Biomedical subjects

S Downes

Publications and source records attributed to S Downes.

At least 19 recordsLinked to original sources

PubMed2004

Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery.

Microporous materials have been produced by gradual precipitation from solutions of poly(epsilon-caprolactone) (PCL) in acetone induced by solvent extraction across a semi-permeable PCL membrane which is formed in situ at the polymer solution/non-solvent interface. Microparticulates of hydroxyapatite and inulin polysaccharide, respectively, were incorporated in precipitation cast PCL matrices to illustrate potential applications in hard tissue repair and macromolecular drug release. Microporous PCL and HA filled PCL materials were found to provide a favourable surface for attachment and growth of primary human osteoblasts in cell culture. The in vitro degradation characteristics of microporous PCL and inulin/PCL materials in PBS at 37 degrees C were monitored over 45 months. Microporous PCL demonstrated zero weight loss, minor changes in molecular weight characteristics and a fairly constant indentation resistance of around 1 MN/m2. Inulin-loaded PCL materials exhibited a total weight loss of approximately 17% after 12 months in PBS. The indentation resistance decreased by 50% from an initial value of 28 MN/m2 in the first 2 months and then remained stable. Precipitation cast materials based on PCL are expected to be useful for formulating long-term, controlled release devices for bioactive molecules such as growth factors and hormones and extended-residence supports for cell growth and tissue development.

Bone Substitutes↗

PubMed2003

Comparison of two reference standards in validating two field mydriatic digital photography as a method of screening for diabetic retinopathy.

AIM: To compare two reference standards when evaluating a method of screening for referable diabetic retinopathy. METHOD: Clinics at Oxford and Norwich Hospitals were used in a two centre prospective study of 239 people with diabetes receiving an ophthalmologist's examination using slit lamp biomicroscopy, seven field 35 mm stereophotography and two field mydriatic digital photography. Patients were selected from those attending clinics when the ophthalmologist and ophthalmic photographer were able to attend. The main outcome measures were the detection of referable diabetic retinopathy as defined by the Gloucestershire adaptation of the European Working Party guidelines. RESULTS: In comparison with seven field stereophotography, the ophthalmologist's examination gave a sensitivity of 87.4% (confidence interval 83.5 to 91.5), a specificity of 94.9% (91.5 to 98.3), and a kappa statistic of 0.80. Two field mydriatic digital photography gave a sensitivity of 80.2% (75.2 to 85.2), specificity of 96.2% (93.2 to 99.2), and a kappa statistic of 0.73. In comparison with the ophthalmologist's examination, two field mydriatic digital photography gave a sensitivity of 82.8% (78.0 to 87.6), specificity of 92.9% (89.6 to 96.2), and a kappa statistic of 0.76. Seven field stereo gave a sensitivity of 96.4% (94.0 to 98.8), a specificity of 82.9% (77.4 to 88.4), and a kappa statistic of 0.80. 15.3% of seven field sets, 1.5% of the two field digital photographs, and none of the ophthalmologist's examinations were ungradeable. CONCLUSION: An ophthalmologist's examination compares favourably with seven field stereophotography, and two field digital photography performs well against both reference standards.

Adult↗

PubMed2002

The role of protein adsorption on chondrocyte adhesion to a heterocyclic methacrylate polymer system.

Chondrocyte adhesion to a polymer system consisting of poly(ethyl methacrylate) and tetrahydrofurfuryl methacrylate (PEMA/THFMA) has been investigated in vitro. The adhesive glycoproteins, fibronectin and vitronectin were studied for their role in promoting cell attachment. Fibronectin was the best substrate for chondrocyte attachment, if it was pre-adsorbed and did not have to compete with other proteins for attachment sites. Chondrocytes began to spread on fibronectin coated glass although they remained rounded on the libronectin coated PEMA/THFMA system. Vitronectin was better at competing with the other proteins in serum and was the main adhesive protein for chondrocyte attachment to TCP and the PEMA/THFMA system in normal serum medium. Serum contains non-adhesive proteins that compete for binding sites and hence reduce cell attachment. The alpha5beta1 and alpha(v)beta3/beta5 integrins were detected on the chondrocytes although there may be a difference in expression between different material surfaces.

Adsorption↗

PubMed2002

Biocomposites of non-crosslinked natural and synthetic polymers.

Biocomposite films comprising a non-crosslinked, natural polymer (collagen) and a synthetic polymer, poly(epsilon-caprolactone) (PCL), have been produced by impregnation of lyophilised collagen mats with a solution of PCL in dichloromethane followed by solvent evaporation. This approach avoids the toxicity problems associated with chemical crosslinking. Distinct changes in film morphology, from continuous surface coating to open porous format, were achieved by variation of processing parameters such as collagen:PCL ratio and the weight of the starting lyophilised collagen mat. Collagenase digestion indicated that the collagen content of 1:4 and 1:8 collagen:PCL biocomposites was almost totally accessible for enzymatic digestion indicating a high degree of collagen exposure for interaction with other ECM proteins or cells contacting the biomaterial surface. Much reduced collagen exposure (around 50%) was measured for the 1:20 collagen:PCL materials. These findings were consistent with the SEM examination of collagen:PCL biocomposites which revealed a highly porous morphology for the 1:4 and 1:8 blends but virtually complete coverage of the collagen component by PCL in the 1:20 samples. Investigations of the attachment and spreading characteristics of human osteoblast (HOB) cells on PCL films and collagen:PCL materials respectively, indicated that HOB cells poorly recognised PCL but attachment and spreading were much improved on the biocomposites. The non-chemically crosslinked, collagen:PCL biocomposites described are expected to provide a useful addition to the range of biomaterials and matrix systems for tissue engineering.

Biocompatible Materials↗

PubMed2002

Cellularity of human annulus tissue: an investigation into the cellularity of tissue of different pathologies.

AIMS: To investigate the cells of the inner annulus and to demonstrate that differences in disc pathology can be identified at the cellular level. METHODS AND RESULTS: Annulus tissue taken from scoliotic, degenerate and prolapsed human disc tissue was processed for histology and transmission electron microscopy. Ki67 antibody was used to identify cells in the active part of the cell cycle and cell surface receptors for the matrix-degrading enzyme urokinase were immunolocalized. More chondron clusters were observed in tissue from prolapsed discs than in degenerate and scoliotic discs. Positive Ki67 staining was detected in cells within chondron clusters. Most cells observed from scoliotic and prolapsed annulus contained distinctive nuclei and organelles, whereas cells from degenerate discs contained very few well-defined organelles but abundant glycogen deposits. Immunolocalization identified urokinase receptors on the surface of cells from degenerate discs but not in the other pathologies. CONCLUSIONS: Cellular differences appear to underlie different types of disc pathology. The annulus tissue taken from prolapsed discs appeared to contain more chondron clusters and more active cells than scoliotic and degenerative tissue, suggesting a possible wound repair response. In contrast, cell and matrix degeneration appeared to be the most significant underlying processes in degenerate discs.

Adolescent↗

PubMed2001

Osteoblast cell death on methacrylate polymers involves apoptosis.

The success of an implant depends on the implant-tissue interface. There are many causes of implant failure, one of which is tissue necrosis. The aim of this in vitro study was to determine whether cell death of primary human osteoblasts (implant site specific cells) occurred by apoptosis (a form of programmed cell death) on two methacrylate polymers. Cells were cultured on poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate and poly(methyl methacrylate in the form of 13-mm discs, in conditioned medium containing leachable monomer and in the presence of various concentrations of monomer itself in the culture medium. It was found that monomer and leached monomer caused apoptosis of human osteoblast cells in this system. Tetrahydrofurfuryl methacrylate monomer was found to be more toxic than currently used monomer methylmethacrylate. Preincubation of polymers in serum containing medium was found to increase the biocompatibility of the polymers. High levels of apoptosis occurred on polymer used directly after polymerization. Apoptosis levels were decreased after polymer was incubated at 60 degrees C overnight or for 3 days. Apoptosis therefore may occur in cells at the implant site in vivo.

Apoptosis↗

PubMed2001

Biodegradable polymer/hydroxyapatite composites: surface analysis and initial attachment of human osteoblasts.

Biodegradable polymer/hydroxyapatite (HA) composites have potential application as bone graft substitutes. Thin films of polymer/HA composites were produced, and the initial attachment of primary human osteoblasts (HOBs) was assessed to investigate the biocompatibility of the materials. Poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLA) were used as matrix materials for two types of HA particles, 50-microm sintered and submicron nonsintered. Using ESEM, cell morphology on the surfaces of samples was investigated after 90 min, 4 h, and 24 h of cell culture. Cell activity and viability were assessed after 24 h of cell culture using Alamar blue and DNA assays. Surface morphology of the polymer/HA composites and HA exposure were investigated using ESEM and EDXA, respectively. ESEM enabled investigation of both cell and material surface morphology in the hydrated condition. Combined with EDXA it permitted chemical and visual examination of the composite. Differences in HA exposure were observed on the different composite surfaces that affected the morphology of attached cells. In the first 4 h of cell culture, the cells were spread to a higher degree on exposed HA regions of the composites and on PLA than they were on PCL. After 24 h the cells were spread equally on all the samples. The cell activity after 24 h was significantly higher on the polymer/HA composites than on the polymer films. There was no significant difference in the activity of the cells on the various composite materials. However, cells on PCL showed higher activity compared to those on PLA. A polymer surface exhibiting "point exposure" of HA appeared to provide a novel and favorable substrate for primary cell attachment. The cell morphology and activity results indicate a favorable cell/material interaction and suggest that PLA and PCL and their composites with HA may be candidate materials for the reconstruction of bony tissue. Further investigations regarding long-term biomaterial/cell interactions and the effects of acidic degradation products from the biodegradable polymers are required to confirm their utility.

Bone Substitutes↗

PubMed2001

Role of the cross-linking enzyme tissue transglutaminase in the biological recognition of synthetic biodegradable polymers.

The calcium-dependent cross-linking enzyme tissue transglutaminase (tTgase, type II) is a potential novel player at the cell surface, where its contribution to cell adhesion and stabilization of the extracellular matrix is becoming increasingly recognized. We investigated whether tTgase enhances the biological recognition of poly (DL lactide co-glycolide) (PLG), poly (epsilon-caprolactone) (PCL), and poly (L lactide) (PLA), biomaterials widely used in medical implants. Three cell-model systems consisting of human osteoblasts, endothelial cells (ECV-304), and Swiss 3T3 fibroblasts were utilized, in which tTgase expression was modulated by gene transfer, and the ability of cells to spread on these polymers was quantified in relation to the altered level of expressed tTGase. Results show that over-expression of tTgase in human osteoblasts positively correlated with cell spreading on PLG, while no attachment and spreading was found on PCL and PLA. Antisense silencing of tTgase in the endothelial cells led to a marked reduction of cell spreading on all polymers. The hydrophobic nature of PLC also appeared to favor endothelial cell attachment. Spreading of Swiss 3T3 fibroblasts on these biomaterials was only slightly affected by increased expression of tTgase, although cell spreading on control glass was increased. We propose that the consideration of tTgase-mediated bioactivity in novel biomaterials may improve cell attachment and promote biocompatibility.

3T3 Cells↗

PubMed2001

Osteoblast growth on titanium foils coated with hydroxyapatite by pulsed laser ablation.

Pulsed laser ablation is a new method for deposition of thin layers of hydroxyapatite (HA) on to biomaterial surfaces. In this paper, we report activity and morphology of osteoblasts grown on HA surfaces fabricated using different laser conditions. Two sets of films were deposited from dense HA targets, at three different laser fluences: 3, 6 and 9 Jcm(-2). One set of the surfaces was annealed at 575 degrees C to increase the crystallinity of the deposited films. Primary human osteoblasts were seeded onto the material surfaces and cytoskeletal actin organisation was examined using confocal laser scanning microscopy. The annealed surfaces supported greater cell attachment and more defined cytoskeletal actin organisation. Cell activity, measured using the alamar Blue assay, was also found to be significantly higher on the annealed samples. In addition, our results show distinct trends that correlate with the laser fluence used for deposition. The cell activity increases with increasing fluence. This pattern was repeated for alkaline phosphatase production by the cells. Differences in cell spreading were apparent which were correlated with the fluence used to deposit the HA. The optimum surface for initial attachment and spreading of osteoblasts was one of the HA films deposited using 9 J cm(-2) laser fluence and subsequently annealed at 575 degrees C.

Alkaline Phosphatase↗

PubMed2001

Water absorption and surface properties of novel poly(ethylmethacrylate) polymer systems for use in bone and cartilage repair.

The surface and bulk properties of novel methacrylate polymers prepared by gelling poly(ethyl methacrylate) (PEMA) powder with different ratios of tetrahydrofurfuryl methacrylate (THFMA) and hydroxyethyl methacrylate (HEMA) monomers were investigated. The water adsorption and desorption characteristics of these polymers were measured in water and phosphate buffered saline (PBS). The desorption diffusion coefficients were higher than the adsorption coefficients in both water and PBS. Linear relationships between the equilibrium mass of water taken up and the mass of water desorbed with the concentration of HEMA in the polymer were established. Polymer surfaces were analysed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Surface features varied with polymer composition; during hydration only selective areas of the surface hydrated indicating a heterogeneous surface. Contact angle data showed no trend between the different polymers indicating that contact angles are not an acceptable method of assessing hydrophobicity/wettability of a material which does not have a homogeneous surface. The effect of these bulk and surface characteristics on biological interactions were examined using bovine chondrocytes and human osteoblast (HOB) cell cultures. Cell attachment decreased when HEMA was present in the copolymer.

Absorption↗

PubMed2001

The influence of gamma irradiation and aging on degradation mechanisms of ultra-high molecular weight polyethylene.

The aging behavior of ultra-high molecular weight polyethylene (UHMWPE) has been studied following gamma irradiation in air. Accelerated aging procedures used elevated temperature (70 degrees C), pressurized oxygen (5 bar) and applied stress. Shelf and in vivo aged components have also been investigated. The variation in polymer properties with depth into the polymer was determined using density measurements, infra-red spectroscopy and differential scanning calorimetry. Accelerated aging in pressurized oxygen resulted in peaks in polymer density and degree of oxidation up to 500 microm below the polymer surface. Shelf and in vivo aging was also found to result in increased density at or below the component surfaces. Changes in density were mainly due to changes in crystallinity within the UHMWPE and, to a smaller extent, due to oxygen incorporation within the polymer. The application of stress did not appear to influence the accelerated aging of UHMWPE. A method for estimating the residual stress distribution in the UHMWPE using the measured changes in density is proposed. This study has indicated that oxidation of UHMWPE may lead to the development of tensile residual stresses, near the component surface, in the region of 1.7 MPa. These stresses may contribute to the failure mechanism of UHMWPE acetabular cups or knee tibial trays during service.

Journal Article↗

PubMed2001

Characterization of tissue transglutaminase in human osteoblast-like cells.

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular epsilon(gamma-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,4 14C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p < 0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

Blotting, Western↗

PubMed2000

An in vitro investigation of the PEMA/THFMA polymer system as a biomaterial for cartilage repair.

A polymer system consisting of poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate (PEMA/THFMA) was investigated as a biomaterial for cartilage repair using chondrocyte culture. The PEMA/THFMA system and Thermanox control were shown to support chondrocytes seeded directly onto the surface for up to 28 days in culture. Differences were seen between the PEMA/THFMA system and Thermanox in DNA content, proliferation and glycosaminoglycon (GAG) synthesis. There was a significantly greater medium: cell GAG ratio for the PEMA/THFMA system compared to Thermanox. A greater number of chondrocytes isolated from the superficial zone of bovine cartilage attached to the PEMA/THFMA system compared to cells isolated from the deep zone, whereas the converse was seen for Thermanox. Matrix constituents including collagen type II were synthesised indicating that the differentiated phenotype was maintained for some of the chondrocytes, although the production of type I collagen indicated that dedifferentiation of some of the chondrocytes had occurred. In conclusion, this study has shown that the PEMA/THFMA system can support chondrocytes in vitro and together with further investigations could lead to the development of the polymer as an ideal candidate for articular cartilage repair.

Animals↗

PubMed2000

Physical and biocompatibility properties of poly-epsilon-caprolactone produced using in situ polymerisation: a novel manufacturing technique for long-fibre composite materials.

Preliminary investigations into a novel process for the production of poly-epsilon-caprolactone (PCL) to be used as a matrix material in a bioabsorbable composite material are detailed. This material is primarily being developed as a bone substitute for use in maxillofacial reconstructive surgery, however, the technique described could be adapted to other areas where bioabsorbable composite materials may be used. The development of a totally bioabsorbable long-fibre composite material would allow a two-stage degradation to occur with the matrix material degrading first leaving a scaffold structure of degradable fibres which would be absorbed at a later stage. Caprolactone monomer was polymerised in situ within a tool cavity to produce a net shape moulding. Inclusion of a fibre preform within the tool cavity which was impregnated by the liquid monomer produces a long-fibre composite material. PCL with a range of molecular weights has been produced using this liquid moulding technique to assess the physical and biocompatibility properties compared to commercially available PCL. Osteoblast-like cells derived from human craniofacial bone (CFC) have been used to assess the in vitro biocompatibility of the PCL. The results show that high-quality PCL with a narrow molecular weight distribution and properties similar to commercially available PCL can be produced using this technique. Polymerisation of the monomer around a woven fibre preform made of a poly(lactic acid) (PLA)/poly(glycolic acid) (PGA) copolymer (vicryl mesh) produced a bioabsorbable long-fibre composite material. Further work is ongoing to develop this system towards a method for improving craniofacial bone reconstruction.

Biocompatible Materials↗

PubMed2000

[Antiretinal antibodies associated with cystoid macular edema].

AIM: Recently, anti-Enolase and anti-carbonic anhydrase antibodies have been observed in over 60% of patients with retinitis pigmentosa (RP) and cystoid macular oedema (CME). We investigated the presence of these antibodies in a series of patients with CME due to different pathologies. METHODS: In 10 patients with CME serum antibodies against Carbonic anhydrase (CA) II (30 kD) and Enolase (46 kD) were sought using Western Blots, Dot Blots as well as ELISA. RESULTS: Western and dot blotting showed anti-CA II antibodies in all and anti-Enolase antibodies in six of the 10 patients. The average titer measured with ELISA was 0.9 +/- 0.08 OD Units (0.35-1.4) with a dilution of 1:400. CONCLUSION: The presence of anti-retinal antibodies in the serum of all patients confirms the high prevalence of these antibodies in patients with CME. This may suggest that a dysfunction of CA and enolase activity in the retinal pigment epithelium may lie at the root of oedema formation, whereas other mechanisms may be responsible in the absence of these antibodies.

Adult↗

PubMed1999

Attachment of cultured human bone cells to novel polymers.

The initial attachment of human bone-derived cells (HBDC) to several polymer systems has been studied in vitro. A novel polymer system based on poly(ethyl methacrylate) polymer and tetrahydrofurfuryl methacrylate monomer (PEMA/THFMA) was compared with a variant in which THFMA was replaced by 2-hydroxyethyl methacrylate (PEMA/HEMA). Tissue culture polystyrene (TCPS) and polystyrene (PS) were used as reference materials. The ability of the substrates to adsorb the attachment glycoproteins fibronectin (Fn) and vitronectin (Vn) from serum and the subsequent effect on radiolabeled HBDC attachment were examined. Initial cell attachment from the medium containing 10% (v/v) serum was highest on TCPS; on PEMA/THFMA and PEMA/HEMA substrates it was about 25% of this level, and on PS it was only 10% of that on TCPS. Attachment of HBDC to all substrates was dependent on the presence of Vn, which, unlike Fn, was able to adsorb in the face of competition from other serum components. Both Vn and Fn were able to support cell attachment when precoated onto all substrates. In comparison to TCPS, PEMA/THFMA did not show enhanced adsorption of either Fn or Vn from serum, and this was reflected in the level of cell attachment. Interestingly, the potency of preadsorbed Fn for cell attachment was much higher on this substrate than on any other: 45 ng/cm2 Fn when adsorbed to PEMA/THFMA gave a level of cell attachment 1.6-fold higher than the same density of Fn on PS or TCPS. The maximum Fn surface density achieved on HEMA/PEMA was 16 ng/cm2. Cells on PEMA/THFMA showed typical clustering of the alpha5 beta1 Fn receptor, but this was not evident in cells attached to PEMA/HEMA even when precoated with Fn. This study indicates that the initial attachment of HBDC to all substrates was Vn dependent. It also indicates that on PEMA/THFMA the favorable presentation of subsequently adsorbed Fn may assist matrix assembly.

Biocompatible Materials↗

PubMed1999

Influence of packaging conditions on the properties of gamma-irradiated UHMWPE following accelerated ageing and shelf ageing.

The ageing behaviour of ultra-high molecular weight polyethylene (UHMWPE) has been studied following gamma irradiation (25 or 40 kGy) in air or in inert atmosphere (vacuum packed). Accelerated ageing procedures used elevated temperature (70 degrees C) and/or pressurised oxygen (5 bar). Shelf ageing for up to six months was also performed. The variation in polymer properties with depth into the polymer was determined using density measurements, infra-red spectroscopy and differential scanning calorimetry. Tensile properties of the irradiated polymer after accelerated ageing were also determined. Accelerated ageing in air for 20 days or pressurised oxygen for 4 days resulted in peaks in polymer density, crystallinity and degree of oxidation at the polymer surface. Accelerated ageing in pressurised oxygen for 8 days resulted in peaks in these properties 500 microns below the polymer surface. Where gamma irradiation was performed in inert atmosphere the amount of polymer degradation following accelerated ageing (in oxidising conditions) was generally lower but still significant. Differences were also observed in tensile properties between material gamma-irradiated in air and in vacuum. This study indicated that performing gamma sterilisation procedures under inert conditions can reduce the level of UHMWPE degradation when exposed to an oxidising environment.

Biocompatible Materials↗

PubMed1999

Effects of surface-treated cpTi and Ti6Al4V alloy on the initial attachment of human osteoblast cells.

This study concerns the effect of simple surface treatments on the nature of the oxide layer, of commercially pure titanium (cpTi) and Ti6Al4V alloy substrates and their effect on human osteoblast cells (HOBS). After treatment the surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) in order to identify the surface groups responsible for the cell attachment process. The assessment of cell attachment was monitored by the Alamar blue assay (AB), measuring cell activity, in three types of media: phosphate-buffered saline (PBS), serum containing and serum-free Dulbecco's modified Eagle's cell culture medium (SER+ and SERF respectively). XPS analysis of the treated surfaces revealed consistent peaks representative of TiO2 on all surfaces and Ti(0) and Ti2O3 on the non-heat-treated surfaces. The cell activity assays indicated that there were no significant differences in cellular activity caused by surface treatments, but the cellular activity compared between the three types of medium was greatest in the PBS over the initial stages of attachment.

Journal Article↗