, glycation with 3 M MGO resulted in a 4-fold increase in elastic modulus compared with the control group. Notably, the functionalized dentin retained its improved mechanical properties even after collagenase exposure, whereas the control group experienced a significant 68.2% reduction in elastic modulus (p = 0.002). While MGO pretreatment did not influence resin infiltration or initial TBS ({approx}30-35 MPa), it maintained its original bond strength after one month of collagenase challenge. In contrast, the control group exhibited a significant reduction, decreasing to 17 {+/-} 5.5 MPa compared with its initial value (p < 0.01). ConclusionMGO demonstrated efficacy in enhancing the mechanical properties and enzymatic stability of collagen as well as improving the resistance of the bonded interface to enzymatic degradation. SignificanceMGO pretreatment maintains the long-term stability of the resin-dentin interface by protecting dentinal collagen from enzymatic degradation, without compromising initial bonding performance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/701305v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@54ae32org.highwire.dtl.DTLVardef@1787fdforg.highwire.dtl.DTLVardef@130b40org.highwire.dtl.DTLVardef@47c5f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

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..

PurposeDespite advances in oral and injectable HIV prevention options and oral prophylaxis for sexually transmitted infections (STIs) of bacterial origin, there remains a critical need for effective on-demand topical (vaginal/rectal) products for pre- and post-exposure prophylaxis (PrEP and PEP). To fill this gap, we have developed single and first-in-kind multi-active topical inserts for bacterial STIs and HIV/STIs prevention. MethodsWe have formulated two different inserts, one containing doxycycline (DOX) at 10, 50, and 100mg doses for bacterial STI prevention, and a multipurpose prevention product (TED insert) that combines DOX (10mg) with the antiretrovirals tenofovir alafenamide (TAF; 20mg) and elvitegravir (EVG; 16mg) to target both bacterial STIs and HIV. ResultsInserts were manufactured through a simple, cost-effective process. Drug loading was within 95-105% of the labeled amount, confirming a robust manufacturing process. In vitro, they disintegrated within 10min with >95% drug release within 60min. The dissolution behavior of DOX inserts showed surface erosion but was affected by medium volume and drug amount. The inserts met key physicochemical targets: hardness (5-8kg), friability (<1%), moisture content (<2%), and osmolality (<550mOsm/kg). Based on 6-month storage stability, DOX inserts maintained their physicochemical properties, suggesting a shelf life of >2years. Preliminary 1-month stability of TED inserts under accelerated conditions showed preservation of their physicochemical properties. ConclusionThis study represents the first formulation development report on topical inserts containing DOX alone or in combination with antiretrovirals. Both inserts offer a novel, on-demand topical STI prevention option that supports flexible PrEP/PEP use by both women and men.

Acellular Adipose Tissue (AAT) is an off-the-shelf, cadaveric adipose-derived ECM-based biomaterial for soft tissue reconstruction. AAT has been validated preclinically to promote angiogenesis and adipogenesis and demonstrated safety, biocompatibility, and tolerability in a Phase I study. In this study we report the findings for the first ten patients in the Phase II study for permanent reconstruction of modest soft tissue defects. AAT promoted macrophages, CD3+ T cells, and CD34+ progenitor activity. Multiplex immunofluorescence staining using the PhenoCycler (formerly CODEX) imaging platform found that AAT can induce tertiary lymphoid structures (TLS). Nanostring GEOMx spatial transcriptional data analysis found significant differential gene expression between neighboring tissues with EGR1, MCL1, and NR4A1 upregulated in AAT. These genes have roles in angiogenesis, anti-apoptotic processes, and promotion of anti-inflammatory genes, respectively. AAT promoted anti-fibrotic CD74+ adipose-derived stromal cells, confirmed by immunofluorescence staining. Our findings demonstrate that AAT promotes angiogenesis, adipogenesis, and anti-fibrotic remodeling.

In vitro reconstruction of human tissue microenvironments that integrate native biochemical and biomechanical cues is essential for disease modelling, regenerative medicine, and personalized therapeutic approaches. However, most currently available engineered matrices fail to recapitulate the complexity and tissue specificity of the human extracellular matrix (ECM). To address this limitation, we developed a novel hydrogel derived from decellularized human adipose tissue (atdECM) designed to support three-dimensional culture of human cells. The decellularization and delipidation processes were first validated, and the biochemical composition and biomechanical properties of atdECM were comprehensively characterized. Human pancreatic organoids were then cultured within atdECM hydrogel, and their structural organization and transcriptional profiles were analyzed and compared with those obtained in Matrigel, the current gold-standard matrix for organoid culture. Proteomic and cytokine analyses demonstrated efficient decellularization while preserving collagen-rich ECM architecture and a diverse repertoire of soluble bioactive factors. AtdECM exhibited physiological stiffness and retained tissue-specific extracellular cues. Pancreatic organoids cultured in atdECM displayed morphological similarities with those grown in Matrigel but exhibited transcriptional profiles more consistent with physiological epithelial homeostasis, with reduced activation of inflammatory and stress-related pathways. Altogether, these findings indicate that atdECM provides a human-derived, tissue-relevant, and permissive microenvironment for human organoid generation. This platform represents a promising alternative to Matrigel for studying human tissue biology and for developing physiologically relevant in vitro models.