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The quality of platelet-rich fibrin (PRF) is contingent on the surface characteristics interfacing with blood. Titanium's superior platelet activation, surpassing silica, has made Titanium-platelet-rich fibrin (T-PRF) a favored autogenous bone graft material due to its extended degradation time. Pioneering a novel approach, this study aims to achieve an enhanced fibrin structure using glass tubes coated with nano-titanium, marking the surface's debut in our PRF production endeavors. Employing a rapid thermionic vacuum arc (TVA) process under high vacuum, we conducted comprehensive analyses of the tubes. Comprehensive analyses, including X-ray diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS), were conducted on the nano-titanium-coated glass tubes. Three PRF types were formulated: silica-activated leukocyte- and platelet-rich fibrin (L-PRF, control group), machined-surface titanium tubes (T-PRF), and nano-titanium-coated tubes (nanoT-PRF). Analyses unveiled denser fibrin areas in nanoT-PRF than T-PRF, with the least dense areas in L-PRF. Cell distribution paralled between nanoT-PRF and T-PRF groups, while L-PRF cells were embedded in the fibrin border. NanoT-PRF exhibited the densest autogenous fibrin structure, suggesting prolonged in vivo resorption. Additionally, we explore the potential practicality of single-use production for nanoT-PRF tubes, introducing a promising clinical advancement. This study marks a significant stride in innovative biomaterial design, contributing to the progress of regenerative medicine., (© 2024. The Author(s).)

Wound healing is influenced by various factors, including oxidative damage, bacterial infection, and inadequate angiogenesis, which collectively contribute to a protracted healing process. In this work, we designed innovative multifunctional hydrogels based on fibrin integrated with Bletilla striata polysaccharides (BSP) or oxidated Bletilla striata polysaccharides (OBSP) for use as wound dressings. The preliminary structure and bioactivity of BSP and OBSP were investigated. The effect of polysaccharides on the self-assembly process of fibrin hydrogels were also evaluated. BSP and OBSP significantly altered the initial fibrin fibrillogenesis and the ultimate structure of the fibrin network. Relative to pure fibrin hydrogel, the incorporation of BSP and OBSP enhanced water swelling and retention, and decelerated the degradation of hydrogels in PBS. Furthermore, BSP and OBSP augmented the antioxidant, antibacterial, and anti-inflammatory properties of fibrin hydrogels, with OBSP demonstrating superior performance in these aspects. Through the development of a murine wound model, it was observed that the wound healing efficacy of hydrogels incorporating BSP and OBSP surpassed that of the pure fibrin group. Notably, the hydrogel formulated with 25 mg/mL OBSP exhibited the most pronounced therapeutic effect, achieving a healing rate approaching 100 %. Consequently, fibrin-OBSP composite hydrogels demonstrate significant potential as wound dressings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fibrinogen, a blood plasma protein with a key role in hemostasis and thrombosis, is highly susceptible to post-translational modifications (PTMs), that significantly influence clot formation, structure, and stability. These PTMs, which include acetylation, amidation, carbamylation, citrullination, dichlorination, glycation, glycosylation, guanidinylation, hydroxylation, homocysteinylation, malonylation, methylation, nitration, oxidation, phosphorylation and sulphation, can alter fibrinogen biochemical properties and affect its functional behavior in coagulation and fibrinolysis. Oxidation and nitration are notably associated with oxidative stress, impacting fibrin fiber formation and promoting the development of more compact and resistant fibrin networks. Glycosylation and glycation contribute to altered fibrinogen structural properties, often resulting in changes in fibrin clot density and susceptibility to lysis, particularly in metabolic disorders like diabetes. Acetylation and phosphorylation, influenced by medications such as aspirin, modulate clot architecture by affecting fiber thickness and clot permeability. Citrullination and homocysteinylation, although less studied, are linked to autoimmune conditions and cardiovascular diseases, respectively, affecting fibrin formation and stability. Understanding these modifications provides insights into the pathophysiology of thrombotic disorders and highlights potential therapeutic targets. This review comprehensively examines the current literature on fibrinogen PTMs, their specific sites, biochemical pathways, and their consequences on fibrin clot architecture, clot formation and clot lysis., (© 2024. The Author(s).)

Fibrin fibers are important structural elements in blood coagulation. They form a mesh network that acts as a scaffold and imparts mechanical strength to the clot. A review of published work measuring the mechanics of fibrin fibers reveals a range of values for fiber extensibility. This study investigates fibrinogen concentration as a variable responsible for variability in fibrin mechanics. It expands previous work to describe the modulus, strain hardening, extensibility, and the force required for fiber failure when fibers are formed with different fibrinogen concentrations using lateral force atomic force microscopy. Analysis of the mechanical properties showed fibers formed from 1 mg ml -1 and 2 mg ml -1 fibrinogen had significantly different mechanical properties. To help clarify our findings we developed two behavior profiles to describe individual fiber mechanics. The first describes a fiber with low initial modulus and high extensible, that undergoes significant strain hardening, and has moderate strength. Most fibers formed with 1 mg ml -1 fibrinogen had this behavior profile. The second profile describes a fiber with a high initial modulus, minimal strain hardening, high strength, and low extensibility. Most fibrin fibers formed with 2 mg ml -1 fibrinogen were described by this second profile. In conclusion, we see a range of behaviors from fibers formed from native fibrinogen molecules but various fibrinogen concentrations. Potential differences in fiber formation are investigated with SEM. It is likely this range of behaviors also occurs in vivo . Understanding the variability in mechanical properties could contribute to a deeper understanding of pathophysiology of coagulative disorders., (Creative Commons Attribution license.)

Hydrogels derived from decellularized porcine myocardial matrix have demonstrated significant potential as therapeutic delivery platforms for promoting cardiac repair after injury. Our previous study developed a fibrin-enriched cardiac matrix hydrogel to enhance its angiogenic capacities. However, the bulk hydrogel structure may limit their full potential in cell delivery. Recently, granular hydrogels have emerged as a promising class of biomaterials, offering unique features such as a highly interconnected porous structure that facilitates nutrient diffusion and enhances cell viability. Several techniques have been developed for fabricating various types of granular hydrogels, among which extrusion fragmentation is particularly appealing due to its adaptability to many types of hydrogels, low cost, and high scalability. In this study, we first confirmed the effects of the bulk cardiac matrix hydrogel on the viability of encapsulated human umbilical vein endothelial cells and human mesenchymal stem cells. We then tested the feasibility of producing granular hydrogels from both cardiac matrix and fibrin-enriched cardiac matrix through cellular cross-linking of microgels fabricated by extrusion fragmentation. Afterward, we examined the roles of the produced granular hydrogels in the embedded cells and cell spheroids. Our in vitro data demonstrate that cardiac matrix-derived granular hydrogels support optimal viability of encapsulated cells and promote sprouting of human mesenchymal stem cell spheroids. Additionally, granular hydrogel derived from fibrin-enriched cardiac matrix accelerates angiogenic sprouting of embedded human mesenchymal stem cell spheroids. The results obtained from this study lay an important foundation for the future exploration of using cardiac matrix-derived granular hydrogels for cardiac cell therapy.

Blood clotting is vital for preventing bleeding after an injury. Hemostasis is a complex cascade involving numerous plasma proteins. Uncontrolled bleeding leads to mortality. The presence of Ca (calcium) activates and promotes the different phases in the coagulation cascade. Even nonbiological surfaces such as silicates may activate coagulation factor XII (FXII). This causes the clotting of the blood. The exceptional hemostatic ability of the mesoporous calcium-decorated silica nanoparticles (MCSNs) is achieved by stimulating the factors needed to form fibrin mesh, a durable clot, thereby establishing hemostasis. This may be used as a hemostatic agent during an accident surgical procedure and other bleeding-related trauma conditions. This study investigates the mechanistic activation of the coagulation cascade by MCSN through blood coagulation index, clotting time, and coagulation activation studies like PT and aPTT. Our finding demonstrates that MCSN induces platelet adhesion and RBC aggregation and activates thrombin generation through distinct pathways.

Biomaterials play an increasingly critical role in bone tissue engineering. However, achieving effective clinical translation requires a careful choice of biomimetic materials and thorough assessment of their efficacy and safety. Existing in vitro and in vivo models have drawbacks including time and cost constraints, invasive procedures, and discordance between animal models and clinical outcomes. Therefore, there is a demand for an alternative model. We hypothesized that the chick embryo chorioallantoic membrane can serve as a bioreactor to evaluate the initial sign of bone formation on scaffolds. In parallel, we investigated the osteogenic potential of a previously fabricated fibrin-alginate-calcium phosphate biomaterial (FACaP). Blood vessels were observed to infiltrate the scaffolds with early signs of bone formation, confirmed via RUNX-2 and alpha smooth muscle actin markers. The scaffolds' chemical composition was evaluated by Fourier-transform infrared spectroscopy, and ion chromatography was used to assess calcium ion release. Finally, the topography was examined by atomic force microscopy. In conclusion, this system offers simple refinement for in vivo models in bone tissue engineering and highlights the great potential of FACaP as an angiogenic and osteogenic biomaterial for non-load-bearing applications.

Prolonged osteochondral tissue engineering damage can result in osteoarthritis and decreased quality of life. Multiphasic scaffolds, where different layers model different microenvironments, are a promising treatment approach, yet stable joining between layers during fabrication remains challenging. To overcome this problem, in this study, a bilayer scaffold for osteochondral tissue regeneration was fabricated using 3D printing technology which containing a layer of PCL/hydroxyapatite (HA) nanoparticles and another layer of PCL/gelatin with various concentrations of fibrin (10, 20 and 30 wt.%). These printed scaffolds were evaluated with SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared Spectroscopy) and mechanical properties. The results showed that the porous scaffolds fabricated with pore size of 210-255 µm. Following, the ductility increased with the further addition of fibrin in bilayer composites which showed these composites scaffolds are suitable for the cartilage part of osteochondral. Also, the contact angle results demonstrated the incorporation of fibrin in bilayer scaffolds based on PCL matrix, can lead to a decrease in contact angle and result in the improvement of hydrophilicity that confirmed by increasing the degradation rate of scaffolds containing further fibrin percentage. The bioactivity study of bilayer scaffolds indicated that both fibrin and hydroxyapatite can significantly improve the cell attachment on fabricated scaffolds. The MTT assay, DAPI and Alizarin red tests of bilayer composite scaffolds showed that samples containing 30% fibrin have the more biocompatibility than that of samples with 10 and 20% fibrin which indicated the potential of this bilayer scaffold for osteochondral tissue regeneration., (© 2024. The Author(s).)

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

Coagulation factor XIII (FXIII) is an enzyme that strengthens hemostatic clots, and its deficiency can cause life-threatening bleeding. We immunized mice with human plasma-derived FXIII to generate monoclonal antibodies (mAbs) against the B subunit (FXIII-B), which stabilizes the A subunit (FXIII-A) of FXIII, and analyzed their properties. The epitopes of the seven mouse antihuman FXIII-B mAbs obtained were found to be the 3rd, 5th, 6th, 9th, and 10th Sushi domains. One of these mAbs, mAb 5-6C, recognized the 10th Sushi domain and inhibited the fibrin cross-linking reaction without affecting the amine incorporation activity of FXIII. We previously reported that the 10th Sushi domain is the site where FXIII-B binds to fibrin and functions to bring FXIII-A closer to the substrate fibrin. Except for mAb 5-6C, mouse mAbs with high yields were used to measure the amount of FXIII-B antigen by an immunochromatography test (ICT), which showed a high correlation with enzyme-linked immunosorbent assay-obtained results. In addition, we developed a prototype ICT to detect anti-FXIII-B autoantibodies using mAb 1-3C, which showed good results in measuring the amount of FXIII-B antigen. Thus, mouse mAbs may be useful for clinical applications. mAb 5-6C targeting the 10th Sushi domain may also be useful for inhibiting thrombosis progression when humanized as antibody medicines.

Chronic diabetic wounds present formidable challenges, marked by uncontrolled bacterial infections, prolonged inflammation, and impaired angiogenesis. The evolving landscape of photo-responsive antibacterial therapy holds great promise in addressing these multifaceted issues, with a particular focus on leveraging the distinctive properties of 2D heterojunction materials. In this investigation, we engineered composite sprayed hydrogels, seamlessly integrating Bi/MoS 2 nano-heterojunctions. Capitalizing on the synergistic interplay between photocatalytic antibacterial and photothermal antibacterial mechanisms, the Bi/MoS 2 heterojunction, guided by its localized surface plasmon resonance, demonstrated outstanding antibacterial efficacy within a mere 10-minute exposure to 808 nm near-infrared light. This accelerated sterilization both in vitro and in vivo, consequently expediting wound healing. The sprayed composite gel not only furnishes protective shielding for skin tissues but also fosters endothelial cell proliferation, vascularization, and angiogenesis. This safe and ultrafast sterilizing hydrogel presents immense potential for application in antimicrobial dressings, thereby offering a promising avenue for diabetic wound healing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tissue engineering is a steadily growing field of research due to its wide-ranging applicability in the field of regenerative medicine. Application-dependent mechanical properties of a scaffold material as well as its biocompatibility and tailored functionality represent particular challenges. Here the properties of fibrin-based hydrogels reinforced by functional cytocompatible poly(N-vinylcaprolactam)-based (PVCL) microgels are studied and evaluated. The employment of temperature-responsive microgels decorated by epoxy groups for covalent binding to the fibrin is studied as a function of cross-linking degree within the microgels, microgel concentration, as well as temperature. Rheology reveals a strong correlation between the mechanical properties of the reinforced fibrin-based hydrogels and the microgel rigidity and concentration. The incorporated microgels serve as cross-links, which enable temperature-responsive behavior of the hydrogels, and slow down the hydrogel degradation. Microgels can be additionally used as carriers for active drugs, as demonstrated for dexamethasone. The microgels' temperature-responsiveness allows for triggered release of payload, which is monitored using a bioassay. The cytocompatibility of the microgel-reinforced fibrin-based hydrogels is demonstrated by LIVE/DEAD staining experiments using human mesenchymal stem cells. The microgel-reinforced hydrogels are a promising material for tissue engineering, owing to their superior mechanical performance and stability, possibility of drug release, and retained biocompatibility., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Acoustic droplet vaporization (ADV) offers a dynamic approach for generating bubbles on demand, presenting new possibilities in biomedical applications. Although ADV has been investigated in various biomedical applications, its potential in tissue characterization remains unexplored. Here, we investigated the effects of surrounding media on the radial dynamics and acoustic emissions of ADV bubbles using theoretical and experimental methodologies. For theoretical studies, bubble dynamics were combined with the Kelvin-Voigt material constitutive model, accounting for viscoelasticity of the media. The radial dynamics and acoustic emissions of the ADV-bubbles were recorded via ultra-high-speed microscopy and passive cavitation detection, respectively. Perfluoropentane phase-shift droplets were embedded in tissue-mimicking hydrogels of varying fibrin concentrations, representing different elastic moduli. Radial dynamics and the acoustic emissions, both temporal and spectral, of the ADV-bubbles depended significantly on fibrin elastic modulus. For example, an increase in fibrin elastic modulus from ≈0.2 kPa to ≈6 kPa reduced the maximum expansion radius of the ADV-bubbles by 50%. A similar increase in the elastic modulus significantly impacted both linear (e.g., fundamental) and nonlinear (e.g., subharmonic) acoustic responses of the ADV-bubbles, by up to 10 dB. The sensitivity of ADV to the surrounding media was dependent on acoustic parameters such as driving pressure and the droplets concentration. Further analysis of the acoustic emissions revealed distinct ADV signal characteristics, which were significantly influenced by the surrounding media., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hydrogels can serve as local drug delivery depots that protect the biological activity of labile therapeutics. However, drug release from conventional hydrogels is typically rapid, which is not ideal for many therapeutic agents. We developed a composite hydrogel that enables sustained drug release in response to ultrasound. The composite, termed an acoustically responsive scaffold (ARS), consists of a fibrin hydrogel and a phase-shift emulsion. Upon exposure to ultrasound, the emulsion is vaporized into bubbles, which leads to release of drugs contained within the emulsion. Previously, ARSs have been used in regenerative applications to stimulate blood vessel growth. Here, we characterize the release kinetics and mechanisms of ARSs. Release exhibits a triphasic pattern compromising a slow phase prior to ultrasound exposure; a transient, fast phase immediately after ultrasound exposure that follows a sigmoidal profile; and a sustained, steady phase. In each phase, we demonstrate how derived kinetics parameters are impacted by the ARS composition (e.g., fibrin and emulsion concentrations) and ultrasound properties (e.g., acoustic pressure, pulse duration). Using confocal microscopy, protein assays, and B-mode ultrasound imaging, we demonstrate that drug release from an ARS is independent of fibrin degradation and dependent on bubble growth. These results are critical in optimizing ARSs for delivery of therapeutic agents., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As the most prominent and ideal modality in female fertility preservation, ovarian tissue cryopreservation, and transplantation often confront the challenge of ischemic damage and follicular loss from avascular transplantation. To surmount this impediment, we engineered a novel platelet-derived factors-encapsulated fibrin hydrogel (PFH), a paradigmatic biomaterial. PFH encapsulates autologous platelet-derived factors, utilizing the physiological blood coagulation cascade for precise local delivery of bioactive molecules. In our study, PFH markedly bolstered the success of avascular ovarian tissue transplantation. Notably, the quantity and quality of follicles were preserved with improved neovascularization, accompanied by decreased DNA damage, increased ovulation, and superior embryonic development rates under a Low-concentration Platelet-rich plasma-derived factors encapsulated fibrin hydrogel (L-PFH) regimen. At a stabilized point of tissue engraftment, gene expression analysis mirrored normal ovarian tissue profiles, underscoring the effectiveness of L-PFH in mitigating the initial ischemic insult. This autologous blood-derived biomaterial, inspired by nature, capitalizes on the blood coagulation cascade, and combines biodegradability, biocompatibility, safety, and cost-effectiveness. The adjustable properties of this biomaterial, even in injectable form, extend its potential applications into the broader realm of personalized regenerative medicine. PFH emerges as a promising strategy to counter ischemic damage in tissue transplantation, signifying a broader therapeutic prospect. (197 words)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Accurate assessment of fibrin clot stability can predict bleeding risk in coagulopathic conditions such as thrombocytopenia and hypofibrinogenemia. Hyperfibrinolysis - a clinical phenotype characterized by an accelerated breakdown of the fibrin clot - makes such assessments challenging by obfuscating the effect of hemostatic components including platelets or fibrinogen on clot stability. In this work, we present a biofunctionalized, microfluidic, label-free, electronic biosensor to elicit unique, specific, and differential responses from the multifactorial processes of blood coagulation and fibrinolysis ex vivo. The microsensor tracks the temporal variation in the normalized real part of the dielectric permittivity of whole blood (<10 μL) at 1 MHz as the sample coagulates within a three-dimensional, parallel-plate, capacitive sensing area. Surface biofunctionalization of the microsensor's electrodes with physisorption of tissue factor (TF) and aprotinin permits real-time assessment of the coagulation and fibrinolytic outcomes. We show that surface coating with TF and manual addition of TF result in a similar degree of acceleration of coagulation kinetics in human whole blood samples. We also show that surface coating with aprotinin and manual addition of aprotinin yield similar results in inhibiting tissue plasminogen activator (tPA)-induced upregulated fibrinolysis in human whole blood samples. Validated through a clinically relevant, complementary assay - rotational thromboelastometry for clot viscoelasticity - we finally establish that a microsensor dual-coated with both TF and aprotinin detects the hemostatic rescue in the tPA-induced hyperfibrinolytic profile of whole blood and the hemostatic dysfunction due to concurrent platelet depletion in the blood sample, thus featuring enhanced ability in evaluating complex, combinatorial coagulopathies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M. A. Suster and P. Mohseni are inventors of foundational intellectual property related to this study that has been licensed by Case Western Reserve University to XaTek Inc. They report research funding, consulting fees, and royalties from XaTek Inc. during the conduct of this study. S. P. Ahuja reports research funding from XaTek Inc., DOD, and PCORI, as well as personal fees from Genentech, CSL Behring, BioMarin, Sanofi, and Novo Nordisk. M. D. Neal serves as the Chief Medical Officer of Haima Therapeutics and reports personal fees from CSL Behring, Haemonetics, Octapharma, Takeda, and Janssen Pharmaceuticals, as well as grants from Haemonetics, Alexion, Instrumentation Laboratories, NIH, DOD, and DARPA outside of the submitted work. A. Sen Gupta is a co-founder of Haima Therapeutics and an inventor on patents regarding hemostatic therapeutic technologies that are licensed to Haima Therapeutics. S. Pourang, D. Disharoon, and S. Hernandez report no conflict., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As the cornerstone of tissue engineering and regeneration medicine research, developing a cost-effective and bionic extracellular matrix (ECM) that can precisely modulate cellular behavior and form functional tissue remains challenging. An artificial ECM combining polysaccharides and fibrillar proteins to mimic the structure and composition of natural ECM provides a promising solution for cardiac tissue regeneration. In this study, we developed a bionic hydrogel scaffold by combining a quaternized β-chitin derivative (QC) and fibrin-matrigel (FM) in different ratios to mimic a natural ECM. We evaluated the stiffness of those composite hydrogels with different mixing ratios and their effects on the growth of human umbilical vein endothelial cells (HUVECs). The optimal hydrogels, QCFM1 hydrogels were further applied to load HUVECs into nude mice for in vivo angiogenesis. Besides, we encapsulated human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) into QCFM hydrogels and employed 3D bioprinting to achieve batch fabrication of human-engineered heart tissue (hEHT). Finally, the myocardial structure and electrophysiological function of hEHT were evaluated by immunofluorescence and optical mapping. Designed artificial ECM has a tunable modulus (220-1380 Pa), which determines the different cellular behavior of HUVECs when encapsulated in these. QCFM1 composite hydrogels with optimal stiffness (800 Pa) and porous architecture were finally identified, which could adapt for in vitro cell spreading and in vivo angiogenesis of HUVECs. Moreover, QCFM1 hydrogels were applied in 3D bioprinting successfully to achieve batch fabrication of both ring-shaped and patch-shaped hEHT. These QCFM1 hydrogels-based hEHTs possess organized sarcomeres and advanced function characteristics comparable to reported hEHTs. The chitin-derived hydrogels are first used for cardiac tissue engineering and achieve the batch fabrication of functionalized artificial myocardium. Specifically, these novel QCFM1 hydrogels provided a reliable and economical choice serving as ideal ECM for application in tissue engineering and regeneration medicine., (© 2024 Wiley Periodicals LLC.)

Staphylococcus aureus skin and soft tissue infection is a common ailment placing a large burden upon global healthcare infrastructure. These bacteria are growing increasingly recalcitrant to frontline antimicrobial therapeutics like vancomycin due to the prevalence of variant populations such as methicillin-resistant and vancomycin-resistant strains, and there is currently a dearth of novel antibiotics in production. Additionally, S. aureus has the capacity to hijack the host clotting machinery to generate fibrin-based biofilms that confer protection from host antimicrobial mechanisms and antibiotic-based therapies, enabling immune system evasion and significantly reducing antimicrobial efficacy. Emphasis is being placed on improving the effectiveness of therapeutics that are already commercially available through various means. Fibrin-based nanoparticles (FBNs) were developed and found to interact with S. aureus through the clumping factor A (ClfA) fibrinogen receptor and directly integrate into the biofilm matrix. FBNs loaded with antimicrobials such as vancomycin enabled a targeted and sustained release of antibiotic that increased drug contact time and reduced the therapeutic dose required for eradicating the bacteria, both in vitro and in vivo. Collectively, these findings suggest that FBN-antibiotic delivery may be a novel and potent therapeutic tool for the treatment of S. aureus biofilm infections., (© 2024 The Author(s). Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Uncontrolled bleeding during surgery is associated with high mortality and prolonged hospital stay, necessitating the use of hemostatic agents. Fibrin sealant patches offer an efficient solution to achieve hemostasis and improve patient outcomes in liver resection surgery. We have previously demonstrated the efficacy of a nanostructured fibrin-agarose hydrogel (NFAH). However, for the widespread distribution and commercialization of the product, it is necessary to develop an optimal preservation method that allows for prolonged stability and facilitates storage and distribution. We investigated cryopreservation as a potential method for preserving NFAH using trehalose. Structural changes in cryopreserved NFAH (Cryo-NFAH) were investigated and comparative in vitro and in vivo efficacy and safety studies were performed with freshly prepared NFAH. We also examined the long-term safety of Cryo-NFAH versus TachoSil in a rat partial hepatectomy model, including time to hemostasis, intra-abdominal adhesion, hepatic hematoma, inflammatory factors, histopathological variables, temperature and body weight, hemocompatibility and cytotoxicity. Structural analyses demonstrated that Cryo-NFAH retained most of its macro- and microscopic properties after cryopreservation. Likewise, hemostatic efficacy assays showed no significant differences with fresh NFAH. Safety evaluations indicated that Cryo-NFAH had a similar overall profile to TachoSil up to 40 days post-surgery in rats. In addition, Cryo-NFAH demonstrated superior hemostatic efficacy compared with TachoSil while also demonstrating lower levels of erythrolysis and cytotoxicity than both TachoSil and other commercially available hemostatic agents. These results indicate that Cryo-NFAH is highly effective hemostatic patch with a favorable safety and tolerability profile, supporting its potential for clinical use., (© 2024. The Author(s).)

Fibrinolytic activity assay is particularly important for the detection, diagnosis, and treatment of cardiovascular disease and the development of fibrinolytic drugs. A novel efficacious strategy for real-time and label-free dynamic detection of fibrinolytic activity based on ordered porous layer interferometry (OPLI) was developed. Fibrin or a mixture of fibrin and plasminogen (Plg) was loaded into the highly ordered silica colloidal crystal (SCC) film scaffold to construct a fibrinolytic response interference layer to measure fibrinolytic activity with different mechanisms of action. Fibrinolytic enzyme-triggered fibrinolysis led to the migration of interference fringes in the interferogram, which could be represented by optical thickness changes (ΔOT) tracked in real time by the OPLI system. The morphology and optical property of the fibrinolytic response interference layer were characterized, and the Plg content in the fibrinolytic response interference layer and experimental parameters of the system were optimized. The method showed adequate sensitivity for the fibrinolytic activity of lumbrokinase and streptokinase, with wide linear ranges of 12-6000 and 10-2000 U/mL, respectively. Compared with the traditional fibrin plate method, it has a lower detection limit and higher linearity. The whole kinetic process of fibrinolysis by these two fibrinolytic drug models was recorded in real time, and the Michaelis constant and apparent kinetic parameters were calculated. Importantly, some other blood proteins were less interfering with this system, and it showed reliability in fibrin activity detection in real whole blood samples. This study established a better and more targeted research method of in vitro fibrinolysis and provided dynamic monitoring data for the analysis of fibrinolytic activity of whole blood.

Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. A fibrinolytic protease was isolated from fresh leaves of Moringa oleifera Lam. and characterized for its potential to solubilize blood clots and hydrolyse fibrin under in-vitro conditions. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 o C with 50 µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. K m and V max of isolated protease were determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clots by 96 h under in-vitro conditions. In-vitro fibrin hydrolysis and blood clot solubilization activities shown by an isolated protease from leaves of Moringa oleifera Lam. suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Traumatic brain injury (TBI) is a critical public health concern, yet there are no therapeutics available to improve long-term outcomes. Drug delivery to TBI remains a challenge due to the blood-brain barrier and increased intracranial pressure. In this work, a chemical targeting approach to improve delivery of materials to the injured brain, is developed. It is hypothesized that the provisional fibrin matrix can be harnessed as an injury-specific scaffold that can be targeted by materials via click chemistry. To accomplish this, the brain clot is engineered in situ by delivering fibrinogen modified with strained cyclooctyne (SCO) moieties, which incorporated into the injury lesion and is retained there for days. Improved intra-injury capture and retention of diverse, clickable azide-materials including a small molecule azide-dye, 40 kDa azide-PEG nanomaterial, and a therapeutic azide-protein in multiple dosing regimens is subsequently observed. To demonstrate therapeutic translation of this approach, a reduction in reactive oxygen species levels in the injured brain after delivery of the antioxidant catalase, is achieved. Further, colocalization between azide and SCO-fibrinogen is specific to the brain over off-target organs. Taken together, a chemical targeting strategy leveraging endogenous clot formation is established which can be applied to improve therapeutic delivery after TBI., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

The objective for this study is to advance the development of a specialized biomaterial that can effectively facilitate the regeneration of adipose tissue. In prior studies, the assessment of collagen (Col), elastin (Ela), and fibrin (Fib) unary scaffolds has been conducted. However, it is important to note that native adipose tissue is comprised of a diverse array of extracellular matrix (ECM) constituents. To mimic this behavior, binary compositions of collagen, elastin, and fibrin are fabricated in a 1:1 ratio, resulting in the formation of Col/Ela, Col/Fib, and Ela/Fib composites through a customized fabrication procedure. The physical properties of these scaffolds are comprehensively analyzed using a range of material characterization techniques. Additionally, the biological properties of the scaffolds are investigated by examining the survival, proliferation, and phenotype of adipose-derived stem cells. Subsequently, the aforementioned binary scaffolds are implanted into a rodent model for 28 days. the explants are analysed through X-ray microtomography, histology, and immunohistochemistry. The findings of the study demonstrate that the utilization of binary combinations of Col/Ela, Col/Fib, and Ela/Fib has a discernible impact on the physical and biological characteristics of the scaffolds. Nevertheless, Ela/Fib exhibits characteristics that make it a suitable candidate for adipogenesis due to its notable upregulation of caveolin-1 expression in both acellular and cellular cohorts. The combination of two natural polymers in this cell-material interaction has significantly enhanced the comprehension of adipogenesis., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

Local recurrence post-surgery in early-stage triple-negative breast cancer is a major challenge. To control the regrowth of a residual tumor, we have developed an autologous therapeutic hybrid fibrin glue for intra-operative implantation. Using autologous serum proteins as stabilizers, we have optimized high drug-loaded lapatinib-NanoSera (Lap-NS; ∼66% L.C.) and imiquimod-MicroSera (IMQ-MS; ∼92% L.C). Additionally, plasmonic nanosera (PNS) with an ∼67% photothermal conversion efficiency under 980 nm laser irradiation was also developed. While localized monotherapy with either Lap-NS or PNS reduced the tumor regrowth rate, their combination with IMQ-MS amplified the effect of immunogenic cell death with a high level of tumor infiltration by immune cells at the surgical site. The localized combination immunotherapy with a Nano-MicroSera based hybrid fibrin implant showed superior tumor inhibition and survival with significant promise for clinical translation.

In this study, composite gel was prepared from konjac glucomannan (KGM) and fibrin (FN). Composite gels with different concentration ratios were compared in terms of their mechanical properties, rheological properties, water retention, degradation rate, microstructure and biocompatibility. The results showed that the composite gels had better gel strength and other properties than non-composite gels. In particular, composite hydrogels with low Young's modulus formed when the KGM concentration was 0.8% and the FN concentration was 1.2%. The two components were cross linked through hydrogen-bond interaction, which formed a more stable gel structure with excellent water retention and in-vitro degradation rates, which were conducive to myogenic differentiation of ectomesenchymal stem cells (EMSCs). KGM-FN composite gel was applied to the preparation of cell-culture meat, which had similar texture properties and main nutrients to animal meat as well as higher content of dry base protein and dry base carbohydrate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C 41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0-10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 μM, Vmax = 62.91 ± 1.68 μM/s, kcat = 38.45 ± 1.06 s -1 , and kcat/Km = 1.49 × 10 6  M -1  s -1 . In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of -6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/ - OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/ - OCl, 10 μM HOCl/ - OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BβMet367, γMet264, and γMet94, identified in 10 μM HOCl/-OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels., (© 2024. The Author(s).)

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo , single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

Cardiovascular diseases (CVDs), mainly caused by thrombosis complications, are the leading cause of mortality worldwide, making the development of alternative treatments highly desirable. In this study, the thrombolytic potential of green kiwifruit (Actinidia deliciosa cultivar Hayward) was assessed using in-vitro and in-silico approaches. The crude green kiwifruit extract demonstrated the ability to reduce blood clots significantly by 73.0 ± 1.12% (P < 0.01) within 6 h, with rapid degradation of Aα and Bβ fibrin chains followed by the γ chain in fibrinolytic assays. Molecular docking revealed six favorable conformations for the kiwifruit enzyme actinidin (ADHact) and fibrin chains, supported by spontaneous binding energies and distances. Moreover, molecular dynamics simulation confirmed the binding stability of the complexes of these conformations, as indicated by the stable binding affinity, high number of hydrogen bonds, and consistent distances between the catalytic residue Cys25 of ADHact and the peptide bond. The better overall binding affinity of ADHact to fibrin chains Aα and Bβ may contribute to their faster degradation, supporting the fibrinolytic results. In conclusion, this study demonstrated the thrombolytic potential of the green kiwifruit-derived enzyme and highlighted its potential role as a natural plant-based prophylactic and therapeutic agent for CVDs., (© 2024. The Author(s).)

Thromboembolism, a global leading cause of mortality, needs accurate risk assessment for effective prophylaxis and treatment. Current stratification methods fall short in predicting thrombotic events, emphasizing the need for a deeper understanding of clot properties. Fibrin clot permeability, a crucial parameter in hypercoagulable states, impacts clot structure and resistance to lysis. Current clot permeability measurement limitations propel the need for standardized methods. Prior findings underscore the importance of clot permeability in various thrombotic conditions but call for improvements and more precise, repeatable, and standardized methods. Addressing these challenges, our study presents an upgraded, portable, and cost-effective system for measuring blood clot permeability, which utilizes a pressure-based approach that adheres to Darcy's law. By enhancing precision and sensitivity in discerning clot characteristics, this innovation provides a valuable tool for assessing thrombotic risk and associated pathological conditions. In this paper, the authors present a device that is able to automatically perform the permeability measurements on plasma or fibrinogen in vitro-induced clots on specific holders (filters). The proposed device has been tailored to distinguish clot permeability, with high precision and sensitivity, between healthy subjects and high cardiovascular-risk patients. The precise measure of clot permeability represents an excellent indicator of thrombotic risk, thus allowing the clinician, also on the basis of other anamnestic and laboratory data, to attribute a risk score to the subject. The proposed instrument was characterized by performing permeability measurements in plasma and purified fibrinogen clots derived from 17 Behcet patients and 15 sex- and age-matched controls. As expected, our results clearly indicate a significant difference in plasma clot permeability in Behcet patients with respect to controls (0.0533 ± 0.0199 d vs. 0.0976 ± 0.0160 d, p < 0.001). This difference was confirmed in the patient's vs. control fibrin clots (0.0487 ± 0.0170 d vs. 0.1167 ± 0.0487 d, p < 0.001). In conclusion, our study demonstrates the feasibility, efficacy, portability, and cost-effectiveness of a novel device for measuring clot permeability, allowing healthcare providers to better stratify thrombotic risk and tailor interventions, thereby improving patient outcomes and reducing healthcare costs, which could significantly improve the management of thromboembolic diseases.

Due to its structural and functional complexity the heart imposes immense physical, physiological and electromechanical challenges on the engineering of a biological replacement. Therefore, to come closer to clinical translation, the development of a simpler biological assist device is requested. Here, we demonstrate the fabrication of tubular cardiac constructs with substantial dimensions of 6 cm in length and 11 mm in diameter by combining human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and human foreskin fibroblast (hFFs) in human fibrin employing a rotating mold technology. By centrifugal forces employed in the process a cell-dense layer was generated enabling a timely functional coupling of iPSC-CMs demonstrated by a transgenic calcium sensor, rhythmic tissue contractions, and responsiveness to electrical pacing. Adjusting the degree of remodeling as a function of hFF-content and inhibition of fibrinolysis resulted in stable tissue integrity for up to 5 weeks. The rotating mold device developed in frame of this work enabled the production of tubes with clinically relevant dimensions of up to 10 cm in length and 22 mm in diameter which-in combination with advanced bioreactor technology for controlled production of functional iPSC-derivatives-paves the way towards the clinical translation of a biological cardiac assist device., (© 2024. The Author(s).)

Semiflexible fiber gels such as collagen and fibrin have unique nonlinear mechanical properties that play an important role in tissue morphogenesis, wound healing, and cancer metastasis. Optical tweezers microrheology has greatly contributed to the understanding of the mechanics of fibrous gels at the microscale, including its heterogeneity and anisotropy. However, the explicit relationship between micromechanical properties and gel deformation has been largely overlooked. We introduce a unique gel-stretching apparatus and employ it to study the relationship between microscale strain and stiffening in fibrin and collagen gels, focusing on the development of anisotropy in the gel. We find that gels stretched by as much as 15 % stiffen significantly both in parallel and perpendicular to the stretching axis, and that the parallel axis is 2-3 times stiffer than the transverse axis. We also measure the stiffening and anisotropy along bands of aligned fibers created by aggregates of cancer cells, and find similar effects as in gels stretched with the tensile apparatus. Our results illustrate that the extracellular microenvironment is highly sensitive to deformation, with implications for tissue homeostasis and pathology. STATEMENT OF SIGNIFICANCE: The inherent fibrous architecture of the extracellular matrix (ECM) gives rise to unique strain-stiffening mechanics. The micromechanics of fibrous networks has been studied extensively, but the deformations involved in its stiffening at the microscale were not quantified. Here we introduce an apparatus that enables measuring the deformations in the gel as it is being stretched while simultaneously using optical tweezers to measure its microscale anisotropic stiffness. We reveal that fibrin and collagen both stiffen dramatically already at ∼10 % deformation, accompanied by the emergence of significant, yet moderate anisotropy. We measure similar stiffening and anisotropy in the matrix remodeled by the tensile apparatus to those found between cancer cell aggregates. Our results emphasize that small strains are enough to introduce substantial stiffening and anisotropy. These have been shown to result in directional cell migration and enhanced force propagation, and possibly control processes like morphogenesis and cancer metastasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. No conflicts of interest reported., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged β cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged β cells., (© 2024 John Wiley & Sons Ltd.)

Given the broad biological effects of the Hedgehog (Hh) pathway, there is potential clinical value in local application of Hh pathway modulators to restrict pathway activation of target tissues and avoid systemic pathway activation. One option to limit Hh pathway activation is using fibrin hydrogels to deliver pathway modulators directly to tissues of interest, bypassing systemic distribution of the drug. In this study, we loaded the potent Hh pathway agonist, SAG21k, into fibrin hydrogels. We describe the binding between fibrin and SAG21k and achieve sustained release of the drug in vitro. SAG21k-loaded fibrin hydrogels exhibit strong biological activity in vitro, using a pathway-specific reporter cell line. To test in vivo activity, we used a mouse model of facial nerve injury. Application of fibrin hydrogels is a common adjunct to surgical nerve repair, and the Hh pathway is known to play an important role in facial nerve injury and regeneration. Local application of the Hh pathway agonist SAG21k using a fibrin hydrogel applied to the site of facial nerve injury successfully activates the Hh pathway in treated nerve tissue. Importantly, this method appears to avoid systemic pathway activation when Hh-responsive organs are analyzed for transcriptional pathway activation. This method of local tissue Hh pathway agonist administration allows for effective pathway targeting surgically accessible tissues and may have translational value in situations where supranormal pathway activation is therapeutic., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jon-Paul Pepper and Philip Arden Beachy has patent pending to None. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The widespread use of the antifibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, factor (F)XIIIa catalyzes the formation of fibrin-fibrin and fibrin-α 2 -antiplasmin (α 2 AP) cross-links, which increases clot mechanical strength and resistance to fibrinolysis., Objectives: Here, we developed a method to quantify fibrin-fibrin and fibrin-α 2 AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays., Methods: Fibrinogen alpha (FGA) chain (FGA-FGA), fibrinogen gamma (FGG) chain (FGG-FGG), and FGA-α 2 AP cross-links were quantified using liquid chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring in paired plasma samples from trauma patients prefibrinogen and postfibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analyzed., Results: The abundance of cross-links was significantly increased in trauma patients postcryo, but not Fg-C transfusion (P < .0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibers, the peak thrombin concentration, and FXIII antigen (P < .05)., Conclusion: We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach, we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α 2 AP cross-linking when compared with Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients., Competing Interests: Declaration of competing interests N.C. has received funding from CSL Behring for investigator-led studies. G.B.M., S.F., P.D.C., R.H., T.F., Z.M., E.W., J.W., R.F., R.A.S.A., N.J.M., and M.L. have no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Wounding is one of the most common healthcare problems. Bioactive hydrogels have attracted much attention in first-aid hemostasis and wound healing due to their excellent biocompatibility, antibacterial properties, and pro-healing bioactivity. However, their applications are limited by inadequate mechanical properties. In this study, we first prepared edible rose-derived exosome-like nanoparticles (ELNs) and used them to encapsulate antimicrobial peptides (AMP), abbreviated as ELNs(AMP). ELNs(AMP) showed superior intracellular antibacterial activity, 2.5 times greater than AMP, in in vitro cell infection assays. We then prepared and tested an FDA-approved fibrin-gel of fibrinogen and thrombin encapsulating ELNs(AMP) and novobiocin sodium salt (NB) (ELNs(AMP)/NB-fibrin-gels). The fibrin gel showed a sustained release of ELNs(AMP) and NB over the eight days of testing. After spraying onto the skin, the formulation underwent in situ gelation and developed a stable patch with excellent hemostatic performance in a mouse liver injury model with hemostasis in 31 s, only 35.6 % of the PBS group. The fibrin gel exhibited pro-wound healing properties in the mouse-infected skin defect model. The thickness of granulation tissue and collagen of the ELNs(AMP)/NB-fibrin-gels group was 4.00, 6.32 times greater than that of the PBS group. In addition, the ELNs(AMP)/NB-fibrin-gels reduced inflammation (decreased mRNA levels of TNF-α, IL-1β, IL6, MCP1, and CXCL1) at the wound sites and demonstrated a biocompatible and biosafe profile. Thus, we have developed a hydrogel system with excellent hemostatic, antibacterial, and pro-wound healing properties, which may be a candidate for next-generation tissue regeneration with a wide clinical application for first-aid hemostasis and infected wound healing., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The microvascular network plays an important role in providing nutrients to the injured tissue and exchanging various metabolites. However, how to achieve efficient penetration of the injured tissue is an important bottleneck restricting the reconstruction of microvascular network. Herein, the hydrogel precursor solution can efficiently penetrate the damaged tissue area, and ultrasound triggers the release of thrombin from liposomes in the solution to hydrolyze fibrinogen, forming a fibrin solid hydrogel network in situ with calcium ions and transglutaminase as catalysts, effectively solving the penetration impedance bottleneck of damaged tissues and ultimately significantly promoting the formation of microvascular networks within tissues. First, the fibrinogen complex solution is effectively permeated into the injured tissue. Second, ultrasound triggered the release of calcium ions and thrombin, activates transglutaminase, and hydrolyzes fibrinogen. Third, fibrin monomers are catalyzed to form fibrin hydrogels in situ in the damaged tissue area. In vitro studies have shown that the fibrinogen complex solution effectively penetrated the artificial bone tissue within 15 s after ultrasonic triggering, and formed a hydrogel after continuous triggering for 30 s. Overall, this innovative strategy effectively solved the problem of penetration resistance of ultrasound-triggered hydrogels in the injured tissues, and finally activates in situ microvascular networks regeneration., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Intravascular blood clots are subject to hydrodynamic shear and other forces that cause clot deformation and rupture (embolization). A portion of the ruptured clot can block blood flow in downstream vessels. The mechanical stability of blood clots is determined primarily by the 3D polymeric fibrin network that forms a gel. Previous studies have primarily focused on the rupture of blood plasma clots under tensile loading (Mode I), our current study investigates the rupture of fibrin induced by shear loading (Mode II), dominating under physiological conditions induced by blood flow. Using experimental and theoretical approaches, we show that fracture toughness, i.e. the critical energy release rate, is relatively independent of the type of loading and is therefore a fundamental property of the gel. Ultrastructural studies and finite element simulations demonstrate that cracks propagate perpendicular to the direction of maximum stretch at the crack tip. These observations indicate that locally, the mechanism of rupture is predominantly tensile. Knowledge gained from this study will aid in the development of methods for prediction/prevention of thrombotic embolization.

Ultrasensitive, selective, and non-invasive detection of fibrin in human serum is critical for disease diagnosis. So far, the development of high-performance and ultrasensitive biosensors maintains core challenges for biosensing. Herein, we designed a novel ribbon nanoprobe for ultrasensitive detection of fibrin. The probe contains gold nanoparticles (AuNPs) that can not only link with homing peptide Cys-Arg-Glu-Lys-Ala (CREKA) to recognize fibrin but also carry long DNA belts to form G-quadruplex-based DNAzyme, catalyzing the chemiluminescence of luminol-hydrogen peroxide (H 2 O 2 ) reaction. Combined with the second amplification procedure of rolling circle amplification (RCA), the assay exhibits excellent sensitivity with a detection limit of 0.04 fmol L -1 fibrin based on the 3-sigma. Furthermore, the biosensor shows high specificity on fibrin in samples because the structure of antibody-fibrin-homing peptide was employed to double recognize fibrin. Altogether, the simple and inexpensive approach may present a great potential for reliable detection of biomarkers., (© 2024 John Wiley & Sons Ltd.)

Background:  Factor XIII (FXIII) forms covalent crosslinks across plasma and cellular substrates and has roles in hemostasis, wound healing, and bone metabolism. FXIII activity is implicated in venous thromboembolism (VTE) and is a target for developing pharmaceuticals, which requires understanding FXIII - substrate interactions. Previous studies proposed the β-sandwich domain of the FXIII A subunit (FXIII-A) exhibits substrate recognition sites., Material and Methods:  Recombinant FXIII-A proteins (WT, K156E, F157L, R158Q/E, R171Q, and R174E) were generated to identify FXIII-A residues mediating substrate recognition. Proteolytic (FXIII-A*) and non-proteolytic (FXIII-A°) forms were analyzed for activation and crosslinking activities toward physiological substrates using SDS-PAGE and MALDI-TOF MS., Results:  All FXIII-A* variants displayed reduced crosslinking abilities compared to WT for Fbg αC (233 - 425), fibrin, and actin. FXIII-A* WT activity was greater than A°, suggesting the binding site is more exposed in FXIII-A*. With Fbg αC (233 - 425), FXIII-A* variants R158Q/E, R171Q, and R174E exhibited decreased activities approaching those of FXIII-A°. However, with a peptide substrate, FXIII-A* WT and variants showed similar crosslinking suggesting the recognition site is distant from the catalytic site. Surprisingly, FXIII-A R158E and R171Q displayed slower thrombin activation than WT, potentially due to loss of crucial H-bonding with neighboring activation peptide (AP) residues., Conclusion:  In conclusion, FXIII-A residues K156, F157, R158, R171, and R174 are part of a binding site for physiological substrates [fibrin (α and γ) and actin]. Moreover, R158 and R171 control AP cleavage during thrombin activation. These investigations provide new molecular details on FXIII - substrate interactions that control crosslinking abilities., Competing Interests: None declared., (Thieme. All rights reserved.)

Functional regeneration of anisotropically aligned tissues such as ligaments, microvascular networks, myocardium, or skeletal muscle requires a temporal and spatial series of biochemical and biophysical cues to direct cell functions that promote native tissue regeneration. When these cues are lost during traumatic injuries such as volumetric muscle loss (VML), scar formation occurs, limiting the regenerative capacity of the tissue. Currently, autologous tissue transfer is the gold standard for treating injuries such as VML but can result in adverse outcomes including graft failure, donor site morbidity, and excessive scarring. Tissue-engineered scaffolds composed of biomaterials, cells, or both have been investigated to promote functional tissue regeneration but are still limited by inadequate tissue ingrowth. These scaffolds should provide precisely tuned topographies and stiffnesses using proregenerative materials to encourage tissue-specific functions such as myoblast orientation, followed by aligned myotube formation and recovery of functional contraction. In this study, we describe the design and characterization of novel porous fibrin scaffolds with anisotropic microarchitectural features that recapitulate the native tissue microenvironment and offer a promising approach for regeneration of aligned tissues. We used directional freeze-casting with varied fibrin concentrations and freezing temperatures to produce scaffolds with tunable degrees of anisotropy and strut widths. Nanoindentation analyses showed that the moduli of our fibrin scaffolds varied as a function of fibrin concentration and were consistent with native skeletal muscle tissue. Quantitative morphometric analyses of myoblast cytoskeletons on scaffold microarchitectures demonstrated enhanced cell alignment as a function of microarchitectural morphology. The ability to precisely control the anisotropic features of fibrin scaffolds promises to provide a powerful tool for directing aligned tissue ingrowth and enhance functional regeneration of tissues such as skeletal muscle.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly ( N -isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Introduction: Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis., Methods: Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers., Results: No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness., Conclusions: Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture., (© 2023 John Wiley & Sons Ltd.)

Background: Blood clots are primarily composed of red blood cells (RBCs), platelets/thrombocytes, and fibrin. Despite the similarities observed between mammals and zebrafish, the composition of fish thrombi is not as well known., Objectives: To analyze the formation of zebrafish blood clots ex vivo and arterial and venous thrombi in vivo., Methods: Transgenic zebrafish lines and laser-mediated endothelial injury were used to determine the relative ratio of RBCs and thrombocytes in clots. Scanning electron and confocal microscopy provided high-resolution images of the structure of adult and larval clots. Adult and larval thrombocyte spreading on fibrinogen was evaluated ex vivo., Results: RBCs were present in arterial and venous thrombi, making up the majority of cells in both circulations. However, bloodless mutant fish demonstrated that fibrin clots can form in vivo in the absence of blood cells. Scanning electron and confocal microscopy showed that larval and adult zebrafish thrombi and mammalian thrombi look surprisingly similar externally and internally, even though the former have nucleated RBCs and thrombocytes. Although adult thrombocytes spread on fibrinogen, we found that larval cells do not fully activate without the addition of plasma from adult fish, suggesting a developmental deficiency of a plasma activating factor. Finally, mutants lacking αIIbβ3 demonstrated that this integrin mediates thrombocyte spreading on fibrinogen., Conclusion: Our data showed strong conservation of arterial and venous and clot/thrombus formation across species, including developmental regulation of thrombocyte function. This correlation supports the possibility that mammals also do not absolutely require circulating cells to form fibrin clots in vivo., Competing Interests: Declaration of competing interests J.A.S. has been a consultant for Sanofi, Takeda, Genentech, CSL Behring, and HEMA Biologics. M.H. is a consultant and equity holder for Veralox Therapeutics and Cereno Scientific. M.S.G., A.R.D., C.N., K.K., C.M., R.I.L., R.F., R.J.F., M.N.-A., J.W.W., and R.A., have no conflicts of interest to declare., (Copyright © 2023. Published by Elsevier Inc.)

There is a significant clinical need to develop effective vascularization strategies for tissue engineering and the treatment of ischemic pathologies. In patients afflicted with critical limb ischemia, comorbidities may limit common revascularization strategies. Cell-encapsulating modular microbeads possess a variety of advantageous properties, including the ability to support prevascularization in vitro while retaining the ability to be injected in a minimally invasive manner in vivo. Here, fibrin microbeads containing human umbilical vein endothelial cells (HUVEC) and bone marrow-derived mesenchymal stromal cells (MSC) were cultured in suspension for 3 days (D3 PC microbeads) before being implanted within intramuscular pockets in a SCID mouse model of hindlimb ischemia. By 14 days post-surgery, animals treated with D3 PC microbeads showed increased macroscopic reperfusion of ischemic foot pads and improved limb salvage compared to the cellular controls. Delivery of HUVEC and MSC via microbeads led to the formation of extensive microvascular networks throughout the implants. Engineered vessels of human origins showed evidence of inosculation with host vasculature, as indicated by erythrocytes present in hCD31+ vessels. Over time, the total number of human-derived vessels within the implant region decreased as networks remodeled and an increase in mature, pericyte-supported vascular structures was observed. Our findings highlight the potential therapeutic benefit of developing modular, prevascularized microbeads as a minimally invasive therapeutic for treating ischemic tissues., (© 2023 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Platelets play a pivotal role in hemostasis and wound healing and conditional shape change is an important component of platelet functionality. In normal circumstances, platelets travel through the circulatory system in an inactive rounded state, which enables platelets to easily move to vessel walls for attachment. When an injury occurs, platelets are prompted by molecules, such as thrombin, to shift into a stellate shape and increase exposure of fibrin-binding receptors. When active, platelets promote hemostasis and clot retraction, which enhances clot stability and promotes healing. However, in conditions where platelets are depleted or hyporeactive, these functions are diminished and lead to inhibited hemostasis and healing. To treat platelet depletion, our group developed platelet-like particles (PLPs) which consist of highly deformable microgels coupled to fibrin binding motif. However, first generation PLPs do not exhibit wound-triggered shape change like native platelets. Thus, the objective of these studies was to develop a PLP formulation that changes shape when prompted by thrombin. To create thrombin-sensitive PLPs (TS-PLPs), we incorporated a thrombin-cleavable peptide into the microgel body and then evaluated PLP properties before and after exposure to thrombin including morphology, size, and in vitro clot retraction. Once thrombin-prompted shape change ability was confirmed, the TS-PLPs were tested in vivo for hemostatic ability and subsequent wound healing outcomes in a murine liver trauma model. We found that TS-PLPs exhibit a wound-triggered shape change, induce significant clot retraction following exposure to thrombin and promote hemostasis and healing in vivo after trauma., (© 2023 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Studies have long sought to develop engineered heart tissue for the surgical correction of structural heart defects, as well as other applications and vascularization of this tissue has presented a challenge. Recent studies suggest that vascular cells and a vascular network may have regenerative effects on implanted cardiomyocytes (CM) and nearby heart tissue separate from perfusion of oxygen and nutrients. The goal of this study was to test whether vascular cells or a formed vascular network in a fibrin-based hydrogel would alter the proliferation of human iPSC-derived CM. First, vascular network formation in a slowly degrading PEGylated fibrin hydrogel was optimized by altering the cell ratio of human umbilical vein endothelial cells to human dermal fibroblasts, the inclusion of growth factors, and the total cell concentration. An endothelial to fibroblast ratio of 5:1 and a total cell concentration of 1.1 × 10 6 cells/mL without additional growth factors generated robust vascular networks while minimizing the number of cells required. Using this optimized system, human iPSC-derived CM were cultured on hydrogels without vascular cells, hydrogels with unorganized encapsulated vascular cells, or hydrogels with encapsulated vascular cells organized into networks for 7 days. CM proliferation and gene expression were assayed following 7 days of culture on the hydrogels. The presence of vascular cells in the hydrogel, whether unorganized or in vascular networks, significantly increased CM proliferation compared to an acellular hydrogel. Hydrogels with unorganized vascular cells resulted in lower CM maturity evidenced by decreased expression of cardiac troponin t (TNNT2), myosin light chain 7, and phospholamban compared to hydrogels without vascular cells and hydrogels with vascular networks. Altogether, this study details a robust method of forming rudimentary vascular networks in a fibrin-based hydrogel and shows that a hydrogel containing endothelial cells and fibroblasts can induce proliferation in adjacent CM, and these cells do not hinder CM gene expression when organized into a vascular network., (© 2023 Wiley Periodicals LLC.)

The development of stable (bio)hybrid constructs composed of scaffolds and (bio)matrices is a major challenge in the field of tissue engineering. In the present work, the adhesion of fibrin-based hydrogels to the surface of polythioether-based polymers relevant to the 3D printing of polymer scaffolds produced by thiol-ene click chemistry was investigated. Adhesion properties were characterized by single-lap tensile shear testing. Both the sample preparation and the test method were optimized for the analysis of fibrin gel bonding to the polythioether surface. Our experimental results show that even without further modification, an adhesion between the fibrin hydrogel and polythioether is substantial, with an adhesion strength of 4.9 ± 1.0 kPa. To further improve the bonding, linear functional poly( N -vinylpyrrolidone- co -glycidyl methacrylate) (PVP- co -GMA) copolymers were used that are known for covalently binding to fibrin. The maximum adhesion strength in our study was found to be 18.4 ± 3.4 kPa. The pure PVP- co -GMA copolymers also demonstrate covalent binding to the thiol-ene-based polymers with a maximum adhesion strength of 32.2 ± 2.7 kPa. Therefore, compared to pure fibrin, the presence of copolymer coating both on the polythioether surface and in the fibrin gel led to a significant increase of the adhesion strength by a factor of 1.6.