Internattonal Endodmtic Journal {199\) 24,317-325
The effect of citric acid treatment on periodontal healing after replantation of permanent teeth p. ZERVAS, T. LAMBRIANIDIS*&I. KARABOUTA-VULGAROPOULOUt * Department ofDental Pathology and Therapeutics, Urtiversity o/Thessaloniki, and 1^Department of Oral and Maxillofaoal Surgery, University of Tkessaiomki, Thessaloniki, Greece Summary. The aim of this study was to evaluate the effect of surface demineralization on the healing of 120 replanted teeth in fifteen S- to 16-month-old dogs. Replanted teeth were divided into two groups (A and B). In group A, teeth were replanted following planing of the entire root surface. In group B, teeth were replanted following planing and surface demineraiization with citric add. Replanted teeth were splinted for 7 days, and observation periods were 3,14 and 56 days. A total of 95 teeth and their surrounding tissues were examined histologically under a light microscope. Histological examitiation of specimens 3 days after replantation revealed that initial reunion between the root and the alveolar periodontal ligament fibres was via a fibrin-like network in both groups. Fourteen days after replantation histological observations showed superior healing in group B with regard to epithelial condition, periodontal ligament fibre reatuchment to the root surface, resorption and functional arrangement of the periodonul ligament fibres. By 56 days after replantation, healing was superior in group A; group B was characterized by severe inflammatory and replacement resorption. These results indicated that surface detnineralization had in initial positive effect, but later led to impaired healing.
Introduction The most important factor influencing the prognosis of replanted teeth is the status of the periodontal ligament (Andreasen I98fla, Andreasen & Kristerson 1981, Lindskog et al. 1983), Several authors have attempted to find the most appropriate treatment for avulsed teeth which had a destroyed or necrotic periodonul ligament. Their suggestions could be summarized as follows. (i) Attempts have been made to substitute the periodontal ligament veith bioCorrespondence: Dr Panos Zervas, 6 Maroneias St, 543 51 Thessaloitiki, Greece,
compatible materials (Huebsch 1967, Hardy et al. 1981), proper autografts (Keller et al. Vill, Andreasen 1981), or following culture (Soder et al. 1979). Regeneration of the periodontal ligament in culture media prior to replantation (Reinholdt et al. 1977, Andreasen et at. 1978), as well as in-vive heterotrophic transplantation in soft tissues before replantation (Andreasen 1980b, Birman & de Araujo 1975) have bran tested, (ii) The treatment of the root surfaces with chemical substances to facilitate reattachmenr of collagen fibres. Acids (Nordenram et at. 1973), enzymes (Nevins et al. 1980) and fluoride solutions (Coccia 1980) have been suggested. Lindskog et al. (1985) suggested a sodium hypochlorite solution for removing the necrotic periodontal ligament remnants from the root surface. An interesting and controversial subject concerns the effects of citric acid on the healing of periodontal tissues. Because several experimental studies have shown that citric acid has a positive effect on the reatuchment of periodontal fibres (Register 1973, Register & Burdick 1975, Crigger et al. 1978, Nilveus & Egelberg 19B0, Nilveus ef al. 1980, Ririe et al. 1980, Selvig et al. 1981, Nalbandian & Cote 1982, Poison & Proye 1982, 1983, Proye & Poison 1982, Hanes et al. 1985, Caton et al. 1986, Poison et al. 1986, Poison & Hanes 1987), we decided to investigate the hypothesis that surface demineralization could be a valid procedure in cases of avulsed teeth with destroyed periodontal ligament. The aim of this study was to evaluate the healing 317
p. Zervas, T, Lamhrianidis (S I, Karahouta-Vulgaropoulou
Materials and methods Fifteen healthy dogs, aged 8-16 months, were used in this study. A total of 120 single-rooted lateral incisors and first premolars of both jaws were extracted and replanted. Clinical and radiographic examination ensured that prior to the experimental procedures all teeth were intact, caries free, completely formed and with no signs of chronic gingival inflammation. The citric acid solution used was always freshly prepared by adding hydrated crystalline citric acid' to distilled water under continuous mixing at room temperature, until the solution was almost saturated. The pH was tested with chromatographic paper. Replantations were performed under general anaesthesia. The animals were anaesthetized with Pentotha! (20-25 mg kg"' body weight) and intubated with a cuffed endotracheal tube, receiving a mixture of halothane and oxygen. Clinical crowns of the teeth and their gingivae were disinfected by application of a 2 per cent chlorexidine solution. After an initial radiograph, extraction was performed with Bein elevators and forceps. The entire root surface was planed with a periodontal curette. Preiiminarj tests showed that 10-12 strokes for each part of the root surface with a Gracey curette 13/14" resulted in removal of the periodontal ligament from the entire root surface, and cementum from the coronal and mesial third of the root. A layer of cementum remained around the apical third, but no additional planing was performed in this area, in order to avoid major modifications in root morphology and dimensions. During the root planing procedures, the tooth was held between the beaks of the extraction forceps. Teeth in group A were replanted after rinsing with normal saline. The planed root surfaces of teeth in group B were demineralized for 3 minutes in citric acid on a saturated piece of cotton wool folded around the root and then rinsed with normal
saline. The time between extraction and replantation was less than 15 minutes in every case. Teeth from the right side ofthe jaws were treated as described for group A and those on the left side as for group B. Replanted teeth were splinted with wire and cold-cure acrylic resin. The procedure was completed with a final radiograph and an intramuscular injection of 1.2 million units of benzathine penicillin'. Replantation of teeth and killing of the animals were scheduled to provide 20 specimens for each experimental group at observation periods of 3, 14 and 56 days. Two additional non-extracted, non-replanted teeth from each animal (third upper incisors) were used as controls. Postoperatively, the animals were fed a soft diet. Splints were removed on the 7th postoperative day. During the splinting period a daily cleaning procedure was performed using water rinsing and removal of food remnants with a wet piece of gauze. After removing the splint, plaque control was managed by tooth brushing, three times per week, Endodontic treatment was performed on the I4th day for teeth which had a 56-day observation period. Teeth were isolated with cotton wool rolls. After access opening and pulp extirpation, root canals were prepared with reamers and Hedstrom files, using 5 per cent sodium hypochlorite as irrigant. Root canals were obturated with gutta-percha and Roth sealer"* by the lateral condensation technique. The animals were killed under general ana^thesia with an endocardial perfusion of alcohol. Jaws were resected and placed in a 10 per cent buffered formalin solution for 7-10 days. Following demineraltzation with 6 per cent nitric acid, dehydration and clearing in xylol, the teeth were embedded in paraffin. Step-serial buccolingual sections, 6-8 (im thick, were taken and stained with haematoxylin and eosin. From the total of 120 teeth, nine were discarded during or after extraaion, eight were lost during the observation periods, and eight were discarded during br after histological preparation. Finally, 95 teeth and their
' Matlincrond Chemical Works, St Louis, Missouri, USA. ^ Hu-FHedy, Chicago, Illinois, USA.
•'Pcnadur LA, Wyeth, Amsterdam, The Netherlands. * Roth 811, Roth, Chicago, Illinois, USA.
of replanted dogs' teeth which had been demineralized with citric acid prior to replantation.
319
Repttintation folktming citric acid treatment
.-
.-•
l^i^. 1. Ciroup A, .1 day.s. Reunion with '.In- rosi; surhiLc. P;ic):' ceil population, mainlv polymorphs. Tlu- Jark sunned layer (arrows) is characteristic of LMiiup \ . H.icmatosylin and eosin stain, magnification X 1.14.
Fig. 2. Group B, 3 days. Reunion against dentine surface (large arrows). The demineralized zone appears as a thin eosinophilic layer (small arrows). Haematoxylin and eosin stain, magnification X 126.
surrounding tissues were examined under a light microscope (45 teeth in group A and 50 teeth in group B). The following criteria were used for evaluation of rcattachment and resorption. A. Reattachment (1) Epithelium at the nortnal level. Rcattachment of periodonta! ligament fibres. (2) Epithelial downgrowth or absence of reattachment, < 25 per cent (3) Epithelial downgrowth or absence of reattachment, 25-50 per cent (4) Epithelial downgrowth or absence of reattachment, > 50 per cent B. Surface and inflammatory resorption (1) No resorption (2) Surface resorption, 25 per cent. Inflammatory resorption, 25 per cent C. Replacement resorption (1) No resorption (2) Initial replacement resorption (3) Replacement resorption, < 25 percent (4) Replacement resorption, > 25 per cent Histologicai analysis was performed using 8-10 sections from each specimen. These sections were selected to represent the maximum width of the areas examined. The percentage per tooth was the mean of these sections. Histoiogical data were subjected to statistical analysis using the Mann-Whitney-Wilcoxon non-parametric test at a 5 per cent probability level (P = 0.05). Results 3-day groups
Three days after replantation, the apical extent of the junctional epithelium was
320
P. Zervas, T. Lamtrianidis CS I. Karahouta-Vulgaropoulou
Table I, Reattachment of epithelium Observation period 56 days
14 days Group A
Group B
Group A
Group B
Reattachment score*
No. (per cent)
No. (per cent)
No. (per cent)
No. (per cent)
1 2 3 4
7(47) 1(7) 7(47) 0(0)
11(65) 3(18) 0(0) 3(18)
2(14) 7(50) 1(7) 4(29)
0(0) 3(19) 7(43) 6(37)
*See text for details.
approximately at the original level of the amelo-cemental junction more frequently in group B. In the periodontal ligament space there was an obvious tendency for reunion of the collagenfibreswith the root surface in both groups. This reunion was via afibrin-likezone containing varying numbers of erythrocytes, polymorphs and mononuclear cells. The fibrin-like zone extended approximately half the width of the periodontal ligament space, between the collagen fibres of the alveolus and the planed root surface. The root surface in group A was characterized by a dark basophilic zone, while a light eosinophilic zone was seen in group B (Figs 1 and 2). The density of the initial attachment varied in both groups. The early reactions in both groups were similar. However, the following qualitative differences were noticed. Reunion between the remaining alveolar periodontal ligament fibres and the root surface via the fibrin-like network was seen more frequently in group B than in group A, Reunion of the periodontal ligament fibres with dentine or cennentum surfaces was apparent along the root in group B, while in group A only reunion with cementum could be observed, mainly in the apical third area. Artefactual separation during laboratory procedures was noticed more frequently in group A. 14-day groups
Fourteen days after replantation the apical extent of the epithelium was significantly
closer to normal in group B. Histological examination revealed that the number and extension of resorption cavities in tbe alveolar bone and root surfaces were higher in group A (Table II). Inflammatory resorption was in progress (Figs 3 and 4) via dentinoclasts (targe resorbing cells); polymorphs and monocytes were also present. Ankylosis was seen, usually as thin osseous bridges. Collagen fibres showed marked variation with regard to their maturity, orientation and density. Artefactual separation during laboratory preparation was again a frequent finding in group A. In group B, only a few cavities with active resorption in progress were present- Reattachment was the main charaaeristic, and periodontal ligament fibres showed a normal arrangement (Fig. 5, Tables I to III). When the two groups were compared 14 days after replantation, reattachment was more favourable in group B, but the difference was not statistically significant (Table I). On the other hand, there was a statistically significant difference in the extent of resorption (Table II). 5(h-day groups
The findings 8 weeks after replantation were in contrast with those at t4 days, the epithelium being significantly closer to normal in group A. In most cases inflammatory resorption was reversed or replaced, partially with new cementum apposition (Fig, 6). Replacement resorption was seen, either as compact osseous unions b^nveen root and
Replantation fallowing citric acid iretUmeM
111
Table II. Surface and lnrtammatory resorption Observation period 14davs Surface and infiammatory rcsorption score*
Group A (n = ]5) No. (per cent)
1 2 3 4
0(0) 7(47) 7(47) 1(6)
56 davs Group B
Group A
(B=17)
(«=14)
(K=I6)
No. (per cent)
No. (per cent)
No. (per cent)
6(35) 7(41)
«(0) 1(7) 7(50) 6(3)
0(0) 0(0) 2(13) 14(87)
im
3(18)
Group B
*Sce text for details.
„•
A
" W-'
• •
K-
4 Fig. 3. Group '\, 14 day.-;. Xctui; ;nlUmma5or\ resorption cavit) (arrows.), and some rcpUcciiicni resorption. 1 laematoxylin and eosin stain, magnification X 13.
F i s . 4. U r o u r -\, 14 d.iys. M,-i;.;n:tk'iiiion of the ;K'ti^ e intl.nv,niJHirv resorpliim civiry sliown in Fiir .". DL-niinocla^Is are oiiscrval'ie. Hacniatowlin ar.J. L'osin .slain, mairniricuion ^ S4.
alveolus, or as osseotis bridges between them. In some cases reattachment, inflammatory and replacement resorptioti were present
simultaneously in the same area. In most cases collagen fibres showed a normal arrangement. In group B, brge active inflammatory
322
. Zervas, T. Lamhriamdis & L Kiirabmtta-Vulgarifpouhu
^ /l^^vjij f-.^ I -.fey ' ^ . ^
Fig. 5. Group B, 14 days. Epithelial reattachment at the normal level (arrows). Reattachment of the periodontal ligamentfibres,lack of inflammatory or replacement resorption. Haemataxylin and eosin stain, magnification x2.5. resorptive lesions were seen in several specimens. The main histological characteristic was replacement resorption (Fig. 7). In the alveolar bone active or reversed resorption activit}' was present. Collagen fibres showed variation iti density, maturity atid orientatioti. When the two groups were compared, healing was obviously closer to normal in group A (Tables I to III). There were statistically significant differences in reattachment and resorptioti. Disctission In the present study removal of periodontal ligament and eemetitum by root platiing affected the etitire root surface, and not just the coronal part of the root, as in previous studies (Poison & Proye 1982, I9S3, NymaJi et at 19SS, Catoti et ul 19%). Planing of the
entire root surface was performed to enable evaluation of the cftccts of demineralization on the healing of replanted teeth, without interference from the remaining healthy periodontal ligament. The results of the present study, 3 days after replantation, are in agreement with those of Poison & Proye (1982), iti that surface demineraiizaaort with citric acid had an effect in preventitig epitheiia! downgrowth, but this did not occur in every case. During the first few days after replantation a zone of organized blood ciot was formed between the planed and demineralized root surface and the socket. Poison & Proye (1982, 1983) foutid that this zone was attached to demineralized surfaces, while in denuded surfaces a less good apposition was seen. In contrast, we observed similar histological characteristics in both groups, A and B. The differences observed led to the conclusion that demineralization had a generally positive effect on initial healing after repkntatiot!, bul the qualitative characteristics remained the same as those observed with denuded and non-demineraiized roots. .4tiy difference between the two groups could be attributed to the simultaneous influence of two factors affecting the root surfaces: in group B, following demineralization, the smear layer was removed, and collagen fibrils were exposed. By 14 days after replatitation it was obvious that surface demineralizatioti had a positive influence on healing as assessed b)' reattachment, extent or resorption, and collagen fibres being at a higher level in group B compared with group A. Further evidence was the presence of artefactual separations in group A. These observations support the view that surface demineralization accelerated the biological process of reattachmetjt. However, by 8 weeks after replantation, there was clear evidence against the citric acid group. In group A, the condition of the epithelium was better, there was a higher level of reattachtnent and less resorption than in group B. Collagen fibres were closer to normal in group A. Our results are generally in agreement with those of Nyman et al. (I98.S). They used replantation and atitotransplantation as experimental models for studying the effect
323
Replaittiitton following citric acid treatment
Table III. Replacement resorption Observation period 14 davs Resorption score* 1 2 3 4
56 davs
Group A
Group B
(K=15)
(11=11)
No. (per cent) 11(7.3) 1(7) 3(20) (1(0)
No. (per cent)
Group A («=14) .No. (per cent)
Group B No. (per cent)
15(88) !(6) 1(6) 0(0)
5(36) 1(7) 4(28) 4(28)
6(38) 0(0) 1(6) 9(56)
(« = 16)
•Set text for details.
•.
(
I iii
~
I I ' l !,
• I ,,''P,r.!
,1 ••I"' • • V -v. .
Fig. ,4i:^~420. CRIGGEK, M . , BOGLE, G . , NILVEUS, R., EGELBERG,
J. 8c SELVtG, K.A. (1978) The effect of topical citric acid application on the healing of experimental furcation defects in dogs. Journal of Periodontal Research, 13, 538-549. HAMMAKSTROM, L.E., BLOMLCW, L.B., FEICH-IN,
B. & LtNDSKOG, S.F. (1986) Effect of calcium hydroxide treatment on periodontal repair and root resorption. Endodontics and Dental Traumatology, 2,184-189. HANES, P J . , POLSON, A . M . & LADENHEIM, S.
(1985) Cell and fiber attachment to demineralized dentin from normal root surfaces. Journal of Periodontohgy, 56,752-765. HARDY, L . B , , O'NEAL, R . B . & DEL RIO, C.E.
(198!) Effect of polylactic acid on replanted teeth in dtigs. Oral Surgery, Oral Medicine and Oral Pathology, Sl,Sfi-B2. HtJEBScw, R. (1967) Implanting teeth with methyl-2-cyanoacrylate adhesive. Journal of Dental Research, 46,337-339. KELLER, E.B., HAYWARD, J , R , , NASjLETt, C.E.
& CASTELLI, W . A . (1972) Venous tissue replanted on roots of teeth in monkeys. Oral Surgery, Oral Medicine and Oral Pathology, 34, 352-363. LiNDScoG, S., BLONOXIF, L . & HAMMARSTROM, L .
(1983) Repair of periodontal tissues m vi'tx) atid
Replantation follomng citric acid treatment in vitro. Journal of Clinical Periodontohgy, 10, 188-205. LtNDSKOG,
S . , PlHtCE,
A . M . , BLOMLOF,
L. &
325
POLSON, A.M. & PROYE, M.P. (1983) Fibrin
linkage: a precursor for new attachment. JaBma/ of Periodontohgy, 54,141-147.
HAMMARSTROM, L . (1985) The role of necrodc POLSON, A.M. & HANES, P.J. (1987) CeU and fiber periodontal membrane in cementum resorpattachment to demineralized dentin. A comparition and ankylosis. Endodontics and Dental son between norma! and periodontitis-affected Traumatology, 1,96-101, root surfaces. Journal of Clinical Periodontokgy, NALBANDIAN, J . & COTE, N . (1982) Direct histo14,357-365. logicai comparison of periodontal wound healing POLSON, A.M., LADENHEIM, S . & HANES, P.J. in the beagle dog with and without citric add (1986) Cell and fiber attachment to demineralCfm&t\onin%. Journal of Periodontal Research, 17, ised dentin from penodondtis-affected root sur552-562. faces. >urBa/o//>(rrioii»i«o/o^, 57, 235-246. NEVINS,
A.J.,
LAPORTA, R.F.,
BORDEN, B . G .
& LORENZO, P. (1980) Replantation of enzymatically treated teeth in monkeys. Part 1. Oral Surgery, Oral Medicine and Oral Pathology, 50, 277-28!. NILVEUS, R. & EGELBERG, J. (1980) The efTect of
topical eitric acid application on the healing of experimental furcation defect in dogs. III. The relative importance of co^ulum support, flap design and systemic antibiotics. Journal of Periodontai Research, 15,551-560. NILVEUS, R . , BOGLE, G . , CRIGGER, M . , EGELBERG,
j . & SELVIG, K.A. (1980) The effect of topical citric add application on the healing of experimental furcation defects in dogs. II. Healing after repeated surgery. Journal of Periodontal Research, 15, 544-550. N(MIDE.NRAM,
.\.,
BANG,
G.
&
ANNEROTH,
G.
(1973) A histopathoiogic study of replanted teeth with superfidaliy demineralized root surfaces in Java monkeys. Scandinavian Journal of Dental Research, 81,294-302. NYMAN, S., HOUSTON, F . , SARHED, G . , LINDHE,
J. & KARRING, T . (1985) Healing following reimplantation of teeth subjected to root planing and citric add treatment. Journal of Clinical Periodontology, 12,294-305. PETTERSSON, E . C . & AUKHIL, L (1986) Citric add
conditioning of roots affects guided tissue regeneration in experimental periodontal wounds. Journal of Periodontai Researck, 21,543-552. POLSON, A.M. & PROVE, M . P . (1982) Effect of root surface alterations on periodontal healing. H. Citric acid treatment of denuded root. Journal of Clinical Periodontotogy, 9,441^54.
PROYE, M.P. & POLSON, A.M. (1982) Effect of
root surface alterations on periodontal healing. 1. Surgical denudiaon. Journal of Clinical Periodmtology, 9,428-440. REGISTER, A.A. (1973) Bone and cementum induction by dentin, demineralized in situ. Journal ofPeriodontology, 44,49-54. REGISTER,
A.A.
&
BUKDICK,
F.A.
(1975)
Accelerated reattachment with cementogenesis to dentin, demineralized in situ. I. Optimum range. Journal ofPeriodontology, 46, 646-655. REINHOLDT, J . , ANDREASEN, J.O., SODER, P.O.,
OTTESKOG, P., DYBDAHL, R. & Riis, I. (1977)
Cultivation of periodontal ligament fibroblasts of extracted monkey indsors. A histologic study of three culturing methods. International Journal of Oral Surgery, (i,2\^225. RiRiE, CM., CRIGGER, M . & SELVIG, K.A. (1980)
Healing of periodontal connective tissues following surgical wounding and application of citric acid in dogs. Journal of Periodontal Research, 15, 314-327. SELVIG,
K.A.,
RIRIE,
CM.,
NILVEUS,
R. &
EGEI^ERG, J. (1981) Fine structure of new connective tissue attachment following add treatment of experimental furcation pockets in dogs. Journal of Periodontal Research, 16,123-129. SODER,
P.O.,
OTTESKOG,
L-,
FRITHIOT,
L.,
BLOMLOF, L . & ANDREASEN, J.O. (1979) Culti-
vation of fibroblasts on human teeth. Ultrastructural observations of cells cultivated in multilayers. International Journal of Oral Surgery, 8, 234-240.
The effect of citric acid treatment on periodontal healing after replantation of permanent teeth p. ZERVAS, T. LAMBRIANIDIS*&I. KARABOUTA-VULGAROPOULOUt * Department ofDental Pathology and Therapeutics, Urtiversity o/Thessaloniki, and 1^Department of Oral and Maxillofaoal Surgery, University of Tkessaiomki, Thessaloniki, Greece Summary. The aim of this study was to evaluate the effect of surface demineralization on the healing of 120 replanted teeth in fifteen S- to 16-month-old dogs. Replanted teeth were divided into two groups (A and B). In group A, teeth were replanted following planing of the entire root surface. In group B, teeth were replanted following planing and surface demineraiization with citric add. Replanted teeth were splinted for 7 days, and observation periods were 3,14 and 56 days. A total of 95 teeth and their surrounding tissues were examined histologically under a light microscope. Histological examitiation of specimens 3 days after replantation revealed that initial reunion between the root and the alveolar periodontal ligament fibres was via a fibrin-like network in both groups. Fourteen days after replantation histological observations showed superior healing in group B with regard to epithelial condition, periodontal ligament fibre reatuchment to the root surface, resorption and functional arrangement of the periodonul ligament fibres. By 56 days after replantation, healing was superior in group A; group B was characterized by severe inflammatory and replacement resorption. These results indicated that surface detnineralization had in initial positive effect, but later led to impaired healing.
Introduction The most important factor influencing the prognosis of replanted teeth is the status of the periodontal ligament (Andreasen I98fla, Andreasen & Kristerson 1981, Lindskog et al. 1983), Several authors have attempted to find the most appropriate treatment for avulsed teeth which had a destroyed or necrotic periodonul ligament. Their suggestions could be summarized as follows. (i) Attempts have been made to substitute the periodontal ligament veith bioCorrespondence: Dr Panos Zervas, 6 Maroneias St, 543 51 Thessaloitiki, Greece,
compatible materials (Huebsch 1967, Hardy et al. 1981), proper autografts (Keller et al. Vill, Andreasen 1981), or following culture (Soder et al. 1979). Regeneration of the periodontal ligament in culture media prior to replantation (Reinholdt et al. 1977, Andreasen et at. 1978), as well as in-vive heterotrophic transplantation in soft tissues before replantation (Andreasen 1980b, Birman & de Araujo 1975) have bran tested, (ii) The treatment of the root surfaces with chemical substances to facilitate reattachmenr of collagen fibres. Acids (Nordenram et at. 1973), enzymes (Nevins et al. 1980) and fluoride solutions (Coccia 1980) have been suggested. Lindskog et al. (1985) suggested a sodium hypochlorite solution for removing the necrotic periodontal ligament remnants from the root surface. An interesting and controversial subject concerns the effects of citric acid on the healing of periodontal tissues. Because several experimental studies have shown that citric acid has a positive effect on the reatuchment of periodontal fibres (Register 1973, Register & Burdick 1975, Crigger et al. 1978, Nilveus & Egelberg 19B0, Nilveus ef al. 1980, Ririe et al. 1980, Selvig et al. 1981, Nalbandian & Cote 1982, Poison & Proye 1982, 1983, Proye & Poison 1982, Hanes et al. 1985, Caton et al. 1986, Poison et al. 1986, Poison & Hanes 1987), we decided to investigate the hypothesis that surface demineralization could be a valid procedure in cases of avulsed teeth with destroyed periodontal ligament. The aim of this study was to evaluate the healing 317
p. Zervas, T, Lamhrianidis (S I, Karahouta-Vulgaropoulou
Materials and methods Fifteen healthy dogs, aged 8-16 months, were used in this study. A total of 120 single-rooted lateral incisors and first premolars of both jaws were extracted and replanted. Clinical and radiographic examination ensured that prior to the experimental procedures all teeth were intact, caries free, completely formed and with no signs of chronic gingival inflammation. The citric acid solution used was always freshly prepared by adding hydrated crystalline citric acid' to distilled water under continuous mixing at room temperature, until the solution was almost saturated. The pH was tested with chromatographic paper. Replantations were performed under general anaesthesia. The animals were anaesthetized with Pentotha! (20-25 mg kg"' body weight) and intubated with a cuffed endotracheal tube, receiving a mixture of halothane and oxygen. Clinical crowns of the teeth and their gingivae were disinfected by application of a 2 per cent chlorexidine solution. After an initial radiograph, extraction was performed with Bein elevators and forceps. The entire root surface was planed with a periodontal curette. Preiiminarj tests showed that 10-12 strokes for each part of the root surface with a Gracey curette 13/14" resulted in removal of the periodontal ligament from the entire root surface, and cementum from the coronal and mesial third of the root. A layer of cementum remained around the apical third, but no additional planing was performed in this area, in order to avoid major modifications in root morphology and dimensions. During the root planing procedures, the tooth was held between the beaks of the extraction forceps. Teeth in group A were replanted after rinsing with normal saline. The planed root surfaces of teeth in group B were demineralized for 3 minutes in citric acid on a saturated piece of cotton wool folded around the root and then rinsed with normal
saline. The time between extraction and replantation was less than 15 minutes in every case. Teeth from the right side ofthe jaws were treated as described for group A and those on the left side as for group B. Replanted teeth were splinted with wire and cold-cure acrylic resin. The procedure was completed with a final radiograph and an intramuscular injection of 1.2 million units of benzathine penicillin'. Replantation of teeth and killing of the animals were scheduled to provide 20 specimens for each experimental group at observation periods of 3, 14 and 56 days. Two additional non-extracted, non-replanted teeth from each animal (third upper incisors) were used as controls. Postoperatively, the animals were fed a soft diet. Splints were removed on the 7th postoperative day. During the splinting period a daily cleaning procedure was performed using water rinsing and removal of food remnants with a wet piece of gauze. After removing the splint, plaque control was managed by tooth brushing, three times per week, Endodontic treatment was performed on the I4th day for teeth which had a 56-day observation period. Teeth were isolated with cotton wool rolls. After access opening and pulp extirpation, root canals were prepared with reamers and Hedstrom files, using 5 per cent sodium hypochlorite as irrigant. Root canals were obturated with gutta-percha and Roth sealer"* by the lateral condensation technique. The animals were killed under general ana^thesia with an endocardial perfusion of alcohol. Jaws were resected and placed in a 10 per cent buffered formalin solution for 7-10 days. Following demineraltzation with 6 per cent nitric acid, dehydration and clearing in xylol, the teeth were embedded in paraffin. Step-serial buccolingual sections, 6-8 (im thick, were taken and stained with haematoxylin and eosin. From the total of 120 teeth, nine were discarded during or after extraaion, eight were lost during the observation periods, and eight were discarded during br after histological preparation. Finally, 95 teeth and their
' Matlincrond Chemical Works, St Louis, Missouri, USA. ^ Hu-FHedy, Chicago, Illinois, USA.
•'Pcnadur LA, Wyeth, Amsterdam, The Netherlands. * Roth 811, Roth, Chicago, Illinois, USA.
of replanted dogs' teeth which had been demineralized with citric acid prior to replantation.
319
Repttintation folktming citric acid treatment
.-
.-•
l^i^. 1. Ciroup A, .1 day.s. Reunion with '.In- rosi; surhiLc. P;ic):' ceil population, mainlv polymorphs. Tlu- Jark sunned layer (arrows) is characteristic of LMiiup \ . H.icmatosylin and eosin stain, magnification X 1.14.
Fig. 2. Group B, 3 days. Reunion against dentine surface (large arrows). The demineralized zone appears as a thin eosinophilic layer (small arrows). Haematoxylin and eosin stain, magnification X 126.
surrounding tissues were examined under a light microscope (45 teeth in group A and 50 teeth in group B). The following criteria were used for evaluation of rcattachment and resorption. A. Reattachment (1) Epithelium at the nortnal level. Rcattachment of periodonta! ligament fibres. (2) Epithelial downgrowth or absence of reattachment, < 25 per cent (3) Epithelial downgrowth or absence of reattachment, 25-50 per cent (4) Epithelial downgrowth or absence of reattachment, > 50 per cent B. Surface and inflammatory resorption (1) No resorption (2) Surface resorption, 25 per cent. Inflammatory resorption, 25 per cent C. Replacement resorption (1) No resorption (2) Initial replacement resorption (3) Replacement resorption, < 25 percent (4) Replacement resorption, > 25 per cent Histologicai analysis was performed using 8-10 sections from each specimen. These sections were selected to represent the maximum width of the areas examined. The percentage per tooth was the mean of these sections. Histoiogical data were subjected to statistical analysis using the Mann-Whitney-Wilcoxon non-parametric test at a 5 per cent probability level (P = 0.05). Results 3-day groups
Three days after replantation, the apical extent of the junctional epithelium was
320
P. Zervas, T. Lamtrianidis CS I. Karahouta-Vulgaropoulou
Table I, Reattachment of epithelium Observation period 56 days
14 days Group A
Group B
Group A
Group B
Reattachment score*
No. (per cent)
No. (per cent)
No. (per cent)
No. (per cent)
1 2 3 4
7(47) 1(7) 7(47) 0(0)
11(65) 3(18) 0(0) 3(18)
2(14) 7(50) 1(7) 4(29)
0(0) 3(19) 7(43) 6(37)
*See text for details.
approximately at the original level of the amelo-cemental junction more frequently in group B. In the periodontal ligament space there was an obvious tendency for reunion of the collagenfibreswith the root surface in both groups. This reunion was via afibrin-likezone containing varying numbers of erythrocytes, polymorphs and mononuclear cells. The fibrin-like zone extended approximately half the width of the periodontal ligament space, between the collagen fibres of the alveolus and the planed root surface. The root surface in group A was characterized by a dark basophilic zone, while a light eosinophilic zone was seen in group B (Figs 1 and 2). The density of the initial attachment varied in both groups. The early reactions in both groups were similar. However, the following qualitative differences were noticed. Reunion between the remaining alveolar periodontal ligament fibres and the root surface via the fibrin-like network was seen more frequently in group B than in group A, Reunion of the periodontal ligament fibres with dentine or cennentum surfaces was apparent along the root in group B, while in group A only reunion with cementum could be observed, mainly in the apical third area. Artefactual separation during laboratory procedures was noticed more frequently in group A. 14-day groups
Fourteen days after replantation the apical extent of the epithelium was significantly
closer to normal in group B. Histological examination revealed that the number and extension of resorption cavities in tbe alveolar bone and root surfaces were higher in group A (Table II). Inflammatory resorption was in progress (Figs 3 and 4) via dentinoclasts (targe resorbing cells); polymorphs and monocytes were also present. Ankylosis was seen, usually as thin osseous bridges. Collagen fibres showed marked variation with regard to their maturity, orientation and density. Artefactual separation during laboratory preparation was again a frequent finding in group A. In group B, only a few cavities with active resorption in progress were present- Reattachment was the main charaaeristic, and periodontal ligament fibres showed a normal arrangement (Fig. 5, Tables I to III). When the two groups were compared 14 days after replantation, reattachment was more favourable in group B, but the difference was not statistically significant (Table I). On the other hand, there was a statistically significant difference in the extent of resorption (Table II). 5(h-day groups
The findings 8 weeks after replantation were in contrast with those at t4 days, the epithelium being significantly closer to normal in group A. In most cases inflammatory resorption was reversed or replaced, partially with new cementum apposition (Fig, 6). Replacement resorption was seen, either as compact osseous unions b^nveen root and
Replantation fallowing citric acid iretUmeM
111
Table II. Surface and lnrtammatory resorption Observation period 14davs Surface and infiammatory rcsorption score*
Group A (n = ]5) No. (per cent)
1 2 3 4
0(0) 7(47) 7(47) 1(6)
56 davs Group B
Group A
(B=17)
(«=14)
(K=I6)
No. (per cent)
No. (per cent)
No. (per cent)
6(35) 7(41)
«(0) 1(7) 7(50) 6(3)
0(0) 0(0) 2(13) 14(87)
im
3(18)
Group B
*Sce text for details.
„•
A
" W-'
• •
K-
4 Fig. 3. Group '\, 14 day.-;. Xctui; ;nlUmma5or\ resorption cavit) (arrows.), and some rcpUcciiicni resorption. 1 laematoxylin and eosin stain, magnification X 13.
F i s . 4. U r o u r -\, 14 d.iys. M,-i;.;n:tk'iiiion of the ;K'ti^ e intl.nv,niJHirv resorpliim civiry sliown in Fiir .". DL-niinocla^Is are oiiscrval'ie. Hacniatowlin ar.J. L'osin .slain, mairniricuion ^ S4.
alveolus, or as osseotis bridges between them. In some cases reattachment, inflammatory and replacement resorptioti were present
simultaneously in the same area. In most cases collagen fibres showed a normal arrangement. In group B, brge active inflammatory
322
. Zervas, T. Lamhriamdis & L Kiirabmtta-Vulgarifpouhu
^ /l^^vjij f-.^ I -.fey ' ^ . ^
Fig. 5. Group B, 14 days. Epithelial reattachment at the normal level (arrows). Reattachment of the periodontal ligamentfibres,lack of inflammatory or replacement resorption. Haemataxylin and eosin stain, magnification x2.5. resorptive lesions were seen in several specimens. The main histological characteristic was replacement resorption (Fig. 7). In the alveolar bone active or reversed resorption activit}' was present. Collagen fibres showed variation iti density, maturity atid orientatioti. When the two groups were compared, healing was obviously closer to normal in group A (Tables I to III). There were statistically significant differences in reattachment and resorptioti. Disctission In the present study removal of periodontal ligament and eemetitum by root platiing affected the etitire root surface, and not just the coronal part of the root, as in previous studies (Poison & Proye 1982, I9S3, NymaJi et at 19SS, Catoti et ul 19%). Planing of the
entire root surface was performed to enable evaluation of the cftccts of demineralization on the healing of replanted teeth, without interference from the remaining healthy periodontal ligament. The results of the present study, 3 days after replantation, are in agreement with those of Poison & Proye (1982), iti that surface demineraiizaaort with citric acid had an effect in preventitig epitheiia! downgrowth, but this did not occur in every case. During the first few days after replantation a zone of organized blood ciot was formed between the planed and demineralized root surface and the socket. Poison & Proye (1982, 1983) foutid that this zone was attached to demineralized surfaces, while in denuded surfaces a less good apposition was seen. In contrast, we observed similar histological characteristics in both groups, A and B. The differences observed led to the conclusion that demineralization had a generally positive effect on initial healing after repkntatiot!, bul the qualitative characteristics remained the same as those observed with denuded and non-demineraiized roots. .4tiy difference between the two groups could be attributed to the simultaneous influence of two factors affecting the root surfaces: in group B, following demineralization, the smear layer was removed, and collagen fibrils were exposed. By 14 days after replatitation it was obvious that surface demineralizatioti had a positive influence on healing as assessed b)' reattachment, extent or resorption, and collagen fibres being at a higher level in group B compared with group A. Further evidence was the presence of artefactual separations in group A. These observations support the view that surface demineralization accelerated the biological process of reattachmetjt. However, by 8 weeks after replantation, there was clear evidence against the citric acid group. In group A, the condition of the epithelium was better, there was a higher level of reattachtnent and less resorption than in group B. Collagen fibres were closer to normal in group A. Our results are generally in agreement with those of Nyman et al. (I98.S). They used replantation and atitotransplantation as experimental models for studying the effect
323
Replaittiitton following citric acid treatment
Table III. Replacement resorption Observation period 14 davs Resorption score* 1 2 3 4
56 davs
Group A
Group B
(K=15)
(11=11)
No. (per cent) 11(7.3) 1(7) 3(20) (1(0)
No. (per cent)
Group A («=14) .No. (per cent)
Group B No. (per cent)
15(88) !(6) 1(6) 0(0)
5(36) 1(7) 4(28) 4(28)
6(38) 0(0) 1(6) 9(56)
(« = 16)
•Set text for details.
•.
(
I iii
~
I I ' l !,
• I ,,''P,r.!
,1 ••I"' • • V -v. .
Fig. ,4i:^~420. CRIGGEK, M . , BOGLE, G . , NILVEUS, R., EGELBERG,
J. 8c SELVtG, K.A. (1978) The effect of topical citric acid application on the healing of experimental furcation defects in dogs. Journal of Periodontal Research, 13, 538-549. HAMMAKSTROM, L.E., BLOMLCW, L.B., FEICH-IN,
B. & LtNDSKOG, S.F. (1986) Effect of calcium hydroxide treatment on periodontal repair and root resorption. Endodontics and Dental Traumatology, 2,184-189. HANES, P J . , POLSON, A . M . & LADENHEIM, S.
(1985) Cell and fiber attachment to demineralized dentin from normal root surfaces. Journal of Periodontohgy, 56,752-765. HARDY, L . B , , O'NEAL, R . B . & DEL RIO, C.E.
(198!) Effect of polylactic acid on replanted teeth in dtigs. Oral Surgery, Oral Medicine and Oral Pathology, Sl,Sfi-B2. HtJEBScw, R. (1967) Implanting teeth with methyl-2-cyanoacrylate adhesive. Journal of Dental Research, 46,337-339. KELLER, E.B., HAYWARD, J , R , , NASjLETt, C.E.
& CASTELLI, W . A . (1972) Venous tissue replanted on roots of teeth in monkeys. Oral Surgery, Oral Medicine and Oral Pathology, 34, 352-363. LiNDScoG, S., BLONOXIF, L . & HAMMARSTROM, L .
(1983) Repair of periodontal tissues m vi'tx) atid
Replantation follomng citric acid treatment in vitro. Journal of Clinical Periodontohgy, 10, 188-205. LtNDSKOG,
S . , PlHtCE,
A . M . , BLOMLOF,
L. &
325
POLSON, A.M. & PROYE, M.P. (1983) Fibrin
linkage: a precursor for new attachment. JaBma/ of Periodontohgy, 54,141-147.
HAMMARSTROM, L . (1985) The role of necrodc POLSON, A.M. & HANES, P.J. (1987) CeU and fiber periodontal membrane in cementum resorpattachment to demineralized dentin. A comparition and ankylosis. Endodontics and Dental son between norma! and periodontitis-affected Traumatology, 1,96-101, root surfaces. Journal of Clinical Periodontokgy, NALBANDIAN, J . & COTE, N . (1982) Direct histo14,357-365. logicai comparison of periodontal wound healing POLSON, A.M., LADENHEIM, S . & HANES, P.J. in the beagle dog with and without citric add (1986) Cell and fiber attachment to demineralCfm&t\onin%. Journal of Periodontal Research, 17, ised dentin from penodondtis-affected root sur552-562. faces. >urBa/o//>(rrioii»i«o/o^, 57, 235-246. NEVINS,
A.J.,
LAPORTA, R.F.,
BORDEN, B . G .
& LORENZO, P. (1980) Replantation of enzymatically treated teeth in monkeys. Part 1. Oral Surgery, Oral Medicine and Oral Pathology, 50, 277-28!. NILVEUS, R. & EGELBERG, J. (1980) The efTect of
topical eitric acid application on the healing of experimental furcation defect in dogs. III. The relative importance of co^ulum support, flap design and systemic antibiotics. Journal of Periodontai Research, 15,551-560. NILVEUS, R . , BOGLE, G . , CRIGGER, M . , EGELBERG,
j . & SELVIG, K.A. (1980) The effect of topical citric add application on the healing of experimental furcation defects in dogs. II. Healing after repeated surgery. Journal of Periodontal Research, 15, 544-550. N(MIDE.NRAM,
.\.,
BANG,
G.
&
ANNEROTH,
G.
(1973) A histopathoiogic study of replanted teeth with superfidaliy demineralized root surfaces in Java monkeys. Scandinavian Journal of Dental Research, 81,294-302. NYMAN, S., HOUSTON, F . , SARHED, G . , LINDHE,
J. & KARRING, T . (1985) Healing following reimplantation of teeth subjected to root planing and citric add treatment. Journal of Clinical Periodontology, 12,294-305. PETTERSSON, E . C . & AUKHIL, L (1986) Citric add
conditioning of roots affects guided tissue regeneration in experimental periodontal wounds. Journal of Periodontai Researck, 21,543-552. POLSON, A.M. & PROVE, M . P . (1982) Effect of root surface alterations on periodontal healing. H. Citric acid treatment of denuded root. Journal of Clinical Periodontotogy, 9,441^54.
PROYE, M.P. & POLSON, A.M. (1982) Effect of
root surface alterations on periodontal healing. 1. Surgical denudiaon. Journal of Clinical Periodmtology, 9,428-440. REGISTER, A.A. (1973) Bone and cementum induction by dentin, demineralized in situ. Journal ofPeriodontology, 44,49-54. REGISTER,
A.A.
&
BUKDICK,
F.A.
(1975)
Accelerated reattachment with cementogenesis to dentin, demineralized in situ. I. Optimum range. Journal ofPeriodontology, 46, 646-655. REINHOLDT, J . , ANDREASEN, J.O., SODER, P.O.,
OTTESKOG, P., DYBDAHL, R. & Riis, I. (1977)
Cultivation of periodontal ligament fibroblasts of extracted monkey indsors. A histologic study of three culturing methods. International Journal of Oral Surgery, (i,2\^225. RiRiE, CM., CRIGGER, M . & SELVIG, K.A. (1980)
Healing of periodontal connective tissues following surgical wounding and application of citric acid in dogs. Journal of Periodontal Research, 15, 314-327. SELVIG,
K.A.,
RIRIE,
CM.,
NILVEUS,
R. &
EGEI^ERG, J. (1981) Fine structure of new connective tissue attachment following add treatment of experimental furcation pockets in dogs. Journal of Periodontal Research, 16,123-129. SODER,
P.O.,
OTTESKOG,
L-,
FRITHIOT,
L.,
BLOMLOF, L . & ANDREASEN, J.O. (1979) Culti-
vation of fibroblasts on human teeth. Ultrastructural observations of cells cultivated in multilayers. International Journal of Oral Surgery, 8, 234-240.
