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Bioactive-glass ceramic with two crystalline phases (BioS-2P) for bone tissue engineering

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2017 Biomed. Mater. 12 045018

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Biomed.Mater. 12 (2017) 045018 https://doi.org/10.1088/1748-605X/aa768e

PAPER

Bioactive-glass ceramic with two crystalline phases (BioS-2P) forbone tissue engineering

Emanuela Prado Ferraz1, Gileade Pereira Freitas1,MuriloCamuri Crovace2, Oscar Peitl2,EdgarDutra Zanotto2, Paulo Tambasco deOliveira1,MarcioMateus Beloti1 andAdalberto Luiz Rosa1

1 Cell Culture Laboratory, School ofDentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil2 VitreousMaterials Laboratory, Department ofMaterials Engineering, Federal University of SãoCarlos, SãoCarlos, SP, Brazil

E-mail: adalrosa@forp.usp.br

Keywords: bioactive glass-ceramic, Biosilicate®, bone,mesenchymal stem cell, osteoblast, scaffold

AbstractWeaimed to evaluate the in vitro osteogenic and osteoinductive potentials of BioS-2P and its ability topromote in vivo bone repair. To investigate osteogenic potential, UMR-106 osteoblastic cells werecultured onBioS-2P andBioglass 45S5 discs in osteogenicmedium. The osteoinductive potential wasevaluated usingmesenchymal stem cells (MSCs) cultured onBioS-2P, Bioglass 45S5 and polystyrenein non-osteogenicmedium. Rat bone calvarial defects were implantedwith BioS-2P scaffolds alone orseededwithMSCs. UMR-106 proliferationwas similar for bothmaterials, while alkaline phosphatase(ALP) activity andmineralizationwere higher for BioS-2P. Bone sialoprotein (BSP), RUNX2 andosteopontin (OPN) gene expression andBSP,OPN, ALP andRUNX2protein expressionwere higheronBioS-2P. ForMSCs, ALP activity was higher on Bioglass 45S5 than onBioS-2P andwas lower onpolystyrene. All genes were highly expressed on bioactive glasses compared to polystyrene. BioS-2Pscaffolds promoted in vivo bone formationwithout differences in themorphometric parameters at 4,8 and 12weeks. After 8weeks, the combination of BioS-2PwithMSCs did not increase the quantity ofnew bone compared to the BioS-2P alone. To stimulate osteoblast activity, driveMSCdifferentiationand promote bone formation, BioS-2P is a good choice as a scaffold for bone tissue engineering.

1. Introduction

Over the past decade, tissue engineering based oncombinations of cells and biomaterials has beeninvestigated as a feasible strategy for bone regenera-tion. One of the frontiers for this cell-based therapy isthe development of scaffolds that act as templates toguide bone growth. The ideal scaffold should havefavourable biological and mechanical properties witha suitable surface to stimulate cell adhesion, prolifera-tion and differentiation, biodegradability and ade-quatemechanical strength [1–4].

Since 1969, when Hench discovered the melt-derived bioactive glass, which is called Bioglass 45S5,many investigations have been performed aimed at itsdifferent biological applications [5, 6]. A major stepdevelopment in this field was the demonstration thatbioactive glasses can bond to living bone [7]. Morerecently, the term ‘osteostimulation’ was coined torefer to the mechanisms triggered by bioactive glassesin the body to repair bone [5, 8]. Despite the

outstanding biological properties of Bioglass 45S5,which is available in several forms including porousblocks, the generation of scaffolds with improvedmechanical strength without affecting biocompat-ibility has been a key challenge in the scientific com-munity [9–13].

In this scenario, the development of new glass for-mulations to overcome this mechanical limitationwould be very welcome. A glass-ceramic with thecomposition 23.75Na2O–23.75CaO–48.5SiO2–4P2O5

(wt%) named Biosilicate® was created to combinebioactivity properties with mechanical strength [14].Under controlled heat treatment, Biosilicate® can beproducedwith one (BioS-1P) or two (BioS-2P) crystal-line phases that have an associated increase in the elas-tic modulus and fracture strength [15, 16]. BioS-1Pincreases in vitro extracellularmatrixmineralization inan osteogenic cell culture system and preserves thedental alveolar ridge height, which allowed for tita-nium implant osseointegration in a pre-clinical ani-mal model [17, 18]. The BioS-2P can be produced as

RECEIVED

13March 2017

REVISED

17May 2017

ACCEPTED FOR PUBLICATION

2 June 2017

PUBLISHED

9August 2017

© 2017 IOPPublishing Ltd

scaffolds and exhibits a similar response to BioS-1PandBioglass 45S5 in terms of contact with bone [19].

Several biological and mechanical features makeBiosilicate® an appealing candidate to fabricate scaf-folds for bone regeneration. However, the effects ofthis secondary crystalline phase are underexploredand need to be investigated. Thus, the aim of this studywas to evaluate both the in vitro osteogenic andosteoinductive potentials of BioS-2P and their effectson in vivo bone repair. Furthermore, we evaluated ifBioS-2P scaffold combined with mesenchymal stemcells (MSCs) could improve bone formation.

2.Materials andmethods

2.1. Sample preparation and characterizationHigh-purity silica (Zetasil 3, Santa Rosa Mineração,Pequeri, MG, Brazil) and reagent grade calciumcarbonate (JT Baker, Center Valley, PA, USA), sodiumcarbonate (JT Baker), and monosodium phosphate(JT Baker) were used to produce Biosilicate® parentglass and Bioglass 45S5. The chemicals were blendedovernight and then they were melted in a platinumcrucible at 1450 °C for 4 h, splat-cooled and re-meltedthree times (every 1 h). Finally, themelts were cast intoa cylindrical stainless steel mould 50 mm in height by12 mm in diameter. Biosilicate® parent glass was fullycrystallized using a double stage heat-treatment firstfor crystal nucleation (565 °C/100 h) and then forcrystal growth (665 °C/1 h). To obtain Biosilicate®

containing two crystalline phases (BioS-2P), an addi-tional heat-treatment was performed at 800 °C/1 h.The cylinders of BioS-2P glass-ceramic and Bioglass45S5 were cut into 3 mm thick discs. The discs wereground using silicon carbide paper to a grit of 400(∼35 μm) and cleaned with isopropyl alcohol (99.8%)in an ultrasonic cleaner for 1 min. The scaffolds wereobtained from BioS-2P as previously described [20].Briefly, a suspension containing BioS-2P powdermixed with carbon black was dried, pressed andsintered (975 °C/5 h) to produce porous scaffoldswith5 mm in diameter and 2 mm in height. Prior to cellculture experiments and surgical procedures, allsamples were submitted to sterilization using dry heatat 180 °C for 2 h.

2.1.1. X-ray diffraction (XRD) and opticalmicroscopyThe ground solid discs (grit 400) of BioS-2P wereanalysed via XRD (Ultima IV, Rigaku, Japan). A stepscan mode (0.01° s−1), i.e., a count time of 4 s, from10° to 90° was used. The crystalline phases present inBioS-2P were identified using CrystallographicaSearch-Match software (version 2.1.1.1, Oxford CryoSystems,MurrayHill,NJ,USA). After surface-polishing,BioS-2Pdiscswere analysedusing an opticalmicroscope(Eclipse LV100NPOL, Nikon, Japan) attached to adigital camera (DSFi2, Nikon). On the surface, images

were captured using reflected light. From 1.5 to 8.5 μmbelow the surface, images were automatically capturedevery 0.1 μm using polarized light. The images weremerged, and a focused 3D image was created using theimaging softwareNISElements, version 4.20 (Nikon).

2.1.2.Microtomography andmorphometric analysisBioS-2P scaffolds were evaluated by micro-CT usingthe SkyScan 1172 system (Bruker, Sky-Scan, Belgium).The images were generated at 60 kV and 200 mAusingisotropic voxel 5.88 μm and the reconstructions weremade usingNRecon software (1.6.10.4 version, Bruker)with smoothing set at 1, ring artefact correction set at 5and beam hardening correction set at 20%. Threesampleswere scanned to evaluate thepercentage of totalporosity andpore size distribution.

2.2.Osteogenic potential of BioS-2P2.2.1. Culture ofUMR-106 osteoblastic cell lineThe rat osteoblastic cell line UMR-106 (AmericanType Culture Collection, Manassas, VA, USA) wascultivated in growth medium (non-osteogenic condi-tions) constituted by alpha-minimum essential med-ium (α-MEM, Gibco-Life Technologies, GrandIsland, NY, USA) supplemented with 10% foetalbovine serum (Gibco-Life Technologies), 50 μg ml−1

penicillin/streptomycin (Gibco-Life Technologies)and 0.3 μg ml−1 fungizone (Gibco-Life Technologies).After subconfluence, cells from first passage werecultured in 24-well culture plates on BioS-2P andBioglass 45S5 discs at a cell density of 2 ×104 cells/well in osteogenic medium. This mediumwas composed by growthmedium supplemented with5 μg ml−1 ascorbic acid (Gibco-Life Technologies)and 7 mM β-glycerophosphate (Sigma-Aldrich, SaintLouis, MO, USA). The cultures were kept at 37 °C in ahumidified atmosphere of 5% CO2 and 95% air, themedium was replaced every 48 h, and all assays weredone on day 5.

2.2.2. Cell culture growthCell culture growth was assessed with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide(MTT, Sigma-Aldrich) assay. The MTT absorbance wasmeasured as optical density at 570 nm using a μQuantplate reader (Biotek,Winooski, VT,USA). The data werecollected inquintuplicate (n= 5).

2.2.3. In situ ALP activityIn situ ALP activity was analysed with a Fast Red assay.The cultures were washed in Hank’s balanced saltsolution (Sigma-Aldrich) and incubated for 30 min at37 °C with 0.5 ml of 120 mM Tris buffer (Sigma-Aldrich), pH 8.4, containing 0.9 mMnaphthol AS-MXphosphate (Sigma-Aldrich) and 1.8 mM Fast red TR(Sigma-Aldrich). The cells were washed and dried atroom temperature. The images were obtained using ahigh-resolution camera (Canon EOS Digital Rebel,

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Canon, Tokyo, Japan), processed and then quantifiedusing Image J software [21]. The data were collected inquintuplicate (n= 5).

2.2.4. Gene expression of osteoblast markersGene expression of ALP, BSP, runt-related transcrip-tion factor 2 (RUNX2) and OPN was evaluated withreal-time PCR. Total RNA (1 μg) was used to synthe-size complementary DNA in a reverse transcriptionreaction (M–MLV reverse transcriptase, PromegaCorporation, Madison, WI, USA). Real-time PCR wasdone using a CFX96 Real-Time PCRDetection System(Bio-Rad Laboratories, Philadelphia, PA, USA). Rela-tive gene expression, which was collected in quadru-plicate (n = 4), was normalized to the constitutivegene GAPDH, and the real change was relative to thegene expression of cells cultivated onBioglass 45S5.

2.2.5. Detection of bone-related proteinsThe proteins BSP andOPNwere detected by immuno-fluorescence labelling. The cells were fixed, permeabi-lized and blocked with 5% non-fat powdered milk(Bio-Rad Laboratories). Then, the cells were incubated(1 h each incubation) with primary monoclonal anti-bodies anti-BSP (1:200, WVID1-9C5, DevelopmentalStudies Hybridoma Bank—DSHB, Iowa City, IA,USA) and anti-OPN (1:200, MPIIIB10, DSHB). Afterthat, cells were incubated with a mixture of the AlexaFluor 594 (red fluorescence)-conjugated goat anti-mouse secondary antibody (1:200, Molecular Probes,Eugene, OR, USA) and Alexa Fluor 488 (greenfluorescence)-conjugated phalloidin (1:200, Molecu-lar Probes) to detect the actin cytoskeleton. Cell nucleiwere stained with 300 nM4′,6-diamidino-2-phenylin-dole, dihydrochloride (DAPI, Molecular Probes). Thediscs were observed using a fluorescence microscopeAxio Imager M2 (Carl Zeiss), and the captured imageswere treated usingAdobe Photoshop software.

The proteins ALP andRUNX2were detected usingWestern blotting. A sample of total protein (70 μg)was submitted to electrophoresis in a denaturing 8.5%polyacrylamide gel. Then, proteins were transferred toa Hybond C-Extra membrane (GE Healthcare LifeScience, Piscataway, NJ, USA) using a semidry transfersystem (Bio-Rad Laboratories). ALP protein wasdetected after incubation of the membrane withmouse monoclonal anti-ALP antibody (1:2000, SantaCruz Biotechnology, Santa Cruz, CA, USA), followedby a goat anti-mouse IgG HRP secondary antibody(1:2000, Santa Cruz Biotechnology). For RUNX2detection, the incubation was made with rabbit mono-clonal anti-RUNX2 (1:2500, Cell Signaling Technology,Danvers, MA, USA) followed by a goat anti-rabbit IgGsecondary antibody (1:2500, Santa Cruz Biotechnology).Rabbit polyclonal anti-GAPDH (1:1000, Santa Cruz Bio-technology) followed by a goat anti-rabbit IgG HRPsecondary antibody (1:3000, Santa Cruz Biotechnology)was used as a control. For detection of secondary anti-bodies, the western lightning chemiluminescence reagent

(Perkin Elmer Life Sciences, Waltham, MA, USA)was used and the images were obtained using G-Box gelimaging (Syngene, Cambridge, United Kingdom). Thequantification of ALP and RUNX2 expressions were per-formedby counting the number of pixels andnormalizedto GAPDH expression. The fold change was relative totheprotein expressionof cells cultivatedonBioglass 45S5.

2.2.6. ExtracellularmatrixmineralizationTheVonKossa staining was used to detectmineralizedmatrix. The cultures were incubated with silver nitrate(5 mgml−1) (Sigma-Aldrich), followed by hydroqui-none (5 mgml−1) (Sigma-Aldrich) and sodium thio-sulphate (50 mgml−1) (Sigma-Aldrich). After washingand drying at room temperature, the images wereobtained using a camera (Canon EOS Digital Rebel),processed and quantified using Image J software [21].The datawere collected in quintuplicate (n= 5).

2.3.Osteoinductive potential of BioS-2P2.3.1. Culture ofMSCsThe Committee of Ethics in Animal Research of theSchool of Dentistry of Ribeirão Preto, University ofSão Paulo, Brazil, approved all of the experimentalprocedures performed with animals. Bone marrowMSCs harvested from the femur of two male Wistarrats weighing 150 g were cultivated in growthmedium(non-osteogenic conditions) until subconfluence.MSCs offirst passagewere cultivated in 24-well cultureplates on BioS-2P and Bioglass 45S5 discs andon polystyrene (control) at a cell density of 2 ×104 cells/well in growth medium. The cultures werekept at 37 °C in a humidified atmosphere of 5% CO2

and 95% air. The medium was changed every 48 h,and all assayswere done on day 10.

2.3.2. ALP activityA commercial kit (Labtest Diagnostica SA, LagoaSanta, MG, Brazil) was used to measure ALP activityby the release of thymolphthalein from thymolphtha-lein monophosphate. Absorbance was read at 590 nmusing aμQuant plate reader (BioTek), andALP activitywas determined from a thymolphthalein standardcurve. The data were collected in quintuplicate (n= 5)and are presented as ALP activity normalized to thetotal protein content, whichwas quantified in the samecell lysates using the Lowrymethod [22].

2.3.3. Gene expression of osteoblast markersGene expression of ALP, RUNX2, OPN and osteocal-cin (OC) was evaluated in quadruplicate (n = 4) withreal-time PCR, as described above (2.2.4). The cellscultured on polystyrene were used to calculate the realchanges of gene expression.

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2.4. Effects of BioS-2P scaffolds on bone repair2.4.1. Surgical procedures for BioS-2P scaffold implantationFifteenmaleWistar rats weighing 200–300 gwere usedfor the following research protocols, which wereapproved by the Committee of Ethics in Researchfrom the University of São Paulo. The animals wereanesthetized with ketamine (7 mg/100 g body weight;Agener União, SP, Brazil) and xylazine (0.6 mg/100 gbody weight; Calier, MG, Brazil). Calvarial bonedefects that were 5 mm in diameter were unilaterallycreated with a trephine and implanted with BioS-2Pscaffolds followed by a skin suture with nylon 4.0(Ethicon Ltd, NJ, USA). Then, single doses of anti-biotics and analgesics were administered. Four, 8 and12 weeks post-implantation, the animals (n = 5 perperiod) were euthanized, and the samples wereharvested and prepared for morphometric and histo-logical analyses.

2.4.2.Morphometric and histological analysesAfter harvesting, the samples were maintained in 10%formalin buffered with 0.1 M sodium cacodylate,pH 7.0. Morphometric analysis was performed withmicro-CT using the SkyScan 1172 system (Bruker).The images and reconstructions were obtained asdescribed above (2.1.2). Bone volume (BV), bonesurface (BS), BS/BV ratio (BS/BV) BS/volume ratio(BV/VT), trabecular thickness (Tb.Th) and trabecularseparation (Tb.Sp) of the newly formed bone werequantified using the 3D Ctan software 1.13.1.1.0version (Bruker).

Histological analysis was carried out following apreviously described protocol [23]. After micro-CTanalysis, samples were kept in a solution of 70% etha-nol for 72 h, followed by embedding in resin (LRWhite Hard Grade, London, UK) and sectioned usingthe Exakt Cutting System (Exakt, Norderstedt, Ger-many). The sections were polished, mounted onacrylic slides using the Exakt Grinding System (Exakt)and stained with Stevenel’s blue and Alizarin red S.The tissues formed in the bone defects were histologi-cally described using micrographs obtained with aLeica DMLB light microscope (Leica, Bensheim,Germany).

2.5. Effects of the association of BioS-2PwithMSCsonbone repair2.5.1. Cell incorporation in BioS-2P scaffoldsFirst-passage MSCs were obtained as described above(2.3.1), seeded by pipetting (2× 105 cells/100 μl) intoBioS-2P scaffolds, which were placed into 48-well andcultured for 18 h to allow cell adhesion. Cell incor-poration was detected under epifluorescence using anApoTome system and an Axiocam MRm digitalcamera (Zeiss). The efficiency of cell incorporationwas evaluated by counting the non-incorporated cellsthat were enzymatically released from the 48-wellsafter BioS-2P scaffold removal. During the culture

period, scaffolds were incubated at 37 °C in a humidi-fied atmosphere of 5% CO2 and 95% air for 24 h.BioS-2P scaffolds without cells were used as controls.

2.5.2. Cell trackingMSCs were transduced using a lentiviral vector withluciferase 3–5 d after passage at a multiplicity ofinfection of 8. After 2 d, themediumwas changed, andthe cells were selected using puromycin during 6 d.These cells were seeded into BioS-2P scaffolds andsurgically implanted into bone defects by following thesame protocols described above (2.5.1 and 2.4.1,respectively). For cell tracking, luciferase-positive cellswere detected from6 h to 28 d after implantation usingthe IVIS imaging system (Xenogem Corporation, CA,USA) after 100 μl of a luciferin (mgml−1) local injection.Signal intensity was quantified as the photon flux(photons/s) within the region of interest by using IVISImage 3.0 software (Caliper Life Sciences).

2.5.3. Surgical procedures for BioS-2P scaffolds withMSCs implantationFivemaleWistar rats weighing 200–300 g were used asdescribed above (2.4.1). BioS-2P scaffolds seeded withunlabelled MSCs were implanted in rat calvarialdefects, and 8 weeks post-implantation, the animalswere euthanized, and the samples were collected andprocessed for histomorphometric and histologicalanalyses. These data were compared to data from ratsimplanted with BioS-2P scaffolds without cells for 8weeks.

2.6. Statistical analysisQuantitative data were collected in quintuplicate(n = 5) for all parameters except gene expression,which were collected in quadruplicate (n = 4). Thecomparisons between two groups (BioS-2P versusBioglass 45S5 and BioS-2P scaffold with MSCs versusBioS-2P without MSCs) were done using the Mann–Whitney test, whereas the Kruskal–Wallis test fol-lowed by the Student–Newman–Keuls test was used tocompare three groups (BioS-2P, Bioglass 45S5 andpolystyrene; 4, 8 and 12 weeks). The significance levelwas set at 5%.

3. Results

3.1. Characterization of BioS-2PThe nominal compositions of Na2O, CaO, SiO2 andP2O5 (mol %) of BioS-2P and Bioglass 45S5 aredetailed in table 1. The XRD spectra showed that the

Table 1.Nominal compositions of BioS-2P andBioglass 45S5.

Composition (mol%) Na2O CaO SiO2 P2O5

BioS-2P 23.3 25.8 49.2 1.7

Bioglass 45S5 24.4 26.9 46.1 2.6

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main crystalline phase in BioS-2P was a sodiumcalcium silicate (Na2CaSi2O6–ICSD 060502) followedby a secondary crystalline phase (NaCaPO4–ICSD035629) that presented three main diffraction peaks at23.3°, 32.7° and 47.5°, (figure 1(A)). The lower peakintensity of the secondary crystalline phase indicatedthat there was a lower crystallized volume fraction.Optical micrographs showed grains of Na2CaSi2O6 atthe surface of the BioS-2P (figure 1(B)) but no crystalsof the secondary crystalline phase. However, at asubmicron scale (approximately 0.2–0.4 μm) andbelow the surface, it was possible to observe thesecondary crystalline phase due to its distinct opticalbehaviour. The bright spots that consisted ofNaCaPO4 were uniformly distributed in a matrixcomposed ofNa2CaSi2O6 crystals (figure 1(C)).

Themicro-CT3Dand2D images of theBioS-2P scaf-folds showed a porous structure resembling a cancellous

bone (figures 1(D)–(G)). The BioS-2P scaffold presented76% ± 5% of porosity and pores with sizes rangingbetween 10 and 800μm, whereas the majority of openporeswerewithin100 and600μm (figure 1(H)).

3.2.Osteogenic potential of BioS-2PTo determine osteogenic potential, UMR-106 osteo-blastic cells were cultured on BioS-2P and Bioglass45S5 discs under osteogenic conditions for 5 d. Nodifference in culture growth was detected betweenBioS-2P and Bioglass 45S5 (p = 0.886; figure 2(A)),whereas in situ ALP activity increased in cellscultured on BioS-2P compared to Bioglass 45S5(p = 0.029; figure 2(B)). Higher gene expression forBSP (p = 0.029), RUNX2 (p = 0.029) and OPN(p= 0.050) was detected in cells cultured on BioS-2Pcompared to Bioglass 45S5, while no statisticallysignificant difference in the expression of ALP was

Figure 1.Characterization of the surfaces and scaffolds of Biosilicate®with two crystalline phases (BioS-2P). X-ray diffraction patternof the BioS-2P surface (A). Opticalmicroscopy of the surface of the BioS-2P disc (B) and a 3D-focused image obtained below thesurface (C).Microtomography axial and sagittal images of the BioS-2P scaffold obtained from 3D reconstructions (D), (E) and 2Dreconstructions (F), (G). Average pore distribution of BioS-2P scaffolds according to size (H). Scale bar B–C= 10 μm.

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observed between the bioactive surfaces (p = 0.200)(figure 2(C)). BSP andOPN proteins were detected incells cultivated on both surfaces with marked expres-sion on BioS-2P compared to Bioglass 45S5(figure 2(D)). Higher protein expression of ALP andRUNX2 was also observed in cells cultured on BioS-2P compared to Bioglass 45S5 (figure 2(E)). Extra-cellular matrix mineralization increased in cellscultured on BioS-2P compared to Bioglass 45S5(p= 0.029; figure 2(F)).

3.3.Osteoinductive potential of BioS-2PTo determine osteoinductive potential, MSCs werecultured on BioS-2P, Bioglass 45S5 and polystyreneunder non-osteogenic conditions for 10 d. The ALPactivity was greater in cells cultured on Bioglass 45S5compared to BioS-2P and lower in cells cultured onpolystyrene (p= 0.001; figure 3(A)). Furthermore, thegene expression of all evaluated osteoblast markerswas higher on bioactive glasses than on polystyrene(p= 0.001 for all genes;figures 3(B)–(E)). By comparing

Figure 2.Osteogenic potential of Biosilicate®with two crystalline phases (BioS-2P). Culture growth (A), in situ alkaline phosphatase(ALP) activity (B), gene expression of ALP, bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2) and osteopontin(OPN) (C), BSP andOPNprotein detection (D), ALP andRUNX2 protein detection (E), and extracellularmatrixmineralization (F) ofUMR-106 osteoblastic cells cultured on Bioglass 45S5 andBioS-2P on day 5. The data are showed as themean± standard deviation(A)–(C) and (F), and statistically significant differences are indicated by asterisks (p� 0.05).D: redfluorescence—BSP orOPNprotein, as indicated; green fluorescence—actin cytoskeleton; blue fluorescence—nucleus. Scale barD= 50 μm.

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only the bioactivematerials, the gene expression of ALPwas higher in cells cultured on BioS-2P, whereasRUNX2, OPN and OC expression was higher onBioglass 45S5 (figure 3(B)).

3.4. Effects of the BioS-2P scaffold on bone repairThree-dimensional micro-CT images demonstratedthat BioS-2P promoted bone repair (figures 4(A)–(C)).Morphometric analysis showed there were no differ-ences in all evaluated parameters, irrespective of time(BS p= 0.733; BV p= 0.121; BS/BV p= 0.208; BV/TV

p = 0.904; Tb.Th p = 0.274; Tb.Sp p = 0.482)(figures 4(D)–(I)). The histological observations con-firmed the micro-CT findings, and although the axialimages showed bone formation in the centre of thebone defect (figures 5(A)–(C)), the transversal slicesrevealed that BioS-2P scaffolds remarkably improvedbone formation that was integrated with the parentlamellar bone at the defect margins (figures 5(D)–(F)).Higher magnifications showed cancellous bone indirect contactwithBioS-2P (figures 5(G)–(I)).

Figure 3.Osteoinductive potential of Biosilicate®with two crystalline phases (BioS-2P). Alkaline phosphatase (ALP) activity (A) andgene expression of ALP (B), runt-related transcription factor 2 (RUNX2) (C), osteopontin (OPN) (D) and osteocalcin (OC) (E) ofmesenchymal stem cells (MSCs) cultured on polystyrene, Bioglass 45S5 andBioS-2P on day 10. The data are showed as themean±standard deviation, and statistically significant differences are indicated by asterisks (p� 0.05).

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Figure 4. 3D-reconstructedmicrotomography images of rat calvarial bone defects implantedwith BioS-2P after 4 (A), 8 (B) and 12 (C)weeks were used to evaluate the bone surface (BS) (D), bone volume (BV) (E), bone surface/bone volume (BS/BV) (F), bone surface/tissue volume (BS/TV) (G), trabecular thickness (Tb.Th) (H) and trabecular separation (Tb.Sp) (I). The data are showed as themean± standard deviation, and there were no statistically significant differences for any evaluated parameter (p� 0.05). Scale barA–C= 1 mm.

Figure 5. Lightmicroscopy images of rat calvarial bone defects implantedwith BioS-2P after 4 (A), (D), (G), 8 (B), (E), (H) and 12 (C),(F), (I)weeks. Asterisks indicate the newly formed bone and arrows indicate this newbone in close contact with thematerial surface.Scale bar= 100 μm.

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3.5. Effects of the association of BioS-2P scaffold andMSCs onbone repairMSCs could be seen within the pores of BioS-2Pscaffolds (figure 6(A)), and around of 80% of theseeded cells were incorporated into the scaffolds(figure 6(B)). Cell tracking showed that MSCs weredetected in the bone defects for 4 weeks with a peak at1 week (figure 6(C)). Reconstructed images generatedby micro-CT showed that the BioS-2P scaffold aloneor combinedwithMSCs promoted similar bone repair(figures 7(A)–(B)). Morphometric analysis confirmedthis observation because no differences in all evaluatedparameters were found despite the presence of cells(BS p = 0.589; BV p = 0.180; BS/BV p = 0.589;BV/TV = 0.180; Tb.Th p = 0.818; Tb.Sp p = 0.937)(figures 7(C)–(H)). The histological observations con-firmed the micro-CT findings. The axial imagesshowed bone formation in the centre and at theborder of the bone defects (figures 8(A)–(B)). Higher

magnifications showed a direct contact between bonetissue andBioS-2P (figures 8(C)–(D)).

4.Discussion

Here, we reported that BioS-2P had a secondarycrystalline phase and presented an architecture thatresembled a cancellous bone. BioS-2P displayedin vitro osteogenic and osteoinductive properties andenhanced the repair of bone tissue in a rat calvarialdefect model. Under standard osteogenic conditions,BioS-2P stimulated the osteoblast activity of theUMR-106 cell line and generated amicroenvironmentthat promoted the induction of osteoblast phenotypeand genotype expression in MSCs grown withoutosteogenic factors. We also noticed that the BioS-2Pscaffold is able to stimulate bone formation in suchmagnitude that its association withMSCs was not ableto increase.

Figure 6. Fluorescence images ofmesenchymal stem cells (MSCs) incorporated in the BioS-2P scaffold (A) and the efficiency of thecell incorporation (B). Bioluminescence images positive forMSC-luciferase after administration of luciferin from6 to 672 h. Signalintensity was quantified as photons per secondwithin the region of interest (C).

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We confirmed that the secondary crystalline phasepresent in BioS-2P was a sodium-calcium phosphate(NaCaPO4) that was difficult to identify due to its lowcrystallized volume fraction. This phase was alsodetected in Bioglass 45S5 as well as in other glass-cera-mics after heat treatment at temperatures of approxi-mately 1000 °C, and the phase was identified as α- orβ-rhenanite [24–27]. The secondary crystalline phasewas highly reactive in simulated body fluid and

acted as a heterogeneous nuclei for hydroxycarbonateapatite growth at glass-ceramic surfaces, includingBiosilicate® [16, 27, 28], and it improved the mechanicalstrength, elasticmodulus and fracture toughnesswithoutaffecting the bioactivity index [14]. These featuresfavoured scaffold fabrication with ideal porosity and aporousnetwork,whichwere able tomaintain their integ-rity [29], while cell activities such as migration, grownanddifferentiationoccurred [29–31].

Figure 7. 3D-reconstructedmicrotomography images of rat calvarial bone defects implantedwith BioS-2P (A) or BioS-2P associatedwithmesenchymal stem cells (MSCs, B) after 8weeks. These images were used to evaluate bone surface (BS) (C), bone volume (BV)(D), bone surface/bone volume (BV/BV) (E), bone surface/tissue volume (BS/TV) (F), trabecular thickness (Tb.Th) (G) andtrabecular separation (Tb.Sp) (H). The data are showed as themean± standard deviation, and therewere no statistically significantdifferences in any evaluated parameters (p� 0.05). Scale bar A–B= 1 mm.

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The ability of scaffolds to be populated by osteo-blasts is very important for therapies that aim to regen-erate bone tissue. Our results showed a similar patternof culture growth on BioS-2P and on Bioglass 45S5discs. In a previous study, it was observed that osteo-blasts derived from rat calvarial cells grown on BioS-1Pand Bioglass 45S5 exhibited the same proliferation rate[17]. These findings strongly suggested that the pre-sence of one or two crystalline phases didnot impact thecapacity of Biosilicate® to support cell attachment andproliferation. In both situations, the dynamics ofgrowth followed the growth forBioglass 45S5.

Bioactive glasses can induce osteoblast differentia-tion and extracellular matrix mineralization [17, 32].To track the role of BioS-2P in driving osteoblast func-tions in UMR-106 cells, we analysed the activity ofALP, which is a key enzyme in the process of miner-alization [33]. Higher ALP activity was detected inBioS-2P, which could lead to a higher extracellularmatrix mineralization because of a direct positive cor-relation between these two parameters in differentexperimental designs [34, 35]. Additionally, the releaseof ionic dissolution products by bioactive glasses andglass-ceramics may affect the gene expression profileof osteogenic cells [36–39]. Thus, to further investigatethe osteoblast phenotype and genotype regulationinduced by BioS-2P, we examined the gene and pro-tein expressions of ALP, BSP, RUNX2 and OPN,which are key markers expressed during the timecourse of osteoblast differentiation. In general, our

results showed that BioS-2P increased the gene andprotein expressions of all evaluatedmarkers comparedto Bioglass 45S5, which suggested that BioS-2P pre-sented higher osteogenic potential than Bioglass 45S5.

Among different bioactive glass compositions, theglass-ceramic in the Na2O–CaO–SiO2–P2O5 systemmimicked the microenvironment created by theosteogenic medium and may consequently dictate thefate of MSCs [32]. Based on this possibility and on theosteogenic potential of BioS-2P, we cultured MSCs innon-osteogenic medium to investigate the hypothesisthat BioS-2P by itself could affect osteoblast differ-entiation. In addition to using polystyrene as a control,BioS-2P was also compared to Bioglass 45S5 to deter-mine the osteoinductive potential of both materials.We observed that BioS-2P upregulated ALP activityand the gene expression of ALP, RUNX2, OPN andOC compared to polystyrene. Although the ALP activ-ity and the expression of the majority of genes werehigher for Bioglass 45S5 compared to BioS-2P, ourresults clearly showed the ability of BioS-2P to induceosteoblast differentiation in MSCs. Similar to theseresults, another study showed that Bioglass 45S5-con-ditioned medium favours osteoblast differentiation inthe absence of osteogenic factors [40].

Beyond the in vitro osteogenic and osteoinductiveproperties, the BioS-2P scaffold promoted in vivobone formation. It was noteworthy that many areas ofbone formation were seen at the centre of the defectsand were not related to the parent bone, which

Figure 8. Lightmicroscopy images of rat calvarial bone defects implantedwith BioS-2P andBioS-2P associatedwithmesenchymalstem cells (B) for 8weeks. Asterisks indicate the newly formed bone and arrows indicate this new bone in close contact with thematerial surface. Scale bar= 100 μm.

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suggested BioS-2P had a stimulatory effect. Addition-ally, the observation of direct contact between theBioS-2P surface and bone corroborated the beneficialeffects of BioS-2P. However, the effects of BioS-2Pwere somewhat limited, so no fully defective regenera-tion was observed, and there was no increase in boneformation during the implantation time. Similar tothis outcome, meaningful bone formation wasobserved for BioS-1P and BioS-2P implanted in rabbitfemurs and rat tibias [19, 41].

The effects of combining scaffolds and cells onbone formation have produced contradictory out-comes. Some studies reported synergistic effects[42–46], while others failed to show improved boneformation when MSCs were combined with scaffolds[31, 47, 48]. Our results aligned with the latter studiesbecause the combination of BioS-2P with MSCs didnot increase bone repair. This result could have arisen,at least in part, due to the absence of cells, the scaffoldarchitecture or the number of seeded cells [47]. How-ever, it is most likely that none of these factors played arole in this study. The BioS-2P scaffold presented phy-sical characteristics close to the ideal in terms of poros-ity and pore size. The incorporation method allowedfor seeding approximately 80% of the cells to the scaf-folds, and the cell tracking showed that the cells werepresent in the defects for a long time. It seems that thestimulatory effect of the BioS-2P on bone healingcould not be enhanced by the addition ofMSCs due tomodifications in the microenvironment caused by thedissolution products of glassy materials that may havemasked the cellular effects [39, 49]. On the other hand,it is not possible to rule out that by using a higher celldensity, a synergistic effect could be detected.

5. Conclusion

We showed that BioS-2P displayed a sodium-calciumsilicate and a sodium-calcium phosphate phase, andBioS-2Pcouldbeused tomanufacture three-dimensionalscaffolds with an architecture resembling cancellousbone. BioS-2P permitted culture growth and stimulatedosteoblast activity under osteogenic conditions, whilegenerating an osteoinductive microenvironment thatdrove MSC differentiation into osteoblasts under non-osteogenic conditions. BioS-2P was able to stimulatemeaningful formation of bone tissue in rat calvarialdefects despite the presence of cells. Considering thesuitability of this new biomaterial for use in the design ofscaffolds, these findings open windows for evaluating theeffectiveness of BioS-2P in bone regeneration based ontissue engineering concepts.

Acknowledgments

This study was supported by the São Paulo ResearchFoundation (Grants# 2012/23879-5 and 2012/23525-9,

2013/07793-6, FAPESP,Brazil). Theauthors thankRafaelPeixoto dos Santos for his assistance in the preparation ofthe glass and glass-ceramic discs as well as FabiolaSingaretti de Oliveira and Roger Rodrigo Fernandes fortheir technical assistance in thebiological experiments.

Conflicts of interest

No conflicts of interest are declared.

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