α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis

Background: Ovarian cancer is characterized by a wide-spread intra-abdominal metastases which represents a major clinical hurdle in the prognosis and management of the disease. A significant proportion of ovarian cancer cells in peritoneal ascites exist as multicellular aggregates or spheroids. We hypothesize that these cellular aggregates or spheroids are invasive with the capacity to survive and implant on the peritoneal surface. This study was designed to elucidate early inherent mechanism(s) of spheroid survival, growth and disaggregation required for peritoneal metastases.

Methods: In this study, we determined the growth pattern and adhesive capacity of ovarian cancer cell lines (HEY and OVHS1) grown as spheroids, using the well established liquid overlay technique, and compared them to a normal ovarian cell line (IOSE29) and cancer cells grown as a monolayer. The proteolytic capacity of these spheroids was compared with cells grown as a monolayer using a gelatin zymography assay to analyze secreted MMP-2/9 in conditioned serum-free medium. The disaggregation of cancer cell line spheroids was determined on extracellular matrices (ECM) such as laminin (LM), fibronectin (FN) and collagen (CI) and the expression of α2, α3, αv, α6 and β1 interin was determined by flow cytometric analysis. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin was used to inhibit disaggregation as well as activation of MMPs in spheroids.

Results: We demonstrate that ovarian cancer cell lines grown as spheroids can sustain growth for 10 days while the normal ovarian cell line failed to grow beyond 2 days. Compared to cells grown as a monolayer, cancer cells grown as spheroids demonstrated no change in adhesion for up to 4 days, while IOSE29 cells had a 2–4-fold loss of adhesion within 2 days. Cancer cell spheroids disaggregated on extracellular matrices (ECM) and demonstrated enhanced expression of secreted pro-MMP2 as well as activated MMP2/MMP9 with no such activation of MMP's observed in monolayer cells. Flow cytometric analysis demonstrated enhanced expression of α2 and diminution of α6 integrin subunits in spheroids
versus monolayer cells. No change in the expression of α3, αv and β1 subunits was evident. Conversely, except for αv integrin, a 1.5–7.5-fold decrease in α2, α3, α6 and β1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin inhibited disaggregation as well as activation of
MMPs in spheroids.

Conclusion: Our results suggest that enhanced expression of α2β1 integrin may influence spheroid disaggregation and
proteolysis responsible for the peritoneal dissemination of ovarian carcinoma. This may indicate a new therapeutic target
for the suppression of the peritoneal metastasis associated with advanced ovarian carcinomas.

Endothelialisation and cell retention on gelation-chitosan coated electrospun polyurethane, poly (lactide-co-glycolide) and collagen-coated pericardium

Arterial bypass and heart valve replacements are two of the most common surgical treatments in cardiovascular surgery today. Currently, artificial materials are used as substitute for these cardiac tissues. However, these foreign materials do not have the ability to grow, repair or remodel and are thrombogenic, leading to stenosis. With the aid of tissue engineering, it is possible to develop functional identical copies of healthy heart valves and arteries, which are biocompatible. Although much effort has been made into this area, there are still inconsistencies with respect to
endothelialisation and cell retention on synthetic biological grafts. These variations may be attributed to differences in factors such as cell seeding density, incubation periods and effects of shear stress. In this study, we have compared the endothelialisation and cell retention between gelain chitosan-coated electrospun polyurethane (PU), poly (lactide co-glycolide) (PGA/PLA) and collagen-coated pericardium. Endothelial cells adhered to all of the materials as early as 1–day post seeding. After 7-day of seeding, the coverage on PU was almost 45% and that on PGA/PLA was about 25% and the least was on collagen-coated pericardium of approximately 15%. It was observed that the PU showed superior cell coverage and cell retention in comparison to the PGA/PLA and collagen-coated pericardium.

Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient

Histopathological scoring of disease stage uses descriptive categories without measuring the amount of fibrosis. Collagen, the major component of fibrous tissue, can be quantified by computer-assisted digital image analysis (DIA) using histological sections. We determined relationships between DIA, Ishak stage, and hepatic venous pressure gradient (HVPG) reflecting severity of fibrosis. One hundred fifteen patients with hepatitis C virus (HCV) who had undergone transplantation had 250 consecutive transjugular liver biopsies combined with HVPG (median length, 22 mm; median total portal tracts, 12), evaluated using the Ishak system and stained with Sirus red for DIA. Liver collagen was expressed as collagen proportionate area (CPA). Median CPA was 6% (0.2-45), correlating with Ishak stage (stage 6 range, 13%-45%), and with HVPG (r = 0.62; P < 0.001). Median CPA was 4.1% when HVPG was less than 6 mm Hg and 13.8% when HVPG was 6 mm Hg or more (P < 0.0001) and 6% when HVPG was less than 10 mm Hg and 17.3% when HVPG was 10 mm Hg or higher (P < 0.0001). Only CPA, not Ishak stage/grade, was independently associated by logistic regression, with HVPG of 6 mm Hg or more [odds ratio, 1.206; 95% confidence interval (CI), 1.094-1.331; P < 0.001], or HVPG of 10 mm Hg or more (odds ratio, 1.105; 95% CI, 1.026-1.191; P = 0.009). CPA increased by 50% (3.6%) compared with 20% in HVPG (1 mm Hg) in 38 patients with repeated biopsies. Conclusion: CPA assessed by DIA correlated with Ishak stage scores and HVPG measured contemporaneously. CPA was a better histological correlate with HVPG than Ishak stage, had a greater numerical change when HVPG was low, and resulted in further quantitation of fibrosis in cirrhosis.

Matrices representation of multi soft-sets and its application

In previous paper, we introduced a concept of multi-soft sets and used it for finding reducts. However, the comparison of the proposed reduct has not been presented yet, especially with rough-set based reduct. In this paper, we present matrices representation of multi-soft sets. We define AND and OR operations on a collection of such matrices and apply it for finding reducts and core of attributes in a multi-valued information system. Finally, we prove that our proposed technique for reduct is equivalent to Pawlak’s rough reduct.

Three-dimensional tissue scaffolds from interbonded poly(e-caprolactone) fibrous matrices with controlled porosity

In this article, we report on the preparation and cell culture performance of a novel fibrous matrix that has an interbonded fiber architecture, excellent pore interconnectivity, and controlled pore size and porosity. The fibrous matrices were prepared by combining melt-bonding of short synthetic fibers with a template leaching technique. The microcomputed tomography and scanning electron microscopy imaging verified that the fibers in the matrix were highly bonded, forming unique isotropic pore architectures. The average pore size and porosity of the fibrous matrices were controlled by the fiber/template ratio. The matrices having the average pore size of 120, 207, 813, and 994 mm, with the respective porosity of 73%, 88%, 96%, and 97%, were investigated. The applicability of the matrix as a three-dimensional (3D) tissue scaffold for cell culture was demonstrated with two cell lines, rat skin fibroblast and Chinese hamster ovary, and the influences of the matrix porosity and surface area on the cell culture performance were examined. Both cell lines grew successfully in the matrices, but they showed different preferences in pore size and porosity. Compared with two-dimensional tissue culture plates, the cell number on 3D fibrous matrices was increased by 97.27% for the Chinese hamster ovary cells and 49.46% for the fibroblasts after 21 days of culture. The fibroblasts in the matrices not only grew along the fiber surface but also bridged among the fibers, which was much different from those on two-dimensional scaffolds. Such an interbonded fibrous matrix may be useful for developing new fiber-based 3D tissue scaffolds for various cell culture applications.

Inter-bonded fibrous matrices for 3D tissue engineering scaffolds

The thesis established a stable three-dimensional fibrous tissue scaffold that has controlled pore structure and inter-bonded fibrous structure, and also examined the effects of the 3D fibrous matrices and functional surfaces including nano-scale topography, bioactive CaP coating and antibacterial treatment on the cell growth behavior for tissue engineering application.

Elastin and collagen enhances electrospun aligned polyurethane as scaffolds for vascular graft

Mismatch in mechanical properties between synthetic vascular graft and arteries contribute to graft failure. The viscoelastic properties of arteries are conferred by elastin and collagen. In this study, the mechanical properties and cellular interactions of aligned nanofibrous polyurethane (PU) scaffolds blended with elastin, collagen or a mixture of both proteins were examined. Elastin softened PU to a peak stress and strain of 7.86 MPa and 112.28 % respectively, which are similar to those observed in blood vessels. Collagen-blended PU increased in peak stress to 28.14 MPa. The growth of smooth muscle cells (SMCs) on both collagen-blended and elastin/collagen-blended scaffold increased by 283 and 224 % respectively when compared to PU. Smooth muscle myosin staining indicated that the cells are contractile SMCs which are favored in vascular tissue engineering. Elastin and collagen are beneficial for creating compliant synthetic vascular grafts as elastin provided the necessary viscoelastic properties while collagen enhanced the cellular interactions.

Surface-sensitive method to determine calcium carbonate filler contents in cellulose matrices

This paper describes a method using attenuated total reflectance infra-red spectroscopy to determine the surface concentration of calcium carbonate in paper samples, by applying the linear relationship between the relative infra-red absorption integrals and the concentration. The method was able to detect micro-variations in the surface concentration and could also distinguish between different sheets as well as between the top and bottom side of one sheet. The samples were also split and the calcium carbonate concentration was determined within and compared to bulk calcium carbonate concentration determined from ash testing. The surface results were also compared with analysed scanning electron microscopy images generated from back-scattering electrons. The comparison shows that both sets of results are in excellent agreement. Depending on the sample, large errors (95% confidence) were observed. These, however, are caused by micro-variations of the surface concentration, rather than by inaccuracies of the technique, which is estimated to be less than 1%. Furthermore, measurements of various sample orientations suggest that anisotropic polarisation effects can be neglected. The method can be applied to paper and cellulose matrices having calcium carbonate filler contents of less than 50%. Due to spectral overlaps it is not suitable to determine kaolin filler contents.