Direct monitoring of single-cell response to biomaterials by Raman spectroscopy.

There is continued focus on the development of new biomaterials and associated biological testing methods needed to reduce the time taken for their entry to clinical use. The application of Raman spectroscopy to the study of individual cells that have been in contact with biomaterials offers enhanced in vitro information in a potentially non-destructive testing regime. The work presented here reports the Raman spectral analysis of discreet U-2 OS bone cells after exposure to hydroxyapatite (HA) coated titanium (Ti) substrates in both the as-deposited and thermally annealed states. These data show that cells that were in contact with the bioactive HA surface for 7 days had spectral markers similar to those cultured on the Ti substrate control for the same period. However, the spectral features for those cells that were in contact with the annealed HA surface had indicators of significant differentiation at day 21 while cells on the as-deposited surface did not show these Raman changes until day 28. The cells adhered to pristine Ti control surface showed no spectral changes at any of the timepoints studied. The validity of these spectroscopic results has been confirmed using data from standard in vitro cell viability, adhesion, and proliferation assays over the same 28-day culture period. In this case, cell maturation was evidenced by the formation of natural bone apatite, which precipitated intracellularly for cells exposed to both types of HA-coated Ti at 21 and 28 days, respectively. The properties of the intracellular apatite were markedly different from that of the synthetic HA used to coat the Ti substrate with an average particle size of 230 nm, a crystalline-like shape and Ca/P ratio of 1.63 ± 0.5 as determined by SEM-EDX analysis. By comparison, the synthetic HA particles used as a control had an average size of 372 nm and were more-rounded in shape with a Ca/P ratio of 0.8 by XPS analysis and 1.28 by SEM-EDX analysis. This study shows that Raman spectroscopy can be employed to monitor single U-2 OS cell response to biomaterials that promote cell maturation towards de novo bone thereby offering a label-free in vitro testing method that allows for non-destructive analyses.

The deposition of strontium-substituted hydroxyapatite coatings.

Strontium substituted hydroxyapatite (SrHA) coatings have received a lot of interest recently as strontium (Sr) has been shown to have the dual benefit of promoting bone formation and reducing bone resorption, in vivo. In this work, SrHA coatings were deposited onto polycrystalline titanium surfaces using radio frequency (RF) magnetron co-sputtering and compared to those deposited from HA alone. In particular, the influence of different levels of Sr-substitution of the sputtering targets (5 and 13% Sr-substituted HA targets) on the properties of the deposited coatings produced at a low discharge power level (150 W) were investigated using FTIR, XPS, XRD, ToFSIMS and AFM techniques (both before and after annealing at 500 °C). The results show that Sr could be successfully incorporated into the HA lattice to form SrHA coatings and that they contained no other impurities. However, the coating produced from the 13% Sr-substituted target had a higher Ca+Sr/P ratio (1.95±0.14) and Sr content when compared to the coating produced from the 5% Sr-substituted target (1.58±0.20). The deposition rate also decreased with increasing Sr content of the sputtering targets. Furthermore, as the Sr content of the coatings increased, so did the preferred 002 orientation of the coating along with increased surface roughness and heterogeneity of the surface features. Therefore, this study has shown that RF magnetron sputtering offers a means to control attendant properties of Sr-substituted HA, such as the crystallinity, stoichiometry, phase purity and surface topography.

Nanotopography of Polystyrene/Poly(methyl methacrylate) for the Promotion of Patient Specific Von Willebrand Factor Entrapment and Platelet Adhesion in a Whole Blood Microfluidic Assay.

Platelet function testing is essential for the diagnosis of patients with bleeding disorders. Specifically, there is a need for a whole blood assay that is capable of analysing platelet behaviour in contact with a patient-specific autologous von Willebrand factor (vWF), under physiologically relevant conditions. The creation of surface topography capable of entrapping and uncoiling vWF for the support of subsequent platelet adhesion within the same blood sample offers a potential basis for such an assay. In this study, spin coating of polystyrene/poly (methyl methacrylate) (PS/PMMA) demixed solutions onto glass substrates in air has been used to attain surfaces with well-defined topographical features. The effect of augmenting the PS/PMMA solution with uniform 50 µm PS microspheres that can moderate the demixing process on the resultant surface features has also been investigated. The topographical features created here by spin coating under ambient air pressure conditions, rather than in nitrogen, which previous work reports, produces substrate surfaces with the ability to entrap vWF from flowing blood and facilitate platelet adhesion. The direct optical visualisation of fluorescently-labelled platelets indicates that topography resulting from inclusion of PS microspheres in the PS/PMMA spin coating solution increases the total number of platelets that adhere to the substrate surface over the period of the microfluidic assay. However, a detailed analysis of the adhesion rate, mean translocating velocity, mean translocation distance, and fraction of the stably adhered platelets measured during blood flow under arterial equivalent mechanical shear conditions indicates no significant difference for topographies created with or without inclusion of the PS microspheres.

Assessment of an osteoblast-like cell line as a model for human primary osteoblasts using Raman spectroscopy.

Raman spectroscopy is employed to determine the suitability of the U20S osteoblast-like cell line for use as a model for human primary osteoblasts, with emphasis on the ability of these cell types to replicate their tissue of origin. It was found that both cell types demonstrated early stage mineral deposition that followed significantly different growth patterns. Analysis of the growth pattern and spectral data from primary cells revealed increasing bone quality ratios and a high crystallinity, consistent with previous reports. Conversely the investigation of the U20S osteoblast-like cell line provided evidence of dense multilayered mineralised regions that corresponded more closely to native bone in terms of its crystallinity and bone quality ratios. This finding contradicts previous reports on U20S osteoblast-like cells which have consistently described them as non-osteoinductive when cultured in various conditions on a number of substrates. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analysis for the investigation of osteoblast-like U20S cells and human primary osteoblasts, specifically with focus on the osteoinductive ability of the osteoblast-like cell line and the comparative differences in relation to the primary osteoblasts.

Osteoblast-like cell response to calcium phosphate coating chemistry and morphology on etched silicon surfaces.

Being able to control the behaviour of osteoblast-like cells on a surface may provide a genuine insight into the material surface characteristics and help in creating a successful coating/cell interface. The possibility of creating a micro-environment that can induce proliferation, differentiation and mineralisation of bone cells in vitro, by successfully combining both chemistry and topography of a micro-fabricated substrate is an area that requires a multi-disciplinary approach. Utilising sputter deposition, a process that lends itself to high processability, in conjunction with photolithography allowing for the creation of highly repeatable etched surfaces, we aim to provide a successful combination of chemistry and topography. Correlating the substrate conditions with resultant osteoblast biological function and activity can ultimately be used with a view to modulating the behavior of osteoblast-like cells in vitro.

Nanoindentation and nano-scratching of hydroxyapatite coatings for resorbable magnesium alloy bone implant applications.

The corrosion rate of Mg alloys is currently too high for viable resorbable implant applications. One possible solution is to coat the alloy with a hydroxyapatite (HA) layer to slow the corrosion and promote bone growth. As such coatings can be under severe stresses during implant insertion, we present a nano-mechanical and nano-tribological investigation of RF-sputtered HA films on AZ31 Mg alloy substrates. EDX and XRD analysis indicate that as-deposited coatings are amorphous and Ca-deficient whereas rapid thermal annealing results in c-axis orientation and near-stoichiometric composition. Analysis of the nanoindentation data using a thin film model shows that annealing increases the coating's intrinsic hardness (H) and strain at break (H/E) values, from 2.7 GPa to 9.4 GPa and from 0.043 to 0.079, respectively. In addition, despite being rougher, the annealed samples display better wear resistance; a sign that the rapid thermal annealing does not compromise their interfacial strength and that these systems have potential for resorbable bone implant applications.

3D Fabrication and Characterisation of Electrically Receptive PCL-Graphene Scaffolds for Bioengineered In Vitro Tissue Models.

Polycaprolactone (PCL) is a well-established biomaterial, offering extensive mechanical attributes along with low cost, biocompatibility, and biodegradability; however, it lacks hydrophilicity, bioactivity, and electrical conductivity. Advances in 3D fabrication technologies allow for these sought-after attributes to be incorporated into the scaffolds during fabrication. In this study, solvent-free Fused Deposition Modelling was employed to fabricate 3D scaffolds from PCL with increasing amounts of graphene (G), in the concentrations of 0.75, 1.5, 3, and 6% (w/w). The PCL+G scaffolds created were characterised physico-chemically, electrically, and biologically. Raman spectroscopy demonstrated that the scaffold outer surface contained both PCL and G, with the G component relatively uniformly distributed. Water contact angle measurement demonstrated that as the amount of G in the scaffold increases (0.75-6% w/w), hydrophobicity decreases; mean contact angle for pure PCL was recorded as 107.22 ± 9.39°, and that with 6% G (PCL+6G) as 77.56 ± 6.75°. Electrochemical Impedance Spectroscopy demonstrated a marked increase in electroactivity potential with increasing G concentration. Cell viability results indicated that even the smallest addition of G (0.75%) resulted in a significant improvement in electroactivity potential and bioactivity compared with that for pure PCL, with 1.5 and 3% exhibiting the highest statistically significant increases in cell proliferation.

Raman spectroscopic monitoring of the osteogenic differentiation of human mesenchymal stem cells.

The differentiation of stem cells into multi-lineages is essential to aid the development of tissue engineered materials that replicate the functionality of their tissue of origin. For this study, Raman spectroscopy was used to monitor the formation of a bone-like apatite mineral during the differentiation of human mesenchymal stem cells (hMSCs) towards an osteogenic lineage. Raman spectroscopy observed dramatic changes in the region dominated by the stretching of phosphate groups (950-970 cm(-1)) during the period of 7-28 days. Changes were also seen at 1030 cm(-1) and 1070 cm(-1), which are associated with the P-O symmetric stretch of PO(4)(3-) and the C-O vibration in the plane stretch of CO(3)(2-). Multivariate factor analysis revealed the presence of various mineral species throughout the 28 day culture period. Bone mineral formation was observed first at day 14 and was identified as a crystalline, non-substituted apatite. During the later stages of culture, different mineral species were observed, namely an amorphous apatite and a carbonate, substituted apatite, all of which are known to be Raman markers for a bone-like material. Band area ratios revealed that both the carbonate-to-phosphate and mineral-to-matrix ratios increased with age. When taken together, these findings suggest that the osteogenic differentiation of hMSCs at early stages resembles endochondral ossification. Due to the various mineral species observed, namely a disordered amorphous apatite, a B-type carbonate-substituted apatite and a crystalline non-substituted hydroxyapatite, it is suggested that the bone-like mineral observed here can be compared to native bone. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analyses for monitoring the various mineral species, degree of mineralisation and the crystallinity of hMSCs as they differentiate into osteoblasts.

The Direct 3D Printing of Functional PEEK/Hydroxyapatite Composites via a Fused Filament Fabrication Approach.

The manufacture of polyetheretherketone/hydroxyapatite (PEEK/HA) composites is seen as a viable approach to help enhance direct bone apposition in orthopaedic implants. A range of methods have been used to produce composites, including Selective Laser Sintering and injection moulding. Such techniques have drawbacks and lack flexibility to manufacture complex, custom-designed implants. 3D printing gets around many of the restraints and provides new opportunities for innovative solutions that are structurally suited to meet the needs of the patient. This work reports the direct 3D printing of extruded PEEK/HA composite filaments via a Fused Filament Fabrication (FFF) approach. In this work samples are 3D printed by a custom modified commercial printer Ultimaker 2+ (UM2+). SEM-EDX and µCT analyses show that HA particles are evenly distributed throughout the bulk and across the surface of the native 3D printed samples, with XRD highlighting up to 50% crystallinity and crystalline domains clearly observed in SEM and HR-TEM analyses. This highlights the favourable temperature conditions during 3D printing. The yield stress and ultimate tensile strength obtained for all the samples are comparable to human femoral cortical bone. The results show how FFF 3D printing of PEEK/HA composites up to 30 wt% HA can be achieved.

The Surface Characterisation of Fused Filament Fabricated (FFF) 3D Printed PEEK/Hydroxyapatite Composites.

Polyetheretherketone (PEEK) is a high-performance thermoplastic polymer which has found increasing application in orthopaedics and has shown a lot of promise for 'made-to-measure' implants via additive manufacturing approaches. However, PEEK is bioinert and needs to undergo surface modification to make it at least osteoconductive to ensure a more rapid, improved, and stable fixation that will last longer in vivo. One approach to solving this issue is to modify PEEK with bioactive agents such as hydroxyapatite (HA). The work reported in this study demonstrates the direct 3D printing of PEEK/HA composites of up to 30 weight percent (wt%) HA using a Fused Filament Fabrication (FFF) approach. The surface characteristics and in vitro properties of the composite materials were investigated. X-ray diffraction revealed the samples to be semi-crystalline in nature, with X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry revealing HA materials were available in the uppermost surface of all the 3D printed samples. In vitro testing of the samples at 7 days demonstrated that the PEEK/HA composite surfaces supported the adherence and growth of viable U-2 OS osteoblast like cells. These results demonstrate that FFF can deliver bioactive HA on the surface of PEEK bio-composites in a one-step 3D printing process.

Special issue: select papers from the 29th Northern Ireland Biomedical Engineering Conference, Belfast, UK, April 2009.

Each year, NIBES hosts a spring conference that is jointly organised by Queen's University of Belfast and University of Ulster. The 29th NIBES Spring meeting took place on 8th April 2009 at Queen's University of Belfast. NIBES 2009 had an impressive scientific program with two international leading plenary speakers and 28 oral presentations.

Controlling Fluid Diffusion and Release through Mixed-Molecular-Weight Poly(ethylene) Glycol Diacrylate (PEGDA) Hydrogels.

Due to their inherent ability to swell in the presence of aqueous solutions, hydrogels offer a means for the delivery of therapeutic agents in a range of applications. In the context of designing functional tissue-engineering scaffolds, their role in providing for the diffusion of nutrients to cells is of specific interest. In particular, the facility to provide such nutrients over a prolonged period within the core of a 3D scaffold is a critical consideration for the prevention of cell death and associated tissue-scaffold failure. The work reported here seeks to address this issue via fabrication of hybrid 3D scaffolds with a component fabricated from mixed-molecular-weight hydrogel formulations capable of storing and releasing nutrient solutions over a predetermined time period. To this end, poly(ethylene) glycol diacrylate hydrogel blends comprising mixtures of PEGDA-575 Mw and PEGDA-2000 Mw were prepared via UV polymerization. The effects of addition of the higher-molecular-weight component and the associated photoinitiator concentration on mesh size and corresponding fluid permeability have been investigated by diffusion and release measurements using a Theophylline as an aqueous nutrient model solution. Fluid permeability across the hydrogel films has also been determined using a Rhodamine B solution and associated fluorescence measurements. The results indicate that addition of PEGDA-2000 Mw to PEGDA-575 Mw coupled with the use of a specific photoinitiator concentration provides a means to change mesh size in a hydrogel network while still retaining an overall microporous material structure. The range of mesh sizes created and their distribution in a 3D construct provides for the conditions required for a more prolonged nutrient release profile for tissue-engineering applications.

The association between specific types of childhood adversity and attenuated psychotic symptoms in a community sample.

AIM: This study aimed to examine the relationship between types of childhood adversity and attenuated psychotic symptoms in a general population sample, and to determine the predictive role of other psychosocial factors such as resilience, coping style and social support. METHODS: An online survey was conducted with a US-based general population sample of 748 participants (aged 18 to 35 years) using Amazon's Mechanical Turk (an online crowd-sourcing service). Participants completed the Adverse Childhood Experiences Questionnaire (ACE-Q), the Prodromal Questionnaire (PQ-16), the Brief COPE Scale, the Brief Resilience Scale (BRS), the Multidimensional Scale of Perceived Social Support and the Neighbourhood Cohesion Scale. A series of backwards stepwise hierarchical regression analyses was employed to determine the predictors of PQ-16 score. RESULTS: Participants reported an average of 2.99 attenuated psychotic symptoms (from a total of 16), and an average of 2.77 childhood adversities (from a total of 10). In the final model, the specific types of childhood adversity which significantly predicted PQ-16 score were verbal abuse, sexual abuse and physical neglect. Resilience, as well as the coping styles of self-distraction, denial, substance use, emotional support, venting, religion and self-blame, were significant predictors; perceived social support and neighbourhood cohesion were not. This model predicted 33% of the variance in PQ-16 score. CONCLUSIONS: The results of this study support the association between childhood adversity and attenuated psychotic symptoms in the general population. Resilience and coping styles were also important predictive factors. These data suggest potential strategies on which to focus for early intervention and prevention.

An investigation of associations between experience of childhood trauma and political violence and theory of mind impairments in schizophrenia.

There has been little examination of the association between trauma and cognitive deficits seen in individuals diagnosed with schizophrenia. Theory of Mind (ToM) impairments are a significant feature of schizophrenia but it remains unclear as to why these deficits are so prevalent in this population. This study aimed to explore associations between ToM deficits and specific forms of childhood adversities in a schizophrenia population. The study sample comprised of 66 adults with a confirmed diagnosis of schizophrenia, attending mental health services in Northern Ireland. Assessments were completed to ascertain if individuals had prior experience of sexual or physical abuse, emotional neglect or experience of the political violence of Northern Ireland's "Troubles", and the Gardner Hinting Test was applied to assess ToM ability. Backwards stepwise regression analyses demonstrated that emotional neglect, specifically during early childhood (0-6 years) predicted ToM impairments in this group. Conflict-related trauma was also associated with ToM but was not an independent significant predictor of ToM deficits. This is the first study to examine links between specific forms of childhood adversity and ToM impairments in individuals diagnosed with schizophrenia. Potential underpinning psychological mechanisms are considered and implications for clinical practice are discussed.

A qualitative phenomenological analysis of the subjective experience and understanding of the at risk mental state.

Over recent years there has been a growing interest in identifying the early stages of psychosis. The At Risk Mental State (ARMS) is characteristic of the prodromal stages of psychosis and its identification gives rise to a number of clinical and research opportunities including early intervention and prevention of psychosis. This study employs interpretative phenomenological analysis to gain insights into the subjective experience and individuals understanding of the development of their ARMS. Five participants took part and provided information on the experience of symptoms, life prior to onset of their ARMS and their understanding of symptoms and their development through a semi structured interview. From the analysis of transcripts eight themes emerged which were common across participants accounts. Three themes of experience (disturbed world/disturbed self, disconnection with the world, thunderstruck) and five themes of understanding (absence of understanding, use of others, identity, forming links, fragmented understanding) were identified. Themes are explored in detail and discussed in relation to existing literature and theory. Clinical implications, directions for future research, and limitations are discussed within.

Entrapment of Autologous von Willebrand Factor on Polystyrene/Poly(methyl methacrylate) Demixed Surfaces.

Human platelets play a vital role in haemostasis, pathological bleeding and thrombosis. The haemostatic mechanism is concerned with the control of bleeding from injured blood vessels, whereby platelets interact with the damaged inner vessel wall to form a clot (thrombus) at the site of injury. This adhesion of platelets and their subsequent aggregation is dependent on the presence of the blood protein von Willebrand Factor (vWF). It is proposed here that the entrapment of vWF on a substrate surface offers the opportunity to assess an individual's platelet function in a clinical diagnostic context. Spin coating from demixed solutions of polystyrene (PS) and poly(methyl methacrylate) (PMMA) onto glass slides has been shown previously to support platelet adhesion but the mechanism by which this interaction occurs, including the role of vWF, is not fully understood. In this work, we report a study of the interaction of platelets in whole blood with surfaces produced by spin coating from a solution of a weight/weight mixture of a 25% PS and 75% PMMA (25PS/75PMMA) in chloroform in the context of the properties required for their use as a Dynamic Platelet Function Assay (DPFA) substrate. Atomic Force Microscopy (AFM) indicates the presence of topographical features on the polymer demixed surfaces in the sub-micron to nanometer range. X-ray Photoelectron Spectroscopy (XPS) analysis confirms that the uppermost surface chemistry of the coatings is solely that of PMMA. The deliberate addition of various amounts of 50 µm diameter PS microspheres to the 25PS/75PMMA system has been shown to maintain the PMMA chemistry, but to significantly change the surface topography and to subsequently effect the scale of the resultant platelet interactions. By blocking specific platelet binding sites, it has been shown that their interaction with these surfaces is a consequence of the entrapment and build-up of vWF from the same whole blood sample.

Osteoblastic differentiation of periodontal ligament stem cells on non-stoichiometric calcium phosphate and titanium surfaces.

Bioactive materials offer particular clinical benefits in the field of dental implantology, where differentiation of stem cells towards an osteoblastic lineage is required for osseointegration and appropriate function of implants in vivo. The aim of this study was to evaluate the osteoblastic response of Stro-1 +ve periodontal ligament stem cells (PDLSCs) to three well-characterized biomaterial surfaces: an abraded titanium surface (cpTi) control; a polycrystalline titanium surface, with both micro and nanotopography produced by radio frequency magnetron sputtering (TiTi); and the same surface incorporating a sputter deposited calcium phosphate coating (CaP-TiTi). The CaP-TiTi surfaces were nonstoichiometric, carbonated, and calcium rich with a Ca/P ratio of 1.74. PDLSCs were grown on each surface in the absence of supplementary osteogneic-inducing agents. Osteoblastic responses were assessed for up to 21 days in culture by measuring gene expression using real time q-PCR and via assessment of intracellular alkaline phosphatase (ALP) activity. Gene expression analysis for the CaP-TiTi surfaces showed a significant late stage up-regulation of Secreted Phosphoprotein 1. Additionally, there was a significant up-regulation of the Wnt signaling genes ß-catenin and Wnt Family Member 5 A on days 14 and 21, respectively for the CaP-TiTi surface. A significant increase in intracellular ALP at day 21 for the CaP-TiTi surface was also observed. These data suggest that the CaP-TiTi surfaces provide the bioactive conditions required for direct osteoblastic differentiation of PDLSCs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1692-1702, 2017.

Surface modification of poly(epsilon-caprolactone) using a dielectric barrier discharge in atmospheric pressure glow discharge mode.

The role of roughening and functionalization processes involved in modifying the wettability of poly(epsilon-caprolactone) (PCL) after treatment by an atmospheric pressure glow discharge plasma is discussed. The change in the ratio of CO/C-O bonds is a significant factor influencing the wettability of PCL. As the contact angle decreases, the level of CO bonds tends to rise. Surface roughness alterations are the driving force for lasting increases in wettability, while the surface functional species are shorter lived. We can approximate from ageing that the increase in wettability for PCL after plasma treatment is 55-60% due to roughening and 40-45% due to surface functionalization for the plasma device investigated.