Xiang Wang

Surface modification can regulate surface properties through the design of surface structures and chemical compositions. Heparin has good anticoagulant properties and is widely used in the surface modification of blood-contact materials. However, the molecular structure of heparin has often been overlooked in terms of its application and further utilization in the design process. In this study, heparin was grafted onto substrate materials through polydopamine/polyethyleneimine co-deposition coating and connected with cysteine to obtain PP-Hep-Cys. The sulfhydryl content of PP-Hep-Cys reached about 2.06 nmol/cm², and the NO generation was about 0.2415 nmol/cm². In the absence of NO donors, the introduction of heparin contributed to the inhibition of platelets, but when NO donors were added, the inhibition of platelets was caused by the synergistic effect of heparinization and NO generation. In addition, the clotting time of PP-Hep-Cys was significantly prolonged compared with PP, and the hemolysis rate declined to 0.33 ± 0.01%. Regarding the adsorption performance of Cu²⁺, the adsorption capacity of PP-Hep-Cys was higher than that of PP-Hep and PP-Cys, and monolayer adsorption was dominant. Overall, these results indicated that heparin, as a spacer, and cysteine, as a ligand, exerted a synergistic effect on blood compatibility and adsorption.

Red blood cell (RBC) production from bone marrow hematopoietic stem cells (BMHSCs) in vitro overlooks the mechanical signals of the bone marrow niche and overly relies on growth factors. Considering that the fate of hematopoietic stem cells (HSCs) is determined by the natural bone marrow microenvironment, differences in mechanical microenvironments provide a reference for the regulation of HSC differentiation. This study seek to reveal the role of mechanobiology cues in erythropoiesis and provide a new perspective for the design of in vitro erythropoiesis platforms. The hydrogel platforms we designed simulate the stiffness gradient of the bone marrow niche to culture HSCs and induce their differentiation into the erythroid system. Cells on the low-stiffness scaffold have higher potential for erythrocyte differentiation and faster differentiation efficiency and promote erythrocyte differentiation after erythropoietin (EPO) restriction. In vivo transplantation experiments demonstrated that these cells have the ability for continuous proliferation and differentiation into mature erythrocytes. By combining mechanical cues with in vitro erythrocyte production, this method is expected to provide insights for in vitro hematopoietic design and offer a scalable cell manufacturing platform for transfusion medicine.

Introduction: The integrity of the erythrocyte membrane cytoskeletal network controls the morphology, specific surface area, material exchange, and state of erythrocytes in the blood circulation. The antioxidant properties of resveratrol have been reported, but studies on the effect of resveratrol on the hypoxia-induced mechanical properties of erythrocytes are rare.Methods: In this study, the effects of different concentrations of resveratrol on the protection of red blood cell mor-phology and changes in intracellular redox levels were examined to select an appropriate concentration for further study. The Young’s modulus and surface roughness of the red blood cells and blood viscosity were measured via atomic force microsco-py and a blood rheometer, respectively. Flow cytometry, free hemoglobin levels, and membrane lipid peroxidation levels were used to characterize cell membrane damage in the presence and absence of resveratrol after hypoxia. The effects of oxida-tive stress on the erythrocyte membrane proteins band 3 and spectrin were further investigated by immunofluorescent label-ing and Western blotting.Results and discussion: Resveratrol changed the surface roughness and Young’s modulus of the erythrocyte mem-brane, reduced the rate of eryptosis in erythrocytes after hypoxia, and stabilized the intracellular redox level. Further data showed that resveratrol protected the erythrocyte membrane proteins band 3 and spectrin. Moreover, resistance to band 3 pro-tein tyrosine phosphorylation and sulfhydryl oxidation can protect the stability of the erythrocyte membrane skeleton net-work, thereby protecting erythrocyte deformability under hypoxia. The results of the present study may provide new insights into the roles of resveratrol in the prevention of hypoxia and as an antioxidant.

Activated carbon (AC) is an effective adsorbent for creatinine removal in hemoperfusion. However, the hemocompatibility and adsorption capacity of AC was required to be improved further. Heparin has different anticoagulant mechanisms due to its different molecular weights. Thus, it was necessary to study the surface modification with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) on improvement of hemocompatibility and adsorption. In this study, UFH and LMWH were, respectively, grafted on AC through polyethyleneimine as an intermediate layer. The modification of AC regarding morphology, mechanical strength, and pore structure was characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), texture analyzer (TA), and surface area analyzer. It was found that, compared with AC, the morphology and mechanical strength of AC-UFH and AC-LMWH could be well maintained, but the specific surface area was decreased due to the grafting of macromolecules. Furthermore, AC-UFH and AC-LMWH showed better hemocompatibility on protein adsorption, clotting time, and platelet activation compared with AC, in which AC-LMWH had lower fibrinogen adsorption and longer clotting time than AC-UFH. In addition, it was found that AC, AC-UFH, and AC-LMWH had no significant effect on blood cell composition. Finally, the adsorption capacity of adsorbents for creatinine was evaluated. Although there was no significant difference between AC-UFH and AC-LMWH, it was found that heparin could be interacted with creatinine to enhance the adsorption capacity when compared with polyethyleneimine-modified AC. This study deepened the understanding of anticoagulation of heparinized surface and provided a theoretical basis for adsorption in hemoperfusion.

Activated carbon (AC) is an effective adsorbent for creatinine removal in hemoperfusion. However, the hemocompatibility and adsorption capacity of AC was required to be improved furthermore. Heparin, a widely used anticoagulant biomacromolecule, could improve the hemocompatibility of the absorbents in different ways based on its molecular weight. Thus, it was necessary to study the surface modification with unfractionated heparin (UFH) or low molecular weight heparin (LMWH) on improvement of hemocompatibility and adsorption. In this study, UFH and LMWH were respectively grafted on AC through polyethyleneimine as an intermediate layer. The modification of AC about morphology, mechanical strength and pore structure was characterized by XPS, SEM, TA and BET. It was found that compared with AC, the morphology and mechanical strength AC-UFH and AC-LMWH could be well maintained, but the BET surface was decreased due to the grafting of macromolecules. Furthermore, AC-UFH and AC-LMWH showed the better hemocompatibility on protein adsorption, APTT and platelet activation compared with AC, in which AC-LMWH had lower fibrinogen adsorption and longer APTT than AC-UFH. Besides, it was found that AC, AC-UFH and AC-LMWH had no significant effect on blood cell composition. Finally, the adsorption capacity of adsorbents for creatinine was evaluated. Although there was no significant difference between AC-UFH and AC-LMWH, it was found that heparin could be interacted with creatinine to enhance the adsorption capacity when compared with AC-PEI. This study deepened the understanding of anticoagulation of heparinized surface and provided a theoretical basis for adsorbents in hemoperfusion.

We provide a facile and low-cost method (F-L) to fabricate a two-dimensional positive master using a milling technique for polydimethylsiloxane (PDMS)-based microchannel molding. This method comprises the following steps: (1) a positive microscale master of the geometry is milled on to an acrylic block; (2) pre-cured PDMS is used to mold the microscale positive master; (3) the PDMS plate is peeled off from the master and punctured with a blunt needle; and (4) the PDMS plate is O₂ plasma bonded to a glass slide. Using this technique, we can fabricate microchannels with very simple protocols quickly and inexpensively. This method also avoids breakage of the end mill (ϕ = 0.4 mm) of the computerized numerical control (CNC) system when fabricating the narrow channels (width < 50 µm). The prominent surface roughness of the milled bottom-layer could be overcomed by pre-cured PDMS with size trade-off in design. Finally, emulsion formation successfully demonstrates the validity of the proposed fabrication protocol. This work represents an important step toward the use of a milling technique for PDMS-based microfabrication.

Our previous study showed that the membrane viscosity of human umbilical vein endothelial cells (HUVECs) increased with lipopolysaccharide (LPS) concentration after direct LPS damaging, implying that LPS could change the membrane structure and composition of HUVECs. The purpose of the present study was to investigate the modification of HUVECs membrane lipid by LPS under direct LPS injury to HUVECs. Phospholipid components of HUVEC membrane were determined with high performance capillary electrophoresis (HPCE), and HUVECs were directly damaged by LPS of different concentrations: 0, 0.313, 0.625, 1.25, 2.5, 5 and 10 microgram/ml for 3 h, and by 0.625 microgram/ml for 1, 3, 6, 12, 24 and 48 h. The results show that total phospholipid content of HUVECs increased with the increase of LPS concentration at 3 h, and decreased with the duration of LPS effect at 0.625 microgram/ml. Phosphatidylinositol (PI) and phosphatidylcholine (PC) contents increased with the increase of LPS concentration and decreased with the duration of LPS effect; LPS contents and its effect duration had slight effect on sphingomyelin (SM), phosphatidylethanolamine (PE) or phosphatidylserine (PS) contents. The LPS activating effect of HUVEC membrane lipid showed a typical property of enzymatic dynamics. These experimental results indicate that LPS directly activates the metabolism of HUVEC membrane lipid and induces the modification of HUVEC membrane phospholipid, which implies that the mechanism of the impairment of HUVECs by LPS may be related to the membrane structure of HUVECs and the metabolism of HUVEC membrane lipid.