Acta Biomaterialia 10 (2014) 477–485

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Acta Biomaterialia

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Zinc in calcium phosphate mediates bone induction: In vitro and in vivomodel

1742-7061/$ - see front matter � 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.actbio.2013.10.011

⇑ Corresponding author at: Xpand Biotechnology BV, Prof. Bronkhorstlaan 10, Bld48, 3723 MB Bilthoven, The Netherlands. Tel.: +31 30 2297292; fax: +31 30 2297299.

E-mail address: h.yuan@tnw.utwente.nl (H. Yuan).

Xiaoman Luo a, Davide Barbieri a, Noel Davison a, Yonggang Yan b, Joost D. de Bruijn a,c,d, Huipin Yuan a,c,⇑a Xpand Biotechnology BV, Prof. Bronkhorstlaan 10, Bld 48, 3723 MB Bilthoven, The Netherlandsb College of Physical Science and Technology, Sichuan University, No. 24 South Section 1, Yihuan Road, 610065 Chengdu, Chinac MIRA Institute, University of Twente, Drienerlolaan 5, 7522 NB Enschede, The Netherlandsd School of Engineering and Materials Science, Queen Mary University of London, Mile End Rd, London E1 4NS, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 July 2013Received in revised form 10 October 2013Accepted 10 October 2013Available online 17 October 2013

Keywords:ZincCalcium phosphateMacrophageStem cellOsteogenesis

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteo-blastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ionswere readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkalinephosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activ-ity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cul-tured with different dosages of zinc supplements in medium with or without zinc-free TCP showedthat zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-weekimplantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increasedwith increasing zinc content in Zn-TCP – up to 52% bone in the free space. However, TCP without zincinduced no bone formation. Although the observed bone induction cannot be attributed to zinc releasealone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and renderTCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graftmaterial.

� 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Bone mineral comprises carbonated apatite crystals that con-tain a series of trace elements [1,2] such as magnesium (Mg), fluo-ride (F), manganese (Mn), zinc (Zn), silicon (Si) and strontium (Sr).Although they represent only a small fraction of bone mineral, theyare considered essential for bone metabolism [3]. For example, Mgand Zn have been reported to influence osteoblast and osteoclastactivity [4,5], while strontium ranelate is used clinically to reduceosteoporosis [6].

For the repair of bone defects, calcium phosphate ceramics(CaPs) such as hydroxyapatite (HA), tricalcium phosphate (TCP)and their combination (biphasic calcium phosphate, BCP) arewidely used due to their chemical similarity to bone mineral. Afterimplantation, they allow migration of bone cells on their surfacefollowed by bone deposition, in a phenomenon called osteocon-duction [7]. However, when a bone defect exceeds a critical size,osteoconductive materials are not able to fully regenerate the

defect and osteoinductive materials, which trigger the osteogene-sis of mesenchymal stem cells [8]; thus inducing bone formation,even in non-osseous tissues, is needed. Osteoinduction can beachieved by adding growth factors such as bone morphogeneticproteins (BMPs) or osteogenic cells to the material.

Inspired by the idea that calcium phosphate ceramics mimicbone mineral chemistry and that trace ions regulate bone metabo-lism, the introduction of trace elements in CaP was expected toprovide a novel method to enhance the bone-forming ability of abone graft. An example of such a material is silicon substitutedHA [9], although there is debate whether silicon plays a direct rolein the functioning of these materials [10].

Zn, another important trace element, is considered as an impor-tant mineral in the normal growth and development of the skeletalsystem. Bone tissue contains �30% of the total zinc in the body[11]. Indeed, Zn deficiency impairs the development of maximalbone mass [12]. It is associated with the pathogenesis of osteopo-rosis [13,14], while dietary supplementation of zinc showed posi-tive effects on bone metabolism [12].

It has been shown that zinc can influence the behavior of(pre)osteoblast [15–17] and osteoclast [5,18,19], indicating thatzinc plays an important role in bone metabolism. Thereafter, zinchas been introduced in various calcium phosphate ceramics in

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expectation that the release of zinc may promote the bone-formingability of synthetic materials. Naturally it was found that zinc-containing materials would release zinc, and influence thedifferentiation of osteogenic cells [20] and hence promote theregeneration of nearby host bone [21,22], suggesting their potentialrole in improving conductive bone formation at osseous sites.

Mineralized Achilles tendon and cartilage calcification havebeen associated with high Zn tissue levels [23]. Furthermore, someresearchers showed that zinc could induce osteogenic differentia-tion of mesenchymal stem cells (i.e. bone marrow stromal cells)[24,25]. These facts indicated a possible role of zinc in ectopic boneformation (osteoinduction). We hypothesized that the incorpora-tion of Zn in CaP may render the ceramic osteoinductive due tothe stimulation of osteogenic differentiation of mesenchymal stemcells (originating from either the local tissues or the blood stream[26,27]). To investigate the hypothesis, zinc-containing TCP(Zn-TCP) was synthesized and evaluated as a model.

As mentioned above, zinc plays its role not only in bone forma-tion but also in bone resorption, which should continuously influ-ence the ossification mass in the osteoinductive model since bonemetabolism is an equilibrium between ossification and boneresorption. It is clear that zinc-containing ceramics can inhibitthe resorption activity of osteoclast [28,29] while bone resorptionalso depends on the formation of osteoclasts. Osteoclasts are differ-entiated from monocyte/macrophages, which can fuse to formmultinucleated, TRAP-positive, bone-resorbing giant cells in thepresence of certain cytokines, in particular the receptor activatorof NF-jB ligand (RANKL) [30]. As a matter of fact, zinc is involvedin this osteoclastic differentiation too [15,31]. Thus zinc mayinfluences bone resorption not only via the resorption activityof osteoclast but also via the formation of osteoclast frommacrophage. To address the possible influence of Zn-TCP onosteoclastogenesis, monocyte/macrophages (RAW264.7) were cul-tured with the presence of RANKL on Zn-TCP and on zinc-free TCPwith zinc supplement in culture medium.

2. Materials and methods

2.1. Preparation and characterization of Zn-TCP

Tricalcium phosphate powder was synthesized by wet precipi-tation using Ca(OH)2 (Fluka) and H3PO4 (Fluka) at a Ca/P molarratio of 1.5. Zn-TCP slurries were made by mixing TCP powder withaqueous solutions containing various concentration of ZnCl2

(Fluka): 0, 5, 15 and 45 mmol ZnCl2/100 g TCP (‘‘TZ00’’ control,‘‘TZ05’’, ‘‘TZ15’’ and ‘‘TZ45’’, respectively). Slurries were vacuumedto remove air bubbles, dried at room temperature and sintered at1100 �C for 480 min, resulting in dense Zn-TCP blocks.

Zn-TCP discs (10 � 1 mm) were machined from dense blocksusing a diamond saw microtome (Leica SP1600); granules (212-300 lm) were prepared by grinding and sieving the residual blockparticulate. Discs and granules were ultrasonically cleaned insequential baths of acetone (Sigma), 70% ethanol (PROLABO) anddemineralized water for 15 min each. Materials were dried at80 �C, steam sterilized at a pressure of 2–4 bar (121 �C) for30 min and dried at 80 �C afterwards.

Materials were characterized on the basis of their crystal chem-istry, surface structure and release of Zn ions. Chemical analysiswas conducted using an X-ray diffractometer (MiniFlexII, Rigaku)with Cu Ka radiation (k = 1.5405 Å, 30 kV, 15 mA) over the 2hrange of 25–45�. The microstructure of the ceramics was analyzedusing a scanning electron microscope (SEM) (Jeol JSM-5600). Grainand pore size were quantified using ImageJ software (NIH) by mea-suring and averaging the vertical length of >40 grains and pores ina representative micrograph taken at 2500�. Ion release was

measured as follows: discs were soaked in 1 ml culture mediumfor 4 days at 37 �C with 5% CO2 (1 ml medium per disc, n = 5 permaterial, more details are given in Section 2.2) and the zinc con-centration in medium was measured by atomic absorption spec-troscopy (AAS) (SpectrAA 220FS). Ca2+ and PO3�

4 concentrationsin the medium were measured using QuantiChrom (BioAssay Sys-tems, USA) and PhosphoWorks (AAT BioQuest, USA) colorimetricassay kits, respectively. Assays were carried out following the man-ufacturers’ instructions and absorbance (620 nm) was measuredusing a multimode plate reader (Zenyth 3100).

2.2. In vitro experiments

All discs were pre-incubated in corresponding basic medium for4 h prior to cell culture. Before the cell seeding, these media werediscarded to prevent potential pH impact to cells (also applied toion release experiments with basic medium used in Section 2.2.2).

Suspension culture plates (Greiner Bio-One Cat. 677102) wereused for cells cultured on discs while cell culture plates (GreinerBio-One Cat. 677180) were used for cells cultured on plastic.

2.2.1. Osteoblast differentiation of hBMSCThe basic medium was a-MEM (minimal essential medium;

Invitrogen, UK) supplemented with 10% fetal bovine serum (FBS)(Lonza, Germany), 2 mM L-glutamine (Invitrogen, UK), 0.2 mML-ascorbic acid 2-phosphate (AsAP, Sigma), 100 IU ml–1 penicillin(Invitrogen, UK) and 100 lg ml–1 streptomycin (Invitrogen, UK).The growth medium was basic medium supplemented with1 ng ml–1 basic fibroblasts growth factors (bFGF, Instruchemie,the Netherlands). The osetogenic medium was basic mediumsupplemented with 10 nM dexamethasone (Sigma).

Human bone marrow stromal cells (Passage 3, PT-2501, Lonza,Netherlands) were expanded in flasks containing growth mediumunder a humidified atmosphere of 5% CO2 at 37 �C for 1 week untilthey were harvested with 0.25% trypsin (Invitrogen, UK).2.5 � 104 cells per well were cultured on Zn-TCP discs (n = 5 permaterial at each time point) with osteogenic medium in 48 wellsfor up to 14 days. The medium was refreshed twice a week andsamples were harvested after 1, 4, 7 and 14 days.

2.2.2. Osteoclast differentiation of RAW264.7The basic medium was a-MEM (Lonza, Germany) supple-

mented with 10% FBS (Peprotech) and 1% penicillin and streptomy-cin (Invitrogen, UK).

Monocytes/macrophage (RAW264.7) cells were seeded in basicmedium for 1 week. Cells were then harvested by using a cell scra-per (BD Falcon) and further cultured on discs with or without zinccontent or on plastic (n = 5 per material at each time point) withbasic medium in 48 wells at the density of 2.5 � 104 cells per wellfor 4 days in the presence of 20 ng ml–1 RANKL (Peprotech). Zincsupplement was applied on the zinc-free group by adding zincchloride in the medium range from 0 to 18 ppm. These media wererefreshed once at day 1.

2.2.3. Biochemistry2.2.3.1. Cell lysis. Cells were rinsed one time with warm phosphatebuffered saline (PBS; Lonza) and frozen at �80 �C for 24 h. Thenthey were thawed to 37 �C. 400 ll Cyquant lysis buffer was addedand ultrasonicated for 2 min followed with 5 min shaking at400 rpm.

2.2.3.2. DNA content. The proliferation of hBMSC was measuredusing a CyQuant cell proliferation assay kit (Invitrogen) followingthe manufacturer’s guidelines. In brief, 100 ll cell lysate wasmixed with 100 ll 400-time-diluted GR reagent and further incu-bated at room temperature in the dark for 5 min. The fluorescent

Fig. 1. XRD patterns of Zn-TCP: TZ00 (A), TZ05 (B), TZ15(C), TZ45 (D) and HA (E),apatite phase presented in TZ15 and TZ45.

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analysis was performed with wavelength of 480 nm for excitationand 520 nm for emission using a spectrophotometer (Zenith 3100).

2.2.3.3. Alkaline phosphatase (ALP) activity. Diethanolamine bufferwas prepared by dissolving 10% (v/v) diethanolamine (Sigma) indemineralized water. Afterwards the pH of diethanolamine bufferwas adjusted with HCl (Sigma) to 9.8. 150 ll buffer containing5 mM p-nitrophenyl phosphate disodium salt hexahydrate (PNPP,Sigma) and 1 mM MgCl2 (Merck) was added to 50 ll cell lysate,and then incubated at 37 �C for 15 min. The absorbance was mea-sured at 405 nm. The ALP activity was defined as lmol of p-nitro-phenol released after a 15 min incubation normalized to the DNAcontent in each well (lmol ng–1).

2.2.3.4. Tartrate-resistant acid phosphatase (TRAP) activity. 10 ll Celllysate was added to 90 ll reagent solution (100 mM NaAc buffer(pH 5.8) supplement with 0.15 mM KCl, 1.0 mM ascorbic acid,0.1 mM FeCl3, 10 mM potassium sodium tartrate and 10 mMPNPP), incubated in the dark at 37 �C for 1 h. To stop the reaction,100 ll 0.3 M NaOH was added and the absorbance was measuredat 405 nm.

2.2.3.5. TRAP staining. The TRAP expression of RAW264.7 cells cul-tured on plastic is stained using a commercial kit (Leukocyte AcidPhosphatase Kit, Sigma). Cells were first rinsed with PBS, fixed with4% formaldehyde for 10 min and then stained following the manu-facturer’s instructions. Staining was visualized using a NikonSMZ800 stereomicroscope equipped with a Nikon camera.

2.2.3.6. Osteoclast size determination. Cells were rinsed with PBS,fixed in 2.5% glutaraldehyde in PBS for 10 min and then dehydratedwith a graded ethanol series (25%, 50%, 70%, 80%, 90%, 95%, 100%�2) following with a graded HMDS (Alfa Cesar) series (33%, 50%,75%, 100%). Cells were then sputter-coated with gold for SEMobservation.

The size of osteoclasts on TCP discs was measured with SEMimages (>200 cells) and quantified by calculating the mean surfacearea of cells using automated threshold, edge detection and parti-cle analysis functions in ImageJ software (NIH).

2.3. In vivo experiments

Four adult male mongrel canines (average body weight 12.5 kg)were used. All surgeries were conducted in Sichuan University un-der general anesthesia by abdominal injection of sodium pentobar-bital (Sigma, 30 mg kg–1 body weight) with the permission of thelocal animal ethics committee. Zn-TCP granules were prepared as1 cm3 aliquots under sterile conditions in glass vials prior to theimplantation. 1 cm3 of Zn-TCP granules was implanted in the sameside (two canines in the left, two canines in the right) of theparaspinal muscle along the direction of spine. In brief, muscularpouches were created by scalpel incision and blunt dissection,and then the granules were inserted into the pouch with a syringe.The distance between each implant was �2–3 cm. The pocketswere then closed with non-resorbable sutures for identificationat harvest. Following the surgeries, animals were given buprenor-phine intramuscularly (0.1 mg per animal) for two days to relievepain and penicillin (40 mg kg–1) by intramuscular injection forthree consecutive days to prevent infection. After operation, ani-mals were fully weight bearing and received a normal diet. Calcein(10 mg kg–1 body weight, Sigma) and xylenol orange (100 mg kg–1

body weight, Sigma) were intravenously injected 6 and 9 weeksafter implantation, respectively. Subsequently, animals were sacri-ficed with a celiac injection of excessive amounts of sodium pento-barbital 12 weeks after implantation. Implants were fixed with 4%formaldehyde, embedded in PMMA after gradient ethanol

dehydration. Three non-decalcified sections were made acrossthe middle of the explants using a diamond saw microtome (LeicaSP-1600, Germany) and stained with 1% methylene blue (Sigma)and 0.3% basic fuchsin (Sigma). Histological observation was per-formed with light microscopy. The histological slides were scannedwith a scanner (DIMAGE Scan Elite5400II, KONICA MINOLTA). His-tomorphometry of bone formation in the stained sections was per-formed by pseudo-coloring pixels representing formed bone (B)and remaining material (M) in a region of interest (ROI, with sur-rounding soft tissues outside the implants excluded) using Photo-shop (Adobe Photoshop Elements 2.0). Bone formation wascalculated as follows:

B% ¼ B=ðROI�MÞ � 100

2.4. Statistical analysis

Multiple comparisons were performed with one-way ANOVAusing Turkey comparisons (osteoclast size determination withthe Kruskal–Wallis test) and p values smaller than 0.05 were con-sidered statistically significant. Data are expressed and representthe mean ± standard deviation of five (four for in vivo samples)replicates. Asterisks were marked in Figs. 3–6 if significance differ-ences were found.

3. Results

3.1. X-ray diffraction (XRD)

The crystalline phases of the obtained TCPs with various zincconcentrations were examined with powder XRD (Fig. 1). Newpeaks were seen at the 2h position of 31.8� and 32.4� on the pat-terns of TZ15 and TZ45. Such peaks increased in intensity withthe rising of zinc content, and indicate the increasing presence ofhydroxyapatite (HA) phase (according to standard PDF 09-0432).No crystalline phases other than b-TCP and HA were detected.

3.2. Scanning electron microscopy analysis (SEM)

The surface morphology of the samples was observed with SEM.It was seen that the surface of the granules had micropores (Fig. 2)and the grains shrunk in size with increasing zinc content. Statis-tics showed a decreasing trend of grain size and pore size withincreasing zinc content (Fig. 3D). TZ15 has significantly smallergrain size than TZ00 (p < 1 � 10�4) while significantly smallergrains (p < 1 � 10�7) and pores (p 1 � 10�5) were seen in TZ45 ascompared to all other materials.

Fig. 3. Ion release of Zn-TCP discs after 4 days’ immersion in culture medium: Ca2+ (A), PO3�4 (B) and Zn2+ (C); (D) a summary of grain size and pore size of Zn-TCP

(⁄significantly different than other samples; #significantly different as compared to TZ00).

Fig. 4. hBMSc cultured on Zn-TCP discs. (A) cell proliferation of hBMSC on Zn-TCP discs as shown with DNA production; (B) osteogenic differentiation of hBMSC on Zn-TCPdiscs as shown with the ALP/DNA ratio (After 14 days’ culture, the difference of hBMSC proliferation was not significant but the TZ45 showed higher ALP expression than TZ00with significant difference, ⁄p 0.05).

Fig. 2. SEM images of TZ00 (A), TZ05 (B), TZ15 (C), TZ45 (D); the grain size and micropore size decreased with the increasing zinc content.

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Fig. 5. The proliferation (A, B, C) and TRAP expression (D, E, F) of murine macrophage RAW264.7 cultured in three different models: on Zn-TCP discs (A, D); on zinc-free discswith different zinc supplement in medium (B, E); on plastic culture plates with different zinc supplement in medium (C, F) (⁄significantly different than other samples;#significantly different as compared to 0 ppm; $significantly different as compared to 18 ppm).

Fig. 6. (A) TRAP staining light microscopy images of RAW264.7 cells cultured on plastic culture plates supplemented with zinc-free medium, 0.18 ppm zinc, 1.8 ppm zinc and18 ppm zinc; (B) MNGC size on Zn-TCP discs; (C) MNGC size on zinc-free TCP discs (⁄significantly different as compared to other samples, #significantly different as comparedto the first sample).

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3.3. Ion release

After 4 days’ immersion a significantly higher concentration ofzinc ions was released from TZ45, compared with other samples(p < 0.001, Fig. 3C) while the Ca2+ (Fig. 3A) and PO3�

4 (Fig. 3B)concentration in the medium was similar (p > 0.05).

3.4. Human bone marrow stem cells (hBMCs) culture

3.4.1. Cell proliferationAn increasing number of hBMCs cells was seen on all ceramic

discs over time. However, hBMSC on TZ45 discs showed higherproliferation than others at day 7 (Fig. 4A, p < 0.02), indicating apositive effect of zinc on hBMSC proliferation. After 14 days theDNA production was similar on all TCP discs, which may due to fullcell confluence on the disc surfaces.

3.4.2. Osteogenic differentiationOsteogenic differentiation of hMSCs indicated by the value of

ALP/DNA did not differ with the zinc content in TCP at day 4 andday 7, while at day 14, the ALP/DNA value increased with zinc con-tent with a significantly higher osteogenic differentiation on TZ45as compared to TZ00 (p < 0.02) (Fig. 4B).

3.5. Murine macrophage RAW264.7 culture

3.5.1. RAW264.7 cultured on Zn-TCP discsDNA data did not show a different proliferation of RAW264.7

cells on TZ00, TZ05 and TZ15; conversely the proliferation was sig-nificantly enhanced on TZ45 (p < 0.001, Fig. 5A). In addition,RAW264.7 cells on TZ45 produced significantly more TRAP thanthose on discs with lower zinc content (p < 0.01, Fig. 5D) whileno differences were seen among TZ00, TZ05 and TZ15.

3.5.2. RAW264.7 cultured on zinc-free TCP with zinc supplement inmedium

When cells were cultured on zinc-free TCP, the zinc supple-mented to the medium did not influence the proliferation ofRAW264.7 until its concentration reached 18 ppm (p < 0.001, Fig.5B). Cells cultured with a supplement of 1.8 ppm zinc expressedhigher TRAP than other groups (p < 0.001, Fig. 5E).

3.5.3. RAW264.7 cultured on plastic with zinc supplement in mediumWhen RAW264.7 cells were cultured on plastic (culture dishes)

with a supplement of zinc in the culture medium, the proliferationincreased gradually along with the zinc supplement. Groups withzinc-containing medium had higher DNA production than thegroup with zinc-free medium (p < 10�5, Fig. 5C) and a concentra-tion of 18 ppm zinc induced more proliferation than the groupwith 0.18 ppm zinc (p < 0.01, Fig. 5C), indicating that zinc up to18 ppm might promote the proliferation of RAW264.7. However,TRAP activity was dramatically inhibited when the zinc concentra-tion was as high as 18 ppm (p < 10�8, Fig. 5F), while it was stimu-lated by lower concentration (i.e. 0.18 ppm and 1.8 ppm) (p < 0.03,Fig. 5F).

3.5.4. Cell morphology on plastic with zinc supplement in mediumWhen RAW264.7 cells were cultured on plastic with zinc sup-

plement in the culture medium, the fusion of cells into multiplenuclear giant cells (MNGCs) was observed with zinc lower than18 ppm (Fig. 6A). The size of these MNGCs was bigger with1.8 ppm zinc as compared to the zinc-free control, indicating thatzinc promotes the fusion of mononuclear cells to MNGC. However,fusion did not occur in the presence of 18 ppm zinc (Fig. 6A), indi-cating that excessive zinc suppresses the fusion.

3.5.5. Size characterization of MNGC fused on TCP discsThe morphological variation of MNGC with zinc content was

also observed with RAW264.7 cells on Zn-TCP or zinc-free TCPsupplemented with zinc in culture medium. The size of MNGCon Zn-TCP discs are bigger than those cultured on TZ00(p < 0.02, Fig. 6B), while the biggest MNGC were seen on TZ45(p < 10�8, Fig. 6B), showing that the fusion of mononuclear cellsto MNGC is promoted with the increase of zinc content in TCP.When zinc is supplemented in medium, the MNGC size on zinc-free TCP was gradually increased until a zinc concentration of1.8 ppm (p < 0.002, Fig. 6C). A suppressing effect was observedthereafter with 18 ppm zinc (p < 10�4, Fig. 6C), corroboratingwhat concluded earlier.

3.6. Intramuscular bone formation

A histological overview after 12 weeks’ implantation in caninemuscle showed different bone-forming abilities of Zn-TCP with atrend following zinc content (Fig. 7A–D). In fact, bone volume in-creased significantly with zinc content (p < 0.02, Table 1).

Detailed histological observations showed no inflammationadjacent to implants while fibroblasts and blood vessels were evi-dent in soft tissue matrix. Bone was directly bonded to TCP gran-ules and bridges of mature bone were formed between granules.Osteoblasts were observed on the surface of bone matrix, whilemultinuclear giant cells were found around the resorption areason TCP (Fig. 7a–d).

Calcein deposition was observed in bone formed in TZ45 im-plants (Fig. 8B) but not that in TZ15 implants (Fig. 8A), indicatingthat bone formation started in TZ45 between 6 and 9 weeks, earlierthan in TZ15. Xylenol Orange deposition was observed on bone tis-sue in TZ15 and TZ45 implants, indicating that bone was continu-ously forming after 9 weeks in both materials.

4. Discussion

Zn-TCP induced ectopic bone formation in canine muscle re-lated to the concentration of zinc they contained; however, TCPcontaining no zinc formed no bone (Table 1, Fig. 7). In additionto the observation that increased zinc content in TCP augmentedectopic bone formation in terms of bone incidence and volume,higher zinc content also appeared to accelerate the bone formationprocess based on fluorescent bone labeling (Fig. 8). These in vivodata suggest that the addition of zinc may be a useful strategy torender a non-inductive TCP osteoinductive.

We speculate that the observed bone formation was due to zincrelease from the ceramics after implantation, which was shown tooccur passively in physiological conditions (Fig. 3C). Moreover, invivo, numerous multinuclear giant cells with internalized ceramicparticles in close proximity to material resorption areas could beobserved in histological sections and may have also facilitated zincrelease (Fig. 7A–D). Thus, elevated zinc concentration in the im-plant microenvironment may occur by both hydrolytic and cell-mediated material degradation, resulting in ectopic boneformation.

In agreement with this hypothesis, we found that Zn-TCP posi-tively influenced osteogenic differentiation of hBMCs (Fig. 4), alsopresumably due to elevated zinc in the medium. These findingsare in line with the finding of Ito et al., who observed that bothrat and human bone marrow stromal cells (BMSc) cultured onzinc-containing BCP differentiated into osteoblast-like cells morethan cells on zinc-free ceramics [21]. Elevated zinc released inthe medium would potentially play an important role in cell differ-entiation since it has been reported that supplementing zinc in themedium significantly increases the osteogenic differentiation of

Fig. 7. Top: histology overviews after 12 weeks’ intramuscular implantation in canines ((A) TZ00, (B) TZ05, (C) TZ15, (D) TZ45). Bottom: the squares in A–D observed withlight microscopy ((a) TZ00, (b) TZ05, (c) TZ15, (d) TZ45), showing that osteoblasts (thin arrow) were secreting osteoid while multinuclear giant cells (thick arrow) wereresorbing the material and bone.

Table 1A summary of ectopic bone formation in intramuscular implants.

Samples TZ00 TZ05 TZ15 TZ45

Implants 4 4 4 4Bone incidence 0/4 1/4 4/4 4/4Bone formation (B%) 0 ± 0 0.1 ± 0.2 20.1 ± 21.9 51.8 ± 11.8

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both rat and human BMSc [32]. Thus, it is possible that blood/bonemarrow stem cells homing to the implantation site due to theinflammatory foreign body reaction may have infiltrated the mate-rials and then differentiated into osteoblasts due to elevated zincreleased by the ceramics.

Zn has also been reported to promote the proliferation of othermineralizing cells in addition to stimulating osteogenic differenti-ation of stem cells. For instance, osteoblast precursor MC3T3-E1was found to have higher proliferation and ALP expression onzinc-containing ceramics than those without [17,33]. Osteoblast-like line MG63, as well as osteoblasts, had improved adhesionand proliferation in the presence of zinc [16,34]. In view of all ofthese findings, we speculate that Zn-TCP may first trigger

osteogenic differentiation of stem cells and then stimulate the pro-liferation of their mineralizing osteoblast progeny. It follows thatwith more osteogenic cells at the site, more bone could be formedheterotopically, resulting in large bone mass (Fig. 7C and D). Thisassumption is demonstrated by our observation that with the in-crease of zinc content, bone formed earlier and its incidence in-creased (Table 1), enveloping the granules.

Because bone formation and metabolism are tightly coupledprocesses, it is also important to consider that Zn may also play arole in osteoclastogenesis. Using the cell line RAW264.7 culturedwith RANKL as an in vitro model of osteoclastogenesis, the prolif-eration and TRAP activity of RAW264.7 cultured on TZ45 was sig-nificantly higher than those cultured on zinc-free TCP (Fig. 5A andD). Additionally, increasing zinc content augmented osteoclastsize, presumably due to higher cell fusion (Fig. 6B). Taken together,we surmised that elevated zinc content can promote both osteo-clast formation and activity. In order to address the confoundinginfluence of the smaller surface microstructure of Zn-TCP accom-panying increasing zinc concentrations, cells were cultured on tis-sue culture plastic and zinc-free TCP discs in the presence of RANKLbut various concentrations of zinc doped into the culture medium.

Fig. 8. Microscopy images of TZ15 (A) and TZ45 (B) obtained by overlapping the fluorescent and transmitted light images taken in the same position. Bone formation startedin TZ45 between 6 and 9 weeks after the implantation (calcein, green) and after 9 weeks (xylenol orange, red), while it appeared in TZ15 only after 9 weeks (xylenol orange,red).

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In both models, proliferation, TRAP activity and osteoclast size wasdependent on zinc concentration. For instance, proliferation wasslightly increased with zinc concentration in both models (Fig. 5Band C). TRAP activity was also increased with zinc concentrationbut was attenuated at an excessive concentration (18 ppm, Fig.5E and F). Corresponding with this, osteoclast size increased withzinc content up until an excessive concentration (18 ppm), where-upon mononuclear cells no longer fused (Fig. 6A). These resultssuggest that when the factor of surface microstructure is removed,zinc concentration can still regulate the differentiation of mono-cyte/macrophages into osteoclasts. Furthermore, because the high-est concentration of zinc released from the synthesized Zn-TCP wasfound to be 1.8 ppm (TZ45, Fig. 3C) after 4 days’ culture and thisconcentration also elicited similar effects when doped into med-ium containing Zn-free TCP, it is probable that osteoclast formationand activity were modulated by zinc content rather than differ-ences in surface microstructure.

As TRAP activity and giant cell fusion are hallmarks of osteoclas-togenic differentiation of mononuclear precursors, our results indi-cate that zinc may regulate the osteoclastogenic differentiation ofmacrophages and osteoclastic precursors. However, this effectmay either be a stimulation or a suppression, depending on thezinc concentration in the environment. The results presented here-in are partly consistent with a finding from an RAW264.7 study inzinc-containing medium [15], which indicated a zinc concentrationbetween 10 lM and 250 lM (i.e. 0.65 ppm and 16.25 ppm) as themain cause of osteoclastogenesis suppression. Furthermore, zinchas been reported to inhibit resorption activity of rat osteoclasts[5]. Holloway et al. noticed that treatment with zinc concentrationlower than 10�4 mol l–1 (6.5 ppm) had no effect on osteoclast activ-ity, while higher zinc amounts increased the number of TRAP-positive cells. Interestingly, it was also seen that osteoclast boneresorption was dramatically suppressed by high zinc contents [19].

Although zinc release from the ceramics reached a bioactivelevel after four days in vitro, the situation is more complex in vivoand zinc levels in the implant tissue were not measured. The circu-lation of body fluids certainly would influence the local ion concen-tration near the implants, likely dispersing the elevated ions. Still,it stands to reason that ions released in the core of the implantswould be more shielded by the circulation, where the body fluidflow is obstructed. Thus we assume that the zinc concentrationin the middle of the implant could reach an effective level duringthe implantation time to modulate osteogenesis.

Beside ion release, differences in chemistry and surface micro-structure may have also played a role in the bone formation. Thechemical composition varied as shown in XRD, where apatitephase formation was provoked by the presence of zinc in TCP(Fig. 1). The influence of calcium phosphate chemistry on osteo-genic differentiation, osteoblast behavior and bone-forming ability

is reported in various studies with respect to the HA/TCP ratio [35–37]. However, many findings are controversial. Moreover, HA andTCP are physicochemically different and thus the HA/TCP contentratio leads to many other differences like calcium and phosphateion release rate and surface topography, though these sub-factorswere not studied. Thus it is difficult to determine whether suchfactors are responsible for improved bone-forming ability.

We observed that the grain and pore size of TCP decreased withzinc addition (Fig. 2). This difference in surface topography be-tween Zn-TCP might be the reason for the difference in the sizeof MNGC between TZ45 and the zinc-free control with 1.8 ppmzinc supplement although they experienced equivalent zinc re-lease (Fig. 6), since surface topography (i.e. surface roughness)alone can have an influence on the cell behavior of osteoclasts[38–40]. Not limited to osteoclasts, various cells were sensitiveto surface topographical cues regarding cell behaviours of attach-ment, adhesion, spreading, migration, proliferation and differenti-ation [41]. For instance, surface topography was able to directlytrigger or enhance osteogenic differentiation of hBMSC andMG63 cells [42–44]. It is therefore difficult to attribute the variousbone formation in Zn-TCP implants only to the release of zinc, sincethe different surface topography may play roles in bone formationas well.

It is important to note that the trace-element-substituted cera-mic is not a simple overlay of trace element and the ceramic. Manyphysicochemical properties of the ceramic, such as the chemistryand surface topography, can be changed because of ion substitu-tion. Thus, it is a complex issue to directly link the effect of ionsto the biological response [10]. We have shown that, using zinc-containing tricalcium phosphate ceramic as a model, incorporatingtrace elements in bone mineral has the potential to induce boneformation. However, the mechanism behind this phenomenon isstill not fully understood. Carefully designed materials that containonly one variable parameter would be useful in more clearlyexplaining the role of ions, or other materials’ factors in osteoin-duction [45].

5. Conclusion

Zn-TCP enhanced osteogenic differentiation of hBMSC and reg-ulated osteoclastic differentiation of monocyte/macrophages invitro. Incorporation of high zinc content in TCP led to osteoinductivematerials capable of forming abundant de novo bone in the muscle.The in vivo responses may be correlated with the release of zinc assupported by our in vitro cellular results. However, other factors suchas the chemical composition and surface topography of TCP, whichare modified by the zinc addition, may also have played roles in thisintriguing phenomenon.

X. Luo et al. / Acta Biomaterialia 10 (2014) 477–485 485

Disclosures

Xiaoman Luo, Davide Barbieri, Noel Davison, Joost D. de Bruijnand Huipin Yuan are employees of Xpand Biotechnology.

Acknowledgements

The authors acknowledge University of Twente and SichuanUniversity for their help in getting the data that have been usedin this manuscript.

Appendix Figures. with essential color discrimination

Certain figures in this article, particularly Figs. 1 and 6, are dif-ficult to interpret in black and white. The full color images can befound in the on-line version, at doi: http://dx.doi.org/10.1016/j.actbio.2013.10.011.

References

[1] Beattie JH, Avenell A. Trace element nutrition and bone metabolism. Nutr ResRev 1992;5:167–88.

[2] Mitchell HH, Hamilton TS, Steggerda FR, Bean HW. The chemical compositionof the adult human body and its bearing on the biochemistry of growth. J BiolChem 1945;158:625–37.

[3] Zofková I, Nemcikova P, Matucha P. Trace elements and bone health. Clin ChemLab Med 2013:1–7.

[4] Percival M. Bone health and osteoporosis. Appl Nutr Sci Rep 1999;5:1–6.[5] Moonga BS, Dempster DW. Zinc is a potent inhibitor of osteoclastic bone

resorption in vitro. J Bone Miner Res Official J Am Soc Bone Miner Res1995;10:453–7.

[6] Reginster JY, Seeman E, De Vernejoul MC, Adami S, Compston J, Phenekos C,et al. Strontium ranelate reduces the risk of nonvertebral fractures inpostmenopausal women with osteoporosis: treatment of PeripheralOsteoporosis (TROPOS) study. J Clin Endocrinol Metab 2005;90:2816–22.

[7] Cao W, Hench LL. Bioactive materials. Ceram Int 1996;22:493–507.[8] Kokubo T. Bioceramics and their clinical applications. London: CRC Press;

2008.[9] Hing KA, Revell PA, Smith N, Buckland T. Effect of silicon level on rate, quality

and progression of bone healing within silicate-substituted poroushydroxyapatite scaffolds. Biomaterials 2006;27:5014–26.

[10] Bohner M. Silicon-substituted calcium phosphates – a critical view.Biomaterials 2009;30:6403–6.

[11] Vitamin and mineral requirements in human nutrition: report of a joint FAO/WHO expert consultation chapter 12. Bangkok, Thailand; 1998.

[12] Eberle J, Schmidmayer S, Erben RG, Stangassinger M, Roth HP. Skeletal effectsof zinc deficiency in growing rats. J Trace Elem Med Biol 1999;13:21–6.

[13] Relea P, Revilla M, Ripoll E, Arribas I, Villa LF, Rico H. Zinc, Biochemical markersof nutrition, and type I osteoporosis. Age Ageing 1995;24:303–7.

[14] Atik OS. Zinc and senile osteoporosis. J Am Geriatr Soc 1983;31:790–1.[15] Yamaguchi M, Weitzmann MN. Zinc stimulates osteoblastogenesis and

suppresses osteoclastogenesis by antagonizing NF-jB activation. Mol CellBiochem 2011;355:179–86.

[16] Cen X, Wang R, Wu Z. Zinc promotes proliferation and differentiation ofosteoblast in rats in vitro. Chin J Prev Med 1999;33:221–3.

[17] Seo H-J, Cho Y-E, Kim T, Shin H-I, Kwun I-S. Zinc may increase bone formationthrough stimulating cell proliferation, alkaline phosphatase activity andcollagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract2010;4:356–61.

[18] Li X, Senda K, Ito A, Sogo Y, Yamazaki A. Effect of Zn and Mg in tricalciumphosphate and in culture medium on apoptosis and actin ring formation ofmature osteoclasts. Biomed Mater Bristol Engl 2008;3:045002.

[19] Holloway WR, Collier FM, Herbst RE, Hodge JM, Nicholson GC. Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of boneresorption and enhancement of osteoclast number. Bone 1996;19:137–42.

[20] Ishikawa K, Miyamoto Y, Yuasa T, Ito A, Nagayama M, Suzuki K. Fabrication ofZn containing apatite cement and its initial evaluation using humanosteoblastic cells. Biomaterials 2002;23:423–8.

[21] Ito A, Otsuka M, Kawamura H, Ikeuchi M, Ohgushi H, Sogo Y, et al. Zinc-containing tricalcium phosphate and related materials for promoting boneformation. Curr Appl Phys 2005;5:402–6.

[22] Li X, Sogo Y, Ito A, Mutsuzaki H, Ochiai N, Kobayashi T, et al. The optimum zinccontent in set calcium phosphate cement for promoting bone formation invivo. Mater Sci Eng C Mater Biol Appl 2009;29:969–75.

[23] Doty SB, Jones KW, Kraner HW, Shroy RE, Hanson AL. Proton microprobeanalysis of zinc in skeletal tissues. Nucl Instrum Methods 1981;181:159–64.

[24] Oh S-A, Kim S-H, Won J-E, Kim J-J, Shin US, Kim H-W. Effects on growth andosteogenic differentiation of mesenchymal stem cells by the zinc-added sol–gel bioactive glass granules. J Tissue Eng 2011;2010:475260.

[25] Oh S, Won J, Kim H. Composite membranes of poly(lactic acid) with zinc-added bioactive glass as a guiding matrix for osteogenic differentiation of bonemarrow mesenchymal stem cells. J Biomater Appl 2012;27:413–22.

[26] Mansilla E, Marín GH, Drago H, Sturla F, Salas E, Gardiner C, et al. Bloodstreamcells phenotypically identical to human mesenchymal bone marrow stem cellscirculate in large amounts under the influence of acute large skin damage:new evidence for their use in regenerative medicine. Transplant Proc2006;38:967–9.

[27] Minguell JJ, Erices A, Conget P, Onget PAC. Mesenchymal stem cells. Exp BiolMed 2001;226:507–20.

[28] Li X, Senda K, Ito A, Sogo Y, Yamazaki A. Difference in osteoclast responses totricalcium phosphate in culture medium supplemented with zinc and to zinc-containing tricalcium phosphate. Bioceram Dev Appl 2010;1:1–4.

[29] Yamada Y, Ito A, Kojima H, Sakane M, Miyakawa S, Uemura T, et al. Inhibitoryeffect of Zn2+ in zinc-containing b-tricalcium phosphate on resorbing activityof mature osteoclasts. J Biomed Mater Res A 2008;84:344–52.

[30] Boyce BF, Yao Z, Zhang Q, Guo R, Lu Y, Schwarz EM, et al. New roles forosteoclasts in bone. Ann N Y Acad Sci 2007;1116:245–54.

[31] Roy M, Fielding GA, Bandyopadhyay A, Bose S. Effects of zinc and strontiumsubstitution in tricalcium phosphate on osteoclast differentiation andresorption. Biomater Sci 2013;1:74.

[32] Ikeuchi M, Ito A, Dohi Y, Ohgushi H, Shimaoka H, Yonemasu K, et al. Osteogenicdifferentiation of cultured rat and human bone marrow cells on the surface ofzinc-releasing calcium phosphate ceramics. J Biomed Mater Res Part A2003;67:1115–22.

[33] Ito A, Kawamura H, Otsuka M, Ikeuchi M, Ohgushi H, Ishikawa K, et al. Zinc-releasing calcium phosphate for stimulating bone formation. Mater Sci Eng C2002;22:21–5.

[34] Paul W, Sharma CP. Effect of calcium, zinc and magnesium on the attachmentand spreading of osteoblast like cells onto ceramic matrices. J Mater Sci MaterMed 2007;18:699–703.

[35] Arinzeh TL, Tran T, Mcalary J, Daculsi G. A comparative study of biphasiccalcium phosphate ceramics for human mesenchymal stem-cell-induced boneformation. Biomaterials 2005;26:3631–8.

[36] Wang C, Duan Y, Markovic B, Barbara J, Howlett CR, Zhang X, et al. Phenotypicexpression of bone-related genes in osteoblasts grown on calcium phosphateceramics with different phase compositions. Biomaterials 2004;25:2507–14.

[37] Kurashina K, Kurita H, Wu Q, Ohtsuka A, Kobayashi H. Ectopic osteogenesiswith biphasic ceramics of hydroxyapatite and tricalcium phosphate in rabbits.Biomaterials 2002;23:407–12.

[38] Costa-Rodrigues J, Fernandes A, Lopes MA, Fernandes MH. Hydroxyapatitesurface roughness: complex modulation of the osteoclastogenesis of humanprecursor cells. Acta Biomater 2012;8:1137–45.

[39] Marchisio M, Di Carmine M, Pagone R, Piattelli A, Miscia S. Implant surfaceroughness influences osteoclast proliferation and differentiation. J BiomedMater Res B Appl Biomater 2005;75:251–6.

[40] Brinkmann J, Hefti T, Schlottig F, Spencer ND, Hall H. Response of osteoclasts totitanium surfaces with increasing surface roughness: an in vitro study.Biointerphases 2012;7:34.

[41] Ross AM, Jiang Z, Bastmeyer M, Lahann J. Physical aspects of cell culturesubstrates: topography, roughness, and elasticity. Small 2012;8:336–55.

[42] Watari S, Hayashi K, Wood JA, Russell P, Nealey PF, Murphy CJ, et al.Modulation of osteogenic differentiation in hMSCs cells by submicrontopographically-patterned ridges and grooves. Biomaterials 2012;33:128–36.

[43] McNamara LE, McMurray RJ, Biggs MJP, Kantawong F, Oreffo ROC, Dalby MJ.Nanotopographical control of stem cell differentiation. J Tissue Eng2010;2010:120623.

[44] Schwartz Z, Lohmann CH, Oefinger J, Bonewald LF, Dean DD, Boyan BD.Implant surface characteristics modulate differentiation behavior of cells inthe osteoblastic lineage. Adv Dent Res 1999;13:38–48.

[45] Barradas AMC, Yuan H, Van Blitterswijk CA, Habibovic P. Osteoinductivebiomaterials: current knowledge of properties, experimental models andbiological mechanisms. Eur Cells Mater 2011;21:407–29 [Discussion 429].