Local delivery of platelet-derived factors mitigates ischemia and preserves ovarian function through angiogenic modulation: A personalized regenerative strategy for fertility preservation.

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).

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels.

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.

A modular approach to 3D-printed bilayer composite scaffolds for osteochondral tissue engineering.

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.

Ex Ovo Chorioallantoic Membrane Assay as a Model of Bone Formation by Biomaterials.

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.

Modulating Coagulation via Bioinspired Mesoporous Calcium-Decorated Silica Nanoparticles for Efficient Fibrin Clot Formation.

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.

Development and Epitope Mapping of Seven Mouse Anti-Human Coagulation Factor XIII-B Subunit Monoclonal Antibodies.

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.

Effect of oxidized Bletilla striata polysaccharide on fibrin hydrogel formation and its application in wound healing dressing.

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.

Acoustically responsive scaffolds: Unraveling release kinetics and mechanisms for sustained, steady drug delivery.

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.

Cryopreserved nanostructured fibrin-agarose hydrogels are efficient and safe hemostatic agents.

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.

Development of a Methodology Based on Optical Interferometry for Measuring Fibrinolytic Activity.

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.

LSPR-enhanced photoresponsive antibacterial efficiency of Bi/MoS2-loaded fibrin gel for management of diabetic wounds.

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/MoS2 nano-heterojunctions. Capitalizing on the synergistic interplay between photocatalytic antibacterial and photothermal antibacterial mechanisms, the Bi/MoS2 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.

Shielding against breast tumor relapse with an autologous chemo-photo-immune active Nano-Micro-Sera based fibrin implant.

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.

Fabrication of chitin-fibrin hydrogels to construct the 3D artificial extracellular matrix scaffold for vascular regeneration and cardiac tissue engineering.

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 ß-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.

The radial dynamics and acoustic emissions of phase-shift droplets are impacted by mechanical properties of tissue-mimicking hydrogels.

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.

A Protease from Moringa oleifera Lam. Exhibits In-vitro Blood Clot Solubilization and Fibrin Hydrolysis.

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 oC with 50 µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. Km and Vmax 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.

Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator.

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.

Fibrin Hydrogels Reinforced by Reactive Microgels for Stimulus-Triggered Drug Administration.

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.

Advancing Thrombosis Research: A Novel Device for Measuring Clot Permeability.

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.

Fabrication of heart tubes from iPSC derived cardiomyocytes and human fibrinogen by rotating mold technology.

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.

In-vitro and in-silico analyses of the thrombolytic potential of green kiwifruit.

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 Bß 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 Bß 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.