scholarly journals Cellular Fragments as Biomaterial for Rapid In Vitro Bone-Like Tissue Synthesis

2020 ◽  
Vol 21 (15) ◽  
pp. 5327
Author(s):  
Mst Nahid Akhter ◽  
Emilio Satoshi Hara ◽  
Koichi Kadoya ◽  
Masahiro Okada ◽  
Takuya Matsumoto
Keyword(s):  
Collagen Gel ◽  
Culture Conditions ◽  
Substrate Material ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Freeze Dried ◽  
Ionic Detergent ◽  
3D Collagen ◽  
Fetal Bovine

Current stem cell-based techniques for bone-like tissue synthesis require at least two to three weeks. Therefore, novel techniques to promote rapid 3D bone-like tissue synthesis in vitro are still required. In this study, we explored the concept of using cell nanofragments as a substrate material to promote rapid bone formation in vitro. The methods for cell nanofragment fabrication were ultrasonication (30 s and 3 min), non-ionic detergent (triton 0.1% and 1%), or freeze-dried powder. The results showed that ultrasonication for 3 min allowed the fabrication of homogeneous nanofragments of less than 150 nm in length, which mineralized surprisingly in just one day, faster than the fragments obtained from all other methods. Further optimization of culture conditions indicated that a concentration of 10 mM or 100 mM of β-glycerophosphate enhanced, whereas fetal bovine serum (FBS) inhibited in a concentration-dependent manner, the mineralization of the cell nanofragments. Finally, a 3D collagen-cell nanofragment-mineral complex mimicking a bone-like structure was generated in just two days by combining the cell nanofragments in collagen gel. In conclusion, sonication for three min could be applied as a novel method to fabricate cell nanofragments of less than 150 nm in length, which can be used as a material for in vitro bone tissue engineering.

Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

2008 ◽  
Vol 20 (5) ◽  
pp. 579 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Buthionine Sulfoximine ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Maturation Medium

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix

1997 ◽  
Vol 110 (7) ◽  
pp. 861-870 ◽  
Author(s):  
D. Greiling ◽  
R.A. Clark
Keyword(s):  
Wound Healing ◽  
In Vitro Model ◽  
Collagen Gel ◽  
Fibrin Clot ◽  
Dependent Manner ◽  
Fibrin Gel ◽  
Concentration Dependent Manner ◽  
Vitro Model ◽  
Early Wound

After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

scholarly journals In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum- deprived cultures

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 248-256 ◽  
Author(s):  
G Migliaccio ◽  
AR Migliaccio ◽  
JW Adamson
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Colony Growth ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Concentration Dependent Manner ◽  
Gm Csf ◽  
Human Erythropoietin ◽  
Normal Humans

Abstract The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.

scholarly journals Interaction of the New Ketolide ABT-773 (Cethromycin) with Human Polymorphonuclear Neutrophils and the Phagocytic Cell Line PLB-985 In Vitro

2004 ◽  
Vol 48 (4) ◽  
pp. 1096-1104 ◽  
Author(s):  
Marie Thérèse Labro ◽  
Houria Abdelghaffar ◽  
Catherine Babin-Chevaye
Keyword(s):  
Cell Line ◽  
Active Transport ◽  
Transport System ◽  
Culture Conditions ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Erythromycin A ◽  
Active Transport System

ABSTRACT A classical velocity centrifugation technique was used to study the in vitro uptake of the new ketolide ABT-773 by human polymorphonuclear neutrophils (PMNs) and a myelomonoblastic cell line, PLB-985, which can be differentiated into PMNs under certain culture conditions, compared to that of HMR 3004. ABT-773 was rapidly taken up by PMNs (cellular concentration to extracellular concentration ratio [C/E], about 34 at 30 s and up to 207 at 5 min), and uptake plateaued from 30 to 180 min (C/E, about 300). ABT-773 was accumulated significantly better than HMR 3004 from 5 to 180 min. Nondifferentiated PLB-985 cells (ND-PLB) accumulated significantly less ABT-773 and HMR 3004 than PMNs and PLB-985 cells differentiated into PMNs (D-PLB). Whatever the cell type and in contrast to the results obtained with HMR 3004, ABT-773 was mainly located in the cytosol (about 75%) and was rapidly released from loaded cells (about 40% at 5 min), followed by a plateau, likely owing to avid reuptake. Verapamil and H89, an inhibitor of protein kinase A, increased drug efflux. Uptake was sensitive to external pH, and the activation energy was moderate (about 50 kJ/mol). The existence of an active transport system on the PMN membrane was suggested by the following findings: concentration-dependent and saturable uptake (V max, about 10 000 ng/2.5 × 106 PMNs/5 min; Km , about 60 μg/ml) the inhibitory effects of PMN activators or inhibitors (phorbol myristate acetate, verapamil, Ni2+) and the significantly decreased levels of accumulation by killed cells and cells treated at low temperatures. In addition, various macrolides impaired ABT-773 uptake, contrary to the findings for the quinolone levofloxacin. ND- and D-PLB also presented saturation kinetics that defined an active transport system (V max and Km values were similar to those obtained with PMNs), but the activation pathway of the carrier system did not seem to be fully functional in ND-PLB. As has been observed with other erythromycin A derivatives, ABT-773 impaired oxidant production by phagocytes in a time- and concentration-dependent manner. These data extend our previous results on the existence of an active transport system common to all macrolides and ketolides, at least in PMNs.

Microvessel formation from mouse embryonic aortic explants is oxygen and VEGF dependent

2002 ◽  
Vol 283 (2) ◽  
pp. R487-R495 ◽  
Author(s):  
Tetsu Akimoto ◽  
Helen Liapis ◽  
Marc R. Hammerman
Keyword(s):  
Type I Collagen ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Thoracic Level ◽  
Aortic Explants ◽  
Endothelial Growth Factor

To delineate the roles of O2 and vascular endothelial growth factor (VEGF) in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O2, but not room air, the addition of VEGF to explants stimulated the formation of CD31-positive, Flk-1-positive, Gs-IB4-positive structures in a concentration-dependent manner. Electron microscopy showed the structures to be capillary-like. VEGF-induced capillary-like structure formation was inhibited by sequestration of VEGF via addition of soluble Flt-1 fusion protein or anti-VEGF antibodies. Expression of Flk-1, but not Flt-1, was increased in embryonic aorta cultured under 5% O2 relative to room air. Our data suggest that low O2 upregulates Flk-1 expression in embryonic aorta in vitro and renders it more responsive to VEGF.

scholarly journals In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum- deprived cultures

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 248-256
Author(s):  
G Migliaccio ◽  
AR Migliaccio ◽  
JW Adamson
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Colony Growth ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Concentration Dependent Manner ◽  
Gm Csf ◽  
Human Erythropoietin ◽  
Normal Humans

The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.

Fibroblast growth factor 2 promotes microvessel formation from mouse embryonic aorta

2003 ◽  
Vol 284 (2) ◽  
pp. C371-C377 ◽  
Author(s):  
Tetsu Akimoto ◽  
Marc R. Hammerman
Keyword(s):  
Type I Collagen ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Type I ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Low Oxygen ◽  
Concentration Dependent Manner ◽  
Fgf Receptor

To delineate the roles that oxygen and fibroblast growth factors (FGFs) play in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O2, but not room air, the addition of FGF2 to explants stimulated the formation of Gs-IB4-positive, CD31-positive, and Flk-1-positive microvessels in a concentration-dependent manner. FGF2-stimulated microvessel formation was inhibited by sequestration of FGF2 via addition of soluble FGF receptor (FGFR) chimera protein or anti-FGF2 antibodies. FGFR1 and FGFR2 were present on explants. Levels of FGFR1, but not FGFR2, were increased in embryonic aorta cultured under 5% O2 relative to room air. Our data suggest that low oxygen upregulates FGFR1 expression in embryonic aorta in vitro and renders it more responsive to FGF2.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Improved Method for Obtaining of Arctigenin from Arctium Lappa L. and its Antiproliferative Effect on Human Hepatocarcinoma HepG2 Cells

2020 ◽  
Vol 16 (3) ◽  
pp. 358-362
Author(s):  
Renan S. Teixeira ◽  
Paulo H.D. Carvalho ◽  
Jair A.K. Aguiar ◽  
Valquíria P. Medeiros ◽  
Ademar A. Da Silva Filho ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Crude Extract ◽  
Hepg2 Cells ◽  
Liver Carcinoma ◽  
Adhesion Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Arctium Lappa ◽  
Nih 3T3

Background: Arctigenin is a lignan found in Arctium lappa L. (Asteraceae) that displays anti-inflammatory activities. Previous studies showed that the crude extract of A. Lappa has antitumor activity in human liver carcinoma, lung and stomach cancer cells. The aim of this study was to obtain arctigenin from A. lappa L., as well as to evaluate its antiproliferative effects in cells of liver carcinoma (HepG2) and fibroblasts (NIH/3T3). Methods: Arctigenin was obtained from the hydrolysis of arctiin, which was isolated from the crude extract of A. lappa. The effects of arctigenin and arctiin on HepG2 cell viability and cell adhesion were analyzed by MTT method. Adhesion assay was also carried out to evaluate the antitumor activity. Results: Our results showed that the analytical process to obtain arctigenin was fast and easy. In vitro experiments showed that arctigenin (107-269 μM) decreased HepG2 cells viability and did not cause cytotoxicity on NIH/3T3 cells. Arctigenin (27-269 μM) demonstrated anti-adhesion in HepG2 cells in a concentration-dependent manner, when compared with control. Conclusion: These results suggest a promising pharmacological activity for arctigenin as an antiproliferative compound.

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