Healing of Periodontal Tissues Following Transplantation of Cells in a Rat Orthodontic Tooth Movement Model

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1 Originl Article Heling of Periodontl Tissues Following Trnsplnttion of Cells in Rt Orthodontic Tooth Movement Model Bob N. Nyk ; Willim A. Wiltshire b ; Ben Gnss c ; Howrd Tenenbum d ; Christopher A.G. McCulloch e ; Chrles Lekic f ABSTRACT Objective: To determine the fte nd differentition of trnsplnted periodontl ligment (PL) precursor cells nd mouse embryonic stem (ES) cells nd their reltive cpcity to regenerte wounded periodontium. Mterils nd Methods: Orthodontic tooth movement ws introduced 24 hours before trnsplnttion of PL or ES cells, nd rts were euthnized either 24 hours or 72 hours fter cell trnsplnttion. The control rts received either no tooth movement nd no cell trnsplnttion or tooth movement nd no cell trnsplnttion. Differentition of trnsplnted cells ws ssessed from mndibulr periodontl histologicl tissue sections by immunohistochemicl methods using monoclonl ntibodies ginst PL cell differentition mrkers. Dt were nlyzed using Student s t-test t significnce level of P.05. Results: Trnsplnttion of PL nd ES cells resulted in higher number of osteopontin, bone siloprotein, nd -smooth muscle ctin lbeled trnsplnted cells, predominntly round the blood vessels of the periodontium in study rts compred with control rts (cell trnsplnttion but no orthodontic tooth movement, P.05). Combined tretments of tooth movement nd cell trnsplnttion resulted in enhnced regenertion of the periodontium s result of tooth movement. Trnsplnttion of PL cells induced higher number of differentiting cells in the PL nd lveolr bone thn did trnsplnttion of ES cells. Conclusions: Orthodontic tooth movement promotes the differentition of trnsplnted cells, nd the differentition occurs predominntly in the prvsculr res of the periodontium. In terms of regenertion of wounded periodontium, trnsplnttion of PL cells produced higher level of regenertion thn ES cells, possibly becuse of PL cell plsticity nd the cpcity to undergo effective differentition in the periodontl cellulr microenvironment. KEY WORDS: Orthodontic tooth movement; Cell trnsplnttion; Differentition; Bone mrrow; Periodontl ligment; Embryonic stem cells INTRODUCTION Periodontl tissue heling is restricted s most periodontl ligment (PL) cells do not undergo renewl 1 Reserch Fellow, Medicl Genetics nd Biochemistry/Mnitob Institute of Cell Biology, University of Mnitob, Winnipeg, Mnitob, Cnd. b Professor nd Deprtment Chir, Preventive Dentl Science, University of Mnitob, Winnipeg, Mnitob, Cnd. c Associte Professor, Biologicl nd Dignostic Sciences, University of Toronto, Toronto, Ontrio, Cnd. d Professor, Biologicl nd Dignostic Sciences, University of Toronto, Toronto, Ontrio, Cnd. e Professor, Biologicl nd Dignostic Sciences, University of Toronto, Toronto, Ontrio, Cnd. f Professor, Preventive Dentl Science, University of Mnitob, Winnipeg, Mnitob, Cnd. Corresponding uthor: Dr Chrles Lekic, Deprtment of Prend hve limited mitotic ctivity. 2 Vrious therpeutic pproches hve been developed to regulte nd promote PL cell prolifertion nd differentition (eg, ppliction of extrcellulr mtrices, growth fctors, nd cell trnsplnts 3,4 ) in order to fcilitte restortion of periodontl tissue homeostsis. In spite of the encourging results obtined fter trnsplnttion of epithelil cells, 5 7 it is difficult to interpret these results nd to distinguish between the recruitment nd differentition of host nd trnsplnted cells. 8,9 ventive Dentl Science, University of Mnitob, 780 Bnntyne Ave, Winnipeg, Mnitob R3E 0W2 Cnd (e-mil: lekicpc@cc.umnitob.c) Accepted: October Submitted: August by The EH Angle Eduction nd Reserch Foundtion, Inc. 826 DOI: /

2 HEALING OF TISSUES IN A TOOTH MOVEMENT MODEL One chllenge of the dt interprettion in these investigtions ws tht the trnsplnted cells were not lbeled nd therefore it ws impossible to compre their contributions to periodontl heling with those of the host cells. Previously, cell-trcking studies hve demonstrted migrtory behvior of lc-z-positive cells trnsplnted into rt brin 10 or dult rt liver. 11 These results indicte tht this lbeling method could be used in the periodontium for ssessing the fte nd differentition of trnsplnted cells. The study of PL cells in stedy-stte conditions hs limittions becuse of the reltively smll number of proliferting cells, n overlp in the temporl expression of phenotypic mrkers of PL cell popultions, 3 nd the existence of cells t different stges of differentition. 12 If PL cells could be synchronously stimulted to proliferte, then the temporl expression of mtrix proteins could be more ccurtely relted to the developmentl stge of nscent cell popultions in repopulting wound sites. In this context, tooth movement could be used to induce cell differentition in the PL s previously described. 13 A tooth-movement model lso permits reproducible experimentl mnipultions nd excludes bcteril infection nd epithelil down-growth. Collectively, the objectives of the study were to determine the extent of differentition of PL nd ES cells compred the quiescent tissue remodeling nd the cpcity of these cells to promote heling of periodontl tissues undergoing orthodontic tooth movement. MATERIALS AND METHODS After the initition of orthodontic tooth movement, lbeled PL cells (lc-z positive cells) or ES cells (green fluorescent protein [GFP] positive cells) were trnsplnted using the periodontl window wound model. 14 Isoltion nd Culturing of PL Cells Trnsgenic mle C57BL/6J B26; 129, week old, g crrying Escherichi coli -glctosidse lc-z genes (Jckson Lbortories, Br Hrbor, Me) were used to generte lbeled PL cells. 14 (GFP-lbeled mouse ES cells were gift from Dr Ngy s lb, University of Toronto, Toronto, Ontrio). Briefly, cells were isolted nd cultured s described previously, 14 except tht the glucose concentrtion in the culture medium ws incresed from 1000 mg/l to 1500 mg/l s high glucose concentrtion improves the cell growth of PL nd ES cells. Animls Thirty Sprgue-Dwley mle rts, 6 8 weeks old, weighing g (Centrl Animl Breeding Fcility, 827 University of Mnitob) were used s recipients (Animl Protocol ). Five nimls were used per group. The control groups received one of two tretments: (1) no tooth movement nd no cell trnsplnttion or (2) tooth movement nd no cell trnsplnttion. The experimentl groups were subjected to tooth movement nd received either lc-z-positive cells (Groups 3 nd 5) or ES cells (Groups 4 nd 6). Twentyfour hours before cell trnsplnttion, tooth movement ws initited by plcing elstic orthodontic ligture (Ormco, Glendor, Clif) between the first nd second mndibulr molr. 13,14 Rts were euthnized either 24 hours (Dy 1, Groups 3 nd 4) or 72 hours (Dy 3, Groups 5 nd 6) fter cell trnsplnttion. Trnsplnttion Procedure Periodontl wounding ws performed under generl inhltion nesthesi using 1 3% hlothne nd oxygen:nitrous oxide (O 2 :N 2 O; 2:1). About of lc Z ( -glctosidse) positive PL or GFP-positive ES cells were trnsplnted t the periodontl wound site under dissecting microscope, nd the tissue ws closed with 4.0-gut suture. 11 Rts were euthnized fter intrperitonel dministrtion of pentobrbitl t 115 mg/kg of body weight or by crbon dioxide (CO 2 ) sphyxi. Tissue Preprtion The declcified specimens were wshed in phosphte-buffered sline (PBS) nd stined clorimetriclly for -glctosidse ctivity. 14 After dehydrting specimens in cold ethnol, the plstic sections were infiltrted nd embedded using Immunobed solutions nd instructions (Polyscience, Wrrington, P). The plstic sections were briefly wshed with 100% ethnol nd stined with either hemtoxylin nd eosin or 5% toluidine blue. Immunohistochemistry The plstic sections were briefly dehydrted nd treted with 1% csein blocking solution, nd then in 0.1% norml mouse serum for 1 hour in moist chmber t room temperture. The slides were treted with 3% hydrogen peroxide in PBS for 30 minutes. They were wshed in PBS for 5 minutes ech nd incubted with primry ntibodies (ie, for osteopontin [OPN] nd bone siloprotein [BSP] mouse monoclonl ntirt ntibodies [Developmentl Studies Hybridom Bnk, University of Iow, Iow City, Iow, USA], dilutions of 1:500 nd 1:200, respectively, were used; for smooth muscle ctin [ -SMA] mouse ntirt [Sigm Chemicl Co, St Louis, Mo], dilution of 1:100 ws used) nd slides were then incubted in moist chmber t 4 C overnight. 14

3 828 NAYAK, WILTSHIRE, GANSS, TENENBAUM, MCCULLOCH, LEKIC Tble 1. The Rtio of Trnsplnted Cells nd the Totl Number of Cells (Expressed s Percentge SD) in the Prvsculr Ares of the Heling PL nd AB Trnsplnted Cells Periodontium Dy 1/Dy 3 AB Dy 1/Dy 3 Prvsculr Dy 1/Dy 3 PL / * / * / * ES / * / / * AB indictes lveolr bone; PL, periodontl ligment; ES, embryonic stem. * Significnt differences in the percentge of PL trnsplnted cells between Dy 1 nd Dy 3 groups for the periodontium, AB, nd prvsculr tissues (P.05). * Significnt difference in the percentge of ES trnsplnted cells in the periodontium nd prvsculr tissues (P.05). (ph 9.5) for 4 5 hours t 30 C. The slides were wshed in distilled wter nd dehydrted in 70% nd 100% ethnol. They were then clered in two chnges of xylol, mounted in permount, nd nlyzed with n Olympus digitl photomicroscope, Nikon Cnd, Mississug, Ontrio, Cnd). Figure 1. Photomicrogrph of Dy 1 group showing bsence of trnsplnted cells t the implnt site (IS). Note tht trnsplnted cells (rrows) hve not migrted into the pulp tissue (P) but only in the wounded periodontl ligment (PL). Br 120 m. The slides were wshed in PBS nd incubted with either vidin-biotin complex (ABC) or ABC lkline phosphtse (ABC-AP) regents (Vector Lbortories, Burlington, Ontrio) for 30 minutes t room temperture nd then wshed in two chnges of PBS. The color rection ws chieved with diminobenzidine (DAB) for 8 15 minutes. The ABC-AP color rection ws llowed to develop for 25 minutes. Immunochemicl Detection of Ribo-Probe Treted Sections Slides were incubted with 5% blocking solution in mlete buffer for 30 minutes followed by tretment with 1.25 units of lkline phosphtse conjugted to nti-digoxigenin (DIG) Fb frgments per 1 ml of blocking solution. The slides were wshed twice in Tris buffer (100 mm sodium chloride [NCl], 50 mm mgnesium chloride [MgCl 2 ], 0.1% Tween 20, ph 9.5) to remove unbound ntibody. The colorimetric rection ws crried out using 0.2 mm 5-bromo-4-chloro-3-indolyl phosphte (BCIP) nd 0.2 mm nitro blue tetrzolium slt (NBT) (Boehringer Mnnheim Lvl, Quebec, Cnd). About 125 L of BCIP/NBT mixture ws dded to 5 ml of Tris buffer Assessment of Immunostining The intensity nd distribution of lbeled cells were evluted under light microscope with n introculr grid eyepiece nd n Olympus digitl microscope ttched with DP 12 digitl cmer (Olympus, Nikon Cnd, Mississug, Ontrio). The stining intensity ptterns were clssified s negtive or positive in the event tht the stining intensity clerly demonstrted presence of intrcellulr expression for the immunostined protein. The number of trnsplnted nd immunolbeled cells in vrious lndmrk sites of PL nd lveolr bone (AB; coronl, middle, nd picl) nd in prvsulr res of the periodontium ws determined under 100 oil immersion lens. The GFP-positive trnsplnted ES cells were clculted using Zeiss epi-fluorescence microscope (Zeiss Cnd, Toronto, Ontrio, Cnd) nd the number of lc-z-positive cells ws determined by the use of Olympus digitl microscope. Dt were nlyzed using Student s t-test t significnce level of P.05. RESULTS Tble 1 shows the rtio of trnsplnted cells nd the totl number of cells (expressed s percentges stndrd devition [SD]) in the periodontium, AB, nd prvsculr res of the periodontium of rts undergoing orthodontic tooth movement. The Dy 1 group hd higher number of trnsplnted cells in the periodontium thn the Dy 3 group. The Dy 3 group, in contrst, hd higher percentge of trnsplnted cells in AB thn the Dy 1 group. The prvsculr res in the PL hd significntly higher number of trnsplnted cells. Notbly, ll trnsplnted cells entered systemic circultion in less thn 24 hours fter the implnttion of cells (Figure 1). Figure 2 shows the differentited -SMA lbeled

4 HEALING OF TISSUES IN A TOOTH MOVEMENT MODEL 829 Figure 2. Photomicrogrph of -smooth muscle ctin blue lbeled trnsplnted cells (red rrow) round the blood vessel (BV) in the heling periodontl ligment (PL). Note the presence of trnsplnted cell in the blood vessel (green rrow). Br 10 m. Figure 3. Immunostined sections showing trnsplnted (green rrows) nd host cells (blck rrows) expressing osteopontin in wounded periodontl ligment (PL). C cementum of the root, AB lveolr bone. Br 60 m. Figure 4. Immunostined sections showing migrtion of trnsplnted cells expressing bone siloprotein (rrows) from the blood vessel (BV) to the remodeling lveolr bone (AB). Br 25 m. trnsplnted cells round blood vessels (prvsculr res) in the PL. The differentited OPN nd BSP lbeled trnsplnted cells in the prvsculr res re shown in Figures 3 nd 4, respectively. Trnsplnted cells were lso found within the blood vessels of the PL s well s in the periostel nd endostel tissues of the AB. In the Dy 1 group only, number of multinucleted osteoclsts were found t the resorption sites of AB. Percentges of OPN, BSP, nd -SMA lbeled trnsplnted nd host cells nd corresponding SDs for coronl, middle, nd picl segments of PL nd prvsculr res in the periodontium in Dy 1 nd Dy 3 groups undergoing tooth movement re presented in Tble 2. There ws significnt (P.05) increse in the percentge of lbeled cells in the prvsculr res in both Dy 1 nd Dy 3 groups. There ws lso response of host cells s result of tooth movement nd trnsplnttion. This cellulr response might be Tble 2. Percentge of Trnsplnted nd Host Cells ( SD) Expressing OPN, BSP, nd -SMA Phenotypic Mrkers in the PL of Animls Undergoing Tooth Movement PL Segments Dy 1 OPN Dy 3 Dy 1 BSP Dy 3 Dy 1 -SMA Dy 3 Coronl / / / / / / Middle / / / / / / Apicl / / / / / / Prvsculr b */ */ */ */ */ */ OPN indictes osteopontin; BSP, bone siloprotein; -SMA, -smooth muscle ctin; PL, periodontl ligment;, trnsplnted cells/host cells. b Prvsculr tissues show higher number of differentiting cells thn the host cells. * Sttisticlly significnt t P.05.

5 830 NAYAK, WILTSHIRE, GANSS, TENENBAUM, MCCULLOCH, LEKIC Tble 3. The PL nd AB Widths in Control Rts Treted with Orthodontic Tooth Movement only nd Rts Treted with Cell Trnsplnttion nd Orthodontic Tooth Movement (in Millimeters) Segments Control Group Dy 1 Dy 3 Experimentl Group Dy 1 Dy 3 PL Coronl Middle 0.20* 0.21** 0.45* 0.42** Apicl 0.28* 0.27** 0.40* 0.38** AB Coronl 0.18* 0.20** 0.35* 0.30** Middle 0.30* 0.28** 1.26* 1.80** Apicl 1.44* 1.48** 1.75* 2.40** PL indictes periodontl ligment; AB, lveolr bone. * Significnt difference (P.05) between Dy 1 control group nd Dy 1 experimentl group. ** Significnt difference (P.05) between Dy 3 control group nd Dy 3 experimentl group. ttributble to the recruitment nd ctivtion of PL stem cells. Rts tht received only trnsplnttion without tooth movement showed bout 6% of differentited cells. Trnsplnttion of PL nd ES cells in produced n incresed percentge of OPN, BSP, nd -SMA expressing cells in the PL nd the AB compred with the control rts. Time-point nlysis showed tht within the first 6 hours fter tooth movement nd cell trnsplnttion, there ws n increse in the number of BSP nd -SMA lbeled cells in the PL (dt not shown). As time progressed, more BSP nd -SMA lbeled cells were found in the PL without pprecible chnges in the number of OPN lbeled cells. Anlysis of distribution of lbeled cells in the coronl, middle, nd picl segments of PL of treted rts indicte tht ech segment of PL hd n increse in the number of OPN, BSP, nd -SMA lbeled cells compred to the control rts (Tble 2). At the sme time there ws significnt increse in the percentge of cells expressing -SMA compred with the BSP expression in the cervicl region of the PL. Heling of wounded PL nd AB ws ssessed by mesuring the coronl, middle, nd picl segment width of PL nd AB in control rts nd rts undergoing trnsplnttion. These dt hve shown tht there is n increse in the width of the AB in rts receiving cell trnsplnttion nd orthodontic tooth movement compred to the control (Tble 3). DISCUSSION PL cells ply preeminent role in the regenertion of wounded periodontium nd re of centrl importnce in determining the outcome of subsequent heling. 15 Notbly, the min finding of this study ws higher degree of regenertion of the periodontium in trnsplnted PL cells compred with ES cells, likely becuse of the plsticity, level of differentition, nd tissue microenvironment of the PL cells. These findings correlte with previous studies in which trnsplnted cells hve been shown to differentite to linege-specific functionl cell types Tooth movement cuses disruption of tissue domins in the entire PL nd the surrounding AB. 13 Shortly fter ppliction of orthodontic force, some portions of PL strt to exhibit chnges in width (ie, mesure of PL homeostsis 14 ). In ddition, intensive tooth movement vi hevy force ppliction my cuse cell deth. 14 This study hs demonstrted tht the PL cells re more suitble to replce destroyed periodontl ligment cells thn ES cells. The fct tht there ws much greter number of trnsplnted thn repopulting host cells in wounded periodontl tissues indictes tht the heling ws more due to the trnsplnted cellulr response rther thn the quiescent tissue remodeling. Similrly, studies hve shown tht stroml mesenchyml cells, isolted from the trget tissue or orgn, my in fct be effective vi trnsplntble cells for their own regenertion. 19 Notbly, the present tooth-movement model fcilittes the study of the physiology of PL cell popultions becuse synchronized cohorts of proliferting nd differentiting cells re generted shortly fter stimultion. 13 As result, in tooth-movement model, trnsplnted cells undergo migrtion, prolifertion, nd differentition, thus repopulting lost cell popultions nd restoring tissue homeostsis. 20 A previous study showed tht fter systemic migrtion trnsplnted cells home to lveolr bone mrrow spces, nd then proliferte nd migrte to repopulte wounded periodontl tissues. 14 Cells in periodontl tissues include multiple cellulr phenotypes 3 nd re derived from proliferting precursors locted t prvsculr sites in the body of the PL 21,22 or in the endostel spces of the lveolr bone. 23 Subsequently, PL fibroblsts express -SMA 24 s well s collgen XII, 25 nd in wounded periodontl tissues expression of these proteins is n indictor of soft-tissue regenertion. Notbly, during erly stges of wound heling, OPN 26 is expressed by minerlized nd nonminerlized tissue-forming PL cells (reviewed by Denhrdt nd Guo 27 ). Previous work hs shown tht the expression of BSP nd osteoclcin 28 by minerlized tissue-forming cells is not normlly detected until the cells hve differentited into osteoblsts, cementoblsts, or odontoblsts. However, we found tht smll number of BSP lbeled cells were expressed in the erly stge fter tooth movement nd in the proximity of blood vessels. Notbly, this protein is expressed lmost exclusively by differentited minerlized tissue-forming cells, nd indeed our recent dt indicte tht trnsplnttion of lbeled PL cells into periodontl wounds leds to the differentition of these

6 HEALING OF TISSUES IN A TOOTH MOVEMENT MODEL cells into osteoblsts, 13 which hve the potentil of enhncing the repir process generted by tooth movement. It is conceivble tht the presence of lrge number of cells round the blood vessels in heling PL nd AB, thus expressing lte-stge mrkers of osteogenic cell differentition (ie, BSP), ctully demonstrte tht cell differentition occurs before cell repopultion of heling sites (ie, wounded periodontium) nd tht trnsplnted cells undergo cell-type differentition in the prvsculr res of the periodontium. CONCLUSIONS In tooth-movement model, trnsplnted PL or ES cells differentite into osteogenic nd nonosteogenic cell types, nd this differentition strts t or round the prvsculr res of blood vessels in the periodontium t the time or before the cells enter the wounded tissue. Collectively, in tissues wounded with extensive tooth movement, trnsplnttion of undifferentited PL progenitor/stem cells cn be used to promote the heling nd the restortion of cell nd tissue domins. ACKNOWLEDGMENT This reserch ws supported by grnt from CIHR, Ottw, Ontrio, Cnd to Drs B. Gnss nd P.C. Lekic (CIHR operting Grnt IMH 60913). REFERENCES 1. Dvidson D, McCulloch CA. Proliferte behvior of periodontl ligment cell popultions. J Periodontl Res. 1986; 21: Nemeth E, McCulloch CA, Melcher AH. Coordinted regultion of endothelil nd fibroblst cell prolifertion nd mtrix synthesis in periodontl ligment djcent to ppositionl nd resorptive bone surfces. Antomic Rec. 1989;223: McCulloch CA, Bordin S. Role of fibroblst subpopultions in periodontl physiology nd pthology. J Periodont Res. 1991;26(3 prt 1): Rutherford RB, Ryn ME, Kennedy JE, Tucker MM, Chrette MF. Pltelet-derived growth fctor nd dexmethsone combined with collgen mtrix induce regenertion of the periodontium in monkeys. 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