Margaret D. M. Evans

Mechanism of initial attachment of cells derived from human bone to commonly used prosthetic materials during cell culture

The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in cell culture. In this study the contribution that serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min of culture was determined for metallic and ceramic surfaces. The requirement for Fn or Vn for attachment and spreading of bone-derived cells onto stainless steel 316 (SS), titanium (Ti) and alumina (Al2O3) and to polyethyleneterephthalate (PET) was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. Attachment and spreading of bone-derived cells onto SS, Ti and Al2O3 surfaces were reduced by 73-83% when the cells were seeded in medium containing serum from which the Vn had been removed. Cell attachment and spreading on these surfaces when seeded in medium containing Fn-depleted serum (which contained Vn) were not reduced to the same extent as in the medium containing Vn-depleted serum. The bone-derived cells failed to attach to the surfaces to the same extent when seeded in medium containing serum depleted of both Vn and Fn. Our results show that for human bone-derived cells, the attachment and spreading of cells onto SS, Ti and Al2O3 as well as PET during the first 90 min of a cell culture attachment assay are a function of adsorption of serum Vn onto the surface.

Attachment of human bone cells to tissue culture polystyrene and to unmodified polystyrene: the effect of surface chemistry upon initial cell attachment

Cell culture studies have often been used in the determination of the suitability of biomaterials as surfaces for the attachment and growth of cells. For such studies of surfaces for potential use in bone implants, cells derived from bone may be maintained in culture on tissue culture polystyrene (TCPS). We have determined the contribution that serum fibronectin (FN) or vitronectin (VN) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min following seeding on culture surfaces. The attachment of bone-derived cells to TCPS was simulated two-fold by the addition of 10% (v/v) fetal bovine serum (FBS) to the seeding culture medium. The roles of FN and VN were determined by selective removal of the FN or VN from the FBS prior to addition to the culture medium. FBS from which the VN had been removed did not have this stimulatory activity. In contrast, the attachment of bone-derived cells onto TCPS from medium containing FN-depleted serum (which contained VN) was the same as when intact FBS was used. There was incomplete attachment of bone-derived cells (27% of cells) when seeded in medium containing FBS depleted of both VN and FN. Our results show that for human bone-derived cells, the attachment onto TCPS of cells planted in medium containing FBS during the first 90 min of culture is principally as a result of adsorption onto the surface of serum VN. As unmodified polystyrene (PS) has also been used previously as a model biomaterial surface, PS was compared to TCPS for attachment of the bone-derived cells. Attachment of bone-derived cells to TCPS was twice that onto PS, both when the medium was serum-free and when it contained FBS. Bone-derived cells attached to TCPS or PS onto which purified VN or FN had been precoated, with VN adsorbed onto PS being as effective as was VN adsorbed onto TCPS. With FN, there was an effect of the polystyrene surface chemistry which was evident in that suboptimal concentrations of FN had a slightly higher potency when adsorbed onto TCPS than did the same concentrations of FN coated onto PS. When preadsorbed onto TCPS, the potency of FN for attachment of bone-derived cells was at least equal to that of VN.

Atmospheric gas plasma–induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis

Atmospheric gas plasmas (AGPs) up-regulate intracellular ROS levels and induce apoptosis in melanoma cells. Evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

Modulation of corneal epithelial stratification by polymer surface topography

The topography and porosity of a polymer may affect the epithelialization of a corneal implant. We used an in vitro model to examine the effect of polymer surface topography on corneal epithelial tissue stratification and the deposition of proteins associated with epithelial adhesion. A range of topographies was provided by polycarbonate membranes with nominal pore diameters of 0.1, 0.4, 0.8, 1.0, 2.0, or 3.0 microm and a nonporous surface. Stratification of epithelial tissue outgrowth on these surfaces was evaluated using light and electron microscopy. Deposition of proteins associated with basement membrane and adhesion complex formation at the tissue-polymer interface was assessed using immunohistochemistry. Surfaces with pores in the 0.1-0.8-microm-diameter range supported superior stratification and protein deposition compared with those containing pores of > or = 1.0 microm. Cytoplasmic processes penetrated single pores 2.0 and 3.0 microm in diameter and fused pores 1.0 microm in diameter. Tissue on the nonporous surface had a lower level of stratification compared with surfaces with pores 0.1-0.8 microm in diameter. These results point to the significance of surface topography in biomaterial applications that require persistent epithelialization.

Polymer surface chemistry and a novel attachment mechanism in corneal epithelial cells.

The initial attachment reaction of most cultured cell types to polymers is based on a linkage of integrin receptors to serum-derived fibronectin and vitronectin that adsorb onto the polymer surface. Recently isolated corneal epithelial cells have an additional attachment mechanism, known to operate on tissue culture polystyrene, which involves endogenous protein synthesis and an intact system of microtubules. Here, we determine if this novel attachment mechanism is operative on polymers of different surface chemistries. The attachment, growth, and deposition of basement membrane proteins by corneal epithelial cells was compared on two hydrophilic surfaces (tissue culture polystyrene and Primaria) and one relatively hydrophobic surface (unmodified polystyrene). Superior levels of cell attachment were found on the hydrophilic polymers, but cells also attached effectively to the hydrophobic surface. Growth rates showed that the cells were able to overcome the differential effects of polymer surface chemistry during a 7-day time period. Polymer surface chemistry had subtle effects on the temporal pattern of biosynthesis of extracellular matrix proteins likely to be involved in cell adhesion. These results show that effective attachment and growth can occur on a hydrophobic polymer when corneal epithelial cells use the endogenous attachment mechanism.

Persistent adhesion of epithelial tissue is sensitive to polymer topography

The persistent adhesion of corneal epithelial tissue to the surface of a porous polymer is of interest in the development of a corneal onlay. Using an in vitro model system, this study examined the effect of polymer surface topography on the assembly of basement membrane and hemidesmosomes. Corneal epithelial tissue was grown on polycarbonate surfaces with a range of pore sizes (0.1-3.0 micron, pore diameter) and an equivalent nonporous surface. The ultrastructure of the tissue-polymer interface was evaluated using electron microscopy. On the porous surfaces, the tissue responded to a balance between the size of the discontinuity (pores) and the amount of polymer surface between the pores. Continuous basement membrane and a regular pattern of hemidesmosomal plaque occurred only on the 0.1 micron surface, where both the pores and the total surface area covered by pores were relatively small. The assembly of adhesive structures on surfaces with pore diameters between 0.4-2.0 microns was restricted to regions of polymer between pores. No adhesive structures assembled on the nonporous or on the 3.0-micron surface. These results demonstrate that, in addition to porosity, surface topography is a significant factor in the formation of structures involved in the persistent adhesion of stratified epithelial tissue on a polymer.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system.

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Encapsulated pancreatic progenitors derived from human embryonic stem cells as a therapy for insulin-dependent diabetes.

Cellular‐based therapies for insulin‐dependent diabetes are potential means of achieving and maintaining normal blood glucose levels (BGL) without the need for insulin administration. Islets isolated from donor pancreases have been the most common tissue used to date, but supply is a limiting factor. The use of human embryonic stem cells (hESC) as a therapy became a possibility with the report that these cells could be differentiated to pancreatic progenitors (PP) over 12 days in vitro. Conversion of PP to glucose‐responsive insulin‐secreting cells can be achieved by transplanting the progenitors in vivo where cell maturation occurs. To date this step has not been shown under in vitro conditions.

Synthetic corneal inlays

This review is based on the activities of the Vision Cooperative Research Centre (previously Cooperative Research Centre for Eye Research and Technology) Corneal Implant team from 1991 to 2007. The development of a synthetic polymer of perfluoropolyether (PFPE), meeting essential physical and biological requirements, for use as a corneal inlay is presented. Each inlay was placed in a corneal flap created with a microkeratome and monitored over a two‐year period in a rabbit model. The results indicate that the PFPE implant shows excellent biocompatibility and biostability. As a result, a Phase 1 clinical trial is being conducted. Three years post‐implantation, the PFPE inlays are exhibiting continued excellent biocompatibility. Corneal inlays made from PFPE are biocompatible with corneal tissue in the long term and offer a safe and biologically‐acceptable alternative to other forms of refractive surgery.

A review of the development of a synthetic corneal onlay for refractive correction

A synthetic corneal onlay, or implantable contact lens, could obviate the need for spectacles or conventional contact lenses in patients who seek convenient, reversible correction of refractive error. Several research groups have attempted to develop such a product in the past but much of the data from these studies remains unpublished due to commercial interests. This article reviews relevant papers and patents in the corneal implant field and discusses our efforts to develop a synthetic corneal onlay using a perfluoropolyether-based polymer.