Polymerization and adhesion, dynamic processes that are hallmarks of sickle cell disease (SCD), have thus far been studied in vitro only separately. Here, we present quantitative results of the ...simultaneous and synergistic effects of adhesion and polymerization of deoxygenated sickle hemoglobin (HbS) in the human red blood cell (RBC) on the mechanisms underlying vasoocclusive pain crisis. For this purpose, we employ a specially developed hypoxic microfluidic platform, which is capable of inducing sickling and unsickling of RBCs in vitro, to test blood samples from eight patients with SCD. We supplemented these experimental results with detailed molecular-level computational simulations of cytoadherence and biorheology using dissipative particle dynamics. By recourse to image analysis techniques, we characterize sickle RBC maturation stages in the following order of the degree of adhesion susceptibility under hypoxia: sickle reticulocytes in circulation (SRs) → sickle mature erythrocytes (SMEs) → irreversibly sickled cells (ISCs). We show that (i) hypoxia significantly enhances sickle RBC adherence; (ii) HbS polymerization enhances sickle cell adherence in SRs and SMEs, but not in ISCs; (iii) SRs exhibit unique adhesion dynamics where HbS fiber projections growing outward from the cell surface create multiple sites of adhesion; and (iv) polymerization stimulates adhesion and vice versa, thereby establishing the bidirectional coupling between the two processes. These findings offer insights into possible mechanistic pathways leading to vasoocclusion crisis. They also elucidate the processes underlying the onset of occlusion that may involve circulating reticulocytes, which are more abundant in hemolytic anemias due to robust compensatory erythropoiesis.
Cell mechanics is closely associated with cellular structure and function. However, the inability to measure both cellular force and viscoelasticity of statistically significant number of cells ...noninvasively remains a challenge for quantitative characterizations of various cellular functions and practical applications. Here a double resonator piezoelectric cytometry (DRPC), using AT and BT cut quartz crystals of the same frequency and surface morphology is developed to simultaneously quantify the cells‐generated forces (ΔS) and viscoelastic moduli (G′, G″) of a population of isolated single cells or cells with different degrees of cell‐cell interactions in a non‐invasive and real time manner. DRPC captures the dynamic mechanical parameters ΔS and G′, G″ during the adhesions of human umbilical vein endothelial cells (HUVECs) under different ligand densities of adhesion molecules fibronectin or Arg‐Gly‐Asp (RGD) modified on the gold surfaces of 9 MHz AT and BT cut quartz crystals, and different seeding densities of HUVECs. It is found that both the ligand density and cell seeding density affect the magnitudes of ΔS and G′, G″ and their correlations are revealed for the first time by DRPC. The validity of DRPC is further verified by mechanical changes of the cells in response to treatments with cytoskeleton regulators.
Double resonator piezoelectric cytometry (DRPC) is developed to simultaneously quantify cells’ generated forces ΔS and viscoelastic moduli G′, G″ of a population of isolated single cells or cells with different cell‐cell contacts adhered to the AT and BT cut quartz crystals of the same frequency and morphology by measuring their respective changes in resonant frequency and motional resistance.
Assessment of cell adhesion and cell size provides valuable information on surface biocompatibility. However, most investigations on cell morphology dynamics are time and resource consuming, of ...rather descriptive character and lack procedures for appropriate quantification. The aim of the study was to develop a software programme which allows automated cell segmentation and identification as well as calculation and further processing of cell size in low-contrast images. The software utilises modified edge detection and morphologic operations for automatic cell analysis in light microscopy images. In an application study, osteogenic cell-adhesion dynamics were quantified for the ECM proteins collagen type I (COL) and fibronectin (FIB) over a period of 12 hrs. Untreated tissue culture polystyrene (TCPS) served as control. The software programme proofed full function in automatic cell tracking and quantification of cell size. After 11 h, cell sizes were highest for COL (6391 ± 1167 µm
2
) and FIB (6036 ± 411 µm
2
) compared with TCPS (3261 ± 693 µm
2
). The developed software allows quantification of initial cell size changes on translucent surface modifications and is suitable as a reliable tool for fast biocompatibility screening. Osteogenic cell adhesion was significantly promoted by COL and FIB indicating the potential of respective functionalized biomaterial surfaces.
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