Colin A. Scotchford

Growth of human osteoblast‐like cells on alkanethiol on gold self‐assembled monolayers: The effect of surface chemistry

Primary human osteoblasts were cultured on self-assembled monolayers (SAMs) of alkylthiols on gold with carboxylic acid and methyl termini, and the kinetics of cell attachment and proliferation were measured. Over 90 min approximately twice as many cells attached to carboxylic-acid-terminated monolayers as attached to methyl-terminated monolayers. After 24 h the number of cells attached to carboxylic-acid-terminated monolayers was ten times that attached to the methyl-terminated monolayers. Cell morphology and cytoskeletal actin organization also were found to be different. Osteoblasts were cultured on SAMs that were patterned by photolithographic techniques. Cells attached almost exclusively to carboxylic-acid-functionalized areas of the patterned surfaces, leaving methyl-functionalized regions bare. The patterns strongly influenced the morphology of the attached cells. After 24 h, cells were observed to bridge between carboxylic-acid-terminated regions separated by 75 microns, methyl-terminated regions but not those separated by 150 microns methyl-terminated regions. After 6 days in culture osteoblasts formed multilayers on the carboxylic-acid-terminated regions of the pattern.

Osteoblast responses to collagen-PVA bioartificial polymers in vitro: the effects of cross-linking method and collagen content

A range of bioartificial collagen/poly(vinyl alcohol) blends have been produced, cast as films and cross-linked using either glutaraldehyde or a dehydrothermal treatment (DHT). Films were used as substrates for the culture of osteoblast-like cells. The attachment, adhesion and proliferative responses of these cells to the range of films were examined using proliferation assays, light, electron and confocal microscopy. There was an inverse relationship between collagen content of gluataraldehyde cross-linked films and the extent of cell proliferation on them. A cytotoxicity assay demonstrated no toxic effect related to increasing collagen content. The greatest differences in cell responses observed were associated with the choice of cross-linking method. Films cross-linked with glutaraldehyde showed variation related to collagen content in cell adhesion, proliferation and morphology. Such differences were not apparent with the DHT cross-linked films. Collagen/PVA bioartificial films can be dehydrothermally cross-linked to increase biological stability and reduce water solubility. The method of cross-linking employed is the greater influence in determining osteoblast compatibility with these materials. The DHT cross-linking method is a preferable alternative to the use of glutaraldehyde. Collagen/PVA bioartificial films cross-linked by the DHT method have shown potential for biocompatibility with osteoblasts.

Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions.

Protein adsorption and adhesion of primary human osteoblasts on chemically patterned, metal-oxide-based surfaces comprising combinations of titanium, aluminium, vanadium and niobium were investigated. Single metal samples with a homogeneous surface and bimetal samples with a surface pattern produced by photolithographic techniques were used. The physical and chemical properties of the samples have been extensively characterised and are presented in a companion paper. Here, we describe their properties in terms of cell responses during the initial 24h of cell culture. Regarding the cell number and activity there was no significant difference between any of the single metal surfaces. However the morphology of cells on vanadium surfaces became spindle-like. In contrast to the behaviour on single metal samples, cells exhibited a pronounced reaction on bimetallic surfaces that contained aluminium. Cells tended to stay away from aluminium, which was the least favoured metal in all two-metal combinations. An initial cell alignment relative to the pattern geometry was detectable after 2h and was fully developed after 18h of incubation. The organisation of f-actin and microtubules as well as the localisation of vinculin were all more pronounced on non-aluminium regions. We hypothesised that the differences in cell response could be associated with differences in the adsorption of serum proteins onto the various metal oxides. Protein adsorption experiments were performed using microscopy in conjunction with immunofluorescent stains. They indicated that both fibronectin and albumin adsorption were significantly greater on the non-aluminium regions, suggesting that differences in cellular response correlate with a modulation of the concentration of serum proteins on the surface.

Physico-chemical and mechanical properties of nanocomposites prepared using cellulose nanowhiskers and poly(lactic acid)

A range of nanocomposites were prepared using cellulose nanowhiskers (CNWs) and poly(lactic acid) (PLA) via a solvent casting process. Acid hydrolysis process was used to produce CNWs from bleached cotton. Structural morphology and surface topography of the CNWs and nanocomposites were examined using transmission (TEM) and scanning electron microscopy. TEM images revealed rod-like whiskers in the nano-scale region which were dispersed within the PLA matrix. The presence of the functional groups of CNWs and PLA were confirmed via FTIR analysis. Tensile tests were conducted on thin films and the nanocomposites containing 1xa0wt% CNWs showed a 34 and 31% increase in tensile strength and modulus, respectively, compared to pure PLA. The dynamic mechanical analysis showed that the tensile storage modulus also increased in the visco-elastic temperature region with increasing CNWs content in the nanocomposites. Thermogravimetric analysis showed that all the materials investigated were thermally stable from room temperature to 210xa0°C. A positive effect of CNWs on the crystal nucleation of PLA polymer in the nanocomposites was observed using differential scanning calorimetry and X-ray diffraction analysis. The degradation profiles of the nanocomposites in deionised water over 1xa0week revealed a mass loss of 1.5–5.6% at alternate temperatures (25, 37 and 50xa0°C) and at the same conditions the swelling ratio and water uptake were seen to increase with CNWs content in the nanocomposites, which was strongly influenced by the presence of crystalline CNWs.

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.

Use of a novel carbon fibre composite material for the femoral stem component of a THR system: in vitro biological assessment.

A novel, low elastic modulus femoral component for THR has been developed using a composite of polyetheretherketone and carbon fibre. The objectives of this study were to investigate human osteoblast-like cell and macrophage responses to this material in vitro. Cells were grown on composite discs and controls. Osteoblast attachment and proliferation was not significantly different to that on Ti6Al4V. The levels of alkaline phosphatase activity, Type I collagen production and osteocalcin production were not significantly different to that on Ti6Al4V by the end of the experimental period. Hydrogen peroxide production by macrophages was significantly less than that detected for cells cultured on copper, but was still greater than that detected for cells cultured on tissue culture plastic and Ti6Al4V. Beta-glucoronidase activity was not significantly different to that detected for cells cultured on tissue culture plastic. The in vitro biocompatibility assessment of this composite undertaken in this study showed initial osteoblast attachment at least comparable to that of the tissue culture plastic and Ti6Al4V controls, with proliferation similar to the controls at all time points up to 11 days. Alkaline phosphatase activity was similar to that of Ti6Al4V but reduced compared to tissue culture plastic controls. Whilst hydrogen peroxide production by macrophages was raised on composite surfaces compared to controls, beta-glucoronidase activity and osteoblastic production of Type I collagen and osteocalcin were similar to levels detected on Ti6Al4V.

Cytocompatibility and effect of increasing MgO content in a range of quaternary invert phosphate-based glasses.

Recently, phosphate-based glass (PBG) fibers have been used to reinforce the biodegradable polymers polycaprolactone and polylactic acid, in order to fabricate materials suitable for use as resorbable bone fracture fixation devices. However, the PBG fibers investigated tended to degrade too quickly for application. Therefore, more durable PBG formulations were sought with emphasis remaining firmly placed on their biocompatibility. In this study, four invert PBG formulations (in the system P2O5—CaO—MgO—Na 2O) were produced with fixed phosphate and calcium content at 40 and 25 mol%, respectively. MgO was added at 10—30 mol% in place of Na 2O and the maximum divalent cation to phosphate ratio obtained was 1.375. Thermal analyses showed a linear increase in Tg with increasing MgO content. This was proposed to be due to an increase in the cross-link density of the glass network, which also improved the chemical durability of the glass. EDX analyses were also conducted to verify the final composition of the glass. XRD analyses confirmed the amorphous nature of the glasses investigated. Rapid quenching of the Mg30 glass revealed a degree of surface crystallization, which was shown to be a CaMgP2O7 phase. The degradation rates of the glasses investigated decreased with increasing MgO content. The decrease in rate seen was almost two orders of magnitude (a ×50 difference was seen between glass Mg0 and Mg30). The cytocompatibility studies of the formulations investigated showed good cellular response over time for up to 14 days. Statistical analysis revealed that the formulations investigated gave a response comparable to the tissue culture plastic control. It is suggested that invert PBG provide degradation profiles and the cytocompatibility response desired to make these glasses useful for bone repair applications.

Composites for bone repair: phosphate glass fibre reinforced PLA with varying fibre architecture.

Internal fixation for bone fractures with rigid metallic plates, screws and pins is a proven operative technique. However, refracture’s have been observed after rigid internal fixation with metal plates and plate fixation has been known to cause localised osteopenia under and near the plate. In the present study, resorbable composites comprising a PLA matrix reinforced with iron doped phosphate glass fibres were investigated. Non-woven random mat laminates of approximately 30% and 45% fibre volume fraction (Vf) were produced, along with unidirectional and 0°–90° samples of approximately 20% Vf. The non-woven composite laminates achieved maximum values of 10xa0GPa modulus and 120xa0MPa strength. The 0–90º samples showed unexpectedly low strengths close to matrix value (~50xa0MPa) although with a modulus of 7xa0GPa. The UD specimens exhibited values of 130xa0MPa and 11.5xa0GPa for strength and modulus respectively. All the modulus values observed were close to that expected from the rule of mixtures. Samples immersed in deionised water at 37°C revealed rapid mechanical property loss, more so for the UD and 0–90º samples. It was suggested that continuous fibres wicked the degradation media into the composite plates which sped up the deterioration of the fibre-matrix interface. The effect was less pronounced in the non-woven random mat laminates due to the discontinuous arrangement of fibres within the composite, making it less prone to wicking. Random mat composites revealed a higher mass loss than the UD and 0°–90° specimens, it was suggested this was due to the higher fibre volume fractions of these composites and SEM studies revealed voidage around the fibres by day 3. Studies of pH of the degradation media showed similar profiles for all the composites investigated. An initial decrease in pH was attributed to the release of phosphate ions into solution followed by a gradual return back to neutral.

Effect of Boron Addition on the Thermal, Degradation, and Cytocompatibility Properties of Phosphate-Based Glasses

In this study eight different phosphate-based glass compositions were prepared by melt-quenching: four in the (P2O5)45-(CaO)16-(Na2O)15-x -(MgO)24-(B2O3)x system and four in the system (P2O5)50-(CaO)16-(Na2O)10-x-(MgO)24-(B2O3)x, where x = 0,1, 5 and 10u2009mol%. The effect of B2O3 addition on the thermal properties, density, molar volume, dissolution rates, and cytocompatibility were studied for both glass systems. Addition of B2O3 increased the glass transition (T g), crystallisation (T c), melting (T m), Liquidus (T L) and dilatometric softening (T d) temperature and molar volume (V m). The thermal expansion coefficient (α) and density (ρ) were seen to decrease. An assessment of the thermal stability of the glasses was made in terms of their processing window (crystallisation onset, T c,ons minus glass transition temperature, T g), and an increase in the processing window was observed with increasing B2O3 content. Degradation studies of the glasses revealed that the rates decreased with increasing B2O3 content and a decrease in degradation rates was also observed as the P2O5 content reduced from 50 to 45u2009mol%. MG63 osteoblast-like cells cultured in direct contact with the glass samples for 14 days revealed comparative data to the positive control for the cell metabolic activity, proliferation, ALP activity, and morphology for glasses containing up to 5u2009mol% of B2O3.

Supercritical carbon dioxide foaming of elastomer/heterocyclic methacrylate blends as scaffolds for tissue engineering

This study reports on the supercritical carbon dioxide (scCO2) foaming of rubber toughened heterocyclic methacrylates for potential applications as non-degradable scaffolds in tissue repair and engineering. Porous blends of styrene–isoprene–styrene copolymer elastomer (SIS) and tetrahydrofurfuryl methacrylate (THFMA) at three SIS/THFMA compositions that ranged from methacrylate to elastomer rich were foamed and characterised in terms of their morphological, mechanical and biological properties. The results showed that the foaming factor (FF) was dependent on blend composition and the foaming conditions demonstrating that the process was tuneable. A greater FF, resulting in higher open and total porosities, was obtained for THFMA rich formulations, which were demonstrated by a predominantly open pore structure. Quasi-static and dynamic mechanical analysis (DMA) showed that the foamed SIS/THFMA blends gave distinct behaviours according to their compositions which were in the range of mechanical properties of soft tissues. The loss modulus and mechanical loss tangent through DMA gave two transition regions associated with the glass transition temperatures of poly(THFMA) and polystyrene components in the blends, along with a reduction in storage modulus. Cell adhesion and spreading in terms of neuroblastoma (human neuron-like SH-SY5Y) cells and ovine meniscal chondrocytes were demonstrated for scaffolds with THFMA rich formulations confirming their suitability for tissue engineering.