Influence of osteocytes in the in vitro and in vivo ...

Influence of osteocytes in the in vitro and in vivo b-tricalciumphosphate-stimulated osteogenesis

Zetao Chen,1 Chengtie Wu,2 Jones Yuen,1 Travis Klein,1 Ross Crawford,1 Yin Xiao1

1Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Brisbane, Queensland 4059,

Australia2State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics,

Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China

Received 8 June 2013; revised 5 September 2013; accepted 6 September 2013

Published online 30 September 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34954

Abstract: Osteocytes, known to act as the main regulators of

bone homeostasis, have become a major focus in the field of

bone research. Bioactive ceramics have been widely used for

bone regeneration. However, there are few studies about the

interaction of osteocytes with bioceramics. The effects of

osteocytes on the in vitro and in vivo osteogenesis of biocer-

amics are also unclear. The aim of this study was to investi-

gate the role of osteocytes on the b-tricalcium phosphate

(b-TCP) stimulated osteogenesis. It was found that osteocytes

responded to the b-TCP stimulation, leading to the release of

Wnt (wingless-related MMTV integration site), which

enhanced osteogenic differentiation of bone marrow stromal

cells via Wnt signaling pathway. Receptor activator of nuclear

factor kappa B ligand, an osteoclast inducer, was also upreg-

ulated, indicating that osteocytes would also participated in

activation of osteoclasts, which played a major role in the

degradation process of b-TCP and new bone remodeling. In

vivo studies further demonstrated that when the material

was completely embedded by newly formed bone, the only

cell contacting with the material was osteocyte. However, the

material would eventually be degraded and replaced by the

new bone, requiring the participation of osteoclasts and

osteoblasts, which were demonstrated by using immuno-

staining in this study. As the only cell contacting with the

material, osteocytes probably acted in a regulatory role to

regulate the surrounding osteoclasts and osteoblasts. Osteo-

cytes were also found to participate in the maturation of

osteoblasts and the mineralization process of biomaterials,

by upregulating E11 (podoplanin) and dentin matrix protein 1

expression. These findings indicated that osteocytes involved

in bone biomaterial-mediated osteogenesis and biomaterial

degradation, providing valuable insights into the mechanism

of material-stimulated osteogenesis, and a novel strategy to

optimize the evaluating system for the biological properties

of biomaterials. VC 2013 Wiley Periodicals, Inc. J Biomed Mater Res

Part A: 102A: 2813–2823, 2014.

Key Words: osteocyte, bone substitute, b-TCP, osteogenesis

How to cite this article: Chen Z, Wu C, Yuen J, Klein T, Crawford R, Xiao Y. 2014. Influence of osteocytes in the in vitro andin vivo b-tricalcium phosphate-stimulated osteogenesis. J Biomed Mater Res Part A 2014:102A:2813–2823.

INTRODUCTION

Osteocytes, comprising more than 95% of all cells of matureadult bone, have a cellular surface around 400 times largerthan that of the entire Haversian system, and more than100 times larger than the surface of trabecular bone.1–3

They are star shaped and extend dendritic processes widelywithin calcified matrix inside microscopic canaliculi, formingthe lacuna–canalicular network.4 Through this network,osteocytes actively communicate with surrounding cells andmatrix.5 In reaction to local environment change, osteocytescan send signals through the canalicular network to the sur-face of the bone tissue and influence the activities of bonecells.6 Osteocytes appear to be centrally involved in phos-phate homeostasis via dentin matrix protein 1 (DMP1)

phosphate-regulating endopeptidase homolog, X-linked(Phex), and fibroblast growth factor 23 (Fgf 23).7 They alsohave some effects in controlling calcium homeostasis,through the expression of parathyroid hormone type 1 recep-tor.8 Osteocytes can control the osteoblastic differentiation ofmesenchymal precursors by activating Wnt signaling path-ways.3,9 Additionally, osteocytes can induce osteoclast activa-tion,5 apparently through the receptor activator of nuclearfactor kappa-B ligand (RANKL) pathway.10 Therefore, it canbe concluded that osteocytes act as a primary regulator ofbone formation and remodeling.

Bone marrow stromal cells (BMSCs) are stem cells hav-ing the potentials to differentiate into multiple cell typesunder specific differentiating conditions. Under osteogenic

Correspondence to: C. Wu; e-mail: [email protected] or Y. Xiao; e-mail: [email protected] grant sponsor: Discovery project of Australian Research Council; contract grant number: DP120103697

Contract grant sponsor: Recruitment Program of Global Young Talent, China (Dr. Wu) and Natural Science Foundation of China; contract grant

number: 81201202 and 51061160499

VC 2013 WILEY PERIODICALS, INC. 2813

condition, they can differentiate into preosteoblasts orosteoblasts. Osteoblasts can secrete nonmineralized bonematrix, to accumulating mineral ions and assemble theminto mineralized bone matrix. During this process, osteo-blasts are buried in bone matrix and change into osteo-cytes.1 As a terminal osteoblastic cell, osteocyte used to besupposed as silence cell, without any meaningful functionbut just resting in bone lacuna. Therefore, for the biologicalevaluation of bone substitute materials, the strategy usuallywas using BMSCs or osteoblasts to test the osteogenesiscapacity of materials11,12; those potentially osteoconductivematerials would then be evaluated in an in vivo scenario.13

However, the in vitro and in vivo biological response ofbiomaterials was not always consistent due to the signifi-cant difference between two microenvironments.14,15 It wassuggested that in vivo osteogenesis was a consequence ofinteractions among multiple bone cells.16 It is inaccurate toonly focus on the simple interactions between bone substi-tute materials and osteoblastic or osteoclastic cells.Although derived from osteoblasts, osteocytes play a differ-ent role in bone dynamics, acting as a regulatory cell ratherthan forming bone matrix as described before. It is reasona-ble to speculate that the neglecting of osteocytes may beone of important reasons responsible for the inconsistentresults in vitro and in vivo. Nevertheless, the interactionsbetween osteocytes and bone substitute materials have notbeen well studied. It is a logical extension to investigate theinteractions of biomaterials and osteocytes, as well as thebiological effects of osteocytes on BMSCs during the processof biomaterials stimulated bone regeneration. Therefore,the aim of this study was to investigate the interactionsbetween bone substitute materials and osteocytes, to revealwhether osteocytes act as a potential regulator of material-stimulated bone formation and the degradation of materials.b-Tricalcium phosphate (b-TCP) has been well recognized asan osteoconductive material and has been widely used inclinics with certain clinical success.17–19 In the currentstudy, b-TCP is used as a model biomaterial to investigatethe osteocyte response with respect to bone formation andbiomaterials degradation.

MATERIALS AND METHODS

Cell cultureThe murine-derived osteocyte cell line MLO-Y4 (provided byProf. Lynda Bonewald, University of Missouri-Kansas City)and human bone marrow mesenchymal stem cells (BMSCs)were used in this study. MLO-Y4 cells were cultured as pre-viously described.20,21 They were maintained on rat tailtype I collagen (Becton Dickson Bioscience, Franklin Lakes,NJ) coated plates in Dulbecco’s modified eagle medium(DMEM; Gibco-Invitrogen, Carlsbad, CA) supplemented with10% fetal bovine serum (FBS; Thermo Scientific, Waltham,MA), and 1% penicillin/streptomycin (Gibco-Invitrogen) at37�C in a humidified CO2 incubator. Cells were passaged atapproximately 70% confluence and expanded through twopassages before being used for the study.

BMSCs were isolated and cultured based on protocolsfrom previous studies.22,23 Briefly, bone marrow was

obtained from patients (50–60 years old) undergoing hipand knee replacement surgery with informed consent givenby all donors and the procedure was approved by the EthicsCommittee of Queensland University of Technology. Lympho-prep was added to isolate the mononuclear cells from thebone marrow by density gradient centrifugation (Axis-ShieldPoC AS, Oslo, Norway). The obtained cells were seeded intothe tissue culture flasks containing DMEM supplementedwith 10% FBS and 1% penicillin/streptomycin and incu-bated at 37�C in a humidified CO2 incubator. The culturemedium was changed every 3 days until the primary mes-enchymal cells reached 80% confluence. The unattachedhematopoietic cells were removed through medium change.The confluent cells were routinely subcultured by trypsini-zation. Only early passages (p3–5) of cells were used in thisstudy.

Metabolic activity of osteocytes with b-TCP extractsb-TCP was synthesized by a chemical precipitation methodas previously described.24 The prepared powders weresieved with 60–80 mesh to obtain the particle size of 180–250 lm before further experiment. Based on the standardISO/EN 10993-5, the cells were cultured in b-TCP powderextract medium.24,25 The powder extracts were prepared bysoaking powders in serum-free DMEM at a series of solid/liquid ratio (200, 100, 50, 25, 12.5, 6.25, and 0 mg/mL).After incubation at 37�C for 24 h, the mixture was centri-fuged and the supernatant was collected and sterilized byfiltration through 0.2 mm filter membranes (Pall Corpora-tion, Port Washington, NY).

MLO-Y4 cells were seeded in a 96-well tissue cultureplate at a density of 1000 cells per well, with each groupincluding three wells. After 24 h of incubation, the culturemedium was removed and replaced by 200 mL of materialextracts containing 10% FBS. DMEM without extract supple-mented with 10% FBS was used as a control. At days 1, 3,and 5, a 3-(4,5-dimethylthiazol-2-yl)22,5-diphenyl tetrazo-lium bromide (MTT) assay was applied to test the overallmetabolic activity. The culture media was changed at day 3for the day 5 groups. 20 mL of 5 mg/mL MTT solution wasadded into each well. After incubation for a further 4 h, theDMEM-MTT solution was carefully removed and replaced by100 mL of dimethyl sulfoxide to dissolve the formazan crys-tals. The absorbance was read twice at the wavelength of495 nm by a microplate spectrophotometer (BenchmarkPlus, Tacoma, WA).

Osteogenesis-related gene expression of osteocytes withb-TCP extractsMLO-Y4 cells were seeded in a 6-well plate at a density of50,000 cells per well. After 24 h of incubation, the culturemedium was removed and replaced by material extract con-taining 10% FBS. Culture medium with 10% FBS was usedas a negative control. After three days, media from cells cul-tured in material extract or culture medium were collected,and centrifuged at 1500 rpm to gain the supernatants forfurther conditioned media experiment and ions concentra-tion detection. Total RNA of both groups were extracted

2814 CHEN ET AL. INFLUENCE OF OSTEOCYTES

using TRIzol reagent (Invitrogen) for Reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) detection.

Total RNA of 500 ng was used for the synthesis of com-plementary DNA (cDNA) using DyNAmoTM cDNA SynthesisKit (Finnzymes; Thermo Scientific) following the manufac-turer’s instructions. RT-qPCR primers (Table I) weredesigned based on cDNA sequences from the NCBI Sequencedatabase. SYBR Green qPCR Master Mix (Invitrogen) wasused for detection and the target messenger RNA (mRNA)expressions were assayed on the ABI Prism 7300 ThermalCycler (Applied Biosystems, Foster City, CA). Each samplewas performed in triplicate. The mean cycle threshold (Ct)value of each target gene was normalized against Ct valueof the house keeping gene (glyceraldehyde 3-phosphatedehydrogenase) and the relative expression calculated usingthe following formula: 22(normalized average Cts) 3 104.

Changes in Ca21 and PO432 ionic concentrations

To understand the mechanism of the gene expressionchanges, the ionic concentrations of Ca21, and PO4

32 incomplete culture medium, b-TCP extracts and osteocyte con-ditioned media were quantified by inductive-coupled plasmaatomic emission spectrometry (ICP-AES; PerkinElmer, Wal-tham, MA). To investigate the mechanism of the ion concen-tration changes before and after immersion, 10 mg b-TCPpowders were immersed in 50 mL DMEM. After incubationat 37�C for 24 h, the mixture was centrifuged and thesupernatant was removed. The gained powers were madedry, and then dissolved in 50 mL HNO3. The ionic concen-trations of Ca21, and PO4

32 were detected using ICP-AES.The functional groups in surface of the b-TCP powders afterimmersion were identified using Fourier transform infraredspectroscopy (NicoletTM iSTM50 FT-IR Spectrometer; Thermo

Scientific), with the attenuated total reflectance samplingtechnique. Raw b-TCP powders were used for comparison.

Effects of the osteocyte conditioned b-TCP extracts onthe osteogenic differentiation of BMSCsTo investigate whether osteocytes could regulate osteogene-sis of BMSCs in response to material extracts, the BMSC cul-ture medium was supplemented with the collectedsupernatants detailed in the previous section at the ratio of1:2 to obtain the conditioned media. The pure materialextract and culture medium without being conditioned byosteocytes were used as controls. BMSCs were plated at adensity of 20,000 cells per well in separate 6-well plates.After 24 h of incubation, the culture medium was removedand replaced by conditioned media or control media. TotalRNA of all four groups were extracted using TRIzol reagentafter 7 days of culture for the RT-qPCR detection as thesame method in the previous section.

Animal surgeryTo further investigate the osteocyte roles in the in vivo osteo-genesis of b-TCP bioceramics, adult Wistar rats at 10 weeksof age were used in the study. Animal surgery protocols wereapproved by the Animal Ethics Committee of QueenslandUniversity of Technology. The animals were operated undergeneral anesthetic using intramuscular injections with 10mg/300 g xylazine, and 17.5 mg/300 g ketamine. The medialfemoral condyles were used for the test of materials in bonedefect model, while empty defects in the contralateral legwere used as control. Following a knee arthrotomy, a drilledbone defect of 3 mm diameter and a 3-mm depth was madeon the medial condyles of the femur. b-TCP powders weremixed with little saline before filled to the defects. Gentamy-cin (5 mg/kg) was administered intramuscularly to avoid any

TABLE I. Primer Pairs Used in the RT-qPCR

Genes Primer Sequences

E11 Forward: 50-AAACGCAGACAACAGATAAGAAAGAT-30

Reverse: 50-GTTCTGTTTAGCTCTTTAGGGCGA-30

SOST Forward: 50-TATGACGCCAAAGATGTGTC-30

Reverse: 50-GAGCACACCAACTCGGTGAC-30

DMP1 Forward: 50-CCAGTTGCCAGATACCACAATAC-30

Reverse: 50-GGTCACTATTTGCCTGTCCC-30

WNT 5A Forward: 50-CAACTGGCAGGACTTTCTCAA-30

Reverse: 50-CCTGATACAAGTGGCAGAGTTTC-30

WNT 5B Forward: 50-CACCAGTTTCGACAGAGGC-30

Reverse: 50-GCAGTCTCTCGGCTACCTATCT-30

RANKL Forward: 50-CGCTCTGTTCCTGTACTTTCG-30

Reverse: 50-GAGTCCTGCAAATCTGCGTT-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

GAPDH (mouse) Forward: 50-TGACCACAGTCCATGCCATC-30

Reverse: 50-GACGGACACATTGGGGGTAG-30

GAPDH (human) Forward: 50-TCAGCAATGCCTCCTGCAC-30

Reverse: 50-TCTGGGTGGCAGTGATGGC-30

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | AUG 2014 VOL 102A, ISSUE 8 2815

infection to the wound. All animals were sacrificed after 4weeks by means of barbiturate intracardiac injection. Aftersacrifice, the soft tissues were removed and the femoral con-dyles were obtained. The specimens were fixed in 4% para-formaldehyde at room temperature (RT).

Histological hematoxylin and eosin stainingThe femoral samples were decalcified in 10% EDTA for upto 4 weeks, embedded in paraffin and 5-mm thick serial sec-tions were cut from the paraffin blocks with a microtome.Selected slides from each sample were stained with hema-toxylin and eosin (H&E; HD Scientific, Wetherill Park, NewSouth Wales, Australia). Briefly, following dewaxing andhydration, Mayer’s hematoxylin was applied for nuclearstaining. After dehydration in increasing alcohol concentra-tions, cell plasma and extracellular matrix were stainedwith eosin. Pictures of the stained slides were then capturedusing Axion software under a light microscope (Carl ZeissMicroimaging GmbH, Gottingen, Germany), using variousmagnifications.

Tartrate resistant acid phosphatase stainingAfter being dewaxed and hydrated, samples were incubatedin tartrate resistant acid phosphatase (TRAP) buffer fortwenty minutes at RT. TRAP buffer was made by mixing theacetate solution, tartarte solution, napthol AS-BI phosphate,and fast garnet GBC salt in 50 mL of distilled water, follow-ing the manufacturer’s instructions (Sigma-Aldrich, St. Louis,MO). TRAP buffer without tartrate solution was used ascontrol. Samples were then incubated for 1 h at 37�C in ahumidity chamber. After being rinsed by distilled water, thesamples were counterstained with acid hematoxylin solution(Sigma-Aldrich). Pictures of the stained slides were thencaptured using Axion software under a light microscope, toshow the osteoclasts and their relationship with materials.

Immunohistochemical stainingSelected slides were used for immunohistochemical staining.Osteoblastic and osteoclastic cells were detected using alka-line phosphatize (ALP) and CD6826,27 (Abcam, Cambridge,UK). Osteocytes and the secreted matrixes were detectedusing antibodies against sclerostin (SOST), DMP1 and E11(Abcam). Tissue slices were dewaxed and hydrated. Theendogenous peroxidase activity was eliminated by incubat-ing in 3% H2O2 for 15 min. Nonspecific proteins wereremoved with the incubation of 10% swine serum for 1 h.Samples were incubated with ALP, SOST, DMP1, and E11antibody (1:300, 1:200, 1:1000, and 1:100, respectively)overnight at 4�C, followed by incubation with a biotinylatedswine-anti-mouse, rabbit, goat secondary antibody (DAKOMultilink, CA) for 15 min at RT, and then with steptavidinperoxidate (DAKO Multilink) for 15 min. Diaminobenzidinesolution was then added for 3 min to visualize the antibodycomplexes. The samples were counter stained with Mayer’shematoxylin for 15 s. Pictures of the stained slides werethen captured using Axion software under a light micro-scope, to show the osteoblastic cells and their relationship

with materials, also the maturity of osteocytes and theirsecreting matrixes related to mineralization.

Statistical analysisAll the analyses were performed using SPSS software (IBMSPSS, Armonk, NY). All the data were shown as mean-6 standard deviation and analyzed using one-way analysisof variance followed by least significant difference post hoctest. A level of significance was set at p< 0.05.

RESULTS

Metabolic activity of osteocytes with b-TCP extractsMTT analysis showed that the overall metabolic activity ofall groups with different solid/liquid ratios of materialextracts increased with the incubation time (Fig. 1). Overall,the cells grew well in the medium with different solid/liquid ratios of material extracts, with no significant differ-ences between the groups.

Osteogenesis-related gene expression of osteocytes withb-TCP extractsThe expressions of all osteocyte markers (E11, DMP1, andSOST) were studied after stimulation by material extract.Both E11 and DMP1 showed significant upregulation of rel-ative mRNA expression [p<0.05; Fig. 2(A,B)]. However, theexpression of SOST showed significant downregulation withsame treatment [p< 0.05; Fig. 2(C)].

WNT5A, WNT5B, and RANKL were all upregulated by thematerial stimulation (p< 0.05) in a dose dependent mannerof solid/liquid ratios of material extracts [Fig. 3(A–C)]. Highersolid/liquid ratios of material extracts led to higher upregula-tion of the gene expressions.

FIGURE 1. The overall metabolic activity of MLO-Y4 cells in medium

with different concentration of material extracts, detected by MTT.

[Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


The ionic concentration analysisThe ionic concentrations of Ca21 and PO4

32 ions in all col-lected samples are shown in Table II. After soaking withb-TCP powders (200 mg/mL), both Ca21 and PO4

32 ions con-centrations in the culture medium were decreased. Althoughboth Ca21 and PO4

32 ions concentrations in the materialextract were increased after being conditioned by osteocyte,they were still lower than that of osteocyte conditioned com-plete culture medium.

After immersion, Ca21 and PO432 ions in the b-TCP

powders were increased (Table III). Although the functionalgroups in surface of the b-TCP powders after immersionshowed no significant change for the wavelength of the cor-responding characteristic peaks, the intensity of peaks atboth 560–610 cm21 and 1000–1100 cm21 were enhancedwith same treatment (Fig. 4).

Effects of the osteocyte conditioned media on theosteogenic differentiation of BMSCsExpression of all three measured mineralization-relatedgene by BMSCs stimulated by osteocyte conditioned b-TCPextract were significantly upregulated relative to b-TCP

extract alone [p<0.05; Fig. 5(A–C)]. However, osteocyteconditioned complete culture medium alone showed nosignificant changes in expression of ALP and OPN, whencompared with complete culture medium [p< 0.05;Fig. 5(A,C)].

Osteocytes interaction with b-TCP particles in vivoAfter 4 weeks of implantation, new bone formation wasnoted around b-TCP materials and bone integration withthe remaining b-TCP particles was observed [Fig. 6(C,D)].Direct contact of osteocytes with materials was observedand mature bone matrix was formed surrounding b-TCPparticles [Fig. 6(B)]. At this stage, osteoblasts and osteo-clasts were also present around the surface of the remain-ing materials (Figs. 6 and 7).

Osteocytes in the newly formed bone were stained posi-tively by E11, indicating early formed osteocytes around b-TCP particles [Fig. 8(A)]. The DMP1 was detected in theosteocytes surrounding the materials, however, no SOSTexpression was detected [Fig. 8(B,C)]. It was noted thatthere was an accumulation of DMP1 protein in the materialsclose to the new bone formed around b-TCP particles

FIGURE 2. Relative mRNA expressions of E11 (A), DMP1 (B), and SOST (C) by MLO-Y4 cells with or without stimulation of material extract.

*Significant difference (p<0.05) for b-TCP extract groups compared to the blank group. [Color figure can be viewed in the online issue, which is

available at wileyonlinelibrary.com.]

FIGURE 3. Relative mRNA expressions of RANKL (A), WNT5A (B), and WNT5B (C) by MLO-Y4 cells with or without stimulation of material

extract. *Significant difference (p< 0.05) for b-TCP extract groups compared to the blank group. [Color figure can be viewed in the online issue,

which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


[Fig. 8(B)]. Osteoblastic cells and intermediate osteocytesboth stained positively for E11 [Fig. 8(A)].

DISCUSSION

Biodegradable materials, after implantation, will undergodegradation either by physicochemical dissolution, cell-medicated dissolution, hydrolysis, enzymatic decomposition,or corrosion.28 During this process, degraded components ofbiomaterials are released to the local environment. Whetherosteocytes could respond to the environmental changecaused by biomaterials and elicit some effects on the sur-rounding cells was still unknown, despite the well-documented importance of osteocyte in bone dynamics. Ourstudy demonstrated that osteocytes responded to the b-TCPstimulation, leading to the release of RANKL and Wnt, whichenhanced the material degradation by osteoclastic cells andosteogenesis by osteoblastic cells, respectively. We also foundthat osteocytes participated in the mineralization process ofbiomaterials.

b-TCP significantly affected the biological behaviors ofosteocytes in osteogenesisAlthough osteocytes have been found to contact with theintermediate osteocyte directly in our study, the matureosteocytes, buried in the bone matrix, contact with thematerial indirectly through the lacuna–canalicular network.The ions released from b-TCP were more important thanthe material particle itself, which could elicit some effectson the osteocyte through the lacuna–canalicular network. Tomimic this in vivo environment, we used material extractsrather than the material particle itself. It was found that,when the biocompatible ceramics was immersed in culturemedium, dissolved Ca and P in the medium were removedfrom the solution, forming a deposition on surface of theceramics.29 Our studies further found that Ca and P concen-trations of culture medium were decreased, which might berelated to reprecipitation and the formation Ca–P depositionon the surface of b-TCP.

Ca has been known to play an important role in Wntpathway.30 It was reported that an increase in intracellular

calcium concentration led to inhibition of the Wnt path-way.31 The decrease of extracellular calcium ions resulted inthe efflux of Ca, releasing the inhibition effects of intracellu-lar calcium on Wnt pathway. In addition, the upregulation ofWnts might also relate to the downregulation of SOST. SOSTgene encodes sclerostin, the Wnt signaling antagonist.32

This protein is secreted by osteocytes and acts in a para-crine or autocrine manner to inhibit bone formation byantagonizing the prodifferentiation and survival actions ofWnts in osteoblasts.33 Downregulation of sclerostin resultsin the increasing of Wnt signaling and enhances bone for-mation.34 In this study, mRNA expression of Wnt5a andWnt5b were both significantly upregulated by the stimula-tion of b-TCP extract, which might be related to the changein Ca concentration via b-TCP extracts and the downregu-lated of SOST.

As to the effects of phosphate, high extracellular inorganicphosphate concentration was reported to inhibit RANK–RANKL signaling in osteoclast-like cells through inhibit theRANKL-induced JNK and Akt activation. The decreased PO4

32

concentration of culture medium might activate the RANK–RANKL signaling, resulting in the observed upregulation ofRANKL from osteocytes.

b-TCP stimulated osteocytes with stimulatory effects onosteoblastic and osteoclastic cellsThe Wnt signaling pathway, known as a critical regulator ofosteocyte biology,3,9 participate in the maintenance of bonehomeostasis and regeneration.35 It is known that osteocytes,

TABLE II. The Ionic Concentrations of b-TCP Powder Extract (200 mg/mL) and Osteocyte Conditioned Media

Material immersed 2 2 1 1

Osteocyte conditioned 2 1 2 1

Ionic concentrations (mg/L)Ca21 76 6 1.95 96 6 4.26 52 6 1.67 76 6 1.36PO4

32 36 6 1.47 44 6 4.05 19 6 1.96 32 6 1.02

TABLE III. The Ca21 and PO432 Ionic Concentrations of 10 mg

b-TCP Powders Before and After Immersion

Before After

Ionic concentrations (mg/L)Ca21 68.6 6 0.8 75.5 6 2.9PO4

32 33 6 0.7 38.6 6 1.7

FIGURE 4. FTIR spectra of b-TCP powders before and after immersion.

[Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


when stimulated by mechanical loading, show evidences ofWnt/b-catenin signaling activation, and the signal is propa-gated to the surrounding osteocytes and then to the bone sur-face.36 Fluid flow also enhances the expression of b-catenin

target genes and activates the PKA signaling pathway inMC3T3-E1 osteoblasts, which can be a downstream target ofnoncanonical Wnts.37–39 As secreted glycoproteins, Wnt couldbe released and elicit effects on the surrounding osteocytes or

FIGURE 5. Relative mRNA expressions of ALP (A), OCN (B), and OPN (C). BMSCs cultured in complete culture medium, material immersed

medium, or MLO-Y4 conditioned media. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 6. H&E staining, 4 weeks after implantation of b-TCP particles in the femurs of Wistar rats. The figure showed the interaction between

osteocytes and implanted b-TCP particles at different stages: (A) the remaining material was surrounded by fibro-vascular tissue only; (B) osteo-

clasts began to degrade the material (black arrow) and new bone began to be formed on the surface of material; some intermediate osteocytes

contacted the material directly (white arrow); (C) more than half of the material was surrounded by new bone; osteoclasts continued to degrade

the material when osteoblasts formed more bone; and (D) the material was completely embedded by new formed bone; only osteocytes were

found surrounding the material. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


FIGURE 7. A and C, H&E staining, (B) ALP immunohistochemical staining, (D) TRAP staining, and (E and F) CD68 immunohistochemical staining.

A, osteoblasts lined on the surface of material; (B) ALP antibody bound to the lining cells around the material; (C) adjacent multinuclear cell was

found (arrow); (D) the multinuclear cells were positively stained by TRAP; and (E and F) CD68 positive cells were found in the surface of both

new-formed bone and remaining materials. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 8. Immunohistochemical staining, 4 weeks after implantation of b-TCP particles in the femurs of Wistar rats. A, E11 staining: osteoblas-

tic cells, intermediate osteocytes, osteocytes were all strongly positively stained; (B) DMP1 staining: some of the osteocytes were positively

stained (arrow); the material area was strongly stained; and (C) SOST staining: osteocytes were negatively stained. [Color figure can be viewed

in the online issue, which is available at wileyonlinelibrary.com.]


other cells through the lacuna–canalicular network. Wnt5abinds to the receptor tyrosine kinase-like orphan receptor 2(Ror2) on target cells, leading to activation of the Wnt5a/Ror2 signaling pathway,40 which is known to be a substantialconstituent for osteoblastic differentiation.41 In this study, b-TCP extract upregulated Wnt expression in osteocytes, andthe conditioned medium from the stimulated osteocytes ledto the osteogenic differentiation of BMSCs, even though b-TCPextract alone could not directly induce the osteogenic differ-entiation of BMSCs under the observation of this study. Theseresults indicate that osteocytes may play an important regula-tory role in b-TCP inducing osteogenesis.

In normal bone homeostasis, osteocytes are the majorsource of RANKL, which is essential for osteoclast differen-tiation during cancellous bone remodeling.42 The releasedRANKL can bind to RANK, a receptor on the surface ofosteoclast precursors, activating signaling through nuclearfactor kappa B, activator protein 1, and nuclear factor ofactivated T cells 2 to induce expression of genes for survivaland differentiation of osteoclasts.43 The upregulation ofRANKL in osteocytes may be responsible for the recruit-ment of osteoclasts to the biomaterials surface leading tobiomaterial degradation and the remodeling of new-formedbone.

b-TCP stimulated osteocytes with stimulatory effects onthe maturation of osteoblasts and the mineralizationDuring osteogenesis, osteoblasts are embedded by mineral-ized matrix to eventually form osteocytes. Several criticalgenes are involved in the osteocyte maturation and matrix

mineralization. E11, a transmembrane mucin-type glycopro-tein with O-glycosylation and high sialic acid content,44,45 isexpressed by the intermediate cell types (mature osteo-blasts and newly formed osteocytes that have recentlyembedded or are in the process of embedding).45,46 Whenthe osteocytes become more mature with the formation ofdendrites, E11 expression is reduced46; however, SOST andDMP1, markers for the mature osteocytes, are increased.44

Low SOST and DMP1 gene expression were observed, whichwas consistent with the previous study indicating that MLO-Y4 expressed low levels of SOST and DMP1 although osteo-cytes were known to express these genes in vivo.47,48 b-TCPextract significantly affected the expression of E11, SOST,and DMP1, indicating its effect on osteocyte maturation.

It was also found that the remaining material particlesseemed to accumulate DMP1 proteins, which can enhancehydroxyapatite formation and control osteogenesis and den-tinogenesis.49 DMP1 is particularly important in bone min-eralization. Clusters in DMP1 provide the molecular designnecessary for controlling the formation of oriented calciumphosphate crystals.50 DMP1 can also accelerate the nuclea-tion of hydroxyapatite.51 This suggests that osteocytes maysecrete DMP1 proteins to participate in the mineralizationof implanted material.

Possible effects of osteocytes in the process of b-TCPmaterial inducing osteogenesis are summarized in Figure 9.Degradation products released from the materials elicitsome effects on the osteocytes through the lacuna–canalicu-lar network. The stimulated osteocytes downregulate theSOST gene expression, resulting in the increase of Wnt,

FIGURE 9. Summary of possible regulation effects of osteocytes in the process of b-TCP material inducing osteogenesis. [Color figure can be

viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


which leads to the osteogenic differentiation of BMSCs viaactivating the Wnt pathways. Osteocytes also elicit somepositive effects on the maturation of osteoblasts and themineralization process of biomaterials, by upregulating E11and DMP1 gene expression. Osteocytes also enhance the dif-ferentiation of osteoclasts by the release of RANKL, whichfacilitate the material degradation and new bone remodeling.

The confirmation that osteocytes behaved actively duringthe process of osteogenesis caused by implanted biomateri-als is meaningful. Materials failing to activate osteocytes mayresult in the prevention of timely degradation and integra-tion between bone and materials, or inadequately activateosteocyte might causing the imbalance between osteoblasto-genesis and osteoclastogenesis, leading to undesired boneloss. The reaction of osteocytes should be added into themultiple bone cells testing system for evaluating the biologi-cal properties of bone substitute materials.

It is known that in vivo material/host body interactionis a complex process, involving multiple systems, includingthe skeletal system, immune system, coagulation system,and so forth. Therefore, the interaction between culturemedium and material could not completely mimic the invivo material/host body interaction. We further carried outin vivo studies, trying to prove the in vitro findings in an invivo environment, and we found supporting results as indi-cated before. However, the in vivo evidence for osteocytereleasing factors to regulate the surrounding osteoclastsand osteoblasts were limited by study methods used for thein vivo studies of osteocytes. In addition, particles size andother properties may affect the osteocyte indirectly byaffecting other cell types, which then release cytokines andeventually affect osteocyte. Further studies should be car-ried out to make the conclusions more strong.

CONCLUSIONS

This study demonstrated that osteocytes responded to b-TCPbone substitute materials by mediation of osteogenesis andbiomaterial degradation, which provides valuable insights forunderstanding the mechanisms of material-stimulated osteo-genesis and a novel approach for the optimization and evalu-ation of materials properties.

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