Objectives: This study characterised the bioactive properties (i.e., ion release, pH rise, and apatite formation) of a newly developed Voco Profluorid + BioMin F varnish. Three additional varnishes were investigated for comparison: Clinpro™ White Varnish (3M™, St. Paul, MN, USA), MI Varnish (GC, Tokyo, Japan), and Profluorid varnish (VOCO GmbH, Cuxhaven, Germany). The Clinpro™ White and MI varnishes were chosen for comparison due to their similar composition of active ingredients. Profluorid served as a standard fluoride-only varnish reference. Methods: Dental varnish ingredients were characterised using ATR-FTIR, XRD, and 19F and 31P MAS-NMR. Coated coverslips were immersed in Tris buffer and artificial saliva (pH 4.0 and 7.0) for 2-24 h. Ion release was analysed using ICP-OES and a fluoride ion-selective electrode whilst monitoring pH changes. Post-immersion, coverslips were analysed by XRD and MAS-NMR to assess possible apatite formation. Results: XRD and 19F MAS-NMR detected NaF in all four varnishes. BioMin F varnish showed a 31P peak matching BioMin F glass, with an additional brushite peak, indicating partial reaction of the bioactive glass (BAG) with rosin resin water. All varnishes released fluoride and calcium, but only BioMin F and MI varnishes released phosphate, which is essential for the formation of calcium fluorapatite. Post-immersion analysis confirmed fluorapatite formation in BioMin F and, to a lesser extent, the Profluorid varnish. No apatite formation was observed in the other two varnishes. MI varnish exhibited calcium fluoride formation before and after immersion, as evidenced by XRD and 19F MAS-NMR analysis. Conclusions: The novel BioMin F varnish potentially promotes remineralisation by providing a sustained and slow release of therapeutic ions that are essential for the formation of fluorapatite.

This study investigates the structural location of silicon present in 'silicate-substituted' apatite.It has been hypothesized that silicon is present in the form of silicate groups site-specifically substituted in the hydroxyapatite lattice for phosphate groups, or that there may be an amorphous silicon-rich phase in the vicinity of the grain boundaries.Solid-state nuclear magnetic resonance (SSNMR) spectroscopy was carried out to view the elemental interactions using phosphorus ( 31 P) and silicon ( 29 Si) probes to confirm or refute the presence of silicate groups in relation to phosphate groups in the bulk material structure.The analysis was performed on stoichiometric hydroxyapatite (HA) and silicate-substituted hydroxyapatite (SA) with a nominal silicon content of 0.8wt%, in asprecipitated, calcined (700°C), and sintered (1,250 and 1,300°C, respectively) powder forms.XRD confirmed all forms of powder were phase pure.FTIR confirmed both hydroxyl and phosphate group functionalities in all forms of HA and SA, while silicate group functionality was only observed in all forms of SA.SSNMR using 31 P-and 29 Si-coupled probes demonstrated that as the crystallinity of the powders increased from the precipitate to the sintered form, the signal associated with the presence of a silicate group in the phosphate environment developed as the crystal structure became more ordered.These results support the hypothesis that in SA containing 0.8wt% silicon, silicate groups are site-specifically substituted in the hydroxyapatite lattice for phosphate groups.This observation may be key to understanding the mechanisms by which the introduction of 0.8wt% silicon enhances bone regeneration in apatitic bone graft substitute materials.

A photoelectrochemical imaging system (PEIS) using α-Fe 2 O 3 (hematite) thin films as the sensor substrate has been demonstrated for mapping the dynamic responses of multiple independent cells simultaneously. Herein, one-dimensional (1D) hematite nanorods (NRs) synthesized via a simple hydrothermal method are proposed as a platform for PEIS with enhanced photocurrent responses, good stability and excellent spatial resolution for applications requiring concurrent imaging of live cells and ion sensing applications. The photoelectrochemical sensing capability of hematite NRs was demonstrated by recording live cell images and by a high pH sensitivity without any surface modification. In contrast to potentiometric field-effect sensors based on silicon substrates, the deposition of a ion-sensitive PVC membrane on the highly structured hematite surface resulted in enhanced adhesion and an amperometric response to calcium ions (Ca 2+ ).

α-Fe2O3 (hematite) thin films have been shown to be a robust sensor substrate for photoelectrochemical imaging with good stability and high spatial resolution. Herein, one-dimensional (1D) hematite nanorods (NRs) synthesized via a simple hydrothermal method are proposed as a substrate which provides nanostructured surfaces with enhanced photocurrent responses compared to previously described hematite films, good stability, and excellent spatial resolution for potential imaging applications. The photoelectrochemical sensing capability of hematite NRs was demonstrated by a high pH sensitivity without modification. The modification of the hematite NRs with a thin poly(vinyl chloride) (PVC)-based ion-selective film allowed highly reversible amperometric detection of calcium ions with sensor materials traditionally employed in potentiometric devices.

Photoelectrochemical imaging has great potential in the label-free investigation of cellular processes. Herein, we report a new fast photoelectrochemical imaging system (PEIS) for DC photocurrent imaging of live cells, which combines high speed with excellent lateral resolution and high photocurrent stability, which are all crucial for studying dynamic cellular processes. An analog micromirror was adopted to raster the sensor substrate, enabling high-speed imaging. α-Fe2O3 (hematite) thin films synthesized via electrodeposition were used as a robust substrate with high photocurrent and good spatial resolution. The capabilities of this system were demonstrated by monitoring cell responses to permeabilization with Triton X-100. The ability to carry out dynamic functional imaging of multiple cells simultaneously provides improved confidence in the data than could be achieved with the slower electrochemical single-cell imaging techniques described previously. When monitoring pH changes, the PEIS can achieve frame rates of 8 frames per second.

Aims To evaluate graft healing of decellularized porcine superflexor tendon (pSFT) xenograft in an ovine anterior cruciate ligament (ACL) reconstruction model using two femoral fixation devices. Also, to determine if pSFT allows functional recovery of gait as compared with the preoperative measurements. Methods A total of 12 sheep underwent unilateral single-bundle ACL reconstruction using pSFT. Two femoral fixation devices were investigated: Group 1 (n = 6) used cortical suspensory fixation (Endobutton CL) and Group 2 (n = 6) used cross-pin fixation (Stratis ST). A soft screw was used for tibial fixation. Functional recovery was quantified using force plate analysis at weeks 5, 8, and 11. The sheep were euthanized after 12 weeks and comprehensive histological analysis characterized graft healing at the graft-bone interface and the intra-articular graft (ligamentization). Results The pSFT remodelled into a ligament-like structure and no adverse inflammatory reaction was seen. The ground reaction force in the operated leg of the Endobutton group was higher at 11 weeks (p < 0.05). An indirect insertion was seen at the graft-bone interface characterized by Sharpey-like fibres. Qualitative differences in tendon remodelling were seen between the two groups, with greater crimp-like organization and more aligned collagen fibres seen with Endobutton fixation. One graft rupture occurred in the cross-pin group, which histologically showed low collagen organization. Conclusion Decellularized pSFT xenograft remodels into a ligament-like structure after 12 weeks and regenerates an indirect-type insertion with Sharpey-like fibres. No adverse inflammatory reaction was observed. Cortical suspensory femoral fixation was associated with more enhanced graft remodelling and earlier functional recovery when compared with the stiffer cross-pin fixation.

Aims To evaluate graft healing of decellularized porcine superflexor tendon (pSFT) xenograft in an ovine anterior cruciate ligament (ACL) reconstruction model using two femoral fixation devices. Also, to determine if pSFT allows functional recovery of gait as compared with the preoperative measurements. Methods A total of 12 sheep underwent unilateral single-bundle ACL reconstruction using pSFT. Two femoral fixation devices were investigated: Group 1 (n = 6) used cortical suspensory fixation (Endobutton CL) and Group 2 (n = 6) used cross-pin fixation (Stratis ST). A soft screw was used for tibial fixation. Functional recovery was quantified using force plate analysis at weeks 5, 8, and 11. The sheep were euthanized after 12 weeks and comprehensive histological analysis characterized graft healing at the graft-bone interface and the intra-articular graft (ligamentization). Results The pSFT remodelled into a ligament-like structure and no adverse inflammatory reaction was seen. The ground reaction force in the operated leg of the Endobutton group was higher at 11 weeks (p < 0.05). An indirect insertion was seen at the graft-bone interface characterized by Sharpey-like fibres. Qualitative differences in tendon remodelling were seen between the two groups, with greater crimp-like organization and more aligned collagen fibres seen with Endobutton fixation. One graft rupture occurred in the cross-pin group, which histologically showed low collagen organization. Conclusion Decellularized pSFT xenograft remodels into a ligament-like structure after 12 weeks and regenerates an indirect-type insertion with Sharpey-like fibres. No adverse inflammatory reaction was observed. Cortical suspensory femoral fixation was associated with more enhanced graft remodelling and earlier functional recovery when compared with the stiffer cross-pin fixation.

The surface charge of cells affects cell signaling, cell metabolic processes, adherence to surfaces, and cell proliferation. Our understanding of the role of membrane charges is limited due to the inability to observe changes without interfering, chemically or physically, with the cell or its membrane. Here, we report that a photoelectrochemical imaging system (PEIS) based on label-free ac-photocurrent measurements at indium tin oxide (ITO) coated glass substrates can be used to map the basal surface charge of single live cells under physiological conditions. Cells were cultured on the ITO substrate. Photocurrent images were generated by scanning a focused, modulated laser beam across the back of the ITO coated glass substrate under an applied bias voltage. The photocurrent was shown to be sensitive to the negative surface charge of the substrate facing, basal side of a single living cell-an area not accessible to other electrochemical or electrophysiological imaging techniques. The PEIS was used to monitor the lysis of mesenchymal stem cells.

Summary StatementThe structure of bone inside a porous bone graft substitute can be quantified and compared by using a combination of novel measurements of surface area and connectivity. This allows for a numerical representation of the bone structure to be calculated.IntroductionVariation in absolute bone volume as a function of bone graft porosity has been well documented. However quantification of the 3D shape of bone and it's connectivity has always been difficult to assess let alone quantify. By use of novel computational methods the shape and connectivity of the bone can be characterised giving more insight to the relative quality of the bone ingrowth within the different porous grafts.Materials & MethodsCylindrical monoliths of hydroxyapatite (HA) of varying total porosities (60, 70 and 80% total) were implanted into a lapin model (subchondral distal femur) and the implants removed and XMT (resolution ∼30μm) scans taken at 3, 6, 12 and 24 weeks. The regions of bone and HA were defined using a modif...

This study compared the bone forming capacity of the same formulation of silicate-substituted bone graft substitute materials with different microporosity in an instrumented posterolateral spinal fusion ovine model. Materials with a strut porosity of (i) 22.5% (SiCaP) or (ii) 36.0% (SiCaP(+)) were packed along either side of the spine. Bone apposition rates, % new bone formation, % bone-implant contact, and % graft resorption were quantified at 8, 12, and 24 weeks post surgery. Computed Tomography (CT) was used to grade the formation of fusion bridges between vertebrae. Results showed no significant difference in bone apposition rates, % new bone formation, and % bone-implant contact when the two materials were compared. However, at 8 weeks, a significantly higher CT score was obtained in the SiCaP(+) group (0.83 ± 0.17) when compared with the SiCaP group (0.17 ± 0.17; p = 0.027). Significantly less scaffold remained in the SiCaP(+) group at 12 weeks (p = 0.018). Both SiCaP and SiCaP(+) formulations augmented bone formation. Increasing the strut porosity did not significantly increase bone formation however, at 8 weeks it promoted the formation of more highly mineralized bone resulting in a significantly higher CT score, suggesting the bone tissue formed was more mature. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 805-814, 2017.

A comparative investigation was undertaken on 1-2mm sized granules of two forms of synthetic bone graft substitute (SBG) with identical pore structure but varied bulk chemistry, stoichiometric hydroxyapatite (HA) and silicate substituted (0.8wt% Si) hydroxyapatite (SA), to assess the influence of SBG chemistry on the relative affinity of an osteogenic growth factor (GF), recombinant human bone morphogenetic protein-2 (rhBMP-2). A previously described novel fluorescent probe, fluoresceinthioureidoaminocaproic acid (FTCA), was covalently attached to rhBMP-2 to give FTCA-rhBMP-2 and facilitate the quantitative monitoring of GF uptake and release from the two chemistries of SBG. The relative affinity of rhBMP-2 for the HA and SA granules was assessed at a physiologically relevant concentration of 300ngmL-1 from three (increasingly complex) environments; phosphate buffered saline (PBS), minimum Eagles' medium (MEM) and serum supplemented MEM (SCEM) in order to closely mimic clinical bone repair procedures. The results demonstrated that rhBMP-2 affinity to SBGs was highly sensitive to both SBG chemistry and the composition of the local environment. Under the most physiologically relevant competitive conditions of SCEM, rhBMP-2 showed greater affinity to SA (P<0.05) such that 50% of the rhBMP-2 in solution was adsorbed to the SA granules after only 15min, as compared to 30% adsorbed to the HA granules. Subsequent investigation of the desorption of adsorbed GF from the SBGs demonstrated that a significantly higher percentage of the adsorbed rhBMP-2 was desorbed from HA as compared to SA granules. Together, these observations suggested that at physiologically relevant concentrations and conditions, rhBMP-2 has a greater affinity to silicate-substituted hydroxyapatite as compared to stoichiometric hydroxyapatite, which may in part explain the enhanced osteoconductivity and reported osteoinductivity for silicate-substituted hydroxyapatite based SBGs.

Recently, the porous bioactive glasses have attracted a lot of attention for use as scaffolds for tissue engineering bone; such glasses include the high phosphate, strontium containing glass (Stronbone P). However, the previous studies suggest that strontium can have a detrimental effect on the ability of apatite-like phase formation of the glass. The previously studied high phosphate all Sr glass showed an unidentified phase rather than an apatite-like phase upon immersion. Octa-calcium phosphate (OCP) is believed to be a precursor phase to apatite, however octa-strontium phosphate does not exist. Fluoride is known to knock out the OCP formation and promotes fluorapatite formation. This work presents the incorporation of a small amount of fluoride into calcium/strontium bioactive glasses. Differential scanning calorimetry was used to estimate the glass thermal properties. All of the studied glass compositions were subjected to bioactivity studies in Tris buffer (pH=7.4) for up to 21days. The initial glasses and the resultant precipitates were analysed using Fourier transform infrared spectroscopy, X-ray diffraction and magic angle spinning-nuclear magnetic resonance. The findings showed that all the fluoride containing glasses were amorphous and there was a marked increase in the rate of apatite formation in vitro compared to the equivalent fluoride free glasses, particularly for the all strontium containing glass. This indicates that the presence of fluoride affects the pathway of apatite formation, forming fluorapatite directly instead of via the transformation from OCP to hydroxyapatite. Therefore, fluoride may have potential future clinical applications as an additive to increase apatite formation.

A synthetic bone graft substitute consisting of silicate-substituted calcium phosphate with increased strut porosity (SiCaP EP) was evaluated in an ovine distal femoral critical sized metaphyseal defect as a standalone bone graft, as an autologous iliac crest bone graft (ICBG) extender (SiCaP EP/ICBG), and when mixed with bone marrow aspirate (SiCaP EP/BMA). Defects were evaluated after 4, 8, and 12 weeks with radiography, decalcified paraffin-embedded histopathology, non-decalcified resin-embedded histomorphometry, and mechanical indentation testing. All test groups exhibited excellent biocompatibility and osseous healing as evidenced by an initial mild inflammatory response followed by neovascularization, bone growth, and marrow infiltration throughout all SiCaP EP-treated defects. SiCaP EP/ICBG produced more bone at early time points, while all groups produced similar amounts of bone at later time points. SiCaP EP/ICBG likewise showed more favorable mechanical properties at early time points, but was equivalent to SiCaP EP and SiCaP EP/BMA at later time points. This study demonstrates that SiCaP EP is efficacious as a standalone bone graft substitute, mixed with BMA, and as an autograft extender.

Porous bioactive glasses are attractive for use as bone scaffolds. There is increasing interest in strontium containing bone grafts, since strontium ions are known to up-regulate osteoblasts and down regulate osteoclasts. This paper investigates the influence of partial to full substitution of strontium for calcium on the dissolution and phase formation of a multicomponent high phosphate content bioactive glass. The glasses were synthesised by a high temperature melt quench route and ground to a powder of <38 microns. The dissolution of this powder and its ability to form apatite like phases after immersion in Tris buffer (pH 7.4) and simulated body fluid (SBF) was followed by inductively coupled plasma optical emission spectroscopy (ICP), Fourier transform infra red spectroscopy (FTIR), X-ray powder diffraction (XRD) and (31)P solid state nuclear magnetic resonance spectroscopy up to 42 days of immersion. ICP indicated that all three glasses dissolved at approximately the same rate. The all calcium (SP-0Sr-35Ca) glass showed evidence of apatite like phase formation in both Tris buffer and SBF, as demonstrated after 3 days by FTIR and XRD analysis of the precipitate that formed during the acellular dissolution bioactivity studies. The strontium substituted SP-17Sr-17Ca glass showed no clear evidence of apatite like phase formation in Tris, but evidence of an apatite like phase was observed after 7 days incubation in SBF. The SP-35Sr-0Ca glass formed a new crystalline phase termed "X Phase" in Tris buffer which FTIR indicated was a form of crystalline orthophosphate. The SP-35Sr-0Ca glass appeared to support apatite like phase formation in SBF by 28 days incubation. The results indicate that strontium substitution for calcium in high phosphate content bioactive glasses can retard apatite like phase formation. It is proposed that apatite formation with high phosphate bioactive glasses occurs via an octacalcium phosphate (OCP) precursor phase that subsequently transforms to apatite. The equivalent octa-strontium phosphate does not exist and consequently in the absence of calcium, apatite formation does not occur. The amount of strontium that can be substituted for calcium in OCP probably determines the amount of strontium in the final apatite phase and the speed with which it forms.

Five bioactive glass compositions in the SiO2–P2O5–CaO–Na2O–CaF2 system (0–32mol% CaF2) and Bioglass® 45S5 were evaluated for their apatite forming ability in serum-free and serum-containing cell culture media for up to seven days. While F− ions in low concentrations were found to enhance apatite formation, higher fluoride content caused formation of fluorite and calcite. The presence of serum proteins delayed apatite precipitation for all compositions, while Bioglass® 45S5, despite considerably higher phosphate content (2.6 vs. ≤1.1mol% P2O5) and high concentrations of Ca2+ and PO43− in solution, formed only amorphous calcium phosphate.

Bioactive glasses are used clinically for bone regeneration, and their bioactivity and cell compatibility are often characterized in vitro, using physiologically relevant test solutions. The aim of this study was to show the influence of varying medium characteristics (pH, composition, presence of proteins) on glass dissolution and apatite formation. The dissolution behavior of a fluoride-containing bioactive glass (BG) was investigated over a period of one week in Eagle's Minimal Essential Medium with Earle's Salts (MEM), supplemented with either, (a) acetate buffer, (b) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, (c) HEPES + carbonate, or (d) HEPES + carbonate + fetal bovine serum. Results show pronounced differences in pH, ion release, and apatite formation over 1 week: Despite its acidic pH (pH 5.8 after BG immersion, as compared to pH 7.4-8.3 for HEPES-containing media), apatite formation was fastest in acetate buffered (HEPES-free) MEM. Presence of carbonate resulted in formation of calcite (calcium carbonate). Presence of serum proteins, on the other hand, delayed apatite formation significantly. These results confirm that the composition and properties of a tissue culture medium are important factors during in vitro experiments and need to be taken into consideration when interpreting results from dissolution or cell culture studies.

The dissolution re-precipitation behaviour of a series of fluoride-containing bioactive glasses was investigated under physiologically relevant conditions of low pH. All formulations including the classic fluoride-free 45S5 Bioglass® dissolved in acetate-buffered MEM, and formed apatite despite the pH remaining acidic throughout. Fluoride incorporation into the glasses and subsequent fluoride release resulted in the formation of an apatite (fluorapatite) of higher crystallinity; however, with increasing fluoride content in the glass, fluorite was formed at the expense of apatite. Results suggest that low fluoride content BG form apatite well at low pH conditions, representative of those present during the inflammation phase of normal bone healing or within the oral environment during caries or an acid-erosive challenge.