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Effect of duration of force application on blood vessels in young and adult rats

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Effect of duration of force application on blood vessels in young and adult rats
  SHORT COMMUNICATION Effect of duration of force application onblood vessels in young and adult rats  Yijin Ren, a Jaap C. Maltha, b Ietse Stokroos, c Robert S. B. Liem, d and Anne Marie Kuijpers-Jagtman e Groningen and Nijmegen, the Netherlands Introduction:  Age effects on orthodontically induced periodontal vascular reactions have not been studied.The aim of the present study was to test the hypothesis that prolonged tooth movement induces age-relatedincreases in periodontal vascularity. Methods:  A standardized orthodontic appliance was placed in 2 groupsof 30 rats aged 6 weeks and 9 to12 months. At 1, 2, 4, 8, and 12 weeks, animals were killed. Blood vessels(BV) were identified based on their morphology and by immunohistochemical staining for alpha-smoothmuscle actin. At each study region, surface areas (SA) of the periodontal ligament space and each BV weremeasured; BV mean SA, BV relative SA (the summed BV SA as a percentage of the periodontal ligament SA),and BV numbers were calculated. Results: Pressure and tension regions showed similar vascular changes.Young rats had lower BV relative SA and BV mean SA in the early phase of force application (   4 weeks); thisincreased in the late phase, reaching the same level as adult rats. In the late phase (4-12 weeks), young ratshad increases of both small- and large-sized BV that did not affect the BV mean SA; adult rats had anincrease of small-sized BV only; this resulted in decreased BV mean SA. Conclusions: The hypothesis wasconfirmed that prolonged tooth movement increases periodontal vascularity, which is age related. Theseresults suggest that clinicians should consider age-related difference in tissue reactions during orthodontictooth movement. (Am J Orthod Dentofacial Orthop 2008;133:752-7) T he blood vessels (BV) in the periodontalligament (PDL) are involved in the regulationof tissue remodeling during orthodontic inter-vention. In areas of tension, PDL vascularity in-creases; subsequently, osteoclast precursors migratefrom the PDL capillaries and produce various sig-naling molecules involved in force-induced tissueremodeling. 1-3  In areas of compression, the PDLshows leakage of blood constituents into the ex-travascular space, gradual obliteration of the BV, andbreakdown of the walls of veins. 4-6  Regarding thetiming of vascular reactions, packing of erythrocytesin dilated blood vessels occurred within 30 minutes,fragmentation of erythrocytes after 2 to 3 hours, anddisintegration of BV walls and extravasation of theircontents after 1 to 7 days. 1  Studies longer than 1week showed increased vascular activity, 7  sproutingof microvessels, 8,9  and increased BV density 10,11  atboth regions. Observations on vascular changes dur-ing tooth movement longer than 2 to 3 weeks arelacking. The PDL BV showed dynamic changes inresponse to force regimen 11 : the vascular density atboth tension and pressure areas increased after forceapplication, decreased on force removal, and in-creased again during distal movement of the molars(distal drift is a normal physiologic phenomenon inrats). 9  This responsiveness of the vasculature tochanges in tissue strain caused us to look intovascular reactions during prolonged force adminis-tration after initial adaptation of the periodontalmicroenvironment. Moreover, our previous studiesshowed an age-related pattern in both the rate of tooth movement and osteoclast recruitment overtime. 12,13  However, age-related vasculature reactionshave not been quantified.Therefore, the aim of this study was two-fold: (1) toevaluate changes in vasculature with short- and long-term orthodontic force application, and (2) to analyzethe change in the nature of blood vessels in young andadult rats with orthodontic force application. Becausethe recruitment of osteoclasts depends on vasculature,and prolonged tooth movement requires continuous a Professor, Department of Orthodontics, University Medical Centre Groningen,University of Groningen, Groningen, the Netherlands. b Associate professor, Department of Orthodontics and Oral Biology, RadboudUniversity Nijmegen Medical Centre, Nijmegen, the Netherlands. c Research associate, Department of Cell Biology, Section Electronmicroscopy,University Medical Centre Groningen, University of Groningen, Groningen,the Netherlands. d Senior researcher, Department of Cell Biology, Section Electronmicroscopy,University Medical Centre Groningen, University of Groningen, Groningen,the Netherlands. e Professor and chair, Department of Orthodontics and Oral Biology, RadboudUniversity Nijmegen Medical Centre, Nijmegen, the Netherlands.Reprint requests to: Yijin Ren, Department of Orthodontics, University MedicalCentreGroningen,UniversityofGroningen,TriadegebouwIngang24,Hanzeplein1, 9700 RB Groningen, the Netherlands; e-mail, Submitted, August 2007; revised and accepted, October 2007.0889-5406/$34.00Copyright © 2008 by the American Association of Orthodontists.doi:10.1016/j.ajodo.2007.10.030 752  recruitment of osteoclasts, 14  we hypothesized that pro-longed force application induces long-lasting increasedperiodontal vascularity that is age related. MATERIALANDMETHODS Two groups of 30 male Wistar rats aged 6 weeks(150-250 g) and 9 to 12 months (400-550 g) were used.The animals were acclimatized for 2 weeks before theexperiment. They were housed under normal laboratoryconditions and fed powdered rat chow (Sniff, Soest,The Netherlands) and water ad libitum. Ethical permis-sion was obtained from Radboud University NijmegenMedical Centre, The Netherlands.A split-mouth design was used with the experimen- Fig.  Alightmicrographindicatingthestudyvariables.ThePDLspacewasdividedbyalinethroughthelongaxisoftherootinto2parts:tensionandpressureareasdependingonthedirectionoftoothmovement.(Atexperimentalsides,teethweremovedmesially;thusthemesialaspectwasthepressureregion,andthedistalaspectwasthetensionregion.Atthecontrolsides,teethunderwentphysiologicdistaldrift;therefore,thedistalaspectwasthepressureregion,andthemesialaspectwasthetensionregion.)TheupperborderofthePDLspacewasdefinedasthecementodentinjunctionlevel.Ateachregion,3variableswerecalculated:BVrelativeSAisthesummedSAofallBVsinpercentagesofthePDLSA;BVmeanSAisthemeanSAvalueofallBVs;BVnumberisthecountofallBVs,small-sizedBV(SA    500   m 2  )andlarge-sizedBV(SA    500   m 2  ). TableI . BV relative SA during tooth movement (medians and ranges)  BV relative SA (%)Control Experimental Early phase Late phase Early phase Late phasePressure Tension Pressure Tension Pressure Tension Pressure Tension Young 6 † (4-13) 6 † (3-9) 8 † (2-12) 11 † (2-18) 5 † * (1-9) 5 † * (2-11) 10 ‡ (5-24) 8 (5-10)Adult 12 (9-20) 11 (9-16) 14 (8-23) 13 (6-21) 11 (8-22) 11 (6-20) 8 ‡ (4-22) 11 ‡ (6-16)*Between early and late phases; † between young and adult rats; ‡ between experimental and control sides ( P  0.05).  American Journal of Orthodontics and Dentofacial OrthopedicsVolume  133,  Number   5  Ren et al 753  tal side randomly chosen and the contralateral side asthe control. An orthodontic appliance was placed onlyon the experimental side after general anesthesia withan intraperitoneal injection containing fentanyl citrate(0.079 mg per milliliter), fluanisone (2.5 mg per milli-liter), and midazolam (2.5 mg per milliliter) (Janssen,Beerse, Belgium) in a dosage of 2.7 mL per kilogram of body weight. The appliance was described extensivelyelsewhere. 15  Briefly, a transverse hole was drilledthrough the alveolar bone and the maxillary incisors atthe midroot level. A preformed ligature wire enclosingall 3 molars was bonded on the experimental side. Acoil spring was attached to a ligature wire through thehole to move the molars mesially with a force of 10  2cN. 12  At 1, 2, 4, and 8 weeks, 5 or 6 rats from eachgroup were killed, and, at 12 weeks, the remaininganimals were killed.The rats received an overdose of anesthetic beforethey were killed. They were then perfused with 4%formaldehyde solution at 37°C. The maxillae weredissected and immersed in the same fixative for 24hours at 4°C. After decalcification in 10% EDTA andparaffin embedding, serial parasagittal 7-  m sectionsincluding all 3 molars were cut. Every 25th section wascollected on slides and stained with hematoxylin andeosin.BVs were identified by their morphology and byimmunohistochemical staining for alpha-smooth mus-cle actin. Before staining, the sections were deparaffi-nated and rehydrated. Sections were preincubated with0.1% trypsine in Tris/HCL buffer for 10 minutes at37°C for antigen retrieval. They were pretreated with3% hydrogen peroxide followed by incubation with 5%PBSA. Subsequently, the sections were first incubatedwith mouse  -SMA monoclonal antibody (R&D, Min-neapolis, Minn) overnight and then with biotin-SP-conjugated affinipure donkey anti-mouse lgG (Jackson,Westgrove, Pa) for 45 minutes. The sections were thentreated with ABC-peroxidase (Vector, Burlingame, Pa).The staining was enhanced by incubating the sampleswith 0.5% copper sulphate in a 0.9% sodium chloridesolution. Immunohistochemical controls included re-placement of the primary antibody with PBS.Three roots per section and 3 sections near thelargest longitudinal root surfaces were selected torepresent each animal. The sections were digitized, andthe borders of the BV and the PDL were highlightedand measured (Quantimet 520, Cambridge, England).Twenty sections were randomly chosen from both agegroups. The same observer (Y.R.) made the measure-ments twice on these 20 sections with a 3-week interval. Intraobserver agreement was tested by kappastatistics (    0.91). The upper border of the PDLspace was defined as the cementodentin junction. Ateach region, surface areas (SA) of the PDL space andeach BV were measured, and BV mean SA (the averageSA of all BV), BV relative SA (the summed BV SA asa percentage of the PDL SA), and BV numbers (thecounts of all BV; small-sized BV: SA  500  m 2 , andlarge-sized BV: SA  500  m 2 ) were calculated (Fig). Statisticalanalysis For each region, the mean of the 9 measurements(3 roots    3 sections) of each variable was calcu-lated, representing 1 animal. Medians were calcu-lated for each region with the number of animals asunits. Comparisons across time were done with theKruskal-Wallis nonparametric (ANOVA) test. Be-cause there were no differences in the early (weeks 1and 2) and the late (weeks 4, 8, and 12) groups, toothmovement was divided into early (  4 weeks) andlate phases (4-12 weeks), 12  and Mann-Whitney testswere used to compare early and late phases, youngand adult groups, and experimental and controlssides. Differences were considered significant if   P  0.05. RESULTS For all 3 variables, the pressure and tension regionsshowed similar vascular changes with time in both agegroups. The changes described below refer to bothregions unless specified. TableII . BV mean SA during tooth movement (medians and ranges)  BV mean SA (  m 2 )Control Early phase Late phasePressure Tension Pressure Tension Young 451 † (341-1096) 670 † (314-1500) 483 † (195-1303) 926 † (294-1914)Adult 459 (231-724) 514 (207-1198) 672 (306-1494) 568 (297-1035)  American Journal of Orthodontics and Dentofacial Orthopedics May 2008 754  Ren et al  In the BV relative SA (Table I), no time-related differences existed in the control samples of eithergroup. In young rats, the BV relative SA was lowerthan in adults in both early and late phases ( P  0.05).At the experimental sides, it increased from early to latephases ( P   0.05) only in the young rats; a differencebetween the 2 age groups existed only in the earlyphase ( P  0.05). Differences between the experimentaland control sides existed only in late phase at pressureregions in young rats and at both regions in adult rats( P  0.05).In the BV mean SA (Table II), at the control sides, no difference existed between the 2 phases in either agegroup. The BV mean SA was lower in youngsters than inadults in both phases ( P   0.05). At the experimentalsides, the BV mean SA in the early phase was higher inadults than in youngsters ( P  0.05), and it decreased inthe late phase ( P  0.05) only in the adults. A significantdifference between experimental and control sides existedin the late phase only in the adult group ( P  0.01).Neither tooth movement-related nor age-relatedchanges of the BV numbers existed at control sides(Table III). At the experimental sides, the total number increased from early to late phases at the pressureregion in both groups ( P   0.05). The BV numbers inthe late phase were higher than those at the controlsides ( P   0.05). The numbers of small-sized BVshowed the same changes as the overall BV numbers.The numbers of large-sized BV increased from early tolate phases only in young rats ( P   0.05). In the earlyphase, it was lower in youngsters than in adults( P   0.05), and in late phase it was higher than thecontrol side only at the pressure region in young rats( P  0.05). DISCUSSION Orthodontically induced periodontal vascular re-actions, previously reported, focused mainly on theearly phase. 6,7,15  Moreover, age effects on vascularchanges during tooth movement have never beeninvestigated. Our study provides a quantitative rep-resentation of the PDL vascular reactions in rats andconfirms the hypothesis that prolonged force appli-cation in rats resulted in increased vascularity, whichis age related. We acknowledge that the variables inour study are affected by the orientations of peri-odontal vasculature and histologic sections. How-ever, this is a point for discussion at all regions, andit might be considered random error equally distrib-uted in all samples.At the physiologic state, the general vascular rela-tive SA and average BV size were larger in adult rats, TableII . Continued  Experimental Early phase Late phasePressure Tension Pressure Tension 1070 † (575-1210) 993 † (709-1584) 1186 (402-1676) 1361 (753-2294)868* (806-2135) 841* (677-1240) 646 ‡ (311-1898) 604 ‡ (375-944)*Between early and late phases; † between young and adult rats; ‡ between experimental and control sides ( P  0.05). TableIII . BV numbers during tooth movement (medians and ranges)  BV (n)Control Experimental Early phase Late phase Early phase Late phasePressure Tension Pressure Tension Pressure Tension Pressure Tension YoungAll 16 (13-21) 13 (8-20) 17 (8-33) 14 (10-29) 12* (5-30) 16 (7-36) 24 ‡ (7-43) 21 ‡ (13-40)Small 11 (8-17) 7 (3-15) 12 (5-19) 9 (4-21) 9* (5-26) 13 (4-27) 16 ‡ (9-36) 15 ‡ (9-41)Large 4 (2-7) 4 (1-8) 4 (3-9) 6 (3-8) 4* † (2-6) 4* † (1-8) 7 ‡ (2-10) 6 (3-11)AdultAll 15 (11-22) 13 (9-16) 14 (18-21) 12 (7-16) 19* (12-37) 18 (7-26) 23 ‡ (11-57) 21 ‡ (16-49)Small 9 (7-15) 8 (5-15) 8 (5-18) 6 (3-11) 12* (4-18) 12 (6-31) 17 ‡ (10-38) 19 ‡ (16-46)Large 6 (4-9) 6 (5-10) 6 (3-10) 5 (3-8) 5 (3-8) 7 (3-9) 7 (4-11) 6 (3-12)  All,  Overall BV numbers;  small,  small-sized BV;  large,  large-sized BV.*Between early and late phases; † between young and adult rats; ‡ between experimental and control sides ( P  0.05).  American Journal of Orthodontics and Dentofacial OrthopedicsVolume  133,  Number   5  Ren et al 755  but the BV numbers did not differ with age. Prolongedforce application induced a significant increase of theBV relative SA only in youngsters, reaching the samelevel as in adults, and induced a significant decrease of the BV mean SA only in adult rats. The differencescould be explained by the BV numbers, which showedapparent age-related and size-related alterations:youngsters had increases in both small and large BV,and adults had an increase only in small BV. Therefore,in youngsters, the BV relative SA increased withoutaffecting the mean SA. In adult rats, it seems contra-dictory that increased BV did not increase its relativeSA; it was even decreased compared with the controls.This might be explained by increased BV permeabil-ity. 12  The BV of the rat PDL is characterized by manyfenestrations. 16  Mechanical loading induces increasednumbers and sizes of these fenestrae, and BV respondwith increased permeability, which enhances extrava-sation of fluid into the interstitial tissue. 2,17,18  Thisleakage might have reduced the relative BV SA in adultrats.Previous studies showed that, at the start of forceapplication, the periodontal vasculature at the pressureand tension regions reacted differently, 7,19  and, in thefirst weeks, the trend of vascular changes was similarunder pressure and tension. 9,10,20  This study demon-strated that this similarity exists also in the late phase.Prolonged force altered PDL vascularity for both tissuedeposition and resorption; this gives rise to an interest-ing question: why did bone resorption occur andcontinue at 1 side and bone formation at the other?Mechanically, the first reaction to orthodontic forceapplication is alteration in the strain-stress distributionin the periodontium; this triggers the reaction of thePDL cells. 21,22  Strain is postulated as the major biome-chanical factor influencing cell behavior. 3  It is plausiblethat vascular reactions at the onset of force applicationalter local stress and strain in the PDL. This triggersdifferential activations of osteoblasts and osteoclasts toinitiate bone resorption and apposition, respectively.Once tooth movement starts, vasculature remodeling isnot dictated by stress or strain in the PDL. CONCLUSIONS Prolonged tooth movement induces increases inperiodontal vascularity that is age related. These resultssuggest that clinicians should consider the age-relateddifferences in tissue reactions during orthodontic toothmovement. These results might also suggest that thevasculature changes in the periodontium can influencethe time-related biomechanical properties of the PDL.This should be taken into account in future finiteelement modeling studies. REFERENCES 1. Rygh P. Ultrastructural vascular changes in pressure zones of ratmolar periodontium incident to orthodontic tooth movement.Scand J Dent Res 1972;80:307-21.2. Lew KK. Orthodontically induced microvascular injuries in thetension zone of the periodontal ligament. J Nihon Univ Sch Dent1989;31:493-501.3. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-levelreactions to orthodontic force. Am J Orthod Dentofacial Orthop2006;129:469.e1-32.4. Kvam E. Cellular dynamics on the pressure side of the ratperiodontium following experimental tooth movement. Scand JDent Res 1972;80:369-83.5. Iida J, Inage S, Kurihara S, Miura F. Changes in the microvas-culature after mechanical pressure on the hamster cheek pouch.J Dent Res 1992;71:1304-9.6. Vandevska-Radunovic V, Kristiansen AB, Heyeraas KJ, Kvinns-land S. Changes in blood circulation in teeth and supportingtissues incident to experimental tooth movement. Eur J Orthod1994;16:361-9.7. Rygh P, Bowling K, Hovlandsdal L, Williams S. Activation of the vascular system: a main mediator of periodontal fiberremodeling in orthodontic tooth movement. Am J Orthod 1986;89:453-68.8. Parlange LM, Sims MR. A T.E.M. stereological analysis of blood vessels and nerves in marmoset periodontal ligamentfollowing endodontics and magnetic incisor extrusion. EurJ Orthod 1993;15:33-44.9. Murrell EF, Yen EH, Johnson RB. Vascular changes in theperiodontal ligament after removal of orthodontic forces. Am JOrthod Dentofacial Orthop 1996;110:280-6.10. Khouw FE, Goldhaber P. Changes in vasculature of the peri-odontium associated with tooth movement in the rhesus monkeyand dog. Arch Oral Biol 1970;15:1125-32.11. Vandevska-Radunovic V, Kvinnsland S, Kvinnsland IH. Effectof experimental tooth movement on nerve fibres immunoreactiveto calcitonin gene-related peptide, protein gene product 9.5, andblood vessel density and distribution in rats. Eur J Orthod1997;19:517-29.12. Ren Y, Maltha JC, Van ’t Hof MA, Kuijpers-Jagtman AM. Ageeffect on orthodontic tooth movement in rats. J Dent Res 2003;82:38-42.13. Ren Y, Kuijpers-Jagtman AM, Maltha JC. Immunohistochemicalevaluation of osteoclast recruitment during experimental toothmovement in young and adult rats. Arch Oral Biol 2005;50:1032-9.14. Nardelli B, Zaritskaya L, McAuliffe W, Ni Y, Lincoln C, ChoYH, et al. Osteostat/tumor necrosis factor superfamily 18 inhibitsosteoclastogenesis and is selectively expressed by vascularendothelial cells. Endocrinology 2006;147:70-8.15. Reitan K. The initial tissue reaction incident to orthodontic toothmovement as related to the influence of function. Acta OdontolScand 1951;9:1-240.16. Moxham BJ, Shore RC, Berkovitz BK. Fenestrated capillaries inthe connective tissues of the periodontal ligament. MicrovascRes 1985;30:116-24.17. Tang MP, Sims MR, Sampson WJ, Dreyer CW. Evidence forendothelial junctions acting as a fluid flux pathway in tensionedperiodontal ligament. Arch Oral Biol 1993;38:273-6.18. Cooper SM, Sims MR. Evidence of acute inflammation in theperiodontal ligament subsequent to orthodontic tooth movementin rats. Aust Orthod J 1989;11:107-9.  American Journal of Orthodontics and Dentofacial Orthopedics May 2008 756  Ren et al
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