Bioengineered Fluorescent Nanoprobe Conjugates for Tracking Human Bone Cells: In Vitro Biocompatibility Analysis

Herein, we validated novel functionalized hybrid semiconductor bioconjugates made of fluorescent quantum dots (QD) with the surface capped by chitosan (polysaccharide) and chemically modified with O-phospho-L-serine (OPS) that are biocompatible with different human cell sources. The conjugation with a directing signaling molecule (OPS) allows preferential accumulation in human bone mesenchymal stromal cells (HBMSC). The chitosan (Chi) shell with the fluorescent CdS core was characterized by spectroscopical (UV spectrophotometry and photoluminescence), by morphological techniques (Transmission Electron Microscopy (TEM)) and showed small size (ø 2.3 nm) and a stable photoluminescence emission band. The in vitro biocompatibility results were not dependent on the polysaccharide chain length (Chi with higher and lower molecular weight) but were remarkably affected by the surface modification (Chi or Chi-OPS). In addition, the efficiency of nanoparticles uptake by the cells was dependent on cells nature (human primary cells or cell lines) and tissue source (bone or skin) in the presence or absence of the OPS modification. The complex cellular uptake pathways involved in the cell labeling with the nanoparticles do not interfere on the normal cellular biology (adhesion and proliferation), osteogenic differentiation, and gene expression. The bone cells particles uptake evaluation showed a possible pathway by Caveolin-1 that regulates cell transduction in the membrane’s Caveolae. Caveolae mediates non-specific endocytosis, and it is upregulated in HBMSC. The OPS-modified nanoparticles promoted an intense intracellular trafficking by the HBMSCs that showed late-osteoblast phenotype with an increase of extracellular matrix (ECM) mineralization (Alizarin red and Von Kossa staining for calcium phosphate crystals). In this work, the OPS modified bioconjugated QD proved to be a reliable and stable fluorescent bioprobe for cell imaging and targeting research that could also help in clarifying some cellular mechanisms of particles intracellular traffic through the cytoplasmic membrane and osteogenic differentiation induction. The in vitro HBMSC’s biocompatibility responses indicated that the OPS-modified chitosan QDs have a prospective future in laboratory and pre-clinical applications such as bioimaging analysis and for ex-vivo cellular evaluation of biomedical implants.     

Uptake of Tritiated Folates by Human Bone Marrow Cells in Vitro

S ummary . Using incubation periods up to 4 hr, it was demonstrated that uptake of tritiated pteroylglutamic acid ([³H]PteGlu) by human bone marrow cells in vitro was in the range of six-fold greater than uptake by reticulocytes. Uptake was temperature-dependent, increasing during 4 hr at 37°C but not at 4°C; a similar temperature dependence was found for the uptake of [³H]methyltetrahydrofolate ([³H-CH₃]H₄PteGlu). The pH optimum for [³H]PteGlu uptake was in the range of 7.4. Percentage uptake decreased as concentration of [³H]PteGlu increased. Preincubation with unlabelled PteGlu reduced uptake of subsequently added [³H]PteGlu by twice as much as did preincubation with methotrexate, suggesting that methotrexate may only partly share the uptake mechanism for [³H]PteGlu. [³H]PteGlu uptake was not affected by preincubation with diphenylhydantoin or a sulphydryl inhibitor. Uptake of [³H-CH₃]H₄PteGlu by human bone marrow cells appeared to be approximately twice the uptake of [³H]PteGlu. The findings support the concept of two mechanisms for folate uptake by human reticulocytes and bone marrow cells: an energy-dependent, active carrier mechanism probably of primary physiologic significance, and a seemingly passive diffusion-like mechanism, probably primarily of pharmacologic significance.     

A Robust Numerical Methodology for Fatigue Damage Evolution Simulation in Composites

Composite materials, like metals, are subject to fatigue effects, representing one of the main causes for component collapse in carbon fiber-reinforced polymers. Indeed, when subject to low stress cyclic loading, carbon fiber-reinforced polymers exhibit gradual degradation of the mechanical properties. The numerical simulation of this phenomenon, which can strongly reduce time and costs to market, can be extremely expensive in terms of computational effort since a very high number of static analyses need to be run to take into account the real damage propagation due the fatigue effects. In this paper, a novel cycle jump strategy, named Smart Cycle strategy, is introduced in the numerical model to avoid the simulation of every single cycle and save computational resources. This cycle jump strategy can be seen as an enhancement of the empirical model proposed by Shokrieh and Lessard for the evaluation of the fatigue-induced strength and stiffness degradation. Indeed, the Smart Cycle allows quickly obtaining a preliminary assessment of the fatigue behavior of composite structures. It is based on the hypothesis that the stress redistribution, due to the fatigue-induced gradual degradation of the material properties, can be neglected until sudden fiber and/or matrix damage is verified at element/lamina level. The numerical procedure has been implemented in the commercial finite element code ANSYS MECHANICAL, by means of Ansys Parametric Design Languages (APDL). Briefly, the Smart Cycle routine is able to predict cycles where fatigue failure criteria are likely to be satisfied and to limit the numerical simulation to these cycles where a consistent damage propagation in terms of fiber and matrix breakage is expected. The proposed numerical strategy was preliminarily validated, in the frame of this research study, on 30° fiber-oriented unidirectional coupons subjected to tensile–tensile fatigue loading conditions. The numerical results were compared with literature experimental data in terms of number of cycles at failure for different percentage of the static strength. Lastly, in order to assess its potential in terms of computational time saving on more complex structures and different loading conditions, the proposed numerical approach was used to investigate the fatigue behavior of a cross-ply open-hole composite panel under tension–tension fatigue loading conditions.     

双环铂对人主动脉平滑肌细胞和内皮细胞增殖影响的初步体外实验研究

目的:通过体外实验,研究双环铂对人主动脉平滑肌细胞(HASMC)和人主动脉内皮细胞(HAEC)增殖的影响,探讨其成为支架携带药物的可能性。方法:3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基胺)-2-(4-苯磺基)-2氢-四唑盐(MTS)法评价双环铂对HASMC和HAEC增殖的影响。探索不同浓度和不同作用时间条件下双环铂对HASMC和HAEC增殖抑制率的影响;双环铂对HASMC和HAEC半数抑制浓度的差异;以西罗莫斯作为阳性对照组,探索双环铂对HASMC和HAEC抗增殖作用的最低有效浓度。结果:双环铂对HASMC和HAEC的增殖抑制作用呈时间和浓度依赖性。双环铂对HASMC和HAEC的半数增殖抑制浓度分别为3.47μg/ml和72.44μg/ml。双环铂浓度为10 ng/ml～10μg/ml时,对HASMC有显著抑制作用(P<0.05)。双环铂浓度为10μg/ml时,对HAEC有显著抑制作用(P<0.01)。阳性对照药物西罗莫斯在纳克(ng)水平对HASMC和HAEC的增殖也有明显的抑制作用。结论:双环铂对HASMC和HAEC的增殖抑制作用呈时间和浓度依赖性。双环铂对HAEC的最低抑制浓度比对HASMC的最低抑制浓度高1000倍。尽管双环铂对细胞增殖的作用弱于西罗莫斯,但是其明显抑制血管平滑肌细胞增生而较少抑制血管内皮细胞增生的特性,使其有可能成为药物洗脱支架所携带的药物。     

Erythroid Colony Growth in Vitro from Human Peripheral Blood Null Cells: Evidence for Regulation by T-Lymphocytes and Monocytes

S ummary. Colony assays in methylcellulose of primitive erythroid precursors (BFU-E) were carried out from the null cell fraction of normal human peripheral blood lymphoid cells. There was little proliferation or maturation of BFU-E as assessed by both the number and the size of colonies formed, when null cells alone were cultured. Culture of null cells with up to 8 × 105 autologous T-lymphocytes per ml led to considerable stimulation of colony growth and maturation. Culture of null cells with peripheral blood monocytes also resulted in the induction of BFU-E growth, although the response was inferior to that seen with T-cells. Co-culture of null cells together with both T-cells and monocytes resulted in a uniformly greater response than with either alone, and this was shown to be due to a positive interaction between these two cell types.     

Colony Growth of Human Leukemic Peripheral Blood Cells In Vitro

The colony-forming potential of peripheral white blood cells from patientswith acute leukemia on normal humanperipheral WBC feeder layers has beenstudied. White blood cells from 12 of 20patients with acute granulocytic leukemiagave rise to large numbers of colonies,while WBC from eight patients withAGL, four patients with acute lymphocytic leukemia and three patients withacute stem cell leukemia gave rise to nocolonies or only small numbers. Coloniesformed from WBC of patients with AGLappear to go through a process of morphologic maturation to segmented granulocyte forms. Leukemic WBC will notserve as feeder layers in this system, butare not inhibitory in this respect. Thesignificance of these findings is discussed.

Submitted on February 19, 1971 Revised on May 3, 1971 Accepted on May 10, 1971     

β2-Agonist-Elevated Stress Response in Human Bronchial Epithelial Cells in Vivo and in Vitro

Recent studies report high baseline levels of stress (heat shock) proteins in bronchial epithelial cells from asthmatic individuals. The promoter of the gene encoding the 72-kDa heat shock protein has an element responsive to cAMP, which may be affected by β-agonists. This study examined stress protein levels in subjects enrolled in a segmental lung allergen challenge study to determine whether β-agonist medication could contribute to a stress response. Subjects were divided on the basis of no premedication (n= 17), metered dose inhalations of albuterol (n= 24), or placebo inhalation (n= 3) prior to bronchoscopy. Levels of the inducible stress protein Hsp72 and constitutive Hsp73 were quantitated in bronchial epithelial cells from brush biopsy of allergic nonasthmatic, allergic asthmatic, and normal individuals. Mean levels were increased significantly (p < 0.003 and p < 0.004, respectively) in those subjects who received albuterol premedication. No significant differences were found between clinical groups of individuals or for placebo inhalation vs nonpremedication. Albuterol in vitro increased the levels of Hsp72 and Hsp73 in epithelial cells from either nonpremedicated or placebo-treated donors; the Hsp72 levels correlated linearly with increased albuterol concentration (r= 0.81, p < 0.01). Therefore, β-agonists elevate or prolong an elevated stress response in epithelial cells, possibly through cAMP-mediated effects. Accepted for publication: 20 December 1996     

In Vitro Toxicity of Bone Graft Materials to Human Mineralizing Cells

Bone graft materials from synthetic, bovine, and human sources were analyzed and tested for in vitro cytotoxicity on dental pulp stem cells (DPSCs) and osteosarcoma cells (Saos-2). Raman spectroscopy indicated significant amounts of collagen only in human bone-derived materials, where the mineral to protein ratio was 3.55 ± 0.45, consistent with bone. X-ray fluorescence revealed tungsten (W) concentrations of 463 ± 73, 400 ± 77, and 92 ± 42 ppm in synthetic, bovine, and human bone chips, respectively. When these chips were added to DPSCs on tissue culture plastic, the doubling times after two days were the same as the controls, 16.5 ± 0.5 h. Those cultured with synthetic or bovine chips were 96.5 ± 8.1 and 25.2 ± 1.4 h, respectively. Saos-2 was more sensitive. During the first two days with allogeneic or bovine graft materials, cell numbers declined. When DPSC were cultured on collagen, allogeneic and bovine bone chips did not increase doubling times. We propose cytotoxicity was associated with tungsten, where only the concentration in human bone chips was below 184 ppm, the value reported as cytotoxic in vitro. Cells on collagen were resistant to bone chips, possibly due to tungsten adsorption by collagen.     

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.     

Increased Potassium Permeability Induced In Vitro by Menadione in Normal Human Red Cells

Normal red blood cells, preincubated for 75 min with 1.15 mM menadione sodium bisulfite lose potassium and water on subsequent incubation at 37° C for 24 h without menadione. The potassium loss is increased by addition of calcium and prevented by addition of glucose. Since normal red cells treated with menadione behave like untreated hypochromic cells, both from β-thalassaemia or iron deficiency anaemia in respect to membrane permeability to potassium, it may be supposed that menadione induces in normal red cells an abnormality similar to that naturally occurring in hypochromic cells.     

In Vitro Growth of Granulocytic Colonies From Circulating Cells in Human Cord Blood

Human umbilical cord blood cells from 26newborn infants and peripheral blood cellsfrom 18 adults were cultured in vitro byusing the agar-gel method of humanhemopoietic cell culture. An increasedconcentration of colony-forming cells wasseen in the cord blood cultures. Between17 and 385 colonies, with a mean of 122,were formed in these cultures per 2 x10⁵ nucleated cells plated. The peripheralblood cell cultures from adults gave rise to0-11 colonies, with a mean of 3, per 2 x10⁵ nucleated cells plated. The averagenumber of cells per colony was 1000-1500cells after 14 days of culture, predominantly granulocytic.

Submitted on April 10, 1973 Revised on August 27, 1973 Accepted on August 29, 1973     

Berberine and Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.     

Adaptive Cytoprotection Against Deoxycholate-Induced Injury in Human Gastric Cells In Vitro: Is There a Role for Endogenous Prostaglandins?

The majority of previous work investigatingadaptive cytoprotection has involved in vivo studies,which have suggested that this protective response is inlarge part mediated by endogenous prostaglandins (PGs). The aim of this study was to investigateadaptive cytoprotection under in vitro conditions inhuman gastric cells and to better delineate the role ofendogenous PGs in this protective response. AGS cells (a human gastric carcinoma cell line)were characterized morphologically and subsequently usedfor all experiments. Sodium deoxycholate was used asboth the mild irritant and the damaging agent, and cell injury was quantified using both acommercial viability/cytotoxicity kit as well astransepithelial permeability studies. Finally,endogenous PG synthesis in response to varyingconcentrations of deoxycholate was determined. AGS cells were determined to bemorphologically similar to gastric mucous cells.Pretreatment of cells with low-dose deoxycholatesignificantly attenuated injury upon subsequent exposure to damaging concentrations of deoxycholate, andthis protection was determined to be dependent upon bothconcentration and duration of mild irritant exposure.Preincubation of AGS cells with indomethacin reversed protection induced by mild irritantpretreatment and also significantly increased cellularsusceptibility to injury. Results of the permeabilitystudies closely paralleled those assessing cell mortality. While deoxycholate exposureincreased PG synthesis, the concentrations required weremuch higher than those needed to initiate protection.Adaptive cytoprotection exists in AGS cells under in vitro conditions independent of intact bloodflow, neural innervation, or circulating humoralmediators. While this protection is reversed byindomethacin, it appears that this reversal results fromincreased cellular injury secondary to diminished basalPGs, rather than inhibition of endogenous PGsynthesis.     

Defibrination of Normal Human Blood in Vitro: a Method giving a High Recovery of Untraumatized Cells

A new automatic apparatus is described for the defibrination of blood in vitro at laboratory temperature which results in only small (<10%) losses of white cells. Freshly-drawn blood is mixed smoothly at high speed (>1200 rpm) and fibrin is removed rapidly as formed. The apparatus is designed to produce minimum mechanical trauma to the blood. Subsequent in vitro incubation of red cells, granulocytes and lymphocytes demonstrated their viability to be comparable with cells obtained by other methods of defibrination.     

The Chronology of the Mitotic Cycle of Human Granulocytopoietic Cells Phase Contrast Studies on Living Cells in Vitro

Data on the mitotic index of human granulocytopoietic cells are presented.From these and from the duration of mitosis directly measured in living cells byphase contrast microscope, the weighted average generation time and themean compartment transit time are computed. Maturation in granulocytopoieticcells appears to induce a reduction of mitotic indices and mitotic rate and anincrease in mitotic time and in mean compartment transit time. Part of theincrement in mitotic duration may be due to the acquisition by a part of thegranulocytopoietic cells of cytoplasmic peripheral motility or other specializedactivities, thus distracting part of the energies destined to mitosis.

Submitted on June 1, 1966 Accepted on May 17, 1967     

In Vitro DNA and RNA Synthesis in Human Bone Marrow Cells: A Study of 12 Normal Subjects and 12 Patients with Lymphoplasmocytic Disorders

In 12 normal subjects, DNA and RNA synthesis by individual marrow cellswas studied in vitro by autoradiography after short-term incubation withtritiated nucleosides. The DNA labeling index and RNA synthesis rate werehighest in normal immature cells. The DNA index was zero in all maturecells, including small lymphocytes and plasma cells. In the most activelyproliferating normal immature cells, the RNA synthesis rate was about 5 timeshigher than in normal plasma cells. Since both normal immature cells andplasma cells are characterized by a high content of RNA and active proteinsynthesis, the results of the present study suggest that a high RNA turnovermay not be necessary for protein synthesis to proceed in nonproliferating,protein-producing marrow cells.

In 10 patients with myeloma and in two patients with primary macroglobulinemia, the abnormal plasma cells were found to be labeled with H³thymidine in each instance. The RNA synthesis rate was about 3 times greaterin plasmocytoid myeloma cells than in mature plasma cells of normal marrows,and also about 3 times greater in lymphocytoid myeloma cells than in maturelymphocytes of normal marrows. The abnormal cells in the lymphoplasmocyticdisorders apparently differ from their counterparts in normal marrows notonly by a higher proliferative potential, but also by a much greater rate ofRNA synthesis.

In actively dividing normal bone marrow cells, high values for both DNAand RNA synthesis were found. By contrast, in the neoplastic lymphoplasmacells studied, a discrepancy was noted between relatively low values of DNAsynthesis and high values of RNA synthesis. Production of abnormal proteinswithin long-lived neoplastic plasmocytic cells may account for this finding.

Submitted on March 26, 1965 Accepted on September 11, 1965     

Stimulation and Inhibition by Normal Human Serum of Colony Formation in Vitro by Bone Marrow Cells

S ummary . Normal human sera were able to stimulate granulocyte and macrophage colony formation in vitro by mouse bone marrow cells, but only following removal of inhibitory material either by dialysis or by heat- or ether-treatment. Inhibitors were shown to be present in all normal human sera tested, to exhibit partial species specificity, to be multiple and to be probably lipoprotein in nature. The in vivo function of these inhibitors is unknown but they may modulate the action of the colony stimulating factor (CSF) in stimulating granulopoiesis and macrophage formation.