Fabrication of a novel triphasic and bioactive ceramic and evaluation of its in vitro and in vivo cytocompatibility and osteogenesis

Journal ofMaterials Chemistry B

PAPER

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aBiomaterials and Tissue Engineering Res

University of Sydney, Sydney, 2006, AustralbMesenchymal Stem Cell Laboratory, Schoo

Sciences, University of Adelaide, Australi

Fax: +61-2-93517060; Tel: +61-2-93512392cColgate Australian Clinical Dental Research

Adelaide, AustraliadSchool of Chemical and Bimolecular Engine

Cite this: J. Mater. Chem. B, 2014, 2,1866

Received 25th October 2013Accepted 8th January 2014

DOI: 10.1039/c3tb21504k

www.rsc.org/MaterialsB

1866 | J. Mater. Chem. B, 2014, 2, 186

Fabrication of a novel triphasic and bioactiveceramic and evaluation of its in vitro and in vivocytocompatibility and osteogenesis

Seyed-Iman Roohani-Esfahani,a Kai Yuen Wong,a Zufu Lu,a Yong Juan Chen,a

Jiao Jiao Li,a Stan Gronthos,b Danijela Menicanin,c Jeffrey Shi,d Colin Dunstana

and Hala Zreiqat*a

We report, for the first time, the synthesis of a novel triphasic and crystalline bioactive ceramic (MSM-10)

with the ability to simultaneously release three types of bioactive ions (strontium (Sr), silicon (Si) and

magnesium (Mg)) to the surrounding microenvironment. An MSM-10 powder with a nominal

composition (wt%) of 54 Mg2SiO4, 36 Si3Sr5 and 10 MgO was prepared by the sol–gel method and

fabricated as porous scaffolds using the foam replication method. The effects of the different amounts

of the phases in the ceramics on the mechanical and physical properties of the scaffolds as well as their

in vitro and in vivo behaviors were comprehensively investigated. Biphasic calcium phosphate (BCP,

b-tricalcium phosphate (60 wt%)/hydroxyapatite (40 wt%)) scaffolds were used as the control material.

The attachment, morphology, proliferation and differentiation of primary human osteoblasts (HOBs)

were investigated after cell culturing on the various scaffolds. In vitro cytotoxicity (ISO/EN 10993-5)

results not only indicated the biocompatibility of MSM-10, but also its positive effects on inducing the

proliferation of HOBs. Our results showed significant enhancement in osteogenic gene expression levels

(Runx2, osteocalcin, osteopontin and bone sialoprotein), when HOBs were cultured on MSM-10,

compared to those for BCP and other generated ceramic scaffolds. For the in vivo studies, the different

types of the materials were seeded with cultured human mesenchymal stem cells (hMSC) and then

subcutaneously transplanted into the dorsal surface of eight-week-old immunocompromised (NOD/

SCID) mice. MSM-10 demonstrated a significant amount of new bone formation compared to the other

groups tested with no macroscopic signs of inflammation or toxicity in the tissue surrounding the

implants. The novel MSM-10 ceramic presents promising potential for bone regeneration in orthopaedic

and maxillofacial applications.

1. Introduction

The bone mineral, while frequently described as hydroxyapa-tite, is in fact highly modied by the substitution of calcium bycations such as strontium (Sr) and magnesium (Mg), and ofphosphate and hydroxyl groups by anions such as silicate andcarbonate.1 Development of biomaterials that contain and canrelease Sr, silicate (Si) and Mg to the bone sites has receivedintense interest in recent years.1–41

earch Lab Unit, School of AMME, the

ia

l of Medical Sciences, Faculty of Health

a. E-mail: [email protected];

Centre, School of Dentistry, University of

ering, the University of Sydney, Australia

6–1878

Sr is highly bioactive with both antiresorptive and anaboliceffects,38,42 with its ranelate salt being used for treatment forosteoporosis. In vitro studies revealed that the presence of Srions resulted in increased collagen and non-collagenousprotein synthesis during early osteoblast differentiation and inthe inhibition of osteoclast differentiation and function.38,43–45

Moreover, Sr has also been found to induce an antibacterialeffect.46,47 Si is an essential element for the metabolic processesassociated with the formation and calcication of bonetissue48,49 and high Si content has been detected in early stagesof bone matrix calcication.50,51 Mg is the fourth most abundantcation in the human body, reported to make up 0.44 wt% ofenamel, 1.23 wt% of dentin and 0.72% of bone52 and a link issuggested between Mg deciency and osteoporosis.53 Mg hasbeen shown to be involved in bone remodelling and metabo-lism; in the promotion of angiogenesis; and in the growth andmineralization of bone tissue.54–58

Research efforts have been focused on incorporating thesetherapeutic ions (Sr, Si and Mg) into different biomaterials

This journal is © The Royal Society of Chemistry 2014

http://dx.doi.org/10.1039/c3tb21504k

http://pubs.rsc.org/en/journals/journal/TB

http://pubs.rsc.org/en/journals/journal/TB?issueid=TB002013

Tab

le1

Designationan

dmolarratiosofelements

usedin

theso

l–gelp

roce

ssan

dfinal

compositionofstudymaterials

Designation

Molar

ratioof

elem

ents

insol–gelprocess

Ceram

iccompo

sition

aer

calcination(w

t%)

MMg

Si¼

2;

SrSr

þMg¼

0Mg 2SiO4

M-1

Mg

Si¼

2;

SrSr

þMg¼

0:01

Mg 2SiO4

M-2

Mg

Si¼

2;Sr

SrþMg¼

0:02

Mg 2SiO4

MSM

-5Mg

Si¼

2;Sr

SrþMg¼

0:1

Mg 2SiO4(70),S

i 3Sr

5(25),M

gO(5)

MSM

-10

Mg

Si¼

2;Sr

SrþMg¼

0:2

Mg 2SiO4(54),S

i 3Sr

5(36),M

gO(10)

MSM

-20

Mg

Si¼

2;Sr

SrþMg¼

0:4

Mg 2SiO4(11),S

i 3Sr

5(69),M

gO(20)

Paper Journal of Materials Chemistry B

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including calcium phosphates, bioactive glasses and calciumsilicates.3,9–11,24,59–62 However concerns regarding the efficacy ofthese devices include: (1) controllability of the ion release rate;(2) ability to simultaneously releasemultiple ions, and (3) abilityto reach therapeutic concentrations in adjacent tissues.39 In thisstudy we synthesised and developed a new triphasic ceramic(Australian patent # 2013000498) designed to simultaneouslyrelease the three bioactive ions (Sr, Si and Mg). We assessed thechemical, physical and mechanical properties of this materialand evaluated its in vitro and in vivo bioactivity.

2. Materials and methods2.1. Preparation and characterization of ceramic powdersand scaffolds

To design a ceramic with the ability to release Sr, Mg and Sisimultaneously into the microenvironment, we selectedMg2SiO4, with Mg/Si atomic ratio of 2, as the referencecomposition and matrix for introducing the Sr atoms. Mg2SiO4

is the only available ceramic with a simple structure thatcontains Mg2+ and SiO4

4� groups in an orthorhombic crystalstructure. Magnesium nitrate hexahydrate (Mg(NO3)2$6H2O,Sigma-Aldrich, >99.0%), tetraethyl orthosilicate (TEOS,Si(OC2H5)4, Sigma-Aldrich, >99.0%) and strontium nitrate(Sr(NO3)2, Sigma-Aldrich, >99.0%) were used as the precursors.

The sol–gel method was used to prepare a solution (sol) byhydrolyzation of TEOS in ethanol at the volume ratio of TEOS–ethanol ¼ 1 : 3. Then, Mg(NO3)2$6H2O was dissolved in thehydrolyzed TEOS to obtain a sol with an Mg/Si atomic ratio of 2.At this point, except for the rst group (labelled as M, Table 1)where the sol was aged and calcined to obtain a pure Mg2SiO4,strontium nitrates with different molar ratios XSr¼ Sr/(Sr + Mg)were added to the sol and then the sol aged at 65 �C for 12 h toform a gel. Subsequently, the gel was dried at 100 �C for 24 hand calcined at 1400 �C for 3 h in air using an electrical furnaceto obtain ceramic powders containing Sr, Mg and Si (labelled asM1, M2, MSM-5, MSM-10 and MSM-20, Table 1). Table 1 showsdesignation, molar ratio of elements used in the sol–gel processand nal composition of different study groups. Phase structureanalyses of obtained powders and determination of latticeparameters were carried out by an X-ray diffractometer (XRD,Siemens D6000, Germany) using Cu Ka radiation with a scan-ning speed of 1� min�1 and step size of 0.01� over a 2q range of10 to 80�. BCP, the control group used in the biological study,was prepared as reported previously.63 Fully reticulated poly-urethane foam was used as a sacricial template for scaffoldreplication via the polymer sponge method. The ceramic slurrywas prepared by adding powders to a polyvinyl alcohol (PVA)solution to prepare a 30 wt% suspension. Foam templates werecut to appropriate dimensions and treated in a NaOH solutionfor 30 min to improve surface hydrophilicity. Aer cleaning anddrying, foams were immersed in the slurry and compressedslightly to facilitate slurry penetration. The excess slurry wassqueezed out and the foam was subsequently blown withcompressed air to ensure uniform ceramic coating on the foamsurface and achieving 85% of porosity aer burning the foam.To obtain scaffolds with less porosity (74% and 66%), more

This journal is © The Royal Society of Chemistry 2014 J. Mater. Chem. B, 2014, 2, 1866–1878 | 1867


Journal of Materials Chemistry B Paper

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slurry was kept in the foam structure to decrease the number ofopen pores.

Aer drying at 37 �C for 48 h, coated foams were red in airin an electric furnace using a 4-stage schedule: (i) heating from25 �C to 600 �C at a heating rate of 1�C min�1, (ii) furtherheating from 600 �C to 1200 �C at 2�C min�1 for BCP and from600 �C to 1450 �C at 2�Cmin�1 for M, M-1, M-2, MSM-5, MSM-10and MSM-20 scaffolds, (iii) holding the temperature at 1200 �Cfor 2 h for HA/TCP and at 1450 �C for 3 h for M, M-1, M-2, MSM-5, MSM-10 and MSM-20, and (iv) cooling to 25 �C at a coolingrate of 5 �C min�1. The microstructure of the scaffolds wascharacterized by eld emission scanning electron microscopy(FE-SEM) (Zeiss; Carl Zeiss, Germany). Internal structure,porosity and interconnectivity of the scaffolds were evaluated bymicro-computerized tomography (Skyscan 1076, Micro-Computed Tomography).

2.2. Degradation study in different solutions

For a comprehensive investigation of efficacy of developedscaffolds in the release of bioactive ions to their microenvi-ronments, three buffered solutions (simulated body uid(SBF)), culture medium (a-Minimal Essential Medium[a-MEM], Gibco Laboratories, USA) and phosphate bufferedsaline (PBS) were used. SBF has chemical composition and pHsimilar to human blood plasma. PBS is a commonly usedbuffer solution in biological studies and has isotonic proper-ties similar to the body uids. The culture medium is a buffersolution and was used to simulate the in vitro cell expansionmicroenvironment.

Cubic scaffolds (8 mm � 8 mm � 8 mm) of M, M-1, M-2,MSM-5, MSM-10 and, MSM-20 were immersed in solutions at37 �C for 1, 3, 7, 14, 21 and 28 days at a solid/liquid ratio of150 mg L�1. All scaffolds were held in plastic asks and sealed.At each time point, the scaffolds were removed, rinsed withMilli-Q water and dried at 100 �C for 1 day, aer which the nalweight of each scaffold was measured. The concentration of theions in the solutions aer soaking was tested using inductivelycoupled plasma atomic emission spectroscopy (ICP-AES; PerkinElmer, Optima 3000DV, USA). The weight loss (calculatedaccording to the percentage of initial weight before soaking inSBF) and pH change results were expressed as means� SD. Fivesamples of each type of scaffold were tested per time point forstatistical analysis.

2.3. Mechanical properties of the scaffolds

Mechanical properties of the M, M-1, M-2, MSM-5, MSM-10,MSM-20 and BCP scaffolds were determined under dry and wetconditions. For wet conditions, the scaffolds were rst soakedin SBF for 1, 3, 7, 14, 21 and 28 days at 37 �C. The compressivestrength was determined by crushing the cubic scaffolds (6 mm� 6 mm � 12 mm) between two at plates using a computer-controlled universal testing machine (Instron 8874, UK) at aramp rate of 0.5 mmmin�1. Ten identical specimens from eachsample group were used for compressive testing under dry andwet conditions.

1868 | J. Mater. Chem. B, 2014, 2, 1866–1878

2.4. Cytocompatibility and osteogenic induction property ofthe scaffolds

2.4.1. Scaffold sterilization. Cubic scaffolds 5 � 5 � 5 mmwere sterilized before cell culture using an autoclave (121 �C;20 min).

2.4.2. Isolation seeding and culturing of primary humanosteoblasts. Permission to use discarded human tissue wasgranted by the Human Ethics Committee of the University ofSydney and informed consent was obtained. An establishedmethod for culturing osteoblast cells was used. Primary humanosteoblasts (HOBs) were isolated from a normal humantrabecular bone. The bone was divided into 1 mm3 pieces,washed several times in PBS, and digested for 90 min at 37 �Cwith 0.02% (w/v) trypsin (Sigma-Aldrich, USA) in PBS. Digestedcells were cultured in a complete medium containing a-MEM,supplemented with 10 vol% heat-inactivated fetal calf serum(FCS) (Gibco Laboratories, USA), 2 mM L-glutamine (GibcoLaboratories, USA), 25 mM Hepes buffer (Gibco Laboratories,USA), 2 mM sodium pyruvate, 100 U ml�1 penicillin, 100 mgml�1 streptomycin (Gibco Laboratories, USA) and 1 mML-ascorbic acid phosphate magnesium salt (Wako Pure Chem-icals, Japan). The cells were cultured at 37 �C with 5% CO2 andcomplete medium changes were performed every 3 days. AllHOBs used in the experiments were at passage three. Aer thecells reached 80–90% conuence they were trypsinized withTrypLE™ Express (Invitrogen) and subsequently suspended inthe complete medium. For HOB attachment and proliferationstudies cells were seeded on the scaffolds at initial cell densitiesof 5� 104 cells per scaffold, in 90 ml of a cell suspension. For thegene expression study cells were seeded on the scaffolds at aninitial cell density of 2 � 105 cells per scaffold in 100 ml of thecell suspension. A suspension of HOBs was gently dropped ontothe scaffolds (n ¼ 4) placed in 24-well plates (untreated, NUNC)and incubated for 90 min at 37 �C to allow the cells to attach.Then each scaffold was transferred to a new well and 1.5 ml ofthe culture medium was added for culturing. At the designatedtime points, HOBs on the scaffolds were analysed for attach-ment, viability and gene expression. If we observed that HOBmigrated from the scaffolds and were growing on the wells, thescaffolds were transferred to a new well for examination.

2.4.3. HOB attachment and proliferation. HOB attachmentwas evaluated aer 2 and 24 h culture. At each time point M, M-1, M-2, MSM-5, MSM-10, MSM-20 and BCP scaffolds wereprepared for scanning electron microscopy (SEM) (Carl Zeiss,Germany) examination. Scaffolds with cells were xed with a 4%paraformaldehyde solution, post-xed with 1% osmiumtetroxide in PBS for 1 h, dehydrated in graded ethanol (30%,50%, 70%, 95% and 100%), dried in hexamethyldisilizane for 3min and then desiccated overnight. The scaffolds were goldsputtered prior to SEM examination. To evaluate HOB prolif-eration the CellTiter 96 Aqueous Assay (Promega, USA) was usedto determine the number of viable cells on the cultured scaf-folds via a colorimetric method. The assay solution is acombination of a tetrazolium compound (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium), MTS) with an electron coupling reagent




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(phenazine methosulfate, PMS) at a volume ratio of 20 : 1. Theformer compound can be bio-reduced by viable cells into for-mazan, which is soluble in the cell culture medium, and theabsorbance of formazan at 490 nm is directly proportional tothe number of viable cells present. HOB proliferation wasevaluated aer 1 and 7 days' culture. At each time point testedthe culture medium was replaced by 1.5 ml of the MTS workingsolution, which consisted of the CellTiter 96 Aqueous Assaysolution diluted in PBS at a volume ratio of 1:5. Aer 4 hincubation at 37 �C 100 ml of the working solution was trans-ferred to a 96-well cell culture plate and the absorbance at490 nm was recorded using a microplate reader (PathTech,Australia) using the soware Accent (Australia).

2.4.4. HOB gene expression. Quantitative real time poly-merase chain reaction (qRT-PCR) was used to evaluate osteo-genic gene expression on the cultured scaffolds. Total RNA wasisolated from HOBs cultured on each scaffold using Trizol(Sigma-Aldrich, USA) and puried using the RNeasy Mini Kit(Qiagen, USA) according to the manufacturer's instructions.First strand cDNA was synthesized from 0.7 mg total RNA usingan Omniscript RT Kit (Qiagen, USA) according to the manu-facturer's instructions. The cDNA was analyzed for the expres-sion of osteoblast-specic genes, specically Runx2, collagentype I, bone sialoprotein and osteocalcin, and their expressionwas normalized to glyceraldehyde 3-phosphate dehydrogenase(GAPDH) to obtain relative gene expression. The primers for theselected genes are listed in Table 2.

2.4.5. Cytotoxicity test. The dissolution extracts of theceramics were prepared in a serum-free culture mediumfollowing the International Standard Organization (ISO/EN10993-5) protocol. In brief, M, M-1, M-2, MSM-5, MSM-10 andMSM-20 scaffolds were ground and sieved to achieve a nepowder with an average particle size of 40 mm. The obtainedceramic powder for each group was soaked in a serum-freea-MEM culture medium (Gibco Laboratories, USA) at a ratio of

Table 2 Primers used for qRT-PCR osteogenesis-related genes

Gene Sequence (50–30) Melting temperature (�C)

GAPDH F ACCCAGAAGACTGTGGATGG

60

R CAGTGAGCTTCCCGTTCAG

Runx-2 F ATGCTTCATTCGCCTCAC

60

R ACTGCTTGCAGCCTTAAAT

Osteopontin F TTCCAAGTAAGTCCAACGAAG

60

R GTGACCAGTTCATCAGATTCAT

Osteocalcin F ATGAGAGCCCTCACACTCCTCG

60

R GTCAGCCAACTCGTCACAGTCC

Bone sialoprotein F ATGGCCTGTGCTTTCTCAATG

60

R GGATAAAAGTAGGCATGCTTG


200 mg ml�1 (w/v) and incubated at 37 �C for 24, 48 and 72 h. Ateach time point, the mixture was mixed and centrifuged for5 minutes at 300 � g, and then the supernatant was collected forion analysis and cytotoxicity test using human osteoblasts(HOBs). Serial dilutions of the extract were carried out in aserum-free a-MEMmedium at the concentrations of 100, 50, 25,12.5 and 6.25 mg ml�1. HOBs were seeded at the cell density of1 � 104 cells per cm2 in 96-well plates and incubated for 24 h at37 �C in a humidied atmosphere of 95% air and 5% CO2. Ineach well, the culture medium was removed and replaced with50 ml of a-MEM supplemented with 20% fetal calf serum (FCS)and 50 ml of appropriate concentration of extracts. 50 ml ofserum-free a-MEM and 50 ml of a-MEM supplemented with 20%FCS were used as a blank control. A negative control wasincluded by using 50 ml of serum-free a-MEMwith 0.2% Triton X-100 and 50 ml of a-MEM supplemented with 20% FCS. Aer cellswere cultured for 1, 3 and 7 days, cell viability was analyzed usingtheMTT assay (Sigma Chemical, St Louis, MO, USA) according tothe manufacturer's instructions. Briey, the cell culture mediumwas removed and replaced with 25 ml of a 2.5 mg ml�1 MTTsolution to each well. Aer incubation for 2 h at 37 �C, the MTTsolution was removed and 100 ml DMSO was added to each welland mixed for 10 minutes on a shaker. The absorbance was readat 570 nm in the microplate reader of Thermo Scientic Multi-scan EX (Thermo Fisher Scientic, USA).

2.5 Transplantation of human MSC intoimmunocompromised mice

All procedures were conducted in accordance with guidelines ofan approved small-animal protocol (SA Pathology AEC #141/12and University of Adelaide AEC # MSM-2012-207). BCP, M-1,MSM-5 and MSM-10 scaffolds were ground and sieved to ach-ieve uniform sized granules (�450 mm). Approximately, 4 � 106

bone marrow derived MSC (2 donor populations) were mixedwith 40 mg BCP, M-1, MSM-5 and MSM-10 granules, thenclotted with 20 ml mouse brinogen (30 mg ml�1 in PBS) and20 ml mouse thrombin (100 U ml�1 in 2% CaCl2) to form a plugand then subcutaneously transplanted into the dorsal surface ofeight-week-old immunocompromised (NOD/SCID) mice aspreviously described.64 Replicate transplants for each donorwere performed. All transplants were recovered 8 weeks aertransplantation.

2.5.1. Histological assessment. Specimens were dehy-drated through graded ethanol, cleared in xylene at roomtemperature and then inltrated with, and embedded in, poly-methyl-methacrylate resin using a standard processing inl-tration schedule as previously described.65 Uponpolymerization, multiple equidistant transverse sections wereprepared from the embedded resin block using a water-cooledslow-speed Buehler isomet saw (Buehler, Germany) and subse-quently polished and stained with toluidine blue for histolog-ical assessment.

2.6. Statistics

All data are presented as means � SD and were derived from atleast four independent samples. For statistical analysis Levene's

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test was performed to determine the homogeneity of variance ofthe data, and then either Tukey's HSD or Tamhane's post hoctests were used. The PASW statistics program was employed forall statistical analyses and differences were considered assignicant for p < 0.05.

3. Results

The X-ray diffraction patterns and crystallographic analysis ofthe prepared materials are shown in Fig. 1(a–f). Characteristicpeaks of pure Mg2SiO4 were detected for M, M-1 and M-2 scaf-folds. Diffraction peaks for M-1 andM-2 were shied to lower 2qvalues (Fig. 1b) compared to those for M, indicating an increasein d-spacings and hence lattice parameters. The addition of Sr(up to 2 mol%) caused a linear increase of lattice parameters (a–c) as well as unit cell volume (V) (Fig. 1c–e) (R2 values > 0.9). Moreimportantly, the aspect ratios of a Mg2SiO4 unit cell (b/a and b/c)increased linearly (R2 > 0.9) by increasing Sr up to 2 mol%(Fig. 1f), and the amount of this increase for (b/a) was signi-cantly higher (�5 times) than that for (b/c). Additional phases ofMgO and Sr3Si5 were detected for MSM-5, MSM-10 and MSM-20as shown in the X-ray patterns (Fig. 1a) with incremental increasein peak intensity of MgO and Sr3Sr5 from MSM-5 to MSM-20,while that for Mg2SiO4 decreased. MSM-5 had a composition of(70 wt%) Mg2SiO4, (25 wt%) Si3Sr5 and (5 wt%) MgO; MSM-10had a composition of (54 wt%) Mg2SiO4, (36 wt%) Si3Sr5 and(10 wt%) MgO; MSM-20 had a composition of (11 wt%) Mg2SiO4,(69 wt%) Si3Sr5 and (20 wt%) MgO.

SEM examination revealed a highly porous (porosity: �85%and pore size: �400 mm) structure for all the prepared scaffoldswith �100% pore interconnectivity (Fig. 2a–c). Fig. 2d–f showtypical strut microstructure of M, M-2 and MSM-10 (MSM-5 andMSM-20 had microstructure features similar to MSM-10) scaf-folds, respectively. The M microstructure consisted of theequiaxed grains of Mg2SiO4 with an average size of 1.5 mm

Fig. 1 (a and b) X-ray diffraction patterns for M, M-1, M-2, MSM-5, MSM-a, (d) b, (e) c and (f) aspect ratios of the Mg2SiO4 crystal.

1870 | J. Mater. Chem. B, 2014, 2, 1866–1878

(Fig. 2d); however the M-2 microstructure consisted of elon-gatedMg2SiO4 grains with a preferred growth direction (Fig. 2e).Three different crystalline grains could be observed in the MSM-10 microstructure (Fig. 2d): I – cuboidal-shaped grains of MgOwhich are scattered in small amounts in the triple junctions ofother grains with an average size of 350 nm; II – large prismshaped grains of Si3Sr5 with an average size of 3 mm and III –equiaxed grains of Mg2SiO4 with an average size of 700 nmdispersed between the Si3Sr5 grains. Elemental distributionmapping was carried out to evaluate the qualitative distributionof Si, Mg and Sr elements in the MSM-10 microstructure(Fig. 2g–i). Results demonstrated a striking difference in Sr andMg distribution, where Mg was localized in type I (MgO) and III(Mg2SiO4) grains but Sr only existed in the type II grains (Si3Sr5).Themap shows the presence of Si elements in themajor parts ofthe microstructure except for type I grains (MgO). Fig. 3 andTable 3 depict the degradation behaviour of M, M-1, M-2, MSM-5, MSM-10 and MSM-20 scaffolds when incubated in culturemedium, PBS and SBF solutions for different time periods.

For brevity, we have included the ion concentrations derivedfrom the culture medium in Fig. 3 and those for PBS and SBF inTable 3. Fig. 3a–c depict the concentrations of Sr, Mg and Sireleased from all the scaffolds incubated in the culture mediumfor 0, 7, 14 and 21 days. For MSM-5, MSM-10 and MSM-20scaffolds, concentrations of Sr, Mg and Si released into theculture medium were signicantly higher than those for M, M-1and M-2 scaffolds at the time points tested. This trend wasmaintained aer incubating the scaffolds in PBS or SBF solu-tions (Table 3) in the following order: MSM-20 > MSM-10 >MSM-5 >M-2 >�M-1 >M (Table 3). M scaffolds showed the leastdegradability with Si and Mg concentrations in the culturemedium reaching 2.6 � 0.1 and 4.1 � 0.2 ppm, respectively,compared to 10.2 � 01 and 81 � 4 respectively for MSM-10scaffolds at 28 days. For MSM-5, MSM-10 andMSM-20 scaffolds,the initial fast release can be observed; however aer that initial

10 and MSM-20 scaffolds. Effects of strontium on lattice parameters (c)



Fig. 2 Pore morphology and strut microstructure of: (a and d) M, (b and e) M-2 and (c and f) MSM-10 scaffolds. SEM images taken with asecondary electron detector and EDX spectral maps of (g) Si (green), (h) Mg (pink) and (i) Sr (yellow), over a 60 � 40 mm2 area on MSM-10scaffolds.


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burst, a sustained slow and relatively linear release of ions wasobserved.

A slight increase in the ion release rate was found for M-1and M-2, compared to M scaffolds; however the valuesremained markedly lower than those for MSM-5, MSM-10 andMSM-20 scaffolds. Between 1 and 28 days, M-1, M-2, MSM-10and MSM-20 scaffolds released the Sr into the culture medium

Fig. 3 Concentrations of (a) Sr, (b) Mg and (c) Si released from the M, M-1after 1, 3, 7, 14, 21 and 28 days of soaking. (d) pH changes of SBF and (e)periods.


according to their Sr content (M-1: from 0.5 � 0.05 to 1.1 � 0.07ppm; M-2: from 0.8 � 0.12 to 2.4 � 0.22 ppm; MSM-5: from 2.3� 0.16 to 4.9 � 0.20 ppm; MSM-10: from 4.5 � 0.49 to 10 � 0.60ppm and MSM-20: from 6.5 � 0.57 to 17 � 0.87 ppm). The pHvariation patterns of the SBF solution containing M, M-1, M-2,MSM-5, MSM-10 andMSM-20 scaffolds as a function of time aredepicted in Fig. 3d.

, M-2, MSM-5, MSM-10 and MSM-20 scaffolds into the culture mediumweight loss of the scaffolds after soaking the scaffolds at different time

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Tab

le3

Conce

ntrations(ppm)ofSr,M

gan

dSi

releasedfrom

theM,M

-1,M

-2,M

SM-5

,MSM

-10an

dMSM

-20scaff

oldsinto

(a)PBSan

d(b)SB

Fafter1,3,7

,14,2

1an

d28daysofso

aking

a1

37

1421

28b

13

714

2128

MSr

0.0

0.0

0.0

0.0

0.0

0.0

MSr

0.0

0.0

0.0

0.0

0.0

0.0

Mg

0.0

0.0

0.0

0.1�

0.04

0.2�

0.08

0.3�

0.1

Mg

0.0

0.4�

0.2

1.1�

0.9

2.0�

0.5

2.5�

0.3

3.3�

0.8

Si0.1�

0.02

0.2�

0.08

0.2�

0.1

0.3�

0.09

0.3�

0.1

0.3�

0.2

Si2.3�

0.3

2.5�

0.4

2.9�

0.7

2.2�

0.1

3.0�

0.5

3.1�

0.9

M-1

Sr0.5�

0.1

0.6�

0.1

0.9�

0.3

2.0�

12.7�

0.5

3.8�

0.4

M-1

Sr0.5�

0.2

0.5�

0.4

0.9�

0.1

2.0�

0.4

2.7�

0.2

3.8�

0.1

Mg

0.0

0.7�

0.05

0.9�

0.2

1.0�

0.07

0.9�

0.2

1.1�

0.1

Mg

0.0

0.8�

0.3

2.8�

0.2

3.1�

0.3

3.9�

0.1

4.0�

0.8

Si0.5�

0.09

0.5�

0.1

0.6�

0.4

0.9�

0.3

0.9�

0.1

1.0�

0.3

Si2.7�

1.2

2.6�

1.5

3.7�

1.1

3.7�

1.7

3.8�

0.4

4.2�

0.3

M-2

Sr0.6�

0.2

1.0�

0.05

1.2�

0.6

1.8�

0.6

3.8�

0.9

4.0�

0.1

M-2

Sr0.9�

0.1

2.0�

0.3

3.1�

0.4

4.3�

0.2

4.8�

0.7

7.0�

0.2

Mg

0.0

2.0�

0.06

3.0�

13�

23.9�

0.6

4.2�

0.3

Mg

1.2�

0.7

2.3�

0.9

3.1�

0.3

4.2�

0.5

4.8�

0.2

5.1�

0.6

Si0.2�

0.08

0.3�

0.08

0.5�

0.1

0.5�

0.2

0.7�

0.3

0.9�

0.1

Si2.9�

1.5

2.5�

1.1

3.9�

0.9

4.7�

0.3

4.6�

0.1

4.8�

0.3

MSM

-5Sr

3.4�

0.3

3�

13.1�

0.4

3.6�

0.2

4.2�

0.7

5.8�

0.4

MSM

-5Sr

4.1�

0.2

5.2�

0.3

5.8�

0.3

8.2�

0.6

12�

213

.9�

0.1

Mg

26�

528

�6

30�

234

�3

40�

442

�1

Mg

15�

118

�2

25�

329

�1

31�

234

.2�

0.4

Si1.0�

0.06

1.0�

0.02

1.0�

0.02

1.3�

0.1

1.7�

0.3

1.9�

0.3

Si4.3�

0.6

5.1�

0.8

6.8�

0.6

7.3�

0.2

7.8�

0.4

8.1�

0.2

MSM

-10

Sr4�

17�

18�

28�

111

�2

12�

1MSM

-10

Sr10

�1

13�

114

.2�

0.2

14.2

�0.4

18�

120

.1�

0.2

Mg

56�

610

0�

1718

0�

2321

3�

1023

0�

624

0�

5Mg

46�

573

�2

78�

193

�5

140�

1016

0�

7Si

1.1�

0.2

1.0�

0.04

0.8�

0.1

1.0�

0.01

1.5�

0.1

1.6�

0.1

Si3.4�

1.3

6�

17.2�

0.7

7.8�

0.2

8.2�

0.1

8.9�

0.1

MSM

-20

Sr6�

211

�3

10�

314

�1

17�

218

�4

MSM

-20

Sr15

�2

16�

0.3

18.6

�0.4

26�

325

�1

29.1

�0.6

Mg

64�

1121

0�

1430

0�

1030

3�

732

0�

1034

0�

5Mg

58�

613

2�

1018

9�

1519

0�

821

0�

522

0�

4Si

1.3�

0.7

1.4�

0.9

1.5�

0.7

1.6�

11.8�

0.4

2.0�

0.1

Si3.8�

1.4

5.1�

0.8

7.6�

1.2

8.2�

0.2

8.8�

0.1

9.2�

0.3

1872 | J. Mater. Chem. B, 2014, 2, 1866–1878 This journal is © The Royal Society of Chemistry 2014


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In agreement with the degradation results, the compositionof MSM-5, MSM-10 and MSM-20 scaffolds signicantly affectedthe pH values of the SBF solution with pH remaining almostunchanged at 7.4 for M, M-1 and M-2 scaffolds during the28 days of soaking.

In contrast, the pH of the SBF containing MSM-10 and MSM-20 scaffolds increased to �8 and �8.6 respectively, aer 7 days(Fig 3). For MSM-10 scaffolds the pH stabilised to �8.2 aer28 days whereas the pH for MSM-20 scaffolds showed a gradualincrease to �9.

Except for M scaffolds, the weights of all the scaffoldsdecreased signicantly aer soaking in SBF and this decreasecorresponded exponentially to the soaking time (Fig. 3e). Aer28 days, weight loss for M, M-1, M-2, MSM-5, MSM-10 andMSM-20 reached �1.1%, �2.2%, 3.2%, 6.3%, 14.2% and 18.8%,respectively. The order of weight loss seen paralleled thatobserved for the release of Sr, Si and Mg ions. Fig. 4 shows thesurface microstructure of M and MSM-10 scaffolds aer 28 daysof soaking in an SBF solution. The surface microstructure of M-1 and M-2 was similar to that of M and the surface micro-structure of MSM-5 and MSM-20 was similar to that of MSM-10scaffolds aer soaking in SBF.

The M, M-1 and M-2 surfaces were free of mineral precipi-tates and remained almost intact during the 28 days of soakingin SBF (Fig. 4a). The surface of MSM-10 contained submicronholes, grooves and nanoscale precipitates providing evidence ofa degradation process (Fig. 4b). The holes were formed due todissolution of MgO grains in triple junctions and inside the

Fig. 4 The microstructure of (a) M and (b–d) MSM-10 scaffolds after 28

Fig. 5 Compressive strength of M, M-1, M-2, MSM-5, MSM-10 and MScompressive strength of the scaffolds at 85% porosity after soaking in SB


Sr3Si5 grains (Fig. 4c). EDS analysis showed that the precipitates(Fig. 4d) consisted of Ca, P, Na, Cl and Mg elements.

The compressive strength values of M, M-1, M-2, MSM-5,MSM-10 and MSM-20 scaffolds were measured at differentporosities (85%, 74% and 66%) (Fig. 5a). Compressive strengthvalues for all of the scaffolds decreased markedly as theporosity increased. At a porosity of 85%, the compressivestrength values of M, M-1, and M-2 scaffolds were 1.15 MPa,0.9 MPa and 0.95 MPa respectively, and increased to 14.1 MPaand 12.1 MPa and 21.7 MPa when the porosity decreased to66%. Compressive strength values of MSM-5 and MSM-10scaffolds at 85% porosity were 0.8 MPa and 0.85 MPa respec-tively, and increased to 10.2 MPa and 11 MPa at the 66%porosity. MSM-20 scaffolds had the lowest compressivestrength, 0.6 to 6.1 MPa, at porosities of 85% and 66%,respectively. However the compressive strength of all scaffoldstested (regardless of their porosities) remained signicantlyhigher than that of BCP scaffolds.

Compressive strength of the scaffolds with 85% porosity wasmeasured aer soaking in SBF up to 28 days. M, M-1 and M-2scaffolds showed the least decrease in compressive strength(Fig. 5b), compared to the marked decrease (from 0.6 MPa to0.2MPa) seen for MSM-20 scaffolds and themild decrease (from0.8 MPa to 0.62) for MSM-10 scaffolds aer 28 days of soaking inSBF. Therefore, the MSM-20 scaffolds were subsequentlyomitted from the in vitro and in vivo bioactivity tests.

HOB attachment and morphology on the M, M-2 and MSM-10 were examined by SEM (Fig. 6a–b). Aer 2 h and 24 h culture,

days' soaking in SBF.

M-20 scaffolds (a) at different porosities (85%, 74% and 66%) and (b)F for different time periods.

J. Mater. Chem. B, 2014, 2, 1866–1878 | 1873


Fig. 6 Morphology of cultured HOBs on (a) M, (b) M-2 and (c) MSM-10 after 24 h showing close adhesion and spreading of the HOBs across theceramic surface (insets: formation of granules on the cell surfaces of MSM-10 scaffolds). (d) Proliferation of HOB on BCP, M, M-1, M-2, MSM-5and MSM-10 after 3 and 7 days' culture (*p < 0.05).


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HOB attached to the surface of the various scaffolds with almostsimilar morphology with a attened appearance. At 24 h,attached HOB cells on scaffolds were spread out and exhibitedan elongated morphology with the formation of extended lo-podia. Close observation by SEM showed that some granuleswere formed on the cell surfaces of MSM-10 scaffolds whichmay be an indication of early biomineralization. Fig. 6 showsproliferation of HOBs on the BCP, M, M-1, M-2, MSM-5 and

Fig. 7 The effects of (a) M, (b) M-1, (c) M-2, (d) MSM-5 and (e) MSM-proliferation (*p < 0.05, number of the cells increased significantly from d(the control group is the culture medium without a ceramic extract).

1874 | J. Mater. Chem. B, 2014, 2, 1866–1878

MSM-10 over the 3 and 7 days of culture. For all of the groups,cell activity increased signicantly from 3 to 7 days of culture.Aer 3 days, M-1 and M-2 scaffolds exhibited a signicantincrease in cell proliferation compared to MSM-5 and MSM-10scaffolds where proliferation has slowed down. Fig. 7 shows theresults of the cytotoxicity test of the extracts of M, M-1, M-2,MSM-5 andMSM-10 on HOB cells aer incubation for 1, 3 and 7days. The extracts of M, M-1, M-2, MSM-5 and MSM-10 did not

10 extracts with different extract concentrations (mg ml�1) on HOBay 1 to day 3 and day 3 to day 7, regardless of the extract concentration)



Fig. 8 HOB osteogenic gene expression profiles cultured on BCP, M, M-2 and MSM-10 scaffolds. Gene expression levels for (a) osteopontin and(b) osteocalcin were significantly higher on MSM-10 scaffolds than those on other scaffolds on days 1 and 7 (for osteopontin) and on day 7 (forosteocalcin); osteocalcin levels were significantly higher on M-1 and M, compared to that for BCP scaffolds on days 1 and 7; (c) Runx2 wassignificantly higher in MSM-10 scaffolds compared to the other scaffolds on days 1 and 7 and on M-1 and M scaffolds Runx2 was significantlyhigher, compared to levels on BCP scaffolds on days 1 and 7 and (d) BSP onMSM-10 scaffolds was significantly higher than that for other scaffoldson day 7. BSP gene expression on M-1 and M scaffolds was significantly higher than that on BCP scaffolds on days 1 and 7 (*p < 0.05).


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show any toxic effects on HOBs even at the high concentrationswhich indicated the good biocompatibility of these materials.

Fig. 8(a–d) show osteogenic gene expression proles forHOBs cultured on BCP, M, M-2 and MSM-10 scaffolds. qRT-PCRresults showed that the HOB seeded for 3 and/or 7 days onMSM-10 scaffolds expressed signicantly higher levels ofosteopontin and Runx2 than those on HA/TCP, M and M-2scaffolds. Moreover, MSM-10 scaffolds expressed signicantlyhigher levels of BSP (bone sialoprotein) and osteocalcin thanthose on BCP, M and M-2 scaffolds at day 7. M-1 and M-2

Fig. 9 Representative images of toluidine blue stained plastic embeddeweeks of implant subcutaneous transplantation into the dorsal surface i


scaffolds expressed signicantly higher levels of osteopontin,osteocalcin, Runx2 and BSP than those on BCP at 3 and/or7 days.

For the in vivo studies, BCP, M-1, MSM-5 and MSM-10 in thegranule form were seeded with human mesenchymal stem cells(hMSC) and subcutaneously transplanted into the dorsalsurface of eight-week-old immunocompromised (NOD/SCID)mice to characterise the local tissue response to these materials.At the 8 week time point, no macroscopic signs of inammationor toxicity were evident in the tissue surrounding the implants

d sections of the (a) BCP, (b) M-1, (c) MSM-5 and (d) MSM-10 after 8n 8 week old immunocompromised (NOD/SCID) mice.

J. Mater. Chem. B, 2014, 2, 1866–1878 | 1875



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(Fig 9). Furthermore, animals looked healthy at all times.Interestingly, the tissue formed with the triphasic ceramicsvaried signicantly. Using light microscopy we conrmed thatwhen M-1 was implanted, dispersed broblastic cells andpartially degraded particles (Fig 9a, white arrows) were foundthroughout the tissue, with the appearance of a small amount ofbone (Fig 9a, black arrows). When the MSM-5 ceramic wasimplanted a noticeable amount of a new bone was formedintegrating throughout the ceramic (Fig 9b, white arrow heads),compared to a signicant amount of new bone formationevident when the MSM-10 was implanted (Fig 9d, black arrows).BCP induced new bone formation within the implanted pelletsof 2.4% � 2.8 vs. 6.3 � 6.4% and 5.4 � 3.1% for the M-5 and M-10 respectively.

4. Discussion

In this study we successfully developed a new and bioactiveceramic (MSM-10) that has the ability to simultaneously releasethree bioactive ions, essential for providing the necessarysignals required for bone formation, into the surroundingenvironment. A series of ceramics was prepared by introducingdifferent molar percentages of Sr (1 to 20 mol%) into a solution(sol) containing a xed ratio of Mg/Si atoms (Table 1). Intro-ducing Sr up to 2 mol% (M, M-1 and M-2) resulted in formationof pure Mg2SiO4. Mg2SiO4 has a dense structure consisting ofthe anion SiO4

4� and the cation Mg2+ in an orthorhombiccrystal structure. Mg2+ ions are located in two different spaces ofthe lattice where one space is larger and uniform (in b direction)compared to that in the c and a direction, and therefore can be apotential site for substitution by larger ions. However the strongrepulsion forces between the oxygen atoms result in the crystalstructure being adapted in a manner that minimizes theseforces. Although introducing Sr up to 2% did not result in theformation of any new phases, the crystal aspect ratio andvolume parameters of Mg2SiO4 increased linearly withincreasing amounts of Sr and also grain morphology changedsignicantly compared to that of pure Mg2SiO4. This is due tothe substitution of small atoms by larger atoms with the ionicradius of Sr being approximately 1.5 times higher than that ofMg. The increasing aspect ratio of the crystal (b/a and b/c) can beattributed to a preferred substitution of Mg ions located in the bdirection.

Introducing 5 to 20 mol% Sr (MSM-5, MSM-10, and MSM-20)resulted in the formation of Si3Sr5 and MgO phases upon sin-tering the scaffolds. M-1 and M-2 had a slight increase in theirdegradation rate (marginal increase in ion release pattern andweight loss) compared to M, probably due to the disruption ofthe uniformity of the Mg2SiO4 crystal structure. Formation ofSi3Sr5 and MgO in MSM-5, MSM-10 and MSM-20 was associatedwith an increase in the degradation rate and a decrease inmechanical properties.

MSM-10 demonstrated a balanced composition withcombined degradability and good mechanical properties in ahighly porous form. MSM-20 showed the lowest mechanicalproperties especially under wet conditions and MSM-5 showeda compromised degradability.

1876 | J. Mater. Chem. B, 2014, 2, 1866–1878

BCP scaffolds compared with other scaffolds had theweakest compressive strength under both dry and wet condi-tions. BCP has a poor thermal stability therefore sinteringparameters (temperature and time) must be adjusted in a way toobtain a right ratio of b-TCP to HA (60 wt% b-TCP/40 wt% HAwas used in this study) without compromising the densicationprocess; a process in which the ceramic structure becomesstronger through atomic diffusionmechanisms and it is directlydependent on the sintering time and temperature. In contrast,the developed materials have a high thermal stability; hencethey can be fully sintered at high temperatures which will resultin a much stronger and denser microstructure.

Mg, Sr and Si ions were actively released fromMSM-10 due tothe dissolution of MgO, Si3Sr5 and Mg2SiO4 phases. The releaseof these ions has the potential to enhance osteogenesis and insome cases angiogenesis within scaffold materials enhancingtheir potential as therapeutic agents.10,59 It is well documentedthat these ions, at specic concentrations, are involved in bonemetabolism and play physiological roles in angiogenesis and inthe growth and mineralization of bone tissue. Previous studieshave shown that ionic dissolution products from bioactiveglasses possess the capacity to stimulate osteoblast prolifera-tion and differentiation.66,67 In this study we demonstrated thatit is possible to control the simultaneous release of threeimportant ions (Sr, Mg and Si) by changing the fractions ofSi3Sr5 (25 to 69 wt%), MgO (5 to 20 wt%) and Mg2SiO4 (70 to11 wt%) in the microstructure. The release rates of Sr fromMSM-5, MSM-10 and MSM-20 in the culture medium wereapproximately 0.6, 1.2 and 1.7 ppm per day, respectively. Therelease rates for Si were approximately 0.18, 0.3, and 0.4 ppmper day, respectively and those for Mg were 0.7, 2.1 and 3.9 ppmper day, respectively. Thus it is reasonable to expect that, in vivo,concentration gradients may be formed in the tissue around thedeveloped materials to levels sufficient to enhance materialbioactivity. Degradation trends of materials in SBF, PBS andculture medium were quite similar where MSM-20 showed thehighest and M the least degradation rate (MSM-20 > MSM-10 >MSM-5 > M-2 > M-1 > M). However, there was a signicantdifference in degradation values of a material at a specic timepoint by using different solutions. For example, aer soakingMSM-10 for 28 days in SBF, PBS and culture medium, Mgconcentrations in these solutions reached 160.2, 240 and 80,respectively. These solutions not only have a different initial pH(SBF: 7.4, PBS: 7.3 and culture medium: 7.5) but also a distinctchemical composition and saturation state. This will result inhaving different interactions with the surface of the materialand hence variation in the degradation values.

We note that in this study, MSM-10 scaffolds markedlyincreased the pH of the surrounding in vitro environment tovalues around 8. While increasing pH in vivo has the potential tocause toxicity, recently it has been reported that osteoblastactivity was signicantly enhanced with a modest increase in pHto 8–8.5, where the positive effect of strontium on osteoblastswas further increased.20 Bioactive glasses are well-knownbiocompatible bioceramics with an excellent in vivo bioactivityand they can increase the pH of an aqueous environment to verybasic values. Thus small increases in local pH around an




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implant may not be detrimental. In agreement with this, ourresults further demonstrate that HOBs cultured on MSM-10scaffolds expressed signicantly higher levels of osteopontin,osteocalcin, Runx2 and bone sialoprotein than those on BCP, Mand M-2 scaffolds aer 7 days. These are genetic markers forosteoblast differentiation, with osteocalcin in particular being amarker of late stages of osteoblast maturation. The observationof slowing of proliferation of HOBs grown with MSM-10 is alsoconsistent with differentiation as proliferation and differentia-tion tend to change inversely. Moreover, cell proliferation andcytotoxicity results did not show any harmful response from theMSM-10 ceramic. In vivo new bone formation was evident whenthe MSM-10 ceramic was implanted into eight-week-old immu-nocompromised (NOD/SCID) mice. Together these resultssuggest that MSM-10 promotes the in vitro and in vivo formationof a new bone. However, it remains unclear which ionic products(and at what concentration) were responsible for cell activationand the exact mechanism(s) of interaction between the ionicproducts and cells. If dissolution rates of bioactive materials aretoo rapid, the ionic concentrations of the uid environmentaround the cells will be too high, which will be detrimental tocells. If the rates are too slow, the ion concentrations are too lowto stimulate cellular activity. Other characteristic features of theceramic (topography, grain size, crystallinity and surface chem-istry) might contribute to the overall bioactivity of this mate-rial.39 Identifying an optimum release rate of bioactive ions isdifficult given that it is not possible to determine local ionconcentrations in vivo. In vitro, accumulation of ions over time,pH changes and re-precipitation are complicating factors. It hasbeen reported that Si in the range of 50–103 ppm has aninhibitory effect on growth of osteoblast-like cells; however arange of 0.02–103 ppm results in enhancement of cell activity.68

Other studies demonstrated that less than 2.50 mM Si in theculture medium enhances the proliferation, protein synthesisand ALP activity of osteoblasts, as well as the formation anddevelopment of bone tissues;69 however, a high Si dose mayresult in in vitro toxicity. Sr concentrations from 8.7 to 87.6 ppmwere found to have a stimulatory effect on osteoblasts, andinhibitory effects (at 8.7 to 2102.8 ppm) on osteoclast action.70

The combination of Si and Sr at certain concentration range (Si:1.87–0.12 mM and Sr: 0.12–3.75 mM) was found to exhibit astimulatory effect on mesenchymal stem cells24,32 and Sr ionsshowed a stimulatory effect on osteoblastic activity in vitro whenSr ion concentrations were below 87.6 ppm.70

In this study we successfully developed a triphasic ceramic(MSM-10) with the capability for the simultaneous multiple ionrelease (Sr, Mg and Si) at rates that can be varied, providing thetool for optimisation. We demonstrated that this developedceramic not only has a potential to be used as a carrier forreleasing the bioactive ions but also it attained a compressivestrength within and above the range of human cancellous bone(0.6–15 MPa) at the corresponding porosity of 85 to 66%. Takentogether, the developed triphasic ceramic with its suitablemechanical, physical and biological properties may have a widerange of applications in orthopaedic and maxillofacial areasincluding use as bone void llers, injectable and porous scaf-folds for bone tissue engineering applications.


5. Conclusion

We successfully developed a novel triphasic and bioactive MSM-10 ceramic (containing Mg2SiO4, Si3Sr5 and MgO phases) whichcan simultaneously release Sr, Mg and Si ions into the micro-environment. We demonstrated the in vitro and in vivo bioac-tivity of the MSM-10 ceramic suggesting the potential of thisceramic for use in orthopaedic/maxillofacial applicationsrequiring bone regeneration.

Conflict of interest

There is no conict of interest.

Acknowledgements

The authors acknowledge the Australia National Health andMedical Research Council, Australian Research Council,Australian Orthopedic Association and the Rebecca CooperFoundation. We acknowledge the Australian Center forMicroscopy and Microanalysis (ACMM) at the University ofSydney for their help with microscopic analysis.

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