noncanonicalwnt-4signalingenhancesboneregeneration ...canonical wnt signaling plays a critical role...

Noncanonical Wnt-4 Signaling Enhances Bone Regenerationof Mesenchymal Stem Cells in Craniofacial Defects throughActivation of p38 MAPK*□S

Received for publication, March 20, 2007, and in revised form, August 23, 2007 Published, JBC Papers in Press, August 24, 2007, DOI 10.1074/jbc.M702391200

Jia Chang‡§, Wataru Sonoyama¶, Zhuo Wang‡, Qiming Jin�, Chengfei Zhang‡**, Paul H. Krebsbach‡,William Giannobile�, Songtao Shi¶, and Cun-Yu Wang‡§1

From the ‡Department of Biologic and Materials Sciences, �Department of Periodontics and Oral Medicine, School of Dentistry, andDepartment of Biomedical Engineering, College of Engineering, University of Michigan, Ann Arbor, Michigan 48109, the **Schooland Hospital of Stomatology, Peking University Health Science Center, Beijing 100081, China, the ¶Center for CraniofacialMolecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90033, and the §Laboratory ofMolecular Signaling, Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, California 90095

Mesenchymal stemcells (MSCs) aremultipotent cells that canbe differentiated into osteoblasts and provide an excellent cellsource for bone regeneration and repair. Recently, the canonicalWnt/�-catenin signaling pathway has been found to play a crit-ical role in skeletal development andosteogenesis, implying thatWnts can be utilized to improve de novo bone formation medi-ated by MSCs. However, it is unknown whether noncanonicalWnt signaling regulates osteogenic differentiation. Here, wefind that Wnt-4 enhanced in vitro osteogenic differentiation ofMSCs isolated from human adult craniofacial tissues and pro-moted bone formation in vivo. WhereasWnt-4 did not stabilize�-catenin, it activated p38 MAPK in a novel noncanonical sig-naling pathway. The activation of p38 was dependent on Axinand was required for the enhancement of MSC differentiationbyWnt-4. Moreover, using two different models of craniofacialbone injury, we found that MSCs genetically engineered toexpress Wnt-4 enhanced osteogenesis and improved the repairof craniofacial defects in vivo. Taken together, our results revealthat noncanonicalWnt signaling could also play a role in osteo-genic differentiation.Wnt-4mayhave a potential use in improv-ing bone regeneration and repair of craniofacial defects.

Human adult mesenchymal stem cells (MSCs)2 are multipo-tent cells originally isolated from the bonemarrow that are ableto differentiate into several cell types, including osteoblasts,chondrocytes, adipocytes, andmyocytes (1–5). Because of theirosteogenic potential, MSCs could be potentially used for

repairing critical size bone defects that normally cannotundergo spontaneous healing (2, 3). Recently, growing evidencehas suggested that MSCs also exist in a number of nonmarrowtissues. For example, MSCs isolated from adipose tissue andmuscle express some common cell surface markers that areidentified in marrow MSCs from bone marrow and can differ-entiate into a variety of cell types, including osteoblasts, musclecells, and neural cells (6–8). Recently, based on the primarycharacteristics of MSCs from bone marrow, we have isolatedMSCs from craniofacial tissues, such as the dental pulp andperiodontal ligament (9, 10). Like MSCs isolated from bonemarrow, these cells are self-renewing, multipotent, and clono-genic. They can be induced to differentiate into osteoblast-likecells and adipocytes in vitro. When implanted into immunod-eficient mice, these cells formed mineralized tissues or relatedcraniofacial structures, suggesting that they can be used in thereplacement of tooth and craniofacial (9, 10).Although bone regeneration mediated by MSCs shows

potential utility, the regenerative capacity of these cells is rela-tively lowwhen taking into account the number of cells utilizedfor transplantation in vivo. Moreover, critical size defects inpatients often require a large number of MSCs. To obtainenoughMSCs for cell transplantation,MSCsmust be expandedex vivo (2). However, the potential problem for ex vivo expan-sion is thatMSCs have a limited life span andmay gradually losetheir osteogenic potential. To overcome these potential prob-lems, several strategies have been explored.We and others havefound that overexpression of telomerase significantly extendedthe life span ofMSCs and enhanced bone formation potential ofMSCs both in vitro and in vivo (11). A variety of growth factorsthat promote osteogenic differentiation have also been utilizedto enhance bone regenerationmediated byMSCs. For example,studies by Peng et al. (7) have demonstrated that vascular endo-thelial growth factor and bonemorphogenetic protein (BMP)-4could significantly improve bone formation and healing byMSCs isolated from muscle.The canonical Wnt/�-catenin signaling pathway has

recently been found to play an essential role in bone develop-ment and postnatal maintenance of bone mass (12–20). SinceWnts are secreted growth factors, they may potentially be uti-lized as recombinant factors to improve bone regeneration.

* This work was supported by NIDCR, National Institutes of Health, GrantsDE16513 (to C. Y. W.) and DE13397 (to W. V. G.) and by the Department ofDefense (to C. Y. W. and P. K.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1– 4.

1 To whom correspondence should be addressed: Laboratory of MolecularSignaling, Division of Oral Biology and Medicine, UCLA, 10833 Le ConteAve., Los Angeles, CA 90095. Tel.: 310-825-4415; Fax: 310-794-7109; E-mail:[email protected].

2 The abbreviations used are: MSC, mesenchymal stem cell; BMP, bone mor-phogenetic protein; MAPK, mitogen-activating protein kinase; JNK, c-JunN-terminal kinase; ALP, alkaline phosphatase; HA, hemagglutinin; siRNA,small interfering RNA.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 42, pp. 30938 –30948, October 19, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

30938 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 42 • OCTOBER 19, 2007

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There are 19 Wnt family proteins in total that are primarilydivided into twomain categories based on their role in cytosolic�-catenin stabilization: canonical and noncanonical (21–24).Canonical Wnts transduce their signals through intracellular�-catenin. In the absence of Wnt proteins, �-catenin is associ-ated with a cytoplasmic complex containing adenomatous pol-yposis coli, glycogen synthase kinase-3�, Axin, and caseinkinase 1. In this complex, glycogen synthase kinase-3� consti-tutively phosphorylates �-catenin, resulting in its ubiquitina-tion and the degradation by the 26 S proteasome. In the pres-ence of Wnt stimulation, a frizzled receptor and the Wntco-receptor LRP5 (low density lipoprotein receptor-relatedprotein 5) or LRP6 transduce signals to inhibit Axin/adenoma-tous polyposis coli/glycogen synthase kinase-3� activity. Thisinhibition leads to the accumulation of free cytosolic�-catenin.The elevated cytosolic�-catenin can translocate to the nucleus,forma complexwithmembers of theTcf family of transcriptionfactors, and activate the expression of Wnt target genes (21–24). In contrast, noncanonical Wnts transduce their signalindependent of �-catenin. The noncanonical Wnt signalingpathway has been found to be associated with gastrulationmovements, heart induction, dorsoventral patterning, tissueseparation, and neuronal migration (21). Unlike the canonicWnt signaling pathway, the noncanonical Wnt signaling path-way is quite diverse. It has been reported that noncanonicalWnts can activate calcium flux, G proteins, Rho GTPases, orc-Jun N-terminal kinase (JNK) (21, 25, 26).Initial observations on the association between human LRP5

gene mutation and osteoporosis-pseudoglioma syndrome haveprovided a connection betweenWnt signaling and bone forma-tion (13, 14). Subsequently, studies from knock-out mice havedemonstrated that the canonical Wnt/�-catenin signalingpathway plays a critical role in bone formation (15–20). Thispathway might stimulate bone formation through severalmolecular mechanisms, including stem cell renewal, the stim-ulation of preosteoblast proliferation and/or differentiation,and the inhibition of apoptosis (15–20). These important dis-coveries provide amolecular basis to explore whether targetingWnt/�-catenin signaling can improve the efficiency of boneregeneration byMSCs (27–30). Although some studies demon-strated that the activation of theWnt/�-catenin signaling path-way promoted osteogenic differentiation, paradoxically, anumber of works recently claimed that Wnt/�-catenin signal-ing inhibited osteogenic differentiation and mineralization ofMSCs in vitro (31–37). For example, Boer et al. (31) and Bolandet al. (32) have demonstrated that the canonical Wnt-3a andWnt-1 inhibited osteogenic differentiation and promoted pro-liferation of MSCs in vitro. Cho et al. (33) found that Wnt/�-catenin signaling promoted proliferation and suppressedosteogenic differentiation of human adipose-derived MSCs.However, the underlying mechanism that is responsible for theinhibition of osteogenic differentiation of MSCs is not clear.Nevertheless, these findings suggest at least that, althoughcanonical Wnt signaling plays a critical role in bone develop-ment, targeting Wnt/�-catenin may be not useful for improv-ing osteogenic potential of MSCs for bone tissue engineering.During the course of studying the role of Wnts in craniofacialbone regeneration, consistent with the studies of Boer et al.

(31), we also observed that several canonical Wnts stronglyinhibited osteogenic differentiation of MSCs in vitro and invivo.3 Unexpectedly, we identified that Wnt-4, a noncanonicalWnt member, played a novel role in promoting osteogenic dif-ferentiation of MSCs isolated from craniofacial tissues. Usingtwo different models of craniofacial bone injury, we found thatMSCs thatwere genetically engineered to expressWnt-4 exhib-ited robust bone formation capacities and improved the repairof craniofacial bone defects. We found that p38 MAPK wasactivated by Wnt-4 in a novel noncanonical signaling pathwayin MSCs and played a critical role in enhancing osteogenic dif-ferentiation of MSCs. Our results suggest that Wnt-4 may bepotentially utilized for enhancing the properties of MSCs incraniofacial bone regeneration and repair.

EXPERIMENTAL PROCEDURES

Cell Culture and Retroviral Infection—C2C12 cells were pur-chased from ATCC and maintained in Dulbecco’s modifiedEagle’s medium (Invitrogen) supplemented with 15% fetalbovine serum. MSCs from human periodontal ligaments weregrown as described previously. BMP4 was purchased fromR & D Systems. To engineer C2C12 cells or MSCs stablyexpressing Wnt-4, the retrovirus-mediated infection was per-formed as described previously (38). Briefly, retroviruses wereproduced by transfecting the retroviral vector encodingWnt-4or control vector into 293T cells by the calcium phosphatemethod. Forty-eight h after transfection, retrovirus-containingsupernatants were collected, filtered with 0.45-�m filters, andstored in �70 °C. C2C12 cells or MSCs were infected with ret-roviruses in the presence of 6 �g/ml Polybrene (Sigma). Forty-eight h after infection, cells were selected with neomycin (2�g/ml) for 10 days. The surviving cells were pooled, and cellsexpressing Wnt-4 were confirmed by Western blot analysis.Western Blot Analysis—Cells were either untreated or

treated with BMP4 (50 ng/ml) and then harvested and washedonce with PBS. Cells were pelleted and lysed with cell lysisbuffer containing 1%Nonidet P-40, 5% sodium deoxycholate, 1mM phenylmethylsulfonyl fluoride, 100mM sodium orthovana-date, and 1:100 protease inhibitor mixtures (Sigma). The pro-tein concentrationwasmeasured according to themanufactur-er’s instruction (Bio-Rad). 30–50 aliquots of protein extractswere subjected to 10% SDS-PAGE and transferred to a polyvi-nylidene difluoride membrane (Bio-Rad) using a semidry geltransfer cell. Themembraneswere blockedwith 5%nonfatmilkovernight at 4 °C and then probed with the primary antibodies.The immunocomplexes were visualized with horseradish per-oxidase-coupled goat anti-rabbit or anti-mouse IgG (Promega)using the SuperSignal reagents (Pierce), as described previously(38). The primary antibodies were from the following sources:anti-�-tubulin monoclonal antibodies from Sigma; anti-HAmonoclonal antibodies were from Convance; anti-�-cateninmonoclonal antibodies were fromClontech; anti-phospho-p38polyclonal antibodies were from Cell Signaling; and anti-p38and anti-Axin polyclonal antibodies were from Santa Cruz Bio-technology, Inc. (Santa Cruz, CA).

3 J. Chang, W. Sonoyama, Z. Wang, Q. Jin, C. Zhang, P. H. Krebsbach, W. Gian-nobile, S. Shi, and C.-Y. Wang, unpublished observation.

Wnt-4 Enhances Mesenchymal Stem Cell Differentiation

OCTOBER 19, 2007 • VOLUME 282 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 30939


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Northern Blot Analysis—Cells were lysed with TRIzol rea-gent (Invitrogen). Total RNAs were extracted according to themanufacturer’s protocol. 5–10-�g aliquots of RNA sampleswere separated on a 1.4% agarose-formaldehyde gel and trans-ferred onto a nylon membrane for 16–24 h (Bio-Rad). RNAswere cross-linkedwith aUV cross-linker (Promega). Blots wereprehybridized with PerfectHyb (Sigma) buffer containingsalmon sperm DNA (100 �g/ml) for 20 min and then hybrid-ized with PerfectHyb buffer containing 32P-labeled osterix(Osx), type I collagen, or glyceraldehyde-3-phosphate dehydro-genase cDNA probes. The probes were labeled with a randomprimed labeling kit (Amersham Biosciences) in the presence of[�-32P]dCTP (ICN). The probes were purified with a micro-G50 Sephadex column (Amersham Biosciences). After hybrid-ization, the blots were washed twice in 2� SSC, 0.1% SDS for 10min at room temperature and twice in 0.1� SSC, 0.1% SDS for20 min at 42 °C, as described previously (38).Alkaline Phosphatase (ALP), von Kossa, Alizarin Red Stain-

ing, and Transplantation in Nude Mice—Cells were grown indifferentiation-inducing media containing 100 �g/ml ascorbicacid, 5 mM �-glycerophosphate, and 1 �M dexamethasone. 1–7days after induction, cells were fixed with 70% ethanol andstained with an ALP staining kit according to the manufactur-er’s protocol (Sigma). For detecting mineralization, cells wereinduced for 2–3 weeks, fixed with 70% ETOH, and stained with2% alizarin red or 1% silver nitrate (11) (Sigma).Approximately 4.0 � 106 of the cells were mixed with 40 mg

of hydroxyapatite/tricalcium phosphate ceramic particles(Zimmer Inc., Warsaw, IN) and then transplanted subcutane-ously into the dorsal surface of 10-week-old immunocompro-mised beigemice (bg-nu/nu-xid; Harlan Sprague-Dawley, Indi-anapolis, IN) as previously described (11). 8 weeks aftertransplantation, the transplants were harvested, fixed with 10%formalin, decalcified with buffered 10% EDTA (pH 8.0), andembedded in paraffin. Sections were then deparaffinized,hydrated, and stained with hematoxylin and eosin.Periodontal Bone Defect Model—A nude rat periodontal

defect model was utilized as previously described (44–46). Inbrief, athymic nude rats (approximate weight 240–260 g) (Har-lan, Indianapolis, IN) were anesthetized with ketamine (90mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection. A2-cm skin incision was made at the lower border of the mandi-ble, and the tissues were exposed by separating the superficialfascia, masseter muscle, and periosteum. A 3 � 2-mm defectwas made using a high speed dental handpiece on the buccalplate of the alveolar bone between the mandibular first andsecond molar teeth. The periodontal ligament, cementum, andalveolar bone were removed from the mesial and distobuccalroots of themandibular first molar and themesiobuccal root ofthemandibular secondmolar teethwithin the defect area. Poly-lactic co-glycolide polymer scaffolds (3 � 2 � 1 mm) wereseeded overnight with 2.5 � 105 MSC/V or MSC/Wnt-4 cellsand placed within the respective defects, as previouslydescribed for other tissue engineering applications. This studywas performed under protocols approved by the UniversityCommittee on Use and Care of Animals of the University ofMichigan and was in compliance with state and federal laws.

Five weeks postsurgery, rats were sacrificed by CO2 euthani-zation, and the mandibulae were harvested, immediately fixedin 10% neutral buffered formalin for 48 h, and then scanned bymicrocomputed tomography (�CT) (model Pxs5–928EA; GEHealthcare, London, Canada) to evaluate osseous structures.The mandibulae were subsequently decalcified with a 10%EDTA solution for�2–3weeks, dehydratedwith gradient alco-hols, and embedded in paraffin. Coronal sections �5 �m inthickness were cut and stained with hematoxylin and eosin.�CT scanning produced three-dimensional images, allowingfor detailed visualization and analysis of hard tissue architec-ture and anisotropy. The samples were scanned at 18 �m reso-lution formineralized tissuewith accelerated potential of 80 kVand a beam current of 80 �A. The eXplore Microview version2.0 software provides image analyzing tools with coronal, sag-gital, and transversal views two- and three-dimensionally. Lin-ear distances of roots were measured (mm) from the root fur-cation to root apex on the first molar (M1) distal root of thebuccal side of mandibulae by a single calibrated examiner. The�CT can create sections with 18-�m thickness from a three-dimensionally reconstructed image. Tooth furcations and rootapices were determined through a coronal view, and the num-ber of frames was counted. The exposed root length was meas-ured between furcation and the root apex. The fractions ofbone-covered root surface were calculated with the fractionexposed root length/total root length, as previously described(44–46). All results from the scans were statistically calculatedwith SPSS version 12 (SPSS Inc. Chicago, IL) with a statisticalsignificance at p � 0.05.Craniofacial Defect Model—Eighteen 5-week-old female

SCID mice (N:NIH-bg-nu-xid; Charles River Laboratories,Raleigh,NC)were divided into three groups randomly.Animalswere anesthetized with intraperitoneal injections of ketamineand xylazine. A linear scalp incision was made from the nasalbone to the occiput, and full-thickness flaps were elevated. Theperiosteum overlying the calvarial bone was completelyresected. A trephine was used to create a 5-mm craniotomydefect centered on the sagittal sinus, and thewoundswere copi-ously irrigatedwithHanks’ balanced salt solutionwhile drilling.The calvarial diskwas removed carefully in order to avoid injuryto the underlying dura or brain. After careful hemostasis, poly-mer scaffolds previously loaded with 5 � 105 cells were placedinto the defects. The scaffolds filled the entire defect andattached the bone edges around the entire periphery. The inci-sions were closed with 4-0 Chromic Gut suture (Ethicon/John-son & Johnson, Sommerville, NJ), and the mice recovered fromanesthesia on a heating pad. All mice were sacrificed 5 weeksafter the implantation (47). Calvaria were harvested, immedi-ately fixed in 10% neutral buffered formalin for 48 h, and thenscanned for �CT analysis. Calvaria were subsequently decalci-fied with a 10% EDTA solution for �2–3 weeks, dehydratedwith gradient alcohols, and embedded in paraffin. Coronal sec-tions �5 �m in thickness were cut and stained with hematox-ylin and eosin.For �CT scanning, the specimens were fitted in a cylindrical

sample holder, 15.4 mm in diameter, with the coronal aspect ofthe calvarial bone in a horizontal position. Specimens werescanned with the scanning direction parallel to the coronal




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aspect of the calvarial bone. High resolution scanning, with anin-plane pixel size and slice thickness of 24�m,was preformed.To cover the entire thickness of the calvarial bone, the numberof slices was set at 400. GEMS MicroView� software was usedto make a three-dimensional reconstruction form the set ofscans. To obtain the three-dimensional image, a thresholdvalue of 500 was used. On three-dimensional images of thespecimen, bone volume (mm3) and bone mineral density in thedefect site were measured directly.

RESULTS

OsteogenicDifferentiation ofMesenchymalCells Enhanced byWnt-4—C2C12 mouse mesenchymal cells are well character-ized cells that can be induced to differentiate into osteoblast-like cells in vitro following BMP stimulation (38). SinceWnt/�-catenin signaling has been found to play an essential role inosteoblast differentiation, we screened several Wnts to deter-mine if they could enhance BMP-4-induced osteogenic differ-entiation of C2C12 cells. C2C12 cells were engineered toexpress Wnt-4 by retroviral transduction as described previ-ously (39). As shown in Fig. 1A, Western blot analysis detectedectopic Wnt-4 from the cell lysates of C2C12 cells expressingWnt-4 (C2C12/Wnt-4), but not from control cells expressingempty vector (C2C12/V). Also, Wnt-4 could be detected fromthe conditioned media from C2C12/Wnt-4 cells, but notC2C12/V cells, suggesting that Wnt-4 could be secreted (datanot shown). To determine whether Wnt-4 promoted osteo-genic differentiation, both C2C12/V and C2C12/Wnt-4 cellswere stimulated with BMP-4. The induction of ALP activity byBMP4, an early marker for osteogenic differentiation (40, 41),was significantly enhanced in C2C12/Wnt-4 cells comparedwith C2C12/V cells (Fig. 1B). Northern blot analysis revealedthatWnt-4 and BMP-4 also synergistically induced the expres-sion of Osterix, a specific transcription factor for osteoblastdifferentiation (38) (Fig. 1C). Consistently, we found thatWnt-4 also enhanced the expression of type I collagen inducedby BMP-4.Wnt-4 Enhances Osteogenic Differentiation of MSCs Isolated

from Human Craniofacial Tissues—Recently, we isolated sev-eralMSCs from human adult oral and craniofacial tissues, suchas periodontal ligament and dental pulp. These cells are capableof forming bone-like tissues following in vivo transplantation,suggesting that they may be utilized to repair craniofacial bonedefects (9, 10). Therefore, we explored whether Wnt-4 couldenhance osteogenic differentiation of MSCs from periodontalligament. MSCs were also engineered to express Wnt-4 usingretrovirus-mediated transduction. As shown in Fig. 2, Westernblot analysis confirmed that MSCs stably expressedWnt-4. Toinduce osteogenic differentiation ofMSCs, bothMSCs express-ing Wnt-4 (MSC/Wnt-4) and control cells (MSC/V) weretreated with differentiation-inducing medium containingascorbic acid, �-glycerophosphate, and dexamethasone. Asshown in Fig. 2B, ALP activities were more significantlyinduced in MSC/Wnt-4 cells than in MSC/V cells upon stimu-lation with differentiation-inducing medium. Northern blotanalysis and Western blot analysis also found that Wnt-4enhanced the expression of specific bone matrix proteins, typeI collagen and OCN (Fig. 2, C and D). von Kossa staining

revealed that Wnt-4 potentiated mineralization of MSCs invitro (Fig. 2E). Of note, Wnt-4 expression did not significantlyaffect cell proliferation during the induction of cell differentia-tion or cell death (supplemental Fig. 1).Wnt-4 Enhances Osteogenic Formation ofMSCs in Vivo—We

have previously found that transplants of MSCs mixed with ahydroxyapatite/tricalcium phosphate carrier could generatemineralized tissues in immunodeficient mice, although theirosteogenic potentials were relatively weaker compared withMSCs from human bone marrow (9, 10). Thus, we examinedwhether Wnt-4 could enhance the formation of mineralizedtissues in vivo. Both MSC/Wnt-4 and MSC/V cells wereimplanted into nude mice for 6 weeks. In these experiments,MSC/Wnt-4 cells formed more mineralized tissues thanMSC/V cells in vivo (Fig. 3, A, D, and F). Immunostainingrevealed that the osteoblast-like cells lining the surface of min-eralized tissues reacted with human specific mitochondrialantibodies, suggesting that themineralized tissues were formedby the transplanted MSCs (Fig. 3, B and E).

FIGURE 1. Wnt-4 enhances osteoblastic differentiation of C2C12 mesen-chymal cells induced by BMP-4. A, establishment of C2C12 cells stablyexpressing Wnt-4. C2C12 cells were infected with retroviruses expressing HA-tagged Wnt-4 or a control vector and selected with G418 (600 �g/ml) for 10days. Cell lysates were probed with anti-HA monoclonal antibodies. �-Tubu-lin was utilized as an internal control. B, Wnt-4 enhanced BMP-4-induced ALPactivities in C2C12 cells. Both C2C12/V and C2C12/Wnt-4 cells were treatedwith BMP-4 for 0, 24, and 48 h. ALP activity was measured with a kit fromSigma. The assay was performed in duplicate, and the results represent aver-age value from three independent experiments. C, Wnt-4 enhanced BMP-4-induced Osx in C2C12 cells. Both C2C12/V and C2C12/Wnt-4 cells weretreated with BMP-4 for the indicated time periods. Total RNAs were extractedwith TRIzol reagent. Five-�g aliquots of total RNAs were probed with a 32P-labeled Osx cDNA probe. For a loading control, the membrane was strippedand reprobed with 32P-labeled glyceraldehyde-3-phosphate dehydrogenase(GAPDH). D, Wnt-4 enhanced BMP-4-induced type I collagen. Five-�g aliquotsof total RNAs were probed with 32P-labeled type I collagen cDNA probes. Fora loading control, the membrane was stripped and reprobed with 32P-labeled18 S RNA.




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Activation of the Noncanonical Wnt Signaling Pathway byWnt-4—Since the canonical Wnt signaling pathway regulatesskeletal development through �-catenin, we first examinedwhether or notWnt-4 could enhance osteogenic differentiationby increasing the cytosolic level of �-catenin. Subcellular frac-tionation was performed to determine the cytosolic level of�-catenin, as described previously. As shown in Fig. 4A, thecytosolic level of �-catenin was identical in both C2C12/Wnt-4and C2C12/V cells. Similarly, we found that Wnt-4 did notincrease the cytosolic level of �-catenin in MSCs under unin-duced and induced conditions (Fig. 4B). As a positive control,overexpression of Wnt-1 in MSCs significantly increased thecytosolic level of �-catenin (Fig. 4B). Additionally, Wnt-4 didalso not induce the transcription activity of �-catenin/Tcf asdetermined by Topflash luciferase reporter assays (supplemen-tal Fig. 2). These results suggest that Wnt-4 might promoteosteogenic differentiation of MSCs independent of �-catenin.We further screened other signaling pathways associated with

osteogenic differentiation that wereactivated by Wnt-4. Since JNKsignaling can be activated by thenoncanonical Wnts (26), we firstexamined whether Wnt-4 couldpotentiate JNK activation inducedby BMP-4 in C2C12 cells.We foundthat BMP-4 and Wnt-4 negligiblyinduced JNK activation in C2C12cells (data not shown). In contrast,as shown in Fig. 4C, the basal level ofp38 phosphorylation was signifi-cantly higher in C2C12/Wnt-4 cellsthan in C2C12/V cells, as deter-mined byWestern blot analysis (Fig.4C; compare lane 1 with lane 5).Moreover, BMP-4 induced p38phosphorylation to a greater extentin C2C12/Wnt-4 cells when com-pared with C2C12/V cells. We alsoobserved that the basal level of p38activity in MSC/Wnt-4 cells washigher than that in MSC/V cells.Upon the addition of differentia-tion-inducing medium, p38 phos-phorylation in MSC/Wnt-4 cellswas further enhanced as comparedwith MSC/V cells (Fig. 4D).Our results suggest that Wnt-4

might directly stimulate p38 activa-tion to promote osteogenic differ-entiation. Given that the purifiedrecombinant Wnt-4 proteins arenot available, we could not directlystimulate MSCs with Wnt-4 toexamine p38 activation. To over-come this problem, we immunode-pleted Wnt-4 proteins from theconditioned media from C2C12/Wnt-4 cells with anti-HA mono-

clonal antibodies, since Wnt-4 was HA-tagged (Fig. 4E). Asshown in Fig. 4F, although the Wnt-4-conditioned mediaimmunodepleted with control IgG retained the capacities tostrongly induce p38 phosphorylation, the removal of Wnt-4from the conditioned media abolished p38 phosphorylation inMSCs. These results suggest that Wnt-4-conditioned mediadid not contain the inducible secreted factors that could stim-ulate p38. Since the activation of p38 was rapidly induced in5–10 min by the Wnt-4-conditioned media, it was likely thatWnt-4 directly activated p38. Additionally, we found that theconditioned media from MSC/Wnt-4 cells also stronglyinduced p38 phosphorylation in MSCs. The removal of Wnt-4from the conditioned media also eliminated p38 activation inMSCs (data not shown). These results suggest that Wnt-4 canactivate the noncanonical p38 pathway in MSCs.Previously, it has been reported that the overexpression of

Axin induced the activation of JNK (26). Because JNK and p38are two closely related MAPK pathways (42, 43), although

FIGURE 2. Wnt-4 enhances osteoblastic differentiation of MSCs. A, MSCs were engineered to express Wnt-4.MSCs were infected with retroviruses expressing Wnt-4 or a control vector and selected with G418 (600 �g/ml)for 10 days. Cell lysates were probed with anti-HA monoclonal antibodies. �-Tubulin was utilized as an internalcontrol. B, Wnt-4 enhanced ALP activities induced by mineralization-inducing medium in MSCs. Both MSC/Vand MSC/Wnt-4 cells were treated with differentiation-inducing media for 10 days. ALP activities were meas-ured with a kit from Sigma. The assay was performed in duplicate, and the experiments were repeated twice.C, Wnt-4 enhanced type I collagen expression in MSCs. Both HMSC/V and HMSC/Wnt-4 cells were treated withdifferentiation-inducing medium for 0, 2, 7, and 14 days. Total RNAs were extracted with TRIzol reagent.Five-�g aliquots of total RNAs were probed with 32P-labeled type I collagen cDNA probes. For a loading control,the membrane was stripped and reprobed with 32P-labeled 18 S RNA. D, Wnt-4 enhanced OCN expression.Both MSC/V and MSC/Wnt-4 cells were induced for 28 days. Fifty-�g aliquots of protein lysates were probedwith polyclonal antibodies against OCN. For loading control, the membrane was stripped and reprobed withanti-HSP90. E, Wnt-4 enhanced mineralization of MSC in vitro. Both MSC/V and MSC/Wnt-4 cells were inducedfor 28 days. Mineralization was examined by von Kossa staining.




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Wnt-4 did not activate JNK at MSCs, we explored whetherAxin played a role inWnt-4-mediated p38 activation. As shownin Fig. 4G, overexpression of Axin strongly induced p38 phos-phorylation. Since C2C12 cells were more readily transfectedwith small interference RNA (siRNA) than MSCs, we also uti-lized siRNA to knock down Axin to determine if endogenousAxin played a role in p38 activation induced by Wnt-4. Asshown in Fig. 4H, we were able to partially reduce Axin expres-sion over 60%. This partial reduction of Axin significantlyreduced p38 phosphorylation induced by Wnt-4-conditionedmedia (Fig. 4I). Taken together, our results suggest that Wnt-4signals through Axin to activate p38.To determine if the activation of p38 played a role in the

potentiation of osteogenic differentiation induced by Wnt-4,we utilized a specific p38 inhibitor, SB-203580, to suppress p38activity. Western blot analysis demonstrated that SB-203580not only inhibited low p38 activity induced by BMP4 but alsoabolished enhancing p38 activity stimulated by both BMP-4and Wnt-4 (Fig. 5A). The ALP activity assay revealed that theinhibition of p38 by SB-203580 significantly attenuated theincreased ALP activity induced by BMP-4 andWnt-4 (Fig. 5B).Moreover, SB-203580 also abolished p38 phosphorylationinduced by Wnt-4-conditioned media in MSCs (Fig. 5C). Sim-ilarly, the treatment with SB-203580 suppressed ALP activityand mineralization in MSC/Wnt-4 cells upon the stimulationof differentiation-inducingmedia (Fig. 5D). Of note, SB-203580did not significant affect cell proliferation and cell death duringthe induction of cell differentiation. Taken together, our resultssuggest thatWnt-4 activated a new noncanonical p38 signalingpathway to enhance osteogenic differentiation independent of�-catenin.Wnt-4 Promotes Healing of Periodontal Bone Defects—

Chronic periodontitis is a common inflammatory disease thatoften destroys alveolar bone and periodontal tissues, resultingin loss of tooth support (44–46). To date, there are no effectivetherapies to restore alveolar bone loss. Since our MSCs wereoriginally isolated from human periodontal ligament, we

sought to determine whether MSCs engineered to expressWnt-4 could promote the repair of periodontal bone defects.As described in Fig. 3, we have found that Wnt-4-expressingMSCs generated significantlymore bonelike tissue inmice thanMSC/Vcells aftermixingwith hydroxyapatite/tricalciumphos-phate. Toward clinical application, we tested whether usingpolymer scaffolds seeded with transplanted MSCs, which canbe conveniently handled in the clinical setting to deliverMSCs,could also generate bonelike tissues. A nude rat model of peri-odontal bone defect was utilized as previously described (44–46). The periodontal ligament and alveolar bone were removedfrom the mesial and distobuccal roots of the mandibular firstmolar and the mesiobuccal root of the mandibular secondmolar teeth within the defect area. Polylactic co-glycolide poly-mer scaffolds were seeded overnight with MSC/Wnt-4or MSC/V cells and placed within the respective defects. Fiveweeks after operation, rats were sacrificed, and mandibulaewere subjected to �CT and histological analysis. Minimal heal-ing was observed in the control group, whereas extensive peri-odontal bone regeneration was found in the group implantedwith MSC/Wnt-4 cells (Fig. 6A). Specifically, alveolar bonehealing with complete bridging was seen only in the MSC/Wnt-4 group. Importantly, the newly formed bone tissues werewell vascularized, and no significant inflammatory reactionwasdetected in these tissue areas.�CT analysis revealed thatMSC/Wnt-4 cells generated 3–5-fold greater alveolar bone thanMSC/V cells (Fig. 6B).Wnt-4 Enhances Bone Generation in Critical Size Calvarial

Bone Defects—Since Wnt-4-expressing MSCs exhibited astrong osteogenic potential in the periodontal bone defectmodel, we further tested whether Wnt-4-expressing MSCscould improve repair for significantly larger craniofacial bonedefects. In addition to periodontal bone defects due to peri-odontitis, craniofacial bone defects are also frequently causedby bone injuries, tumor growth, or genetic diseases (47). Thecritical size calvarial defect was generated as described previ-ously (47). The polymer scaffolds loaded withMSC/Wnt-4 andMSC/V cells were placed into the defects. Five weeks after theoperation, x-ray analysis found that the calvarial defects cov-ered with the scaffolds alone hadminimal repair in comparisonwith those with cell implantation, and a clear bone defect wasstill evident (Fig. 7A). Histological examination revealed thatthe calvarial defects were filled with fibrous-like tissues (datanot shown). In contrast, radioopaque regions appeared in boththeMSC/V and theMSC/Wnt-4 group, and significantly moreabundant new bone formation was observed in the MSC/Wnt-4 group than in the MSC/V group. MSC/Wnt-4 trans-plantation showed new bone formation at the center andperiphery of the calvarial defect, as determined by x-ray and�CT, indicating that new bone was osseointegrated with theperiphery (Fig. 7A). Histological evaluation of the newly formedbone within the defect revealed that solid bone nodulesoccurred only in the center of the defects in the MSC/V group,whereas fibrous tissue separated the new bone nodule from themargins of the calvarial defect. In contrast, the MSC/Wnt-4group displayed extensive new bone formation bridging thedefect, with excellent osteointegration at many of the woundmargins. Interestingly, abundant bone marrow cavities in the

FIGURE 3. Wnt-4 enhances mineralized tissue formation in vivo by MSCs.MSC/V and MSC/Wnt-4 cells were mixed with hydroxyapatite/tricalciumphosphate (HA/TCP) ceramic particles and then transplanted subcutane-ously into nude mice. After 8 weeks of transplantation, MSC/Wnt-4 cells(D) formed thicker mineralized tissues than MSC/V cells (A) did. Immuno-histochemical staining showed that osteoblast-like cells (arrows) werepositive for the human-specific mitochondria antibody in both HMSC/Vand MSC/Wnt-4 groups (B and E). Isotype control for mouse primary anti-body did not show any positive staining (C). Mineralized tissue area ratewas calculated (F). The graph represents the mean � S.D. (n � 4; *, p �0.0001).




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new bone nodules were observed (Fig. 7B). Moreover, �CTanalysis revealed that the bone mineral density values of newbone at the calvarial defect generated byMSC/Wnt-4 cells weresignificantly higher than that by MSC/V cells (178.00 � 13.18versus 138.20 � 15.10 mg/cm3; p � 0.01), suggesting thatWnt-4 can strongly enhance mineralization of MSCs in largecalvarial defects (Table 1).

DISCUSSION

Our results reported here are the first demonstration thatWnt-4, a noncanonical member of Wnt family proteins,potently enhanced osteogenic differentiation of MSCs iso-lated from human adult craniofacial tissues in vitro and boneformation in vivo. Intriguingly, whereas Wnt-4 did notincrease the cytosolic level of �-catenin, we revealed thatWnt-4 activated a novel noncanonical signaling pathway,

p38 MAPK, which is known to positively regulate osteogenicdifferentiation induced by BMPs and other growth factors(42, 43). The siRNA knockdown of Axin significantlyreduced p38 activation induced by Wnt-4, suggesting thatWnt-4 signals through Axin. The inhibition of p38 abolishedosteogenic differentiation of MSCs promoted by Wnt-4.Moreover, using two different models of craniofacial boneinjury, MSCs genetically engineered to expressWnt-4 signif-icantly regenerated more bone tissues and promoted therepair of craniofacial bone defects in vivo. Our resultssuggest that Wnt-4 may have a novel application in improv-ing bone regeneration and repair for craniofacial bonedefects.Recent work from several groups has elucidated the impor-

tance of the canonical Wnt/�-catenin signaling pathway in theskeletal development and the postnatal maintenance of bone

FIGURE 4. Wnt-4 induces the noncanonical p38 signaling pathway in MSCs. A, Wnt-4 did not increase the cytosolic level of �-catenin in C2C12 cells.Subcellular fractionation was performed. 40-�g aliquots of protein lysates were probed with anti-�-catenin. For a loading control, the membrane was strippedand reprobed with anti-�-tubulin. B, Wnt-4 did not increase the cytosolic level of �-catenin in MSCs. 20-�g aliquots of protein lysates were probed withanti-�-catenin. MSC/Wnt-1 cells were utilized as a positive control. �-Tubulin was utilized as a loading control. C, Wnt-4 enhanced p38 activation in C2C12 cells.Both C2C12/V and C2C12/Wnt-4 cells were treated with BMP-4 for the indicated time periods, and the p38 phosphorylation was examined with anti-phospho-p38 antibodies. For an internal control, the membrane was stripped and reprobed with anti-p38 or anti-�-tubulin. D, Wnt-4 enhanced p38 activation in MSCs.Both MSC/V and MSC/Wnt-4 cells were induced for the indicated times, and the p38 phosphorylation was examined with anti-phospho-p38 antibodies. For aninternal control, the membrane was stripped and reprobed with anti-p38 or anti-�-tubulin. E, immunodepletion of Wnt-4 in Wnt-4-conditioned media isolatedfrom C2C12/Wnt-4 cells. C2C12/Wnt-4 cells were grown in serum-free media for 24 h, and Wnt-4-conditioned media were harvested. The Wnt-4-conditionedmedia were concentrated and incubated with anti-HA or control IgG. The immunocomplexes were pulled down with protein G-agarose beads. The depletionof Wnt-4 in Wnt-4-conditioned media was confirmed by Western blot analysis. F, Wnt-4 induced p38 phosphorylation in MSCs. MSCs were treated withWnt-4-conditioned media or Wnt-4-depleted media. p38 phosphorylation was probed anti-phospho-p38 antibodies. For a loading control, the membrane wasstripped and reprobed with anti-p38 or anti-�-tubulin. HA, Wnt-4-conditioned media depleted with anti-HA; IgG, Wnt-4-conditioned media depleted with IgGcontrol. G, overexpression of Axin induced p38 phosphorylation. Cells were transfected with pcDNA-FLAG-p38 and pcDNA-HA-Axin or control vector for 24 h.p38 phosphorylation was probed with anti-phospho-p38 antibodies. H, knockdown of Axin by Axin siRNA. Cells were transfected with Axin siRNA or controlluciferase (Luc) siRNA. 48 h after transfection, the protein level of Axin was examined by Western blot analysis. �-Tubulin was utilized as an internal control.I, the knockdown of Axin attenuated p38 phosphorylation induced by Wnt-4. C2C12 cells were transfected with Axin siRNA or Luc siRNA. 48 h after transfection,the cells were treated with Wnt-4 for 10 min. p38 phosphorylation was probed with anti-phospho-p38 antibodies. For a loading control, the membrane wasstripped and reprobed with anti-p38 or anti-�-tubulin.




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mass (12). Because human adult MSCs are one of the mostpromising types of adult stem cells for bone regeneration andrepair, a number of studies have examined whether the canon-ical Wnt proteins, such as Wnt-1 or Wnt-3a, could enhanceosteogenic differentiation of MSCs. Paradoxically, the treat-ment with Wnt-1 or Wnt-3a strongly inhibited osteogenic dif-ferentiation ofMSCs (31–37). Currently, the underlyingmech-anisms that are responsible for these conflicting results areunknown. It is possible thatWnt/�-cateninmay regulate osteo-genic differentiation at a specific stage or may be cell-specific.Since the Wnt/�-catenin signaling pathway also plays a role inregulating adult stem cell properties (5, 48, 49), it is possiblethat the canonical Wnts may help to maintain MSCs in an

undifferentiated state, therebyinhibiting their osteogenic differ-entiation. Nevertheless, these stud-ies strongly suggest that targetingthe canonical Wnt/�-catenin sig-naling pathway to improve osteo-genic potential of MSCs mayrequire further investigation.A number of Wnts have been

identified to be expressed in bone orto be associated with bone develop-ment. For example, Wnt-1, Wnt-4,Wnt-7, and Wnt-14 have beenfound to be expressed by osteo-blasts, and Wnt-1 and Wnt-3a areinduced by BMP in MSCs (12, 16,29). We also found that Wnt-4 wasinduced during osteogenic differen-tiation of MSCs (supplemental Fig.3). However, it should be noted thatendogenous Wnt-4 expression wasinduced at a fairly late stage duringMSC differentiation. Thus, it islikely that other endogenousWnt(s)may regulate osteogenic differentia-tion of the MSCs. Although thecanonical Wnt/�-catenin signalingpathway has been extensively exam-ined in bone, little is known aboutwhether or not the noncanonicalWnt signaling pathway plays a rolein skeletal development and thepostnatal maintenance of bonemass. Moreover, results from stud-ies with Dkk2 knockout mice sug-gest that regulation of bone forma-tion by Wnt signaling is morecomplex (37). Dkk2 is known to bean inhibitor of Wnt signaling and isinduced byWnts as one of the feed-back mechanisms to attenuate Wntstimulation. Interestingly, Dkk2�/�

mice have increased secreted bonematrix but impairedmineralization,resulting in an osteopenic pheno-

type (37). Our studies demonstrated that Wnt-4 enhancedosteogenic differentiation ofMSCs, suggesting that noncanoni-cal Wnts may also play a role in osteoblast differentiation andbone formation. We observed that Wnt-4 induced p38 activa-tion to promote osteogenic differentiation. p38 activation isa known pathway that is associated with osteogenic differenti-ation (42, 43). Using the combination of the conditionedmediaand an immunodepletion approach, we demonstrated thatWnt-4 could directly activate p38 through its receptor. First,theWnt-4-conditioned medium rapidly stimulated p38 activa-tion in 5–10 min, indicating that it was unlikely that Wnt-4induced a new protein(s) to stimulate p38 activation throughautocrine mechanisms. Unfortunately, we were unable to use

FIGURE 5. The inhibition of p38 activation suppresses osteogenic differentiation promoted by Wnt-4.A, SB-203580 inhibitors suppressed p38 phosphorylation induced by BMP-4 and Wnt-4 in C2C12 cells. BothC2C12/V cells and C2C12/Wnt-4 cells were pretreated with or without SB-203580 for 30 min and then treatedwith BMP-4 for the indicated times. p38 phosphorylation was examined with anti-phospho-p38. p38 and�-tubulin were utilized as an internal control. B, SB-203580 inhibited ALP activities induced by Wnt-4 andBMP-4. Both C2C12/V and C2C12/Wnt-4 cells were pretreated with or without SB-203580 and then treated withBMP-4 for 48 h. The assay was performed in duplicate, and the result represents an average value from twoindependent experiments. C, SB-203580 suppressed p38 phosphorylation induced by Wnt-4 in MSCs. MSCswere pretreated with SB-203580 for 30 min and then treated with Wnt-4-conditioned media for 10 min. D, theinhibition of p38 activation suppressed the osteogenic differentiation of MSCs promoted by Wnt-4. BothMSC/V and MSC/Wnt-4 cells were pretreated with SB-203580 for 30 min and then induced with differentiation-inducing media for 0, 10, and 21 days. ALP activities were stained with an ALP kit, and the mineralization wasstained with alizarin red.




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protein or transcription synthesis inhibitors to determinewhether the inhibition of new protein synthesis would affectp38 activation induced by Wnt-4, because these inhibitorsalone activated p38 in MSCs. Second, the immunodepletion ofWnt-4 for theWnt-4-conditioned medium abolished p38 acti-vation, suggesting that there were no Wnt-4-induced secretedp38 activators present in the Wnt-4-conditioned media.Finally, we found that the knockdown of Axin significantlyreduced p38 activation induced byWnt-4. Until now, it has notbeen shown that p38 is activated byWnt-4 or other noncanoni-cal Wnts. Axin is a key scaffold protein involved in the inhibi-tion of glycogen synthase kinase-3� and the degradation of�-catenin. Canonical Wnts binding to their co-receptor LRP5or -6 have been found to recruit Axin to the membrane, result-ing in stabilization of �-catenin (50, 51). Since noncanonicalWnts do not signal through the co-receptor LRP5 or -6, it sug-gests that Wnt-4 may transduce signals to Axin through friz-zled receptors. Currently, we do not know how Wnt-4 signalsthrough Axin to activate p38. We found that Wnt-4 did notaffect the protein level of Axin (supplemental Fig. 4). Interest-ingly, Axin has been found to form a complex with MEKK1through a novel domain and to promote JNK activation when

Axin was overexpressed (26). Since p38 also is downstream ofMEKK1, it is possible that Wnt-4 may stimulate interactionbetween Axin and MEKK1 and thereby activate p38 in MSCs.The isolation ofMSCs from various tissue sources, including

dental and craniofacial tissues, has provided an excellent ther-apeutic approach for bone tissue regeneration and repair(5–10). The bone loss in dental, oral, and craniofacial regions isvery common due to injuries, infection, and cancer-associateddamage. To date, restoration of these defects or damaged tis-sues has mainly relied on implanting structural substitutes,often with little or no reparative potential. Our previous workhas suggested that MSCs isolated from periodontal ligamenthave a potential to repair periodontal tissue damage (9).Although we were able to demonstrate that MSCs attached tothe surface of alveolar bone and teeth when implanted in thedefect regions, minimal alveolar bone repair was detected in anude rat model (9). In this study, we found that MSCs engi-

FIGURE 6. Wnt-4 promotes healing periodontal alveolar bone defectsmediated by MSCs in vivo. A, Wnt-4 promoted the repair of alveolar bonedefect mediated by MSCs in vivo. Polylactic co-glycolide polymer scaffoldswere seeded overnight with MSC/V cells or MSC/Wnt-4 cells and transplantedinto mandibular osseous defects. Five weeks following surgery and cell trans-plantation, the biopsies were harvested for histology and �CT analysis. Theleft panel shows coronal slide orientations of MSC/Wnt-4 and MSC/V cell treat-ments of the defect at low power (2�) magnification; the middle panel showshigh power (10�) magnification of corresponding treatment groups; and theright panel shows �CT images of corresponding treatment groups. After 5weeks, minimal healing was observed in the control group, whereas exten-sive periodontal bone regeneration was found in the group with MSC/Wnt-4cell treatment. Specifically, alveolar bone healing with complete bridgingwas seen only in the MSC/Wnt-4 group. The arrows indicate the borders of theosseous defects. In the �CT panel, the red color indicates tooth crowns (yellow,tooth roots; blue, newly regenerated alveolar bone). B, linear measures ofalveolar bone repair by �CT. Linear measures of alveolar bone repair on theexposed root surfaces were assessed by �CT. The values reveal the amount ofalveolar bone regeneration in mm on the bone surfaces as measured from�CT scans of the biopsies. Results from analysis of variance showed that thefractions were significantly different between the two groups (*, p � 0.011).

FIGURE 7. Wnt-4 enhances the repair of calvarial bone defect mediated byMSCs in vivo. A, analysis of bone regeneration by soft x-ray and �CT. The5-mm diameter calvarial defects were transplanted with MSC/V cells, MSC/Wnt-4 cells, or gelatin control for 5 weeks in SCID mice. Left, control animalswere transplanted with gelatin only; middle, animals transplanted withMSC/V cells; right, animals transplanted with MSC/Wnt-4. B, histologicalexamination of bone regeneration mediated by MSC/V and MSC/Wnt-4 cells.Left, magnification (�40); arrows indicate the borders of the calvarial bonedefects. Middle, high magnification (200�) of the border bone repair; bonehealing with complete bridging was seen only in the MSC/Wnt-4 group. Right,high magnification (�200) of the center of bone repair. NB, new bone.

TABLE 1�CT analysis of the calvarial bone regenerationValues are shown as mean � S.D. BFV, bone volume/total tissue volume; BMD,bone mineral density.

Group n BFV BMD% mg/cm3

Control 6 19.50 � 3.45 81.97 � 11.63MSC/V 6 33.17 � 4.79 138.20 � 15.10MSC/Wnt-4 6 45.67 � 8.91 178.00 � 13.18




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neered to express Wnt-4 regenerated more bone tissues andrepaired large alveolar bone defectswhen comparedwithMSCsalone. Importantly, the new bone tissues generated by MSC/Wnt-4 cells were well integrated with the host bones at thewound margins in both periodontal and calvarial bone defectmodels. Although new bone was well vascularized, we did notobserve that Wnt-4 induced inflammatory cell infiltrations. Ithas been reported that vascular endothelial growth factorenhanced bone formation and promoted recruiting mesenchy-mal stem cells (7). Currently, we do not know whether Wnt-4also helps to recruit MSCs from the host to promote bonerepair in vivo. Wnt-4 has been reported to play a role in bloodvessel development. Therefore, it was possible that Wnt-4might enhance bone regeneration by promoting angiogenesisin vivo, which could further enhance bone formation. Weobserved that there was abundant bone marrow present inthe new bones generated by MSC/Wnt-4 cells, which wasquite different from bone tissues generated by otherapproaches in which bone marrows was barely observed.Our results suggest that Wnt-4 may promote MSCs to forma suitable microenvironment resembling natural bone andtherefore recruit hemotopoietic cells to generate an entirebone/bone marrow structure.Finally, it should be pointed out that Wnt signaling has been

found to be associated with tumor development. The abnormalactivation of the canonical Wnt/�-catenin signaling pathwaycauses several human cancers, including colorectal cancers andmelanoma (21–23, 39). Although the canonical Wnt signalingpathway plays an essential role in skeletal development andpostnatal maintenance of bone mass, the overactivation of thispathway for tissue engineering may have a risk in increasingtumorigenesis. According to our studies, noncanonical Wntsmay also play a role in bone formation. Given the fact thatnoncanonical Wnts activate diverse signaling pathways, it ispossible that they may utilize different mechanisms to stimu-late osteogenic differentiation. It is worth investigatingwhetherother noncanonical Wnts also activate p38 to stimulate osteo-genic differentiation. Importantly, we have observed thatMSCsengineered to express Wnt-4 did not form tumors in nudemice. Taken together, our results suggest that Wnt-4 may beutilized for improving craniofacial bone regeneration andrepair.

Acknowledgment—We thank Chan Ho Park for assistance in per-forming �CT imaging.

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Krebsbach, William Giannobile, Songtao Shi and Cun-Yu WangJia Chang, Wataru Sonoyama, Zhuo Wang, Qiming Jin, Chengfei Zhang, Paul H.

Cells in Craniofacial Defects through Activation of p38 MAPKNoncanonical Wnt-4 Signaling Enhances Bone Regeneration of Mesenchymal Stem

doi: 10.1074/jbc.M702391200 originally published online August 24, 20072007, 282:30938-30948.J. Biol. Chem.

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noncanonicalwnt-4signalingenhancesboneregeneration ...canonical wnt signaling plays a critical role...

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