Cell Proliferation. 2018;51:e12479. wileyonlinelibrary.com/journal/cpr  | 1 of 9https://doi.org/10.1111/cpr.12479

© 2018 John Wiley & Sons Ltd

Received:3April2018  |  Accepted:2May2018DOI: 10.1111/cpr.12479

O R I G I N A L A R T I C L E

The cell re- association- based whole- tooth regeneration strategies in large animal, Sus scrofa

Fu Wang1,2  | Zhifang Wu1 | Zhipeng Fan3  | Tingting Wu1 | Jinsong Wang4 | Chunmei Zhang1 | Songlin Wang1,4

Fu Wang and Zhifang Wu contributed equally to this work.

1MolecularLaboratoryforGeneTherapy&ToothRegeneration,BeijingKeyLaboratoryofToothRegenerationandFunctionReconstruction,SchoolofStomatology,CapitalMedicalUniversity,Beijing,China2SchoolofStomatology,DalianMedicalUniversity,Liaoning,China3LaboratoryofMolecularSignalingandStemCellsTherapy,BeijingKeyLaboratoryofToothRegenerationandFunctionReconstruction,SchoolofStomatology,CapitalMedicalUniversity,Beijing,China4DepartmentofBiochemistryandMolecularBiology,SchoolofBasicMedicalSciences,CapitalMedicalUniversity,Beijing,China

CorrespondenceSonglinWang,SchoolofStomatology,CapitalMedicalUniversity,Beijing,China.Email: slwang@ccmu.edu.cn

Funding informationBeijingMunicipalityGovernment,Grant/AwardNumber:PXM2016_014226_000034,PXM2016_014226_000006,PXM2015_ 014226_000116,PXM2015_014226_000055,PXM2015_ 014226_000052,PXM2014_014226_000048,PXM2014_014226_000013,PXM2014_ 014226_000053,Z121100005212004,PXM2013_014226_000055,PXM2013_ 014226_000021,PXM2013_014226_07_000080andTJSHG201310025005;NationalNaturalScienceFoundationofChina,Grant/AwardNumber:91649124and81771032

AbstractObjectives: Whole- tooth regeneration for tooth loss has long been a goal of den-tistry.Thereisalsoanincreasingdemandtocarryoutpre-clinicalinvitroandinvivoresearchmethods in large animal model similar to human. Theminiature pig hasproven to be an alternative as a large mammal model owing to its many similarities to human. However, whole-tooth regeneration in large animal remains a challenge.Here,weinvestigatedthefeasibilityofcellre-association-basedwhole-toothregen-eration in miniature pigs.Materials and methods:Singlecellsfromtheforthdeciduousmolargerms(p4)ofpigwere reconstituted to bioengineered tooth bud using different treatment for in vitro culture and in vivo transplantation in mouse subrenal capsules and jawbones.Results:Thebioengineeredtoothbudfromre-aggregatedepithelialtomesenchymalsinglecellswithandwithoutcompartmentalizationrestoredthemorphogenesis,in-teractions or self-sorting between 2 cells in vitro culture. The pig bioengineeredtooth bud transplanted in mouse subrenal capsules and jawbones restored odon-togenesis and developed into large size tooth.Conclusions: We characterized the morphogenesis and interaction of single- tooth germ cells in vitro, and first addressed efficient long-term survival and growththrough transplantation of pig bioengineered tooth bud under mouse subrenal cap-sulesor inmouse jawbones,where itcandevelop into largesizetooth.Ourstudyextends the feasibility of whole- tooth regeneration in large animal.

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1  | INTRODUC TION

Theultimategoalofwhole-tooth regeneration for tooth loss is toprovideliving,functionalandbiocompatibletissuethatismoreinlinewith the human desire for a third dentition that represents an attrac-tive alternative to classical prosthesis- based therapies.1,2Currently,denovoodontogenesisinhumanshasbeenchallenging,withmanyobstacles, although some regenerative attempts have beenmadeusing cells from human donors.3-7Theepithelialandmesenchymalinteraction- based whole- tooth regenerative approaches in rodents andcaninemodel,holdgreatpromiseasastrategyfordevelopinga functional substitute for lost teeth.8-13 Whether de novo odon-togenesisis feasible in humans remains elusive. An essential step is required to move this tooth regeneration strategy from rodents to a large animal model before these regenerative properties are intro-duced into humans.

Pigs serve as a promising large animal model for studying human diseases and contribute to overcome the shortage of human donor organs.14-17 Theminiaturepighasproven tobe avaluable animal model for diphyodont development and regen-eration owing to its many similarities to human including the morphology, number and size of teeth, particularly its hetero-dontdentition (incisors, canines,premolars andmolars) anddi-phyodontdentition,whicharenotavailable in rodents.18,19Themorphology and chronology of diphyodont dentition in miniature pigs have been well characterized by our previous studies and other reports.20-22Moreover,recentbreakthroughinporcinege-nome engineering aiming to overcome immunological challenges andpotentialriskofporcineendogenousretrovirus(PERV)trans-mission make safe clinical xenotransplantation possible.14,17,23 However,studyingwhole-toothregenerationusingminiaturepigas a model remains a significant obstacle. The morphogenesisand interaction of single cells from pig tooth germ in vitro cul-ture remainundefined. Some issues alsoneed tobeovercome,such as longer process required for growth and replacement of swine teethwith larger size and dynamic tracking. There is anincreasing demand to seek alternative approaches to promote pre- clinical study.

As in vitro organ culture only provides short term growth and limited functional cytodifferentiation, transplantation of graftunder the renal capsule is used for study of development and dif-ferentiation of tissue recombinants owing to its high degree of vas-cularity, suitability for xenografts and convenient examinationoforganogenesis.However,whether the subrenal capsulemicroen-vironment can bear long- term growth of pig bioengineered tooth germ remains unknown.

Inthisstudy,wetracedthemorphogenesis,interactionsorself-sortingofcellsfrompigtoothgerms.Thepigbioengineeredtoothbudachievedlong-termsurvivalandgrowth,anddevelopedatooththrough transplantation in mouse subrenal capsules and jawbones. Our pilot study for whole- tooth regeneration in large animal has the potential to be clinically applied and will further promote the use of pig as a diphyodont model similar to humans.

2  | MATERIAL S AND METHODS

2.1 | Animals

Ten pregnant miniature pigs were obtained from the Institute ofAnimal Science of the Chinese Agriculture University (Beijing,China).Theminiaturepigembryos (85)wereobtainedas reportedpreviously.20 Briefly, the pregnantminiature pigswere verified byB-typeultrasonicinspection,andthestagedminiaturepigembryoswere obtained by caesarean section.

Theadulthostimmunocompromised(SCID)mice(5weekold)were obtained from the Institute of LaboratoryAnimal Science,ChineseAcademyofMedicalSciences,andmaintained ina spe-cific pathogen- free animal facility with free access to water and food.

All experimental animal procedures were reviewed and ap-provedbytheAnimalCareandUseCommitteeofCapitalMedicalUniversity. (Permit Number: CMU-2012-x-102), and the methodswere carried out in accordance with the approved guidelines.

2.2 | Isolation of tooth germs from miniature pigs

Theforthdeciduousmolargerms(p4) inmandiblesfromthesamelitter of stagedminiature pig embryos (embryonic day [E] E40 orE70)wereisolatedandpooledunderstereomicroscopywithanat-tachedOlympusDP72digitalcamerasystem(OlympusCorporation,Tokyo, Japan). Themorphological stages of the p4 at E40 or E70correspondedtothecapstageandsecretorystage,respectively,andwere verified by serial histological sections as previously described.20

2.3 | Dissociation of single- tooth germ cells

Single-toothgermcellsfromminiaturepigswereobtainedaspre-viously reported.10,11 Briefly, the epithelium andmesenchyme ofisolatedlowerdeciduousmolargermswereincubatedinPBScon-taining Dispase II (1.2U/mL, Sigma-Aldrich, St. Louis, MO, USA)andDNaseI(20U/mL,TakaraBio,Shiga,Japan)for15minatroomtemperature,thenseparatedunderastereomicroscope.Theepi-thelium and mesenchyme were each dissociated into single cells inPBS(−)supplementedwithCollagenasetypeI(3mg/mL,Sigma,WorthingtonBiochemicalCorp.,Lakewood,NJ,USA)andDispaseII(4mg/mL,Sigma,RocheDiagnosticsCorp.,Indianapolis,IN,USA)and filtered through a 70- μm cell strainer (BD Biosciences, SanJose,CA,USA).

2.4 | Reconstitution of single- tooth germ cells and tissue culture in vitro

Single cells of epithelial and mesenchymal origin were then, re-spectively, pelleted by centrifugation. To characterize the inter-action between epithelial and mesenchymal single cells and test whole-tooth regeneration potential of single-tooth germ cells, thesingle- tooth germ cells were processed using three strategies for

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bioengineeredtoothgerms.Forre-association,singlecellsofepithe-lial and mesenchymal origin were pelleted separately by centrifuga-tion.Bioengineeredtoothgermswereconstructedbythesequentialinjection of 0.25 μLmesenchymaland0.25μLepithelialsinglecellspellets into a 50 μL collagen gel drop (Cellmatrix type I-A, Nittagelatin,Osaka,Japan)(1:1ratio,eachpelletincludingapproximately1 × 105cells)toformcompartmentcontactbetweenthetwopellettypes.10,11Formesenchymalcellsalone,0.5μLmesenchymalsinglecellspellet (approximately2×105cells)were injected intoa50μLcollagengeldrop.Forre-aggregationoftoothgermcells,thetoothgerms were directly disassociated into mixed single- tooth germ cells and were filtered and pelleted, then 0.5μL cells pellet (approxi-mately 2 × 105cells) were injected into a 50μL collagen gel dropwithout compartmentalization.

The above-reconstituted pellets embedded into collagen geldrops were placed on a cell culture insert (0.4-μm pore size, BD,FranklinLakes,NJ,USA)in12-wellcellcultureplates.Theexplantswereculturedfor3-8daysinDMEMmedium(GIBCO,GrandIsland,NY,USA)supplementedwith10%FCS(GIBCO),100μg/mLascorbicacid(Sigma),2mML-glutamine(Sigma).

2.5 | Transplantation of reconstituted explants into SCID mice

The bioengineered tooth germs cultured in vitro for 3days weretransplanted into subrenal capsules or maxillary diastema of adult SCIDmice.Forsubrenalcapsuletransplantion,asmallincisionwasmade with scissors in the skin and body wall over the kidney after SCIDmicewasanaesthetized.Thenthekidneywaspoppedoutoftheholeinthebodywall,anincisionwasmadetocreateapocketbetween the capsule and the underlying renal parenchyma. Thebioengineeredtoothgermwasplacedintothepocket.Theincisionwas sutured after return of the kidneys to the peritoneal cavity. For jawbone transplantation, an incision of 2mm in lengthwasmadethrough the oral mucosa of maxillary diastema and a 0.5- 1.0 mm bony holewasmade in alveolar bone surface. The bioengineeredtoothgermswerethentransplantedintothebonyhole.Theincisionwasnextsutured.Thehostmicewerekilledafter8or16weeks,andperfusionfixedwith4%neutralparaformaldehyde.

2.6 | Radiographic analysis and three- dimensional reconstruction

Thespecimenswereexaminedwithconebeamcomputed tomog-raphy (CBCT, NewTom 3G tomography, Summer, Italy). The dataanalysis and three- dimensional reconstruction were carried out with Mimics(Materialise,Version11).

2.7 | Histochemical and immunohistochemical analysis

For the histology study, specimens were decalcified, dehydrated,and then embedded in paraffin. Serial sections (5μm thick) werestainedwithhaematoxylin-eosin(H&E).

For immunohistochemistry of regenerated tooth, briefly, afterthe sampleswere fixedwith4%PFA,decalcified,dehydratedandembedded in paraffin, they were cut into 10-μm thick sections. Serialsectionswerepermeabilizedin0.4%TritonX-100andblockedinPBScontaining5%BSA.Sectionswereincubatedwithprimaryan-tibodiesovernightat4°C,thenwashedandincubatedfor1hat37°Cwith the respective secondary antibodies followed by haematoxylin orDAPInuclearstain.Sliceswereanalysedusingamicroscope(BX43Olympus)withanattachedOlympusDP72digitalcamerasystem.

3  | RESULTS

3.1 | Isolation of tooth germs from miniature pigs

According to our previous research,20,24 the staged miniature pig embryos and foetuses at E40 and E70 were obtained by caesarean section.Basedonconsiderationsofcellularacquisition,wechoosetheforthdeciduousmolargerms (p4) inmandibleswith largesize.Thep4wasatcapstageatE40(Figure1A-E)andreachedsecretory

F IGURE  1  Isolation of deciduous molar germs from miniature pigsatE40.(A),SchematicbuccalviewofthemandibleofminiaturepigatE40.(B),TheisolatedminiaturepigembryosatE40.(C),MacroviewofmandiblefromminiaturepigatE40.Blackdottedlineindicatedthelocationofp4,scalebar=200μm.(D),Frontalsectionsshowingp4atcapstage(H&E),scalebar=200μm.(E),Theisolatedp4atcapstage

(A)

(B) (C)

(D) (E)

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F IGURE  2 Bioengineeredtoothgermsfromsingle-toothgermcellsinvitroculture.(A),Schematicrepresentationsoftreatmentoftoothgermcellsforinvitroorganculture.Theepitheliumandmesenchymeoftoothgermsweredissociatedtosinglecells.Thesinglecellswerepelletedtoreconstructtoothgermswith3waysandculturedinvitrofor8d.(B),Ep-Meinvitrocultureandhistologicalanalysis(H&Estaining,rightpanels),bioengineeredtoothgermsfromre-associatedepithelialandmesenchymalsinglecellswithcompartment.(C),TGcellsinvitrocultureandhistologicalanalysis(H&Estaining,rightpanels),bioengineeredtoothgermsfrommixedtoothgermsinglecells.(D),Meinvitrocultureandhistologicalanalysis(H&Estaining,rightpanels),bioengineeredtoothgermsfromre-aggregatedmesenchymalcellsalone.Scalebar=200μm

F IGURE  3  In vitro development of re-aggregatedEp-Mecellsfromp4atsecretorystage.(A),Thedissociatedepitheliumandmesenchymeofp4(E70,left),Ep-Mebioengineeredtoothgerminvitrocultureat3rdday(middle)and8thday(right),Scalebar=500μm.(B),Morphogenesisandinteractionbetweenepithelialcells(red)andmesenchymalcells(green),Scalebar=100μm

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F I GUR E   4  Invivodevelopmentofre-aggregatedEp-Mecellsfromtoothgermsatcapstageinmousesubrenalcapsule.(A),Schematicdiagramoftheectopictransplantationofbioengineeredtoothgermsintoamousesubrenalcapsuleafterculturingfor3dinvitro.(B),There-associatedexplantswithepithelialandmesenchymalcellcompartmentalization(E40Ep-Me)restorethedenovoodontogenesisat8wkpost-transplantation.Leftpanel:macroviewofregeneratedtissues(reddottedline).Rightpanels:histologicalanalysis(H&Estaining)andimmunostainingoftheameloblast-specificmarkerameloblastin(AMBN,green).(C),There-associatedexplantswithepithelialandmesenchymalcellcompartmentalization(E40Ep-Me)developedintoatoothaftertransplantationfor16wkinthesubrenalcapsule,Leftpanel:macroview.Reddottedlineindicatesthetoothandsupportingtissues.Middlepanel:crossviewofprofileoftoothandsupportingtissuesattheblackdottedcutlineintheleftpanel.Rightpanels:histologicalanalysis(H&Estain)correspondingtoboxedareasinmiddlepanel.(D),Re-aggregationofmixedtoothepithelialandmesenchymalcells(TGcells)regeneratedtoothcrownstructuresbyself-sortingofepithelialandmesenchymalcellsat8wkpost-transplantation.Leftpanel:macroviewofregeneratedtissues(reddottedline).Middleandrightpanels:histologicalanalysis(H&Estain).(E),Re-aggregationofmesenchymalcellsalone(Mecells)formedbonetissueat8wkpost-transplantation.ab,alveolarbone;am,ameloblast;b,bone;d,dentin;dp,dentalpulp;e,enamel;od,odontoblast;rt,renaltissue.Scalebars:grossview,1mm;histology,100μm

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stageatE70(FigureS1A-C).Themorphogeneticstageswereveri-fied by serial histological sections.

3.2 | Reconstructed single- tooth germ cells from cap stage in vitro culture

To characterize theodontogenesis of dissociated single cells fromtoothgermsofminiaturepigsatcapstage,wetreatedsinglecellsfrom tooth germs with three methods for in vitro organ culture (Figure2A).We isolated thep4at cap stage frommandibles fromthe same litter of staged miniature pig embryos and separated the epithelium from the mesenchyme. After sequential enzymatic treat-ment,theepithelialandmesenchymaltissuesweredissociatedintosingle cells.

We tracked the in vitro morphogenesis of re- associated cell pel-letswithepithelialcellandmesenchymalcellcompartment(Ep-Me)in our organ culture system. Interactions were visible after culture for3days,andapparentmorphogenesisaroseatthe8thday,imply-ingtheinitiationofodontogenesisidentifiedbyhistologicalsections,and polarized epithelial cells were separated from concentrated mes-enchymalcellsbybasementmembrane (Figure2B).Re-aggregatedepithelial and mesenchymal single cells mixed from tooth germs without compartmentalization (TG cells) exhibited a self-sortingfeature; the epithelial cells were able to sort from the mesenchymal cells and organized into a well- defined dental epithelial structure envelopingamesenchymalcellmass(Figure2C).Incontrast,theag-gregatedmesenchymalcellsalone(Mealone)exhibitednoobviousmorphogenesis(Figure2D).

We further characterized the reciprocal interaction between the epithelial and mesenchymal cells by re- associating epithelial cells with mesenchymal cells marked with enhanced green fluorescent

protein(eGFP)toidentifythemorphogenesisoftheepithelialcom-partment(FigureS2A,B).

3.3 | Reconstructed single- tooth germ cells from secretory stage in vitro culture

We also tested the de novo odontogenesis of cells from tooth germ atthesecretorystage(E70).Ep-Mecellspelletsfromp4atE70alsorestoredmorphogenesislikeEp-Mecellspelletsfromp4atcapstage(Figure3A).Themorphogenesisandinteractionbetweenepithelialcells(markedwithanti-CK14)andmesenchymalcells(markedwithanti-vimentin)wasvisibleafterculturefor8days(Figure3B).

3.4 | Growth and survival of bioengineered tooth germs in subrenal capsules of SCID mice

Toinvestigatetheregenerationpotentialoftheconstructedtoothgerms described above, the bioengineered explantswere trans-planted into a SCID mouse subrenal capsule after culturing for3daysinvitro(Figure4A).There-associatedexplantsfromcellsoftooth germ at cap stage with epithelial and mesenchymal cell com-partmentalization in subrenal capsule restored the de novo odon-togenesis,asdemonstratedbyexaminationofexplantsat8weeksafter implantation, the differentiated ameloblasts expressedameloblastin, andvisibleHertwigepithelial root sheaths impliedthe initiationof rootdevelopment (5 in6grafts,Figure4B), anddeveloped into tooth- like structures with crown nearly completed inbony tissuesafter transplantation for16weeks (4 in6grafts,Figure4C). It isnoteworthy thatno secondary toothwasvisiblein all bioengineered tooth germ grafts. All 4 re- aggregation grafts from mixed epithelial and mesenchymal single cells from tooth

F IGURE  5  Invivodevelopmentofre-aggregatedEp-Mecellsfromp4atsecretorystageinmousesubrenalcapsule.(A),There-associatedexplantswithepithelialandmesenchymalcellcompartmentalization(E70Ep-Me)restorethedenovoodontogenesisat8wkpost-transplantation.Toppanel:macroviewofregeneratedtissues(reddottedline).Bottompanels:histologicalanalysis(H&Estaining).(B),There-associatedexplantswithepithelialandmesenchymalcellcompartmentalization(E70Ep-Me)developedintotooth-likestructures(reddottedline)aftertransplantationfor16wkinthesubrenalcapsule,viewedbyX-raysandH&Estaining.b,bone;d,dentin;dp,dentalpulp.Scalebars:grossview,1mm;histology,100μm

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germ at cap stage without compartmentalization in our experi-ment were able to form tooth- like tissues with correctly arranged tooth components, but without normal tooth appearance at8weeks after transplantation (Figure4D).While, re-aggregationof mesenchymal cells alone from cells of tooth germ at cap stage (Mecells)only formedbone tissueat8weeksafter transplanta-tion(Figure4E).

Then,wetestedtheregenerationpotentialofbioengineeredex-plantsfromcellsoftoothgermatsecretorystage.Themostofre-associated explants from cells of tooth germ at E70 with epithelial

and mesenchymal cell compartmentalization also restored the de novo odontogenesis in subrenal capsule at 8 weeks after implanta-tion(4/5,Figure5A)andformedtooth-liketissuesat16weeksafterimplantation(3/5,Figure5B).

3.5 | Growth of re- associated explants in jawbone of SCID mouse

A prior study has verified that a bioengineered tooth germ can develop into a fully functioning tooth in mouse jawbone.11Next,

F IGURE  6 Orthotopic regeneration of re- associated explants in the maxilla ofSCIDmice.(A),Schematicdiagramofexperimentaldesign.(B),Macroviewand3D reconstruction of a regenerated tooth (reddottedline)inmurinemaxillaat8wkpost-transplantation.(C),Coronal,sagittaland axial views of regenerated tooth with CBCT.AandP,anteriorandposterior;TandB,topandbottom;RandL,rightandleft.(D),Crossviewofprofilein(B)andhistologicalsections.(E),Histologicalsectionsoftheboxedareasin(D)andimmunostaining of the amnioblast- specific markerameloblastin(AMBN,green),the odontoblast- specific marker dentin sialophosphoprotein(DSPP,green)andDAPI(blue),showingtheregeneratedtooth-likestructure.c,cementum;d,dentin;dp,dentalpulp;Od,odontoblast.Scalebars:100μm

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we investigated whether mouse jawbone is also suitable for grow-ing a re- associated bioengineered tooth germ from miniature pig. We transplanted the re- associated explants from single- tooth germ cells at cap stage from miniature pigs into the maxillary dias-temain5SCIDmice(Figure6A).At8weekspost-transplantation,we found a large bulge in maxillary diastema in 4 mice, whichwas illustrated with cone beam computed tomography (CBCT)(Figure6B,C). Histological analysis (haematoxylin/eosin) revealedregenerated tooth tissue with normally arranged structure and tissue elements, includingodontoblasts, dentin, dentinal tubules,dentalpulp,rootanalogue,cementum,bloodvesselsandalveolarbone(Figure6D,E).

4  | DISCUSSION

The restorationof lost teeth throughwhole-tooth regeneration inhumans is the ultimate goal.3,11,12,25-29Thesuccessesofdenovood-ontogenesis in mice provide an attractive alternative to the future manipulation of cell- based whole- tooth regeneration in humans for the recovery of tooth function.10,11,30,31 Despite some significant advances,thecell-basedrecombinationapproachtoclinicalwhole-toothregenerationremainsachallenge,includinglargertoothsize,longer developmental time and so on.32 It is essential for pre- clinical study in large animal model similar to human for tooth regeneration. Compared with mice, Pigs (Sus scrofa) serve as a promising largeanimal model for studying human diseases and pre- clinical thera-pies owing to its comparability with humans in many respects.17,33 Previous studies have proven that miniature pigs can serve as an at-tractive alternative to rodents for understanding tooth development and replacement.18,20,21,34Studyingtoothregenerationinminiaturepigs can contribute to understanding processes of human tooth de-velopment that are applicable to regenerative medicine.

Theminiaturepig’sdeciduousmolarsaresimilartohumans’;bothare larger and require a longer developmental period than those of mice. Our previous results showed that approximately 3 months were required to grow p4 from the cap stage at E40 to eruption withnearlycompletedrootsatpostnatalday20(P20).20 It is criti-cal to develop an environment using alternative method where can contribute to long- term observation for large size tooth growth. Currently,ectopictoothregenerationispredominant.Theomentum,anterior eye chamber, subcutaneous tissues, jawbone and kidneycapsule of rodents have traditionally been used for the long- term culture of tooth germ grafts.35-37Thesubrenalcapsulegraftissuit-able for tissue recombinants which permits dynamic examination of development from morphogenesis to functional differentiation and overcomes the inaccessible restrictions in pig embryos. Our results suggested that the subrenal capsules of mice are sufficient to sustain the growth and survival of bioengineered tooth germs.

Previous study has suggested that a fully functioning tooth can be achieved by transplantation of a bioengineered tooth germ into the alveolar bone in an adult mouse.11 Our results also indicate that the orthotopic transplantation of bioengineered tooth bud

fromlargeanimalsintothejawboneinSCIDmouseisalsofeasible.However, we did not obtain functioning tooth at 16weeks post-transplantation in jawbone, due to the large size and longgrowthstage of regenerated tissue which increase the potential for damage in the mice.

Previous study in mice suggested that the odontogenesis po-tential of single cells from tooth germ after cap stage reduced and develops into teeth at a lower frequency.12 Our results suggested that cells from pig late- stage tooth germ seemed to still retain better odontogenesispotential than it inmouse,moreworkneeds tobedone for studying the differences in cell properties from both animal toothgerms.In3Dcellculture,thepelletfrommixedepithelialandmesenchymal cells can form highly ordered structure by selective aggregation of cells (homing) or by rearrangement of the relativepositionsofcellswithinthestructure (self-sorting),suggestingtheself- sorting and homing ability of two cell types.38-40

We used deciduous tooth germ cells from the miniature pig for ectopic and orthotopic whole-tooth regeneration, suggesting thefeasibility of whole- tooth regeneration in a large animal model. Our pilot study to manipulate swine tooth germ cells in vitro and in vivo offer an alternative model and a reference for whole- tooth regener-ation with large size and long growth stage in large animals.

ACKNOWLEDG EMENTS

We thank J. Xiao and J. He for their helpful suggestions. This studywas supported by grants from Beijing Municipality Governmentgrants (Beijing Scholar Program PXM2016_014226_000034,PXM2016_014226_000006, PXM2015_014226_000116, PXM2015_ 014226_000055, PXM2015_014226_000052, PXM2014_014226_ 000048, PXM2014_014226_000013, PXM2014_014226_000053,Z121100005212004,PXM2013_014226_000055,PXM2013_014226_ 000021, PXM2013_014226_07_000080 and TJSHG201310025005);theNationalNaturalScienceFoundationofChina(91649124toS.W.);theNationalNaturalScienceFoundationofChina(81771032toF.W.).

CONFLIC T OF INTERE S T

Theauthorsdeclarenocompetingfinancialinterestsorconflictsinrelation to the work described.

AUTHOR CONTRIBUTIONS

FWandZWperformed studies and interpreteddata. ZF andTWparticipatedininvivoexperimentsandperformedanalysis.JWandCZassistedwithinvitroexperiments.SWdesignedstudy,analysedandinterpreteddata,obtainedfunding.SWandFWdraftedmanu-script. All authors reviewed and approved manuscript.

ORCID

Fu Wang http://orcid.org/0000-0001-9615-1038

Zhipeng Fan http://orcid.org/0000-0003-0629-3476

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SUPPORTING INFORMATION

Additional supporting information may be found online in the SupportingInformationsectionattheendofthearticle.

How to cite this article:WangF,WuZ,FanZ,etal.Thecellre- association- based whole- tooth regeneration strategies in largeanimal,Sus scrofa. Cell Prolif. 2018;51:e12479. https://doi.org/10.1111/cpr.12479