http://jdr.sagepub.com/Journal of Dental Research

http://jdr.sagepub.com/content/75/10/1761The online version of this article can be found at:

 DOI: 10.1177/00220345960750100901

1996 75: 1761J DENT RESK.A. Derringer, D.C. Jaggers and R.W.A. Linden

Angiogenesis in Human Dental Pulp Following Orthodontic Tooth Movement  

Published by:

http://www.sagepublications.com

On behalf of: 

International and American Associations for Dental Research

can be found at:Journal of Dental ResearchAdditional services and information for    

  http://jdr.sagepub.com/cgi/alertsEmail Alerts:

 

http://jdr.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

http://jdr.sagepub.com/content/75/10/1761.refs.htmlCitations:  

What is This? 

- Oct 1, 1996Version of Record >>

at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

J Dent Res 75(10): 1761-1766, October, 1996

Angiogenesis in Human Dental PulpFollowing Orthodontic Tooth MovementK.A. Derringerl*, D.C. Jaggers3, and R.W.A. Linden2' 3

Clinical Craniofacial Biology Research Unit, 'Departments of Orthodontics and 2Conservative Dentistry, King's College School of Medicineand Dentistry, Caldecot Road, London SE5 9RW; and 3Biomedical Sciences Division, King's College, Strand, London WC2R 2LS, UK; *towhom correspondence should be addressed

Abstract. The pulpal response to orthodontic force isthought to involve cell damage, inflammation, and woundhealing. These situations are likely to be associated with therelease of angiogenic growth factors. We thereforeinvestigated human dental pulps to determine if angiogenicchanges could be detected after orthodontic forceapplication. Fifteen premolar teeth were treated withstraight-wire fixed orthodontic appliances for two weeks,and comparisons were made with 15 untreated controlpremolar teeth from the same subjects. The teeth wereextracted and sectioned. The pulps were removed, dividedinto 1-mm sections, embedded in collagen, and cultured ingrowth media for up to four weeks. Cultures wereexamined daily, by light microscopy, for growth andnumber of microvessels. Apparent microvessels wereobserved within five days. Confirmation of microvesselidentification was by electron microscopy for endothelialcell morphology. There were significantly greater numbersof microvessels at day five and day ten of culture in thepulp explants from orthodontically treated teeth comparedwith those from the pulps of control teeth. These results areconsistent with the hypothesis that there is an increase inangiogenic growth factors in the pulp of orthodonticallymov,ed teeth.

Key words: angiogenesis, angiogenic growth factors, dentalpulp, orthodontic tooth movement.

Received January 11, 1996; Accepted May 22, 1996

Introduction

The angiogenic changes in human dental pulp associatedwith orthodontic tooth movement are unknown.Angiogenesis is the formation of new capillary structuresultimately leading to the organization of larger structuresby a process of neovascularization (Maciag, 1990). It isfound in the developing embryo, developing or growingtissue, wound healing and repair, and in inflammatory andpathological processes involving growth of blood vessels.However, it has not yet been investigated inorthodontically moved teeth. The stages invTolved inangiogenesis are vascular membrane breakdown,endothelial cell mitosis and migration to form a capillarysprout, folding of cells to form a vessel lumen, andcapillary loop formation. The initiation of angiogenesis is acomplex multistep process (Folkman and Klagsbrun, 1987).Growth factors and cytokines may either act directly tomodulate endothelial cell growth and differentiation orindirectly by diverting target helper cells (macrophages,monocytes, mast cells, pericytes, vascular smooth musclecells) to express growth factors and cytokines. Severalpolypeptide growth factors are involved; these may bestimulators or inhibitors of the system. Regulation isbrought about by a balance of these factors.

Lack of collateral circulation in the pulp makes it one ofthe most sensitive tissues in the body, yet the specific pulpalreactions to orthodontic force remain unclear. Evidencesuggests that orthodontic tooth movement may causereduced oxygen levels of the pulp (Hamersky et a!., 1980;Unsterseher et al., 1987), cell damage and circulatorydisturbances (Mostafa et al., 1991; Nixon et al., 1993), andinflammatory changes. These situations are analogous towound healing and ischemic/hypoxic conditions describedelsewhere in the body where angiogenic growth factorshave been identified (Knighton et al., 1983; Schultz andGrant, 1991; Millauer et al., 1994; Harik et al., 1995). The aimof this study was to determine if these angiogenic changestook place in the human dental pulp following orthodontictooth movement.

1761 at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

1762 Derringer et al.

Materials and methods

MaterialsAll materials were purchased from Sigma Chemical Co. (Poole,UK) except where otherwise stated in the text.

Human dental pulp culture assayAssays for angiogenesis can be used to detect either of the twostages of migration and proliferation of endothelial cells.Evidence indicates that the use of a three-dimensional assay isimportant (Williams, 1993). The three-dimensional proliferativeassay technique described by Nicosia and Ottinetti (1990) andreported by Jaggers and Milligan (1993) was used in this studyas a basis for investigating angiogenesis of the pulp followingorthodontic tooth movement. Sections of pulp were used inplace of the rat aorta described in the previous assay techniques.

The material consisted of 30 premolar teeth planned forextraction in orthodontic patients requiring fixed-appliancetherapy. Only patients who agreed to participate with informedparental consent were used, and ethical approval was given forthe study by the Research Ethics Committee at King's CollegeHospital. The teeth used were dentally healthy (free of caries,restorations, and pathology). Patients were aged in the range of11 to 14 years.

Orthodontic force (in the range of 0.5 N to 1 N) was appliedto two premolar teeth, while the contralateral premolar teethwere used as untreated controls. Fixed-appliance (Andrews0.022 in, Forestadent, Milton Keynes, UK) straight-wireorthodontic brackets were direct-bonded (Concise, DentalExpress, Kent, UK) to the upper and lower teeth from secondpremolar to second premolar except for the two controlpremolar teeth. Straight-wire orthodontic bands were cemented(Ketac, Baxter Dental, Watford, UK) onto all first permanentmolar teeth. Test tooth position was carefully assessed andbrackets bonded in a position so that archwires placed gave therequired force to the test teeth. After two weeks, test and controlpremolar teeth were extracted under local anesthesia. The teethwere immediately placed in Dulbecco's modified Eagle'smedium with HAM F12 (1:1 DMEM/HAM F12) andsubsequently sectioned vertically through the buccolingualaspect by means of a high-speed water-cooled diamond bur.The pulps were removed, placed in DMEM/HAM F12,sectioned horizontally into 1-mm sections, and embedded incollagen gel (rat tail type 1), supported by an agarose ring. Thesections were uniformly oriented on their sides so that, whenviewed from above, two cut surfaces were seen on the top andbottom edges of the section. The location of each 1-mm sectionwithin the tooth, from crown to root apex, for each tooth wasrecorded. Each gel was surrounded by serum-freeDMEM/HAM F12 medium supplemented with glutamine (2mM), penicillin (100 units/mL), streptomycin (100 pg/mL), andamphotericin B (2.5 pg/mL) and incubated at 37°C and 5% CO2in a humidified atmosphere. After 24 hours, the collagen gelswere released from the supporting agarose rings, transferred toindividual 35 mm x 10 mm culture dishes, and allowed to floatfreely in the serum-free medium. The medium was changedevery three days and the gels kept in the humidified incubatorfor up to four weeks.

Each individual section from all parts of each pulp wasexamined daily for angiogenic changes in the form ofmicrovessel growth. A record of the shapes of the individualsections facilitated orientation when changes were examined onsubsequent days. Growth of microvessels for each explant wasmeasured quantitatively and qualitatively. We carried out aquantitative examination by counting numbers of microvesselsusing direct observation under a brightfield phase invertedmicroscope. A count was recorded on day five and day ten ofculture, and explants were coded so that we would not knowtheir source at the time of counting. The three-dimensionalgrowth was most accurately counted by focusing the microscopeso that microvessels could be followed through planes of view.Vessels were counted radiating out from the two cut surfacesand two intact pulpal surfaces of each explant, while the upperand lower surfaces could not be accurately visualized and wereexcluded from the count. The area of collagen base support intowhich the vessels could grow was horizontally wide butvertically shallow, thereby encouraging vessel growth in thehorizontal plane, where it could be viewed readily and recorded.A qualitative examination was carried out by the use ofinstantaneous video prints for recording and assessing growthchanges. Prints were not used for counting due to the inaccuracyof the two-dimensional image (microscope attached to CCDvideo camera module connected to a video monitor [Panasonic]and videographic printer [Sony]).

Identification of microvesselsApparent microvessels were identified by light microscopy;explant cultures were examined by means of a brightfield phaseinverted microscope. Identification was confirmed by electronmicroscopic examination of a few sample explants. For this partof the study, explant gels were fixed in 4% formaldehyde, 5%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 48 hoursand post-fixed in 1% osmium tetroxide in 0.1 M phosphatebuffer, pH 7.4, for 2 hours, then dehydrated through gradedethanols and embedded in Araldite resin. Blocks were trimmedand sectioned at 2 iim on an ultramicrotome. Sections werestained with 1% toluidine blue in 1% Borax, mounted onAraldite, and examined. Ultrathin sections were cut byultramicrotome, stained with uranyl acetate and lead citrate,and examined by transmission electron microscopy.

Results

Quantitative examinationMicrovessels were observed in both treated and controlgroups within five days, with numbers reaching a peak ateight to ten days. Greater numbers of microvessels wereseen in the pulps of orthodontically moved teeth than in thepulps of control teeth in each individual patient.Comparison of the total numbers of microvessels inorthodontic and control groups by paired t tests showedsignificantly greater growth at day five (p < 0.001) and dayten (p < 0.005) in the pulps of orthodontically moved teethcompared with those of the control teeth (Fig. 1; Table).We compared microvessel numbers between different

j Dent Res 7500) 1996

at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

Angiogenesis in the Dental Pulp

-c

0

(I,

£2)

00

(U)2)

500

400

300

200

100

0DAY 5 DAY 10

Figure 1. Comparison of mean number and SEM of total pulpmicrovessel growths in the pulps of 15 orthodontically treated teethwith the pulps of 15 control teeth at day five and day ten of culture.

areas of the pulp by grouping the 1-mm horizontal sectionsaccording to origin into crown, middle, and root areas foreach pulp. There was significantly greater growth in theorthodontic group compared with the control group in allthree areas at day five (p < 0.01) but only in the crown androot areas at day ten (p < 0.01). These three areas of the pulpwere also compared within the orthodontically treated toothpulp group and within the control tooth pulp group. Whenintragroup microvessel numbers were examined,microvessel growth was observed in all areas of the pulp;howvever, there was individual variation in growth frompatient to patient. There was a trend to a higher count in theroot and crown sections of the pulp within bothorthodontically treated and control groups. Significantintragroup differences in the orthodontic group werereflected in a greater number of microvessels in the root areacompared with the middle area (p < 0.01 at day five and p <0.05 at day ten, by paired t tests) and in the crown areacompared with the middle area (p < 0.05 at day five and dayten). However, in the control group, the only difference thatbarely reached significance (p < 0.05) occurred between themicidle and root areas of the pulp at day five (Fig. 2; Table).

Qualitative examinationEach explant was examined daily by light microscope andanm changes recorded on video print. Each area of explantwas identified and recorded on sequential video prints,giv ing a record of growth, thickness, and morphologicalchanges of the microvessels and facilitating reorientation ofareas on subsequent days. Microvessel growth occurredfrom both the cut edges and intact surfaces of the pulpsections, with most growth tending to occur at the sides ofthe cut edges. Growth was evident in both treated andcontrol group pulps within a few days, microvessels becameobx\ious by five days, and their number and lengthincreased in most explants to a peak at eight to ten days. Thethickness of some microvessels continued to increase, whilethe numbers remained similar for up to 14 days. After 14dax s, microvessels started to degenerate, and a few thicker

wCA0

.-0

£-(to*v

0

C)

co0)

140

120

100

80

60

40

20

0

Root

Crown Root

Middle

ORTHO CONTROL

DAY 5

Ca

0

£0

£4

0.-

00

C

0

280260240220'200180'160140120100806040200

Root

ORTHO

RootCrown

Middle

CONTROL

DAY 10

Figure 2. Comparison of the mean number and SEM of microvesselgrowths in crown, middle, and root areas of the pulp in 15orthodontically treated teeth and in 15 control teeth at day five andday ten of culture.

vessels remained in some cases, mainly in theorthodontically treated group, for up to 21 days. By 28 days,degeneration had occurred in most of the explants.

Microvessels were identified by their tube-likeappearance under the brightfield phase inverted microscopeand were observed in fine branching networks radiating outfrom the explants (Fig. 3). Microvessels were distinguishedfrom fibroblasts on the basis of their morphological features,being thicker and of more uniform width along their length(Nicosia and Ottinetti, 1990). Electron microscopicexamination of the microvessels showed the presence ofpatent lumina (Fig. 4) and confirmed the capillary-vessel-like appearance of the outgrowths from the pulp explants.

DiscussionThe appearance of microvessel networks indicated anangiogenic response in the pulp explants. This response wassignificantly greater in the pulps from orthodontically

1 763I Doit Res 175(10) 1996

at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

1764 Derringer et al.

Table. Comparison of mean number and SEM of (a) total pulp microvessel growths and (b) microvesselgrowths in crown, middle, and root areas of the pulp in 15 orthodontically treated teeth and in 15 controlteeth at days five and ten of culture, by paired t tests

Day 5 Day 10Mean SEM t Mean SEM t

(a) Total pulpOrtho 249 ± 53 405 ± 93 **Control 119 ± 33 194 + 51

(b) Areas of pulpOrtho crown 100.5 ± 26.47 ** 132.6 + 35.5 **Control crown 48.0 ± 18.27 70.7 ± 23.1

Ortho middle 53.8 ± 13.93 68.1 ± 20.8Control middle 23.9 ± 6.21 ** 41.5 ± 11.7 NS

Ortho root 95.1 ± 18.81 205.2 ± 59.0Control root 48.0 ± 14.10 ** 82.5 ± 25.8 **

Ortho crownOrtho middle * *

Ortho crownOrtho root NS NS

Ortho middleOrtho root ** *

Control crownControl middle NS NS

Control crownControl root NS NS

Control middleControl root * NS

Significant at p < 0.001; ** significant at p < 0.01; * significant at p< 0.05; NS, not significant.

treated teeth than in those from control teeth, suggesting anincreased release of angiogenic growth factors associatedwith the application of orthodontic force. A 100% increase inthe total number of microvessel growths found in this studyin the pulps of teeth subjected to orthodontic force correlateswith a 230% increase in microvessels reported in the rataorta cultured in media conditioned with a powerful knownangiogenic agent, sarcoma 180 (Nicosia and Ottinetti, 1990).

Proliferation in the form of apparent microvessels wasobserved within five days of culture, with numbersincreasing to a peak around day ten and then reducing.These findings are similar to the microvessel growth curvesreported in the rat aorta, where a peak occurred at seven toeight days followed by degenerative changes (Nicosia andOttinetti, 1990). Microvessel networks produced by thepulps of orthodontically treated teeth in this studyappeared, by microscopic observation, to be similar to thosefound in the rat aorta (Nicosia and Ottinetti, 1990), althoughvessel size was smaller.

The anatomy of normal pulp vasculature and thedistribution of pulp capillaries in different areas of the pulp

have been reported in electron microscopy studies (Bishop,1987). In the present study, microvessel growth occurredfrom both cut and intact surfaces of the pulp sections and inall areas of the pulp from both orthodontic and controlgroups. Microvessel numbers were higher in the crown androot areas compared with the middle areas of the pulp inboth groups. This was most significant when the root andmiddle areas of the pulp were compared in the orthodonticgroup. Although there was individual patient variation inthe amount of microvessel growth in different areas of thepulp, total pulp microvessel numbers in all patients weresignificantly greater following orthodontic tooth movement.

Orthodontic forces are known to produce mechanicaldamage and inflammatory reactions in the periodontium(Rygh et al., 1986) and cell damage, inflammatory changes, andcirculatory disturbances in the pulp (Mostafa et al., 1991).These conditions are closely analogous to those elsewhere inthe body where angiogenic growth factors have been reported(Schultz and Grant, 1991). Repeated orthodontic force couldlead to cycles of trauma, ischemia, inflammation, and woundhealing and, therefore, to repeated stimulation of angiogenic

j Dent Res 75(10) 1996

at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

Angiogenesis in the Dental Pulp

Figure 3. Light micrographs of orthodontic pulp explant culture at ten days, showing microvessels radiating out from the explant at (a). Lowmagnification (bar = 0.1 mm); and (b) high magnification (bar = 0.01 mm).

factors. The origin of these factors is unclear. They may beprimarily blood-borne or synthesized locally by vascularendothelial cells. In wound healing, angiogenesis probablyoriginates in activated platelets and macrophages and may betriggered by changes in oxygen tension or factors releasedfrom cell lysis in ischemic tissues. (Platelet-derived growthfactor [PDGF], epidermal growth factor [EGF], transforminggrowth factor beta [TGF-41, and fibroblast growth factor [FGF]have been implicated [Schultz and Grant, 1991].) Theseangiogenic growth factors have also been identified in theperiodontal ligament in wound healing ([bFGF] Terranova etal., 1987, 1989; Tweden et al., 1989) and during orthodontictooth movement in cats ([TGF-P1] Davidovitch, 1995) and inthe pulp following endodontic injury ([TGF-P] Shirakawa etal., 1994) and during tooth development and eruption ([EGF]Klein et al., 1994). Vasoactive neuropeptides have beendetected proximal to pulpal blood vessel walls afterorthodontic force (Nicolay et al., 1990) and links with tumornecrosis factor alpha (TNF-o) production suggested(Haegerstrand et al., 1990; Trantor et al., 1995). It appears thatreactions of the pulp to orthodontic force involve changeswhich are likely to result in the production of angiogenicgrowth factors, a hypothesis supported by the results of thisstudy. These elaborate repair processes and powers ofneovascularization in the pulp and supporting tissue wouldenable the tooth to be moved extensive distances,withstanding heavy orthodontic forces applied, and couldaccount for the perhaps surprising fact that considerably moreiatrogenic clinical problems are not encountered during acourse of fixed-appliance orthodontic treatment.

It is difficult to define appropriate force levels for usewith fixed appliances to a great degree of accuracy in theclinical situation (Burstone and Koenig, 1974; Drenker, 1988)because of the variables of individual patient reaction, root-surface area, and frictional losses within the appliance. Inthis study, we reduced the first two variables by using thecontralateral teeth as controls within the patient. Lightforces in the range of 0.5 N to 1 N were used; however, forcemagnitude was difficult to standardize, since this studyattempted to maintain parity with the normal clinical

situation where teeth were bonded and a flexible archwireengaged into the bracket, giving a continuous but reducingactive force as tooth alignment occurred. This study,therefore, accurately reproduces the clinical effects of twoweeks of initial archwire forces, which inevitably would beof a jiggling and multidirectional nature and are of directclinical relevance.

In conclusion, the results of this study are, therefore,consistent with the hypothesis that there is an increase inangiogenic growth factors in the pulp of orthodontically movedteeth. Further research will determine the nature of thisangiogenic response and identify the principal factors involved.

AcknowledgmentsWe wish to thank Dr. M.A. Bishop of the Department ofAnatomy, Queen Mary and Westfield College, London, UK,for carrying out the electronmicroscopy part of this study.This study was financially supported in part by a grant fromthe Research Committee of the Biomedical SciencesDivision, King's College, University of London, UK.

Figure 4. Electron micrograph from orthodontic pulp explantshowing microvessel with patent lumen (bar = 2 pm).

I Dent Res 7500) 1996

at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

1766 Derringer et al.

ReferencesBishop MA (1987). An investigation of pulp capillaries and tight

junctions between odontoblasts in cats. Anat Embryol177:131-138.

Burstone CJ, Koenig HA (1974). Force systems from an idealarch. Am J Orthod 65:270-289.

Davidovitch Z (1995). Cell biology associated with orthodontictooth movement. In: The periodontal ligament in healthand disease. Berkovitz BKB, Moxham BJ, Newman HN,editors. London: Mosby, pp. 259-278.

Drenker E (1988). Calculating continuous archwire forces. AngleOrthod 58:59-70.

Folkman J, Klagsburn M (1987). Angiogenic factors. Science235:442-447.

Haegerstrand A, Dalsgaard CJ, Jonzon B, Larsson 0, Nilsson J(1990). Calcitonin gene-related peptide stimulatesproliferation of human endothelial cells. Proc Natl Acad SciUSA 87:3299-3303.

Hamersky PA, Weimer AD, Taintor JF (1980). The effect oforthodontic force application on the pulpal tissuerespiration rate in the human premolar. Am J Orthod77:368-378.

Harik SI, Hritz MA, LaManna JC (1995). Hypoxia-induced brainangiogenesis in the adult rat. J Physiol 485:525-530.

Jaggers D, Milligan SR (1993). Is oestradiol an angiostaticsteroid? (abstract). J Reprod Fert Abstr Series 11:17.

Klein RM, Chiego DJ, Sonneborn AA, Topham RT, Gattone VH(1994). Effects of growth factors on tooth eruption andrelated developmental processes. In: Biological mechanismsof tooth eruption and resorption and replacement byimplants. Davidovitch Z, editor. Boston: Harvard Societyfor the Advancement of Orthodontics, pp. 407-428.

Knighton DR, Hunt TK, Scheuenstuhl H, Halliday BJ, Werb Z,Banda MJ (1983). Oxygen tension regulates the expression ofangiogenesis factor by macrophages. Science 221:1283-1285.

Maciag T (1990). Molecular and cellular mechanisms ofangiogenesis. Important Adv Oncol 85-98.

Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A (1994).Glioblastoma growth inhibited in vivo by a dominant-negative FLK-1 mutant. Nature 367:576-579.

Mostafa YA, Iskander K, el-Mangoury NH (1991). Iatrogenicpulpal reactions to orthodontic extrusion. Am J Ortho

Dentofac Orthop 99:30-34.Nicolay OF, Davidovitch Z, Shanfeld JL, Alley K (1990).

Substance P immunoreactivity in periodontal tissuesduring orthodontic tooth movement. Bone Miner 11:19-29.

Nicosia RF, Ottinetti A (1990). Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay ofangiogenesis in vitro. Lab Invest 63:115-122.

Nixon CE, Saviano JA, King GJ, Keeling SD (1993).Histomorphometric study of dental pulp duringorthodontic tooth movement. J Endodont 19:13-16.

Rygh P, Bowling K, Hovlandsdal L, Williams S (1986).Activation of the vascular system: a main mediator ofperiodontal fiber remodeling in orthodontic toothmovement. Am J Orthod 89:453-468.

Schultz GS, Grant MB (1991). Neovascular growth factors. Eye5:170-180.

Shirakawa M, Shiba H, Nakanishi K, Ogawa T, Okamoto H,Nakashima K, et al. (1994). Transforming growth factorbeta-1 reduces alkaline phosphatase mRNA activity andstimulates cell proliferation in cultures of human pulpcells. J Dent Res 73:1509-1514.

Terranova VP, Hic S, Franzetti LC, Lyall R, Wikesjo UM (1987).A biochemical approach to periodontal regeneration.AFSCM: assays for specific cell migration. J Periodontol58:247-257.

Terranova VP, Odziemiec C, Tweden KS, Spadone DP (1989).Repopulation of dentin surfaces by periodontal ligamentcells and endothelial cells. Effect of basic fibroblast growthfactor. J Periodontol 60:293-301.

Trantor IR, Messer HH, Birner R (1995). The effects ofneuropeptides (calcitonin gene-related peptide andsubstance P) on cultured human pulp cells. J Dent Res74:1066-1071.

Tweden KS, Spadone DP, Terranova VP (1989).Neovascularization of surface demineralized dentin. JPeriodontol 60:460-466.

Unsterseher RE, Weimer AD, Nieberg LG, Dyer JK (1987). Theresponse of human pulpal tissue after orthodontic forceapplication. Am J Orthod Dentofac Orthop 92:220-224.

Williams S (1993). Angiogenesis in three-dimensional cultures(editorial; comment). Lab Invest 69:491-493. Comment on:Lab Invest 69:508-517, 1993.

j Dent Res 7500) 1996

at UCSF LIBRARY & CKM on December 8, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from