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Contents lists available atScienceDirectMaterials & Designjournal homepage:www.elsevier.com/locate/matdesNovel bioglasses for bone tissue repair and regeneration: Effect of glassdesign on sintering ability, ion release and biocompatibilityElena Mancusoa,b ,, Oana A. Bretcanua, Martyn Marshallc, Mark A. Birchd, Andrew W. McCaskied,Kenneth W. DalgarnoaaSchool of Mechanical and Systems Engineering, Newcastle University, UKbSchool of Mechanical Engineering, University of Leeds, UKcGlass Technology Services Ltd, Sheffield, UKdDivision of Trauma and Orthopaedic Surgery, University of Cambridge, UKARTICLE INFOKeywords:Glass designSintering abilityIon releaseBiocompatibilityBone substitutesABSTRACTEight novel silicate, phosphate and borate glass compositions (coded as NCLx, where x = 1 to 8), containingdifferent oxides (i.e.MgO, MnO2,Al2O3, CaF2,Fe2O3, ZnO, CuO, Cr2O3) were designed and evaluated alongsideapatite-wollastonite (used as comparison material), as potential biomaterials for bone tissue repair andregeneration. Glass frits of all the formulations were processed to have particle sizes under 53μm, with theirmorphology and dimensions subsequently investigated by scanning electron microscopy (SEM). In order toestablish the nature of the raw glass powders, X-ray diffraction (XRD) analysis was also performed. The sinteringability of the novel materials was determined by using hot stage microscopy (HSM). Ionic release potential wasassessed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Finally, the cytotoxic effect ofthe novel glass powders was evaluated for different glass concentrationsviaa colorimetric assay, on which basisthree formulations are considered promising biomaterials.GRAPHICAL ABSTRACT1. IntroductionThefirst reported use of a glass intended for bone tissue repair datesback to 1969, when Professor L. Hench proposed a composition in theNa2O-CaO-SiO2-P2O5system, designated as bioglass 45S5[1,2], com-mercially known as Bioglass®.Although Bioglass®proved to be an excellent material, consideredfor long time the gold standard bone substitute, it suffers from severaldrawbacks. Specifically, the main difficulties are related to the materialprocessing in form of 3D porous scaffolds, due to the limited ability ofthis glass in sintering[3]. Additionally, other weaknesses include: itsslow degradation kinetic with the consequent difficulties to match theformation rate of new tissue, and the abrupt pH variations of thebiological microenvironment, due to the increase in the concentrationof ions such as Na+and Ca2+, especially in the short term when therelease is faster[46].http://dx.doi.org/10.1016/j.matdes.2017.05.037Received 12 January 2017; Received in revised form 11 May 2017; Accepted 11 May 2017Corresponding author at: School of Mechanical Engineering, University of Leeds, Woodhouse Lane, LS2 9JT Leeds, UK.E-mail address:e.mancuso@leeds.ac.uk(E. Mancuso).Materials & Design 129 (2017) 239–248Available online 12 May 20170264-1275/ © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).MARK
Worldwide many researchers have used the SiO2-Na2O-CaO-P2O5system as a template for developing new silica-based compositions[7].Subsequently, many formulations in the phosphate and borate-basedsystem have been also designed to overcome the Bioglass®and silicate-based glass limitations[811], and thus to meet the set of requirementsthat are both crucial and necessary for optimised tissue-engineeredsubstitutes[12].The possibility to tailor glass properties by doping the maincomposition with network modifiers and/or intermediate oxides[1320] offers significant potential for this class of biomaterials. Inaddition to promoting bone bonding, the release of soluble ions (i.e.Si,Ca, P and Na) from these glasses have been demonstrated to promotecell proliferation, differentiation and activate gene expression[2024].Furthermore, it has been also revealed that even slight changes in theglass main formulation can substantially affect the material behaviour,particularly the physico-chemical and mechanical properties, dissolu-tion rate, bioactivity and bioresorbability[5,16,18,2527].However, there are still several criticisms related to the clinical useof this class of biomaterials in bone repair[12]. Firstly, whether or notglass dissolution products have a positive effect on adult stem cells isstill an open debate[28]. Secondly, they have often proved inadequatewhen used in load-bearing bone defects, due to their low tensilestrength and fracture toughness[29]. Ultimately, there are no large-scale porous bioactive glasses on the market, thus their commercialsuccess as bone scaffolds is limited[6,12].The aim of this work was the development and characterisation ofeight novel silicate, phosphate and borate glass formulations (coded asNCLx, where x = 1 to 8), containing different oxides and in diversemolar percentages as promising biomaterials for the repair andregeneration of bone tissue.2. Materials and methods2.1. Development of novel glass formulationsBased on the current state of the art, the eight bioceramic formula-tions were developed using: silicon dioxide, phosphorous pentoxide andboron trioxide as network formers due to their promising bioactivepotential[1,30,31], distinctive resorbable properties[32,33], andcustomable degradation rate[5,34], along with a range of differentdoping agents (i.e.MgO, MnO2,Al2O3, CaF2,Fe2O3, ZnO, CuO, Cr2O3),which were used to tailor the properties of the main composition[3541]. The rationale and innovative characteristics of the novelmaterials are reported inTable 1. Additionally, considering theexcellent biocompatibility eitherin vitroandin vivoof apatite wollas-tonite (AW)[4244], and the fact that it has been adopted for a broadrange of medical applications, either in the form of powder, porousstructures or bulk material[45,46], AW glass-ceramic was used ascomparison material in this study.2.2. Glass production and processingThe novel glasses were produced and supplied by Glass TechnologyService (GTS) Ltd. (Sheffield, UK) along with AW. Briefly, the indivi-dual components (seeTable 2) of each formulation were weighed outand then mixed together to obtain a uniform blend, which wassubsequently melted in platinum crucibles at temperatures up to1500 °C. The individual melts of glass were cast as solid blocks andthen thermally shocked in de-ionised water to produce the precursormaterials, known as frits.Glass frits of all the compositions were ground in a one-bowlzirconia ball milling machine (Planetary Mono Mill Pulverisette 6,Fritsch GmbH, Germany) using a rotational speed of 400 rpm for30 min, and then sieved using a mechanical sieve shaker (Impact TestEquipment Ltd., UK) to have afinal particle size about 20μm and below53μm.Powders were prepared for pressing through mixing with anisopropanol solution (Sigma Aldrich, UK) in the proportion 1:3 (w/w). Powders were then pressed using an automatic hydraulic press(Specac-Atlas8T, Specac Ltd., UK) to make 10 mm diameter and2.5 mm high pellets. The pressed pellets were then sintered in a furnace(Carbolite 1200 CWF, Carbolite GmbH, Germany), with the sinteringtimes and temperatures defined by the results of the hot stagemicroscopy analysis, reported inSection 2.3.2.2.3. Physico-chemical characterisation2.3.1. Microstructural characterisationPowder glasses and dense pellets were sputtered with a thin layer ofgold (approximately 10 nm, sputter time 40 s at 40 mA), and afterwardanalysed using a Philips XL30 Field-Emission Environmental ScanningElectron Microscope (ESEM FEG), which isfitted with a RontecQuantax system for the Energy-Dispersive Spectroscopy (EDS) analysis.All the images were taken at an operation voltage of 20 kV, andworking distance between 5 and 10 mm.2.3.2. Hot stage microscopy (HSM)The sintering ability of the novel glass powders was determinedusing hot stage microscopy (Misura®, Expert System Solutions, Italy).Tests were performed in air using a heating rate of 10 °C/min up to1200 °C. Glass powders were manually pressed into a small cylindricaldie (2 × 3 mm) and placed on a 10 × 15 × 1 mm alumina support.During the process the specimens were observed by a video camera andimages of the changing sample profile were acquired up to 1450 °C.Afterwards, the sample shrinkage at different temperatures was calcu-lated from the variation of the sample area, using the followingformula:AAshrinkage (%) = × 100T0whereA0(mm2) was the initial area of the specimen at roomtemperature andAT(mm2) was the area of the specimen at thetemperatureT.2.3.3. XRD AnalysisTo investigate the nature of the novel materials. XRD analysis wasTable 1Rationale of the novel glass compositions.CODE MAIN NETWORK FORMER AIMNCL1SiO2To develop a material with osteogenic properties, mainly determined by the presence of a high amount of silica.NCL2SiO2To develop a load-bearing material with osteogenic properties and tailored degradation rate.NCL3B2O3To develop a material with improved degradation rate and appropriate level of bioactivity as well as mechanical propertiesNCL4B2O3To develop a material with tailored degradation rate and osteogenic effects.NCL5P2O5To develop a resorbable glass with controlled degradation rate.NCL6P2O5To develop a resorbable glass with controlled degradation rate, and improved mechanical strengthNCL7SiO2To develop a material with antibacterial properties, mainly determined by the presence of silver oxide, and a good level of bioactivity.NCL8SiO2To develop a material with osteogenic properties and tailored degradation rate for non-load bearing applications.E. Mancuso et al.Materials & Design 129 (2017) 239–248240
performed using a PANalytical X'Pert Pro MPD, powered by a PhilipsPW3040/60 X-ray generator, andfitted with an X'Celerator detector.Diffraction data was acquired by exposing powder samples to Cu-KαX-ray radiation, at 40 kV and 40 mA. The data were collected over a 2θrange between 5 and 80°, with a step size equal to 0.0334°, a countingtime per step of 200 s using the scanning X'Celerator detector. Phaseidentification was carried out using the PANalytical X'Pert HighScorePlus© software.2.3.4. Ion leaching evaluationUn-sintered glass powders with a concentration of 10 mg/ml wereimmersed in deionised water (Veolia Water Technologies, UK) andincubated under an atmosphere of 5% CO2and 95% air at 37 °C. Aftereach storage period (1, 3, 7, 14 and 28 days), the specimens wereremovedviafiltration, andfiltrates retained to analyse the ion releasepotential of each compositions. An inductively coupled plasma opticalemission spectroscopy (ICP-OES) (Specto-Ciros-Vision, SheffieldUniversity, UK), which allows simultaneous multi-element analysisfollowing the calibration of the instrument by standards of knownconcentrations of the elements of interest, was employed.2.4. Biological characterisationThein vitrocytotoxicity of the novel glass formulations wasevaluated according to ISO 109935 [47]using rat calvaria osteoblastcells in indirect contact with glass powders up to 7 days. Each powdersample wasfirstly sterilised using 100% ethanol solution, and afteradded to Dulbecco's modified Eagle medium (DMEM, Gibco®UK) atthree different concentrations (0.1, 1 and 10 mg/ml) and incubated for24 h at 37 °C. After incubation, the glass-conditioned medium wasfiltered through a 0.22μm microbiologicalfilter, and used forin vitrotests.Cells at early passages were provided by Institute of CellularMedicine (Medical School, Newcastle University, UK), and then werecultured in T75flasks at 37 °C in a humidified incubator with 5% CO2,using DMEM supplemented with 10% fetal bovine serum (FBS), 1%penicillin-streptomycin and 1% glutamine (Gibco®, UK). Cells wereseeded at a density of 1x104cells/well in 96-well plates and incubatedat 37 °C. After 24 h culture period the culture media was discarded andreplaced with thefiltered solution for indirect cytotoxicity testing. RatOBs cultured in the absence of glass powders were used as negativecontrol.The culture plates were then incubated for 1 and 7 days. Thecytotoxic effect was measured exposing each well to 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT,SigmaAldrich, UK) solubilisation at a concentration of 0.5 mg/ml.100μl of MTT solution was added to each well and incubated at 37 °Cfor another 4 h. MTT was taken up only by active cells and reduced intheir mitochondria to insoluble purple formazan granules. The mediumwas then removed and 100μl of dimethyl sulfoxide (DMSO) was addedto dissolve the precipitated formazan. The absorbance of the solutionwas evaluated spectrophotometrically at a wavelength of 570 nm, andacquired using a Sunrise microplate reader (Tecan Group Ltd.,Switzerland).The absorbance values from three replicates were averaged andstatistically analysed using twoway analysis of variance (ANOVA)followed by Bonferronipost hocanalysis.P-values < 0.05 were con-sidered statistically significant.3. Results3.1. Glass production and powder processingDuring synthesis, composition NCL5 could not form a glass at anytemperature up to 1500 °C, which was the highest temperature theavailable furnaces could reach. Glass frits from the seven compositionsTable 2Composition of the novel glass formulations.CODEGLASS COMPOSITION (wt%)NCL130.44SiO29P2O56.29Na2O7.10CaO9.54K2O5.56MgO5.52MnO25.16ZnO13.14SrO1.01CuO5.91Bi2O32.02TeO22.31V2O5NCL236.90SiO29.70P2O51.90B2O33.39Na2O11.48CaO3.85K2O4.41MgO2.38MnO26.97Al2O32.13CaF210.92Fe2O30.41Li2O1.97MoO31.52SeO22.07Cr2O3NCL320.03SiO23.79P2O532.52B2O34.97Na2O5.23CaO6.27K2O2.69MgO2.72Al2O33.20TiO210.67Fe2O30.40Li2O2.05BaO1CoO2.43V2O52.03Cr2O3NCL416.28SiO29.63P2O537.77B2O34.21Na2O3.80CaO6.38K2O2.73MgO5.52ZnO7.03SrO2.12CaF21.08CuO1.95MoO31.51SeO2NCL560.28P2O54.09Na2O5.28CaO5.32K2O3.80MgO3.84ZnO4.10MnO29.77SrO0.75TiO22.75Sb2O3NCL64.61SiO268.19P2O56.68B2O34.76Na2O5.38CaO2.71K2O1.55MgO1.50CaF21.67MnO20.76CuO0.72CoO1.46Cr2O3NCL739.96SiO29.46P2O512.39Na2O11.19CaO2.50K2O1.61MgO15.44AgO2.13TiO24.26Fe2O31.06CuONCL838.93SiO210.24P2O52.01B2O38.94Na2O12.11CaO6.78K2O2.91MgO6.27MnO21.17ZnO2.30Fe2O31.49SrO2.81CaF20.57CuO0.54CoO1.04MoO30.80SeO21.10Cr2O3AW4.6 MgO44.7CaO34SiO216.2P2O50.5 CaF2E. Mancuso et al.Materials & Design 129 (2017) 239–248241
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