Biology subjects

Kloxin, A. M.

Publications and source records attributed to Kloxin, A. M..

6 recordsLinked to original sources

bioRxiv2026

Commensal Engineered Living Materials for Sustained Secretome Delivery and Wound Healing

Using living cells as dynamic biological factories offers immense potential for the continuous, localized production of therapeutic proteins within compromised tissues. This strategy is particularly compelling for treating chronic non-healing wounds, which lack therapeutics that are able to function effectively in the inflammatory microenvironment. Commensal bacteria have been shown to promote healing in these hostile microenvironments, yet applying live microbes directly onto compromised tissue is clinically unfeasible due off-target safety risks and engraftment challenges. Herein, we establish a well-defined commensal Engineered Living Material (cELM) that allows encapsulation, retention, function, and delivery of native beneficial bacteria for localized production and release of a pro-healing secretome over weeks. We specifically encapsulated a pro-healing human wound bacterial isolate, Alcaligenes faecalis, using biocompatible, click-reactive hydrogel building blocks (thiol- or vinyl-sulfone-functionalized multi-arm poly(ethylene glycol)) and systematically varied the density of the polymer network and of the encapsulated bacteria to establish design rules for creating pro-healing cELMs. Physical confinement in the hydrogel network ensured bacterial biocontainment, while the hydrogel mesh size still permitted diffusion of the therapeutic secretome. Notably, controlling encapsulation density revealed an inverse relationship wherein lower cell densities yielded significantly enhanced per-cell metabolic activity and provided sustained production of pro-healing secreted factors. In a murine diabetic wound model, these A. faecalis cELMs shielded the encapsulated bacteria from host clearance, maintained biocontainment, accelerated re-epithelialization, and improved wound bed maturity. Together, these findings establish design principles for cELMs that couple bacterial biocontainment with sustained secretome delivery, providing a promising platform for localized treatment of chronic wounds.

bioengineering↗

bioRxiv2026

Thermoresponsive, Shear-Thinning Supramolecular Hydrogels from Peptide-Polymer Conjugates for Bioinspired 3D Cell Culture

Physically assembled, supramolecular hydrogels often are formed by weak bonds, producing useful viscoelastic properties that are reminiscent of soft tissues within the human body yet presenting challenges in stability under physiological conditions. In this work, we establish an approach for creating self-assembling, supramolecular hydrogels based on a peptide-polymer conjugate (PPC) as a three-dimensional (3D) cell culture platform. The viscoelastic properties of these hydrogels, including thermoresponsiveness and shear-thinning, were measured using rheometry. Building on these observations, we then developed a method for the encapsulation and culture of human wound healing cells, fibroblasts, within the PPC hydrogels, observing both excellent cell viability and material stability over time. The modulation of stiffness by temperature variation and the reversible nature of these hydrogels imparts unique properties for both cell encapsulation and harvesting and offers potential for a variety of biomedical applications, from 3D cell culture systems to therapeutic delivery.

bioengineering↗

bioRxiv2026

Matrix stiffening toolbox: dynamic hydrogels for three-dimensional cell culture with real-time cell response

Extracellular matrix (ECM) mechanical properties regulate tissue homeostasis and disease progression, with persistent ECM stiffening serving as a hallmark of fibrosis; yet, the early transition from healthy to diseased tissue remains poorly understood. Dynamic three-dimensional (3D) tissue models that capture early-stage stiffening are needed to investigate cellular responses during disease initiation. This work presents an innovative platform for studying cell responses in 3D environments undergoing active matrix stiffening. A bioinspired synthetic ECM incorporates collagen-mimetic peptides and employs sequential, non-terminal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to enable controlled increases in matrix stiffness over physiologically relevant timescales. Alternating polymer incubations produce a 2.5-fold increase in storage modulus over 72 hours, modeling the mechanical transition from healthy to early-stage fibrotic lung tissue. Live-cell reporter fibroblasts enable real-time monitoring of alpha-smooth muscle actin (SMA) expression, revealing significant upregulation during matrix stiffening that remains transient and difficult to detect via traditional endpoint assays. Active stiffening also modulates fibroblast motility, transiently increasing migration speed while persistently enhancing directional persistence. Complementary computational reaction-diffusion modeling provides mechanistic insight into modulus gradient formation and reaction kinetics. This versatile toolbox enables investigation of early mechanobiological responses to matrix stiffening and may aid identification of markers of fibrotic disease onset.

bioengineering↗

bioRxiv2025

Probing Critical Injury Thresholds for Maladaptive Epithelial Injury and Repair Processes with Photoresponsive Bioinspired Synthetic Basement Membrane

Microinjuries to the lung epithelium are hypothesized to initiate maladaptive processes that lead to fibrosis. Human in vitro models remain a great need for studying this injury-initiation process for mechanistic understanding and therapeutic development. We established a photoresponsive synthetic extracellular matrix (ECM) inspired by the basement membrane that enables triggered injuries of defined size and frequency for probing cellular responses. The synthetic matrix integrated a photolabile bis-coumarin linker for light-triggered injury and relevant integrin-binding peptides for cell function. Bio-orthogonal chemistry was used to create hydrogel-based ECMs with tunable elasticity in transwells, which are traditionally used for epithelial cell culture. Integrin-binding peptide combinations synergistically promoted model epithelial cell layer formation with increased E-cadherin expression and barrier function. An accessible photomasking approach was established for selectively photodegrading the synthetic matrix with cytocompatible visible light and achieving different injury depths and widths. Following a critical injury size, cell responses recapitulated key features of dysregulated re-epithelialization with decreased E-cadherin, proliferation, and barrier function and increased apoptosis. This work provides a new materials-based tool for probing injury and repair processes with tunable control of both the ECM and injury to it with opportunities for future mechanistic and therapeutic insights to address maladaptive wound healing processes.

bioengineering↗

bioRxiv2020

Multiscale Invasion Assay for Probing Macrophage Response to Bacteria

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the gut was established. Workflows were developed for monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe bacterial fragment production and localization in the cell at the carbohydrate level for insights into how our immune system properly senses bacteria. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/385617v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@45cdf1org.highwire.dtl.DTLVardef@d3c3d3org.highwire.dtl.DTLVardef@dd74f2org.highwire.dtl.DTLVardef@128d1da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Success Criteria for Preclinical Testing of Cell-Instructive Hydrogels for Tendon Regeneration

Tendon injuries are difficult to heal in part because intrinsic tendon healing, which is dominated by scar tissue formation, does not effectively regenerate the native structure and function of healthy tendon. Further, many current treatment strategies also fall short of producing regenerated tendon with the native properties of healthy tendon. There is increasing interest in the use of cell-instructive strategies to limit the intrinsic fibrotic response following injury and improve the regenerative capacity of tendon in vivo. We have established multi-functional, cell-instructive hydrogels for treating injured tendon that afford tunable control over the biomechanical, biochemical, and structural properties of the cell microenvironments. Specifically, we incorporated integrin-binding domains (RGDS) and assembled multi-functional collagen mimetic peptides (mfCMPs) that enable cell adhesion and elongation of stem cells within synthetic hydrogels of designed biomechanical properties and evaluated these materials using targeted success criteria developed for testing in mechanically-demanding environments like tendon healing. The in vitro and in situ success criteria were determined based on systematic reviews of the most commonly reported outcome measures of hydrogels for tendon repair and established standards for testing of biomaterials. We then showed, using validation experiments, that multi-functional and synthetic hydrogels meet these criteria. Specifically, these hydrogels have mechanical properties comparable to developing tendon; are non-cytotoxic both in 2D bolus exposure (hydrogel components) and 3D encapsulation (full hydrogel); are formed, retained, and visualized within tendon defects over time (two-weeks); and provide mechanical support to tendon defects at the time of injection and in situ formation. Ultimately, the in vitro and in situ success criteria evaluated in this study were designed for preclinical research to rigorously test the potential to achieve successful tendon repair prior to in vivo testing and indicate the promise of multi-functional and synthetic hydrogels for continued translation. IMPACT STATEMENTTendon healing results in a weak scar that forms due to poor cell-mediated repair of the injured tissue. Treatments that tailor the instructions experienced by cells during healing afford opportunities to regenerate the healthy tendon. Engineered cell-instructive cues, including the biomechanical, biochemical, and structural properties of the cell microenvironment, within multi-functional synthetic hydrogels are promising therapeutic strategies for tissue regeneration. In this paper, the preclinical efficacy of multi-functional synthetic hydrogels for tendon repair is tested against rigorous in vitro and in situ success criteria. This study indicates the promise for continued preclinical translation of synthetic hydrogels for tissue regeneration.

bioengineering↗