Publications by authors named "Kimberly Nellenbach"

13 Publications

  • Page 1 of 1

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 Apr 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
April 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
March 2021

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

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

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

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

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

Peptide Mimetic Drugs for Modulating Thrombosis and Hemostasis.

Drug Dev Res 2017 09 16;78(6):236-244. Epub 2017 Aug 16.

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel-Hill, Raleigh, North Carolina, 27606.

Preclinical Research Hemostasis is the complex physiological process that stems bleeding at an injury site while simultaneously maintaining unobstructed circulation in other areas of the body. This system is kept in balance with finely tuned regulation by pro- and antithrombotic agents. When this balance is thrown out of equilibrium, uncontrolled bleeding, or thrombotic complications can occur. Because of the high number of hemostatic disorders, researchers are continually searching for improved technologies for controlling coagulation. Recently, peptide mimetic strategies have been employed to target and regulate various stages of the coagulation cascade. In this review, we present an overview of the coagulation cascade and provide a summary of various peptide-mimetic approaches for its modulation. Drug Dev Res 78 : 236-244, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ddr.21407DOI Listing
September 2017