Publications by authors named "Ashley C Brown"

51 Publications

Fibrin-Based Biomaterial Systems to Enhance Anterior Cruciate Ligament Healing.

Med Devices Sens 2021 Feb 11;4(1). Epub 2020 Nov 11.

Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, NC 27695.

Anterior cruciate ligament (ACL) tears are a common and potentially career-ending injury, particularly for athletes and soldiers. Partial and complete ruptures of this ligament cause instability in the knee, and the ACL does not have the capacity for healing due, in part, to its position within the highly thrombolytic synovial fluid environment of the knee joint. Traditional methods of ACL reconstruction, such as graft replacement with attached bone anchors for bone integration, restore stability, but do not prevent the development of post-traumatic osteoarthritis. To enhance therapeutic treatment options, novel fibrin-based technologies and repair techniques have been recently explored and show promise for improved patient outcomes. Through modification of existing surgical methods, such as the use of fibrin glues incorporating growth factors and cells and the implementation of scaffolds containing platelet-rich plasma, platelet-rich fibrin, and other blood derivatives, surgeons are attempting to overcome the shortcomings of traditional treatments. This mini-review will detail current efforts using fibrin-based treatments and discuss opportunities to further enhance ACL healing.
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http://dx.doi.org/10.1002/mds3.10147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386506PMC
February 2021

Platelet-like particles reduce coagulopathy-related and neuroinflammatory pathologies post-experimental traumatic brain injury.

J Biomed Mater Res B Appl Biomater 2021 Jun 11. Epub 2021 Jun 11.

School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona, USA.

Coagulopathy may occur following traumatic brain injury (TBI), thereby negatively affecting patient outcomes. Here, we investigate the use of platelet-like particles (PLPs), poly(N-isopropylacrylamide-co-acrylic-acid) microgels conjugated with a fibrin-specific antibody, to improve hemostasis post-TBI. The objective of this study was to diminish coagulopathy in a mouse TBI model (controlled cortical impact) via PLP treatment, and subsequently decrease blood-brain barrier (BBB) permeability and neuroinflammation. Following an acute intravenous injection of PLPs post-TBI, we analyzed BBB permeability, ex vivo coagulation parameters, and neuroinflammation at 24 hr and 7 days post-TBI. Both PLP-treatment and control particle-treatment had significantly decreased BBB permeability and improved clot structure 24 hr post-injury. Additionally, no significant change in tissue sparing was observed between 24 hr and 7 days for PLP-treated cohorts compared to that observed in untreated cohorts. Only PLP-treatment resulted in significant reduction of astrocyte expression at 7 days and percent difference from 24 hr to 7 days. Finally, PLP-treatment significantly reduced the percent difference from 24 hr to 7 days in microglia/macrophage density compared to the untreated control. These results suggest that PLP-treatment addressed acute hypocoagulation and decreased BBB permeability followed by decreased neuroinflammation and fold-change tissue loss by 7 days post-injury. These promising results indicate that PLPs could be a potential therapeutic modality for TBI.
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http://dx.doi.org/10.1002/jbm.b.34888DOI Listing
June 2021

Synthetic platelet microgels containing fibrin knob B mimetic motifs enhance clotting responses.

Adv Ther (Weinh) 2021 May 18;4(5). Epub 2021 Mar 18.

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Raleigh, NC, USA.

Native platelets are crucial players in wound healing. Key to their role is the ability of their surface receptor GPIIb/IIIa to bind fibrin at injury sites, thereby promoting clotting. When platelet activity is impaired as a result of traumatic injury or certain diseases, uncontrolled bleeding can result. To aid clotting and tissue repair in cases of poor platelet activity, our lab has previously developed synthetic platelet-like particles capable of promoting clotting and improving wound healing responses. These are constructed by functionalizing highly deformable hydrogel microparticles (microgels) with fibrin-binding ligands including a fibrin-specific whole antibody or a single-domain variable fragment. To improve the translational potential of these clotting materials, we explored the use of fibrin-binding peptides as cost-effective, robust, high-specificity alternatives to antibodies. Herein, we present the development and characterization of soft microgels decorated with the peptide AHRPYAAK that mimics fibrin knob 'B' and targets fibrin hole 'b'. These "Fibrin-Affine Microgels with Clotting Yield" (FAMCY) were found to significantly increase clot density and decrease bleeding in a rodent trauma model . These results indicate that FAMCYs are capable of recapitulating the platelet-mimetic properties of previous designs while utilizing a less costly, more translational design.
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http://dx.doi.org/10.1002/adtp.202100010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8171167PMC
May 2021

Synthesis of sonicated fibrin nanoparticles that modulate fibrin clot polymerization and enhance angiogenic responses.

Colloids Surf B Biointerfaces 2021 Aug 29;204:111805. Epub 2021 Apr 29.

Joint Department of Biomedical Engineering, NC State University and UNC Chapel-Hill, Raleigh, NC, United States; Comparative Medicine Institute, NC State University, Raleigh, NC, United States. Electronic address:

Chronic wounds can occur when the healing process is disrupted and the wound remains in a prolonged inflammatory stage that leads to severe tissue damage and poor healing outcomes. Clinically used treatments, such as high density, FDA-approved fibrin sealants, do not provide an optimal environment for native cell proliferation and subsequent tissue regeneration. Therefore, new treatments outside the confines of these conventional fibrin bulk gel therapies are required. We have previously developed flowable, low-density fibrin nanoparticles that, when coupled to keratinocyte growth factor, promote cell migration and epithelial wound closure in vivo. Here, we report a new high throughput method for generating the fibrin nanoparticles using probe sonication, which is less time intensive than the previously reported microfluidic method, and investigate the ability of the sonicated fibrin nanoparticles (SFBN) to promote clot formation and cell migration in vitro. The SFBNs can form a fibrin gel when combined with fibrinogen in the absence of exogenous thrombin, and the polymerization rate and fiber density in these fibrin clots is tunable based on SFBN concentration. Furthermore, fibrin gels made with SFBNs support cell migration in an in vitro angiogenic sprouting assay, which is relevant for wound healing. In this report, we show that SFBNs may be a promising wound healing therapy that can be easily produced and delivered in a flowable formulation.
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http://dx.doi.org/10.1016/j.colsurfb.2021.111805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217261PMC
August 2021

Multivalent Aptamer-Functionalized Single-Strand RNA Origami as Effective, Target-Specific Anticoagulants with Corresponding Reversal Agents.

Adv Healthc Mater 2021 06 21;10(11):e2001826. Epub 2021 Apr 21.

Department of Materials Science and Engineering, College of Engineering, North Carolina State University, Raleigh, NC, 27695, USA.

Anticoagulants are commonly utilized during surgeries and to treat thrombotic diseases like stroke and deep vein thrombosis. However, conventional anticoagulants have serious side-effects, narrow therapeutic windows, and lack safe reversal agents (antidotes). Here, an alternative RNA origami displaying RNA aptamers as target-specific anticoagulant is described. Improved design and construction techniques for self-folding, single-molecule RNA origami as a platform for displaying pre-selected RNA aptamers with precise orientational and spatial control are reported. Nuclease resistance is added using 2'-fluoro-modified pyrimidines during in vitro transcription. When four aptamers are displayed on the RNA origami platform, the measured thrombin inhibition and anticoagulation activity is higher than observed for free aptamers, ssRNA-linked RNA aptamers, and RNA origami displaying fewer aptamers. Importantly, thrombin inhibition is immediately switched off by addition of specific reversal agents. Results for single-stranded DNA (ssDNA) and single-stranded peptide nucleic acid (PNA) antidotes show restoration of 63% and 95% coagulation activity, respectively. To demonstrate potential for practical, long-term storage for clinical use, RNA origami is freeze-dried, and stored at room temperature. Freshly produced and freeze-dried RNA show identical levels of activity in coagulation assays. Compared to current commercial intravenous anticoagulants, RNA origami-based molecules show promise as safer alternatives with rapid activity switching for future therapeutic applications.
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http://dx.doi.org/10.1002/adhm.202001826DOI Listing
June 2021

Neonatal coagulopathies: A review of established and emerging treatments.

Exp Biol Med (Maywood) 2021 Jun 15;246(12):1447-1457. Epub 2021 Apr 15.

Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, NC 27695, USA.

Despite the relative frequency of both bleeding and clotting disorders among patients treated in the neonatal intensive care unit, few clear guidelines exist for treatment of neonatal coagulopathies. The study and treatment of neonatal coagulopathies are complicated by the distinct hemostatic balance and clotting components present during this developmental stage as well as the relative scarcity of studies specific to this age group. This mini-review examines the current understanding of neonatal hemostatic balance and treatment of neonatal coagulopathies, with particular emphasis on emerging treatment methods and areas in need of further investigative efforts.
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http://dx.doi.org/10.1177/15353702211006046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8243218PMC
June 2021

Modeling and Parameter Subset Selection for Fibrin Polymerization Kinetics with Applications to Wound Healing.

Bull Math Biol 2021 Mar 22;83(5):47. Epub 2021 Mar 22.

Department of Mathematics, North Carolina State University, Box 8205, Raleigh, NC, 27695-8205, USA.

During the hemostatic phase of wound healing, vascular injury leads to endothelial cell damage, initiation of a coagulation cascade involving platelets, and formation of a fibrin-rich clot. As this cascade culminates, activation of the protease thrombin occurs and soluble fibrinogen is converted into an insoluble polymerized fibrin network. Fibrin polymerization is critical for bleeding cessation and subsequent stages of wound healing. We develop a cooperative enzyme kinetics model for in vitro fibrin matrix polymerization capturing dynamic interactions among fibrinogen, thrombin, fibrin, and intermediate complexes. A tailored parameter subset selection technique is also developed to evaluate parameter identifiability for a representative data curve for fibrin accumulation in a short-duration in vitro polymerization experiment. Our approach is based on systematic analysis of eigenvalues and eigenvectors of the classical information matrix for simulations of accumulating fibrin matrix via optimization based on a least squares objective function. Results demonstrate robustness of our approach in that a significant reduction in objective function cost is achieved relative to a more ad hoc curve-fitting procedure. Capabilities of this approach to integrate non-overlapping subsets of the data to enhance the evaluation of parameter identifiability are also demonstrated. Unidentifiable reaction rate parameters are screened to determine whether individual reactions can be eliminated from the overall system while preserving the low objective cost. These findings demonstrate the high degree of information within a single fibrin accumulation curve, and a tailored model and parameter subset selection approach for improving optimization and reducing model complexity in the context of polymerization experiments.
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http://dx.doi.org/10.1007/s11538-021-00876-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237246PMC
March 2021

Development of novel microenvironments for promoting enhanced wound healing.

Curr Tissue Microenviron Rep 2020 Sep 29;1(3):73-87. Epub 2020 Jul 29.

Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, NC 27695.

Purpose Of Review: Nonhealing wounds are a significant issue facing the healthcare industry. Materials that modulate the wound microenvironment have the potential to improve healing outcomes.

Recent Findings: A variety of acellular and cellular scaffolds have been developed for regulating the wound microenvironment, including materials for controlled release of antimicrobials and growth factors, materials with inherent immunomodulative properties, and novel colloidal-based scaffolds. Scaffold construction methods include electrospinning, 3D printing, decellularization of extracellular matrix, or a combination of techniques. Material application methods include layering or injecting at the wound site.

Summary: Though these techniques show promise for repairing wounds, all material strategies thus far struggle to induce regeneration of features such as sweat glands and hair follicles. Nonetheless, innovative technologies currently in the research phase may facilitate future attainment of these features. Novel methods and materials are constantly arising for the development of microenvironments for enhanced wound healing.
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http://dx.doi.org/10.1007/s43152-020-00009-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968354PMC
September 2020

Fibrin-modulating nanogels for treatment of disseminated intravascular coagulation.

Blood Adv 2021 02;5(3):613-627

Joint Department of Biomedical Engineering of University of North Carolina-Chapel Hill and North Carolina State University, Raleigh, NC.

Disseminated intravascular coagulation (DIC) is a pathological coagulopathy associated with infection that increases mortality. In DIC, excessive thrombin generation causes symptoms from formation of microthrombi to multiorgan failure; bleeding risks can also be a concern because of clotting factor consumption. Different clinical events lead to DIC, including sepsis, trauma, and shock. Treatments for thrombotic episodes or bleeding presentation in DIC oppose each other, thus creating therapeutic dilemmas in management. The objective of this study was to develop fibrin-specific core-shell nanogels (FSNs) loaded with tissue-type plasminogen activator (tPA) to treat the microcirculatory complications of DIC, which would facilitate targeted clot dissolution to manage microthrombi and the potential consumptive coagulopathy that causes bleeding. FSNs enhance formation of actively polymerizing clots by crosslinking fibrin fibers, but they can also target preexisting microthrombi and, when loaded with tPA, facilitate targeted delivery to lyse the microthrombi. We hypothesized that this dual action would simultaneously address bleeding and microthrombi with DIC to improve outcomes. In vivo, tPA-FSNs decreased the presentation of multiorgan microthrombi, recovered platelet counts, and improved bleeding outcomes in a DIC rodent model. When incorporated with human DIC patient plasma, tPA-FSNs restored clot structure and clot growth under flow. Together, these data demonstrate that a fibrinolytic agent loaded into fibrin-targeting nanogels could improve DIC outcomes.
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http://dx.doi.org/10.1182/bloodadvances.2020003046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876887PMC
February 2021

Ultrasound enhanced synthetic platelet therapy for augmented wound repair.

ACS Biomater Sci Eng 2020 05 7;6(5):3026-3036. Epub 2020 Apr 7.

Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, NC.

Native platelets perform a number of functions within the wound healing process, including interacting with fibrin fibers at the wound site to bring about retraction after clot formation. Clot retraction improves clot stability and enhances the function of the fibrin network as a provisional matrix to support cellular infiltration of the wound site, thus facilitating tissue repair and remodeling after hemostasis. In cases of traumatic injury or disease, platelets can become depleted and this process disrupted. To that end, our lab has developed synthetic platelet-like particles (PLPs) that recapitulate the clot retraction abilities of native platelets through a Brownian-wrench driven mechanism that drives fibrin network densification and clot retraction over time, however, this Brownian-motion driven process occurs on a longer time scale than native active actin/myosin-driven platelet-mediated clot retraction. We hypothesized that a combinatorial therapy comprised of ultrasound stimulation of PLP motion within fibrin clots would facilitate a faster induction of clot retraction on a more platelet-mimetic time scale and at a lower dosage than required for PLPs acting alone. We found that application of ultrasound in combination with a subtherapeutic dosage of PLPs resulted in increased clot density and stiffness, improved fibroblast migration and increased epidermal thickness and angiogenesis , indicating that this combination therapy has potential to facilitate multiphase pro-healing outcomes. Additionally, while these particular studies focus on the role of ultrasound in enhancing specific interactions between fibrin-binding synthetic PLPs embedded within fibrin networks, these studies have wide applicability in understanding the role of ultrasound stimulation in enhancing multi-scale colloidal interactions within fibrillar matrices.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725264PMC
May 2020

Development of biomimetic antimicrobial platelet-like particles comprised of microgel nanogold composites.

Regen Eng Transl Med 2020 6;6:299-309. Epub 2019 Aug 6.

Joint Department of Biomedical Engineering at UNC-Chapel Hill and North Carolina State University.

A blood clot is formed in response to bleeding by platelet aggregation and adherence to fibrin fibers. Platelets contract over time, stabilizing the clot, which contributes to wound healing. We have developed platelet-like particles (PLPs) that augment clotting and induce clot retraction by mimicking the fibrin-binding capabilities and morphology of native platelets. Wound repair following hemostasis can be complicated by infection; therefore, we aim to augment wound healing by combining PLPs with antimicrobial gold to develop nanogold composites (NGCs). PLPs were synthesized with N-isopropylacrylamide (NIPAm)/co-acrylic acid in a precipitation polymerization reaction and conjugated to a fibrin-specific antibody. Two methods were employed to create NGCs: 1) noncovalent swelling with aqueous gold nanospheres, and 2) covalent seeding and growth. Since the ability of PLPs to mimic platelet morphology and clot retraction requires a high degree of particle deformability, we investigated how PLPs created from NGCs affected these properties. Cryogenic Scanning Electron Microscopy (cryoSEM) and atomic force microscopy (AFM) demonstrated that particle deformability, platelet-mimetic morphology and clot retraction were maintained in NGC-based PLPs. The effect of NGCs on bacterial adhesion and growth was assessed with antimicrobial assays. These results demonstrate NGCs fabricated through noncovalent and covalent methods retain deformability necessary for clot collapse and exhibit some antimicrobial potential. Therefore, NGCs are promising materials for preventing hemorrhage and infection following trauma.
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http://dx.doi.org/10.1007/s40883-019-00121-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678143PMC
August 2019

Neonatal Fibrin Scaffolds Promote Enhanced Cell Adhesion, Migration, and Wound Healing Compared to Adult Fibrin Scaffolds.

Cell Mol Bioeng 2020 Oct 27;13(5):393-404. Epub 2020 May 27.

Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, NC 27695 USA.

Introduction: Fibrin scaffolds are often utilized to treat chronic wounds. The monomer fibrinogen used to create such scaffolds is typically derived from adult human or porcine plasma. However, our previous studies have identified extensive differences in fibrin network properties between adults and neonates, including higher fiber alignment in neonatal networks. Wound healing outcomes have been linked to fibrin matrix structure, including fiber alignment, which can affect the binding and migration of cells. We hypothesized that fibrin scaffolds derived from neonatal fibrin would enhance wound healing outcomes compared to adult fibrin scaffolds.

Methods: Fibrin scaffolds were formed from purified adult or neonatal fibrinogen and thrombin then structural analysis was conducted confocal microscopy. Human neonatal dermal fibroblast attachment, migration, and morphology on fibrin scaffolds were assessed. A murine full thickness injury model was used to compare healing in the presence of neonatal fibrin, adult fibrin, or saline.

Results: Distinct fibrin architectures were observed between adult and neonatal scaffolds. Significantly higher fibroblast attachment and migration was observed on neonatal scaffolds compared to adults. Cell morphology on neonatal scaffolds exhibited higher spreading compared to adult scaffolds. significantly smaller wound areas and greater epidermal thickness were observed when wounds were treated with neonatal fibrin compared to adult fibrin or a saline control.

Conclusions: Distinctions in neonatal and adult fibrin scaffold properties influence cellular behavior and wound healing. These studies indicate that fibrin scaffolds sourced from neonatal plasma could improve healing outcomes compared to scaffolds sourced from adult plasma.
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http://dx.doi.org/10.1007/s12195-020-00620-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596151PMC
October 2020

Microphysiological systems for the modeling of wound healing and evaluation of pro-healing therapies.

J Mater Chem B 2020 08;8(32):7062-7075

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina, Chapel Hill, 911 Oval Dr., Raleigh, NC 27695, USA. and Comparative Medicine Institute, North Carolina State University, 1060 William Moore Dr., Raleigh, NC 27606, USA and Department of Electrical & Computer Engineering, North Carolina State University, 890 Oval Dr., Raleigh, NC 27695, USA.

Wound healing is a multivariate process involving the coordinated response of numerous proteins and cell types. Accordingly, biomedical research has seen an increased adoption of the use of in vitro wound healing assays with complexity beyond that offered by traditional well-plate constructs. These microphysiological systems (MPS) seek to recapitulate one or more physiological features of the in vivo microenvironment, while retaining the analytical capacity of more reductionist assays. Design efforts to achieve relevant wound healing physiology include the use of dynamic perfusion over static culture, the incorporation of multiple cell types, the arrangement of cells in three dimensions, the addition of biomechanically and biochemically relevant hydrogels, and combinations thereof. This review provides a brief overview of the wound healing process and in vivo assays, and we critically review the current state of MPS and supporting technologies for modelling and studying wound healing. We distinguish between MPS that seek to inform a particular phase of wound healing, and constructs that have the potential to inform multiple phases of wound healing. This distinction is a product of whether analysis of a particular process is prioritized, or a particular physiology is prioritized, during design. Material selection is emphasized throughout, and relevant fabrication techniques discussed.
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http://dx.doi.org/10.1039/d0tb00544dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460719PMC
August 2020

Platelet-like particles improve fibrin network properties in a hemophilic model of provisional matrix structural defects.

J Colloid Interface Sci 2020 Oct 26;577:406-418. Epub 2020 May 26.

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC, United States; Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States. Electronic address:

Following injury, a fibrin-rich provisional matrix is formed to stem blood loss and provide a scaffold for infiltrating cells, which rebuild the damaged tissue. Defects in fibrin network formation contribute to impaired healing outcomes, as evidenced in hemophilia. Platelet-fibrin interactions greatly influence fibrin network structure via clot contraction, which increases fibrin density over time. Previously developed hemostatic platelet-like particles (PLPs) are capable of mimicking platelet functions including binding to fibrin fibers, augmenting clotting, and inducing clot retraction. In this study, we aimed to apply PLPs within a plasma-based in vitro hemophilia B model of deficient fibrin network structure to determine the ability of PLPs to improve fibrin structure and wound healing responses within hemophilia-like abnormal fibrin network formation. PLP impact on structurally deficient clot networks was assessed via confocal microscopy, a micropost deflection model, atomic force microscopy and an in vitro wound healing model of early cell migration within a provisional fibrin matrix. PLPs improved clot network density, force generation, and stiffness, and promoted fibroblast migration within an in vitro model of early wound healing under hemophilic conditions, indicating that PLPs could provide a biomimetic platform for improving wound healing events in disease conditions that cause deficient fibrin network formation.
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http://dx.doi.org/10.1016/j.jcis.2020.05.088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415593PMC
October 2020

Nanosilver composite pNIPAm microgels for the development of antimicrobial platelet-like particles.

J Biomed Mater Res B Appl Biomater 2020 08 26;108(6):2599-2609. Epub 2020 Feb 26.

Joint Department of Biomedical Engineering at the University of North Carolina, Chapel Hill and North Carolina State University, Raleigh, North Carolina.

Platelets crucially facilitate wound healing but can become depleted in traumatic injury or chronic wounds. Previously, our group developed injectable platelet-like particles (PLPs) comprised of highly deformable, ultralow crosslinked pNIPAm microgels (ULCs) coupled to fibrin binding antibodies to treat post-trauma bleeding. PLP fibrin-binding facilitates homing to sites of injury, promotes clot formation, and, due to high particle deformability, induces clot retraction. Clot retraction augments healing by increasing clot stability, enhancing clot stiffness, and promoting cell migration into the wound bed. Because post-traumatic healing is often complicated by infection, the objective of these studies was to develop antimicrobial nanosilver microgel composite PLPs to augment hemostasis, fight infection, and promote healing post-trauma. A key goal was to maintain particle deformability following silver incorporation to preserve PLP-mediated clot retraction. Clot retraction, antimicrobial activity, hemostasis after trauma, and healing after injury were evaluated via confocal microscopy, colony-forming unit assays, a murine liver trauma model, and a murine full-thickness injury model in the absence or presence of infection, respectively. We found that nanosilver incorporation does not affect base PLP performance while bestowing significant antimicrobial activity and enhancing infected wound healing outcomes. Therefore, Ag-PLPs have great promise for treating hemorrhage and improving healing following trauma.
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http://dx.doi.org/10.1002/jbm.b.34592DOI Listing
August 2020

Comparison of Neonatal and Adult Fibrin Clot Properties between Porcine and Human Plasma.

Anesthesiology 2020 05;132(5):1091-1101

From the Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, North Carolina (K.A.N., S.N., A.K., S.S., A.C.B.) the Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina (K.A.N., A.C.B.) the Department of Anesthesiology, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia (N.A.G.).

Background: Recent studies suggest that adult-specific treatment options for fibrinogen replacement during bleeding may be less effective in neonates. This is likely due to structural and functional differences found in the fibrin network between adults and neonates. In this investigation, the authors performed a comparative laboratory-based study between immature and adult human and porcine plasma samples in order to determine if piglets are an appropriate animal model of neonatal coagulopathy.

Methods: Adult and neonatal human and porcine plasma samples were collected from the Children's Hospital of Atlanta and North Carolina State University College of Veterinary Medicine, respectively. Clots were formed for analysis and fibrinogen concentration was quantified. Structure was examined through confocal microscopy and cryogenic scanning electron microscopy. Function was assessed through atomic force microscopy nanoindentation and clotting and fibrinolysis assays. Lastly, novel hemostatic therapies were applied to neonatal porcine samples to simulate treatment.

Results: All sample groups had similar plasma fibrinogen concentrations. Neonatal porcine and human plasma clots were less branched with lower fiber densities than the dense and highly branched networks seen in adult human and porcine clots. Neonatal porcine and human clots had faster degradation rates and lower clot stiffness values than adult clots (stiffness [mmHg] mean ± SD: neonatal human, 12.15 ± 1.35 mmHg vs. adult human, 32.25 ± 7.13 mmHg; P = 0.016; neonatal pig, 10.5 ± 8.25 mmHg vs. adult pigs, 32.55 ± 7.20 mmHg; P = 0.015). The addition of hemostatic therapies to neonatal porcine samples enhanced clot formation.

Conclusions: The authors identified similar age-related patterns in structure, mechanical, and degradation properties between adults and neonates in porcine and human samples. These findings suggest that piglets are an appropriate preclinical model of neonatal coagulopathy. The authors also show the feasibility of in vitro model application through analysis of novel hemostatic therapies as applied to dilute neonatal porcine plasma.
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http://dx.doi.org/10.1097/ALN.0000000000003165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460720PMC
May 2020

Biomimetic antimicrobial material strategies for combating antibiotic resistant bacteria.

Biomater Sci 2020 Feb 28;8(4):1089-1100. Epub 2019 Nov 28.

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina - Chapel Hill, 1001 William Moore Dr., Raleigh, NC 27606, USA. and Comparative Medicine Institute, North Carolina State University, 1001 William Moore Dr., Raleigh, NC 27606, USA.

Antibiotic drugs have revolutionized the field of medicine for almost 90 years. However, continued use has led to the rise of antibiotic resistant bacteria, motivating the need for alternative treatments. Several strategies to combat this phenomenon have been investigated, with biomimetic strategies gaining significant appeal due to inherent compatibility with physiologically relevant environments. In this review, we will discuss current antimicrobial strategies and then present an overview on biomimetic antimicrobial material-based strategies for combating antibiotic resistant bacteria.
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http://dx.doi.org/10.1039/c9bm01393hDOI Listing
February 2020

Clot Structure and Implications for Bleeding and Thrombosis.

Semin Thromb Hemost 2020 Feb 15;46(1):96-104. Epub 2019 Oct 15.

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, Raleigh, North Carolina.

The formation of a fibrin clot matrix plays a critical role in promoting hemostasis and wound healing. Fibrin dynamics can become disadvantageous in the formation of aberrant thrombus development. Structural characteristics of clots, such as fiber diameter, clot density, stiffness, or permeability, can determine overall clot integrity and functional characteristics that have implications on coagulation and fibrinolysis. This review examines known factors that contribute to changes in clot structure and the presence of structural clot changes in various disease states. These insights provide valuable information in forming therapeutic strategies for disease states where alterations in clot structure are observed. Additionally, the implications of structural changes in clot networks on bleeding and thrombus development in terms of disease states and clinical outcomes are also examined in this review.
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http://dx.doi.org/10.1055/s-0039-1696944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460717PMC
February 2020

Mechanical properties of tissue formed in vivo are affected by 3D-bioplotted scaffold microarchitecture and correlate with ECM collagen fiber alignment.

Connect Tissue Res 2020 03 26;61(2):190-204. Epub 2019 Jul 26.

Edward P. Fitts Department of Industrial and Systems Engineering, North Carolina State University, Raleigh, NC, USA.

: Musculoskeletal soft tissues possess highly aligned extracellular collagenous networks that provide structure and strength. Such an organization dictates tissue-specific mechanical properties but can be difficult to replicate by engineered biological substitutes. Nanofibrous electrospun scaffolds have demonstrated the ability to control cell-secreted collagen alignment, but concerns exist regarding their scalability for larger and anatomically relevant applications. Additive manufacturing processes, such as melt extrusion-based 3D-Bioplotting, allow fabrication of structurally relevant scaffolds featuring highly controllable porous microarchitectures.: In this study, we investigate the effects of 3D-bioplotted scaffold design on the compressive elastic modulus of neotissue formed in vivo in a subcutaneous rat model and its correlation with the alignment of ECM collagen fibers. Polycaprolactone scaffolds featuring either 100 or 400 µm interstrand spacing were implanted for 4 or 12 weeks, harvested, cryosectioned, and characterized using atomic-force-microscopy-based force mapping.: The compressive elastic modulus of the neotissue formed within the 100 µm design was significantly higher at 4 weeks (p < 0.05), but no differences were observed at 12 weeks. In general, the tissue stiffness was within the same order of magnitude and range of values measured in native musculoskeletal soft tissues including the porcine meniscus and anterior cruciate ligament. Finally, a significant positive correlation was noted between tissue stiffness and the degree of ECM collagen fiber alignment (p < 0.05) resulting from contact guidance provided by scaffold strands.: These findings demonstrate the significant effects of 3D-bioplotted scaffold microarchitectures in the organization and sub-tissue-level mechanical properties of ECM in vivo.
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http://dx.doi.org/10.1080/03008207.2019.1624733DOI Listing
March 2020

Maintaining Hemostatic Balance in Treating Disseminated Intravascular Coagulation.

Anesthesiology 2019 Sep;131(3):459-461

From the Joint Department of Biomedical Engineering and the Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, (A.C.B.) the Duke University School of Medicine, Department of Anesthesiology and Critical Care, Durham, North Carolina (J.H.L.).

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http://dx.doi.org/10.1097/ALN.0000000000002862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692189PMC
September 2019

Biomimetic Strategies To Treat Traumatic Brain Injury by Leveraging Fibrinogen.

Bioconjug Chem 2019 07 5;30(7):1951-1956. Epub 2019 Jul 5.

School of Biological and Health Systems Engineering , Arizona State University , Tempe , Arizona 85287 , United States.

There were over 27 million new cases of traumatic brain injuries (TBIs) in 2016 across the globe. TBIs are often part of complicated trauma scenarios and may not be diagnosed initially because the primary clinical focus is on stabilizing the patient. Interventions used to stabilize trauma patients may inadvertently impact the outcomes of TBIs. Recently, there has been a strong interest in the trauma community toward administrating fibrinogen-containing solutions intravenously to help stabilize trauma patients. While this interventional shift may benefit general trauma scenarios, fibrinogen is associated with potentially deleterious effects for TBIs. Here, we deconstruct what components of fibrinogen may be beneficial as well as potentially harmful following TBI and extrapolate this to biomimetic approaches to treat bleeding and trauma that may also lead to better outcomes following TBI.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824581PMC
July 2019

Platelet-like particles dynamically stiffen fibrin matrices and improve wound healing outcomes.

Biomater Sci 2019 Jan;7(2):669-682

Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, NC 27695, USA.

Native platelets perform several critical functions within the context of wound healing, including participating in initial hemostasis and interacting with fibrin at the wound site to induce clot retraction. Platelet depletion or dysfunction due to trauma or disease can inhibit robust wound healing responses. There has been a focus recently on developing synthetic, non-immunogenic platelet mimetic technologies for the purpose of augmenting hemostatic responses in cases of deficient native platelet functionality. Here we describe the application of synthetic platelet-like particles (PLPs), capable of recapitulating the deformable platelet body and fibrin specificity found in native platelets, to enhance healing outcomes. We first demonstrate PLPs mimic activated platelet morphology and induce fibrin clot retraction. During clot retraction, native platelets generate forces within a fibrin network to stiffen the fibrin matrix; therefore, we hypothesized that our PLPs will likewise be able to stiffen provisional fibrin matrices. Due to previous studies indicating that increased matrix stiffness is linked to increased cellular migration, we further hypothesize that PLP-mediated fibrin stiffening will enhance cell migration and improve healing outcomes within in vitro and in vivo models of wound healing. PLPs were found to enhance fibroblast migration in in vitro models of early wound healing and enhance healing outcomes in an in vivo murine model of wound healing. These studies demonstrate the utility of PLPs for enhancing wound repair and also provide insight into the role of native platelet-mediated clot retraction in wound healing.
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http://dx.doi.org/10.1039/c8bm01201fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385160PMC
January 2019

Fibrin Nanoparticles Coupled with Keratinocyte Growth Factor Enhance the Dermal Wound-Healing Rate.

ACS Appl Mater Interfaces 2019 Jan 15;11(4):3771-3780. Epub 2019 Jan 15.

Joint Department of Biomedical Engineering , North Carolina State University and University of North Carolina at Chapel Hill , Raleigh 27695 , North Carolina , United States.

Expediting the wound-healing process is critical for patients chronically ill from nonhealing wounds and diseases such as hemophilia or diabetes or who have suffered trauma including easily infected open wounds. FDA-approved external tissue sealants include the topical application of fibrin gels, which can be 500 times denser than natural fibrin clots. With lower clot porosity and higher polymerization rates than physiologically formed fibrin clots, the commercial gels quickly stop blood loss but impede the later clot degradation kinetics and thus retard tissue-healing rates. The fibrin nanoparticles (FBNs) described here are constructed from physiologically relevant fibrin concentrations that support new tissue and dermal wound scaffold formation when coupled with growth factors. The FBNs, synthesized in a microfluidic droplet generator, support cell adhesion and traction generation, and when coupled to keratinocyte growth factor (KGF), support cell migration and in vivo wound healing. The FBN-KGF particles enhance cell migration in vitro greater than FBN alone or free KGF and also improve healing outcomes in a murine full thickness injury model compared to saline, bulk fibrin sealant, free KGF, or bulk fibrin mixed with KGF treatments. Furthermore, FBN can be potentially administered with other tissue-healing factors and inflammatory mediators to improve wound-healing outcomes.
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http://dx.doi.org/10.1021/acsami.8b21056DOI Listing
January 2019

Viscoelastic properties of microgel thin films control fibroblast modes of migration and pro-fibrotic responses.

Biomaterials 2018 12 14;185:371-382. Epub 2018 Sep 14.

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel-Hill, Raleigh, NC, USA; Comparative Medical Institute, North Carolina State University, Raleigh, NC, USA. Electronic address:

Cell behavior is influenced by the biophysical properties of their microenvironments, and the linear elastic properties of substrates strongly influences adhesion, migration, and differentiation responses. Because most biological tissues exhibit non-linear elastic properties, there is a growing interest in understanding how the viscous component of materials and tissues influences cell fate. Here we describe the use of microgel thin films with controllable non-linear elastic properties for investigating the role of material loss tangent on cell adhesion, migration, and myofibroblastic differentiation, which have implications in fibrotic responses. Fibroblast modes of migration are dictated by film loss tangent; high loss tangent induced ROCK-mediated amoeboid migration while low loss tangent induced Rac-mediated mesenchymal cell migration. Low loss tangent films were also associated with higher levels of myofibroblastic differentiation. These findings have implications in fibrosis and indicate that slight changes in tissue viscoelasticity following injury could contribute to early initiation of fibrotic related responses.
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http://dx.doi.org/10.1016/j.biomaterials.2018.09.012DOI Listing
December 2018

Biomimetic microgels with controllable deformability improve healing outcomes.

Adv Biosyst 2018 Oct 14;2(10). Epub 2018 Aug 14.

Joint Department of Biomedical Engineering, North Carolina State University and the University of North Carolina-Chapel Hill, Raleigh, NC USA.

Platelets mediate hemostasis by aggregating and binding to fibrin to promote clotting. Over time, platelets contract the fibrin network to induce clot retraction, which contributes to wound healing outcomes by increasing clot stability and improving blood flow to ischemic tissue. In this study, we describe the development of hollow platelet-like particles (PLPs) that mimic the native platelet function of clot retraction in a controlled manner and demonstrate that clot retraction-inducing PLPs promote healing . PLPs are created by coupling fibrin-binding antibodies to CoreShell (CS) or hollow N-isopropylacrylamide (NIPAm) microgels with varying degrees of shell crosslinking. We demonstrate that hollow microgels with loosely crosslinked shells display a high degree of deformability and mimic activated platelet morphology, while intact CS microgels and hollow microgels with increased crosslinking in the shell do not. When coupled to a fibrin-binding antibody to create PLPs, hollow particles with low degrees of shell crosslinking cause fibrin clot collapse recapitulating the clot retraction function of platelets, while other particle types do not. Furthermore, hollow PLPs with low degrees of shell crosslinking improve some wound healing outcomes .
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http://dx.doi.org/10.1002/adbi.201800042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869964PMC
October 2018

Interdigitated microelectronic bandage augments hemostasis and clot formation at low applied voltage in vitro and in vivo.

Lab Chip 2018 09;18(19):2985-2993

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Hemorrhage or uncontrolled bleeding can arise either due to a medical condition or from a traumatic injury and are typically controlled with the application of a hemostatic agent. Hemostatic agents are currently derived from animal or human products, which carry risks of blood borne infections and immune dysregulation. Therefore, the need exists for novel biomedical therapies not derived from animal or human products to achieve hemostasis. Accordingly, we created an interdigitated microelectronic bandage that applies low voltage electrical stimulation to an injury site, resulting in faster clot formation without excessive heating, accelerated fibrin formation, and hemostasis overall. Our interdigitated microelectronic bandage found fibrin formed 1.5× faster in vitro. In vivo, total cessation of bleeding was 2.5× faster, resulting in 2× less blood loss. Electricity has been used in medical applications such as defibrillation, cauterization, and electrosurgery, but scant research has focused on hemostasis. Here we report a novel surface treatment using an interdigitated microelectronic device that creates rapid hemostasis in both in vitro and in vivo bleeding models with low applied voltages, representing a new and novel class of hemostatic agents that are electrically-based.
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http://dx.doi.org/10.1039/c8lc00573gDOI Listing
September 2018

Targeted Treatment of Ischemic and Fibrotic Complications of Myocardial Infarction Using a Dual-Delivery Microgel Therapeutic.

ACS Nano 2018 08 25;12(8):7826-7837. Epub 2018 Jul 25.

Myocardial infarction (MI), commonly known as a heart attack, affects millions of people worldwide and results in significant death and disabilities. A major cause of MI is fibrin-rich thrombus formation that occludes the coronary arteries, blocking blood flow to the heart and causing fibrin deposition. In treating MI, re-establishing blood flow is critical. However, ischemia reperfusion (I/R) injury itself can also occur and contributes to cardiac fibrosis. Fibrin-specific poly( N-isopropylacrylamide) nanogels (FSNs) comprised of a core-shell colloidal hydrogel architecture are utilized in this study to design a dual-delivery system that simultaneously addresses the need to (1) re-establish blood flow and (2) inhibit cardiac fibrosis following I/R injury. These therapeutic needs are met by controlling the release of a fibrinolytic protein, tissue plasminogen activator (tPA), and a small molecule cell contractility inhibitor (Y-27632). In vitro, tPA and Y-27632-loaded FSNs rapidly degrade fibrin and decrease cardiac cell stress fiber formation and connective tissue growth factor expression, which are both upregulated in cardiac fibrosis. In vivo, FSNs localize to fibrin in injured heart tissue and, when loaded with tPA and Y-27632, showed significant improvement in left ventricular ejection fraction 2 and 4 weeks post-I/R as well as significantly decreased infarct size, α-smooth muscle actin expression, and connective tissue growth factor expression 4 weeks post-I/R. Together, these data demonstrate the feasibility of this targeted therapeutic strategy to improve cardiac function following MI.
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http://dx.doi.org/10.1021/acsnano.8b01977DOI Listing
August 2018

Controlling Fibrin Network Morphology, Polymerization, and Degradation Dynamics in Fibrin Gels for Promoting Tissue Repair.

Methods Mol Biol 2018 ;1758:85-99

Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Raleigh, NC, USA.

Fibrin is an integral part of the clotting cascade and is formed by polymerization of the soluble plasma protein fibrinogen. Following stimulation of the coagulation cascade, thrombin activates fibrinogen, which binds to adjacent fibrin(ogen) molecules resulting in the formation of an insoluble fibrin matrix. This fibrin network is the primary protein component in clots and subsequently provides a scaffold for infiltrating cells during tissue repair. Due to its role in hemostasis and tissue repair, fibrin has been used extensively as a tissue sealant. Clinically used fibrin tissue sealants require supraphysiological concentrations of fibrinogen and thrombin to achieve fast polymerization kinetics, which results in extremely dense fibrin networks that are inhibitory to cell infiltration. Therefore, there is much interest in developing fibrin-modifying strategies to achieve rapid polymerization dynamics while maintaining a network structure that promotes cell infiltration. The properties of fibrin-based materials can be finely controlled through techniques that modulate fibrin polymerization dynamics or through the inclusion of fibrin-modifying biomaterials. Here, we describe methods for characterizing fibrin network morphology, polymerization, and degradation (fibrinolysis) dynamics in fibrin constructs for achieving fast polymerization dynamics while promoting cell infiltration.
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http://dx.doi.org/10.1007/978-1-4939-7741-3_7DOI Listing
January 2019

Material Strategies for Modulating Epithelial to Mesenchymal Transitions.

ACS Biomater Sci Eng 2018 Apr 19;4(4):1149-1161. Epub 2017 Apr 19.

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, Raleigh, North Carolina 27695, United States.

Epithelial to mesenchymal transitions (EMT) involve the phenotypic change of epithelial cells into fibroblast-like cells. This process is accompanied by the loss of cell-cell contacts, increased extracellular matrix (ECM) production, stress fiber alignment, and an increase in cell mobility. While essential for development and wound repair, EMT has also been recognized as a contributing factor to fibrotic diseases and cancer. Both chemical and mechanical cues, such as tumor necrosis factor alpha, NF-κB, Wnt, Notch, interleukin-8, metalloproteinase-3, ECM proteins, and ECM stiffness can determine the degree and duration of EMT events. Additionally, transforming growth factor beta is a primary driver of EMT and, interestingly, can be activated through cell-mediated mechanoactivation. In this review, we highlight recent findings demonstrating the contribution of mechanical stimuli, such as tissue and material stiffness, in driving EMT. We then highlight material strategies for controlling EMT events. Finally, we discuss drivers of the similar process of endothelial to mesenchymal transition (EndoMT) and corresponding material strategies for controlling EndoMT.
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http://dx.doi.org/10.1021/acsbiomaterials.6b00751DOI Listing
April 2018

Enhancing clot properties through fibrin-specific self-cross-linked PEG side-chain microgels.

Colloids Surf B Biointerfaces 2018 Jun 2;166:89-97. Epub 2018 Mar 2.

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA; Schmid College of Science and Technology, Chapman University, Orange, CA 92866, USA. Electronic address:

Excessive bleeding and resulting complications are a major cause of death in both trauma and surgical settings. Recently, there have been a number of investigations into the design of synthetic hemostatic agents with platelet-mimicking activity to effectively treat patients suffering from severe hemorrhage. We developed platelet-like particles from microgels composed of polymers carrying polyethylene glycol (PEG) side-chains and fibrin-targeting single domain variable fragment antibodies (PEG-PLPs). Comparable to natural platelets, PEG-PLPs were found to enhance the fibrin network formation in vitro through strong adhesion to the emerging fibrin clot and physical, non-covalent cross-linking of nascent fibrin fibers. Furthermore, the mechanical reinforcement of the fibrin mesh through the incorporation of particles into the network leads to a ∼three-fold decrease of the overall clot permeability as compared to control clots. However, transport of biomolecules through the fibrin clots, such as peptides and larger proteins is not hindered by the presence of PEG-PLPs and the altered microstructure. Compared to control clots with an elastic modulus of 460+/-260 Pa, PEG-PLP-reinforced fibrin clots exhibit higher degrees of stiffness as demonstrated by the significantly increased average Younǵs modulus of 1770 +/±720 Pa, as measured by AFM force spectroscopy. Furthermore, in vitro degradation studies with plasmin demonstrate that fibrin clots formed in presence of PEG-PLPs withstand hydrolysis for 24 h, indicating enhanced stabilization against exogenous fibrinolysis. The entire set of data suggests that the designed platelet-like particles have high potential for use as hemostatic agents in emergency medicine and surgical settings.
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http://dx.doi.org/10.1016/j.colsurfb.2018.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050065PMC
June 2018
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