Biology subjects

Segura, T.

Publications and source records attributed to Segura, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Physical continuity at biomaterial-ECM interfaces regulates fibroblast activation via NF-κB

Fibrotic responses at biomaterial-tissue interfaces limit implant integration and regenerative healing, yet how the interaction between biomaterials and the extracellular matrix (ECM) regulates fibroblast activation remains poorly understood. Granular hydrogels including microporous annealed particle scaffolds (MAP) reduce fibrosis, while chemically and mechanically matched hydrogels do not, suggesting a dominant role for scaffold architecture. In this model, MAP scaffolds allow collagen infiltration and form physically continuous composites, whereas hydrogels exclude collagen and generate interfacial slip planes. To isolate how biomaterial architecture influences extracellular matrix (ECM) integration and fibroblast activation, we developed a reductionist in vitro model that integrates collagen type I with either microporous annealed particle (MAP) scaffolds or chemically and mechanically matched bulk hydrogels. This physical integration stabilizes collagen architecture, limits fibroblast-mediated matrix compaction, suppresses contractility, and attenuates myofibroblast transition. Fibroblasts in mechanically integrated environments exhibit reduced expression and nuclear localization of NF-{kappa}B and are enriched for quiescent phenotypes. Together, these findings identify biomaterial-ECM physical continuity as a design principle for limiting fibrotic signaling.

bioengineering↗

bioRxiv2025

Polysialic Acid Presentation on Microporous Scaffolds Supports Neural Repair after Ischemic Stroke

Recovery following ischemic stroke remains limited due to insufficient neural regeneration. Polysialic acid (PSA), a glycan prominently expressed during neural development, modulates neural progenitor cell (NPC) plasticity and migration, but its therapeutic potential in biomaterial-based stroke therapies remains underexplored. In this study, microporous annealed particle (MAP) scaffolds conjugated with PSA (PSA-MAP) were engineered to regulate NPC fate and promote neural tissue regeneration after stroke. PSA-MAP increased the presence of Sox2-positive progenitor cells within infarct and peri-infarct regions and elevated axonal content (NF200) in the lesion, while astrocytic and vascular coverage were not detectably changed at this early stage. In addition, 3D NPC cultures in MAP showed that tethered PSA alters NPC behavior over time, with reduced progenitor marker expression and PSA-dependent shifts in morphology, consistent with progression away from a progenitor state. Together, these data identify a glycan-forward, neuro-first repair route in which PSA-MAP enhances early neural regeneration without requiring concomitant angiogenic expansion, establishing PSA-MAP as a targeted biomaterial approach for endogenous neural repair after ischemic stroke.

bioengineering↗

bioRxiv2025

Polysialic Acid-Functionalized MAP Scaffolds Promote Regulatory Immune Responses After Ischemic Stroke

Glycosylation regulates immune and neural functions within the central nervous system (CNS), yet biomaterials rarely leverage glycans due to their structural complexity. Polysialic acid (PSA), comprising 2,8-linked sialic acid residues, is a promising candidate owing to its potent immunomodulatory interactions with inhibitory Siglec receptors. Systematic screening of multiple sialic acid derivatives identifies PSA as uniquely effective in inducing anti-inflammatory polarization of bone marrow-derived macrophages (BMDMs). Based on these findings, an injectable microporous annealed particle (MAP) scaffold presenting PSA covalently via its reducing end (MAP-PSA) is engineered, recapitulating physiological glycan orientation. MAP-PSA exhibits robust mechanical properties, stable glycan immobilization, and resistance to enzymatic degradation. Using ischemic stroke as a CNS injury model, MAP-PSA significantly reduces neutrophil infiltration and inflammatory activation while enhancing reparative macrophage and microglial phenotypes. These immunomodulatory effects persist into subacute stages, characterized by sustained reductions in inflammation and enhanced microglial homeostasis. Overall, MAP-PSA scaffolds demonstrate a novel therapeutic paradigm for CNS injuries such as stroke, with translational potential for broader neuroinflammatory and regenerative applications.

bioengineering↗

bioRxiv2025

Metrics for studying the porous void space of packed particles

Characterizing porosity in packed particle assemblies is a complex task that requires advanced analytical tools. We present a visually rich and extensive library of global, pore-based, and other metrics for analyzing features of porosity in such assemblies. Our library includes over 25 descriptors of "3D pores" that are identified using our LOVAMAP software. By applying our metrics to a set of simulated packings that vary by particle size, shape, and stiffness, we reveal predictable relationships between particle and void space characteristics. We identify two fundamental parameters of a monodisperse particle system - particle diameter ({delta}) and void volume fraction ({phi}) - that govern several void space features, such as the total number of bottlenecks (i.e., doors between pores), the median value of the largest enclosed sphere across all pores in a packing, and the fraction of reaction-center "hotspots." Through regression analyses on transformations of{delta} and{phi} , we quantify multiple packing-descriptor relationships, demonstrating, for example, that packing properties scale linearly with the median values of length-based descriptors across assemblies. We further introduce approaches for computing the number of vertices, edges, and faces of 3D pores, allowing for approximation to simpler polyhedra. Additional metrics explore surface entrances into the particle scaffold, traversable paths through the void space, and size-based accessibility. Together, these descriptors, which have been bundled into LOVAMAP, offer new insights into particle-pore architecture and spatial organization.

bioengineering↗

bioRxiv2025

Comparative proteomic analysis of skin wound healing responses to biomaterial treatments identifies key pathways which govern differential regenerative outcomes

The wound healing cascade is characterized by the steady progression of distinct stages. Though biomaterials are used clinically to enhance wound closure rate and quality of healed tissue, their mechanisms of action are less understood. Here we use proteomic analysis to characterize changes in the wound healing response across three biomaterial treatments: a clinically used collagen hydrogel, and two synthetic biomaterials that are characterized by an increased regenerative response either through decreased fibrosis or through an activation of adaptive immunity. We identified close to 5,000 proteins shared across the biomaterial treatment groups, sampled at timepoints representing the inflammation, proliferation, and resolution phases of wound healing. The collagen hydrogel maintains an enrichment of immune-related pathways throughout the healing process. The fibrosis-suppressing material enriches gene ontology (GO) terms related to increased epidermis development pathways, collagen synthesis, and collagen fibril organization. In contrast, the adaptive immunity-activating biomaterial shows an early enrichment of GO terms related to broad immunity and inflammation. Later, this same material promotes keratinization, muscle and lipid oxidation GO pathways. Taken together, this work determines the key temporal pathways (immunity, keratinization, muscle system process, and ECM organization) mediated by three biomaterials, which result in varying healed tissue structure.

bioengineering↗

bioRxiv2025

Clustered VEGF Nanoparticles in Microporous Annealed Particle (MAP) Hydrogel Accelerates Functional Recovery and Brain Tissue Repair after Stroke

Ischemic stroke, a blockage in the vasculature of the brain that results in insufficient blood flow, is one of the worlds leading causes of disability. The cascade of inflammation and cell death that occurs immediately following stroke drives vascular and functional loss that does not fully recover over time, and no FDA-approved therapies exist that stimulate regeneration post-stroke. We have previously developed a hydrogel scaffold that delivered heparin nanoparticles with and without VEGF bound to their surface to promote angiogenesis and reduce inflammation, respectively. However, the inclusion of the naked heparin nanoparticles warranted concern over the development of bleeding complications. Here, we explore how microporous annealed particle (MAP) scaffolds functionalized with VEGF coated heparin nanoparticles can both reduce inflammation and promote angiogenesis - without the inclusion of free heparin nanoparticles. We show that our updated design not only successfully promotes de novo tissue formation, including the development of mature vessels and neurite sprouting, but it also leads to functional improvement in a photothrombotic stroke model. In addition, we find increased astrocyte infiltration into the infarct site correlated with mature vessel formation. This work demonstrates how our biomaterial design can enhance endogenous regeneration post-stroke while eliminating the need for excess heparin.

bioengineering↗

bioRxiv2024

Highly porous granular hydrogels reinforced by electrospun hydrogel fibers for long-term stability

Granular hydrogels (GHs) formed from hydrogel microparticles (HMPs) possess inherent, microscale porosity. In GHs including microporous annealed particle (MAP) hydrogels as well as unannealed GHs, increases in pore size and total porosity correlate with enhanced cellular infiltration and growth. However, increased porosity or pore size are typically achieved by decreasing HMP density, reducing interparticle contacts and weakening hydrogel structure. Here, to achieve a highly porous GH without compromising stability, we developed an approach using sacrificial HMPs to achieve high, mesoscale porosity and high-aspect ratio hydrogel fibers to reinforce the scaffold. The use of sacrificial HMPs circumvented the need to decrease packing density, creating GHs with up to 60% porosity. The hydrogel fibers span particles in the porous space, stabilizing the porous structure by enhancing interactions among GH components. Upon 5-10% v/v fiber incorporation, highly porous GHs retained their structure over 28 days of in vivo culture, compared to 4 days in GHs without fibers. These scaffolds supported and promoted endothelial cell growth as porosity increased. Overall, this study presents a stable, highly porous granular scaffold system with high processability and cytocompatibility.

bioengineering↗

bioRxiv2023

Engineering the microstructure and spatial bioactivity of MAP scaffolds in vitro instructs neovascularization in vivo

In tissues where the vasculature is either lacking or abnormal, biomaterials can be designed to promote vessel formation and enhance tissue repair. In this work, we independently tune the microstructure and bioactivity of microporous annealed particle (MAP) scaffolds to guide cell patterning in 3D and promote de novo assembly of endothelial progenitor-like cells into vessels. We implement both in silico characterization and in vitro experimentation to elucidate an optimal scaffold formulation for vessel formation. We determine that MAP scaffolds with pore volumes on the same order of magnitude as cells facilitate cell growth and vacuole formation. We achieve spatial control over cell spreading by incorporating adhesive microgels in well-mixed, heterogeneous MAP scaffolds. While we demonstrate that integrin engagement is the primary driver of network formation in these materials, introducing adhesive microgels loaded with heparin nanoparticles leads to the formation of vascular tubes after 3 days in culture. We then show in vivo that this unique scaffold formulation enhances vessel maturation in a wound healing model and instructs differential vascular patterning in the tumor microenvironment. Taken together, this work determines the optimal microstructure and ligand presentation within MAP scaffolds that lead to vascular constructs in vitro and facilitate neovascularization in vivo.

bioengineering↗

bioRxiv2023

SDF-1 Bound Heparin Nanoparticles Recruit Progenitor Cells for Their Differentiation and Promotion of Angiogenesis After Stroke

Angiogenesis after stroke is correlated with enhanced tissue repair and functional outcomes. The existing body of research in biomaterials for stroke focuses on hydrogels for the delivery of stem cells, growth factors, or small molecules or drugs. Despite the ability of hydrogels to enhance all these delivery methods, no material has significantly regrown vasculature within the translatable timeline of days to weeks after stroke. Here we developed 2 novel biomaterials for tissue regeneration after stroke, a highly porous granular hydrogel termed Cryo microgels, and heparin-norbornene nanoparticles with covalently bound SDF-1. The combination of these materials resulted in fully revascularized vessels throughout the stroke core in only 10 days, as well as increased neural progenitor cell migration and maintenance and increased neurons.

bioengineering↗

bioRxiv2023

Exploring the Role of Spatial Confinement in Immune Cell Recruitment and Regeneration of Skin Wounds

Microporous annealed particle (MAP) scaffolds are injectable granular materials comprised of micron sized hydrogel particles (microgels). The diameter of these microgels directly determines the size of the interconnected void space between particles where infiltrating or encapsulated cells reside. This tunable porosity allows us to use MAP scaffolds to study the impact of spatial confinement (SC) on both cellular behaviors and the host response to biomaterials. Despite previous studies showing that pore size and SC influence cellular phenotypes, including mitigating the macrophage inflammatory response, there is still a gap in knowledge regarding how SC within a biomaterial modulates immune cell recruitment in vivo in wounds and implants. Thus, we studied the immune cell profile within confined and unconfined biomaterials using small (40 m), medium (70 m), and large (130 m) diameter spherical microgels, respectively. We discovered that MAP scaffolds imparted regenerative wound healing with an IgG1-biased Th2 response. MAP scaffolds generated from 130 m diameter microgels have a median pore size that can accommodate [~]40 {micro}m diameter spheres induced a more balanced pro-regenerative macrophage response and better wound healing outcomes with more mature collagen regeneration and reduced levels of inflammation.

bioengineering↗

bioRxiv2022

Reactive astrocyte derived extracellular vesicles promote functional repair post stroke

Reactive astrocytes are both neurotoxic and pro-regenerative. Their reparative roles after injury have been demonstrated, but how they play a contributing role to regeneration remains question. Here, we investigate the use of astrocytic extracellular vesicles from primary astrocytes cultured in reactive conditions in promoting repair after ischemic stroke. Our studies show that extracellular vesicles derived from reactive astrocytes that co-express a significant number of reactive genes (155 upregulated including log2 of 9.61 for Lcn2) and axonal outgrowth genes (59 upregulated including log2 of 3.49 Ntn1) are necessary for improved regenerative outcomes, including axonal infiltration, vascularization, and improved behavioral recovery. Proteomic analysis of the extracellular vesicles show that astrocytes enrich pro-reparative proteins in extracellular vesicles with only 30 proteins relating to inflammatory or complement pathways loaded out of a total of 1073 proteins. Further, we show that the use of a biomaterial scaffold is necessary for the improved regeneration observed from reactive astrocyte extracellular vesicles. These studies show that reactive astrocytes use extracellular vesicles enriched with pro-repair proteins to promote recovery after injury.

neuroscience↗

bioRxiv2022

A balance between pro-inflammatory and pro-reparative macrophages is observed in regenerative D-MAPS

Microporous annealed particle scaffolds (MAPS) are a new class of granular materials generated through the interlinking of tunable microgels, which produce an interconnected network of void space. These microgel building blocks can be designed with different mechanical or bio-active parameters to facilitate cell infiltration and modulate host response. Previously, changing the chirality of the microgel crosslinking peptides from L- to D-amino acids led to significant tissue regeneration and functional recovery in D-MAPS-treated cutaneous wounds. In this study, we investigated the immunomodulatory effect of D-MAPS in a subcutaneous implantation model. We uncovered how macrophages are the key antigen-presenting cells to uptake and present these biomaterials to the adaptive immune system. A robust linker-specific IgG2b/IgG1 response to D-MAPS was detected as early as 14 days post-implantation. The fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes was observed in D-MAPS as an indicator for regenerative scaffolds. Our work offers valuable insights into the temporal cellular response to synthetic porous scaffolds and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes.

bioengineering↗

bioRxiv2022

Spatial confinement modulates macrophage response in microporous scaffolds

Macrophages are essential in the initiation, maintenance, and transition of inflammatory processes like foreign body response and wound healing. Mounting evidence suggests that physical factors also modulate macrophage activation in vitro and in vivo. 2D in vitro systems have demonstrated that constraining macrophages to small areas or channels modulates their phenotypes and changes their responses to known inflammatory agents such as lipopolysaccharide. However, how dimensionality and pore size affect macrophage phenotype is less explored. In this work, we studied the change in M1/M2 polarization when macrophages were confined in microporous annealed particle scaffolds (MAP), which are granular hydrogels generated from annealed spherical microgels. We engineered three types of MAP gels comprising 40, 70, and 130 {micro}m diameter particle sizes, respectively. Particles sizes were selected using outputs from software LOVAMAP that analyzes the characteristics of 3-D pores in MAP gels. Since the size of building block particle correlates with pore size inside the final scaffolds, our three scaffold types allowed us to study how the degree of spatial confinement modulated the behavior of embedded macrophages. Spatially confining macrophages in scaffolds with pore size on the scale of cells led to a reduced level of the inflammatory response, which was correlated with a change in cell morphology and motility.

bioengineering↗

bioRxiv2022

Void volume fraction of granular scaffolds

Void volume fraction (VVF) of granular biomaterials is a global measurement frequently used to characterize void space. There is currently no gold standard for measuring the VVF of granular scaffolds made in lab. To help the biomaterials field, we provide a library of different simulated scaffolds with known VVF for easy look-up. We use our simulated data to explore the relationship between microscope magnification and VVF, and we study the accuracy of approximating VVF using 2-D z-slice images, which is the most common approach for computing VVF of real scaffolds. Lastly, we test four different approaches for computing VVF using microscope images of real granular scaffolds and reveal that VVF may be an unreliable descriptor.

bioengineering↗

bioRxiv2020

Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing.

Biomaterial scaffolds represent a promising approach for material-based tissue regeneration. We previously developed microporous annealed particle (MAP) hydrogels - a flowable, microparticle-based hydrogel in which neighboring hydrogel particles are linked in situ to form a porous scaffold that accelerates wound healing. To promote more extensive tissue ingrowth before scaffold degradation, we aimed to slow scaffold degradation by switching the chirality of the crosslinking peptides from L-peptides to D-peptides. Unexpectedly, despite showing the predicted slower enzymatic degradation in vitro, D-peptide crosslinked MAP hydrogel (D-MAP) hastened material degradation in vivo and imparted significant tissue regeneration to healed cutaneous wounds, including increased tensile strength and hair neogenesis. By themselves, D-chiral peptides were poor activators of macrophage innate immune signaling in vivo, but MAP particles elicit IL-33 type 2 myeloid cell recruitment which is amplified in vivo in the presence of D-peptides. Remarkably, D-MAP elicited significant antigen-specific immunity against the D-chiral peptides, and an intact adaptive immune system was required for the hydrogel-induced skin regeneration. These findings demonstrate that the generation of an adaptive immune response from a biomaterial is sufficient to induce cutaneous regenerative healing despite faster scaffold degradation.

bioengineering↗

bioRxiv2019

Hyaluronic acid particle hydrogels decrease cerebral atrophy and promote pro-reparative astrocyte/axonal infiltration after ischemic stroke

The death rate due to stroke is decreasing, resulting in more individuals living with stroke related disabilities. Following stroke, dying cells contribute to the large influx of highly reactive astrocytes and pro-inflammatory microglia that release cytokines and lead to a cytotoxic environment that causes further brain damage and prevents endogenous repair. Paradoxically, these same cells also activate pro-repair mechanisms that contribute to endogenous repair and brain plasticity. Here, we show that the direct injection of a hyaluronic acid based microporous annealed particle (HA-MAP) hydrogel into the stroke core reduces the percent of highly reactive astrocytes and increases the percent of alternatively activated microglia in and around the lesion. Further, we show that HA-MAP hydrogel promotes reparative astrocyte infiltration into the lesion, which directly coincides with axonal penetration into the lesion. Additionally, HA-MAP injection decreases cerebral atrophy and preserves nigrostriatal bundles after stroke. This work shows that the injection of a porous scaffold into the stroke core can lead to clinically relevant decrease in cerebral atrophy and modulates the phenotype of astrocytes and microglia towards a pro-repair phenotype.

bioengineering↗

bioRxiv2019

Isolation and quantification of blood apoptotic bodies in neurological patients

Dysregulation of apoptosis may contribute to the etiology and/or progression of several prevalent diseases, including stroke and neurodegenerative pathologies. So, detection of the apoptotic processes in patients would be useful in daily clinical practice, However, the in vivo analysis of apoptosis that occurs in tissue has limitations. We therefore propose to use circulating apoptotic bodies as biomarkers for measuring apoptotic death in patients. Since there is no scientific literature establishing the most appropriate method for measuring apoptotic bodies from human blood samples, we here describe a reproducible centrifugation-based method combined with electron microscopy, dynamic light scattering and flow cytometry studies to isolate, characterize and quantify plasma apoptotic bodies of patients with ischemic stroke, multiple sclerosis and Parkinsons disease. The analysis revealed that our isolation protocol achieves notable recovery rates of highly-purified intact apoptotic bodies. This easy and rapid procedure would help physicians to implement the use of plasma apoptotic bodies analysis as a non-invasive tool to monitor apoptotic death in neurological patients for prognostic purposes and for following disease activity and assessing drug effectiveness.

neuroscience↗

bioRxiv2019

Click by Click Microporous Annealed Particle (MAP) Scaffolds

Macroporous scaffolds are being increasingly used in regenerative medicine and tissue repair. While our recently developed microporous annealed particle (MAP) scaffolds have overcome issues with injectability and in situ hydrogel formation, limitations with respect to tunability to be able to manipulate hydrogel strength and rigidity for broad applications still exist. To address these key issues, here we synthesized hydrogel microparticles (HMPs) of hyaluronic acid (HA) using the thiol-norbornene click reaction and then subsequently annealed HMPs into a porous scaffold using the tetrazine-norbornene click reaction. This assembly method allowed for straightforward tuning of bulk scaffold rigidity by varying the tetrazine to norbornene ratio, with increasing tetrazine resulting in increasing scaffold storage modulus, Youngs modulus, and maximum stress. These changes were independent of void fraction. Further incorporation of human dermal fibroblasts (HDFs) throughout the porous scaffold revealed the biocompatibility of this annealing strategy as well as differences in proliferation and cell-occupied volume. Finally, injection of porous HA-Tet MAP scaffolds into an ischemic stroke model showed this chemistry is biocompatible in vivo with reduced levels of inflammation and astrogliosis as previously demonstrated for other crosslinking chemistries.

bioengineering↗