Research Article The Adaptive Nature of the Bone...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 876316 17 pageshttpdxdoiorg1011552013876316

Research ArticleThe Adaptive Nature of the Bone-PeriodontalLigament-Cementum Complex in a Ligature-InducedPeriodontitis Rat Model

Ji-Hyun Lee1 Jeremy D Lin1 Justine I Fong1 Mark I Ryder2 and Sunita P Ho1

1 Division of Biomaterials and Bioengineering Department of Preventive and Restorative Dental Sciences University of CaliforniaSan Francisco CA 94143 USA

2Division of Periodontology Department of Orofacial Sciences University of California San Francisco CA USA

Correspondence should be addressed to Sunita P Ho sunitahoucsfedu

Received 4 January 2013 Revised 18 March 2013 Accepted 24 March 2013

Academic Editor Brian L Foster

Copyright copy 2013 Ji-Hyun Lee et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Thenovel aspect of this study involves illustrating significant adaptation of a functionally loaded bone-PDL-cementum complex in aligature-induced periodontitis rat model Following 4 8 and 15 days of ligation proinflammatory cytokines (TNF-120572 and RANKL)a mineral resorption indicator (TRAP) and a cell migration and adhesionmolecule for tissue regeneration (fibronectin) within thecomplex were localized and correlated with changes in PDL-space (functional space) At 4 days of ligation the functional space ofthe distal complex was widened compared to controls and was positively correlated with an increased expression of TNF-120572 At 8and 15 days the number of RANKL(+) cells decreased near the mesial alveolar bone crest (ABC) but increased at the distal ABCTRAP(+) cells on both sides of the complex significantly increased at 8 days A gradual change in fibronectin expression from thedistal PDL-secondary cementum interfaces through precementum layers was observed when compared to increased and abruptchanges at the mesial PDL-cementum and PDL-bone interfaces in ligated and control groups Based on our results we hypothesizethat compromised strain fields can be created in a diseased periodontium which in response to prolonged function can significantlyalter the original bone and apical cementum formations

1 Introduction

Mechanical loads as a result of chewing or biting promotehomeostasis of the periodontal ligament (PDL) and that ofthe bone-PDL-cementum complex following developmentof the bone-PDL-tooth fibrous joint [1 2] Homeostasisof the joint is maintained by the PDL which containsa heterogeneous population of cells such as fibroblastsosteoblasts cementoblasts and undifferentiated mesenchy-mal cells and its entheses (attachment sites) with bone andcementum These cells are sensitive to mechanical loadswhich manifest into strains and as a result can promotemineral formation or resorption The cell-matrix and cell-cell interactions regulate continuous PDL turnover modelingand remodeling of bone and subsequent adaptation ofprimary and secondary cementum including PDL-bone andPDL-cementum interfaces throughout their physiological

function [2ndash5] Accommodation of functional loads withinthe physiological range continues as long as the bone-PDL-cementum complex is protected from the oral environmentby gingival epithelium and underlying connective tissuesHowever the complex is a target for bacterial infection due tocommensal microorganisms in the oral environment and theunique anatomical feature of this environment [6] As a resultperiodontal tissues are susceptible to bacterial invasion

The relationship between host-microbial interactions andprogression to periodontitis is thought to depend on acombination of the ecological shift in subgingival biofilmcomposition genetic factors and other extraneous influences[7] Despite many extraneous factors the ldquocritical pathwayrdquomodel for periodontal pathogenesis developed by Offen-bacher [8] does not account for mechanical loads that isthe effect of functional mechanics on disease progression inthe load bearing joint of humans Biomechanics and other

2 BioMed Research International

functional perspectives on diseased joints are importantareas of study since the altered homeostasis due to bacterialinvasion can lead to pathological adaptation of unaffectedtissues Hence the combinatorial effect of disease and func-tional (mechanical) loads on disease progression and jointadaptation is an important area of investigation Howeverthe challenge that lies in performing such studies is thelimited access to specimens from humans Hence animalmodels in general and rat models in particular continue tobe used extensively as an experimental system to elucidatethe effect of diverse factors on periodontal pathogenesis andprogression [9 10] Despite the extensive literature regardingthe characteristics of periodontal progression in differentanimal models few studies have described the effects of thedisease from a biomechanical perspective

Numerous human and animal studies have confirmed theinfectious etiology of gingival inflammation [11ndash13] apicalmigration of the gingival epithelial attachment and the even-tual loss of underlying bone and connective tissue support[14 15] that leads to clinical periodontitis In this study weemployed the lipopolysaccharide-soaked rat ligature modelto initiate the clinical features of periodontitis namelyinflammation and loss of PDL attachment and alveolar bone[16]Thismodel is an analog that stimulates the natural occur-rence of periodontitis in humansmdashfood impaction over timefollowed by acute and chronic inflammatory host responses[17ndash19] Once disease is initiated the complex is kept undercontinued function and the changes in clinical features canbe correlated with the distribution of biochemical markersIn this study the presence and distribution of the followingbiochemical markers in sections taken from hemimaxillaeharvested at 4 8 and 15 days of ligation and correspondingcontrols were assessed receptor activator of nuclear factor120581B ligand (RANKL a membrane-bound protein commonlyfound on PDL cells osteoblasts and T cells that inducethe expression of RANK on the surface of osteoclasts) [2021] tartrate-resistant acid phosphatase (TRAP an enzymehighly expressed by osteoclasts) [22] tumor necrosis factor-alpha (TNF-120572 a marker for systemic inflammation) [23]and fibronectin (FN a glycoprotein adhesion molecule forfibroblasts that promotes extracellular matrix production)[24] Moreover changes in biochemical expressions andlocalization were correlated to morphological changes of thecomplex Based on previous studies by others morphologicalalterations of bone and cementum due to inflammation wereassociated with increased expressions of proinflammatorymediators [25ndash28] including a variety of cytokines such asTNF-120572 that are involved in inflammatory bone resorption[29] RANKL which is known to orchestrate osteoclastogen-esis synergistically with TNF-120572 [30] wasmapped throughoutthe bone-PDL-cementum complex and was expected toincrease in inflamed periodontal tissue In addition activeosteoclasts and correlative changes in biochemical distribu-tion and alveolar bone resorption were identified Finallyadaptive changes in secondary cementum marked by FNexpression [31] were discussed within the context of jointfunction Our overall hypothesis was that coronal degrada-tion due to periodontitis can cause a significant change in thebiomechanics of the complex and that the resulting adaptive

effects may not be the same between bone and secondarycementum The objective was to identify the adaptation ofthis load-bearing joint that is the bone-PDL-tooth complexwith the onset of periodontitis by mapping and correlatingmorphological changes of the tooth and the alveolar socketto key biomolecular expressions within the complex

2 Materials and Methods

To minimize the effects of environmentally induced peri-odontitis [32ndash34] 6-week-old male Sprague-Dawley ratswere housed in a germ-free facility (Parnassus Services Build-ing) and fed a hard pelleted diet for the duration of the studyAll animals included in this study were housed in pathogen-free conditions in compliance with the guidelines of theInstitutional Animal Care and Use Committee (IACUC) ofUCSF and the National Institute of Health (NIH)

21 Induction of Periodontitis Using an In Vivo Rat LigatureModel 4ndash0 silk suture threads soaked in 1mg of lipopolysac-charide (LPS) from Escherichia coli serotype 055B5 (Sigma-Aldrich USA) per 1mL of 1x Tris buffer were used to induceperiodontitis (119873 = 5 per time point) Threads were placedbetween the first and second molars and the second andthird molars of both maxillae (Figure 1(a)) Molars werereligated every 2-3 days to ensure retention Control rats(119873 = 5 per time point) were flossed every 2-3 days with4ndash0 silk ligatures without LPS Rats were euthanized after4 8 and 15 days of ligation Maxillae were harvested andhemisected Right hemimaxillae were stored in 70 ethanolfor micro-XCT analysis Left hemimaxillae were fixed in 4paraformaldehyde (PFA) at room temperature overnight forhistology

22 Histological Analysis of Cytokine Expressions UsingImmunohistology Identification of Bone Resorption throughTRAP(+) Osteoclasts and Observation of Changes in Col-lagen Birefringence Following fixation intact hemimaxillae(119873 = 5) were decalcified in 05M ethylenediaminetetraaceticacid (EDTA) solution for 3 weeks The EDTA solutionwas changed every 3 days Specimens were then dehy-drated through 80 95 and 100 Flex Alcohol (Richard-Allan Scientific Kalamazoo MI USA) before embeddingin paraffin (Tissue Prep-II Fisher Scientific Fair Lawn NJUSA) Embedded specimens were sagittally sectioned on arotary microtome (Reichert-Jung Biocut Vienna Austria)using a disposable steel blade (TBF Inc ShurSharp FisherScientific Fair Lawn NJ USA) Paraffin serial sections weremounted on Superfrost Plus microscope slides (Fisher Scien-tific Fair LawnNJ) Sections were deparaffinizedwith xyleneand rehydrated through a descending ethanol series of 10095 and 80 ethanol before further use

221 Immunostaining for RANKL FN and TNF-alpha Theimmunofluorescence staining protocol used for RANKL andFN was based on a previously described protocol [35] Inbrief deparaffinized sections were digested with trypsin(Sigma-Aldrich St Louis MO USA) at 37∘C Following

BioMed Research International 3

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Figure 1 In vivo rat ligature model for the induction of acute periodontitis (a) Photograph illustrates lipopolysaccharide (LPS) soaked4ndash0 braided silk threads in the diastemata flanking leftright second maxillary molars Controls were flossed in the same interproximalregions (b) Schematic illustrates the targeted regions of the fibrous joint within the study (c) 3D tomogram illustrates the lingual-sagittalmid-sagittal and buccal-sagittal 2D virtual sections through a second maxillary molar used for morphometrics Anatomical landmarksused to measure alveolar bone crest recession (CEJ-ABC) are indicated (d) 2D virtual section illustrates anatomical landmarks to measureinterradicular distance and PDLwidth Division of the bone-PDL-cementum complex into coronalmiddle and apical sections for PDL-spacemeasurements is also illustrated

washing the specimens were incubated in blocking buffer(3 goat serum 01 BSA in 1x PBS) and then in primaryantibodies of polyclonal rabbit anti-RANKL (Santa CruzBiotechnology Inc sc-9073 Santa Cruz CA USA) andmonoclonal mouse anti-FN (Santa Cruz Biotechnology Inc

sc-8422 Santa Cruz CA USA) diluted to 1 50 in block-ing buffer Slides were stored at 4∘C followed by washingwith 01 Tween-20 in PBS (PBST) and were incubatedwith secondary antibodies AlexaFluor 594 goat anti-rabbit(Invitrogen A-11012 Carlsbad CA USA) and AlexaFluor


488 goat anti-mouse (Invitrogen A-11029 Carlsbad CAUSA) were used to label polyclonal rabbit anti-RANKL andmonoclonal mouse anti-FN at 1 300 (diluted in blockingbuffer) respectively Sections were washed with PBST andthen stainedwith 1 10000 trihydrochloride trihydrate (Invit-rogen Carlsbad CA USA) for ten minutes in the absenceof light Slides were rinsed twice with PBS and mountedusing Fluoro-Gel (Electron Microscopy Sciences HatfieldPA USA) Stained sections were visualized using EclipseE800 fluorescent microscope (Nikon Inc Melville NY)TRITC filter (540ndash565 nm) was used to excite AlexaFluor594 (abs 590 nm emit 617 nm) FITC filter (465ndash495 nm) toexcite AlexaFluor488 (abs 495 nm emit 519 nm) and DAPIfilter (340ndash380 nm) to excite trihydrochloride trihydrate (abs358 nm emit 461 nm) Images were stitched using MicrosoftResearch Image Composite Editor (Microsoft CorporationRedmond WA USA)

For quantitative analysis for RANKL the number ofRANKL(+) cells in two 125120583m square regions around thesurface of alveolar bone crest was counted using Image J(v144p National Institute of Health USA) Group means(plusmnstandard deviation) were calculated A two-way analysis ofvariance (ANOVA) was used to analyze the effect of time andexperimental conditions A follow-up post-hoc test was usedto analyze the differences between groups119875 lt 005was takento indicate significance For comparative evaluation of FNa 300120583m line plot spanning dentin secondary cementumPDL and alveolar bone were generated for immunofluo-rescence micrographs of each group and gradients of FNintensities were mapped using Image J

331015840-Diaminobenzidine (DAB) staining for detection ofTNF-120572was performed on serial sections Endogenous perox-idases were deactivated with 80 methanol and 06 H

2O2

Following antigen retrieval sections were incubated withnormal serum for 30min to prevent nonspecific binding Forcytokine detection the primary antibody (goat polyclonalanti-rat TNF-120572 sc-1350 Santa Cruz Biotechnology Inc SantaCruz CA USA) was applied on the sections at a dilution of1 100 in PBS and incubated overnight at room temperatureThe sections were incubated for 15min at room temperaturewith the secondary antibody (biotinylated rabbit anti-goatIgG antibody PK-6105 Vector Labs Burlingame CA USA)Antigen-antibody complexeswere visualizedwithDAB tetra-chloride solution (Sigma D3939 St Louis MO USA)washed in distilled water counterstained with hematoxylinGill (3X) (Fisher Scientific KalamazooMI USA) and rinsedin running water Finally the sections were dehydrated inascending concentrations of alcohol cleared with xylene andmounted Negative controls were obtained by substitutionof the primary antibodies with normal goat serum Lungtissues harvested from the same animals were used as positivecontrols The sections were evaluated by a single examinerwho was blinded to the treatment assignment using a lightmicroscope (BX 51 Olympus America Inc San Diego CAUSA)

222 Resorption by Mapping TRAP(+) Osteoclastic CellsTartrate-resistant acid phosphatase (TRAP) staining forosteoclasts was performed by treating rehydrated specimens

with 02M acetate buffer a solution of 02M sodium acetateand 50mM L-(+)-tartaric acid (Sigma-Aldrich St LouisMO USA) After 20 minutes of incubation at room temper-ature naphthol AS-MX phosphate and fast red TR salt wereadded followed by incubation at 37∘C for 1 hour with closemonitoring under the microscope after the first half hour tomonitor the development of a bright red staining for osteo-clastic activityThe stained sections were washed in deionizedwater counterstained with hematoxylin and mounted withImmu-Mount (ThermoScientific Fremont CA USA) forsubsequent examination under light microscopy TRAP(+)stained regions were categorized based on location thesegment from alveolar crest (defined as the curved surfaceconnecting the mesial and distal faces of the alveolar boneproper) to the starting point of secondary cementum as thecoronal segment and from secondary cementum to the apexas the apical segment Criteria for identification of osteoclastswere TRAP(+) staining with greater than three nuclei [36]The number of osteoclasts within each region was manuallycounted along the PDL-bone perimeter using Image-ProPlus v60 data acquisition software (Media CyberneticsInc Bethesda MD USA) and ratios of osteoclast count toperimeter per mesial and distal location were calculated [37]These ratios were tested for statistical differences betweencontrol and ligature groups and across time points usingtwo-way ANOVA followed by post-hoc tests to analyze thedifference between the groups Differences with 119875 lt 005were considered significant

223 Collagen BirefringenceUsing Picrosirius Red (PSR) StainDeparaffinized sections were stained with Sirius red F3B (CI35782) and picric acid (American MasterTech Scientific CoLodi CA USA) Stained sections were analyzed with a lightmicroscope and Image-Pro Plus Polarized light was used toenhance the birefringence of collagen to illustrate changesin collagen fiber orientation and birefringence intensitythroughout the complex [38 39]

23 Changes in Morphometrics of the Bone-PDL-CementumComplex Using Micro-X-Ray Computed Tomography (120583-XCT) Macroscale structural analysis of intact right hemi-maxillae (119873 = 5 each group) was performed using micro-X-ray tomography (120583-XCT Micro XCT-200 Xradia IncPleasanton CA USA) at 2x magnification X-ray imagingwas performed on specimens using a tungsten anode witha setting of 75KVp at 6W at binning 2 and quartz silica(SiO2) filter designed specifically for biological specimens

Specimens were scanned while immersed in 70 ethanolwith the second molar centered in the field of view 2000projections were collected at an exposure time of 9ndash14 s foreach projection

Tomograms were reconstructed (XMReconstructorv816599 Xradia Inc Pleasanton CA USA) and 2D virtualsections were generated (Xradia 3D viewer v116 Xradia IncPleasanton USA) to complete the following measurementsusing Image J To analyze the progression of periodontitisthrough the time points three sagittal sections containingapical foramen were made through each specimen in


the mesiodistal direction through (1) both buccal roots (2)both lingual roots and (3) the interradicular region (Figure1(c)) Alveolar bone crest (ABC) resorption was determinedby measuring the distance from the cementoenamel junction(CEJ) to the adjacent ABC of the second maxillary molaralong mesial and distal roots (Figure 1(c)) The width ofthe PDL-space surrounding the second maxillary molarwas determined using the aforementioned buccal andlingual sagittal sections (119873 = 3 each group) Mesial anddistal roots were divided into fourths from the CEJ to theroot apex (Figure 1(d)) From the three apical fourths fivePDL-space measurements from the alveolar bone to the rootcementum per quarter section were measured using ImageJ Interradicular PDL spaces (or interradicular distances)measured using the midsagittal section as the distancefrom the crest of the interradicular bone to the molar rootfurcation (Figure 1(d)) All statistical analyses for significantdifferences in morphometrics across and within time pointswere performed using two-way ANOVA combined withpost-hoc tests Differences with 119875 lt 005 were consideredsignificant

3 Results

The Naturally Occurring Tension and Compression Fields in aRat Bone-PDL-Tooth Complex [35] It should be noted thatin a rat the distal side is more prone to mineral resorptionwhile the mesial side is prone to mineral formation From abiomechanical perspective this is due to the tensile strains inthe mesial side compared to reactionary compressive strainson the distal side As a result mineral is formed on the mesialside of the alveolar socket while mineral is resorbed on thedistal side of the same alveolar socket thus maintaining auniform functional space within the bone-PDL-tooth fibrousjoint

31 Changes in the Expressions of Biochemical Markers withinthe LPS Soaked Ligature-Induced Periodontitis Model

311 RANKL Expression in the Complex Intense expressionof RANKL in endosteal spaces of alveolar bone and vascula-ture in PDLwas detected (Figure 2(a)) in the ligated group Inaddition osteoclasts in resorption pits at both PDL-bone andPDL-cementum attachment sites strongly expressed RANKL(Figure 2(b))

To correlate RANKL expression with osteoclastic activityRANKL(+) cell number was counted in subepithelial con-nective tissue (CT) near the alveolar crest (Figures 2(c)ndash2(e)) RANKL(+) cell count interestingly exhibited advancingtrends to TRAP(+) staining On themesial side (Figure 2(d))the number of RANKL(+) cells in the ligated group wasgreater than that in the control group However the numberof RANKL(+) cells was reduced with time in both groupsshowing a significant decrease in ligated group from 4 daysto 15 days (119875 lt 005) On the distal side (Figure 2(e)) therewas a significant elevation of RANKL(+) cells in the ligatedgroup compared to the control group (119875 lt 005) at 4 daysbut a decrease was observed at 8 days A higher numberof RANKL(+) cell count was identified on the distal side

compared to mesial side of the ligated group and distal andmesial sides of the control group (Figures 2(d) and 2(e))

312 Osteoclastic Activity in the Complex Morphologicalchanges were also evaluated in mesiodistal stained sec-tions and increased alveolar bone resorption with timewas observed (Figure 3(a)) Consistent with 120583-XCT mor-phometric analysis the CEJ-ABC distance was higher inligated groups (Figures 3(a) and 5(a)(i)) MultinucleatedTRAP(+) cells were observed in endosteal and bone marrowspaces irrespective of experimental conditions or time points(Figure 3(b)(indashix)) Active osteoclasts at the PDL-bone inter-face were also found on the alveolar crest in the 8-day lig-ated group (Figure 3(b)(ii)) Quantification of TRAP(+) cellsshowed preferential distal localization regardless of experi-mental condition or time point (Figures 3(b)(x) and 3(b)(xi))

Data for the 4-day ligated group was discarded due toinadequate samples TRAP(+) cells showed preferential distallocalization regardless of experimental condition or timepoint (Figures 3(b)(x) and 3(b)(xi)) and peaked at 8 daysexcept in apical segments on the distal side On the mesialside osteoclastic activity at 8 days was significantly greaterthan that observed at 4 days in coronal segments and that ofthe 15-day ligated group in apical segments (Figure 3(b)(x))Within control groups no significant increase or decreasewas seen in both coronal and apical segments Howeverthe mesial osteoclastic activity of ligated groups significantlyincreased at 8 days in apical segments compared to respectivecontrol groups before decreasing at 15 days (Figure 3(b)(x))On the distal side osteoclastic activity both at 8 days and 15days was significantly greater than that of the 4-day controlgroup in coronal segments when the 4-day control group wasused as the reference (data not shown) At both 8 and 15 daysligated groups showed an increase in osteoclastic activityin coronal segments compared to corresponding controlgroups while osteoclastic activity in ligated groups decreasedin apical segments (Figure 3(b)(xi)) Between 8 and 15 daysthe distal osteoclastic activity of ligated groups decreased inboth coronal and apical segments (Figure 3(b)(xi))

313 Immunohistochemical Localization of TNF-120572 TNF-120572 was identified in the gingival epithelium subepithelialCT endosteal spaces predentin and secondary cemen-tum (Figure 4(a)) Particularly TNF-120572 expression in ligatedgroups was more intense than that of control groups at bothmesial and distal PDL-cementum interfaces the interradic-ular complex and in endosteal spaces (Figure 4(b)) At ahigher magnification osteoclast-like cells were detected atPDL-bone interfaces in ligated groups (Figure 4(b) arrow-heads)

32 LPS Soaked Ligature-Induced Periodontitis RatModel Stimulated Significant Changes in Alveolar BoneResorption PDL-Space Width Collagen Birefringenceand Fibronectin Expression

321 Alveolar BoneResorption Morphometric analysis using120583-XCT revealed that at all time points the distance from


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Figure 2 Identification of RANKL using immunofluorescence (a) Representative micrographs illustrate immunofluorescence of antibodiesagainst RANKL at 4-day control and 8- and 15-day ligated groups Note RANKL expression around the vasculature (V) and endostealspaces (asterisks) Multinucleated osteoclast-like cells were also observed at the PDL-bone interface (white arrows) (b) Higher magnificationmicrographs show RANKL immunofluorescence in local regions of the complex at 15 days of ligation (c) Schematic of rat periodontaltissue (mesiodistal section) with gray boxes (125120583m times 125120583m) that indicate target areas used to count RANKL(+) cells (d e) Bar graphsillustrate RANKL(+) cell count within specified target areas between control and ligated groups onmesial (d) and distal (e) sides lowastStatisticallysignificant difference at 95 confidence interval was observed Junctional epithelium (JE) epithelium (E) periodontal ligament (PDL)alveolar bone (AB) alveolar bone crest (ABC) dentin (D) and secondary cementum (SC)


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Figure 3 Alveolar bone resorption through TRAP(+) osteoclast identification (a) Mesiodistal histological sections illustrate TRAP(+) cellson distal surfaces The relative height of the alveolar bone crest (ABC) in relation to the cementoenamel junction (CEJ) is shown to decreasewith duration of ligation (b)Magnified images of 3A show alveolar bone crest (ABC) coronal and apical regions of distal surfaces across timepoints and between control and ligated complexes (indashix) The number of multinucleated osteoclasts (OC) located along the bone perimeterwas counted in coronal and apical root segments on mesial (x) and distal (xi) sides lowastStatistically significant difference at 95 confidenceinterval was observed Alveolar bone (AB) periodontal ligament (PDL) dentin (D) and secondary cementum (SC)


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Figure 4 Immunohistochemical staining for identification of TNF-120572 (a) Representative lightmicrograph of the complex at 15 days of ligationillustrates localization of TNF-120572 (b) Representative images illustrate immunohistochemical localization of TNF-120572 at subepithelial connectivetissue coronal and apical PDL spaces and interradicular PDL regions according to experimental conditions For example note TNF-120572expression at the distal coronal PDL-bone interface (black arrow heads) Connective tissue (CT) alveolar bone (AB) periodontal ligament(PDL) dentin (D) and secondary cementum (SC)

CEJ to ABC (CEJ-ABC) was greater in ligated groups whencompared to control groups in both mesial and distal regions(Figure 5(a)(i)) While at 4 days both mesial and distalregions of ligated groups exhibited significant increases inCEJ-ABC compared to controls only the distal regions exhib-ited significant increases in corresponding comparisons Inmesial regions of both control and ligated complexes CEJ-ABC exhibited a decreasing trend between 4 days to 8 daysand an increase from 8 to 15 days however only the ligatedmesial region exhibited a significant increase between 8 and15 days (119875 lt 005) In distal regions the CEJ-ABC exhibitedincreasing trends in both control and ligated complexes withtime increasing significantly at 8 and 15 days when comparedto 4 days (119875 lt 005) The interradicular distance withinthe control complex decreased with time (Figure 5(a)(ii))In contrast the interradicular distance within the ligatedcomplex decreased significantly between 4 and 8 days ofligation (119875 lt 005) and then increased slightly after 15 days ofligation Comparisons of the averages in ligated and controlinterradicular PDL-spaces at each time point showed thatthe ligated complex had a significantly greater interradicularPDL-space at 4 days (119875 lt 005) This significant increase wasnot maintained at 8 days but was reestablished at 15 days

322 PDL-Space PDL-space measurements showed differ-ences in trends across time between control (Figure 5(a)(iii))and ligated complexes (Figure 5(a)(iv)) in both coronal andapical regions In all ligated regions a decreasing followed byan increasing trend in PDL-space was found over time with a

significant decrease in the distal apical region (119875 lt 005) Incontrast control complexes did not exhibit this trendwith theexception of the distal coronal region Instead between 8 and15 days the PDL-space of both mesial and distal sides showeda decreasing trend with time in the control complex with asignificant decrease between 4 and 15 days at themesial apicalregion (119875 lt 005) When comparing the PDL-space betweencontrol and ligated complexes it was interesting to notethat all observed significant differences in the mesial regionsoccurred at the earlier time points of 4 and 8 days (119875 lt 005)The significant differences in distal regions occurred mostlyat later time points of 8 and 15 days (119875 lt 005)

To separate the effect of treatment from that of develop-ment the average control PDL-space was subtracted from theaverage treated PDL-space for each region (Figure 5(b)) Themesial apical and all distal PDL-spaces of the ligated complexincreased at 4 days while mesial coronal and mesial middlePDL-spaces decreased compared to controls The mesialcoronal mesial middle and distal apical regions exhibitedsignificant differences in PDL-space between ligated andcontrol groups Regardless of anatomical location at 8 daysthe difference between ligated and controlmesial PDL-spacesincreased negatively and the difference between ligated andcontrol of distal PDL-spaces increased positively with timerelative to corresponding control regions (119875 lt 005) Assuch there was a significant difference in PDL-space betweenligated and control groups in all regions At 15 days thedistal PDL-spaces of ligated coronal regions remained greaterthan the corresponding control region but decreased in


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SC

TF

SCSCSCSCSCSCSCSCSCSCSSSCSCCCCCSCCSCSCSCSCSCSCSCSCSCSCSSCSSSCSSSCSCCSCSCCCSCSCSCCSCSCSSCSCSSCSCSCSCSCCCSCSCSCCSCSCSSSSSCSCSCSCSCCSCCCCSCSCCCCSSCSCSSSSSCSCCSCCCCCSCSCCCCSCSCSSSSSCSSSCSCSCCSCSCSCSSSSCSSCSCSCSCCCCCSCSSSCSCSCCCCSSCCCCSSSSCSCCCCCCCCCSSSCSCSSSSCSCCCCSCSCSCSSCCCSSSSCSCCCCSCSSSSSSCSCCSCSSCCSCCCCCSCCSCSCCCCSCSSSCCCCSSSCCCSSSSSCCCCCCSSSCCCC

(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m

4d control 4d ligated 8d ligated 15d ligated

(c)

Figure 5 Morphometric comparisons of control and ligated bone-PDL-cementum complexes including changes in collagen birefringence(a) Comparisons of measurements in mesial and distal regions of the CEJ-ABC (i) the interradicular region (ii) and the PDL-space (iii iv)between control and ligated complexes at 4 8 and 15 days Individual graphswere used to compare PDL-spacemeasurements between coronaland apical anatomical locations within mesial (iii) and distal (iv) complexes (b) The differences between average ligated and average controlPDL-space measurements are plotted for each aforementioned anatomical location across time (c) Histological sections show the distalcomplex stained with PSR and visualized under polarized light microscopy Alveolar bone (AB) periodontal ligament (PDL) secondarycementum (SC) and transseptal fibers (TF) Symbols within plots indicate statistically significant differences at 95 confidence intervalampdaggerΨyenSignificant difference between control and ligated groups 120578Dagger120581Significant difference over time +Significant difference between controland ligated groups lowastSignificant difference over time AΒCDEFGHSignificant difference between control and ligated groups 998771998779Significantdifference over time


difference while the distal PDL-spaces of ligated middle andapical regions increased in difference with correspondingcontrol regions Similarly the mesial ligated regions stillremained smaller than the PDL-spaces of controls howeverthey decreased in difference with the corresponding controlregions Interestingly only the distal regions exhibited sig-nificant differences between ligated and control PDL-spacemeasurements at 15 days

323 Collagen Birefringence PSR birefringence changeswere observed on the distal side depending on the dura-tion of the insult (Figure 5(c) see Figure S1 and supple-mental movies in Supplementary Material available onlineat httpdxdoiorg1011552013876316) In coronal regionsthe ligated complex showed compromised transseptal fiberintegrity with decreased birefringence unlike the observedhigh birefringence with straight collagen fibers in the controlcomplex In apical regions the width of increased birefrin-gence in the distal PDL-secondary cementum of the ligatedcomplex widened across 4 8 and 15 days (Figure 5(c))It should be noted that birefringence is dependent on theangle between the PSR-stained specimen and the polarizedlight For a complete representation of the above observationsupplemental movies are included

324 FN Expression at the PDL-Bone and PDL-CementumInterfaces On the mesial side FN expressions were illus-trated as sharp peaks at both PDL-bone and PDL-cementuminterfaces regardless of the experimental condition and timepoint However the width of high FN intensity was widerin the ligated group compared to controls (Figures 6(a1)6(a3) 6(b1) 6(b3) 6(c1) and 6(c3)) On the distal side thedisplay of FN over a larger width of cementum from the PDL-cementum attachment site was observed On the other handonly a sudden drop of intensity with no peak at the PDL-boneattachment site was shown (Figures 6(a2) 6(a4) 6(b2) 6(b4)6(c2) 6(c4) and Figure S2)

4 Discussion

The results of this study are from a commonly used exper-imental rat model for periodontitis [40ndash44] The impetusfor choosing the ligature model is that it promotes scenariossimilar to food impaction between teeth in humans Theligatures between the 1st and 2nd molars and the 2nd and3rd molars (Figure 1(a)) are speculated to promote equal andopposite forces negating or minimizing effects due to theligature itself Additionally LPS was used as a catalyst toaccelerate coronal tissue degeneration by inducing hallmarksof periodontitis such as alveolar crest resorption and PDLdegeneration thus promoting the loss of coronal toothattachment It should be noted that morphometrics thatis PDL-space can increase or decrease As a result thefunctional space is either widened or narrowed which in turncan alter overall biomechanics of the organ

Based on observations from this study it can be inferredthat changes in biochemical expression andor coronal-apical gradients resulting from inflammation could explain

tissue degradation and a subsequent adaptive response drivenby mechanobiology However future studies are needed toinvestigate potential bone formation and resorption effectsduring disease This can be done by temporal mapping ofmineral formation- and resorption-related events to eluci-date PDL-cementum and PDL-bone interfaces under ten-sion andor compression using dynamic histomorphometrythrough a fluorochrome technique Furthermore our inves-tigation is limited to the early stage of periodontitis so amodel for chronic periodontitis should be developed throughobservations made at longer time points by using the pro-posed endotoxin-ligature model The longer ligation timeswould elucidate time-related mechanobiological effects onoverall jointmorphology and soft-hard tissuemicrostructureincluding impairment of joint functionmdasha topic currentlyunder investigation

The results of this study are discussed on the basis thatboth disease and function (continued mastication of hardpellets by the rats) have a concomitant effect on biochemicaland subsequent morphological changes in the organ Thecombined effect can result in positive or negative feedbackthus shifting organ function to impairment Even thoughcomprehensive studies have postulated a cause-and-effectrelationship between bacteria andor inflammatory cytokinesand tissue destruction [44ndash51] they are spatially limitedand describe only alveolar bone destruction As a resultstudies are needed to illustrate effects on the entire bone-PDL-cementum complex in which the role of no one tissuedominates specifically when in function

Hence the two concomitant effects that will be discussedwill include (1) inflammation-induced coronal degenera-tion as a result of LPS soaked threads and (2) potentialmechanobiological effects specifically in the apical regions ofthe tooth attachment apparatus due to coronal degenerationover time To identify a host inflammatory response wemapped expressions of RANKL osteoclastic resorption andTNF-120572 Resulting adaptation was detailed through a changeinmorphology due to increased alveolar bone resorption andnet changes in the PDL-space coronal losses apical increasesin collagen birefringence within the complex and a gain inFN expression specifically at the apical regions that is withinsecondary cementum and bone These observed patterns aresummarized in Figures S2 and S3

41 Inflammation-Related Biomolecular Expressionswithin theComplex Ligature-induced inflammation is a host responseto eliminate the harmful stimuli due to foreign body andbacterial colonization The response triggers recruitmentof immune cells from the blood into various vascularizedconnective tissues predominantly exposed to LPS and lig-ature impaction [27] The recruited inflammatory cells inturn promote a biochemical cascade causing proteinase-induced fibrinolysis osteoclastogenesis and activation oflatent osteoclasts [16] Formation and activity of osteoclastsin vivo are dependent on the expression of RANKL byosteoblastic or bone marrow stromal cells [52 53] As aresult we observed RANKL expression in endosteal spacesand vasculature of PDL In this study the coronal part ofalveolar bone is most affected by LPS due to its proximity


101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D

Higher intensity zone of FN expression at PDL-bone attachment siteHigher intensity zone of FN expression at PDL-cementum attachment site

(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated

Figure 6 Line profiles and micrographs of immunolabeled fibronectin (FN) Representative micrographs illustrate FN immunofluorescencein apical regions of control and ligated complexes at 4 8 and 15 days The intensity of FN expression was measured along the anatomicallocations indicated by the 300120583m long bar For the mesial complex the 119909-axis of the 300 120583m long profile corresponds to alveolar bone (AB)periodontal ligament (PDL) secondary cementum (SC) and dentin (D) Note that the direction for the distal complex is reversed (a1) 4-daycontrol mesial complex (a2) 4-day control distal complex (a3) 4-day ligated mesial complex (a4) 4-day ligated distal complex (b1) 8-daycontrol mesial complex (b2) 8-day control distal complex (b3) 8-day ligated mesial complex (b4) 8-day ligated distal complex (c1) 15-daycontrol mesial complex (c2) 15-day control distal complex (c3) 15-day ligated mesial complex and (c4) 15-day ligated distal complex Dentin(D) secondary cementum (SC) periodontal ligament (PDL) alveolar bone (AB) new bone (NB)


to the ligature and as such the expression of RANKL(+)cells near the alveolar crest was quantified (Figures 2(d) and2(e)) Interestingly the observed RANKL(+) trend opposedthe trend of TRAP(+) osteoclastic activity (Figures 3(b)(x)and 3(b)(xi)) In other words the number of RANKL(+) cellsincreased at 4 days before decreasing and then rebounding at15 days in the distal sides of ligated groups It is well knownthat osteoclastogenesis involves a complex series of sequentialsteps including RANKL-RANK signaling [54] This couldexplain the observed trend of delayed osteoclastic activitythat is TRAP(+) cells

Alongwith RANKL TNF-120572 also plays a role in inflamma-tory bone resorption since TNF-120572 synergizes with RANKLto potentiate osteoclastogenesis [52 55 56] The knownmechanism by which TNF-120572 modulates bone destruction isrelated to RANKL stimulation of osteoclast differentiationthrough autocrine signaling [57] The majority of in vivostudies show that TNF-120572 is mainly produced by activatedmacrophages and acts as an agonist or antagonist to advanceor restrain alveolar bone resorption respectively [47 58ndash60]From our observation TNF-120572 was distributed throughoutthe periodontium (Figure 4(a)) This can be attributed toimmune cells the source of TNF-120572 that is recruited alongthe blood stream in highly vascularized PDL The overallTNF-120572 expression was elevated in ligated groups especiallyat PDL-primary cementum interfaces the interradicularcomplex and endosteal spaces This implies that coronallyplaced ligatures introduced inflammation to the entire bone-PDL-cementum complex and was not just limited to thecoronal area Anatomically TNF-120572 positive osteoclasts werepredominant at the PDL-bone interface on the distal sidein ligated groups This observation was positively correlatedwith increased immunofluorescence of RANKL in conjunc-tion with previous studies that have shown that TNF-120572 sharesthe mechanism by which RANKL exerts its osteoclastogeniceffect [26 57] Additionally TNF-120572 was localized to thejunctional epithelium It is plausible that TNF-120572 releasedextracellularly drifted into the epithelial layer since the inter-cellular space in the junctional epithelium is comparativelywider and contains proportionately fewer desmosomes thanin the oral epithelium [61] allowing cytokines to permeateepithelium

42 Changes in Morphometrics of the Complex CommonHallmarks of Periodontitis Ligated groups showed increasedABC and interradicular resorption compared to controls at 4daysThis observation is most likely caused by the early-stagehost inflammatory events due to LPS as the primary stimulusThe argument can be further corroborated by the observationof a decrease in CEJ-ABC distance in mesial regions and adecrease in interradicular PDL-space at 8 days However thedecrease in bone resorption despite heightened inflammationcould have been caused by age-dependent bone modelingprocesses [42 62] between 4 and 8 days as bone has a rapidrate of turnover By 15 days the resorptive effects of inflam-mation are speculated to overcome growth-related processesin ligated complexes resulting in resumed increases of bothmesial and distal CEJ-ABC as well as interradicular PDL-space It should be noted that within this observed effect

is the effect of osteoclasts in controls due to natural distalresorption in rats [31 62 63] However the ligated complexincluded active osteoclasts on the alveolar crest in addition tothe distal PDL-bone interface Furthermore the osteoclasticactivity peaked at 8 days of ligation This phenomenon canbe explained in terms of the characteristics of inflammationinduced with ligature placement an acute and short-termedinflammation that resulted in the initial shift of equilibriumfrom bacterial endotoxin that is LPS to host responseHowever the osteoclastic activity was not maintained after8 days suggesting that the effect of inflammation induced byligation was not sustained because (1) the location of ligaturewas relatively coronal and (2) the host response that couldhave subsided as the local concentration of LPS was diffused[64] This result is consistent with previous studies [65ndash69]and can be identified as remission of bonemodeling followingacute phase of inflammation

43 Resulting Early Mechanobiological Effects of a DiseasedFibrous Joint Based on observed morphological differences(Figures 5(a) and 5(b)) we predict that the coronal degener-ation could have shifted from physiological to nonphysiolog-ical that is pathological function This shift from physiolog-ical to aberrant loads on the complex is hypothesized to be asignificant deviation from optimum functionThis is becauseincreased jointmobility due to PDLdegeneration and coronalalveolar bone support can cause increased tooth movementwith the alveolar socket Such argument can be furtherreinforced by the fact that the ligature had to be reintroducedevery two to three days in the rats Based on our previouswork regions that are predicted to experience increasedstrain during the initial disease stages include coronal attach-ment sites apical compression-dominant regions and pre-dominantly the interradicular regions [70] (the PDL-space atthe interradicular region is narrower compared to PDL-spacearound the tooth (Figure 5(a))) As such it is conceivable thatwithin ligated groups healthy PDL in interradicular regionsand apical to diseased regions could experience amplifiedstrains during mastication compared to their control coun-terparts Additionally strains could be altered at the PDL-bone and PDL-cementum interfaces [71] in ligated groupsThese altered three-dimensional strain profiles are of specialinterest because deviations from normal physiological straincan lead to pathological function (aberrant loadsfunction)and compromisedmechanotransduction over time [5 72 73]including the soft-hard tissue interfaces where multiple celltypes reside

Ligated groups exhibited different patterns of morpho-logical adaptation over time compared to control groups asindicated by changes in PDL-space Complementary increasein RANKL expression and osteoclast activity an increasein interradicular PDL-space (15 days Figure 5(a)(ii)) couldindicate excessive strains in the interradicular complex at alater time resulting in resorption and shifting the adaptiveeffects of our model to those observed in a hyperocclusionmodel [74 75] As such it is possible that in our study theinterradicular bone commonly known as the fulcrum fortooth rotation [70] shifted thus altering the strain fieldwithinthe entire bone-PDL-cementum complex


The shift toward aberrant function can promote a netchange in the localization and intensity of RANKL RANKLis a mechanosensitive molecule that decreases in expressionwith tension in osteoblasts [76] When comparing alveolarcrests within ligated complexes RANKL expression was seento increase between 8 and 15 days on the distal side butdecreased over time on the mesial side (Figures 2(d) and2(e)) Although RANKL expression increases at each timepoint between ligated and controls both mesially and distallydue to a host inflammatory response the suggested agedominated mesial-tension due to growth may have counter-acted this increase during prolonged mechanical loading Assuch while a RANKL expression increase was seen in 4-dayligated complexes compared to controls a similar numberof RANKL(+) cells were seen between ligated and controlcomplexes at 15 days In contrast the progressive increaseof RANKL(+) cell count from 8 days to 15 days on thedistal side was most likely caused by the compounded effectof compression-induced expression due to distal drift andhost inflammatory response [77 78] Although we correlateRANKL expression increase to osteoclast activation it isimportant to note that a RANKLosteoprotegerin (OPG)ratio should be used as the standard index for formative andresorptive bone [16] Together this data suggests that an earlyhost inflammatory response drives increased biochemicalexpressions coronally However later changes in expressionsof respective biomolecules due to opposing mechanobio-logical effects on mesial and distal sides are induced byaberrant function of the ligated joint Aberrant function-related effects were seen in secondary cementum In thisstudy the complementary data of collagen birefringenceand FN expression seen at the distal secondary cementumis proposed as a compensatory adaptation due to a netincrease in distal bone resorption and maintenance of age-related normal physiological activity at the mesial complex(Figure 5(c)) However birefringence indicated by PSR isonly a complementary marker to the more confirmatory FNexpression This is because collagen birefringence identifiedthrough PSR staining is also a function of section thicknessand is highly dependent on fiber orientation relative to thepolarizers (movies were included to highlight the specificityof our resultsmdashFigure S1 and supplemental movies) and levelof tissue demineralization and collagen integrity

Secondary cementum is hypothesized to adapt to occlu-sion during the posteruption phase of tooth developmentand has been shown to respond to load [35 79] Assuch it is conceivable that secondary cementum adaptationwould follow coronal bone resorption as a mechanism tocompensate for disruption due to inflammation In thisstudy we used two identifiers to detail the adaptation ofsecondary cementumThese included collagen birefringenceand FN expression [24 80 81] The triggering of the localbiochemical effects caused an increase in apical collagenousmatrix organization with time (Figure 5(c)) As a result analtered birefringence was observed at the mesial but higherat the distal PDL-secondary cementum interface in ligatedgroups (Figure S1 supplemental movies) This implies thatinflammation-induced mechanobiology remotely stimulatedorganic matrix within the apical cementum (Figures 5(c)

6 S1 and S2) Although an increased level of birefringenceequivalent to that of controls was observed at 4 days withincreased time both the width and intensity of birefringenceat the distal secondary cementum surface increased It isplausible that the complex has adapted to changes resultingfrom ligature-induced coronal inflammation in the tension-dominant mesial region and the compression-dominant dis-tal region However adaptation manifested itself in apicalregions as new bone formation which was predominant onthe mesial side and new cementum formation on the distalside (Figures 5(c) and 6) within respective groups

The aforementioned secondary cementum adaptationdetailed through collagen birefringence can be furtherstrengthened by FN expressions Nonphysiological tensionsat the mesial complex of ligated rats due to coronaldegeneration of transseptal and periodontal fibers couldhave altered the FN mRNA expression and protein contentapically (Figure 6) [82 83] The localization pattern of a wideFN expression band apically could be from an increased orga-nization around periphery of osteo- or cementoblast cells andincreased density as the collagenous matrix is generated [84]

FN is an important chemotactic protein for the storage ofgrowth factors along with its prolific interactions with cellsurface molecules to facilitate cellular adhesion migrationand regulate cellular differentiation and proliferation [24]Although FN is not a direct marker for hard-tissue formationusing immunohistochemistry it provides evidence that thismultipurpose protein can amplify the response of osteoblastprogenitors to alter the regenerative or reparative potential oforganic matrices during mechanobiological adaptation [83]The same may hold true for cementoblast progenitors onthe tooth side resulting in mesial deposition of secondarycementum While compressive loads have been shown tocause a decrease in FN expression within PDL cells [82 83]when compared to controls the distal regions in ligatedmolars interestingly illustrated increased FN expression atPDL-cementum attachment sites and through the distalprecementum layers of the complex (Figure 6)

We summarize the results by presenting a biomechanicalmodel representative of the measured downstream local-ization and expression level changes of mechanosensitiveproteins and net spatiotemporal changes in the PDL-space(Figure S3) On the distal coronal side temporal trends ofPDL-space were directly correlated with those of osteoclasticactivity As such the net increase in PDL-space on the distalcoronal side in ligated groups could be explained by anincreased osteoclastic activity due to increased compressivestrains within the PDL Increased compression could arisefrom increased whole body distal rotation of the tooth(Figure S3A3) coupled with coronal inflammation In bothcontrol and ligated groups osteoclastic activity increasedsignificantly at 8 days and remained significantly higherat 15 days when compared to 4-day controls While therewas greater osteoclastic activity in ligated groups at 8 dayscompared to the controls the lack of significant differencebetween control and ligated groups at both time points couldbe explained by (1) development-related changes at early-timepoints which may have masked the effect of inflammationand mechanobiological response andor (2) a need for


a larger sample size However it is interesting to note thatthere was a decreased but sustained osteoclastic activity at 15days which could have affected the observed PDL-space

In the apical segment of the distal complex the trendobserved in PDL-space did not correspond to the osteoclas-tic activity change Specifically when correlating trends ofosteoclastic activity and PDL-space over time the increasedosteoclastic activity did not match the decreased PDL-spacein 8-day ligated joints Furthermore the measured increasein width of FN expression adjacent to the distal secondarycementum which indicates new cementum apposition callsfor speculation that the PDL-space in this region experiencedincreased tensile strains during increased distal rotation andvertical displacement (Figure S3A3) Therefore the PDL-space is expected to become narrower than that of controlgroups over time (Figure S3A4) However the oppositemorphological change was observed The increase in PDL-space of ligated joints at 15 days when compared to controlsand 8-day ligated joints suggests that there was a delay inobservable morphological effects (resorption of bone) afterthe increase of osteoclast activity [69] Namely it can bespeculated that 8 days of ligation was closer to the beginningof an increase in osteoclastic activity that carried into 15 daysof ligation resulting in an increased PDL-space As such thenew cementum apposition labeled by FN could have beenmissed in micro-XCT measurements since the FN-labeledband indicates a predominance of organic matrix ThereforePDL-space could be narrowed with prolonged observationas the cementum layer is given time to mineralize Furtherstudies identifying cementum growth using fluorochrometechnique or cementum-specific markers are necessary andcould be used to verify this phenomenon Similarly the trendsin osteoclastic activity on the mesial side reflected oppositemorphological changes in PDL-space at corresponding timepoints

From a morphological standpoint the significantlyreduced PDL-space in more apical unaffected regions ofligated fibrous joints can be attributed to increased ten-sile strains in this area due to coronal degradation [70]Although it is well established that experimentally inducedperiodontitis stimulates inflammation-induced osteoclasticactivity such activity is limited to the coronal regions [85]Therefore the osteoclastic activity observed in more apicalregions of ligated complexes could be the manifestationof the resorptionformation coupling of physiological boneturnover [86] However the amplified strains could haveaccelerated adaptation of the complex with an increased rateof bone turnover From a strain localization perspectivethese normally compression-dominant regions are predictedto be shifted to tension-dominant regions This is due to anapically migrating fulcrum of tooth rotation that stems fromthe increased mobility of the tooth as a result of coronalattachment loss and resorption of interradicular bone (FigureS3A3) Our model is also supported by the increased bandwidth of FN expression observed at the PDL bone attachmentsite at the apical region on the mesial side (Figures 6(a3)6(b3) 6(c3) and S3A4)

From these results we speculate that disease-relatedchanges promote adaptation of bone and apical cementum

which was predominantly observed on the mesial-coronaland distal-apical sides of the tooth It is important to notethat the mechanical strains within the complex of a singleroot are assumed to be similar to strains in the entire complexof a tooth despite the ligature Hence the mesiodistal effectsdiscussed in the bone-PDL-tooth complex should be similarto those observed in the corresponding complexes of themesial and distal roots of the same tooth

5 Conclusions

In this present study we clarified the alteration due to diseaseby distinguishing disease-induced effects from physiologi-cally determined phenomena with unpaired control groupsIn conclusion our results showed that coronal induction of aninflammatory perturbation affected the overall distribution ofbiochemical molecules in the entire complex Furthermoreconcomitant function imposed on the compromised complexcould have accelerated tissue adaptation to meet functionaldemands (Figures S2 and S3) To be specific a net decreasein functional space was identified in the mesial complexwhereas a net increase in functional space was identifiedin the distal complex In addition an adaptive responsein secondary cementum that is predominant cementumformation wasmore apparent distallyThe altered expressionat later time points suggests that prolonged function canmanifest into excessive loads on the diseased fibrous jointshifting physiological function into an impaired functionwhen compared to controls It is imperative that future stud-ies should measure the biomechanical response of joints thathave undergone short- and long-term changes to inflamedand degraded fibrous joints When measured the biome-chanical results will validate the proposed model that earlymorphological changes due to disease can impair functioneventually causing the observed secondary adaptations of thefibrous joint

Authorsrsquo Contribution

Ji-Hyun Lee and Jeremy D Lin contributed equally

Acknowledgments

The authors thank the Biomaterials and BioengineeringMicroCT Imaging Facility UCSF for the use of MicroXCT Support was provided by NIHNIDCR R00DE018212NIHNIDCR R01DE022032 NIHNIDCR T32 DE07306-14NIHNCRR S10RR026645 and Departments of Preventiveand Restorative Dental Sciences and Orofacial SciencesUCSF The authors acknowledge that they do not haveconflict of interests and were fully involved in the study andpreparation of the paper

References

[1] M Shimono T Ishikawa H Ishikawa et al ldquoRegulatory mech-anisms of periodontal regenerationrdquo Microscopy Research andTechnique vol 60 no 5 pp 491ndash502 2003


[2] C A G McCulloch P Lekic and M D McKee ldquoRole of phys-ical forces in regulating the form and function of the periodon-tal ligamentrdquo Periodontology 2000 vol 24 no 1 pp 56ndash722000

[3] W Beertsen C A GMcculloch and J Sodek ldquoThe periodontalligament a unique multifunctional connective tissuerdquo Peri-odontology 2000 vol 14 no 1 pp 20ndash40 1997

[4] P Lekic and C A McCulloch ldquoPeriodontal ligament cellpopulation the central role of fibroblasts in creating a uniquetissuerdquoTheAnatomical Record vol 245 no 2 pp 327ndash341 1996

[5] J M Hurng M P Kurylo G W Marshall S M Webb M IRyder and S P Ho ldquoDiscontinuities in the human bone-PDL-cementum complexrdquo Biomaterials vol 32 no 29 pp 7106ndash71172011

[6] S S Socransky and A D Haffajee ldquoThe nature of periodontaldiseasesrdquo Annals of periodontology vol 2 no 1 pp 3ndash10 1997

[7] K S Kornman ldquoMapping the pathogenesis of periodontitis anew lookrdquo Journal of Periodontology vol 79 no 8 supplementpp 1560ndash1568 2008

[8] S Offenbacher ldquoPeriodontal diseases pathogenesisrdquo Annals ofperiodontology vol 1 no 1 pp 821ndash878 1996

[9] H V Jordan ldquoRodent model systems in periodontal diseaseresearchrdquo Journal of Dental Research vol 50 no 2 pp 236ndash2421971

[10] D T Graves D Fine Y T A Teng T E Van Dyke and GHajishengallis ldquoThe use of rodent models to investigate host-bacteria interactions related to periodontal diseasesrdquo Journal ofClinical Periodontology vol 35 no 2 pp 89ndash105 2008

[11] H Loe E Theilade and S B Jensen ldquoExperimental gingivitisinmanrdquoThe Journal of Periodontology vol 36 pp 177ndash187 1965

[12] P Axelsson and J Lindhe ldquoEffect of controlled oral hygieneprocedures on caries and periodontal disease in adultsrdquo Journalof Clinical Periodontology vol 5 no 2 pp 133ndash151 1978

[13] P Axelsson and J Lindhe ldquoEffect of controlled oral hygieneprocedures on caries and periodontal disease in adults Resultsafter 6 yearsrdquo Journal of Clinical Periodontology vol 8 no 3 pp239ndash248 1981

[14] R C Page and H E Schroeder ldquoPathogenesis of inflammatoryperiodontal disease a summary of current workrdquo LaboratoryInvestigation vol 34 no 3 pp 235ndash249 1976

[15] J Lindhe S E Hamp and H Loe ldquoPlaque induced periodontaldisease in beagle dogs A 4 year clinical roentgenographical andhistometrical studyrdquo Journal of Periodontal Research vol 10 no5 pp 243ndash255 1975

[16] D L Cochran ldquoInflammation and bone loss in periodontaldiseaserdquo Journal of Periodontology vol 79 no 8 supplementpp 1569ndash1576 2008

[17] H S Oz and D A Puleo ldquoAnimal models for periodontaldiseaserdquo Journal of Biomedicine and Biotechnology vol 2011Article ID 754857 8 pages 2011

[18] R Liu H S Bal T Desta et al ldquoDiabetes enhances periodontalbone loss through enhanced resorption and diminished boneformationrdquo Journal of Dental Research vol 85 no 6 pp 510ndash514 2006

[19] C A Genco T Van Dyke and S Amar ldquoAnimal modelsfor Porphyromonas gingivalis-mediated periodontal diseaserdquoTrends in Microbiology vol 6 no 11 pp 444ndash449 1998

[20] T Ogasawara Y Yoshimine T Kiyoshima et al ldquoIn situ expres-sion ofRANKLRANK osteoprotegerin and cytokines in osteo-clasts of rat periodontal tissuerdquo Journal of Periodontal Researchvol 39 no 1 pp 42ndash49 2004

[21] M A Taubman P Valverde X Han and T Kawai ldquoImmuneresponse they key to bone resorption in periodontal diseaserdquoJournal of Periodontology vol 76 no 11 supplement pp 2033ndash2041 2005

[22] C Minkin ldquoBone acid phosphatase tartrate-resistant acidphosphatase as amarker of osteoclast functionrdquoCalcified TissueInternational vol 34 no 3 pp 285ndash290 1982

[23] E F Rossomando J E Kennedy and J Hadjimichael ldquoTumournecrosis factor alpha in gingival crevicular fluid as a possibleindicator of periodontal disease in humansrdquo Archives of OralBiology vol 35 no 6 pp 431ndash434 1990

[24] W J Grzesik andA S Narayanan ldquoCementum and periodontalwound healing and regenerationrdquo Critical Reviews in OralBiology and Medicine vol 13 no 6 pp 474ndash484 2002

[25] J E Rogers F Li D D Coatney et al ldquoActinobacillus actino-mycetemcomitans Lipopolysaccharide-mediated experimentalbone loss model for aggressive periodontitisrdquo Journal of Peri-odontology vol 78 no 3 pp 550ndash558 2007

[26] S Kwan Tat M Padrines S Theoleyre D Heymann andY Fortun ldquoIL-6 RANKL TNF-alphaIL-1 interrelations inbone resorption pathophysiologyrdquo Cytokine and Growth FactorReviews vol 15 no 1 pp 49ndash60 2004

[27] D Graves ldquoCytokines that promote periodontal tissue destruc-tionrdquo Journal of Periodontology vol 79 no 8 supplement pp1585ndash1591 2008

[28] S Ma J Guo X You W Xia and F Yan ldquoExpressions ofinterleukin-1120573 and interleukin-6 within aortas and uteri ofrats with various severities of ligature-induced periodontitisrdquoInflammation vol 34 no 4 pp 260ndash268 2011

[29] K Kobayashi N Takahashi E Jimi et al ldquoTumor necrosisfactor 120572 stimulates osteoclast differentiation by a mechanismindependent of the ODFRANKL-RANK interactionrdquo Journalof Experimental Medicine vol 191 no 2 pp 275ndash285 2000

[30] K Fuller C Murphy B Kirstein S W Fox and T J Cham-bers ldquoTNF120572 potently activates osteoclasts through a directaction independent of and strongly synergistic with RANKLrdquoEndocrinology vol 143 no 3 pp 1108ndash1118 2002

[31] G Anastasi G Cordasco G Matarese et al ldquoAn immunohis-tochemical histological and electron-microscopic study of thehuman periodontal ligament during orthodontic treatmentrdquoInternational Journal of Molecular Medicine vol 21 no 5 pp545ndash554 2008

[32] M J Bjornsson S Velschow K Stoltze A Havemose-PoulsenS Schou and P Holmstrup ldquoThe influence of diet consistencedrinking water and bedding on periodontal disease in Sprague-Dawley ratsrdquo Journal of Periodontal Research vol 38 no 6 pp543ndash550 2003

[33] L Heijl J Wennstrom J Lindhe and S S Socransky ldquoPeri-odontal disease in gnotobiotic ratsrdquo Journal of PeriodontalResearch vol 15 no 4 pp 405ndash419 1980

[34] H Sicher and J P Weinmann ldquoBone growth and physiologictooth movementrdquo The American Journal of Orthodontics andOral Surgery vol 30 no 3 pp C109ndashC132 1944

[35] R P Herber J Fong S A Lucas and S P Ho ldquoImaging anadapted dentoalveolar complexrdquo Anatomy Research Interna-tional vol 2012 Article ID 782571 13 pages 2012

[36] K J Ibbotson G D Roodman L M McManus and GR Mundy ldquoIdentification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrowmononuclear cellsrdquo Journal of Cell Biology vol 99 no 2 pp471ndash480 1984


[37] A Sawyer P Lott J Titrud and J M McDonald ldquoQuantifica-tion of tartrate resistant acid phosphatase distribution inmousetibiae using image analysisrdquo Biotechnic and Histochemistry vol78 no 5 pp 271ndash278 2003

[38] L C U Junqueira G Bignolas and R R Brentani ldquoPicrosiriusstaining plus polarization microscopy a specific method forcollagen detection in tissue sectionsrdquoHistochemical Journal vol11 no 4 pp 447ndash455 1979

[39] S P Ho B Yu W Yun G W Marshall M I Ryder and SJ Marshall ldquoStructure chemical composition and mechanicalproperties of human and rat cementum and its interface withroot dentinrdquo Acta Biomaterialia vol 5 no 2 pp 707ndash718 2009

[40] M P Galvao C K Rosing and M B Ferreira ldquoEffects ofligature-induced periodontitis in pregnant Wistar ratsrdquo Brazil-ian Oral Research vol 17 no 1 pp 51ndash55 2003

[41] A Gyorfi A Fazekas Z Suba F Ender and L RosivallldquoNeurogenic component in ligature-induced periodontitis inthe ratrdquo Journal of Clinical Periodontology vol 21 no 9 pp 601ndash605 1994

[42] A Kuhr A Popa-Wagner H Schmoll C Schwahn and TKocher ldquoObservations on experimental marginal periodontitisin ratsrdquo Journal of Periodontal Research vol 39 no 2 pp 101ndash106 2004

[43] K Sallay F Sanavi I Ring P Pham U H Behling and ANowotny ldquoAlveolar bone destruction in the immunosuppressedratrdquo Journal of Periodontal Research vol 17 no 3 pp 263ndash2741982

[44] S Kimura A Nagai T Onitsuka et al ldquoInduction of experi-mental periodontitis in mice with Porphyromonas gingivalis-adhered ligaturesrdquo Journal of Periodontology vol 71 no 7 pp1167ndash1173 2000

[45] R Achong I Nishimura H Ramachandran T H Howell J PFiorellini and N Y Karimbux ldquoMembrane type (MT) 1-matrixmetalloproteinase (MMP) and MMP-2 expression in ligature-induced periodontitis in the ratrdquo Journal of Periodontology vol74 no 4 pp 494ndash500 2003

[46] J P Bezerra L R F Da Silva V A D A Lemos P M Duarteand M F Bastos ldquoAdministration of high doses of caffeineincreases alveolar bone loss in ligature-induced periodontitis inratsrdquo Journal of Periodontology vol 79 no 12 pp 2356ndash23602008

[47] X Cai C Li G Du and Z Cao ldquoProtective effects of baicalinon ligature-induced periodontitis in ratsrdquo Journal of PeriodontalResearch vol 43 no 1 pp 14ndash21 2008

[48] G D R Nogueira-Filho B T Rosa J B Cesar-Neto R STunes and U D R Tunes ldquoLow- and high-yield cigarettesmoke inhalation potentiates bone loss during ligature-inducedperiodontitisrdquo Journal of Periodontology vol 78 no 4 pp 730ndash735 2007

[49] K Okuda T Kato Y Naito et al ldquoProtective efficacy of activeand passive immunizations against experimental infection withBacteroides gingivalis in ligated hamstersrdquo Journal of DentalResearch vol 67 no 5 pp 807ndash811 1988

[50] Y Samejima S Ebisu and H Okada ldquoEffect of infection withEikenella corrodens on the progression of ligature-inducedperiodontitis in ratsrdquo Journal of Periodontal Research vol 25no 5 pp 308ndash315 1990

[51] F Sanavi M A Listgarten F Boyd K Sallay and A NowotnyldquoThe colonization and establishment of invading bacteriain periodontium of ligature-treated immunosuppressed ratsrdquoJournal of Periodontology vol 56 no 5 pp 273ndash280 1985

[52] T Suda N Takahashi N Udagawa E Jimi M T Gillespieand T J Martin ldquoModulation of osteoclast differentiation andfunction by the new members of the tumor necrosis factorreceptor and ligand familiesrdquo Endocrine Reviews vol 20 no 3pp 345ndash357 1999

[53] T J Chambers ldquoRegulation of the differentiation and functionof osteoclastsrdquoThe Journal of Pathology vol 192 no 1 pp 4ndash132000

[54] T Wada T Nakashima N Hiroshi and J M PenningerldquoRANKL-RANK signaling in osteoclastogenesis and bone dis-easerdquo Trends in Molecular Medicine vol 12 no 1 pp 17ndash252006

[55] J Lam S Takeshita J E Barker O Kanagawa F P Ross andS L Teitelbaum ldquoTNF-120572 induces osteoclastogenesis by directstimulation of macrophages exposed to permissive levels ofRANK ligandrdquo Journal of Clinical Investigation vol 106 no 12pp 1481ndash1488 2000

[56] C T Ritchlin S A Haas-Smith P Li D G Hicks and EM Schwarz ldquoMechanisms of TNF-120572- and RANKL-mediatedosteoclastogenesis and bone resorption in psoriatic arthritisrdquoJournal of Clinical Investigation vol 111 no 6 pp 821ndash831 2003

[57] H Lin S WWang R Y Wang and P S Wang ldquoTumor necro-sis factor-120572 mediates RANK ligand stimulation of osteoclastdifferentiation by an autocrine mechanismrdquo Journal of CellularBiochemistry vol 83 no 1 pp 70ndash83 2001

[58] R Assuma TOates D Cochran S Amar andD TGraves ldquoIL-1 and TNF antagonists inhibit the inflammatory response andbone loss in experimental periodontitisrdquo Journal of Immunol-ogy vol 160 no 1 pp 403ndash409 1998

[59] D T Graves A J Delima R Assuma S Amar T Oates andD Cochran ldquoInterleukin-1 and tumor necrosis factor antago-nists inhibit the progression of inflammatory cell infiltrationtoward alveolar bone in experimental periodontitisrdquo Journal ofPeriodontology vol 69 no 12 pp 1419ndash1425 1998

[60] A J Delima T Oates R Assuma et al ldquoSoluble antagonists tointerleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) inhibitsloss of tissue attachment in experimental periodontitisrdquo Journalof Clinical Periodontology vol 28 no 3 pp 233ndash240 2001

[61] S Hashimoto T Yamamura and M Shimono ldquoMorphometricanalysis of the intercellular space and desmosomes of ratjunctional epitheliumrdquo Journal of periodontal research vol 21no 5 pp 510ndash520 1986

[62] N L Leong J M Hurng S I Djomehri S A Gansky M IRyder and S P Ho ldquoAge-related adaptation of bone-PDL-toothcomplex Rattus-Norvegicus as a model systemrdquo PloS One vol7 no 4 Article ID e35980 2012

[63] C M Belting I Schour J P Weinmann andM J Shepro ldquoAgechanges in the periodontal tissues of the rat molarrdquo Journal ofDental Research vol 32 no 3 pp 332ndash353 1953

[64] J C McLeod and E Balish ldquoEndotoxin in germfree gnoto-biotic or conventional-flora Sprague-Dawley ratsrdquo CanadianJournal of Microbiology vol 24 no 12 pp 1602ndash1606 1978

[65] R A Adams H A Zander and A M Polson ldquoCell popula-tions in the transseptal fiber region before during and afterexperimental periodontitis in squirrel monkeysrdquo Journal ofPeriodontology vol 50 no 1 pp 7ndash12 1979

[66] S Nyman H E Schroeder and J Lindhe ldquoSuppression ofinflammation and bone resorption by indomethacin duringexperimental periodontitis in dogsrdquo Journal of Periodontologyvol 50 no 9 pp 450ndash461 1979


[67] L Heijl B R Rifkin and H A Zander ldquoConversion of chronicgingivitis to periodontitis in squirrel monkeysrdquo Journal ofPeriodontology vol 47 no 12 pp 710ndash716 1976

[68] H E Schroeder and J Lindhe ldquoConversion of stable establishedgingivitis in the dog into destructive periodontitisrdquo Archives ofOral Biology vol 20 no 12 pp 775ndash782 1975

[69] C H Li and S Amar ldquoMorphometric histomorphometric andmicrocomputed tomographic analysis of periodontal inflam-matory lesions in a murine modelrdquo Journal of Periodontologyvol 78 no 6 pp 1120ndash1128 2007

[70] J D Lin H Ozcoban J Greene et al ldquoBiomechanics ofa bone-periodontal ligament-tooth fibrous jointrdquo Journal ofBiomechanics vol 46 no 3 pp 443ndash449 2012

[71] L Qian M Todo Y Morita Y Matsushita and K KoyanoldquoDeformation analysis of the periodontium considering theviscoelasticity of the periodontal ligamentrdquo Dental Materialsvol 25 no 10 pp 1285ndash1292 2009

[72] S P Ho M P Kurylo T K Fong et al ldquoThe biomechanicalcharacteristics of the bone-periodontal ligament-cementumcomplexrdquo Biomaterials vol 31 no 25 pp 6635ndash6646 2010

[73] J D Lin S Aloni V Altoe SMWebbM I Ryder and S P HoldquoElastic discontinuity due to ectopic calcification in a humanfibrous jointrdquo Acta Biomaterialia vol 9 no 1 pp 4787ndash47952012

[74] K Nozaki M Kaku Y Yamashita M Yamauchi and H MiuraldquoEffect of cyclic mechanical loading on osteoclast recruitmentin periodontal tissuerdquo Journal of Periodontal Research vol 45no 1 pp 8ndash15 2010

[75] C G Walker Y Ito S Dangaria X Luan and T G HDiekwisch ldquoRANKL osteopontin and osteoclast homeostasisin a hyperocclusion mouse modelrdquo European Journal of OralSciences vol 116 no 4 pp 312ndash318 2008

[76] Z Zhong X L Zeng J H Ni and X F Huang ldquoComparisonof the biological response of osteoblasts after tension andcompressionrdquo European Journal of Orthodontics vol 35 no 1pp 59ndash65 2011

[77] K Nakao T Goto K K Gunjigake T Konoo S Kobayashiand K Yamaguchi ldquoIntermittent force induces high RANKLexpression in human periodontal ligament cellsrdquo Journal ofDental Research vol 86 no 7 pp 623ndash628 2007

[78] Y Li W Zheng J S Liu et al ldquoExpression of osteoclastogenesisinducers in a tissue model of periodontal ligament undercompressionrdquo Journal of Dental Research vol 90 no 1 pp 115ndash120 2011

[79] B L Foster ldquoMethods for studying tooth root cementum bylight microscopyrdquo International Journal of Oral Science vol 4no 3 pp 119ndash128 2012

[80] M A Matias H Li W G Young and P M Bartold ldquoImmuno-histochemical localisation of extracellularmatrix proteins in theperiodontiumduring cementogenesis in the ratmolarrdquoArchivesof Oral Biology vol 48 no 10 pp 709ndash716 2003

[81] R L MacNeil and M J Somerman ldquoMolecular factors regu-lating development and regeneration of cementumrdquo Journal ofPeriodontal Research vol 28 no 6 pp 550ndash559 1993

[82] Y He E J Macarak J M Korostoff and P S Howard ldquoCom-pression and tension differential effects on matrix accumula-tion by periodontal ligaments fibroblasts in vitrordquo ConnectiveTissue Research vol 45 no 1 pp 28ndash39 2004

[83] P S Howard U Kucich R Taliwal and J M KorostoffldquoMechanical forces alter extracellular matrix synthesis byhuman periodontal ligament fibroblastsrdquo Journal of PeriodontalResearch vol 33 no 8 pp 500ndash508 1998

[84] R L Steward C M Cheng J D Ye R M Bellin and P RLeduc ldquoMechanical stretch and shear flow induced reorgani-zation and recruitment of fibronectin in fibroblastsrdquo ScientificReports vol 1 p 147 2011

[85] T Shibutani Y Murahashi E Tsukada Y Iwayama and J NM Heersche ldquoExperimentally induced periodontitis in beagledogs causes rapid increases in osteoclastic resorption of alveolarbonerdquo Journal of Periodontology vol 68 no 4 pp 385ndash391 1997

[86] A M Parfitt ldquoThe cellular basis of bone remodeling thequantum concept reexamined in light of recent advances in thecell biology of bonerdquo Calcified Tissue International vol 36 no1 pp S37ndashS45 1984

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


functional perspectives on diseased joints are importantareas of study since the altered homeostasis due to bacterialinvasion can lead to pathological adaptation of unaffectedtissues Hence the combinatorial effect of disease and func-tional (mechanical) loads on disease progression and jointadaptation is an important area of investigation Howeverthe challenge that lies in performing such studies is thelimited access to specimens from humans Hence animalmodels in general and rat models in particular continue tobe used extensively as an experimental system to elucidatethe effect of diverse factors on periodontal pathogenesis andprogression [9 10] Despite the extensive literature regardingthe characteristics of periodontal progression in differentanimal models few studies have described the effects of thedisease from a biomechanical perspective

Numerous human and animal studies have confirmed theinfectious etiology of gingival inflammation [11ndash13] apicalmigration of the gingival epithelial attachment and the even-tual loss of underlying bone and connective tissue support[14 15] that leads to clinical periodontitis In this study weemployed the lipopolysaccharide-soaked rat ligature modelto initiate the clinical features of periodontitis namelyinflammation and loss of PDL attachment and alveolar bone[16]Thismodel is an analog that stimulates the natural occur-rence of periodontitis in humansmdashfood impaction over timefollowed by acute and chronic inflammatory host responses[17ndash19] Once disease is initiated the complex is kept undercontinued function and the changes in clinical features canbe correlated with the distribution of biochemical markersIn this study the presence and distribution of the followingbiochemical markers in sections taken from hemimaxillaeharvested at 4 8 and 15 days of ligation and correspondingcontrols were assessed receptor activator of nuclear factor120581B ligand (RANKL a membrane-bound protein commonlyfound on PDL cells osteoblasts and T cells that inducethe expression of RANK on the surface of osteoclasts) [2021] tartrate-resistant acid phosphatase (TRAP an enzymehighly expressed by osteoclasts) [22] tumor necrosis factor-alpha (TNF-120572 a marker for systemic inflammation) [23]and fibronectin (FN a glycoprotein adhesion molecule forfibroblasts that promotes extracellular matrix production)[24] Moreover changes in biochemical expressions andlocalization were correlated to morphological changes of thecomplex Based on previous studies by others morphologicalalterations of bone and cementum due to inflammation wereassociated with increased expressions of proinflammatorymediators [25ndash28] including a variety of cytokines such asTNF-120572 that are involved in inflammatory bone resorption[29] RANKL which is known to orchestrate osteoclastogen-esis synergistically with TNF-120572 [30] wasmapped throughoutthe bone-PDL-cementum complex and was expected toincrease in inflamed periodontal tissue In addition activeosteoclasts and correlative changes in biochemical distribu-tion and alveolar bone resorption were identified Finallyadaptive changes in secondary cementum marked by FNexpression [31] were discussed within the context of jointfunction Our overall hypothesis was that coronal degrada-tion due to periodontitis can cause a significant change in thebiomechanics of the complex and that the resulting adaptive

effects may not be the same between bone and secondarycementum The objective was to identify the adaptation ofthis load-bearing joint that is the bone-PDL-tooth complexwith the onset of periodontitis by mapping and correlatingmorphological changes of the tooth and the alveolar socketto key biomolecular expressions within the complex

2 Materials and Methods

To minimize the effects of environmentally induced peri-odontitis [32ndash34] 6-week-old male Sprague-Dawley ratswere housed in a germ-free facility (Parnassus Services Build-ing) and fed a hard pelleted diet for the duration of the studyAll animals included in this study were housed in pathogen-free conditions in compliance with the guidelines of theInstitutional Animal Care and Use Committee (IACUC) ofUCSF and the National Institute of Health (NIH)

21 Induction of Periodontitis Using an In Vivo Rat LigatureModel 4ndash0 silk suture threads soaked in 1mg of lipopolysac-charide (LPS) from Escherichia coli serotype 055B5 (Sigma-Aldrich USA) per 1mL of 1x Tris buffer were used to induceperiodontitis (119873 = 5 per time point) Threads were placedbetween the first and second molars and the second andthird molars of both maxillae (Figure 1(a)) Molars werereligated every 2-3 days to ensure retention Control rats(119873 = 5 per time point) were flossed every 2-3 days with4ndash0 silk ligatures without LPS Rats were euthanized after4 8 and 15 days of ligation Maxillae were harvested andhemisected Right hemimaxillae were stored in 70 ethanolfor micro-XCT analysis Left hemimaxillae were fixed in 4paraformaldehyde (PFA) at room temperature overnight forhistology

22 Histological Analysis of Cytokine Expressions UsingImmunohistology Identification of Bone Resorption throughTRAP(+) Osteoclasts and Observation of Changes in Col-lagen Birefringence Following fixation intact hemimaxillae(119873 = 5) were decalcified in 05M ethylenediaminetetraaceticacid (EDTA) solution for 3 weeks The EDTA solutionwas changed every 3 days Specimens were then dehy-drated through 80 95 and 100 Flex Alcohol (Richard-Allan Scientific Kalamazoo MI USA) before embeddingin paraffin (Tissue Prep-II Fisher Scientific Fair Lawn NJUSA) Embedded specimens were sagittally sectioned on arotary microtome (Reichert-Jung Biocut Vienna Austria)using a disposable steel blade (TBF Inc ShurSharp FisherScientific Fair Lawn NJ USA) Paraffin serial sections weremounted on Superfrost Plus microscope slides (Fisher Scien-tific Fair LawnNJ) Sections were deparaffinizedwith xyleneand rehydrated through a descending ethanol series of 10095 and 80 ethanol before further use

221 Immunostaining for RANKL FN and TNF-alpha Theimmunofluorescence staining protocol used for RANKL andFN was based on a previously described protocol [35] Inbrief deparaffinized sections were digested with trypsin(Sigma-Aldrich St Louis MO USA) at 37∘C Following


1st

2nd

3rd

Maxillary molars

(a)

Interradicular

Coronal distal

Coronal mesial

Apical mesial

Apical distal

(b)


CEJ-ABC

Tooth

Alveolar bone

Lingual-sagittal

(c)


Coronal

Middle

Apical

PDL-space

Tooth

Alveolar bone

2000

120583m

(d)








2O2










3 Results












JE

ABC

(c)

lowast

0

10

20

30

Control Ligature

RAN

KL(+

) cel

l cou

nt



0

10

20

30

Control Ligature

lowast

RAN

KL(+

) cel

l cou

nt

(e)

4 d8 d15 d

SC

SC SC

AB

AB

ABV

V

EE

V

Ligated

Inte

rrad

icul

arC

oron

al

dista

lC

oron

alm

esia

lAp

ical

di

stal

Apic

al

mes

ial

Control

ABAB

PDL PDL

PDL

PDL

PDLPDL

PDL

PDL

PDLB

B

AB

AB

AB

D

D

PDL ABDD

AB

AB

SC

SC SC

SC

(a) (b)

lowast

lowast

lowast


300 120583m



CEJABC



(a)

0

5

10

15

20

25


OC distal

ABC

Cor

onal

Apic

al

0

5

10

15

20

25


OC

cell

coun

t

OC

cell

coun

t

OC mesial

(x) (xi)

AB

AB

AB

AB

AB

AB

AB

ABAB

D D D

DD

D

SCSC

SC

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

(i) (ii) (iii)

(iv) (v) (vi)

(vii) (viii) (ix)

lowast

lowast

lowast

200ums

100ums


4 d8 d15 d

4 d8 d15 d

(b)



Mesial Distal

500ums

(a)


Inte

rrad

icul

ar

AB AB

Cor

onal

AB ABAB

AB

PDL PDL PDL PDLD DD

D

Apic


PDL SCSC SC

SC

Sube

pith

elial

CT

100ums

(b)







120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1st

2nd

3rd

Maxillary molars

(a)

Interradicular

Coronal distal

Coronal mesial

Apical mesial

Apical distal

(b)


CEJ-ABC

Tooth

Alveolar bone

Lingual-sagittal

(c)


Coronal

Middle

Apical

PDL-space

Tooth

Alveolar bone

2000

120583m

(d)








2O2










3 Results












JE

ABC

(c)

lowast

0

10

20

30

Control Ligature

RAN

KL(+

) cel

l cou

nt



0

10

20

30

Control Ligature

lowast

RAN

KL(+

) cel

l cou

nt

(e)

4 d8 d15 d

SC

SC SC

AB

AB

ABV

V

EE

V

Ligated

Inte

rrad

icul

arC

oron

al

dista

lC

oron

alm

esia

lAp

ical

di

stal

Apic

al

mes

ial

Control

ABAB

PDL PDL

PDL

PDL

PDLPDL

PDL

PDL

PDLB

B

AB

AB

AB

D

D

PDL ABDD

AB

AB

SC

SC SC

SC

(a) (b)

lowast

lowast

lowast


300 120583m



CEJABC



(a)

0

5

10

15

20

25


OC distal

ABC

Cor

onal

Apic

al

0

5

10

15

20

25


OC

cell

coun

t

OC

cell

coun

t

OC mesial

(x) (xi)

AB

AB

AB

AB

AB

AB

AB

ABAB

D D D

DD

D

SCSC

SC

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

(i) (ii) (iii)

(iv) (v) (vi)

(vii) (viii) (ix)

lowast

lowast

lowast

200ums

100ums


4 d8 d15 d

4 d8 d15 d

(b)



Mesial Distal

500ums

(a)


Inte

rrad

icul

ar

AB AB

Cor

onal

AB ABAB

AB

PDL PDL PDL PDLD DD

D

Apic


PDL SCSC SC

SC

Sube

pith

elial

CT

100ums

(b)







120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2O2










3 Results












JE

ABC

(c)

lowast

0

10

20

30

Control Ligature

RAN

KL(+

) cel

l cou

nt



0

10

20

30

Control Ligature

lowast

RAN

KL(+

) cel

l cou

nt

(e)

4 d8 d15 d

SC

SC SC

AB

AB

ABV

V

EE

V

Ligated

Inte

rrad

icul

arC

oron

al

dista

lC

oron

alm

esia

lAp

ical

di

stal

Apic

al

mes

ial

Control

ABAB

PDL PDL

PDL

PDL

PDLPDL

PDL

PDL

PDLB

B

AB

AB

AB

D

D

PDL ABDD

AB

AB

SC

SC SC

SC

(a) (b)

lowast

lowast

lowast


300 120583m



CEJABC



(a)

0

5

10

15

20

25


OC distal

ABC

Cor

onal

Apic

al

0

5

10

15

20

25


OC

cell

coun

t

OC

cell

coun

t

OC mesial

(x) (xi)

AB

AB

AB

AB

AB

AB

AB

ABAB

D D D

DD

D

SCSC

SC

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

(i) (ii) (iii)

(iv) (v) (vi)

(vii) (viii) (ix)

lowast

lowast

lowast

200ums

100ums


4 d8 d15 d

4 d8 d15 d

(b)



Mesial Distal

500ums

(a)


Inte

rrad

icul

ar

AB AB

Cor

onal

AB ABAB

AB

PDL PDL PDL PDLD DD

D

Apic


PDL SCSC SC

SC

Sube

pith

elial

CT

100ums

(b)







120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results












JE

ABC

(c)

lowast

0

10

20

30

Control Ligature

RAN

KL(+

) cel

l cou

nt



0

10

20

30

Control Ligature

lowast

RAN

KL(+

) cel

l cou

nt

(e)

4 d8 d15 d

SC

SC SC

AB

AB

ABV

V

EE

V

Ligated

Inte

rrad

icul

arC

oron

al

dista

lC

oron

alm

esia

lAp

ical

di

stal

Apic

al

mes

ial

Control

ABAB

PDL PDL

PDL

PDL

PDLPDL

PDL

PDL

PDLB

B

AB

AB

AB

D

D

PDL ABDD

AB

AB

SC

SC SC

SC

(a) (b)

lowast

lowast

lowast


300 120583m



CEJABC



(a)

0

5

10

15

20

25


OC distal

ABC

Cor

onal

Apic

al

0

5

10

15

20

25


OC

cell

coun

t

OC

cell

coun

t

OC mesial

(x) (xi)

AB

AB

AB

AB

AB

AB

AB

ABAB

D D D

DD

D

SCSC

SC

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

(i) (ii) (iii)

(iv) (v) (vi)

(vii) (viii) (ix)

lowast

lowast

lowast

200ums

100ums


4 d8 d15 d

4 d8 d15 d

(b)



Mesial Distal

500ums

(a)


Inte

rrad

icul

ar

AB AB

Cor

onal

AB ABAB

AB

PDL PDL PDL PDLD DD

D

Apic


PDL SCSC SC

SC

Sube

pith

elial

CT

100ums

(b)







120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


JE

ABC

(c)

lowast

0

10

20

30

Control Ligature

RAN

KL(+

) cel

l cou

nt



0

10

20

30

Control Ligature

lowast

RAN

KL(+

) cel

l cou

nt

(e)

4 d8 d15 d

SC

SC SC

AB

AB

ABV

V

EE

V

Ligated

Inte

rrad

icul

arC

oron

al

dista

lC

oron

alm

esia

lAp

ical

di

stal

Apic

al

mes

ial

Control

ABAB

PDL PDL

PDL

PDL

PDLPDL

PDL

PDL

PDLB

B

AB

AB

AB

D

D

PDL ABDD

AB

AB

SC

SC SC

SC

(a) (b)

lowast

lowast

lowast


300 120583m



CEJABC



(a)

0

5

10

15

20

25


OC distal

ABC

Cor

onal

Apic

al

0

5

10

15

20

25


OC

cell

coun

t

OC

cell

coun

t

OC mesial

(x) (xi)

AB

AB

AB

AB

AB

AB

AB

ABAB

D D D

DD

D

SCSC

SC

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

(i) (ii) (iii)

(iv) (v) (vi)

(vii) (viii) (ix)

lowast

lowast

lowast

200ums

100ums


4 d8 d15 d

4 d8 d15 d

(b)



Mesial Distal

500ums

(a)


Inte

rrad

icul

ar

AB AB

Cor

onal

AB ABAB

AB

PDL PDL PDL PDLD DD

D

Apic


PDL SCSC SC

SC

Sube

pith

elial

CT

100ums

(b)







120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CEJABC



(a)

0

5

10

15

20

25


OC distal

ABC

Cor

onal

Apic

al

0

5

10

15

20

25


OC

cell

coun

t

OC

cell

coun

t

OC mesial

(x) (xi)

AB

AB

AB

AB

AB

AB

AB

ABAB

D D D

DD

D

SCSC

SC

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

PDL

(i) (ii) (iii)

(iv) (v) (vi)

(vii) (viii) (ix)

lowast

lowast

lowast

200ums

100ums


4 d8 d15 d

4 d8 d15 d

(b)



Mesial Distal

500ums

(a)


Inte

rrad

icul

ar

AB AB

Cor

onal

AB ABAB

AB

PDL PDL PDL PDLD DD

D

Apic


PDL SCSC SC

SC

Sube

pith

elial

CT

100ums

(b)







120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Mesial Distal

500ums

(a)


Inte

rrad

icul

ar

AB AB

Cor

onal

AB ABAB

AB

PDL PDL PDL PDLD DD

D

Apic


PDL SCSC SC

SC

Sube

pith

elial

CT

100ums

(b)







120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


120578

0

200

400

600

800

4 8 15Time (days)



amp

amp

Ψ yen

Ψyen

dagger

dagger

(i)

0

50

100

150

200

250

0 4 8 12 16

ControlLigated

lowastlowast

++

Time (days)

(ii)

0

50

100

150

200

250


A A BB C C

998771

(iii)

0

50

100

150

200

250


GD D E FE HF G H

4 days8 days15 days

4 days8 days15 days

(iv)

Dagger

CEJ-

AC (120583

m)

Dist

al P

DL-

spac

e (120583

m)

Mes

ial P

DL-

spac

e (120583

m)

Inte

rrad

icul

ar sp

ace (

120583m

)120581

(a)

0

20

40

60



Mesial apical

minus60

minus40

minus20



0 4 8 12 16

on 8 and 15 days

ΔAv

erag

e lig

ated

minusav

erag

e con

trol

(b)

(iv)

AB

PDL

TF

SCSC

(iii)

AB

PDL

SC

TF


(i)

AB

PDL

TF

(ii)

AB

PDL

SC

TF

SC300 120583m 300 120583m 300 120583m 300 120583m


(c)






4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4 Discussion








101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


101520253035

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10203040506070

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

10

15

20

25

0 50 100 150 200 250 300

FN in

tens

ity

Mesial Distal

(c3)

D

NBSC

PDL

AB

PDL SC DAB

PDL SC DAB

PDLSCD AB

101520253035

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

(a1)

AB D

SC

PDL

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL SC DAB

SC DAB PDL

(a2)

D

AB

SC PDL

PDL SC DAB

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDLSCD AB

1020304050

0 50 100 150 200 250 300

FN in

tens

ity

PDL

AB

SC

D

(c1)

(b1)

AB

PDLSC

D


(a3)

AB SC

PDL D

AB

PDL

SC

D

(a4)

AB

PDLSC

D

(b2)

(b4)

D AB

SC

PDL

(c2)

PDL

ABSC

D

(c4)

D

ABSC PDL

(b3)

DAB

SC

PDL

4d

cont

rol

4d

ligat

ed8

d co

ntro

l8

d lig

ated

15

d co

ntro

l15

d lig

ated






















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















5 Conclusions




Acknowledgments


References

































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article The Adaptive Nature of the Bone...

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Transcript of Research Article The Adaptive Nature of the Bone...