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Myocardial infarction, stroke, and pulmonary embolism are all deadly conditions associated with excessive thrombus formation. Standard treatment for these conditions involves systemic delivery of thrombolytic agents to break up clots and restore blood flow; however, this treatment can impact the hemostatic balance in other parts of the vasculature, which can lead to excessive bleeding. To avoid this potential danger, targeted thrombolytic treatments that can successfully target thrombi and release an effective therapeutic load are necessary. Because activated platelets and fibrin make up a large proportion of clots, these two components provide ample opportunities for targeting. This review will highlight potential thrombus targeting mechanisms as well as recent advances in thrombolytic therapies which utilize blood cells and clotting proteins to effectively target and lyse clots.

To minimize exposure of healthcare workers and clinical laboratory personnel to infectious liquids, we designed biofluid manipulators for on-demand handling of liquid droplets, in-plane or out-of-plane, in a remote and lossless manner.

Respiratory diseases are a global burden, with millions of deaths attributed to pulmonary illnesses and dysfunctions. Therapeutics have been developed, but they present major limitations regarding pulmonary bioavailability and product stability. To circumvent such limitations, we developed room-temperature-stable inhalable lung-derived extracellular vesicles or exosomes (Lung-Exos) as mRNA and protein drug carriers. Compared with standard synthetic nanoparticle liposomes (Lipos), Lung-Exos exhibited superior distribution to the bronchioles and parenchyma and are deliverable to the lungs of rodents and nonhuman primates (NHPs) by dry powder inhalation. In a vaccine application, severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein encoding mRNA-loaded Lung-Exos (S-Exos) elicited greater immunoglobulin G (IgG) and secretory IgA (SIgA) responses than its loaded liposome (S-Lipo) counterpart. Importantly, S-Exos remained functional at room-temperature storage for one month. Our results suggest that extracellular vesicles can serve as an inhaled mRNA drug-delivery system that is superior to synthetic liposomes.

Neonates possess a molecular variant of fibrinogen, known as fetal fibrinogen, characterized by increased sialic acid, a greater negative charge, and decreased activity compared with adults. Despite these differences, adult fibrinogen is used for the treatment of bleeding in neonates, with mixed efficacy. To determine safe and efficacious bleeding protocols for neonates, more information on neonatal fibrin clot formation and the influence of sialic acid on these processes is needed. Here, we examine the influence of sialic acid on neonatal fibrin polymerization. We hypothesized that the increased sialic acid content of neonatal fibrinogen promotes fibrin B:b knob-hole interactions and consequently influences the structure and function of the neonatal fibrin matrix. We explored this hypothesis through analysis of structural properties and knob:hole polymerization dynamics of normal and desialylated neonatal fibrin networks and compared them with those formed with adult fibrinogen. We then characterized normal neonatal fibrin knob:hole interactions by forming neonatal and adult clots with either thrombin or snake-venom thrombin-like enzymes that preferentially cleave fibrinopeptide A or B. Sialic acid content of neonatal fibrinogen was determined to be a key determinant of resulting clot properties. Experiments analyzing knob:hole dynamics indicated that typical neonatal fibrin clots are formed with the release of more fibrinopeptide B and less fibrinopeptide A than adults. After the removal of sialic acid, fibrinopeptide release was roughly equivalent between adults and neonates, indicating the influence of sialic acid on fibrin neonatal fibrin polymerization mechanisms. These results could inform future studies developing neonatal-specific treatments of bleeding.

A hernia is a pathological condition caused by a defect or opening in the muscle wall, which leads to organs pushing through the opening or defect. Hernia recurrence, seroma, persistent pain, tissue adhesions, and wound infection are common complications following hernia repair surgery. Infection after hernia mesh implantation is the third major complication leading to hernia recurrence. In order to reduce the incidence of late infections, we developed a polypropylene mesh with antibacterial properties. In this study, knitted polypropylene meshes were exposed to radio-frequency plasma to activate their surfaces. The antibacterial monomer diallyldimethylammonium chloride (DADMAC) was then grafted onto the mesh surface using pentaerythritol tetraacrylate as the cross-linker since it is able to engage all four functional groups to form a high-density cross-linked network. The subsequent antibacterial performance showed a 2.9 log reduction toward

Three-dimensional plotting of biomaterials (also known as bioprinting) has been a major milestone for scientists and engineers working in nanobiotechnology, nanoscience, and nanomedicine. It is typically classified into two major categories, depending on the plotting principle, as contact and noncontact techniques. This article focuses on the working principles of contact and noncontact printing methods along with their advantages, disadvantages, applications, and challenges. Contact printing methods include micro-plotter, pen printing, screen printing, nanoimprint printing, flexography printing, and gravure printing. Noncontact printing methods include extrusion printing, droplet printing, laser-based polymerization, and laser-based cell transfer. The wide variety of printable biomaterials, such as DNA, peptides, proteins, lipids, and cells, also are discussed.

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.

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 via 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 vivo 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. In vivo 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-020-00620-5) contains supplementary material, which is available to authorized users.

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 in vitro and increased epidermal thickness and angiogenesis in vivo, 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.

Uncontrolled bleeding is a major problem in trauma and emergency medicine. While materials for trauma applications would certainly find utility in traditional surgical settings, the unique environment of emergency medicine introduces additional design considerations, including the need for materials that are easily deployed in austere environments. Ideally, these materials would be available off the shelf, could be easily transported, and would be able to be stored at room temperature for some amount of time. Both natural and synthetic materials have been explored for the development of hemostatic materials. This review article provides an overview of classes of materials used for topical hemostats and newer developments in the area of injectable hemostats for use in emergency medicine.

Cells maintain tensional homeostasis by monitoring the mechanics of their microenvironment. In order to understand this mechanotransduction phenomenon, hydrogel materials have been developed with either controllable linear elastic or viscoelastic properties. Native biological tissues, and biomaterials used for medical purposes, often have complex mechanical properties. However, due to the difficulty in completely decoupling the elastic and viscous components of hydrogel materials, the effect of complex composite materials on cellular responses has largely gone unreported. Here, we characterize a novel composite hydrogel system capable of decoupling and individually controlling both the bulk stiffness and surface viscoelasticity of the material by combining polyacrylamide (PA) gels with microgel thin films. By taking advantage of the high degree of control over stiffness offered by PA gels and viscoelasticity, in terms of surface loss tangent, of microgel thin films, it is possible to study the influence that bulk substrate stiffness and surface loss tangent have on complex fibroblast responses, including cellular and nuclear morphology and gene expression. This material system provides a facile method for investigating cellular responses to complex material mechanics with great precision and allows for a greater understanding of cellular mechanotransduction mechanisms than previously possible through current model material platforms.

To assure the long-term safety and functional performance after implantation, it is of critical importance to completely sterilize a biomaterial implant. Ineffective sterilization can cause severe inflammation and infection at the implant site, leading to detrimental events of morbidity and even mortality. Macrophages are pivotal players in the inflammatory and foreign body response after implanting a biomaterial in the body. However, the relationship between the sterilization procedure and macrophage response has not been established. In this study, three commonly used sterilization methods, including autoclaving, ethylene oxide gas and ethanol treatment, were used to sterilize a gelatin methacryloyl hydrogel. The impacts of different sterilization methods on the structure and physical properties of the hydrogel were compared. Macrophage responses to the sterilized hydrogel were analyzed based on their morphology, viability and in vitro gene expression. It was found that the sterilization methods only marginally altered the hydrogel morphology, swelling behavior and elastic modulus, but significantly impacted macrophage gene expression within 48 h and over 7 d in vitro. Therefore, when selecting sterilization methods for GelMA hydrogel, not only the sterility and hydrogel properties, such as material destruction and degradation caused by temperature and moisture, should be taken into consideration, but also the cellular responses to the sterilized material which could be substantially different.

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research.

Alginate hydrogels are gaining traction for use in drug delivery, regenerative medicine, and as tissue engineered scaffolds due to their physiological gelation conditions, high tissue biocompatibility, and wide chemical versatility. Traditionally, alginate is decorated at the carboxyl group to carry drug payloads, peptides, or proteins. While low degrees of substitution do not cause noticeable mechanical changes, high degrees of substitution can cause significant losses to alginate properties including complete loss of calcium cross-linking. While most modifications used to decorate alginate deplete the carboxyl groups, we propose that alginate modifications that replenish the carboxyl groups could overcome the loss in gel integrity and mechanics. In this report, we demonstrate that restoring carboxyl groups during functionalization maintains calcium cross-links as well as hydrogel shear-thinning and self-healing properties. In addition, we demonstrate that alginate hydrogels modified to a high degree with azide modifications that restore the carboxyl groups have improved tissue retention at intramuscular injection sites and capture blood-circulating cyclooctynes better than alginate hydrogels modified with azide modifications that deplete the carboxyl groups. Taken together, alginate modifications that restore carboxyl groups could significantly improve alginate hydrogel mechanics for clinical applications. Statement of Significant : Chemical modification of hydrogels provides a powerful tool to regulate cellular adhesion, immune response, and biocompatibility with local tissues. Alginate, due to its biocompatibility and easy chemical modification, is being explored for tissue engineering and drug delivery. Unfortunately, modifying alginate to a high degree of substitution consumes carboxyl group, which are necessary for ionic gelation, leading to poor hydrogel crosslinking. We introduce alginate modifications that restore the alginate's carboxyl groups. We demonstrate that modifications that reintroduce carboxyl groups restore gelation and improve gel mechanics and tissue retention. In addition to contributing to a basic science understanding of hydrogel properties, we anticipate our approach will be useful to create tissue engineered scaffolds and drug delivery platforms.

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.

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.

Naturally-occurring surface topographies abound in nature and endow diverse properties, i.e., superhydrophobicity, adhesion, anti-fouling, self-cleaning, anti-glare, anti-bacterial, and many others. Researchers have attempted to replicate such topographies to create human-made surfaces with desired functionalities. For example, combining the surface topography with judicial chemical composition could provide an effective, non-toxic solution to combat non-specific biofouling. A systematic look at the effect of geometry, modulus, and chemistry on adhesion is warranted. In this work, we use a model system that comprises silica (SiOx) beads interacting with a substrate made of a commercial polydimethylsiloxane kit (PDMS, Sylgard 184) featuring a sinusoidal topography. To examine the impact of interactions on particle settlement, we functionalize the surfaces of both the PDMS substrate and the SiOx beads with polyacrylic acid (PAA) and polyethyleneimine (PEI), respectively. We also use the PDMS commercial kit coated with liquid glass (LG) to study the effect of the substrate modulus on particle settlement. Substrates with a higher aspect ratio (i.e., amplitude/periodicity) encourage adsorption of particles along the sides of the channel compared with substrates with lower aspect ratio. We employ colloidal probe microscopy to demonstrate the effect of interaction between the substrate and the particle. The interplay among the surface modulus, geometry, and interactions between the surface and the particle governs particle settlement on sinusoidally-corrugated substrates.

Targeted drug delivery for maintaining blood fluidity can reduce the risks associated with systemic anticoagulants that can lead to off-target bleeding. Recently, there has been much interest in targeted delivery of tissue-type plasminogen activator (tPA) for treating thrombotic complications. The work presented here characterizes a fibrin-specific nanogel (FSN) design for targeted delivery of tPA to treat thrombotic complications. Fibrin binding and clot degradation were characterized in vitro, and animal models of thrombosis were used to examine nanogel effects on coagulation parameters. In vitro assays showed tPA-FSNs attach to fibrin in a dose-dependent manner independent of tPA loading. In animal models of thrombosis, including an electrolytic injury to monitor clot properties in real time, and a lipopolysaccharide-induced disseminated intravascular coagulation (DIC) animal model, tPA-FSNs modulated fibrin/fibrinogen and platelet incorporation into clots and at optimized dosing could recover consumptive coagulopathy in DIC. Distribution of unloaded and tPA-loaded FSNs showed potential clearance of tPA-FSNs after 24 h, although unloaded FSNs may be retained at sites of fibrin deposits. Maximum tolerated dose studies showed tPA-FSNs have minimal toxicity up to 20 times the optimized therapeutic dose. Overall, these studies demonstrate the therapeutic efficacy of targeted fibrinolysis for systemic microthrombi and begin to evaluate key translational parameters for tPA-FSN therapeutics, including optimal tPA-FSN dosage in a DIC rodent model and safety of intravenous tPA-FSN therapeutics.

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.

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.

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. Following injury, a blood clot is formed by platelets aggregating and binding to fibrin fibers. Platelets contract over time, stabilizing the clot, which contributes to wound healing. We have developed PLPs that enhance clotting and stimulate 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 amplify wound healing by combining PLPs with antimicrobial gold to develop NGCs. These NGC PLPs mimic platelet morphology, generate clot retraction, demonstrate some antimicrobial potential, and are promising materials for preventing blood loss and infection following trauma. Future work will include exploring the application of these particles to treat hemorrhage and infection following traumatic injury.

Material scaffolds that mimic the structure, function, and bioactivity of native biological tissues are in constant development. Recently, material scaffolds composed of microgel particles have shown promise for applications ranging from bone regeneration to spheroid cell growth. Previous studies with poly N-isopropylacrylamide microgel scaffolds utilized a layer-by-layer (LBL) technique where individual, uniform microgel layers are built on top of each other resulting in a multilayer scaffold. However, this technique is limited in its applications due to the inability to control microscale deposition or patterning of multiple particle types within a microgel layer. In this study, an ultrasonic microplotting technique is used to address the limitations of LBL fabrication to create patterned microgel films. Printing parameters, such as bioink formulation, surface contact angle, and print head diameter, are optimized to identify the ideal parameters needed to successfully print microgel films. It was found that bioinks composed of 2 mg/mL of microgels and 20% polyethylene glycol by volume (v/v), on bovine serum albumin-coated glass, with a print head diameter of 50 μm resulted in the highest quality prints. Patterned films were created with a maximum resolution of 50 μm with the potential for finer resolutions to be achieved with alternative bioink compositions and printing parameters. Overall, ultrasonic microplotting can be used to create more complex microgel films than is possible with LBL techniques and offers the possibility of greater printing resolution in 3D with further technology development.

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 ssDNA and ssPNA (peptide nucleic acid) antidotes show restoration of 75% and 95% coagulation activity, respectively. To demonstrate potential for practical, long-term storage for clinical use, RNA origami was 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.

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.

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.

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.

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.

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.

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.

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.

Stem cell transplantation, as used clinically, suffers from low retention and engraftment of the transplanted cells. Inspired by the ability of platelets to recruit stem cells to sites of injury on blood vessels, we hypothesized that platelets might enhance the vascular delivery of cardiac stem cells (CSCs) to sites of myocardial infarction injury. Here, we show that CSCs with platelet nanovesicles fused onto their surface membranes express platelet surface markers that are associated with platelet adhesion to injury sites. We also find that the modified CSCs selectively bind collagen-coated surfaces and endothelium-denuded rat aortas, and that in rat and porcine models of acute myocardial infarction the modified CSCs increase retention in the heart and reduce infarct size. Platelet-nanovesicle-fused CSCs thus possess the natural targeting and repairing ability of their parental cell types. This stem cell manipulation approach is fast, straightforward and safe, does not require genetic alteration of the cells, and should be generalizable to multiple cell types.

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.

Introduction: Bleeding is a serious complication among neonates undergoing cardiopulmonary bypass (CPB) and it is linked to significant morbidity and mortality. Current standard of care treatment for bleeding after CPB focuses on the transfusion of adult blood products, including platelets and cryoprecipitate. However, prior work by Nellenbach et al. has demonstrated structural differences between neonatal and adult clotting components. Importantly, neonatal and adult fibrin do not fully integrate during clot formation which may contribute to ineffective clot formation and/or increased thrombotic risk following transfusion of adult cryoprecipitate to neonates. There has been increased interest in using human fibrinogen concentrate (HFC) in treating bleeding in the post-CPB neonate; however, HFC has not been validated in this population through evidence-based means. This study analyzed structural and degradation properties of post-CPB clots +/- the ex vivo addition of HFC and compared structural and degradation properties of post-CPB clots after the in vivo transfusion of HFC versus cryoprecipitate. Methods: Human neonatal plasma samples were collected from patients undergoing CPB at the Children's Hospital of Atlanta. For ex vivo studies, samples were taken at baseline, post-bypass, and post-transfusion of cryoprecipitate (n = 18 patients). Clots were formed for analysis from samples alone as well as post-bypass samples with the addition of 0.5 or 0.9 mg/mL HFC (RiaSTAP, CSL Behring) and structure was examined through confocal microscopy. Clot degradation was assessed through a microfluidic fibrinolysis assay. For in vivo studies, samples were taken at baseline, post-transfusion of cryoprecipitate or HFC, upon ICU arrival, and at 24 hours post-surgery (n = 36 patients). Clots were formed from samples and structure was examined through confocal microscopy. Clot degradation was assessed through a plate-based fibrinolysis assay. Results: In ex vivo studies, clot structural analysis demonstrated no significant differences in fiber density between samples collected at different time points (baseline = 0.541 ± 0.105, post-bypass = 0.431 ± 0.111, post-transfusion = 0.594 ± 0.170). The addition of 0.5 mg/mL or 0.9 mg/mL HFC to post-bypass samples led to a significant increase in fiber density (0.5 mg/mL HFC=0.654 ± 0.158, p=0.02; 0.9 mg/mL HFC= 0.797 ± 0.193, p Conclusion: We identify patterns in structural properties of clots formed after the transfusion of HFC that are consistent with successful hemostasis. However, caution is warranted regarding potentially thrombotic risks and should be carefully analyzed in future studies. Figure: Effect of Ex Vivo HFC Addition on Clot Structure and Degradation. (A) Representative confocal imaging of clots formed from different samples and HFC dosages (scale = 50 um). (B) Effect of HFC Addition on Clot Fiber Density. Addition of both 0.5 and 0.9 mg/mL HFC dosages to post-bypass sample result in statistically significant increases in fiber density compared to post-bypass samples. (C) Effect of HFC Addition on Clot Degradation Profiles. Addition of 0.9 mg/mL HFC to post-bypass sample leads to statistically significant slower fibrinolysis. Figure 1 Figure 1. Disclosures Brown: Selsym Biotech, Inc.: Other: Co-Founder and CEO. OffLabel Disclosure: RiaSTAP (human fibrinogen concentrate) is FDA approved for the treatment of congenital hypofibrinogenemia.

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.

Fibronectin (Fn) is an extracellular matrix protein that orchestrates complex cell adhesion and signaling through cell surface integrin receptors during tissue development, remodeling, and disease, such as fibrosis. Fn is sensitive to mechanical forces in its tandem type III repeats, resulting in extensive molecular enlongation. As such, it has long been hypothesized that cell- and tissue-derived forces may activate an "integrin switch" within the critical integrin-binding ninth and 10th type III repeats-conferring differential integrin-binding specificity, leading to differential cell responses. Yet, no direct evidence exists to prove the hypothesis nor demonstrate the physiological existence of the switch. We report direct experimental evidence for the Fn integrin switch both in vitro and ex vivo using a scFv engineered to detect the transient, force-induced conformational change, representing an opportunity for detection and targeting of early molecular signatures of cell contractile forces in tissue repair and disease.

Wound healing is a complex, dynamic process required for maintaining homeostasis in an organism. Along with being controlled biochemically, wound healing is also controlled through the transduction of biophysical stimuli through cell interactions with the extracellular matrix (ECM). This review provides an overview of the ECM's role in the wound healing process and subsequently expands on the variety of roles biophysical phenomenon play.

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.

Recent studies have identified several major qualitative and quantitative differences in important hemostatic proteins between adult and neonatal humans, including the primary coagulation protein fibrinogen.Despite these differences, neonates with post-operative bleeding from procedures requiring cardiopulmonary bypass (CPB) or extracorporeal membrane oxygenation (ECMO) are treated with transfusions of adult blood products, namely adult fibrinogen. The effectiveness of such transfusions is inconsistent in neonates and often results in a deficient fibrin matrix structure which may not be sufficient for mitigating bleeding. Our recent studies have also identified differences at the bulk clot level between purified neonatal and adult fibrin clots, including major structural and functional distinctions, which could contribute to these outcomes. Notably, adult fibrinogen degrades slower than neonatal clots, therefore transfusion of adult blood products to neonatal patients could contribute to thrombotic complications. Given the inconsistent results and potential complications from the transfusion of adult blood products to neonatal patients, there is a critical need to better understand the mechanistic differences in hemostatic processes between adults and neonates. Neonates possess a molecular variant of fibrinogen known as fetal fibrinogen. Increased sialic acid concentration compared to adults has been identified in many neonatal glycoproteins across physiological systems, including fetal fibrinogen. Studies have shown that fibrin clot properties are influenced by fibrin polymerization mechanisms and post translational modifications (e.g. sialic acid). For example, an increased sialic acid content in the dysfibrinogenemia associated with liver disease has been associated with an altered fibrin clot structure. Additionally, recent work from our group has found that the increased sialic acid in neonatal fibrin networks results in significantly greater fibroblast attachment than adult networks. Therefore, we hypothesized that differences in neonatal and adult fibrin clot properties are due to mechanistic differences in fibrin polymerization between neonates and adults owing to altered sialic acid concentrations. The activation of fibrinogen and conversion into fibrin by the proteolytic enzyme thrombin is essential for the formation of a stable blood clot and the cessation of bleeding. Thrombin converts soluble fibrinogen to insoluble fibrin via cleavage of fibrinopeptides A and B, exposing fibrin knobs A and B. Fibrin protofibrils are then formed from the noncovalent binding of fibrin knobs to complementary fibrin holes a and b on adjacent proteins. In adults, fibrin A:a knob:hole interactions are critical for polymerization. However, these mechanisms have not been explored in neonates. Therefore, we characterized the influence of sialic acid on the knob:hole interactions in neonatal fibrin polymerization. We first investigated the influence of sialic acid concentration on neonatal fibrin polymerization by removing sialic acid via enzymatic digestion and performing structural and functional analysis on desialylated fibrinogen. Desialylated adult and neonatal fibrinogen had roughly equivalent structure, polymerization kinetics, and clottability results. These results indicate that differential sialylation may at least partially explain functional differences been adult and neonatal clots. Additionally, we investigated the role that sialylation plays on neonatal fibrin polymerization dynamics by comparing neonatal or adult fibrin clots formed with snake venom thrombin-like enzymes (svTLE) that preferentially cleave either A or B fibrinogen fibrinopeptides or thrombin. Structural, mechanical, fibrinolytic, and polymerization assays were conducted. Quantitative release of fibrinopeptides was determined via ELISA. Results indicate neonatal fibrin polymerization mechanisms are more dependent on B:b knob:hole interactions than adult fibrin. Results from this study provide insight into the mechanism of the neonatal clotting process and are a critical contribution for the development of neonatal-specific treatments for bleeding and thrombosis. Disclosures Brown: Selsym Biotech, Inc.: Other: Founder and CEO.

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. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

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.

Purpose: 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.Materials and Methods: 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.Results: 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.Conclusion: These findings demonstrate the significant effects of 3D-bioplotted scaffold microarchitectures in the organization and sub-tissue-level mechanical properties of ECM in vivo.

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.