Bond Strength and Adhesion Mechanisms of Novel Bone Adhesives.

Bone adhesives offer distinct advantages over the use of screws to attached internal fixation plates (IFPs). As the chemical composition of bone is similar to dentine, it is possible that the types of monomers used to make dentine adhesives could be utilised to affix IFPs to bone. The ability to attach a bio-resorbable IFP to porcine bone was assessed for the monomer 10-methacryloyloxydecyl dihydrogen phosphate (MDP), used either as a homopolymer or a copolymer with urethane dimethacrylate (MDP + U). Additionally, the addition of a priming step (MDP + U + P) was evaluated. The chemical interactions of the monomers with bone were assessed using XRD and imaged using TEM, revealing the formation of nano-layered structures with the MDP primer, something we believe has not been reported on bone. In a 6-week artificial aging study both MDP + U and MDP + U + P demonstrated adequate shear bond strength to affix bio-resorbable IFPs. The cytotoxicity profiles of the adhesive formulations were determined using indirect and direct contact with MC3T3 cells, with indirect conditions suggesting the MDP + U + P is as cytocompatible as the resorbable IFP. The findings of this study suggest our newly developed adhesive has the potential to be used as a bone adhesive to affix bioresorbable IFPs.

Influence of PCL and PHBV on PLLA Thermal and Mechanical Properties in Binary and Ternary Polymer Blends.

PLLA, PCL and PHBV are aliphatic polyesters which have been researched and used in a wide range of medical devices, and all three have advantages and disadvantages for specific applications. Blending of these materials is an attractive way to make a material which overcomes the limitations of the individual polymers. Both PCL and PHBV have been evaluated in polymer blends with PLLA in order to provide enhanced properties for specific applications. This paper explores the use of PCL and PHBV together with PLLA in ternary blends with assessment of the thermal, mechanical and processing properties of the resultant polymer blends, with the aim of producing new biomaterials for orthopaedic applications. DSC characterisation is used to demonstrate that the materials can be effectively blended. Blending PCL and PHBV in concentrations of 5-10% with PLLA produces materials with average modulus improved by up to 25%, average strength improved by up to 50% and average elongation at break improved by 4000%, depending on the concentrations of each polymer used. PHBV impacts most on the modulus and strength of the blends, whilst PCL has a greater impact on creep behaviour and viscosity. Blending PCL and PHBV with PLLA offers an effective approach to the development of new polyester-based biomaterials with combinations of mechanical properties which cannot be provided by any of the materials individually.

The effect of magnetic resonance imaging on mercury release from dental amalgam at 3T and 7T.

OBJECTIVES: To measure mercury release from standardised hydroxyapatite/amalgam constructs during MRI scanning and investigate the impact of static field strength and radiofrequency (RF) power on mercury release. METHODS: Amalgam was placed into 140 hydroxyapatite disks and matured for 14-days in artificial saliva. The solution was replaced, and samples split into five groups of 28 immediately prior to MRI. One group had no exposure, and the remainder were exposed to either a 3T or 7T MRI scanner, each at high and low RF power. Mercury concentration was measured by inductively coupled plasma mass spectrometry. Groups were compared using one-way ANOVA, and two-way ANOVA for main effects/ interaction of field strength/ RF power. RESULTS: Mercury concentration was increased in the 7T groups (high/ low: 15.43/ 11.33 ng mL-1) and 3T high group (3.59) compared to control (2.44). MRI field strength significantly increased mercury release (p < .001) as did RF power (p = .030). At 3T, mercury release was 20.3 times lower than during maturation of dental amalgam, and for the average person an estimated 1.50 ng kg-1 of mercury might be released during one 3T investigation; this is substantially lower than the tolerable weekly intake of 4,000 ng kg-1. CONCLUSION: Mercury release from amalgam shows a measurable increase following MRI, and the magnitude changes with magnetic field strength and RF power. The amount of mercury released is small compared to release during amalgam maturation. Amalgam mercury release during MRI is unlikely to be clinically meaningful and highly likely to remain below safe levels.

Developments in resin-based composites.

With the phasing down of dental amalgam use in response to the Minamata Convention, it is likely that resin-based composite restoratives will be the dental material of choice for the direct restoration of compromised dentition in the UK, at least for the foreseeable future. The current materials have a finite lifespan, with failures predominately due to either secondary caries or fracture. Consequently, there is considerable in vitro research reported each year with the intention of producing improved materials. This review describes the recent research in materials designed to have low polymerisation shrinkage and increased mechanical properties. Also described is research into materials that are either antimicrobial or are designed to release ions into the surrounding oral environment, with the aim of stimulating remineralisation of the surrounding dental tissues. It is hoped that by describing this recent research, clinicians will be able to gain some understanding of the current research that will potentially lead to new products that they can use to improve patient treatment in the future.

Strategies for Enhancing Polyester-Based Materials for Bone Fixation Applications.

Polyester-based materials are established options, regarding the manufacturing of bone fixation devices and devices in routine clinical use. This paper reviews the approaches researchers have taken to develop these materials to improve their mechanical and biological performances. Polymer blending, copolymerisation, and the use of particulates and fibre bioceramic materials to make composite materials and surface modifications have all been studied. Polymer blending, copolymerisation, and particulate composite approaches have been adopted commercially, with the primary focus on influencing the in vivo degradation rate. There are emerging opportunities in novel polymer blends and nanoscale particulate systems, to tune bulk properties, and, in terms of surface functionalisation, to optimise the initial interaction of devices with the implanted environment, offering the potential to improve the clinical performances of fracture fixation devices.

Development and characterisation of dental composites containing anisotropic fluorapatite bundles and rods.

OBJECTIVES: To develop dental composites incorporating fluorapatite (FA) crystals as a secondary filler and to characterise degree of conversion, key mechanical properties and fluoride release. METHODS: FA rod-like crystals and bundles were hydrothermally synthesised and characterised by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS), X-ray diffraction (XRD) and 19F MAS-NMR. Composites were formulated containing BisGMA/TEGDMA/BisEMA and barium-aluminium-silicate glass (0FA). FA crystals were incorporated at 10 (10FA), 20 (20FA), 30 (30FA) and 40wt% (40FA) maintaining a filler content of 80wt% (63-67vol%). Degree of conversion (DC), flexural strength (FS), flexural modulus (FM), fracture toughness (K1C), Vickers hardness (HV) and 2-body wear were measured. Fluoride release was measured in neutral and acidic buffers. RESULTS: XRD and 19F MAS-NMR confirmed that only FA was formed, whilst SEM revealed the presence of single rods and bundles of nano-rods. DC ranged between 56-60% (p>0.05). FA composites showed lower FM and lower FS (p<0.05), but comparable wear resistance and HV (p>0.05) to 0FA. 30FA and 40FA showed similar K1C to 0FA (p>0.05), with SEM showing evidence of toughening mechanisms, whereas 10FA and 20FA showed lower K1C (p<0.05). FA containing composites released fluoride that was proportional to the amount of FA incorporated (p<0.05) but only under acidic conditions. SIGNIFICANCE: The addition of FA to the experimental composites reduced strength and stiffness but not the DC, hardness or wear rate. 30FA and 40FA had a higher K1C compared to other FA groups. Fluoride release occurred under an accelerated acidic regime, suggesting potential as a bioactive 'smart' composite.

An in vitro model to assess effects of a desensitising agent on bacterial biofilm formation.

Desensitising agents are added to dentifrices to occlude exposed dentine tubules and reduce pain associated with dentine hypersensitivity. In occluding the tubules these agents may alter the surface layer of the dentine and consequently affect bacterial biofilm formation. This research sought to examine the effects of desensitising agents on dentinal biofilms using an in vitro model. A constant depth film fermenter (CDFF) was selected to mimic the oral environment and human dentine with exposed tubules was analysed. Calcium sodium phosphosilicate (CSPS) was selected as a model desensitising agent. Dentine discs were treated with pumice or CSPS-containing dentifrices with or without fluoride, or left untreated (control). Dual-species biofilms of Streptococcus mutans and Streptococcus sobrinus were grown in artificial saliva and analysed by viable counts, polymerase chain reaction (PCR) and scanning electron microscopy (SEM). SEM images confirmed the presence of occluded tubules after CSPS application and demonstrated the formation of biofilms containing extracellular matrix material. Analysis of PCR and viable count data using a one-way ANOVA showed no significant differences for bacterial composition for any of the four treatments. There were, however, trends towards increased numbers of bacteria for the pumice and CSPS treated samples which was reversed by the addition of fluoride to CSPS. In conclusion, CSPS was not found to have a significant effect on biofilms and an in vitro model for testing desensitising agents has been developed, however, further work is required to improve the reproducibility of the biofilms formed and to explore the trends seen.

Development of an arthroscopically compatible polymer additive layer manufacture technique.

This article describes a proof of concept study designed to evaluate the potential of an in vivo three-dimensional printing route to support minimally invasive repair of the musculoskeletal system. The study uses a photocurable material to additively manufacture in situ a model implant and demonstrates that this can be achieved effectively within a clinically relevant timescale. The approach has the potential to be applied with a wide range of light-curable materials and with development could be applied to create functionally gradient structures in vivo.

Development of a methacrylate-terminated PLGA copolymer for potential use in craniomaxillofacial fracture plates.

We synthesised methacrylate-terminated PLGA (HT-PLGA, 85:15 LA:GA, 169kDa), for potential use as an adhesively attached craniomaxillofacial fracture fixation plate. The in vitro degradation of molecular weight, pH and flexural modulus were measured over 6weeks storage in PBS at 37°C, with commercially available high (225kDa, H-PLGA) and low (116kDa, L-PLGA) molecular weight 85:15 PLGAs used as comparators. Molecular weights of the materials reduced over 6weeks, HT-PLGA by 48%, H-PLGA by 23% and L-PLGA by 81%. HT-PLGA and H-PLGA exhibited a near constant pH (7.35) and had average flexural moduli in excess of 6GPa when produced, similar to that of the mandible. After 1week storage both exhibited a significant reduction in average modulus, however, from weeks 1-6 no further significant changes were observed, the average modulus never dropped significantly below 5.5GPa. In contrast, the L-PLGA caused a pH drop to below 7.3 by week 6 and an average modulus drop to 0.6 from an initial 4.6GPa. Cell culture using rat bone marrow stromal cells, revealed all materials were cytocompatible and exhibited no osteogenic potential. We conclude that our functionalised PLGA retains mechanical properties which are suitable for use in craniofacial fixation plates.

Inhibition of multispecies biofilms by a fluoride-releasing dental prosthesis copolymer.

OBJECTIVES: This study aimed to develop a new mixed-species acidogenic biofilm model and use it to assess the antimicrobial properties of a novel fluoride-releasing copolymer. METHODS: Stubs composed of a copolymer of methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA) with polymethyl methacrylate (PMMA) were produced by chemically-activated free radical polymerization. A fluoride-releasing copolymer was developed by incorporating sodium fluoride in place of a portion of the PMMA. Samples were mounted in polysulfone Modified Robbins Devices (MRDs) and were optimized for single- and mixed-species biofilm formation by Candida albicans, Lactobacillus casei and Streptococcus mutans. RESULTS: Fluoride release was sustained for at least 48h in flowing conditions. Fluoride did not affect the colonization and biofilm growth of any of the microorganisms in monocultures. However, in mixed-species biofilms, cell densities of all three species were reduced approximately ten-fold (p<0.05) on the fluoridated material compared with the non-fluoridated copolymer. CONCLUSIONS: These data demonstrate that intermicrobial interactions in mixed-species acidogenic biofilms are sensitive to fluoride, and that the inclusion of fluoride in a denture lining copolymer reduces the formation of polymicrobial biofilms. CLINICAL SIGNIFICANCE: The growth of acidogenic microorganisms on denture materials is associated with denture stomatitis and dental caries on surrounding teeth. A fluoride-releasing copolymer that inhibits acidogenic mixed-species biofilms, such as the material described in this study, has the potential to control these diseases by limiting biofilm growth.

Investigating the biological response of human mesenchymal stem cells to titanium surfaces.

BACKGROUND: We have investigated the behaviour of a newly characterised population of haemarthrosis fluid-derived human mesenchymal stem cells (HF-hMSCs) with titanium (Ti) surfaces. METHODS: HF-hMSCs were seeded onto round cannulated interference (RCI; Smith and Nephew) screws or control Ti discs and cultured under pro-osteogenic conditions. RESULTS: Electron microscopy showed the attachment and spreading of HF-hMSCs across both Ti surfaces during the early stages of osteogenic culture; however, cells were exclusively localised to the basal regions within the vertex of the Ti screws. In the later stages of culture, an osteoid matrix was deposited on the Ti surfaces with progressive culture expansion and matrix deposition up the sides and the top of the Ti Screws. Quantification of cellular content revealed a significantly higher number of cells within the Ti screw cultures; however, there was no difference in the cellular health. Conversely, alizarin red staining used as both a qualitative and quantitative measure of matrix calcification was significantly increased in Ti disc cultures compared to those of Ti screws. CONCLUSIONS: Our results suggest that the gross topography of the metal implant is able to create microenvironment niches that have an influence on cellular behaviour. These results have implications for the design of advanced tissue engineering strategies that seek to use cellular material to enhance biological remodelling and healing following tissue reconstruction.

Qualifying the lapped enamel surface: a profilometric, electron microscopic and microhardness study using human, bovine and ovine enamel.

OBJECTIVE: When enamel specimens are prepared for erosion and abrasion studies, the assumption is often made that specimens prepared in the same way will have the same baseline surface characteristics. This study aimed to test the null hypothesis that there are no significant differences in baseline surface characteristics of human, ovine and bovine enamel specimens prepared using the same method. DESIGN: Twenty enamel slabs were prepared from bovine, human and ovine incisor crowns and polished with 3µm aluminium oxide paste. Roughness average (Ra), bearing parameters (MR1, MR2, Rpk, Rk, Rvk), surface microhardness and scanning electron microscopy (SEM) were used to compare the different tissues. One way Analysis of Variance (ANOVA) was used to quantitatively compare surface characteristics between tissue types. RESULTS: Human, bovine and ovine enamel roughness and microhardness were significantly different to one another at baseline (P<0.001); ovine enamel was the roughest and softest, and bovine enamel was the smoothest and hardest. SEM allowed a visual comparison to be made between tissue types, confirming the quantitative data. CONCLUSIONS: Enamel from human, bovine and ovine specimens showed significantly different surface characteristics after lapping and polishing. The null hypothesis is rejected, recognising that the same preparation techniques will not necessarily result in consistent baseline roughness or surface characteristics between tissue types. Surface studies should lap and polish samples with a standardised approach, whilst ensuring that baseline data are recorded for comparison.

The effect on cast post dimensions of casting investment and airborne particle abrasion.

Cast posts can sometimes prove difficult to seat fully during fitting. This study compared two different liquid/water dilutions for phosphate bonded investment and the effect of controlled airborne particle abrasion on resulting post diameter. After measuring polymeric post patterns (n = 18), 3 groups were invested using concentrated solution and 3 groups using dilute solution. After casting they were weighed and remeasured then exposed to airborne particle abrasion. Both solutions produced oversized cast posts. Mean diameter reduction during airborne particle abrasion was 8 microm/10s taking an average of 41s to reach precast size. Where a post pattern fits tightly, airborne particle abrasion for 70s should reduce the casting sufficiently to accommodate the cement lute.

Use of micro-Raman spectroscopy to investigate hybrid layer quality in demineralized root dentine.

Treatment of early root caries using resin adhesives to stabilize demineralized dentine and deprive bacteria of nutrients is a recognized and conservative treatment option. Essential to its success is a stable resin-dentine interface, with effective infiltration of demineralized dentine and adequate resin curing. The objective of this study was therefore to examine degree of cure (DoC) and depth of penetration of dentine adhesives in demineralized dentine using micro-Raman spectroscopy. Three commercially available adhesives were applied essentially according to manufacturers' instructions to visibly wet demineralized dentine, with two coats of primer to reduce dilution. Specimens were sectioned perpendicular to the bonded interface and micro-Raman spectroscopy performed on the cross section. Molecules associated with the adhesive were detected to depths greater than 100 µm indicating a thicker hybrid layer than seen in sound dentine. The hybrid layer showed an increase in proportion of aliphatic C=C double bonds at the base of the hybrid layer, which may be a consequence of reduced DoC or phase separation. Micro-Raman spectra suggest deep zones contain an increased concentration of HEMA and a lower concentration of BisGMA, which is likely to be less stable and may contribute to early breakdown of the dentine adhesive interface.

Evaluation of two novel aluminum-free, zinc-based glass polyalkenoate cements as alternatives to PMMA bone cement for use in vertebroplasty and balloon kyphoplasty.

Vertebroplasty (VP) and balloon kyphoplasty (BKP) are now widely used for treating patients in whom the pain due to vertebral compression fractures is severe and has proved to be refractory to conservative treatment. These procedures involve percutaneous delivery of a bolus of an injectable bone cement either directly to the fractured vertebral body, VB (VP) or to a void created in it by an inflatable bone tamp (BKP). Thus, the cement is a vital component of both procedures. In the vast majority of VPs and BKPs, a poly(methyl methacrylate) (PMMA) bone cement is used. This material has many shortcomings, notably lack of bioactivity and very limited resorbability. Thus, there is room for alternative cements. We report here on two variants of a novel, bioactive, Al-free, Zn-based glass polyalkenoate cement (Zn-GPC), and how their properties compare to those of an injectable PMMA bone cement (SIMPL) that is widely used in VP and BKP. The properties determined were injectability, radiopacity, uniaxial compressive strength, and biaxial flexural modulus. In addition, we compared the compression fatigue lives of a validated synthetic osteoporotic VB model (a polyurethane foam cube with an 8 mm-diameter through-thickness cylindrical hole), at 0-2300 N and 3 Hz, when the hole was filled with each of the three cements. A critical review of the results suggests that the performance of each of the Zn-GPCs is comparable to that of SIMPL; thus, the former cements merit further study with a view to being alternatives to an injectable PMMA cement for use in VP and BKP.

Surface detail reproduction of elastomeric impression materials related to rheological properties.

OBJECTIVES: The purpose of this work was to discern, for elastomeric impression materials, the important rheological properties and importance of hydrophilicity for detail reproduction. METHODS: Viscosity, modulus and tan delta were measured using a controlled-stress rheometer in cone/plate configuration. The flow of the materials, immediately after mixing and at the manufacturer's stated working time, was measured using a shark fin test and the interaction with moist surfaces was determined by taking impressions from two different sized grooves in moist gypsum casts. RESULTS: Tan delta was found to be the parameter most indicative of the accuracy of the impression and the flow of the material. Impregum samples, a polyether material, exhibited the highest initial tan delta (7.4), the largest shark fins at both time periods and the most accurate impressions from both grooves. Aquasil, a polyvinylsiloxane material, had similar initial tan delta values (6.9) and impressions taken on the deep groove with this material closely matched the groove. The other two polyvinylsiloxane materials (Affinis and Flexitime) had significantly lower initial tan delta values (3.1 and 2.9, respectively), exhibited much smaller shark fins and a worse ability to accurately reproduce the deep groove. SIGNIFICANCE: For large features, it is clear that the higher the initial tan delta of the impression material the better the ability to replicate larger features. However, with smaller features the relative hydrophobicity of the material becomes an important factor, with more hydrophilic materials better able to reproduce fine detail.

Identifying variables responsible for clustering in discriminant analysis of data from infrared microspectroscopy of a biological sample.

In the biomedical field, infrared (IR) spectroscopic studies can involve the processing of data derived from many samples, divided into classes such as category of tissue (e.g., normal or cancerous) or patient identity. We require reliable methods to identify the class-specific information on which of the wavenumbers, representing various molecular groups, are responsible for observed class groupings. Employing a prostate tissue sample divided into three regions (transition zone, peripheral zone, and adjacent adenocarcinoma), and interrogated using synchrotron Fourier-transform IR microspectroscopy, we compared two statistical methods: (a) a new "cluster vector" version of principal component analysis (PCA) in which the dimensions of the dataset are reduced, followed by linear discriminant analysis (LDA) to reveal clusters, through each of which a vector is constructed that identifies the contributory wavenumbers; and (b) stepwise LDA, which exploits the fact that spectral peaks which identify certain chemical bonds extend over several wavenumbers, and which following classification via either one or two wavenumbers, checks whether the resulting predictions are stable across a range of nearby wavenumbers. Stepwise LDA is the simpler of the two methods; the cluster vector approach can indicate which of the different classes of spectra exhibit the significant differences in signal seen at the "prominent" wavenumbers identified. In situations where IR spectra are found to separate into classes, the excellent agreement between the two quite different methods points to what will prove to be a new and reliable approach to establishing which molecular groups are responsible for such separation.

Copper-mediated formation of hydrogen peroxide from the amylin peptide: a novel mechanism for degeneration of islet cells in type-2 diabetes mellitus?

Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet beta-cells in most cases of type-2 diabetes mellitus (T2Dm). Human amylin 'oligomers' are toxic to these cells. Using two different experimental techniques, we found that H(2)O(2) was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H(2)O(2) and did not interact with copper. We conclude that the formation of H(2)O(2) from amylin could contribute to the progressive degeneration of islet cells in T2Dm.

IR microspectroscopy: potential applications in cervical cancer screening.

Screening exfoliative cytology for early dysplastic cells reduces incidence and mortality from squamous carcinoma of the cervix. In the developed world, screening programmes have adopted a 3-5 years recall system. In its absence, cervical cancer would be the second most common female cancer in these regions; instead, it is currently eleventh. However, there exist a number of limitations to the smear test even given the removal of contaminants using liquid-based cytology. It is prohibitively expensive, labour-intensive and subject to inaccuracies that give rise to significant numbers of false negatives. There remains a need for novel approaches to allow efficient and objective interrogation of exfoliative cytology. Methods that variously exploit infrared (IR) microspectroscopy are one possibility. Using IR microspectroscopy, an integrated 'biochemical-cell fingerprint' of the lipid, protein and carbohydrate composition of a biomolecular entity may be derived in the form of a spectrum via vibrational transitions of individual chemical bonds. Powerful statistical approaches (e.g. principal component analysis) now facilitate the interrogation of large amounts of spectroscopic data to allow the extraction of what may be small but extremely significant biomarker differences between disease-free and pre-malignant or malignant samples. An increasing wealth of literature points to the ability of IR microspectroscopy to allow the segregation of cells based on their disease status. We review the current evidence supporting its diagnostic potential in cancer biology.

ATR microspectroscopy with multivariate analysis segregates grades of exfoliative cervical cytology.

Although cervical cancer screening in the UK has led to reductions in the incidence of invasive disease, this programme remains flawed. We set out to examine the potential of infrared (IR) microspectroscopy to allow the profiling of cellular biochemical constituents associated with disease progression. Attenuated total reflection-Fourier Transform IR (ATR) microspectroscopy was employed to interrogate spectral differences between samples of exfoliative cervical cytology collected into liquid based cytology (LBC). These were histologically characterised as normal (n = 5), low-grade (n = 5), high-grade (n = 5) or severe dyskaryosis (? carcinoma) (n = 5). Examination of resultant spectra was coupled with principal component analysis (PCA) and subsequent linear discriminant analysis (LDA). The interrogation of LBC samples using ATR microspectroscopy with PCA-LDA facilitated the discrimination of different categories of exfoliative cytology and allowed the identification of potential biomarkers of abnormality; these occurred prominently in the IR spectral region 1200 cm(-1) - 950 cm(-1) consisting of carbohydrates, phosphate, and glycogen. Shifts in the centroids of amide I (approximately 1650 cm(-1)) and II (approximately 1530 cm(-1)) absorbance bands, indicating conformational changes to the secondary structure of intracellular proteins and associated with increasing disease progression, were also noted. This work demonstrates the potential of ATR microspectroscopy coupled with multivariate analysis to be an objective alternative to routine cytology.