Biochemical Properties and Antithrombotic Effect of a Serine Protease Isolated from the Medicinal Mushroom Pycnoporus coccineus (Agaricomycetes).

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 µg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.

Konjac glucomannan-fibrin composite hydrogel as a model for ideal scaffolds for cell-culture meat.

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.

Evaluation of Fibrin Clot Interaction with Dental Implant after Different Surface Treatments: An In Vitro Study.

AIM: The current study was carried out to assess the interaction between fibrin clots and dental implants following various surface treatments. MATERIALS AND METHODS: In this investigation, 45 dental implants with dimensions of 16 mm in length and 5 mm in diameter were utilized. They were divided up into three groups, each consisting of fifteen samples. Group I: Control; Group II: Ultraviolet (UV) light treated; and group III: Sandblasted and acid-etching (SLA) treated. Healthy volunteers' venous blood samples were drawn into vacutainer tubes without the use of anticoagulants. The samples were centrifuged for 3 minutes at 2700 rpm in a table centrifuge. The entire implant was submerged in room-temperature liquid fibrinogen for 60 minutes. Then, scanning electronic microscopy (SEM) was used to examine each sample. The inter- and intragroup assessments were obtained using the Mann-Whitney U test and the Kruskal-Wallis test; p-values less than 0.05 were regarded as statistically significant. RESULTS: The maximum adhesion of fibrin clot was found in SLA treated group (2.42 ± 0.10) followed by the UV light-treated group (2.18 ± 0.08) and control group (1.20 ± 0.02). There was a statistically significant difference found between the three surface-treated groups (p < 0.001). CONCLUSION: All surface-treatment methods exhibit adhesion between the implant surface and the fibrin clot. However, the highest adherence of fibrin clot was found in SLA treated group compared to the UV light-treated and control group. CLINICAL SIGNIFICANCE: The physical and chemical characteristics of an implant's surface have a significant impact on the way blood clots organize. At the interface between the implant and the bone, blood clot production can initiate and facilitate the healing process. How to cite this article: Jalaluddin M, Ramanna PK, Swain M, et al. Evaluation of Fibrin Clot Interaction with Dental Implant after Different Surface Treatments: An In Vitro Study. J Contemp Dent Pract 2024;25(3):276-279.

Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

The procoagulant signature of cancer cells drives fibrin network formation in tumor microenvironment and impacts its quality. Implications in cancer cell migration and the resistance to anticancer agents.

INTRODUCTION: Cancer cells induce hypercoagulability in the tumoral microenvironment by expressing Tissue Factor (TF). We aimed to study the impact of the procoagulant signature of cancer cells on the quality and structure of fibrin network. We also studied the impact of fibrin clot shield (FCS) on the efficiency of anticancer agents and the migration of cancer cells. MATERIALS AND METHODS: Pancreatic cancer cells BXPC3 and breast cancer cells MDA-MB231 and MCF7, were cultured in the presence of normal Platelet Poor Plasma (PPP), diluted 10 % in conditioning media. Their potential to induce thrombin generation and their fibrinolytic activity were assessed. The structure of fibrin network was analyzed with Scanning Electron Microscopy (SEM). Cancer cells' mobility with fibrin clot and their interactions with fibrin were observed. Cancer cells were treated with paclitaxel (PTX) or 4-hydroxy-tamoxifen (4OHTam) in the presence or absence of FCS. RESULTS: Cancer cells, in presence of PPP, induced fibrin network formation. High TF-expressing cancer cells (BXPC3 and MDA-MB23 cells), led to dense fibrin network with fine fibers. Low TF expressing cells MCF7 led to thick fibers. Exogenous TF enhanced the density of fibrin network formed by MCF7 cells. Cancer cells through their inherent profibrinolytic potential migrated within the fiber scaffold. The BXPC3 and MCF7 cells moved in clusters whereas the MDA-MB231 cells moved individually within the fibrin network. FCS decreased the efficiency of PTX and 4OHTam on the viability of cancer cells. CONCLUSIONS: The procoagulant signature of cancer cells is determinant for the quality and structure of fibrin network in the microenvironment. Original SEM images show the architecture of "bird's nest"-like fibrin network being in touch with the cell membranes and surrounding cancer cells. Fibrin network constructed by triggering thrombin generation by cancer cells, provides a scaffold for cell migration. Fibrin clot shields protect cancer cells against PTX and 4OHTam.

Water content for clot composition prediction in acute ischemic stroke.

BACKGROUND: Mechanical thrombectomy (MT) has become the gold standard care for treating acute ischemic stroke (AIS) due to large vessel occlusion. Emerging evidence suggests that understanding the composition of clots prior to intervention could be useful for the selection of neuroendovascular techniques, potentially improving the efficacy of treatments. However, current imaging modalities lack the ability to distinguish clot composition accurately and reliably. Since water content can influence signal intensity on CT and MRI scans, its assessment may provide indirect clues about clot composition. This study aimed to elucidate the correlation between water content and clot composition using human clots retrieved from stroke patients and experimentally generated ovine clots. MATERIALS AND METHODS: This study involved an analysis of ten clots retrieved from patients with AIS undergoing MT. Additionally, we created ten red blood cells (RBC)-rich and ten fibrin-rich ovine blood clots, which were placed in a human intracranial vascular model under realistic flow conditions. The water content and compositions of these clots were evaluated, and linear regression analyses were performed to determine the relationship between clot composition and water content. RESULTS: The regression analysis in human stroke clots revealed a significant negative association between RBC concentration and water content. We also observed a positive correlation between water content and both fibrin and platelets in ovine blood clots. Conclusion.

Blood Coagulation-Inspired Fibrin Hydrogel for Portable Detection of Thrombin Based on Personal Glucometer.

As one of the biomarkers of coagulation system-related diseases, the detection of thrombin is of practical importance. Thus, this study developed a portable biosensor based on a personal glucometer for rapid detection of thrombin activity. Fibrinogen was used for the detection of thrombin, and the assay principle was inspired by the blood coagulation process, where thrombin hydrolyzes fibrinogen to produce a fibrin hydrogel, and the amount of invertase encapsulated in the fibrin hydrogel fluctuates in accordance with the activity of thrombin in the sample solution. The quantitative assay is conducted by measuring the amount of unencapsulated invertase available to hydrolyze the substrate sucrose, and the signal readout is recorded using a personal glucometer. A linear detection range of 0-0.8 U/mL of thrombin with a limit of detection of 0.04 U/mL was obtained based on the personal glucometer sensing platform. The results of the selectivity and interference experiments showed that the developed personal glucometer sensing platform is highly selective and accurate for thrombin activity. Finally, the reliability of the portable glucometer method for rapid thrombin detection in serum samples was investigated by measuring the recovery rate, which ranged from 92.8% to 107.7%. In summary, the fibrin hydrogel sensing platform proposed in this study offers a portable and versatile means for detecting thrombin using a personal glucometer. This approach not only simplifies the detection process, but also eliminates the need for large instruments and skilled operators, and substantially reduces detection costs.

Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Cascade encapsulation of antimicrobial peptides, exosomes and antibiotics in fibrin-gel for first-aid hemostasis and infected wound healing.

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-1ß, 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.

A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged ß 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 ß cells.

A Case of Pseudothrombocytopenia due to Pre-Analytical Issues Revealed by the Presence of Fibrin in a Blood Film.

BACKGROUND: Pseudothrombocytopenia (PTCP) can be caused by anticoagulants or pre-analytical issues. The authors present a case of PTCP attributed to pre-analytical issues in a 68-year-old male patient. METHODS: The platelet count results were obtained using both the impedance and fluorescence channels of Sysmex XN-10. The blood film was scanned using both Cellavision DM96 and a microscope. RESULTS: The flag for PLT-Clumps and the scattergram from the PLT-F channel indicated the presence of platelet aggregation. Fibrin could be observed at the feathered end of the blood film. A diagnosis of PTCP resulting from pre-analytical issues was made. CONCLUSIONS: The presence of fibrin in a blood film is a critical indicator for diagnosing PTCP due to pre-analytical issues.

A role of methionines in the functioning of oxidatively modified fibrinogen.

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, BßMet367, 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.

A dual amplified gold nanoparticle-based biosensor for ultrasensitive and selective detection of fibrin.

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 (H2O2) 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.

Thrombus histology does not predict stroke etiological subtype but influences recanalization.

BACKGROUND: There is conflicting data on whether clot retrieved from mechanical thrombectomy can predict stroke etiology or the success of recanalization. We aimed to analyse the relation between thrombus histology and stroke aetiology as well as recanalization. METHODOLOGY: Histopathological analysis of clots retrieved from patients with acute ischemic stroke and large vessel occlusion was done. Quantification of the amount of fibrin, red blood cells(RBC), platelets and white blood cells (WBC) in the clots were done. The clinical, imaging data and recanalization parameters were collected. The correlation between clot composition and stroke etiology as well as recanalization were analysed. RESULTS: Of the 77 patients, the mean age was 58. 67 ± 12.96 years. The stroke etiology were cardioembolism 44(57.1 %), large artery atherosclerosis 13(16.8 %), other determined aetiology 4(5.1 %) and undetermined in 16(20.7 %) patients. There was no significant correlation between the proportions of RBC-rich, platelet-rich and fibrin-rich thrombi and the stroke etiology. The susceptibility vessel sign was associated with RBC-rich clot(92.3 % vs 7.7 %, p = .03). All RBC-rich clots(100 %) had good recanalization(p = .05). Platelet-rich clots needed less number of passes(64.7 % vs 35.3 %, p = .006) and reduced groin puncture to recanalization time(87.9 % vs 12.1 %, p = .033). WBC-rich clots required lesser number of passes(57.5 % vs 42.5 %, P = .044). In multivariate analysis, WBC-rich clots (OR 0.230, CI 0.07-0.78, p = .018) showed an independent association with reduced recanalization attempts, while platelet-rich clots showed reduced recanalization time(OR 0.09, CI 0.01-0.63, p = .016). CONCLUSION: There was no correlation between thrombus histology and the etiological stroke subtype. However, clot composition predicted the degree of recanalization and number of passes.

Polymorphic lymphoproliferative disorder arising in a rheumatoid arthritis patient, presenting as fibrin-associated large B-cell lymphoma-like lesions in aortic and mitral valves.

We herein report a case of methotrexate-associated lymphoproliferative disorder (MTX-LPD) showing fibrin-associated large B-cell lymphoma-like heart valve lesions, and Epstein-Barr virus (EBV)-positive mucocutaneous ulcer-like cutaneous and oral mucosal lesions. MTX-LPD is a critical complication that can occur in RA patients who are treated with MTX. EBV also plays a defining or important role in LPDs. Among the sites of MTX-LPD, 40-50% occur in extranodal sites, including the gastrointestinal tract, skin, liver, lung, and kidney. There are few reports of MTX-LPDs involving the heart valves, and to the best of our knowledge, this is the first case to be reported in the English literature. The possibility of EBV-positive LPD should be considered in RA patients, even in patients with an atypical site, as in this case.

Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays.

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

Fibrin promotes oxidative stress and neuronal loss in traumatic brain injury via innate immune activation.

BACKGROUND: Traumatic brain injury (TBI) causes significant blood-brain barrier (BBB) breakdown, resulting in the extravasation of blood proteins into the brain. The impact of blood proteins, especially fibrinogen, on inflammation and neurodegeneration post-TBI is not fully understood, highlighting a critical gap in our comprehension of TBI pathology and its connection to innate immune activation. METHODS: We combined vascular casting with 3D imaging of solvent-cleared organs (uDISCO) to study the spatial distribution of the blood coagulation protein fibrinogen in large, intact brain volumes and assessed the temporal regulation of the fibrin(ogen) deposition by immunohistochemistry in a murine model of TBI. Fibrin(ogen) deposition and innate immune cell markers were co-localized by immunohistochemistry in mouse and human brains after TBI. We assessed the role of fibrinogen in TBI using unbiased transcriptomics, flow cytometry and immunohistochemistry for innate immune and neuronal markers in Fggγ390-396A knock-in mice, which express a mutant fibrinogen that retains normal clotting function, but lacks the γ390-396 binding motif to CD11b/CD18 integrin receptor. RESULTS: We show that cerebral fibrinogen deposits were associated with activated innate immune cells in both human and murine TBI. Genetic elimination of fibrin-CD11b interaction reduced peripheral monocyte recruitment and the activation of inflammatory and reactive oxygen species (ROS) gene pathways in microglia and macrophages after TBI. Blockade of the fibrin-CD11b interaction was also protective from oxidative stress damage and cortical loss after TBI. CONCLUSIONS: These data suggest that fibrinogen is a regulator of innate immune activation and neurodegeneration in TBI. Abrogating post-injury neuroinflammation by selective blockade of fibrin's inflammatory functions may have implications for long-term neurologic recovery following brain trauma.

Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

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.

Developing Porous Fibrin Scaffolds with Tunable Anisotropic Features to Direct Myoblast Orientation.

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.