Adhesion Of Red Blood Cells Research Articles

Sulfonated polyphenylene oxide-based artificial lung membrane with prominent selectivity of CO2

The primary issues impeding oxygenation efficacy in extracorporeal membrane oxygenation (ECMO) are low CO2/O2 selectivity and insufficient permeability of CO2. Herein, sulfonated polyphenylene oxide (SPPO) was chosen as a candidate for the oxygenation membrane material. The CO2-affinitive sulfonic acid groups on the molecular chain of SPPO enhance the interaction between CO2 and the SPPO membrane; thereby, the CO2/O2 selectivity is effectively enhanced. When the solid content of the casting solution is 23%, the SPPO membrane achieves a CO2/O2 selectivity of 17.0, almost as selective as human alveoli. The blood clotting time, red blood cell adhesion, platelet adhesion, and hemolysis ratio were employed to evaluate the blood compatibility of SPPO materials. The results demonstrated that the manufactured SPPO membranes exhibited good hemocompatibility. This work offers novel thinking for researching and applying effective artificial lung membranes.

Serotonin-functionalized starch-based hemostatic sponges enhance platelet activation in the management of non-compressible hemorrhage

Massive hemorrhage poses a serious threat to human health and even life. Access to rapid and potent hemostatic materials is crucial for lowering mortality rates. Starch-based materials exhibit good biocompatibility and are extensively utilized in hemostatic. However, existing starch-based hemostatic products suffer from limited hemostatic efficacy. Serotonin, an indoleamine naturally occurring in the human body, is recognized for its potent platelet-activating properties. Therefore, this study focused on enhancing the procoagulant activity of starch by incorporating serotonin into the starch backbone via esterification and amidation reactions, yielding a novel serotonin-loaded starch-based hemostatic sponge (SLS sponge). The SLS sponges featured exceptional porosity (≥80 %) and water absorption capacity (≥2000 %), which rapidly initiated the coagulation cascade reaction, promoted the adhesion and aggregation of red blood cells and platelets, and intensified platelet activation. This multifaceted approach synergistically enhanced hemostasis via both active and passive coagulation mechanisms. Notably, the SLS sponges adhered firmly to the wound surfaces without pressure. Compared with gelatin sponges, the SLS sponges significantly reduced blood loss by approximately 40.5 % and shortened the time to achieve hemostasis by approximately 28.9 %. These findings indicate that the newly developed SLS sponges are a promising active hemostatic material, particularly effective in managing non-compressible hemorrhages.

Hemostatic Dressing Immobilized with ε-poly-L-lysine and Alginate Coated Mesoporous Bioactive Glass Prevents Blood Permeation by Pseudo-Dewetting Behavior.

The integration of hemostats with cotton fabrics is recognized as an effective approach to improve the hemostatic performance of dressings. However, concerns regarding the uncontrollable absorption of blood by hydrophilic dressings and the risk of distal thrombosis from shed hemostatic agents are increasingly scrutinized. To address these issues, this work develops an advanced dressing (AQG) with immobilized nano-scale mesoporous bioactive glass (MBG) to safely and durably augment hemostasis. The doubly immobilized MBGs, pre-coated with ε-poly-L-lysine and alginate, demonstrate less than 1% detachment after ultrasonic washing. Notably, this MBG layer significantly promotes the adhesion, aggregation, and activation of red blood cells and platelets, adhered five times more red blood cells and 29 times more platelets than raw dressing, respectively. Specially, with the rapid formation of protein corona and amplification of thrombin, dense fibrin network is built on MBG layer and then blocked blood permeation transversely and longitudinally, showing an autophobic pseudo-dewetting behavior and allowing AQG to concentrate blood in situ and culminate in faster hemostasis with lower blood loss. Furthermore, the potent antibacterial properties of AQG extend its potential for broader application in daily care and clinical setting.

Injectable and rapidly expandable thrombin-decorated cryogels achieve rapid hemostasis and high survival rates in a swine model of lethal junctional hemorrhage

Effective therapies are urgently needed to stabilize patients with marginally compressible junctional hemorrhage long enough to get them to the hospital alive. Herein, we report injectable and rapidly expandable cryogels consisting of polyacrylamide and thrombin (AT cryogels) created by cryo-polymerization for the efficient management of lethal junctional hemorrhage in swine. The produced cryogels have small pore sizes and highly interconnected porous architecture with robust mechanical strength. The cryogels exhibit rapid shape memory properties and prove to be resilient against fatigue. These cryogels also show high water/blood absorption capacity, fast blood clotting effect, and enhanced adhesion of red blood cells and platelets in vitro. Further, in vivo, hemostatic efficacy tests in a lethal swine junctional hemorrhage model suggest that treatment with AT cryogels, especially AT-2 cryogels, achieves the least blood loss and the highest survival rate (100 %) compared to currently employed products such as XStat® and combat gauze. The high hemostatic performance of the cryogels may be attributed to highly interconnected porous architecture with small pore size and the use of thrombin as a pro-coagulant agent. Collectively, injectable and rapidly expandable thrombin-decorated polyacrylamide-based cryogels show significant promise as hemostatic material, offering effective management of marginally compressible junctional hemorrhages in prehospital settings.

RBC aggregation, deformation and adhesion to endothelium: Role of nitric oxide derived from L-Arginine and sodium nitroprusside

Red blood cells (RBCs) are the most abundant human blood cells. RBC aggregation and deformation strongly determine blood viscosity which impacts hemorheology and microcirculation. In turn, RBC properties depend on different endogenous and exogenous factors. One such factor is nitric oxide (NO), which is mainly produced by endothelial cells (EC) from L-arginine amino acid in the circulatory system. Since the mechanisms of the RBC-endothelium interplay are not clear up to date and considering its possible clinical importance, the aims of this study are to investigate in vitro: (1) The effect of L-arginine induced NO on RBC aggregation and adhesion to endothelium; (2) the NO effect on RBC aggregation and deformation induced by L-arginine and sodium nitroprusside without the presence of endothelium in the samples. The RBC aggregation and adhesion to a monolayer of EC were studied using optical tweezers (OT). The RBC deformability and aggregation without endothelium in the samples were studied using the flow chamber method and Myrenne aggregometer. We confirmed that NO increases deformability and decreases aggregation of RBCs. We showed that the soluble guanylate cyclase pathway appears to be the only NO signaling pathway involved. In the samples with the endothelium, the “bell-shaped” dependence of RBC aggregation force on L-arginine concentration was observed, which improves our knowledge about the process of NO production by endothelium. Additionally, data related to L-arginine accumulation by endothelium were obtained: Necessity of the presence of extracellular L-arginine stated by other authors was put under question. In our study, NO decreased the RBC-endothelium adhesion, however, the tendency appeared to be weak and was not confirmed in another set of experiments. To our knowledge, this is the first attempt to measure the forces of RBC adhesion to endothelium monolayer with OT.

Forces of interaction of red blood cells and endothelial cells at different concentrations of fibrinogen: Measurements with laser tweezers in vitro.

Blood microrheology depends on the constituents of blood plasma, the interaction between blood cells resulting in red blood cell (RBC) and platelets aggregation, and adhesion of RBC, platelets and leukocytes to vascular endothelium. The main plasma protein molecule -actuator of RBC aggregation is fibrinogen. In this paper the effect of interaction between the endothelium and RBC at different fibrinogen concentrations on the RBC microrheological properties was investigated in vitro. Laser tweezers were used to measure the RBC-endothelium interaction forces. It was shown for the first time that the interaction forces between RBC and endothelium are comparable with the RBC aggregation forces, they increase with fibrinogen concentration and reach the saturation level of about 4 pN at the concentration of 4 mg/ml. These results are important for better understanding the mechanisms of RBC and endothelium interaction and developing the novel therapeutic protocols of the microrheology correction in different pathologies.

A super hydrophilic and high strength chitosan hemostatic sponge prepared by freeze-drying and alkali treatment for rapid hemostasis

Excessive blood loss caused by natural accidents or major surgical procedures can be life-threatening and requires rapid hemorrhage management. Developing hemostatic materials with high water absorption capacity and high strength properties is still challenging. Super hydrophilic/high-strength chitosan hemostatic sponges (SHHS-CHS) were prepared by freeze-drying and alkali treatment. The freeze-drying technology formed a porous three-dimensional structure of sponges, and alkali treatment increased the hydrophilicity of sponges and the rigidity of the chitosan molecular chain, improving the hydrophilicity and mechanical properties of chitosan sponges. Characterization experiments revealed that SHHS-CHS had high water absorption capacity, rich pore structure, and excellent mechanical properties. The cytotoxicity and hemolysis experiments showed that SHHS-CHS had good biocompatibility. Compared to the commercial hemostatic gelatin sponges and chitosan hemostatic material, SHHS-CHS had better blood coagulation and red blood cell and platelet adhesion ability. Moreover, SHHS-CHS showed better hemostatic ability than gelatin sponges in rat-liver injury and rat leg vein rupture models, significantly reducing the hemostasis time and bleeding volume. In conclusion, SHHS-CHS has broad prospects for clinical application as a hemostatic material.

Multifunctional polyvinyl alcohol/gallic acid functionalized chitosan hydrogels prepared by freeze-thaw method for potential application as wound dressings

The design of multifunctional hydrogel dressings to effectively promote wound healing is highly desirable in clinical trauma and surgery. Inspired by excellent mechanical properties of polyvinyl alcohol (PVA) and strong adhesion of polyphenols, gallic acid was grafted onto chitosan molecular chain (CS-GA) by free radical polymerization in this study, and PVA/CS-GA hydrogels were successfully prepared by repeated freeze-thaw method, without introducing toxic crosslinking agents. The prepared PVA/CS-GA hydrogels could adhere to various substrates, which confirmed excellent universal adhesion properties. The red blood cells adhesion ratio was 67.1%, indicating its potential for promoting wound coagulation. Meanwhile, the mechanical strength of PVA/CS-GA hydrogel was significantly enhanced, exhibiting approximately twice tensile strength and failure stretch compared with PVA hydrogel. Furthermore, attributing to GA molecule, PVA/CS-GA hydrogels not only showed favorable antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), but also could scavenge free radicals, showing effective antioxidant activity. The hydrogels also had good biocompatibility, met the basic requirements of biomedical materials and may promote wound repair. It should be noted that the content of GA had significant impact on cytotoxicity. Therefore, multifunctional PVA/CS-GA hydrogels with excellent mechanical, adhesion, antioxidant, antibacterial and biocompatible properties show promising potential applications in the biomedicine field, especially as functional wound dressings.

Multifunctional coatings of nickel-titanium implant toward promote osseointegration after operation of bone tumor and clinical application: a review.

Metal implants, especially Ni-Ti shape memory alloy (Ni-Ti SMA) implants, have increasingly become the first choice for fracture and massive bone defects after orthopedic bone tumor surgery. In this paper, the internal composition and shape memory properties of Ni-Ti shape memory alloy were studied. In addition, the effects of porous Ni-Ti SMA on osseointegration, and the effects of surface hydrophobicity and hydrophilicity on the osseointegration of Ni-Ti implants were also investigated. In addition, the effect of surface coating modification technology of Ni-Ti shape memory alloy on bone bonding was also studied. Several kinds of Ni-Ti alloy implants commonly used in orthopedic clinic and their advantages and disadvantages were introduced. The surface changes of Ni-Ti alloy implants promote bone fusion, enhance the adhesion of red blood cells and platelets, promote local tissue regeneration and fracture healing. In the field of orthopaedics, the use of Ni-Ti shape memory alloy implants significantly promoted clinical development. Due to the introduction of the coating, the osseointegration and biocompatibility of the implant surface have been enhanced, and the success rate of the implant has been greatly improved.

A novel composition of endogenous metabolic modulators improves red blood cell properties in sickle cell disease.

The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism. The effects of ex vivo treatment with the EMM composition on different RBC properties were studied in SCD (n=9 SCA, n=5 HbSC disease). Dose-dependent improvements were observed in RBC hydration assessed by hemocytometry (MCV, MCHC, dense RBCs) and osmotic gradient ektacytometry (Ohyper). Median (interquartile range [IQR]) increase in Ohyper compared to vehicle was 4.9% (4.0%-5.5%), 7.5% (6.9%-9.4%), and 12.8% (11.5%-14.0%) with increasing 20×, 40×, and 80X concentrations, respectively (all p<0.0001). RBC deformability (EImax using oxygen gradient ektacytometry) increased by 8.1% (2.2%-12.1%; p=0.0012), 9.6% (2.9%-15.1%; p=0.0013), and 13.3% (5.7%-25.5%; p=0.0007), respectively. Besides, RBC adhesion to subendothelial laminin decreased by 43% (6%-68%; p=0.4324), 58% (48%-72%; p=0.0185), and 71% (49%-82%; p=0.0016), respectively. Together, these results provide a rationale for further studies with the EMM composition targeting multiple RBC properties in SCD.

Silk fibroin-gelatine haemostatic sponge loaded with thrombin for wound haemostasis and tissue regeneration.

Wound haemostasis is an important part of clinical treatments, especially treatments for patients with avulsion injury, destructive injury and large-scale soft tissue injury. Therefore, developing fast and effective haemostatic materials is critical. This study aimed to design a novel and efficient silk fibroin-gelatine composite haemostatic sponge loaded with thrombin (SFG@TB) to assist in wound haemostasis. The SFG@TB composite haemostatic sponge was formed with gelatine, silk fibroin and thrombin through a freeze-drying technique. First, the material characteristics of SFG@TB were measured, including the elastic modulus, swelling rate and porosity. Second, in vitro cell coculture experiments, in vivo embedding experiments and haemolytic analyses were performed to evaluate the biocompatibility of SFG@TB. Then, coagulation experiments and femoral artery and liver bleeding models were used to evaluate the haemostatic performance of SFG@TB. Finally, the ability of SFG@TB to promote tissue healing was evaluated through experiments with Sprague-Dawley rat models of injury. Compared with gelatine sponges, SFG@TB exhibited outstanding mechanical properties and water absorption properties. In addition, the excellent biosafety of the composite haemostatic sponge was confirmed by cell experiments, subcutaneous embedding experiments and haemolytic analysis. Based on the in vitro coagulation test results, SFG@TB exhibited greater adhesion of red blood cells and platelets and a shorter dynamic coagulation time. Compared to the use of silk fibroin-gelatine composite haemostatic sponges or gelatine sponges, the introduction of thrombin resulted in a shorter haemostasis time and a smaller bleeding volume, as revealed by in vivo coagulation tests. The experiments with Sprague-Dawley rat models of injury indicated that SFG@TB accelerated the wound healing process and reduced scar width, which was accompanied by thicker granulation tissue. Overall, the SFG@TB composite haemostatic sponge, which exhibits outstanding mechanical properties, good haemostatic performance and high biosafety, promoted wound haemostasis and tissue repair. Therefore, the SFG@TB composite haemostatic sponge could be a promising material for wound haemostasis.

Pyruvate Kinase Thermostability Is Associated with Red Blood Cell Adhesion, Deformability and Oxygen Affinity in Patients with Sickle Cell Disease

Introduction: Sickle cell disease(SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell's structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology. Methods: Homozygous HbS (HbSS) and HbS/ β0 thalassemia patients were eligible to participate. Patients who received blood transfusion (&amp;lt;3 months) were excluded. Dense RBCs (MCHC &amp;gt;41 g/dL, Advia 120/2120 Siemens) and HbF and HbS levels (Tosoh G8) were measured. PK and hexokinase (HK) activity were measured on purified RBCs. PK thermostability (reflected by % residual PK activity) was measured on purified RBCs lysates after one hour of incubation (53°C). RBC adhesion to laminin (Biolamina) was measured using a microfluidic device (IBIDI µ-Slide I 0.4). RBC hydration (Ohyper), RBC deformability (EImax) and point of sickling (PoS) were assessed by osmotic and oxygen gradient ektacytometry using the Laser-Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics). Oxygen affinity (P50) was measured with the Hemox-Analyzer (TCS). Statistical analysis was performed in Graphpad Prism (Pearson's correlation). Results: Fifty-seven patients with SCD (53 HbSS, 4 HbS/β 0 thalassemia) were included of which 17 were children (median age 14 years [range 6-17]) and 40 were adults (median age 36 years [range 18-58]). Baseline characteristics are depicted in Table 1. Figure 1 shows a heatmap with correlation coefficients of all laboratory parameters from Table 1 as measured in this cohort. When focusing on PK properties we found that patients with SCD and lower PK thermostability had significantly higher absolute reticulocyte counts (ARC, p&amp;lt;0.0001), lower hemoglobin (p=0.012) and HbF (p=0.020) levels and increased RBC adhesion to laminin (p=0.007). Furthermore, PK thermostability was significantly correlated with EImax (p=0.017) and inversely correlated with PoS (p=0.011) and P50 (p=0.003), indicating that patients with less stable RBC PK had less deformable RBCs which sickled at a higher oxygen tension. RBC adhesion to laminin was significantly correlated with both ARC (p=0.0003) and dense RBCs (p=0.019). To explore whether our findings were a reflection of reticulocytosis we fractionated whole blood samples of HbSS patients according to cell density. We found that more RBCs from the most dense fraction adhered to laminin than from the less dense, reticulocyte enriched, fraction, even though RBCs from this less dense fraction had reduced PK-thermostability. Additionally, the most dense RBCs had the lowest PK activity, were less deformable and had a higher PoS (data not shown). Altogether these findings indicate that the reduced PK thermostability we found in our SCD cohort is associated with an overall functionally impaired RBC population with increased RBC adhesion and ARC. We found no correlation of markers of RBC dehydration (Ohyper and % dense RBCs) with PK thermostability which suggests that RBCs with reduced PK thermostability are not more dehydrated. Discussion/conclusion: This study shows for the first time a significant correlation between PK thermostability in sickle RBCs and RBC functions, such as adhesion, deformability and oxygen affinity. Our results suggest that enhancing the activity and stability of PK, with PK activators, is an attractive target in SCD that might improve other pathophysiological targets outside RBC metabolism. Future studies are needed to explore how activation of PK will affect these altered sickle RBC properties in vivo.

A Phase 2, Open-Label Study to Assess the Pharmacokinetics, Pharmacodynamics and Safety of Orally Administered Epeleuton in Patients with Sickle Cell Disease

Background. Sickle cell disease (SCD) is a rare hereditary multi-system disorder that is caused by a single mutation in the beta allele of the hemoglobin gene, leading to the production of sickle hemoglobin (HbS). HbS polymerizes when deoxygenated, resulting in red blood cell (RBC) sickling and membrane damage, ultimately leading to a cascade of events which include hemolysis, chronic anemia, inflammation, and vaso-occlusion. Despite recent progress, there is a need for additional disease-modifying therapeutic options. Epeleuton is 15-hydroxy eicosapentaenoic acid (15(S)-HEPE) ethyl ester, a novel synthetic second generation w-3 fatty acid which targets key drivers of hemolysis and multicellular adhesion. In preclinical studies in Townes humanized SCD mice and SCD patient RBCs, epeleuton acts as a disease-modifying agent by simultaneously decreasing (a) systemic and local inflammatory activation, (b) endothelial adhesion, (c) organ damage and (d) intravascular hemolysis and sickling. Epeleuton has been administered to 343 subjects in clinical trials, with a favorable safety and tolerability profile. Objective To evaluate the pharmacokinetics, pharmacodynamics and safety of orally administered epeleuton in patients with SCD. Study Design and Methods. This open-label, phase 2, multicenter study (NCT05861453) will enroll approximately 30 adult patients with a confirmed diagnosis of SCD (HbSS and HbSß0-thalassemia). Participants must have hemoglobin levels of 5.0-9.5g/dL at screening, 2-10 vaso-occlusive crises (VOCs) in the past 12 months, and a high adhesion index at screening defined as RBC laminin adhesion &amp;gt; 400 cells. Concomitant treatment with a stable dose of hydroxyurea is permitted during the study. Key exclusion criteria include receiving an RBC transfusion within three months of enrolment, receiving voxelotor, crizanlizumab or l-glutamine within six weeks of enrolment and, prior hematopoietic stem cell transplant. The trial has a 28-day screening period, followed by a 16-week treatment phase and a 30-day post-treatment follow-up period. Study participants will attend the clinic on 6 occasions at screening, baseline, weeks 4, 8 and 16 (end-of-treatment) and the week 20 post-treatment follow-up visit. Enrolled patients will receive epeleuton 2g BID (4g daily) for 16 weeks. Study endpoints include change from baseline in hemoglobin levels, hemolytic markers, assessments of RBC health, soluble adhesion molecules, RBC adhesion, whole blood adhesion and rates of VOC. Conclusion: Treatments that can simultaneously target hemolysis and cellular adhesion may have an important disease-modifying utility for patients with SCD. The results of this study are anticipated to confirm the potential of epeleuton to address multiple aspects of SCD and to guide the design and conduct of future studies in patients with SCD.

Healthcare Utilization and Cost of Care Analysis of Patients with Sickle Cell Disease with High Vs. Low Biomarkers: A Retrospective Chart Review

Sickle cell disease (SCD) is a red blood cell (RBC) disorder that is associated with frequent and unpredictable vaso-occlusive episodes (VOEs) including pulmonary hypertension, acute chest syndrome, chronic anemia, priapism, splenic and renal dysfunction, and stoke. The total cost to manage a SCD patient with a life expectancy of 64 years is between $1.3-$2.1M. Within the United States alone, up to 67% of adults have ≥3 VOEs per yearthat accounts for most of the healthcare utilization, including 78% of emergency room (ER) visits and 95% of hospitalizations. Despite the same single gene mutation of beta-globin, the frequency of VOEs amongst patients is highly variable. The pathophysiology of VOEs involves a complex interplay between circulating blood cells and elevated plasma factors that promote abnormal interactions to the vascular endothelium. Nearly a half century ago studies demonstrated RBC adhesion strongly correlates with disease severity however only recently standardized testing has become available to assess adhesion clinically. More specifically, we developed a suite of RBC health biomarkers and deployed our flow adhesion bioassays in our landmark, observational study (ELIPSIS). We established that flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA -WB-VCAM) at baseline stratified SCD patients with severe disease phenotypes and validated the clinical predictive value of FA-WB-VCAM two years post-ELIPSIS. The objective of this study was to determine whether baseline FA-WB-VCAM biomarker levels could predict patients more likely to receive direct healthcare contact (ER visits/hospital admissions) to manage SCD complications. This study was a retrospective assessment of clinical FA-WB-VCAM in individuals with SCD during steady state between January 2020 through March 2023. Flow adhesion assays were performed using pulsatile, shear flow (1.67Hz, 1.0 dyne/cm 2). SCD patients that received at least 3 steady state FA-WB-VCAM biomarker evaluations within 1-year were included in this analysis. Clinical site received an IRB waiver to obtain patient-reported ER visits and hospitalizations during relevant time periods. We calculated a composite adhesion index by averaging baseline FA-WB-VCAM biomarker evaluations and established SCD disease severity by quantifying the number of ER visits and hospital admissions one year after the most recent FA-WB-VCAM biomarker evaluation. The non-parametric Wilcoxon matched pairs signed rank test was used to test the statistical differences between groups. Data are presented as mean ± standard error of mean. A p-value &amp;lt; 0.05 was considered statistically significant. Out of a total of 3939 clinical tests performed, 366 samples were obtained for “baseline” indications. Of these, only 28 samples were analyzed from 9 unique patients who met the study criteria described above. The average flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM) in SCD patients with high adhesion phenotypes (&amp;gt;400 cells/mm 2) was significantly higher than the low/moderate SCD cohort (mean high=603±78 cells/mm 2, mean low/moderate=343±28 cells/mm 2, p=0.02). More important, the high adhesion cohort had more ER visits (11 vs. 5) and hospitalizations (24 vs 9) and significantly higher healthcare costs ($35,871 vs. $13,563). This is the first healthcare cost analysis assessing the relationship between SCD patients stratified by their adhesive phenotype using the standardized, clinical adhesion biomarker FA-WB-VCAM, and healthcare utilization. The previous ELIPSIS study established that baseline FA-WB-VCAM stratifies patients based on their risk of developing self-reported VOCs during a 6-month longitudinal observation period and their risk of higher clinical disease severity in a 2-year follow-up study. Consistent with this data, the current study demonstrated that a cohort of SCD patients with elevated baseline adhesion phenotypes based on FA-WB-VCAM had a greater utilization of healthcare resources (ER visits and hospitalizations) and had a higher cost of care during the retrospective observation period. These data suggest that RBC health as indicated by “baseline” FA-WB-VCAM levels may predict lower health care utilization and costs, and that this biomarker may be a plausible surrogate endpoint to assess the pharmaco-economic impact of SCD-modifying therapies.

Visualizing a Nanoscale Lubricant Layer under Blood Flow.

Tethered-liquid perfluorocarbons (TLPs) are a class of liquid-infused surfaces with the ability to reduce blood clot formation (thrombosis) on blood-contacting medical devices. TLP comprises a tethered perfluorocarbon (TP) infused with a liquid perfluorocarbon (LP); this LP must be retained to maintain the antithrombotic properties of the layer. However, the stability of the LP layer remains in question, particularly for medical devices under blood flow. In this study, the lubricant thickness is spatially mapped and quantified in situ through confocal dual-wavelength reflection interference contrast microscopy. TLP coatings prepared on glass substrates are exposed to the flow of 37% glycerol/water mixtures (v/v) or whole blood at a shear strain rate of around 2900 s-1 to mimic physiological conditions (similar to flow conditions found in coronary arteries). Excess lubricant (>2 μm film thickness) is removed upon commencement of flow. For untreated glass, the lubricant is completely depleted after 1 min of shear flow. However, on optimized TLP surfaces, nanoscale films of lubricants (thickness between 100 nm and 2 μm) are retained over many tens of minutes of flow. The nanoscale films conform to the underlying structure of the TP layer and are sufficient to prevent the adhesion of red blood cells and platelets.

Radchip: A Laminin-Lined Red Blood Cell (RBC) Adhesion Device for Functional Characterization and Clinical Evaluation of Sickle RBCs at Steady-State

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the beta-globin gene producing an abnormal hemoglobin (HbS) which, under deoxygenation, polymerizes and causes severe distortion of the red blood cell (RBC). RBCs may be dehydrated, poorly deformable and highly adhesive to the endothelium, with abnormal adhesion being of primary interest to this study. Recently, several new disease modifying drugs have been FDA approved, with more in development. These novel agents target specific aspects of SCD pathophysiology, including adhesion. Changes induced by such therapies are not always detectable by common conventional lab instruments. LORRCA (oxygen-gradient ektacytometry) is the gold-standard for deformability measurement; it has been well described both analytically and clinically in SCD. Adhesion and deformability have a dynamic, yet complementary relationship that we explore here. While these may behave in an interdependent manner, each determinant must be considered independently. There is a growing demand for innovative tools to evaluate rheological properties under physiological conditions, particularly microfluidic devices. However, many current devices are limited in their clinical utility due to, choice of ligand, complexity of design, or reliance on endothelial cell culture (typically HUVEC cells) which are difficult to use, vary with passage number, and do not represent adult SCD endothelium. We developed a Red blood cell Adhesion Chip (RAdChip) to model RBC to endothelial adhesion using laminin, a physiologically relevant ligand, to easily, effectively, and reproducibly measure RBC adhesion. Here, we describe our analytic and clinical validation of our device. Peripheral blood samples were collected in EDTA from individuals with SCD and healthy controls under an IRB approved protocol. RBCs were extracted via centrifugation, washed and resuspended in PBS to a 0.5% HCT. Microfluidic devices were fabricated using polydimethylsiloxane (PDMS). A 5ug/ml solution of recombinant laminin was applied to each channel, then blocked with 1% bovine serum albumin (BSA). Resuspended RBCs were introduced to the device through tubing connected to the inlet port. Harvard Apparatus syringe pumps were used to perfuse samples and wash channels at ~1dyne/cm 2. Adhesion was visualized with a Keyence inverted bright-field microscope at 20x (Itasca, IL). Adhered cells were counted within a 1.8mm 2 region of interest (Roi) using the iCLOTS automated cell quantification software (Fay et al, Nat comm accepted 7/2023). We measured adhesion index via RAdChip for common SCD genotypes: HbSS/SB0: (median (x͂) =756; range (r): 154-1889; n=26) HbSC/SB+ : (x͂ =299; r=: 114-731; n=11 ) HbAA: (x͂) =13; r= 5-90; n=7). RBC adhesion values were independent of age (p=0.65) and gender (p=0.63). An increase of 0.1 unit in EImin and EImax decreases RBC adhesion by 214 and 268 cells/mm 2, respectively. A 1mmHg increase in PoS increased adhesion by 19.5 cells/mm 2. sRBC adhesion had a significant positive association with vaso-occlusive events (VOE) (p=0.005), absolute neutrophil count (ANC) (p=0.005), and EImax (p&amp;lt;0.001). Notably, analytical validation of RAdChip produced a mean CV of 0.13 ± 0.94. RAdChip adds a key component of functional RBC evaluation that conventional lab equipment does not measure. We have analytically validated our device, demonstrating an excellent coefficient of variability. 44 patient samples, each examined in triplicate produced a mean CV of 0.13. We have demonstrated clinical validity of our device by establishing correlations with known markers of severity and pain event frequency. RBC adhesion values increase as the number of VOE events increase, reaffirming the belief that adhesion is a principal element in the multistep VOE cascade. In like manner, improvements in deformability are associated with decreased RBC adhesion, strengthening the interdependent nature of VOE molecular events. Recognizing the dynamic nature of SCD, it is necessary to explore sickle erythrocyte adhesion longitudinally to investigate changes before, during, and after VOE occurrences. Continued and increased integration of adhesion measurements with RAdChip and other rheology devices into routine SCD care may be helpful in assessing disease severity, identifying need for additional disease modifying agents, and assist with second line agent selection and monitoring.

Novel RBC Adhesion and Deformability Assays Reveal Deleterious Effect of Diabetes on RBC Health

Diabetes is one of the most important health issues in the world. The number of people living with diabetes has quadrupled since 1980 1. Cardiovascular disease (CVD), mainly coronary and artery disease, remains the most common cause for morbidity and mortality in people living with diabetes 2,3. A better understanding of the effect of diabetes on the deformability and adhesiveness of red blood cells (RBCs) is essential in assessing disease status and evaluating the risk of CVD, as well as in investigating the effectiveness of potential therapies 4-6. In this work we investigate the deformability of RBCs and their adhesion to erythrocytes using two proprietary microfluidic assays, OcclusionChip 7 and Adhesion Biochip 8 respectively. We previously developed these assays for investigating RBC properties. Occlusion chip assay, consisting of micropillar arrays ranging from 20 µm spacing to 4 µm spacing along the flow direction, creating a gradient of microcapillaries, were fabricated using soft lithography and perfused with isolated RBCs at a constant inlet pressure. Venous samples from diabetic or non-diabetic individuals were collected in EDTA tubes and tested within 48 hours of collection. RBCs were isolated from whole blood and resuspended in PBS at 20% hematocrit, then flown through the chip using a pressure pump for 20 min. The microchannel was then washed and imaged using an inverted microscope. The cells trapped within the micropillar arrays were manually counted to calculate the occlusion index, which we previously established as a biomarker for cell deformability 9. To quantify RBC adhesion to endothelial cells, human umbilical vein endothelial cells (HUVECs) were cultured within a microfluidic channel under flow for 48 h. HUVECs were then activated by heme at a concentration of 40 µM for 1 h at 37 °C. Isolated RBCs resuspended in basal cell culture medium at 20% hematocrit supplemented by 10 mM of HEPES were injected in the microchannel for 15 min. Non-adherent RBCs were rinsed with a basal cell culture medium containing HEPES. The remaining adherent erythrocytes were imaged using an inverted microscope and manually quantified. Our results show that RBCs isolated from individuals with diabetes (A1c &amp;gt; 6.5%) tend to form more occlusion events (OI) than RBCs collected from healthy individuals (A1c &amp;lt; 5.7%), reflecting a loss of deformability (Fig. 1). For comparison we evaluated the elongation index (EI) using ektacytometry (data not shown), and no statistically significant difference was observed. Moreover, RBCs with diabetes tend to be more adhesive to the endothelium than those from healthy individuals, as evident from the count of adherent RBCs (Fig. 2). In conclusion, our assays revealed that diabetes presents a significant adverse effect on the deformability and adhesion of RBCs. These assays provide a much-needed tool for the quantification of these effects for the assessment of CVD risk and the evaluation of treatment efficacy. Future work will focus on conducting a larger-scale study to confirm our preliminary results. We will test samples from patients in all clinically relevant ranges of disease status, i.e. healthy, pre-diabetes, and diabetes.

Inclacumab Reduce Preexisting Red Blood Cell Adhesion to Activated Endothelial Cells: In-Vitro Assessment of the Microfluidic Platform Endothelium-on-a-Chip

Background: In 2019 FDA approved the use of an antibody anti P-selectin (Crizanlizumab) for the treatment of sickle cell disease (SCD) to reduce the vaso occlusive crisis (VOCs). New anti-adhesive treatments are being developed recently. Global Blood Therapeutics, Inc., acquired by Pfizer Inc. in October 2022, acquired Inclacumab from Roche and started two clinical trials now in phase 3 to evaluate the capacity of this humanized IgG4 monoclonal antibody anti P-selectin to reduce the VOCs in patients with SCD. In our recent work presented at EHA 23 in Frankfurt Germany, we showed that Inclacumab prevents the adhesion of RBCs to chronically and acutely heme activated human umbilical vein endothelial cells (HUVECs) with a statistically significant higher effect in comparison to Crizanlizumab. In this work we used a similar approach to test the capacity of Inclacumab in the reduction of preexisting adhesion events on acutely heme activated HUVECs. Methods:Whole blood EDTA samples from six SCD subjects all with HbSS genotypes were collected at University Hospital Cleveland Medical Center, Cleveland, OH, USA. To standardize the samples, we isolated RBCs by centrifugation and subsequential washes in Phospahte buffer s of the whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. HUVECs (Lonza, Morristown, NJ, USA) were introduced and maintained within the endothelialized microfluidic channels at physiological flow for at least 48-72 hours prior to experiments. We performed an acute short-term activation, where the blood samples were supplemented with 40 µM heme +/- 100 µg/ml injected through the microfluidic channels for 15 minutes, after the adhesion took place we washed the non-adherent cells with Inclacumab and Crizanlizumab and we compared with untreated washing buffer (both compounds were provided by Global Blood Therapeutics, Inc., a wholly owned subsidiary of Pfizer Inc. South San Francisco, CA, USA) A phase-contrast images of the remaining RBCs were acquired with an inverted microscope (DMi8 Leica Microsystems Inc. Deerfield, IL, USA) and quantified based on previous published methods (Figure 1A). Paired t-test was used to calculate statistical significance. Results: Acute short-term heme activation induces high level of RBCs adhesion. Both compounds Inclacumab and Crizanlizumab showed a reduction of the number of adherent RBCs to the activated heme, but only Inclacumab results statistically a significant higher reduction compared to untreated RBCs (Figure 1B). Conclusions: Inclacumab is able not only to prevent the adhesion of RBCs to acutely and chronically activated HUVECs but is able to remove the preexisting adhesion and results in significant higher reduction compared to untreated. RBCs adhesion assay on Endothelium-on-a-chip is a novel and efficient way to study the efficacy of the new antiadhesive drug and could be used to monitor patient response to new therapies in SCD. Figure1 : Effect of Inclacumab vs Crizanlizumab on removal of SCD RBC adhesion to heme activated HUVECs. A) Schematic of adhesion experiment and inverted microscope contrast of phase images 10X of adherent RBCs at acutely heme activated HUVECs. B) Inclacumab has statistically significant effect on remove adherent RBCs to acutely heme activated HUVECs (p=0.009, N=7) For all experiments, the heme concentrations were 40 µM and Inclacumab and Crizanlizumab were 100 µg/ml. Scale bare 20 µm. P-values were based on paired t-test. Error bars represent the standard error of the mean (SEM)

Double-layered blood vessels over 3 mm in diameter extruded by the inverse-gravity technique

One of the most promising techniques for treating severe peripheral artery disease is the use of cellular tissue-engineered vascular grafts (TEVGs). This study proposes an inverse-gravity (IG) extrusion technique for creating long double-layered cellular TEVGs with diameters over 3 mm. A three-layered coaxial laminar hydrogel flow in an 8 mm-diameter pipe was realised simply by changing the extrusion direction of the hydrogel from being aligned with the direction of gravity to against it. This technique produced an extruded mixture of human aortic smooth muscle cells (HASMCs) and type-I collagen as a tubular structure with an inner diameter of 3.5 mm. After a 21 day maturation period, the maximal burst pressure, longitudinal breaking force, and circumferential breaking force of the HASMC TEVG were 416 mmHg, 0.69 N, and 0.89 N, respectively. The HASMC TEVG was endothelialised with human umbilical vein endothelial cells to form a tunica intima that simulated human vessels. Besides subcutaneous implantability on mice, the double-layered blood vessels showed a considerably lower adherence of platelets and red blood cells once exposed to heparinised mouse blood and were considered nonhaemolytic. The proposed IG extrusion technique can be applied in various fields requiring multilayered materials with large diameters.

Eco-benign synthesis of nano‑gold chitosan-bacterial cellulose in spent ground coffee kombucha consortium: Characterization, microbiome community, and biological performance

Biomaterials that are mediocre for cell adhesion have been a concern for medical purposes. In this study, we fabricated nano‑gold chitosan-bacterial cellulose (CBC-Au) via a facile in-situ method using spent ground coffee (SGC) in a kombucha consortium. The eco-benign synthesis of monodispersed gold nanoparticles (Au NPs) in modified bacterial cellulose (BC) was successfully achieved in the presence of chitosan (CHI) and a symbiotic culture of bacteria and yeast (SCOBY). The dominant microbiome community in SGC kombucha were Lactobacillaceae and Saccharomycetes. Chitosan–bacterial cellulose (CBC) and CBC-Au affected the microfibril networks in the nano cellulose structures and decreased the porosity. The modified BC maintained its crystallinity up to 80 % after incorporating CHI and Au NPs. Depth profiling using X-ray photoelectron spectroscopy (XPS) indicated that the Au NPs were distributed in the deeper layers of the scaffolds and a limited amount on the surface of the scaffold. Aspergillus niger fungal strains validated the biodegradability of each scaffold as a decomposer. Bacteriostatically CBC-Au showed better antimicrobial activity than BC, in line with the adhesion of NIH-3T3 fibroblast cells and red blood cells (RBCs), which displayed good biocompatibility performance, indicating its potential use as a medical scaffold.