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Biomaterials 35 (2014) 8553e8565

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Biomaterials

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Osteoimmunomodulatory properties of magnesium scaffolds coatedwith b-tricalcium phosphate

Zetao Chen a, b, Xueli Mao b, c, d, *, Lili Tan e, Thor Friis a, Chengtie Wu b, f, Ross Crawford a, b,Yin Xiao a, b, **

a Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove, Brisbane, Queensland 4059, Australiab Australia-China Centre for Tissue Engineering and Regenerative Medicine, Queensland University of Technology, 60 Musk Ave, Kelvin Grove, Brisbane,Queensland 4059, Australiac Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, 56 Ling Yuan RoadWest, Guangzhou 510055, Chinad Guangdong Provincial Key Laboratory of Stomatology, 74 Zhongshan Second RD, Guangzhou 510080, Chinae Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang 110016, Chinaf State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences,1295 Dingxi Road, Shanghai 200050, China

a r t i c l e i n f o

Article history:Received 14 June 2014Accepted 19 June 2014Available online 11 July 2014

Keywords:MacrophageBone substituteOsteoimmunomodulatory propertyb-TCPMagnesiumOsteogenesis

* Corresponding author. Department of OperativeGuanghua School of Stomatology, Hospital of Stomato56 Ling Yuan Road West, Guangzhou 510055, Chinfax: þ86 20 8386 2807.** Corresponding author. Institute of Health and Bioland University of Technology, 60 Musk Ave, Kelvin4059, Australia. Tel.: þ61 7 3138 6240; fax: þ61 7 313

E-mail addresses: maoxuel@mail.sysu.edu.cn (X(Y. Xiao).

http://dx.doi.org/10.1016/j.biomaterials.2014.06.0380142-9612/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The osteoimmunomodulatory property of bone biomaterials is a vital property determining the in vivofate of the implants. Endowing bone biomaterials with favorable osteoimmunomodulatory properties isof great importance in triggering desired immune response and thus supports the bone healing process.Magnesium (Mg) has been recognized as a revolutionary metal for applications in orthopedics due to itbeing biodegradable, biocompatible, and having osteoconductive properties. However, Mg's high rate ofdegradation leads to an excessive inflammatory response and this has restricted its application in bonetissue engineering. In this study, b-tricalcium phosphate (b-TCP) was used to coat Mg scaffolds in aneffort to modulate the detrimental osteoimmunomodulatory properties of Mg scaffolds, due to the re-ported favorable osteoimmunomodulatory properties of b-TCP. It was noted that macrophages switchedto the M2 extreme phenotype in response to the Mg-b-TCP scaffolds, which could be due to the inhi-bition of the toll like receptor (TLR) signaling pathway. VEGF and BMP2 were significantly upregulated inthe macrophages exposed to Mg-b-TCP scaffolds, indicating pro-osteogenic properties of macrophages inb-TCP modified Mg scaffolds. This was further demonstrated by the macrophage-mediated osteogenicdifferentiation of bone marrow stromal cells (BMSCs). When BMSCs were stimulated by conditionedmedium from macrophages cultured on Mg-b-TCP scaffolds, osteogenic differentiation of BMSCs wassignificantly enhanced; whereas osteoclastogenesis was inhibited, as indicated by the downregualtion ofMCSF, TRAP and inhibition of the RANKL/RANK system. These findings suggest that b-TCP coating of Mgscaffolds can modulate the scaffold's osteoimmunomodulatory properties, shift the immune microen-vironment towards one that favors osteogenesis over osteoclastogenesis. Endowing bone biomaterialswith favorable osteoimmunomodulatory properties can be a highly valuable strategy for the develop-ment or modification of advanced bone biomaterials.

© 2014 Elsevier Ltd. All rights reserved.

Dentistry and Endodontics,logy, Sun Yat-sen University,a. Tel.: þ86 20 8386 1544;

medical Innovation, Queens-Grove, Brisbane, Queensland8 6030.. Mao), yin.xiao@qut.edu.au

1. Introduction

Biomaterial implants exert a profound impact on host immuneresponses, which in turn may elicit detrimental or beneficial re-actions that significantly affect the healing and repair process. Thedesign paradigm of biomaterials have shifted accordingly, frombeing relatively inert to having immunomodulatory properties; theemphasis is now that the implantehost interaction should lead to

Z. Chen et al. / Biomaterials 35 (2014) 8553e85658554

an immune response that is beneficial to the healing process [1].The field of osteoimmunology has demonstrated that the immuneand skeletal systems are closely related and share a number ofcytokines, receptors, signaling molecules and transcription factors[1,2]. The immune response is found to play a pivotal role in bonebiomaterial-stimulated osteogenesis [1,3,4]. This implies that theimmunomodulatory properties of bone substitute materials arevery important in determining the outcome of new bone regener-ation. Given the nature of the bone environment with theinvolvement of bone cells, the immunomodulatory properties forbone biomaterials should be classified as osteoimmunomodulatoryproperties. Optimal osteoimmunomodulatory properties should beable to induce an inflammatory response, which creates an overallbalance between osteogenesis and osteoclastogenesis that favorsbone regeneration. The traditional design parameters for bonebiomaterials have tended to mainly focus on the positive regulationof osteoblastic lineage cells; however, this approach fails to takeinto account the role of immune cells in regulating osteogenesisand osteoclastogenesis during the healing process, which followsthe implantation of the material. We have proposed the need toinclude the response of immune cells, as well as bone cells, whenevaluating novel bone biomaterials [4].

Magnesium (Mg) has been found to be mechanically similar tobone, thereby eliminating the effects of stress shielding [5,6] andhas been proposed as a biodegradable metal, which can poten-tially be used for a wide range of orthopedics applications [5].However, there are still a number of challenges that must beaddressed before Mg can be considered as a suitable scaffoldmaterial, most notably its rapid in vivo degradation, which trig-gers an acute and unfavorable inflammatory response. Excessiveinflammation can lead to the destruction of adjacent normal boneand the formation of fibrous tissue, which is likely to account forthe failure of magnesium scaffolds to mediate bone reconstruc-tion and restricts the application of magnesium scaffold in clinicalsettings.

b-tricalcium phosphate (b-TCP) is a well characterized osteo-conductive biomaterial that is already in clinical use for boneregeneration [7-9]. In physiological conditions, calcium phosphate(CaP) materials degrade at a significantly slower rate compared toMg [10]. Using physicochemical analysis methods, Mg scaffoldscoated with b-TCP have been shown to have a slower degradationrate, [11]. b-TCP extract has also been found to elicit a favorableosteoimmunomodulatory response that can switch the macro-phage phenotype to the M2 extreme and release BMP2, whichenhances osteogenic differentiation of bone marrow stromal cells(BMSCs) [4]. These findings led to the hypothesis that coating Mgscaffolds with b-TCP would result in scaffolds that combined thebeneficial properties of both materials and was endowed withosteoimmunomodulatory properties that would favorosteogenesis.

Macrophages play an important role in the innate immuneresponse that is activated by the interaction between host body andbone substitute materials. The classically activated M1 phenotypeparticipates actively in T helper cell 1 (Th1) type inflammation,whereas the alternatively activated M2 phenotype leads to a Th2type inflammation [12e14]. Macrophages are both dynamic andplastic and the various phenotypes can switch between each otherunder specific circumstances [15,16]. Macrophages are required forefficient osteoblast mineralization and depletion of macrophagesleads to the complete loss of in vivo bone formation [17,18]. Theirrole in osteogenesis is by the secretion of osteoinductive moleculessuch as bone morphogenetic protein 2 (BMP2) and transforminggrowth factor b (TGF-b) [19,20]. The heterogeneity and plasticity ofmacrophages make them an obvious target for immune modula-tion [21] and given their roles in the dynamic flux of bone

formation and homeostasis, the response of macrophages to bonebiomaterials was applied for the evaluation of osteoimmunomo-dulatory properties [4].

In the present study, a chemical immersion method wasapplied [22,23] to coat Mg scaffolds with b-TCP (Mg-b-TCP) in aneffort to manipulate their osteoimmunomodulatory properties. Itwas unknown whether the introduction of b-TCP would alter thedetrimental immune response of Mg materials, so the osteoim-munomodulatory properties were evaluated using a bio-mimicking system that included biomaterials, osteoblastic line-age cells and immune cells. First, the interactions between Mg/Mg-b-TCP scaffolds and macrophages were investigated to assess theinflammatory response and release of osteoclastogenic and oste-ogenic cytokines, as a measure of a modulated immune environ-ment. The osteogenesis mediated by the Mg/Mg-b-TCP scaffoldswas further evaluated in the presence of both macrophages andosteoblastic cells, to demonstrate whether the regulated immuneenvironment could positively enhance osteogenesis. In summary,this study applied the concept of osteoimmunomodulation tomanipulate the properties of bone biomaterials in an effort tocreate an immune environment that favors osteogenesis. It soughtto determine if and how b-TCP coating of Mg scaffolds couldpositively affect their osteoimmunomodulatory properties andwhether or not these immune environment changes would influ-ence osteogenesis.

2. Materials and methods

2.1. Preparation of Mg and Mg-b-TCP scaffolds

Porous Mg scaffolds were prepared with the laser perforation technique using aprogrammable multifunctional laser processing machine. The pulse frequency,width and effective output were 1e10 Hz, 0.3 ms and 100W, respectively. The b-TCPcoating of the Mg scaffolds was performed as follows: the scaffolds were immersedin a super-saturated Na2HPO4 solution at 37 �C for 3 h and, after ultrasonic cleaning,heated at 400 �C for 10 h. Next, theywere immersed in a mixture of Na2HPO4$12H2O(47.5 g/L) and Ca(NO3)2 (36.4 g/L) at 70 �C for 24 h, followed by washes with distilledwater and air drying [22,23]. The morphology of the scaffolds was observed by lightmicroscopy (M125, Leica Microsystem, Wetzlar, Germany) and scanning electronmicroscopy (SEM, Zeiss, Oberkochen, Germany).

2.2. Cell culture

The murine-derived macrophage cell line, RAW 264.7 (RAW), and humanBMSCs were used in this study. RAW cell cultures were maintained in Dulbecco'smodified Eagle Medium (DMEM; Life Technologies, VIC, Australia) supplementedwith 5% heat inactivated fetal bovine serum (FBS, Thermo Scientific) and 1% (v/v)penicillin/streptomycin (Life Technologies) at 37 �C in a humidified CO2 incubator.The growing cells were passaged at approximately 80% confluence by scrapingand expanded for two passages before being used for the study.

BMSCs were isolated and cultured based on protocols from previous studies[4,24e26]. Briefly, bone marrow was obtained from patients (50e60 years old)undergoing hip and knee replacement surgery after informed consent was given.The procedure was approved by the Ethics Committee of the Queensland Uni-versity of Technology. Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) was addedto isolate the mononuclear cells from the bone marrow by density gradientcentrifugation. The obtained cells were seeded into the tissue culture flaskscontaining DMEM supplemented with 10% FBS and 1% penicillin/streptomycinand incubated at 37 �C in a humidified CO2 incubator. The culture medium waschanged every 3 days until the primary mesenchymal cells reached 80% conflu-ence; unattached hematopoietic cells were removed by the media changes. Theconfluent cells were expanded and only early passages (p3e5) of cells were usedin this study.

2.3. In vitro degradation of Mg and Mg-b-TCP scaffolds

RAW cells were seeded on the Mg and Mg-b-TCP scaffolds (8 � 8 � 8 mm) at adensity of 1 � 106 cells per scaffold, with scaffolds soaked in the growth mediumserved as controls. The growth medium and scaffolds were collected after 24 h in-cubation. The ionic concentration of Mg, Ca and P in the conditioned media wasdetermined by inductively coupled plasma mass spectrometry (ICP-MS, Perki-nElmer, MA, USA). The scaffolds were then examined by SEM to document degra-dation and changes to the pore size. For this purpose ten pores were chosenrandomly from each sample.

Table 1Primer pairs used in the qRT-PCR.

Genes Primer sequences

IL10 Forward: 50-GAGAAGCATGGCCCAGAAATC-30

Reverse: 50-GAGAAATCGATGACAGCGCC-30

IL1ra Forward: 50-CTCCAGCTGGAGGAAGTTAAC-30

Reverse: 50-CTGACTCAAAGCTGGTGGTG-30

TNFa Forward: 50-CTGAACTTCGGGGTGATCGG-30

Reverse: 50-GGCTTGTCACTCGAATTTTGAGA-30

IL1b Forward: 50-TGGAGAGTGTGGATCCCAAG-30

Reverse: 50-GGTGCTGATGTACCAGTTGG-30

IL6 Forward: 50-ATAGTCCTTCCTACCCCAATTTCC-30

Reverse: 50-GATGAATTGGATGGTCTTGGTCC-30

IFNn Forward: 50-GGCCATCAGCAACAACATAAGC-30

Reverse: 50-GGGTTGTTGACCTCAAACTTGG-30

BMP2 Forward: 50-GCTCCACAAACGAGAAAAGC-30

Reverse: 50-AGCAAGGGGAAAAGGACACT-30

VEGFa Forward: 50-GTCCCATGAAGTGATCAAGTTC-30

Reverse: 50-TCTGCATGGTGATGTTGCTCTCTG-30

TGFb1 Forward: 50-CAGTACAGCAAGGTCCTTGC-30

Reverse: 50-ACGTAGTAGACGATGGGCAG-30

TGFb3 Forward: 50-CAACACCCTGAACCCAGAG-30

Reverse: 50-CTTCACCACCATGTTGGACAG-30

MCSF Forward: 50-AGCAGAACAAGGCCT GTGTC-30

Reverse: 50-AAGCTGTTGTTGCAGTTCTTG G-30

TRAP Forward: 50-CACTCCCACCCTGAGATTTGT-30

Reverse: 50-CATCGTCTGCACGGTTCTG-30

Myd88 Forward: 50-AGGTAAGCAGCAGAACCAGG-30

Reverse: 50-TGTCCTAGGGGGTCATCAAGG-30

Ticam1 Forward: 50-AGATGGTTCAGCTGGGTGTC-30

Reverse: 50-TGGAGTCTCAAGAAGGGGTTC-30

Ticam2 Forward: 50-CTTGGCGCTGCAAACCATC-30

Reverse: 50-GCCTCTCAAATACAGACTCCCG-30

GAPDH(mouse)

Forward: 50-TGACCACAGTCCATGCCATC-30

Reverse: 50-GACGGACACATTGGGGGTAG-30

OPG Forward 50-CGCTCGTGTTTCTGGACATCT-30

Reverse 50-CACACGGTCTTCCACTTTGC-30

RANKL Forward: 50-AGAGCAGAGAAAGCGATGGTG-30

Reverse: 50-GAACCAGATGGGATGTCGGT-30

MCSF (human) Forward: 50-AGCATGACAAGGCCTGCGTC-30

Reverse: 50-AAGCTGTTGTTGCAGTTCTTGC-30

ALP Forward: 50-TCAGAAGCTAACACCAACG-30

Reverse: 50-TTGTACGTCTTGGAGAGGGC-30

OPN Forward: 50- TCACCTGTGCCATACCAGTTAA-30

Reverse: 50-TGAGATGGGTCAGGGTTTAGC-30

OCN Forward: 50-GCAAAGGTGCAGCCTTTGTG-30

Reverse: 50-GGCTCCCAGCCATTGATACAG-30

COL1A1 Forward: 50-AGAACAGCGTGGCCT-30

Reverse: 50-TCCGGTGTGACTCGT-30

IBSP Forward: 50-TTTCTGCTACAACACTGGGCTATG-30

Reverse: 50-TTGTTATATCCCCAGCCTTCTTG-30

SMAD1 Forward: 50-GTATGAGCTTTGTGAAGGGC-30

Reverse: 50-TAAGAACTTTATCCAGCCACTGG-30

SMAD4 Forward: 50-CTCCAGCTATCAGTCTGTCAG-30

Reverse: 50-CCCGGTGTAAGTGAATTTCAAT-30

SMAD5 Forward: 50-TCATCATGGCTTTCATCCCACC-30

Reverse: 50-GCTCCCCAACCCTTGACAAA-30

BMPR2 Forward: 50-GGCAGCAGTATACAGATAGGTG-30

Reverse: 50-CTGCCCTGTTACTGCCATTATT-30

BMP1A Forward: 50-CCTGGGCCTTGCTGTTAAATTCA-30

Reverse: 50-TCCACGATCCCTCCTGTGAT-30

GAPDH (human) Forward: 50-TCAGCAATGCCTCCTGCAC-30

Reverse: 50-TCTGGGTGGCAGTGATGGC-30

Z. Chen et al. / Biomaterials 35 (2014) 8553e8565 8555

2.4. Effects of Mg and Mg-b-TCP scaffolds on RAW cells

2.4.1. The expression of inflammatory genes by RAW cells cultured on Mg and Mg-b-TCP scaffolds

RAW cells were seeded on scaffolds at a density of 1�106 cells per scaffold. Afterincubation for 24 and 36 h, the supernatants were collected and clarified bycentrifugation at 1500 rpm for 5 min. The scaffolds were washed twice with PBS andtotal RNA from the cells extracted using TRIzol reagent (Life Technologies). Fivehundred nanograms of total RNAwas used to synthesize complementary DNA usingthe DyNAmo™ cDNA Synthesis Kit (Genesearch, QLD, Australia) following themanufacturer's instructions. RT-qPCR assay was applied to detect the expression ofinflammatory genes (IL10, IL1ra, TNFa, IL6, IL1b, IFNg). GAPDH was used as house-keeping gene. RT-qPCR primers (Table 1) were designed using the Primer-BLASTprogram on the National Center for Biotechnology Information website. SYBRGreen qPCR Master Mix (Life Technologies) was used for detection, and the targetmRNA expressions were assayed on an ABI Prism 7500 Thermal Cycler (AppliedBiosystems, VIC, Australia). The mean cycle threshold (Ct) value of each target genewas normalized against the Ct value of the housekeeping gene. The DDCt methodwas applied to compare the mRNA expression levels between Mg and Mg-b-TCPscaffolds.

2.4.2. M1 and M2 surface markers changes of RAW cellsExpression of the M1 and M2macrophage cell surface markers CCR7 and CD163

were estimated by flow cytometry to determine phenotypic changes to the RAWcells. Complete culture mediumwas supplemented with the collected supernatantsdetailed in Section 2.4.1 at a ratio of 1:3 to obtain the conditioned medium. RAWcells were seeded on T25 culture flask at a density of 1 � 106 cells. After 24 h ofculture, the culture medium was replaced with conditioned medium and the cellswere incubated for a further 24 h. The cells were detached by cell scraper andnonspecific macrophage proteins were blocked with 1% BSA/PBS. The samples wereincubated with CCR7 (1:25) (GeneTex, Irvine, CA, USA) and CD163 antibody (1:100)(AbD Serotec, Raleigh, NC, USA) for 30 min at 4 �C, followed by incubation withDylight 488-anti-mouse and DyLight 405- anti-goat secondary antibody (DAKO,Multilink, California, USA) for 30 min at 4 �C. After washing with 1% BSA/PBS, theexpression was analyzed on a flow cytometer (FACSAria, BD Biosciences, NSW,Australia). The percentages of positively stained cells were determined andcompared.

2.4.3. Mechanisms of the inflammatory regulationThe Toll-like receptor (TLR) signaling pathway was analyzed to understand the

mechanisms that underlie the observed gene expression changes. RAW cells wereseeded on scaffolds at a density of 1 � 106 cells per scaffold and incubated for 24 h.The culture medium was removed. After washed twice by PBS, TRIzol reagent (LifeTechnologies) was added to the scaffold for extraction of RNA. Gene expressions ofmyeloid differentiation factor 88 (MyD88) and TIR domain containing adapter inducinginterferon-beta (TRIF) were detected following the samemethod described in section2.4.1. The whole cell lysates were collected after 7 h of culture and the proteinexpression of nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor alpha (IkB-a) determined by Western blot analysis. Fifteen micrograms ofprotein from each sample were separated by SDS-PAGE and transferred to a nitro-cellulose membrane (Pall Australia, NSW, Australia). The membranes were blockedin Odyssey blocking buffer for 1 h (LI-COR Biosciences, Millenium Science, VIC,Australia), and then incubatedwith primary antibodies against IkB-a (1:1,000, rabbitanti-human; Cell Signaling Technology, Genesearch, Australia) and a-tubulin(1:5,000, rabbit anti-human; Abcam, Sapphire Bioscience, NSW, Australia) overnightat 4 �C. The membranes were washed three times in TBS-Tween buffer and thenincubated with-anti-mouse/rabbit HRP conjugated secondary antibodies at 1: 4000dilutions for 1 h at room temperature. The protein bands were visualized using theOdyssey infrared imaging system (LI-COR Biosciences). The relative intensity ofprotein bands was quantified using the Image J software.

2.4.4. Osteogenesis-related and osteoclastogenesis-related gene expressionsTo investigate the effect of Mg and Mg-b-TCP scaffolds on osteogenesis and

osteoclastogenesis, osteogenesis-related genes (BMP2, VEGF, TGF-b1 and TGF-b3)and osteoclastogenesis-related genes [macrophage colony-stimulating factor (MCSF)and tartrate-resistant acid phosphatase (TRAP)] were analyzed by RT-qPCR, usingGAPDH as housekeeping gene. RAW cell seeding and treatment were as described inSection 2.4.1.

Given the close relationship between osteoclastic and osteoblastic cells, wefurther investigated the osteoclastogenesis-related factors [osteoprotegerin (OPG),MCSF, receptor activator of nuclear factor kappa-B ligand (RANKL)] release from BMSCsin response to stimulation with conditioned media. The BMSCs were cultured ingrowth medium supplemented with the harvested supernatants as described inSection 2.4.1, at a ratio of 1:3 (the conditioned media). BMSCs were plated at adensity of 1.5 � 105 cells per well in separate 6-well plates. After 24 h of incubation,the culture mediumwas removed and replaced with conditioned or control growthmedium. Total RNA for the RT-qPCR analysis was harvested after 3 days of cultureusing TRIzol reagent.

2.5. Effects of RAW cells-conditioned media on the osteogenic differentiation ofBMSCs

2.5.1. Bone-related gene and protein expression of BMSCsThe ability of RAW cells to regulate osteogenesis in BMSCs in response to ex-

tracts released from the scaffolds was investigated. BMSCs were plated at a densityof 1.5 � 105 cells per well in separate 6-well plates. After 24 h of incubation, theculturemediumwas removed and replaced with conditioned or control media. TotalRNA for RT-qPCR analysis was harvested after 7 days of culture using TRIzol reagentto determine the osteogenesis related gene expressions [Alkaline phosphatase (ALP),Osteopontin (OPN), Osteocalcin (OCN), Bone sialoprotein 2 (IBSP), Collagen type 1(COL1)]. The whole cell lysates were collected after 14 days of culture for WesternBlot analysis. The following primary antibodies were used: ALP (1:10,000, rabbit

Table 2The ionic concentrations of culture medium, scaffold extracts and macrophage/scaffold conditioned medium.

Material e Mg Mg-TCP Mg Mg-TCP

Culture medium þ þ þ þ þMacrophage conditioned e e e þ þIonic concentrations (mg/L) Mg2þ 19.44 ± 2.23 1021 ± 2.13 195.4 ± 0.86 1309 ± 1.41 337.3 ± 1.06

Ca2þ 76.52 ± 1.19 16.42 ± 1.08 129.3 ± 2.09 5.76 ± 2.9 54.3 ± 2.65PO4

3- 29.71 ± 0.5 23.99 ± 1.33 93.61 ± 3.66 22.16 ± 1.01 32.03 ± 2.93

Z. Chen et al. / Biomaterials 35 (2014) 8553e85658556

anti-human; Abcam), OPN (1:1,000, rabbit anti-human; Abcam) and a-tubulin(1:5,000, rabbit anti-human; Abcam).

2.5.2. Alkaline phosphatase activity of BMSCsBMSCs were cultured in 24-well plates at a density of 35,000 cells per well. The

ALP activity was assessed after 10 days of culture in conditioned or control mediawith osteogenic supplements [2 mM b-glycerophosphate, 100 mM L-ascorbic acid 2-phosphate, 10 nM dexamethasone (SigmaeAldrich, NSW, Australia)]. The cellswere lysed in 100 mL of 0.2% Triton X-100 then centrifuged at 14,000 rpm for 15 minat 4 �C. Fifty microliter of supernatant was mixed with 150 mL of working solutionand assayed using the QuantiChromTM Alkaline Phosphatase Assay Kit (BioAssaySystems, Biocore, NSW, Australia). The total protein content wasmeasured using theBCA Protein Assay Kit (Thermo Scientific). The relative ALP activity was calculated asthe changed optical density (OD) values divided by the total protein content.

2.5.3. The mineralization of BMSCsTo identify mineralization nodules, Alizarin Red S stainingwas assayed on day 10

after BMSCs had grown as a monolayer in conditioned or control media with oste-ogenic supplements in a well plate. The mediumwas removed and the cells washedwith ddH2O and fixed in 4% paraformaldehyde for 10 min at room temperature.After gently rinsing with ddH2O, the cells were stained in a solution of 2% AlizarinRed S at pH 4.1 for 20min and thenwashed with ddH2O. The samples were air-dried,and photos were taken under a light microscope.

Quantification of staining was performed as described previously [27]. In brief,350 mL 10% acetic acid was added to each well, and the plate was incubated at roomtemperature for 30 min. The monolayer was then scraped from the plate andtransferred with the acetic acid to a 1.5 mL microcentrifuge tube. After vortexing for30 s, the slurry was overlaid with 300 mL mineral oil (SigmaeAldrich, NSW,

Fig. 1. Macro and microphology of Mg and Mg-b-TCP scaffolds by light microscope and SEM

Australia), heated to 85 �C for 10min, and transferred to ice for 5 min. The slurry wasthen centrifuged at 20,000 g for 15 min and 300 mL of the supernatant was trans-ferred to a fresh 1.5 mL microcentrifuge tube. Then 100 mL of 10% ammonium hy-droxide was added to neutralize the acid. Aliquots (100 mL) of the supernatant wereread in triplicate at the wavelength of 405 nm in a 96-well plate reader.

2.5.4. BMP-related signaling pathway of BMSCsTo investigate the expression of BMP signaling genes in BMSCs, BMP signaling

pathway-related genes [Mothers against decapentaplegic homolog 1/4/5 (SMAD1,SMAD5, and SMAD4, bone morphogenetic protein receptor, type IA (BMPR1A) and Bonemorphogenetic protein receptor type II (BMPR2)] were analyzed by RT-qPCR. Asdescribed in Section 2.5.1, BMSCs were treated for 3 days.

2.6. Statistic analysis

All analyses were performed using the SPSS software (IBM SPSS, Armonk, NewYork, USA). All data are shown as means ± standard deviation (SD) and wereanalyzed using one-way ANOVA. The level of significance was set to P < 0.05.

3. Results

3.1. In vitro degradation of Mg and Mg-b-TCP scaffolds

The concentrations of Mg2þ, Ca2þ and PO3�4 ions from all the

samples are shown in Table 2. After soaking the Mg scaffolds inDMEM for 24 h, the concentrations of Ca2þ and PO3�

4 ionsdecreased to 21% and 81% of their original values, respectively. The

respectively. The scaffolds are 8 � 8 � 8 mm in size and the pore sizes are uniform.

Z. Chen et al. / Biomaterials 35 (2014) 8553e8565 8557

Mg2þ ion concentration, on the other hand, showed a 52 fold in-crease. All three ions concentrations increased in the medium aftersoaking the Mg-b-TCP scaffold in DMEM. The concentration ofMg2þ ions was five times greater in theMg scaffold medium than inthe Mg-b-TCP scaffold medium. Concentrations of both Ca2þ andPO3�

4 ions in the macrophage conditioned culture media decreasedin both the Mg and Mg-b-TCP samples, at 7.5% and 75% of theiroriginal values, respectively. However, the concentration of Mg2þ

ions increased in response to the same treatment with a 67 foldincrease in the Mg scaffold sample and a 17 fold increase in the Mg-b-TCP scaffold sample.

The Mg and Mg-b-TCP scaffolds are shown in Fig. 1. The porediameter of the Mg scaffold was approximately 100 mm (Fig. 2A, E)and decreased to approximately 60 mm after being coated with b-TCP (Fig. 2B, E). Following incubation in culture mediumwith RAWcells, the scaffolds show evidence of being partly degraded by anincrease in pore sizes. The Mg scaffold had a larger increase(147.07 ± 9.55 mm, Fig. 2C, E), compared with that of Mg-b-TCPscaffold (82.69 ± 5.62 mm, Fig. 2D, E).

Fig. 2. Pore size changes of Mg and Mg-b-TCP scaffolds before and after cultured with RAdecreased to approximately 60 mm after coated with b-TCP (B, E). Following incubation147.07 ± 9.55 mm (C, E), while Mg-b-TCP scaffold went up to 82.69 ± 5.62 mm (D, E).

3.2. Inflammatory gene expression and surface marker changes ofRAW cells on scaffolds

Acomparisonof thegeneexpressionofRAWcells grownoneitherthe Mg or Mg-b-TCP scaffolds showed that the most significantchange in inflammatory gene expression occurred with the anti-inflammatory gene IL1ra in Mg-b-TCP scaffolds both in 24 and 36 h(P < 0.05) (Fig. 3). The expressions of other inflammatory genes(TNFa, IL6, IL1b and IFNg) also showed slight increase as a result of thesame treatment (P<0.05) but the fold changeswere significantly lessthan that of IL1ra (Fig. 3). IL10 showed no significant change at all.

Stimulation of RAW cells with the RAW cell/Mg-b-TCP scaffoldconditioned medium (Fig. 4 A, B) resulted in a shift towards the M2phenotype with more cells expressing the surface marker CD163compared to cells stimulated with the RAW cell/Mg scaffoldconditioned medium alone (Fig. 4 C, D). By contrast, the sametreatment resulted in fewer cells expressing the surface markerCCR7 (Fig. 4 E, F) in Mg-b-TCP scaffold group compared to Mgscaffold group (Fig. 4 G, H).

W cells. The pore diameter of the Mg scaffold was approximately 100 mm (A, E) andin culture medium with RAW cells and, the pore size of Mg scaffold increase to

Fig. 3. Relative mRNA expressions of inflammation related genes: IL-10, IL-1ra, TNFa,IL-6, IL-1b and IFNg by RAW cells cultured in Mg and Mg-b-TCP scaffolds at 24 h and36 h. GAPDH was used as housekeeping gene. *: Significant difference (P < 0.05) forMg-b-TCP group compared to Mg group.

Z. Chen et al. / Biomaterials 35 (2014) 8553e85658558

3.3. Possible mechanisms for the changes of inflammatory geneexpression

To explore the mechanisms underlying inflammation-relatedgene expression changes, we examined the TLRs signalingpathway in RAW cells by both the gene and protein expressionassays. We tested three genes (MyD88, Ticam1 and Ticam2) in RAWcells, all of which were significantly downregulated (P < 0.05)(Fig. 5). The downstream molecular IkB-a, by contrast, was upre-gulated (Fig. 6) in Mg-b-TCP scaffolds compared with Mg scaffolds.

3.4. Osteogenesis-related and osteoclastogenesis-related geneexpressions

Osteogenesis and osteoclastogenesis-related gene expressionsin RAW cells cultured on both Mg-b-TCP and Mg scaffolds wereinvestigated by the RT-qPCR. The gene expression for both BMP2and VEGF were significantly upregulated (P < 0.05) in response tothe Mg-b-TCP scaffolds compared with the Mg scaffolds (Fig. 7),whereas both TGF-b1 and TGF-b3 were downregulated in responseto the same treatment (Fig. 7). Moreover, the osteoclastogenesis-related genes MSCF and TRAP were also downregulated in theMg-b-TCP scaffolds compared with the Mg scaffolds.

Although RANKL was also upregulated (2~fold), OPG, a decoyreceptor for RANKL, was more than 4.5~fold upregulation in BMSCsby the stimulation of RAW cell/Mg-b-TCP scaffold conditionedmedium (Fig. 8). MCSFwas downregulated in response to the sametreatment (Fig. 8).

3.5. Effects of RAW 264.7 cells-conditioned media on the osteogenicdifferentiation of BMSCs

BMSCs stimulated with RAW cell/Mg-b-TCP scaffold condi-tioned medium had a significantly upregulated expression of themineralization-related genes (ALP, OPN, OCN, IBSP, COL1) comparedto the Mg scaffold conditioned medium treatment (P < 0.05)(Fig. 8).

The western blot analysis of OPN and ALP protein expressionshowed that both were significantly upregulated in BMSCs

stimulated by RAW cells/Mg-b-TCP scaffold conditioned mediumcompared to that of theMg scaffold conditionedmedium treatment(P < 0.05) (Fig. 9).

The ALP activity of BMSCs stimulated with RAW cell/Mg-b-TCPscaffold conditioned media was significantly enhanced comparedto that of the Mg scaffold-treated group (P < 0.05) and even higherthat of the BMSCs cultured in the osteogenic medium alone(Fig. 10). Alizarin Red S staining showed evidence of calciumdeposition and nodule formation. Mineralized nodules wereobserved in all groups treatedwith osteogenic media (Fig.10). Moredistinct nodules were observed in BMSCs stimulated by RAW cell/Mg-b-TCP scaffold conditioned media (Fig. 10).

3.6. Upregulation of the BMP signaling pathway in BMSCs in thestimulation of the RAW cell/Mg-b-TCP conditioned media

To explore the mechanisms of enhanced osteogenesis in BMSCsstimulated by the RAW cells/Mg-b-TCP conditioned media, wefurther examined the expressions of BMP signaling pathway genes.The expressions of SMAD4, SMAD5 and BMPR1A in BMSCs culturedwith RAW cells/Mg-b-TCP scaffold conditioned media were signif-icantly upregulated compared to those cultured with RAW cells/Mgscaffold conditioned media (P < 0.05) (Fig. 11), indicating the acti-vation of BMP pathways in BMSCs under the stimulation of RAWcells/Mg-b-TCP conditioned media. SMAD1 and BMPR2 showed nosignificant changes under the same treatments.

4. Discussion

In this study, we show that it is possible to manipulate theosteoimmunomodulatory properties of Mg scaffolds by coatingthem with b-TCP. This procedure results in a change in theosteoimmunomodulatory properties of Mg scaffolds thatrendered them significantly more conducive to bone formation.Specifically, b-TCP-coated Mg scaffolds induced macrophages toswitch to the extreme M2 phenotype. This resulted in the releaseof anti-inflammatory cytokines and osteoinductive molecules bythe macrophages that enhanced the osteogenesis of BMSCs, mostlikely through the BMP signaling pathway. In addition, the b-TCPcoating also decreased the rate of degradation of the Mg scaffold,not merely by the physicochemical effects by b-TCP, but also byinhibiting cell-mediated degradation. These findings show thatthe osteoimmunomodulatory properties of the Mg-b-TCP scaffoldwere favorable in terms of osteogenesis and reduced inflamma-tion and osteoclastogenesis. Mg-b-TCP type scaffolds are there-fore a promising bioactive material for bone tissue regeneration,since the b-TCP can endow the otherwise physiologically un-suitable Mg scaffold with favorable osteoimmunomodulatoryproperties. This procedure can therefore be a highly valuablestrategy for development or modification of advanced bonebiomaterials.

4.1. In vitro degradation of Mg-b-TCP scaffolds

Bone substitute materials are normally degraded after implan-tation, either by physicochemical dissolution, cell-medicatingdissolution, hydrolysis, enzymatic decomposition, or corrosion[28]. Mg, being a highly reactive metal, corrodes very quickly,particularly in physiological solutions. Coating of the Mg scaffoldswith b-TCP greatly decreased the direct contact between the sur-face of the metal and culture medium and, thereby slowing downthe rate of corrosion. This was evident by significantly lower con-centration of Mg2þ ions in solution fromMg-b-TCP scaffolds soakedin culture medium compared toMg scaffolds (Table 2). The fact thatboth Ca2þ and PO3�

4 ion concentrations were elevated under the

Fig. 4. FACS results of RAW cells stimulated by macrophage/scaffold conditioning medium: M2 marker CD163 (AeD), M1 marker CCR7 (EeH). A, C, E and G are dot plots, indicatingthe parameters of cell morphology. RAW cells showed similar morphology under different conditioned medium treatments. Same region (red color) was selected in each group forthe histogram analysis (B, D, F, H). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Z. Chen et al. / Biomaterials 35 (2014) 8553e8565 8559

same treatment (Table 2) could account for some of the reducedrate of Mg degradation by raising the pH [11].

Cell-mediated degradation is another key component for thedegradation of a material in a physiological system. Both osteo-clastic and osteoblastic cells participate in this process. Osteoclasticcells are key mediators of cell-mediating degradation. Individual

pre-osteoclast macrophages can engulf particles up to a size of 5 mm[1,29]. If the particle is larger, macrophages attempt to coalesce toform foreign body giant cells (FBGCs), a process that can be inducedby the secreted cytokines IL-4 and IL-13 [30]. With the induction ofRANKL and MCSF, macrophages can differentiate into osteoclasts[31], which attach firmly to the substrate to create a “sealing zone”

Fig. 5. Relative mRNA expressions at 24 h and 36 h of MyD88, Ticam1 and Ticam2 byRAW cells cultured with Mg and Mg-b-TCP scaffolds. GAPDHwas used as housekeepinggene. *: Significant difference (P < 0.05) for Mg-b-TCP group compared to Mg group. Fig. 7. Relative mRNA expressions of BMP2, VEGF, TGF b1, TGF b3, MCSF, and TRAP by

RAW cells cultured in Mg and Mg-b-TCP scaffolds at 24 h and 36 h. GAPDH was used ashousekeeping gene. *: Significant difference (P < 0.05) for Mg-b-TCP group comparedto Mg group.

Z. Chen et al. / Biomaterials 35 (2014) 8553e85658560

into which they secrete Hþ, leading to a locally acidic environmentwith a pH of 4.0e5.0 [32]. In bone, osteoblastic cells are a source ofMCSF and RANKL molecules [33], regulating the differentiation andmaturation of osteoclasts, thereby contributing to cell-mediatingmaterial degradation.

The Mg-b-TCP scaffold appears to dampen cell-inducedmaterialdegradation by the RAW macrophage cells, which was evidencedby the Mg scaffolds having increased pore size (Fig. 2) compared toMg-b-TCP scaffolds. This is in addition to the physiochemical effectsof, for example, lower pH from the greater concentration of Ca2þ

and PO3�4 due to b-TCP [11]. The RANKL/RANK/OPG signaling sys-

tem is a well-known regulator of osteoclastogenesis. RANKL bindsto RANK receptors on the surface of osteoclast precursors to acti-vate signaling through the NF-kB activator protein 1 (AP-1) andnuclear factor of activated T cells 2 (NFAT2), resulting in the

Fig. 6. The western blot analysis of IkB-a expression of RAW cells cultured in Mg andMg-b-TCP scaffolds. *: Significant difference (P < 0.05) for Mg-b-TCP group comparedto Mg group.

expression of genes responsible for the survival and differentiationof osteoclasts [34]. OPG, which was strongly upregulated in BMSCsstimulated with RAW/Mg-b-TCP condition medium (Fig. 8), is adecoy receptor that binds RANKL and interrupt its interaction withRANK receptor to inhibit osteoclastogenesis [35,36]. MCSF isanother key factor for the differentiation of osteoclasts, that bindsto its cognate receptor, c-fms, on osteoclast precursors (e.g.,macrophage), which then activates signaling through the proteinkinase B and mitogen-activated protein kinases (MAPK) pathways[33]. TRAP, which is highly expressed by osteoclasts, is commonlyused as a marker of osteoclast function [37]. The inhibition of theRANKL/RANK system and downregulation of the MCSF and TRAPgenes gives an indication of inhibition of cell-mediated degradationby the b-TCP modified Mg scaffolds. We must point out thatalthough pre-osteoclasts macrophages were used to representosteoclastic cells and BMSCs used to represent osteoblastic celllineages, the use of more committed cell types, such as osteoclastsand osteoblasts, would have allowed more conclusive hypothesesto be made.

Fig. 8. Relative mRNA expressions of OPG, MCSF, RANKL, ALP, OPN, OCN, IBSP, and COL1by BMSCs stimulated by macrophage/scaffold conditioned medium. GAPDH was usedas housekeeping gene. *: Significant difference (P < 0.05) for Mg-b-TCP groupcompared to Mg group.

Fig. 9. The western blotting analysis of OPN and ALP expression by BMSCs cultured in culture medium (Negative) and macrophage/scaffold conditioning medium. *: Significantdifference (P < 0.05) for Mg-b-TCP group compared to Mg group.

Z. Chen et al. / Biomaterials 35 (2014) 8553e8565 8561

4.2. Macrophage phenotype and Mg-b-TCP scaffolds

Macrophage phenotypes follow a continuum between the M1and M2 designations [38]. Three criteria (functional properties,surface markers, and inducers) were used to define a specificmacrophage phenotype [15]. Exposure to the Mg-b-TCP scaffoldsresulted in more macrophages expressing the M2 surface markerCD163 and anti-inflammatory cytokines (IL1ra) compared to cellsexposed to the Mg scaffold. These findings indicate that the Mg-b-TCP scaffolds can influence macrophage polarization by inducing adrift towards the M2 extremity.

Fig. 10. The relative ALP activity of BMSCs cultured in osteogenic medium (OM) and macrstaining of BMSCs cultured in osteogenic medium (OM) and macrophage/scaffold conditioninb-TCP group compared to Mg group.

Macrophages recognize foreign bodies via the toll-like receptor(TLR) pathway, which induces the innate immune response in anattempt to degrade or expel the foreign bodies [1]. MyD88 is one ofthe key components in this pathway. Most of the activated TLRsinteract with MyD88, which then activates the downstreamcascade [39], while some TLRs act via MyD88-independentsignaling pathways, i.e., the toll-like receptor adapter molecule(Ticam), which also known as TIR domain containing adapterinducing IFN b (TRIF) [40]. Although they signal via differentadapter proteins, both MyD88-dependent and Ticam-dependentpathways eventually activate NF-kB, which results in the

ophage/scaffold conditioned medium with osteogenic supplements (A). Alizarin Red Sg mediumwith osteogenic supplements (B). *: Significant difference (P < 0.05) for Mg-

Fig. 11. Relative mRNA expressions of BMP signaling pathway genes SMAD1, SMAD4,SMAD5, BMPR1A, and BMPR2 by BMSCs stimulated by macrophage/scaffold condi-tioned medium. GAPDH was used as housekeeping gene. *: Significant difference(P < 0.05) for Mg-b-TCP group compared to Mg group.

Z. Chen et al. / Biomaterials 35 (2014) 8553e85658562

expression of inflammatory cytokines [41]. In the present study, theexpressions of the MyD88, Ticam1 and Ticam2 genes were alldownregulated, while the expression of the NFkB inhibitor IkB-awas upregulated. This finding indicates that the b-TCP coated Mgscaffolds dampens the inflammatory response potentially byinhibiting the TLR pathway. MgSO4 has been shown to decreaseinflammatory cytokine production via the inhibition of the TLRreceptor pathway, however, the Mg2þ concentration used in therecently published in vitro study by Sugimoto et al.,[42] was only60 mg/L, which is significantly lower than the concentrations of thescaffold extracts used in the present study. The link between Mg2þ

concentration and the inhibition of the TLR receptor pathwaytherefore requires further study.

4.3. Effects of the switch on osteogenesis

Although macrophages have been shown to be involved inosteogenesis, the phenotype that is most beneficial to osteogenesishas not yet been established. M1 macrophages are reported toinduce osteogenesis in mesenchymal stem cells via Oncostatin M[43]. However, the culture medium from LPS-activated macro-phages also induced pre-osteoblast cells to differentiate towardsfibroblasts, even during osteogenic media conditions. This findingmay be related to the upregulation of TNFa, IL-1b and IL-6 [44,45],indicating that the switch to the M1 macrophage phenotype doesnot necessarily enhance osteogenesis. In addition to the secretionof osteoinductive and osteogenic cytokines (BMP2, VEGF), M2macrophages can also secrete fibrous agents (such as TGF-bs) topromote pathological fibrosis [45,46]. An excessive switch to theM2 phenotype may result in scar tissue or a delay inwound healing[47]. Therefore, an effective and adequate switch in the macro-phage phenotype may be a more important means of enhancingosteogenesis.

Angiogenesis and osteogenesis are processes that are closelycoupled during bone formation. BMP2 is an important member ofthe BMP family, and a potent osteoinductive agent [48e50],whereas VEGF is a key pro-angiogenic factor, binding to VEGFR toactivate downstream adapters, which drive angiogenesis [51]. VEGFand BMP2 expression has been found to be tightly related andsynergistic [52]. BMP2 is known to activate endothelial cell growthvia the activation of VEGF/VEGFR2 and angiopoietin/Tie2 signaling,while VEGF reportedly enhances BMP2-induced osteogenesis [53].

The inhibition of VEGF could block BMP2-induced angiogenesis andBMP4-induced bone formation [54].In this study, we found that theMg-b-TCP scaffold significantly upregulated the BMP2 and VEGFgene expression in macrophages, which probably accounted for theactivation of the BMP signaling pathway. BMP2 binds to BMPR2which then recruits BMPR1A. This in turn activates the cytosolicreceptor smads, SMAD5, which then form a complex with SMAD4and translocates to the nucleus to facilitate the expression of dis-talless homeobox5 (Dlx5). Dlx5 induced the expression of Runx2,which results in osteogenic differentiation of BMSCs [55].

The coating of b-TCP led to the decrease of scaffold pore size(from 107 mm to 63 mm). Small pores can relatively negatively affectthe diffusion of nutrients and oxygen supplied from blood andinterstitial fluid, especially in the centre of the scaffold, leading to ahypoxic environment [56]. Hypoxia is stabilizes the hypoxia-inducible factors (HIFs) and subsequently activating HIF targetgenes, in particular VEGF [57,58], which was robustly upreguated inthe Mg-b-TCP scaffold samples (Fig. 7).

Elevated extracellular Ca concentration or CaP crystals havebeen reported to stimulate the secretion of BMP2 by macrophages[19], which could be responsible for the aforementioned obser-vation. The calcium sensing receptor (CaSR) reportedly causesBMP2 synthesis and secretion via phosphatidylinostitol 3-kinaseactivation [59]. The deletion of CaSR completely inhibited BMP2secretion [59]. The upregulation of BMP2 might be related to theupregulation of CaSR, due to the report that elevated extracellularCa can help enhance the expression of CaSR. However, the geneexpression of CaSR was not upregulated (data not shown) by theMg-b-TCP scaffold, although the Ca concentration was elevated.Further investigations are therefore required to understand thismechanism. As profibrotic cytokines, TGF-bs were found to behighly expressed in a wide range of fibrotic tissues [60]. TGF-bsupregulate the synthesis of many extracellular matrix (ECM)proteins that promote the fibrosis process [61]. TGF-b1, whichinduces fibroblast proliferation via the FGF-2/ERK pathway, is akey cytokine in the fibrosis process [62]. TGF-b3 plays an impor-tant role in ECM production by inducing the expression of type Icollagen, fibronectin, laminin, and proteoglycans [63]. We assayedboth TGF-b1 and TGF-b3, which were both downregulated in theMg-b-TCP scaffold samples. This finding indicates that the Mg-b-TCP scaffold-induced macrophage phenotype switch does notenhance inflammatory fibrosis, but was effective and adequate inimproving osteogenesis.

The putative osteoimmunomodulatory effects of the b-TCPcoating on the material mediating osteogenesis are summarized inFig. 12. The degradation products released from the materialsstimulate the macrophages and trigger their phenotype switch toM2 by inhibiting the TLR receptor signaling pathway, which resultsin the upregulation of anti-inflammatory cytokines (IL-1ra). The b-TCP stimulated macrophages upregulate VEGF and BMP2 geneexpression, which activate the BMP2 signaling pathway in BMSCs,resulting in osteogenic differentiation. Expression of the fibrosis-promoting genes TGF-b1 and TGF-b3 are downregulated inresponse to the same treatment, which can account for the possibleinhibition of fibrosis. The net result is a dampening of the inflam-matory response and a commensurate increase of osteogenesis. Aswas stated in the Introduction, given their roles in the dynamic fluxof bone formation and homeostasis, the response of macrophagesto bone biomaterials must be considered when evaluating theosteoimmunomodulatory properties. The inflammatory responseof macrophages can to some extent act as a measure of the overalllevel of inflammation, but it has to be emphasized that inflamma-tion is a complex response that cannot easily be mimicked in vitro.Further in vivo studies must therefore be conducted in order to testthe in vitro findings.

Z. Chen et al. / Biomaterials 35 (2014) 8553e8565 8563

The paradigm shift for developing bone biomaterials (osteoim-munomodulatory biomaterials) emphasizes the importance ofosteoimmunomodulation, indicating that these properties must bean essential parameter for the evaluation and development ofadvanced bone biomaterials. Osteoimmunomodulation, as it isdefined in this study includes not only the regulation of inflam-matory reaction towards the implants, but the effects of the im-munemicroenvironments towards the behaviors of bone cells, suchas the balance of osteogenesis versus osteoclastogenesis. This bal-ance effectively determines the in vivo fate of bone biomaterials,towards either de novo bone formation or fibrosis encapsulation.Traditional strategies for the development of bone biomaterialsmainly focused on the response of osteoblastic lineage cells to theexclusion of immune cells [64e66]. This paradigm does not able toadequately reflect in vivo conditions that involve the early immunereaction following an implantation. This study proposed a novel

Fig. 12. Potential molecular mechanisms of how the interaction of macrophages with Mg-bstimulate the macrophages to switch their phenotype to M2 by inhibiting the TLR receptor1ra). The stimulated macrophages upregulate VEGF and BMP2 gene expression, which activasame time, expression of the fibrosis-promoting genes TGF-b1 and TGF-b3 are downregulaflammatory response and increased osteogenesis.

strategy for the development or modification of bone biomaterialsby the targeted modulation of the osteoimmunomodulatoryproperties of osteoinductive scaffolds.

5. Conclusions

ModifyingMg scaffolds by coating themwith b-TCP altered theirdetrimental osteoimmunomodulatory properties in such a waythat they were more favorable for osteogenesis. Specifically, thecoating induced an effective and adequate switch to the M2macrophage phenotype, which inhibits inflammation and osteo-clastogenesis, and enhances the osteogenic differentiation ofBMSCs. The potential mechanisms of this process are likely to bethe inhibition of the TLR receptor pathway and the activation of theBMP2 signaling pathway. Therefore, b-TCP coating can be a valuablemethod to modify scaffolds with fast degradation rate (e.g. Mg

-TCP scaffolds induces osteogenesis. Degradation products released from the materialssignaling pathway and results in the upregulation of anti-inflammatory cytokines (IL-tes the BMP signaling pathway in BMSCs, resulting in osteogenic differentiation. At theted, which can account for reduced fibrosis. The net effect is a dampening of the in-

Z. Chen et al. / Biomaterials 35 (2014) 8553e85658564

scaffolds) for bone tissue engineering applications. In addition,endowing bone biomaterials with favorable osteoimmunomodu-latory properties can be a valuable strategy for the development ormodification of advanced bone biomaterials.

Acknowledgments

We appreciate technical support from Dr. Wenyi Gu (AustralianInstitute for Bioengineering and Nanotechnology, The University ofQueensland) and Dr. Leonore de Boer (Cell Imaging Facility,Queensland University of Technology) for flow cytometry detection.The study is supported by the Queensland-Chinese Academy of Sci-ences Collaborative Science Fund 2014 in Australia, as well as Na-tional Natural Science Foundation of China (81100728), NationalBasic Research Program of China (973 Program) (2012CB619101),Guangdong Province Science Technology Bureau (2012B050600017)and Fundamental Research Funds for the Central Universities(11ykpy49).

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