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Generation of composites for bone tissue-engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means

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Generation of composites for bone tissue-engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means
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  Generationofcompositesforbonetissue-engineering applicationsconsistingofgellangumhydrogelsmineralizedwithcalciumandmagnesiumphosphatephasesbyenzymaticmeans Timothy E. L. Douglas 1 *, Grzegorz Krawczyk  2 , Elzbieta Pamula 2 , Heidi A. Declercq  3 ,David Schaubroeck  4 , Miroslaw M. Bucko 2 , Lieve Balcaen 5 , Pascal Van Der Voort 6 , Vitaliy Bliznuk  7 ,Natasja M. F. van den Vreken 8 , Mamoni Dash 1 , Rainer Detsch 9 , Aldo R. Boccaccini 9 , Frank Vanhaecke 5 ,Maria Cornelissen 3 and Peter Dubruel 1 1  Polymer Chemistry and Biomaterials (PBM) Group, Department of Organic Chemistry, Ghent University, Belgium 2  Department of Biomaterials, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, Krakow, Poland 3  Department of Basic Medical Science  –  Histology Group, Ghent University, Belgium 4 Centre for Microsystems Technology (CMST), ELIS, Imec, Ghent, Belgium 5  Department of Analytical Chemistry, Ghent University, Belgium 6  Department of Inorganic Chemistry, COMOC, Ghent University, Belgium 7  Department of Materials Science and Engineering, Zwijnaarde, Belgium 8  Department of Basic Medical Science  –  Biomaterials Group, Ghent University, Belgium 9  Department of Materials Science and Engineering, Institute of Biomaterials (WW7), University of Erlangen-Nuremberg, Erlangen, Germany   Abstract Mineralizationofhydrogels,desirableforboneregenerationapplications,maybeachievedenzymatically byincorporationofalkalinephosphatase(ALP).ALP-loadedgellangum(GG)hydrogelsweremineralizedby incubation in mineralization media containing calcium and/or magnesium glycerophosphate (CaGP,MgGP). Mineralization media with CaGP:MgGP concentrations 0.1:0, 0.075:0.025, 0.05:0.05,0.025:0.075and0:0.1(allvaluesmol/dm 3 ,denotedA,B,C,DandE,respectively)werecompared.Mineralformationwascon fi rmedbyIRandRaman,SEM,ICP-OES,XRD,TEM,SAED,TGAandincreasesinthethemassfractionofthehydrogelnotconsistingofwater.CawasincorporatedintomineraltoagreaterextentthanMginsamplesmineralizedinmediaA  – D.Mgcontentandamorphicityofmineralformedincreasedinthe order A  < B < C < D. Mineral formed in media A and B was calcium-de fi cient hydroxyapatite (CDHA).Mineral formed in medium C was a combination of CDHA and an amorphous phase. Mineral formed inmedium D was an amorphous phase. Mineral formed in medium E was a combination of crystalline andamorphousMgP.Young  ’ smoduliandstoragemodulidecreasedindependenceofmineralizationmediumin the order A  > B > C > D, but were signi fi cantly higher for samples mineralized in medium E. Theattachment and vitality of osteoblastic MC3T3-E1 cells were higher on samples mineralized in mediaB – E (containing Mg)than in those mineralizedin medium A(notcontainingMg).Allsamples underwentdegradationandsupportedtheadhesionofRAW264.7monocyticcells,andsamplesmineralizedinmedia A and B supported osteoclast-like cell formation. Copyright © 2014 John Wiley & Sons, Ltd. Received 22 April 2013; Revised 6 November 2013; Accepted 7 January 2014 Keywords hydrogel; composite; calcium phosphate; magnesium phosphate; enzyme; gellan gum; osteo-blast; cytocompatibility  1. Introduction Gellan gum (GG) is an anionic calcium-binding polysaccha-ride produced by bacteria (Fialho  et al ., 2008; Giavasis *Correspondence to: Timothy E. L. Douglas, Polymer Chemistry andBiomaterials(PBM)Group,DepartmentofOrganicChemistry,Ghent University, Krijgslaan 281S4, 9000 Ghent, Belgium. E-mail: Timothy.Douglas@UGent.be Copyright © 2014 John Wiley & Sons, Ltd. JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE  RESEARCH ARTICLE  J Tissue Eng Regen Med  (2014)Published online in Wiley Online Library (wileyonlinelibrary.com)  DOI:  10.1002/term.1875  etal .,2000;OkamotoandKubota,1996),whichhasbeenap-plied in hydrogel form as a tissue-engineering (TE) scaffold(Fan  et al ., 2010; Oliveira  et al ., 2010). Further advantagesof gellan gum are its low cost and the fact that it is not ani-mal-derived, avoiding regulatory concerns. Gelation is in-duced by cooling a GG – CaCl 2  solution to body temperature, while cells and bioactive substances such as enzymes canbe incorporated before gelation at temperatures > 37°C(Oliveira  et al ., 2010; Douglas  et al ., 2012a).Mineralization of hydrogels is desirable for bone TEapplications in order to promote bioactivity, i.e. theformation of a chemical bond with surrounding bonetissue after implantation (LeGeros, 1991). Potentialfurther advantages of mineralization are the promotionof osteoblastic differentiation through increased stiffness(Engler  et al ., 2006; Rowlands  et al ., 2008; Evans  et al .,2009) and enhanced binding of growth factors that stim-ulate bone healing (Ruhe  et al ., 2005). Furthermore, incontrast to traditional ceramics, hydrogels can serve asdelivery vehicles for bioactive substances such as growthfactors and enzymes. As mineralized hydrogels consistmainly of water and contain a polymer component, they are expected to undergo more rapid degradation than ’ traditional ’  calcium phosphates, such as  β  -tricalciumphosphate (  β  -TCP) and hydroxyapatite (HA), whoseresorption remains incomplete after several months oreven years (Draenert  et al ., 2013; Gunther  et al ., 1998;Hwang  et al ., 2012; Moore  et al ., 2001).Hydrogel mineralization can be achieved by addition of enzymes such as alkaline phosphatase (ALP) (Gkioni et al ., 2010), which causes mineralization of bone by cleav-age of phosphate from organic phosphate, thereby increas-ing the local phosphate concentration and enablingprecipitation of insoluble phosphate salts. ALP has beenadded to hydrogel materials to induce their mineralization with calcium phosphate (CaP) during incubation in solu-tionscontainingcalciumandglycerophosphate(GP),whichserves as a substrate for ALP (Beertsen and van den Bos,1992; Douglas  et al ., 2012a, 2012b, 2012c; Filmon  et al .,2000; Spoerke  et al ., 2009; Yamauchi  et al ., 2004). ALPhas been implanted  in vivo  as a coating on titanium-basedimplants(Schouten etal .,2009)andasacomponentofcol-lagen sheets (Beertsen and van den Bos, 1992; Doi  et al .,1996) and synthetic oligo[poly(ethylene glycol)fumarate](OPF)hydrogels(Bongio etal .,2013).Noseverein fl amma-tory reactions were observed in any of these studies,suggesting low immunogenicity of ALP.However, ALP-induced enzymatic mineralization withCaP enriched with Mg or magnesium phosphate (MgP) re-mains unexplored. MgP is gaining interest as an alternativeto CaP, due to its comparable cytocompatibility and ability to supportosteoblast adhesionandexpressionofosteoblas-ticmarkersatthemRNAlevel(Tamimi etal .,2012).Thanksto the cytocompatibility of MgP, bone cements based onMgP have been developed as alternatives to CaP cements(Ewald  et al ., 2011; Klammert  et al ., 2010; Mestres andGinebra, 2011; Moseke  et al ., 2011; Tay   et al ., 2007). Anadditional justi fi cation for the use of magnesiumphosphate-based ceramics is their faster resorption  in vivo compared to calciumphosphate-based ceramics(Klammert et al ., 2011). Mg-substituted hydroxyapatite coatings ontitanium implants have promoted proliferation anddifferentiation of osteoblast-like cells and improved early osseointegration  in vivo  (Zhao  et al ., 2013).Inapreviousstudy,enzymaticmineralizationofGGwithCaP by incubation in a mineralization solution containingcalcium glycerophosphate (CaGP), which served as asource of calcium and organic phosphate, led to enhancedmechanical properties and adhesion and proliferation of osteoblastic cells (Douglas  et al ., 2012a). In this study, thisstrategy was extended to induce enzymatic mineralizationof GG with mineral consisting of CaP and MgP phases, by incubation in solutions of CaGP and magnesiumglycerophosphate (MgGP). The CaGP:MgGP concentrationratiointhemineralizationwasvariedinordertoinvestigateits in fl uence on the nature of mineral formed. Resultingmineralized hydrogels were characterized physicochemi-cally, using infrared (IR) and Raman spectroscopy, X-ray diffraction (XRD), selected area electron diffraction(SAED),transmissionelectronmicroscopy(TEM),scanningelectron microscopy (SEM), ion-coupled plasma opticalemission spectroscopy (ICP-OES), thermogravitationalanalysis (TGA) and increases in the dry mass percentage,i.e. the mass fraction of the hydrogel not consisting of wa-ter. Mechanical properties were evaluated by compressivetesting and rheometry. With a view to evaluating the suit-abilityofmineralizedhydrogelsasscaffoldsforapplicationsin bone TE, their degradation, cytocompatibility and ability to support adhesion and proliferation of osteoblasts andformation of osteoclast-like cells were also evaluated.The present study aimed to  fi ll the following gaps in thescienti fi c literature: (a) the development of composites of hydrogels and mineral containing Mg by enzymatic means;(b) the in fl uence of mineralization medium, i.e. differentCaGP:MgGP concentration ratios, on the incorporation of Mg and Ca into mineral formed; and (c) the effect of  varying Mg and Ca content on composite mechanicalproperties, osteoblast behaviour and osteoclast formation, which is the  fi rst step towards creeping resorption. 2. Materials and methods 2.1. Materials Unless stated otherwise, all materials, including GG(Gelzan ™ CM, G1910), ALP (P7640), CaGP (50043)and MgGP (17766) were obtained from Sigma-Aldrich.The GG preparation used was classi fi ed as  ’ low-acyl ’  by the manufacturer. 2.2. Production and characterization of GGhydrogels containing ALP  ALP was incorporated into GG hydrogels by a modi fi ca-tion of the method of Oliveira  et al . (2010). Brie fl  y, an T. E. L. Douglas  et al. Copyright © 2014 John Wiley & Sons, Ltd.  J Tissue Eng Regen Med  (2014)DOI: 10.1002/term  aqueous GG solution was heated to 90°C and mixed withan aqueous CaCl 2  solution to achieve  fi nal GG and CaCl 2  w/v concentrations of 0.7% and 0.03%, respectively.The resulting solution was mixed and allowed to cool to50°C. At 50°C, ALP solution was added to achieve a  fi nal ALP concentration of 2.5mg/ml. This temperature waschosen in order to avoid the excessive deactivation of  ALP associated with higher temperatures, while permit-ting good mixing and preventing premature gelation dur-ing casting due to cooling. ALP in milk has been reportedto retain more than 80% of its activity after heating for10min at 50°C (Fadilogwlu  et al ., 2004). In anotherstudy, ALP in milk showed no loss of activity after30min at 50°C (Lombardi  et al ., 2000), while ALP in se-rum heated for 30min was not inactivated at all at 45°Cand only by 28% at 50°C (Neale  et al ., 1965). Hydrogelcylinders of volume 250 μ l, diameter 8mm and thickness5mm were prepared by casting 40ml GG – CaCl 2  solutionat 50°C in glass Petri dishes of diameter 10cm at roomtemperature and cutting out cylinders with a hole punch.Casting took place immediately after ALP addition to min-imize ALPactivity loss. Finally, the GG – CaCl 2  solution wasallowed to cool at room temperature for 20min to gelify. 2.3. Mineralization of gels Gel mineralization was induced at room temperature by incubation in mineralization medium containing differentconcentrations of CaGP and MgGP. Five different CaGP:MgGP concentration ratios were compared: 0.1:0,0.075:0.025, 0.05:0.05, 0.025:0.075 and 0:0.1 (all valuesmol/dm 3 ). These mineralization media were denoted as A, B,C,DandE,respectively(see Table 1).Themineraliza-tion medium was changed every day. After conclusion of mineralizationafter7days,thegelswererinsedthreetimesin Milli-Q water and subsequently incubated in Milli-Q water for 1 day with the aim of removing residual CaGP. 2.4. Calculation of mass change due tomineralization by measurement of dry masspercentage The dry mass percentage, i.e. the hydrogel weightpercentage not consisting of water, was calculated as:(weight after incubation and subsequent freeze-drying/ weight after incubation but before freeze-drying)×100.This served as a measure of the extent of mineralformation. Freeze-drying was performed for 24h. Experi-ments were performed in triplicate. 2.5. Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) was performed usinga Hi-Res TGA 2950 Thermogravimetric Analyser (TA In-struments). Lyophilized samples ( n= 3) were heatedfrom 30ºC to 800ºC in a helium atmosphere at a rateof 10ºC/min under constant monitoring of remaining weight percentage, to determine the mass percentage of lyophilized samples attributable to mineral. 2.6. Physicochemical and morphological characterization  After mineralization experiments and subsequent lyophili-zation, the molecular structure of the hydrogels wasexamined using IR and Raman spectroscopy, XRD, TEM,SAED, EDS, ICP-OES and SEM. Samples were lyophilizedfor 48h prior to analysis.IR spectra of the powdered samples dispersed in KBrtablets were recorded using a Galaxy 6030 Fourier trans-form IR spectrophotometer (Mattson, Madison, WI, USA).Raman spectra were recorded using a Kaiser OpticalSystems Rxn1-532 device equipped with a 532nm(Nd:YAG) laser source. Spectra were recorded after 10accumulations of 5s each. X-ray diffraction (XRD) analysis was performed usingan X  ’ Pert Pro diffractometer (Panalytical) with the X  ’ Celerator strip detector, Cu K  α 1  radiation (50kV, 30mA)and the standard Bragg – Brentano geometry. The diffrac-tion patterns were collected in the 2  θ   angle range of 10 – 90º with formal step of 0.001º; the total time of eachmeasurement was 8h. The diffractograms were analysed with the help of ICDD cards 01-084-1147 and 01-089-6440 for bobierrite and hydroxyapatite, respectively.Transmission electron microscopy (TEM) analysis wasperformed using a JEM-2200FS FEG (Jeol) instrumentoperated at 200kV. Conventional TEM bright fi eld(TEM BF), selected area electron diffraction (SAED) andscanning transmission (STEM) modes were used in this work. An in-column omega  fi lter was used to diminishchromatic aberration caused by inelastic scattering of primary electrons in thick areas of the sample. EDSspectrometry was used in combination with STEM modeto measure chemical compositions and produce elementalmappings of the material agglomerates. Lyophilizedhydrogel samples were prepared by chopping them up with a razor blade to obtain powders. A lacey carbonsupport  fi lm on a Cu grid was then repeatedly dipped intothis powder.SEM analysis was performed using a Jeol JSM-5600instrument. The instrument was used in the secondary electron mode (SEI). The SEM instrument was equipped with an electron microprobe JED 2300. Prior to analysis, Table 1. Mineralization medium concentrations used in study  MineralizationmediumConcentrationCaGP (mol/dm 3 ) MgGP (mol/dm 3 )A 0.1 0B 0.075 0.025C 0.05 0.05D 0.025 0.075E 0 0.1 Gellan gum and calcium and magnesium phosphate composites Copyright © 2014 John Wiley & Sons, Ltd.  J Tissue Eng Regen Med  (2014)DOI: 10.1002/term  lyophilized samples were coated with a thin layer of gold(ca. 20nm), using a plasma magnetron sputter coater.The concentrations of Ca, Mg and P were determinedby inductively coupled plasma optical emission spectros-copy (ICP-OES), using a Spectro Arcos Optical EmissionSpectrometer (Spectro, Germany). Before analysis,lyophilized hydrogel samples were dissolved in 1ml14 M  HNO 3  and further diluted (200×) with 0.3 M HNO 3  (HNO 3  of analytical grade; ChemLab, Belgium).The instrument was calibrated by means of six standardsolutions, with Ca, Mg and P concentrations in the range0 – 15mg/l. Yttrium was added to all solutions as an inter-nal standard in order to correct for possible instrumentinstabilities and matrix effects. Measurements wereperformed in triplicate. 2.7. Mechanical testing  Hydrogel samples were subjected to compressive testingusing a Houns fi eld Universal Testing Machine H10KM.Samples were placed between the piston heads at aprede fi ned distance. Displacement was applied at a rateof 4mm/min until the samples were compressed to 50%of their srcinal height, up to a maximum load of 80N.During displacement, force was recorded with a 100Nload cell every 0.5s, using Qmat software. Compressivestress was calculated as the force recorded divided by the cross-sectional area. Compressive strain was calcu-lated as the distance moved during compression dividedby the initial distance between piston heads. Finally, Young ’ s modulus was calculated as the gradient of thestress – strain curve.In addition, the viscoelastic properties of all types of gels were determined using a rheometer (Anton PaarPhysica, MCR 301) equipped with a PP25 rotating headof diameter 25mm. Storage modulus ( G ′ ) and lossmodulus ( G ′′ ) values were recorded at a strain of 0.01%and an angular frequency of 10rad/s. For all hydrogelsevaluated,  n= 5. 2.8. Degradation testing  Degradation of hydrogel samples was evaluated using anextreme solution test and a simulation solution test,according to ISO 10993-14 norm  ’ Identi fi cation and quan-ti fi cation of degradation products from ceramics ’ . Prior totesting, the hydrogel samples were sterilized by autoclav-ing for 20min in 50ml Milli-Q water at 115ºC and 1.2bar.For the extreme solution test, a citric acid buffer solu-tion, pH3, at 37ºC was prepared by dissolution of 21gcitric acid monohydrate in 500ml Milli-Q water in a1000ml volumetric  fl ask, addition of 200ml 1 M  NaOH so-lution and dilution to the mark with Milli-Q water;40.4ml of this solution was mixed with 59.6ml 0.1 M HCl to yield the desired buffered citric acid solution.Hydrogel samples were weighed, placed in glass vesselsand 2ml citric acid solution was added/0.1g sample. After 120h incubation at 37°C, the samples were re-moved from solution and reweighed. The experiments were repeated  fi  ve times/sample group. Samplescontaining 2.5mg ALP/ml hydrogel mineralized in media A  – E were compared. Samples containing 0mg ALP/mlhydrogel mineralized in medium A served as controls.Forthesimulationsolutiontest,aTris – HClbuffer,pH7.4, was prepared by dissolving 13.25g tris(hydroxymethyl)aminomethane in 500ml Milli-Q water and adjusting thepH with an appropriate amount of 1 M  HCl to pH7.4 at atemperature of 37°C. Milli-Q water was added until a  fi nal volume of 1000ml was reached.The hydrogel samples were weighed, placed incontainers and 15.6ml Tris – HCl buffer was added. After120h of incubation at 37°C, the samples were removedfrom the buffer and reweighed. The buffer was retainedand 1ml was mixed with 1ml 14 M  HNO 3  for ICP-OESanalysis, as described in Section 2.6. The experiments were repeated  fi  ve times/sample group. Samplescontaining 2.5mg ALP/ml hydrogel mineralized in media A  – E were compared. 2.9. Cell biological characterization 2.9.1. Sterilization Prior to cell experiments, the samples were sterilized asdescribed in Section 2.8. 2.9.2. Cytocompatibility testing Cytocompatibility was evaluated by determining the via-bility of human  fi broblastic cells HFF-1 (human foreskin fi broblasts; ATCC) after culture in eluate from hydrogelsamples. HFF cells were cultured in Dulbecco ’ s modi fi edEagle ’ s medium (DMEM; Glutamax ™ ) supplemented with15% fetal bovine serum (FBS), 0.1% sodium pyruvate and0.1% penicillin/streptomycin (all Gibco, Invitrogen).The colorimetric MTT assay, using a 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT; Merck Promega) was performed to quantify cell via-bility. The tetrazolium component is reduced in living cellsby mitochondrial dehydrogenase enzymes to a water-insolu-blepurpleformazanproduct,whichcanbesolubilizedbyad-ditionof lysisbufferandmeasuredusingspectrophotometry.Eluate was produced by incubating four hydrogelsamples in 2ml cell culture medium (corresponding to asurface:volume ratio of 5.52cm 2  /ml) for 48h. The eluate was diluted by factors of 1 (undiluted), 2, 4, 6, 8, 16, 32and 64. HFF cells (10 000/well of a 96-well plate) weresubsequently incubated in eluate at the aforementioneddilutions for 72h. Afterwards, the eluate was replacedby 0.2ml (0.5mg/ml) MTT reagent and the cells wereincubated for 4h at 37°C. The MTT reagent was removedand replaced by 0.2ml lysis buffer (1% Triton X-100 inisopropanol/0.04  N  HCl) for 30min. The dissolvedformazan solution was measured spectrophotometri-cally at 580nm (Universal Microplate Reader EL 800, T. E. L. Douglas  et al. Copyright © 2014 John Wiley & Sons, Ltd.  J Tissue Eng Regen Med  (2014)DOI: 10.1002/term  Biotek Instruments). Triplicate measurements wereperformed. The viability was calculated as a percentageof control cultures. 2.9.3. Osteoblast seeding on GG hydrogels Cells of the osteoblastic cell line MC3T3-E1 (ATCC) wereseeded onto hydrogel samples. Each hydrogel sample wasplaced in a well of a 24-well plate and a suspension of 100 000 cells in 1ml cell culture medium [ α -minimalessential medium ( α -MEM) supplemented with 10% FBSand 0.5% penicillin – streptomycin; all Gibco, Invitrogen] was added. After incubation for 4h to allow attachment,1ml supplementary cell culture medium was added. 2.9.4. Osteoblast adhesion To visualize cell attachment and distribution on thehydrogels, the cell cultures were evaluated using  fl uores-cence microscopy, which was performed as follows. A live/dead staining (Calcein AM/propidium iodide) wasperformed to evaluate cell viability. The cells were rinsedand the supernatant was replaced by 1ml phosphate-buff-ered saline (PBS) solution supplemented with 2 μ l (1mg/ml) calcein AM (Anaspec, USA) and 2 μ l (1mg/ml)propidium iodide. The cultures were incubated for 10minat room temperature, washed twice with PBS solution andevaluated by   fl uorescence microscopy (Type U-RFL-T,Olympus,Aartselaar, Belgium).Evaluationswere performed1 day and 6 days post-seeding. 2.9.5. Osteoblast proliferation Cell vitality was investigated using an MTT assay; 2mlMTT reagent was added directly to the hydrogel samplesin 24-well plates and incubated for 4h at 37°C.Subsequently, the samples were transferred to 48-wellplates, 0.5ml lysis buffer was added and incubated for30min at 37°C on a gyratory shaker (70rpm); 0.2ml of the resulting solution was used for the spectrophotomet-ric measurement. Evaluations were performed 1 and 11days post-seeding. The viability was expressed as apercentage of control cultures after 11 days. 2.9.6. Culture of osteoclast precursor cells (RAW 264.7 monocytic cells) Monocytic cells from the RAW 264.7 cell line were cul-tured on samples as described previously (Detsch  et al .,2010, 2011; Schaefer  et al ., 2011). Brie fl  y, 600 μ l of asuspension of 100 000 cells/ml were incubated in RPMImedium on the samples for 14 days and stimulated withmacrophage colony-stimulating factor (M-CSF, 25ng/ml) and receptor activator of NF- κ  B ligand (RANKL;40ng/ml). These factors induce the differentiation of the monocytes into osteoclast-like cells (Wei  et al ., 2001;Jung  et al ., 2002). The formation of actin ring structuresand the multinuclearity of osteoclast-like cells were veri fi ed by immuno fl uorescence microscopy (Axioplan 2Imaging, Zeiss, Germany). The RAW 264.7 cells were fi xed on the samples and stained with phalloidin and4 ′ ,6-diamidino-2-phenylindole (DAPI). 2.10. Statistical analysis Statistical analysis was performed using SPSS statisticssoftware (IBM Corp., USA). The results of dry masspercentage, TGA, compressive testing and rheometricalmeasurements were analysed using a one-way analysisof variance (ANOVA) combined with Tukey  ’ s  post hoc  test.  p < 0.05 was considered signi fi cant. 3. Results 3.1. Physicochemical characterization of mineralized hydrogels Formation of mineral was investigated by IR and Ramanspectroscopy, XRD, SAED, TEM, EDS, ICP-OES and SEM.The IR spectrum of GG without the presence ofALPandmineralized for 7 days in mineralization medium A wasrepresentative for the IR spectrum of GG without ALPand mineralized for 7 days in mineralization medium E(Figure 1). The absorption bands in these IR spectra werecomparable to the absorptions present in the IR spectrum Figure 1. Infrared spectra of gellan gum (GG) hydrogels without(A, control) and with ALP after mineralization for 7 days in me-dia A  – E. The spectra obtained after mineralization in media A and B were indistinguishable; for comparison, the IR spectra of pure GG and CDHA are shown. Dotted lines mark the OH – andHPO 42 – absorptions; transmittance is expressed in arbitrary units Gellan gum and calcium and magnesium phosphate composites Copyright © 2014 John Wiley & Sons, Ltd.  J Tissue Eng Regen Med  (2014)DOI: 10.1002/term
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