Cell Adhesion Ratio Research Articles

Evaluation of nanofibrous scaffolds obtained from blends of chitosan, gelatin and polycaprolactone for skin tissue engineering

Polymer blending is a strategy commonly used to obtain hybrid materials possessing properties better than those of the individual constituents regarding their use in scaffolds for Tissue Engineering. In the present work, the scaffolds produced by electrospinning solutions of polymeric blends obtained using a polyester (polycaprolactone, PCL), a polysaccharide (chitosan, CS) and a protein (gelatin extracted from cold water fish skin, GEL), were investigated. Solutions conductivity, shear viscosity and surface tension were determined. GEL-containing scaffolds were crosslinked with vapour phase glutaraldehyde (GTA). The scaffolds were characterized physico-chemically regarding fibre morphology, porosity, water contact angle, mechanical properties, chemical bonds and fibre and dimensional stability upon immersion in water and cell culture medium. The scaffolds were further tested in vitro for cell adhesion, growth and morphology of human foetal fibroblasts (cell line HFFF2). Results show that the nanofibrous scaffolds are hydrophilic and display the typical porosity of non-woven fibre mats. The CS/PCL and CS/PCL/GEL scaffolds have the highest elastic modulus (48MPa). Dimensional stability is best for the CS/PCL/GEL scaffolds. FTIR spectra confirm the occurrence of cross-linking reactions of GTA with both GEL and CS. Cell adhesion ratio ranked from excellent (close to 100%) to satisfactory (around 50%) in the order PCL/GEL>CS/GEL>CS/PCL/GEL>CS/PCL. Cell populations show an extended lag phase in comparison with the controls but cell proliferation occurs on all scaffolds until confluence is reached. In conclusion, all scaffolds studied possess characteristics that enable them to be used in skin tissue engineering but the CS/PCL/GEL scaffolds have better physical properties whereas the PCL/GEL scaffolds support a higher cell adhesion.

Epidermal growth factor can optimize a serum-free culture system for bone marrow stem cell proliferation in a miniature pig model

Bone marrow-derived mesenchymal stem cells have become an attractive cell source for periodontal ligament regeneration treatment because of their potential to engraft to several tissue types after injury. Most researchers have focused on the transplantation process, but few have paid attention to cell safety concerns and rapid proliferation before transplantation. Using serum-free medium to culture stem cells may be an effective method to avoid problems associated with exogenous serum and the addition of growth factors to promote cell proliferation. Here, we randomly divided our serum-free cultures and treated them with different levels of epidermal growth factor (EGF). We then evaluated changes in rates of cell adhesion, proliferation, apoptosis, and cell cycle ratio as well as their differentiation potential. The data showed that all of these parameters were significantly different when comparing serum-free cultures with and without 10 nM/L EGF (p < 0.05/0.01); however, cells with 10 nM/L EGF did not respond differently than cells grown in standard serum-containing media without EGF (p > 0.05). In summary, our results demonstrate that 10 nM/L EGF was the optimal dose for serum-free culture, which can replace traditional standard serum medium for in vitro expansion of miniature pig bone marrow-derived mesenchymal stem cells.

Corneal Stromal Cell Growth on Gelatin/Chondroitin Sulfate Scaffolds Modified at Different NHS/EDC Molar Ratios

A nanoscale modification strategy that can incorporate chondroitin sulfate (CS) into the cross-linked porous gelatin materials has previously been proposed to give superior performance for designed corneal keratocyte scaffolds. The purpose of this work was to further investigate the influence of carbodiimide chemistry on the characteristics and biofunctionalities of gelatin/CS scaffolds treated with varying N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) molar ratios (0–1) at a constant EDC concentration of 10 mM. Results of Fourier transform infrared spectroscopy and dimethylmethylene blue assays consistently indicated that when the NHS to EDC molar ratio exceeds a critical level (i.e., 0.5), the efficiency of carbodiimide-mediated biomaterial modification is significantly reduced. With the optimum NHS/EDC molar ratio of 0.5, chemical treatment could achieve relatively high CS content in the gelatin scaffolds, thereby enhancing the water content, glucose permeation, and fibronectin adsorption. Live/Dead assays and interleukin-6 mRNA expression analyses demonstrated that all the test samples have good cytocompatibility without causing toxicity and inflammation. In the molar ratio range of NHS to EDC from 0 to 0.5, the cell adhesion ratio and proliferation activity on the chemically modified samples significantly increased, which is attributed to the increasing CS content. Additionally, the materials with highest CS content (0.143 ± 0.007 nmol/10 mg scaffold) showed the greatest stimulatory effect on the biosynthetic activity of cultivated keratocytes. These findings suggest that a positive correlation is noticed between the NHS to EDC molar ratio and the CS content in the biopolymer matrices, thereby greatly affecting the corneal stromal cell growth.

3,4‐dihydroxy‐L‐phenylalanine as a cell adhesion molecule in serum‐free cell culture

In this article, we examined the feasibility of using 3,4-dihydroxy-L-phenylalanine (DOPA) as a cell adhesion molecule in serum-free cultures of anchorage-dependent mammalian cells. DOPA is a critical, functional element in mussel adhesive proteins and is known to bind strongly to various natural or synthetic materials. DOPA coating on culture plates was confirmed using X-ray photoelectron spectroscopy and energy-dispersive spectroscopy. Human dermal fibroblasts (HDFs) were cultured on DOPA-coated, fibronectin-coated, or no material-coated culture plates in serum-free medium. HDFs cultured on DOPA showed the highest cell adhesion ratio, spreading, and viability but the lowest apoptotic activity. Therefore, DOPA may be a useful cell-adhesion molecule for serum-free culture.

Effects of laminin‐coated carbon nanotube/chitosan fibers on guided neurite growth

This study assesses the ability and potential of carbon nanotube (CNT)/chitosan to guide axon re-growth after nerve injuries. The CNT/chitosan fibers were produced via the coagulation and hydrodynamic focusing method. Fiber width and morphology were adjusted using such parameters as syringe pumping rate and the coagulant used. The CNT/chitosan fiber diameters were 50-300 μm for syringe pumping rates of 6-48 mL/h. Polyethylene glycol/NaOH (25%, w/w) solution was a suitable coagulant for forming fibers with small diameters. Physical property tests demonstrate that the CNT/chitosan composites had superior tensile strength and electrical conductivity compared with those of chitosan alone. The MTT and LDH tests reveal that CNT/chitosan composites were not cytotoxic. To improve the neural cell affinity of CNT/chitosan fibers, laminin was incorporated onto fiber surfaces via the oxygen plasma technique; cell adhesion ratio increased significantly from 3.5% to 72.2% with this surface modification. Immunofluorescence staining and SEM imaging indicate that PC12 cells adhered successfully and grew on the laminin (LN)-coated CNT/chitosan films and fibers. Experimental results show that PC12 grown on LN-coated CNT/chitosan fibers in vitro extend longitudinally oriented neurites in a manner similar to that of native peripheral nerves. With the inherent electrical properties of CNTs, oriented CNT/chitosan fibers have a potential for use as nerve conduits in nerve tissue engineering.

Circulating CD56+ cells of diabetic women show deviated homing potential for specific tissues during and following pregnancy

Human uterine natural killer (uNK) cells, the dominant lymphocytes in early pregnancy decidua, are important for spiral arterial remodelling. uNK cells are thought to arise from circulating CD56(bright) NK cells that egress into decidualizing endometrium. Both incomplete spiral arterial modification and aberrant NK cell function have been linked with pre-eclampsia, a syndrome that is more prevalent in diabetic women. Since previous in vitro studies have shown that changes in decidual endothelium induced by type 1 diabetes (T1D) reduce its interactions with circulating leucocytes, we hypothesized that diabetes additionally has direct effects on circulating CD56(+) NK cells that impair their decidual homing potential. Serial blood samples were collected from control, T1D and T2D pregnant women throughout and after pregnancy. In vitro adhesion under shear forces was used to assay the functional capacity of circulating leucocytes and of CD56(+) cells to adhere to endothelium in cryostat sections of gestation day (gd) 7 normal mouse decidua, pancreas and lymph node. Fewer CD56(+) cells from diabetic compared with control women adhered to normal decidual endothelium. The CD56(+) cell/total cell adhesion ratio was also lower in diabetics. More diabetic CD56(+) cells adhered to pancreatic endothelium and their proportion was greater than for controls. Neither absolute nor proportional adhesion of CD56(+) cells to lymph node endothelium differed between diabetics and controls. The CD56(+) cell adhesion patterns of T1D and T2D women differ from those of non-diabetic women and support the hypothesis that diabetes impairs mechanisms that could be used by CD56(+) cells for egress into decidua.

Induction of injury to endothelium of pulmonary artery due to entero superantigen by up regulation of lymphocyte chemokine receptor 5

To observe the injurious effect of T cell activated by Staphylococcus enterotoxin B (SEB) on human pulmonary artery endothelial cell (HPAEC) and explore its possible mechanism. HPAEC was cocultured with SEB-activated T cells supernatant, and the secretion of chemotactic factors from HPAEC was examined. The Transwell inserts was used in chemoattraction assays. After HPAECs were cocultured with T cells and 10 ng/ml SEB for 3 days, HPAEC damage was monitored by microscopy and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. Three kinds of tested chemokines showed a time dependent increase in all supernatant of HPAEC incubated with different concentrations of T cells. After 72 hours, the monocyte chemoattractant protein 1 (MCP 1, ng/ml) in 1×10(-2) , 1×10(-1), 1×10(0) T cell supernatant groups was 1.240±0.103, 4.200±0.305, 6.500±0.500, respectively, macrophage inflammatory protein 1α (MIP 1α, ng/ml) was 0.210±0.015, 0.287±0.012, 0.531±0.037, respectively , and Rantes (ng/ml) was 1.420±0.074, 7.634±0.630, 15.700±1.300, respectively. Rantes presented a two phase secretion mode: in early 6 hours it increased swiftly, but relatively slow at 12, 24, 48, 72 hours. T cell adherent to polycarbonate membrane increased after SEB stimulation in superantigen group compared with control group without SEB stimulation (86.38±14.50 vs. 16.50±2.50, P<0.01). When 10 ng/ml SEB-activated T cell was cocultured with HPAEC, more of originally suspended cultured T cells adhered to HPAEC monolayer [(15.50±1.08)% vs. (1.60±0.22)%, PP<0.01], whereas the cell adhesion ratio decreased markedly in 1 μg/ml Met Rantes group [(4.39±0.66)%, PP<0.01). FACs test of HPAEC adherent T cell showed lymphocyte chemokine receptor 5 (CCR5)/CD4 and CCR5/CD8 increased over 2.5 folds and 2.8 folds compared with 100 ng/ml SEB-activated T cell. Cell death rate of HPAEC was increased when cocultured with SEB-activated T cell in superantigen group compared with HPAEC normal incubation group [(32.50±4.50)% vs. (3.50±0.50)%, P<0.01]. Increased chemoattraction and adherence of SEB-activated T cells to HPAEC could damage HPAEC; this effect was possibly due to up regulation of CCR5 on T cell.

Promoting effect of Chlamydia pneumoniae infection on human laryngeal carcinoma HEp-2 cell adhesion and migration

To explore the effect of Chlamydia pneumoniae (C.pn) infection on human laryngeal carcinoma cell line HEp-2 cell adhesion and migration, to further clarify the role and mechanism of C.pn infection in tumor metastasis. HEp-2 cells were infected with C.pn after the culture and propagation of C.pn. The cytopathic effect was observed by microscopy. Morphological characteristics of C.pn inclusions in HEp-2 cells were examined by fluorescence microscopy and acridine orange staining. The ultrastructural changes of C.pn inclusions in the HEp-2 cells were examined by transmission electron microscopy (TEM). Cell adhesion assay was performed to investigate the effect of C.pn infection on the adhesion of HEp-2 cells to collagen I. Wound-healing assay and transwell assay were performed to explore the effect of C.pn infection on HEp-2 cell migration. At 72 h post-infection, C.pn infected-HEp-2 cells were swollen and partially desquamated. Numerous vacuoles (inclusions) were observed and C.pn inclusions occupied almost the whole cytoplasm of the HEp-2 cells. Grape-like C.pn inclusions were observed in the HEp-2 cells stained with acridine orange under a fluorescence microscope at 72 h after infection. Under TEM, there were more mature pear-shaped elementary bodies, but less larger and round reticulate bodies in the HEp-2 cells infected with C.pn for 72 h. In the cell adhesion assay, the A value in C.pn infection group was 0.669 ± 0.011, significantly higher than that in the control group (0.558 ± 0.005) at 2 h after infection (P < 0.001). The cell adhesion ratio in the C.pn infection group was 119.89%. The migration distance of C.pn infected-HEp-2 cells in the wound-healing assay was significantly longer than that of control cells at 24 h after infection (P < 0.05). HEp-2 cells infected with C.pn for 12 h migrated more than the control cells in the transwell assay (23.40 ± 2.41 vs 10.40 ± 1.67) (P < 0.001). C.pn infection can significantly promote HEp-2 cell adhesion to collagen I and migration of HEp-2 cells, indicating that C.pn infection may play an important role in promoting the metastasis of laryngeal cancer.

Effects of transfection of ICAP-1α and its mutants on adhesion and migration of 2H-11 cells

This study examined the effect of integrin cytoplasmic domain-associated protein 1α (ICAP-1α) and its mutatants T38A and I138A on the adhesion, migration and tube formation of 2H-11 cells. rAAV-ICAP-1α, rAAV-T38A and rAAV-I138A were constructed. After infection, the expression of ICAP-1α and p-ERK1/2, p-c-Jun protein was measured by Western blotting. Adhesion ability was evaluated by using MTT. Cell migration was determined by using Boyden chamber method. Tube formation test was conducted on Matrigel. The results showed that in ICAP-1α, T38A and I138A groups, ICAP-1α protein expression was increased. In T38A and I138A groups, phospho-ERK1/2, phospho-c-Jun protein expressions were significantly increased as compared with the control group and the GFP group. ICAP-1α group protein expression was obviously decreased when compared with the control group and the GFP group. Cell adhesion ratio was 0.1429±0.0080 in control group, 0.1434±0.0077 in GFP group and the ratio in T38A and I138A groups increased to 0.3210±0.0082 and 0.3250±0.0079, respectively. In ICAP-1α group, the ratio was decreased to 0.1005±0.0073. In T38A and I138A groups, the number of migrating 2H-11 cells was increased to 31.45±3.20 and 33.10±5.40 against 18.51±2.80 in control group and 20.47±3.12 in GFP group. In ICAP-1α group, the number was decreased to 12.06±1.72. The number of tube-like structures was increased to 20.41±2.54 in T38A and to 22.26±3.07 in I138A groups as compared to those of control group 12.45±1.84 and GFP group 13.63±2.71. In ICAP-1α group, the number of tube-like structures was decreased to 8.32±1.24. It was suggested that rAAV-T38A and rAAV-I138A transfection can substantially increase 2H-11 cell adhesion, migration and angiogenisis, while rAAV-ICAP-1α can greatly inhibit the effect. These effects might be correlated with ERK1/2 and c-Jun protein phosphorylation.

Morphological and functional studies of rat hepatocytes on a hydrophobic or hydrophilic polydimethylsiloxane surface

This study describes the morphological and functional behavior of rat hepatocytes on a polydimethylsiloxane (PDMS)-coated surface. Hepatocytes were cultured on hydrophobic or hydrophilic PDMS-coated surfaces in serum-free and serum-containing media. In the serum-free medium, almost all hepatocytes adhered onto the surface irrespective of the wettability, with a cell adhesion ratio of >90% at 24h. In the serum-containing medium, although they strongly adhered onto the hydrophilic surface (cell adhesion ratio >85%), the ratio on the hydrophobic surface was <15%. Furthermore, hepatocytes in the serum-free medium gradually formed spheroids irrespective of the surface characteristics; however, on the hydrophilic surface in the serum-containing medium, they maintained a monolayer configuration for up to 10 days, and their numbers gradually decreased over time. Expression levels of the functional activities (albumin secretion and ammonia removal) and the cell–cell adhesion molecules (cadherin and connexin-32) were higher in the hepatocytes that formed spheroids compared to those which assumed a monolayer configuration, and these levels were maintained for at least 10 days. These results suggest that the wettability of PDMS and the composition of the culture medium together control the cell adhesion, morphology and expression of functional genes in hepatocytes.

Steady Measurement of Glucose and Oxygen Consumption Rate and Cell Adhesion Ratio of HepG2 Hepatoma Cells for High Density Culture

High density culture is one of the essential technologies for an engineered tissue where cellular physiological functions such as enzymatic reactions are required. Cellular energy metabolism and adherence to scaffold are the most important factor to design high density culture. In this study, we developed flow chamber with which steady state measurement of glucose and oxygen metabolic rate of HepG2 cell line can be measured. In addition, cell adhesion ratio was measured using the same flow chamber. The results provided not only glucose and oxygen consumption rates to glucose concentration, but also the interaction of both consumption rates on glucose concentration. Cell adhesion ratio under various shear stresses showed that HepG2 cells are detached under the shear stress around 10-60 Pa. This large diversity of detached shear stress suggests that the adhesion force of collagen fibers to substrate may dominate the detachment.

Interaction of Human Lactoferrin with Cell Adhesion Molecules through RGD Motif Elucidated by Lactoferrin-binding Epitopes

Lactoferrin (LF) is an iron-binding secretory protein, which is distributed in the secondary granules of polynuclear lymphocytes as well as in the milk produced by female mammals. Although it has multiple functions, for example antimicrobial, immunomodulatory, antiviral, and anti-tumor metastasis activities, the receptors responsible for these activities are not fully understood. In this study, the binding epitopes for human LF were first isolated from a hexameric random peptide library displayed on T7 phage. Interestingly, two of the four isolated peptides had a representative cell adhesion motif, Arg-Gly-Asp (RGD), implying that human LF interacts with proteins with the RGD motif. We found that human LF bound to the RGD-containing human extracellular matrix proteins, fibronectin and vitronectin. Furthermore, human LF inhibited cell adhesion to these matrix proteins in a concentration-dependent manner but not to the RGD-independent cell adhesion molecule like laminin or collagen. These results indicate that a function of human LF is to block the various interactions between the cell surface and adhesion molecules. This may explain the multifunctionality of LF.

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have less influence on the peptide design. Such rules that are indicative of the nature of the functional peptide sequence can be obtained only by the mass comparison analysis of PIASPAC using peptide array. By following such indicative rules, numerous amino acid combinations can be effectively screened for further examination of novel peptide design.

Vaso-occlusion in Children With Sickle Cell Disease: Clinical Characteristics and Biologic Correlates

Vaso-occlusive pain is a frequent manifestation of sickle cell disease, but most clinical studies have documented only those pain episodes for which patients seek acute care or require hospitalization. Based on limited previous studies, the authors suggest that pain episodes managed at home are more frequent then those resulting in acute care management but likely share a common pathophysiology. The authors determined the characteristics of vaso-occlusive pain managed at home in 30 subjects (ages 6-19 years) using a self-report diary daily for 6 months. A total of 175 pain episodes were reported in 4,885 days, with 51% lasting 1 day or less. Severe pain, rated as 7 to 10 on a 10-point scale, was reported on 12% of pain days, but most pain was of mild to moderate intensity. A combination of baseline hematologic parameters and biomarkers assessing erythrocyte/endothelial cell adhesion, including hematocrit, fetal hemoglobin, and adhesion ratio, were statistically significant predictors of pain frequency in statistical analyses. Given the overlap in clinical features and predictive hematologic parameters of home-managed and acute care-managed pain, both likely represent a continuum of frequency and severity rather than distinct clinical entities. The higher frequency of these home-managed episodes suggests their potential utility as additional outcome measures in studies of vaso-occlusive pain.

Pleiotropic influence of triiodothyronine on thymus physiology.

It is well demonstrated that the thymus gland is under neuroendocrine control. Thymic endocrine function can be modulated by a variety of hormones including those secreted by the thyroid gland. This prompted us to investigate putative influences of T3 in further aspects of thymus physiology. We showed that T3-treated animals exhibited an increase in thymus weight, cellularity and cycling cells. Moreover, Thy1+ thymocytes as well as CD4-CD8 defined subsets were augmented in absolute numbers, whereas PgP.1+ cells increased in both absolute and percentage values. In parallel, the total numbers of thymic nurse cells were also increased. Regarding the expression of extracellular matrix components (ECM) by microenvironmental cells, we observed an enhancement in the intrathymic ECM upon T3 in vivo treatment. Similar effects were found in vitro by treating a thymic epithelial cell line or thymic nurse cell-derived epithelial cultures with T3. This treatment also increased the expression of ECM receptors by thymic epithelial cultures. Interestingly, an enhancement in thymocyte/thymic epithelial cell adhesion ratio was observed after T3 treatment of epithelial cells. Our data suggest that T3 exerts a pleiotropic effect upon thymus physiology, stimulating thymocyte differentiation, not only by modulating epithelial cell hormonal secretion but also their production of ECM proteins and respective receptors.