Pulp revascularization in reimplanted immature monkey incisors - predictability and the effect of antibiotic systemic prophylaxis Cvek M, Cleaton-Jones P, Austin J, Lownie J, Khng M, Fatti R Pulp revascularization in reimplanted immature monkey incisors - predictability and the effect of antibiotic systemic prophylaxis. Endod Dent Traumatol 1990; 6: 157-169. Abstract - In 32 monkeys 105 immature maxillary incisors were extracted and reimplanted either immediately or after 30 or 60 min wet or dry storage. Of the monkeys, 17 (group I) did not receive and 15 (group II) received prophylactic treatment with 4 mg/kg doxycycline before extraction and 2 mg/kg for 5 d after reimplantation. The observation time varied from 6 to 8 weeks. After being histologically processed, the material was evaluated with respect to the amount of vital tissue and presence of microorganisms in the pulpal lumen. A comparison revealed no difference in the results between the groups. The results were therefore pooled and statistically analysed with respect to the significance of apical foramen width, extra-alveolar time, wet or dry storage and presence of micro-organisms in the pulpal lumen for the occurrence of complete pulp revascularization (CPR). The overall frequency of CPR was 18%. Log-hnear analyses (SAS, 1985) of the material as a whole or of separate parameters consistently revealed a relationship between presence of microorganisms and absence of CPR {P = 0.0001). A higher frequency of CPR and a lower frequency of micro-organisms {P = 0.05) was found only for the group of immediately reimplanted teeth. The presence of micro-organisms could be explained for 61 teeth. In 27 of these, blood clots containing bacteria in the apical portion of pulpal lumen indicated contamination during the extra-alveolar time, while in 34, the micro-organisms originated from plaque covered mechanical damage in the cervical part of the root surface.
In immature and occasionally in mature reimplanted teeth the pulp can be completely revascularized, usually within 30 days after reimplantation (1-14). It has therefore been recommended that, in reimplanted immature teeth, endodontic treatment be postponed until infiammatory changes and/or inflammatory root resorption are seen in the control radiographs (15). These changes, however, may progress very rapidly in immature teeth (16), worsening the prognosis of the tooth. Therefore, it would be desirable if, on the basis of clinically available variables, the prognosis of pulp revasculariza-
Miemir Cvek\ Peter Cieaten-Jenes^ Jehn Austin^ Jehn Lewnie^ Margareta K\m\ Paui Fatti^ ^Departmenf of Pedodontics, Eastman Institute, Stockholm, Sweden, ^Dental Research Institute, MRC/University of Witwatersrand, Johannesburg, ^MRC/Laboratory Animal Unite, Johannesburg, "Division of Maxillo-Facial and Oral Department of Surgery, ^Department of Statistics and Actuarial Science, University of the Witwatersrand, Johannesburg, Republic of South Africa
Key words: doxycycline; reimplantation; revascularization; pulpal revascularization; dental pulp. Miomir Cvek, Department of Pedodontics, Eastmaninstltutet, Dalagatan 11, S-113 24 Stockholm, Sweden. Accepted for publication January 31, 1990.
tion could be more distinctly predicted and, eventually, improved by some therapeutic measures. From experimental studies, it can be concluded that some factors, such as the width of apical foramen, i.e. the stage of root development (2, 7, U), duration of extra-alveolar time and storage conditions (5, 6), could indicate the occurrence of pulp revascularization. A recent study of clinically reimplanted incisors, however, could only confirm the predictability of these factors to a hmited extent (14). In this study, pulp revascularization did occur only in immature teeth, but in these teeth it was not related to the width 157
Cvek et ai. of apical foramen, that is, to the stage of root development. Regarding the extent of extra-alveolar time and dry or wet storage, a weak but statistically significant increase in frequency of revascularization was found only in the teeth reimplanted within 45 min after avulsion, irrespective of storage conditions. However, revascularization also occurred in teeth with an extra-alveolar time that exceeded 2 h and included periods of 60 min dry storage. Furthermore, the frequency of revascularization was low (18%), when compared with the results obtained in experimental studies. All teeth in which revascularization did not occur exhibited a periapical radiolucency or an external infiammatory root resorption, i.e. processes known to be closely related to the presence of micro-organisms in the pulpal lumen (17, 18). Micro-organisms have been discussed as a reason for the occurrence of abscesses between vital and necrotic tissue in the pulpal lumen of reimplanted teeth (2), but this has not been experimentally investigated nor the presence of micro-organisms in the pulpal lumen explained. Prophylactic treatment with antibiotics has been recommended after reimplantation of accidentally avulsed teeth (15). The effects of such treatment on pulpal and/or periodontal healing, however, are, controversial. In clinical studies, no difference in the frequency of subsequent infiammatory processes, related to the presence of micro-organisms, could be found between groups of patients that were or were not treated svstemicallv with antibiotics after reimplantation or transplantation of teeth (14, 19). In a recent experimental study, on the other hand, a complete absence of external infiammatory root resorption was reported in mature reimplanted monkey teeth, which were not treated endodontically but the animals received systemic treatment with antibiotics for 6 d after reimplantation (20). As far as we know, no experimental study dealing with the effect of antibiotics on pulpal healing has been recorded. A series of experiments designed to imitate clinical conditions were therefore performed, using maxillary incisors in monkeys, in order further to study healing of the pulp in young reimplanted teeth. The purpose of the present study was to evaluate the significance of width of apical foramen, extent of extra-alveolar time, dry or wet storage, presence of micro-organisms in the pulpal lumen and systemic treatment with doxycycline, on the occurrence of complete pulp revascularization, i.e., the only type of pulpal healing to be aimed for in therapeutically reimplanted teeth. iWateriai and metheds The material consisted of 128 maxillary incisors in 32 young vervet monkeys [Cercopithecus aethiopspyger158
ythrus), primarily used in the production of poliomyelitis vaccine. The animals selected had unerupted permanent maxillary canines or had canines erupted, but not more than about the half of the crown of adjacent lateral incisor. The animals were immobifized with ketamine hydrochloride 10 mg/kg (Parke Davis, Johannesburg, RSA) and then anesthetized with pentobarbitone sodium (Parke Davis). Two intraoral radiographs were taken, one for the left and one for the right incisors, after which the animals were weighed. Thereafter, the maxillary incisors were extracted with forceps and then, in randomly selected animals, reimplanted in the following manner. In one group, the incisors were extracted one after another and placed in a Petri dish. After the last incisor was extracted, the first was reimplanted and followed by the others in order of extraction. For each tooth, this procedure required approximately 60 s, i.e., it corresponded to what in clinical situations is normally regarded as an immediate reimplantation. In the other 2 groups of animals the incisors were extracted and reimplanted after 30 or 60 min extra-alveolar time, respectively. In these groups, all right incisors were kept wet, intraorally in saliva, while all left incisors were kept dry in a Petri dish, with the incisal edge resting at the bottom and the tip of the root leaning against the vertical wall of the dish. The alveolar sockets were not treated and no attempt was made to remove coagulated blood from the tooth socket before reimplantation. After reimplantation, the reimplanted teeth were joined with a band of a composite material, placed over the acid-etched buccal surfaces of the crowns. This type of splinting prevents the postoperative loss of a single tooth but permits a certain mobility of the whole block of teeth during mouth activities. The material was divided into 2 groups. Group I consisted of 17 animals, which received no other treatment than the one described above. Group II consisted of 15 animals that were given 4 mg/kg doxycycline (Vibramycin, Pfizer, Amboise, France), intravenously, at the time of anesthesia, approximately 20 min before extraction and, 2 mg/kg doxycycline (Milvet Ethicals, Pietermaritzburg, RSA) intramuscularly for 5 consecutive days after reimplantation. Doxycycline is a broad-spectrum antibiotic that, after a single therapeutic dose, was found in the fiuid of gingival pockets and in the alveolar serum after tooth extraction, in concentrations that exceeded the inhibitory level for most of the oral bacteria (21, 22). Two monkeys had only central incisors erupted and 7 teeth were lost during various experimental procedures. This left 117 teeth, 63 in the first and 54 in the second group, for evaluation. The distri-
Puip revascuiarizatien: systemic antibietics Table 1. Distribution of 63 reimplanted maxillary incisors (group I), according to the duration of extra-alveolar time, wet or dry storage and the width of the apical foramen Storage and extra-alveolar time (min) Width of apical foramen (mm) 2.1 Total
wet
dry
1
30
6 1 3 5
4 2 2 4 2
5 1 3 1
15
14
10
60
30
60
Total
4 3 2 4
4 2 5
8 20 8 19 8
13
11
63
bution of the material is presented in Tables 1 and 2. The width of the apical foramen was measured from the intraoral radiographs obtained before extraction (Fig. 1). The radiographs were magnified in a photographic enlarger 5 times and the enlarged image of the apical foramen was measured with a ruler graduated in mm to the nearest half or whole mm, so that the reading error was at most 0.25 mm. The values were then divided by the magnifying factor 5, so that the reading error was reduced to 0.05 mm. The width of apical foramen varied from less than 0.5 to 2.8 mm. Four size limits were used: 0.5, 1.0, 1.5 and 2.0 mm. The teeth with a width of apical foramen 0.5 mm or less were considered as mature and the others as immature. For reasons related to vaccine production, the observation time varied from 6 to 8 weeks. The animals were killed with an overdose of barbitone sodium and then the neck and head were retrogradely perfused with physiologic sahne, followed by 10% buffered formafin (23). The teeth were removed in tissue blocks and kept in 10% buffered formalin for 7 d, decalcified in EDTA at pH 6.8, embedded in celloidin-paraffin and then the blocks were serially sectioned at 5-6 |J,m. In each group, 4
blocks were sectioned transversely and the remaining blocks longitudinally to the long axis of the teeth. Each tenth slide, containing 2 or 3 sections, was stained with hematoxylin-eosin and each eleventh with a modified Gram stain, according to Brown & Brenn (24). In addition, the teeth in 9 blocks were stained with a modified Mallory stain according to Puchtler et al. (25). Histologically, the material was evaluated with regard to the presence and amount of vital tissue, occurrence of infiammatory changes and presence of bacteria in the pulpal lumen. The vital tissue in the pulpal lumen was recorded as absent (0) or present. When present, the amount of vital tissue was estimated to occupy: (1) one third of the pulpal lumen, when it was seen only in its apical portion; (2) one half, when it was seen close to but below the level of marginal bone; (3) two thirds, when it was over this level but the revascularization was not complete; and (CPR) the whole pulpal lumen, when mesenchymal cells and capillaries were seen adjacent to the coronal roof of the pulpal lumen in all slides, i.e., the pulpal lumen was completely revascularized (Fig. 2). In the sections cut perpendicularly to the long axis of the teeth, the amount of vital tissue was calculated from all the sections. The micro-organisms were recorded as present
Table 2. Distribution of 54 reimplanted maxillary incisors treated pre- and postoperatively with doxycycline (group II), according to the duration of extraalveolar time, wet or dry storage and the width of the apical foramen Storage and extra-alveolar time (min)
wet
Width of apical foramen (mm)
1
2.1 Total
12
30
dry
60
30
60
Total
6 2 2 1
3 6 1 1
5 3 1 2
7 1 1
4 15 21 9 5
11
11
11
9
54
Fig. 1. Preoperative radiograph of the right incisors. The width of apical foramen for central and lateral incisor was measured to 2.8 and 1.9 mm, respectively ( x 5).
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Fig. 2. A, B. Complete revascularization of the pulpal lumen in an immediately reimplanted incisor, cells and capillaries at the coronal roof of the pulpal lumen. Note the longitudinally arranged collagen fibers, ( x 15 and x 35).
when they were seen in the pulpal lumen and/or in the dentinal tubules adjacent to this cavity. Infiammatory changes were classified as slight (few, scattered infiammatory cells), moderate (accumulation of infiammatory cells) or severe (formation of abscess). The statistical evaluation of the material was performed using a log-linear model analysis via the Catmod procedure of SAS (26) by which the results in the experimental groups were compared. Then, the teeth of both groups were subdivided into those with and without complete pulp revascularization and the significance of investigated variables for the occurrence of complete revascularization was analysed. The significance level chosen was 0.05.
Table 3. Amount of vital tissue in the pulpal lumen of 117 reimplanted maxillary incisors, 63 not treated and 54 treated systemically with doxycyctlne, distributed according to the width of the apical foramen Amount of vital tissue*
Width of apical foramen (mm): 2.1
4 3
The statistical analyses of the material revealed no difference between the results obtained in group I, in which the animals were not treated, and in group II, in which the animals were treated systemically with doxycycline, either when whole groups or separate parameters were compared. The results were therefore pooled and are presented in Tables 3-6. In mature teeth, i.e., in the teeth with the width of apical foramen 0.5 mm or less, none or only an insignificant amount of vital tissue was present in the pulpal lumen (Table 3). The mature teeth were, therefore, excluded from the further statistical evaluation. In the remaining 105 immature teeth, complete pulp revascularization occurred in 19
Table 4. Amount of vital tissue in the pulpal lumen of 105 reimplanted immature maxillary incisors, 55 in not treated and 50 in animals treated systemically with doxycycline, distributed according to the duration of extraalveolar time and dry or wet storage
Total
18%** * 0 = none; 1 = only in the apical area; 2 = to the level of marginal bone; 3 = over the level of marginal bone; CPR = complete pulp revascularization. ** Frequency (%) of CPR in immature teeth.
160
Resuits
Storage and extra-alveolar time (min) Amount of vital tissue*
wet 1
0 1 2 3 CPR
1 4 5 1 12
Total
23
dry
30
60
30
60
Total
9
8 8 3
9 8 2
2
6 10 1 2 1
24 39 18 5 19
21
20
20
105
1 4 21
* For explanation, see Table 3.
Puip revascuiarizatien: systemic antibiotics Table 5. Presence of micro-organisms in the pulpal lumen of 105 reimplanted immature incisors, distributed according to the amount of vital tissue Amount of vital tissue* 2
3
CPR
24
1 38
18 1
CO CM CM OO
Total
1
CM CO
absent present
0
CM CD
Micro-organisms
24
39
18
5
19
105
Total
* For explanation, see Table 3.
teeth (18%). In these teeth the width of the apical foramen, i.e., the stage of root development, could not be shown to be statistically significant, neither for the amount of vital tissue present in the pulpal lumen nor for the occurrence of a complete revascularization. The amount of vital tissue in the pulpal lumen of immature teeth is distributed according to the extra-alveolar time and wet or dry storage in Table 4. No statistically significant difference in the amount of vital tissue or the frequency of complete revascularization could be found between the teeth stored dry or stored wet, either when the whole groups or the time intervals, i.e., 30 or 60 min, were compared. Regarding extra-alveolar time, the only statistically significant increase in the frequency of the complete revascularization (P = 0.05) found was for the group of immediately reimplanted teeth compared with the other groups. Micro-organisms were found in the pulpal lumen of all mature teeth. The presence of micro-organisms in immature teeth, distributed according to the amount of vital tissue in the pulpal lumen, is shown in Table 5. Micro-organisms were present in most of the teeth v/ith the exception of the 19 teeth with a completely revascularized pulp, in which micro-organisms were found only in one. The relationship between the presence of micro-organisms in the pulpal lumen and absence of the complete revascularization was statistically significant {P = 0.0001). No difference in occurrence of micro-organisms was found be-
Table 6. Presence of micro-organisms in the pulpal lumen of 105 reimplanted immature incisors, distributed according to the duration of extra-alveolar time and storage Storage Time (min)
wet 1
30
dry 60
30
60
Total
iVlicro-organisms absent present
13 10
4 17
3 18
2 18
1 19
CO CM CM OO
Total
23
21
21
20
20
105
tween the teeth stored wet or stored dry. Regarding the duration of extra-alveolar time, fewer microorganisms {P = 0.05) were found in the group of immediately reimplanted teeth compared with the other groups. There was no difference between the 30- and 60-min extra-alveolar time intervals (Table 6). The micro-organisms could be seen in all parts of the pulpal lumen. In 2 groups of immature teeth, however, the presence and localization of bacteria could be related to two particular sites. In the apical portion of the pulpal lumen of 27 teeth, 16 in group I and 11 in group II (Table 7), with no or only an insignificant amount of vital tissue present, heavy contaminated sites were seen between abscesses below the vital tissue and the necrotized pulp (Figs. 3, 4). These sites, identified as blood clots, contained tangled fibrin strands and platelets which in the modified Mallory stain (25) were stained blue. Around the bacterial colonies, the clots were either structureless, amorphous and stained darkly in the hematoxylin-eosin stain or the clot had lost its consistency. The microfiora was mixed, but the rods predominated in the superficial and the cocci in the deeper parts of the clots (Figs. 3, 4). The other bacterial site was seen in the cervical area of 65 teeth, at crushed cementum and dentin, damage that apparently was caused by forceps at the time of extraction. The crushed tissue was covered with plaque that contained micro-organisms, which were seen to penetrate from the plaque into the dentinal tubules of 46 teeth. Furthermore, this penetration of micro-organisms could be followed, through serial sections and via dentinal tubules, from the mechanical damage right into the pulpal
Table 7. Inflammatory changes in periapical tissues and in the pulpal lumen, presence of bacteria and findings of contaminated blood clots or contamination via mechanical damage cervically in 105 reimplanted immature incisors, distributed according to the amount of vital tissue in the pulpal lumen Amount of vital tissue in the pulpal lumen* 0
1
24
39
18
24
36
5
1 38
2 1 15
2 1 2
15 4
24
14 38
7 16
2 3
4 1
Demonstrable contamination through blood clot 13 7 mechanical damage**
14 13
12
1
1
Inflammation periapically Inflammation in the pulpal lumen none slight-moderate severe total necrosis dentin resorption Micro-organisms present
CPR
19
24
* For explanation see Table 3. ** Only the teeth in which micro-organisms could be followed from mechanical damage cervically to the pulpal lumen.
161
Cvei( et ai.
Fig. 3. A. An incisor stored 60 min wet before reimplantation. a: remnants of coagulated pulp tissue ( x8). B. Apical area of the tooth, a: periapical osteitis; b: abscess; c: blood clot ( x 15). C. a: leukocytes; b: blood clot, tangled strands and platelets; c: darkly stained area of the clot containing micro-organisms (x 140). D. The same site stained according to Brown & Brenn. a: leukocytes; b, c: micro-organisms in the blood clot, rods prevailing at periphery and cocci deeper in the clot ( x 140).
162
Pulp revascuiarizatien: systemic antibiotics
Fig. 4. A. Apical area of an incisor stored 30 min wet. a: abscess; b: blood clot; c: coagulated pulp tissue (x45). B. Appearance of coagulated pulp tissue ( x 145). C. a: accumulation of leukocytes; b: appearance of the blood clot ( x 145). D. The same site stained with Brown & Brenn stain, a: leukocytes; b and c: rods and cocci in the blood clot (x 170).
163
Cvek et al. lumen of 34 teeth (Fig. 5). Among these, 14 exhibited ingrowth of vital tissue to the half or more of the pulpal lumen, and one had a completely revascularized pulp (Fig. 6). There were no differences in the frequency of contaminated blood clots or contamination from cervical damage on the root surface between group I and group H. Histologic observations regarding inflammatory changes in the pulpal lumen and its relationship to the presence of micro-organisms are presented in Table 7. With the exception of the completely revascularized teeth, most of the inflammatory changes were severe, i.e., abscesses had formed between vital and necrotic tissue in the pulpal lumen. In these, polymorphonuclear leukocytes were the predominant cells, while other inflammatory cells were only occasionally seen. Of the completely revascularized teeth, 4 showed slight or moderate inflammation that, in one tooth, could be related to the presence of bacteria in the adjacent dentinal tubules (Fig. 6). With regard to inflammatory changes, internal resorption of dentin with adjacent multinucleated clast cells were noted in 25 teeth. The resorptions were limited and localized to the predentin and superficial layers of dentin (Fig. 7), with the exception of one tooth, in which the process was more extensive. Among 23 teeth, in which the vital tissue occupied from one half to two thirds of the pulpal lumen, 5 showed periapical inflammation. The remaining showed apical reparative processes, such as formation of hard tissue or ingrowth of bone, without inflammation periapically, although an abscess was present coronally in the pulpal lumen. In most of these teeth, the accumulation of leukocytes could be followed from a coronal abscess through vital tissue into the apical or periapical area of the tooth (Figs. 7, 8). It is also clear that inflammatory changes were closely related to the presence of micro-organisms (Table 7). Discussion The results were in accordance with previous clinical findings (16). In mature teeth complete pulp revascularization did not occur, whereas in immature teeth it occurred with the same frequency as in the clinically reimplanted teeth and, furthermore, it did not depend on the width of the apical foramen, i.e., the degree of root development, or storage conditions. A significantly increased frequency of complete pulp revascularization was only found in the group of immediately reimplanted teeth, but in these the frequency of bacteria found in the pulpal lumen was also significantly decreased. In the material as a whole, the occurrence of a complete pulp revascularization was highly dependent on the 164
absence of bacteria in the pulpal lumen {P = 0.0001). Such a dominant influence of bacteria, i.e., the interference of inflammatory changes with the process of revascularization, render any discussion about the significance of other factors academic. However, in most of incompletely revascularized immature teeth, vital tissue was present in a part of the pulpal lumen. In these teeth, the amount of vital tissue was not related to the width of apical foramen, extra-alveolar time or storage conditions but possibly was determined by the level at which ingrowing vital tissue was confronted with microorganisms in the pulpal lumen. One may, therefore, speculate that, in absence of micro-organisms, complete revascularization might have taken place in all immature teeth. When the teeth, for example, were experimentally transplanted in a way that diminished possibilities of contamination, complete revascularization also occurred in the mature teeth, although compared with immature teeth it required a longer time (9). Extra-alveolar time and storage conditions may not be critical for the occurrence of revascularization per se but, on the other hand, these factors could be decisive for the type of tissue that replaces necrotized pulp and thus for the type of hard tissue formed in the pulpal lumen after revascularization has been completed (14). These aspects will be discussed in a following article. The prophylactic, systemic treatment with doxycycline had no effect on the frequency of pulp revascularization nor on the occurrence of inflammatory changes. This is in agreement with clinical experience as well as with the results in retrospective reimplantation and transplantation studies (14, 19). In this study, the monkeys were given doxycycline shortly before extraction and for 5- consecutive days after reimplantation. The absence of effect on the frequency of bacteria present in the pulpal lumen seems best discussed in relation to the pathways for its contamination. One of these is anachoretic, by blood-borne bacteria. Such contamination of freshly damaged tissue could be demonstrated when bacteremia was created in experimental animals by intravenous injections of a bacterial suspension (27). Whether similar bacteremia occurred in some animals of the present material and the doxycycline treatment had prevented contamination appears unlikely but is a possibility that cannot be excluded. The root surface of extracted teeth may be contaminated during the extra-alveolar time (28), which in the present material is illustrated by the finding of contaminated blood clots, formed at the time of extraction or pushed into the pulpal lumen during the reimplantation. Regarding preoperative administration of doxycycline, it has been shown that, after a single therapeutic oral dose, the inhibi-
Puip revascuiarizatien: systemic antiiiietics
B
%^
Fig. 5. Penetration of micro-organisms from mechanical damage on the root surface cervically into the pulpal lumen ii m an incisor reimplanted after 30 min wet storage. Micro-organisms could be via dentinal tubules followed in serial sections (stained according to Brown & Brenn) from cervical damage right into the pulpal lumen. A, B. Mechanical damage on the root surface covered with plaque and penetration of bacteria into dentinal tubules ( x 30 and x 140). C. Micro-organisms in dentinal wall, close to the pulpal lumen ( x 150). D, E. Micro-organisms in the pulpal lumen ( x 25 and 180). F. Formation of bacterial colonies in the necrotized pulp (p) tissue (x 150).
tory concentration in the serum is reached after 3 h (29). This may indicate that after a dose given shortly before extraction, the concentration in tissues of the extracted teeth in the present material was not high enough to prevent bacterial establishment on the root surface. Thus, contaminated teeth were reimplanted into the alveoli, most of which were filled with coagulated blood, known as an
excellent substrate for the growth of micro-organisms. With respect to the postoperative treatment, it has been shown that systemic penicillin may penetrate into the blood clot formed in alveolar sockets after extraction (30), but opinions about its prophylactic effect, for example, after extraction of third molars, are not unanimous (31-33). As far as we know, the effect of systemic treatment with anti165
Cvek et ai.
Fig. 6. A. Cervical part of an immediately reimplanted and completely revascularized incisor, dilated blood vessels and resorption (arrows) of the dentinal wall ( x40). B. Clast cell in the resorption lesion and a few, scattered leukocytes (arrows) in the pulp tissue (x 145). C. Brown & Brenn stain of the resorption site, micro-organisms in the adjacent dentinal tubules (x 145). D. Mechanical damage on the root surface, penetration of micro-organisms which via dentinal tubules could be followed to the site of resorption. Brown & Brenn stain ( x 135).
166
Puip revascuiarizatien: systemic antibiotics
Fig. 7. A. Apical portion of an incisor reimplanted after 30 min dry storage, a: reparative hard tissue formation at the apex; b: ingrowing, inflamed vital tissue; c: abscess and resorption (arrow) of dentin ( x 15). Insert: clast cell at the resorption site ( x 140). B. Coronal portion of the tooth, mechanical damage on the root surface (arrows), a: colonies of micro-organisms in the necrotized pulp tissue ( X 15).
biotics on the bacteria already entrapped in a blood clot has not been investigated. However, an essential component of a blood clot is fibrin and it can, therefore, be compared with implanted fibrin clots. It has been shown that systemic antibiotics may diffuse into implanted fibrin clots but for most antibiotics, concentrations found in the clots were significantly lower than those in the serum (34—36). Thus, when the contaminated fibrin clots were implanted, systemic treatment with antibiotics prevented dissemination of the bacteria but not the formation of the local abscesses (37, 38). This may explain the finding of abscesses close to contaminated blood clots in the teeth of animals treated with doxycycline in this material. Another source of pulp contamination demonstrated as a new finding is the mechanical damage of cementum and dentin in the cervical area of the tooth. This damage, found in more than half of the teeth, was caused by fbrceps at the time of extraction. In accidentally luxated teeth similar damage can be caused by the fulcrum effect from the margin 01 alveolar bone. The crushed tissue was covered by plaque from which micro-organisms, probably attracted by the disintegration products from necrot d pulp, penetrated into the dentinal tubules.
This penetration could be followed in serial sections of 33 teeth, from damage to the pulpal lumen. It is probable that the same occurred in other teeth with cervical damage, though it could not be verified, as the histological sectioning and staining may have missed some of the micro-organisms present in the tissues. Regarding the effect of doxycycline, inhibitory concentrations of the drug were found in the alveolar serum after oral administration (21). It is, therefore, possible that, in this material, some preventive effect on the formation of plaque was exerted during the postoperative treatment but after this, plaque could form in the crushed tissues and from it, micro-organisms could penetrate to the necrotized pulp. It is difficult to know whether prolonged treatment with doxycycline or some other antibiotic would decrease the frequency of this contamination pattern. However, if any effect is to be expected, the treatment should be extended over an interval that includes the time needed for revascularization as well as for the subsequent apposition of hard tissue on the dentinal walls which, by sealing off the dentinal tubules, might prevent access of bacteria to the pulpal lumen. This study confirms previous clinical findings about frequency and predictability of pulp revascul-
167
CvBk et al.
w.n> '• ir^f#^sj z^. 5. An incisor reimplanted after 30 min wet storage. A. Penetration of micrO-organisms to coronal roof of the pulpal lumen, colonies in the necrotized pulp (p) (Brown & Brenn stain, x 100). B. Coronal part of the pulpal lumen, p: necrotic, contaminated pulp; a: abscess formation apically. C-E. Progression of the abscess (a) through grown-in or original vital tissue in the pulpal lumen towards apical area of the tooth, (B-E x 15). F. Note the dentin formed after reimplantation: arrow points the line between the original and new formed dentin ( x 33).
arization in reimplanted immature teeth. The dominant factor preventing occurrence of a complete pulp revascularization was presence of micro-organisms in 166
the pulpal lumen and the main pathway for its contamination seem to have been from a mechanical damage on the root surface in the cervical area of
Puip revascuiarization: systemic antibiotics the tooth. Prophylactic treatment with doxycycline failed to prevent the contamination of the infarcted pulp tissue or to eliminate micro-organisms from the pulpal lumen and other prophylactic methods should, therefore, be tested. However, the present results should not exclude administration of antibiotics after reimplantation of teeth. This may not increase the frequency of pulp revascularization in reimplanted teeth, but it may be of help in a separate tooth and may also enhance the healing of other injuries, such as those in periodontal tissues. The process of revascularization and the following formation of hard tissues in the pulpal lumen of reimplanted immature teeth will be discussed in a subsequent article.
I. Radiographic and clinical study of 110 human teeth replanted after accidental loss. Acta Odontol Scand 1966; 24: 263-86. 17. SuNDqviST C. Bacteriological studies of necrotic dental pulps. Umea: Umea University Odontol Diss No. 7, 1976. 18. ANDREASEN JO. Relationship between surface and inflammatory resorption and changes in the pulp after replantation of permanent incisors in monkeys. J Endod 1981; 7: 294-301. 19. SCHWARTZ O , BERGMAN P, KLAUSEN B. Autotransplantation of human teeth. A life-table analysis of prognostic factors. Int J Oral Surg 1985; 14: 245-58. 20. HAMMARSTROM E , BLOMLOF E , FEIGLIN B, ANDERSSON E ,
EiNDSKOG S. Replantation of teeth and antibiotic treatment. Endod Dent Traumatol 1986; 2: 51-7. 21. BYSTEDT H , DAHLBAGK A, NORD C - E . Concentration of azidocillin, erythromycin, doxycycline and clindamycin in dental alveolar serum after single oral doses. Int J Oral Surg 1977; 6: 65-74. 22. PASCALE D, CORDON J,
EAMSTER I, MANN P, SEIGER
M,
Concentration of doxycycline in human gingival fluid. J Clin Periodontol 1986; 13: 841-4. RETIEF DH, AUSTIN J C . The vervet monkey {Cercopithecus aethiops) as an experimental model for pulpal studies. J Dent Assoc S Afr 1973; 28: 98-103. BROWN J H , BRENN E. Method for differential staining of gram-positive and gram-negative bacteria in tissue sections. Bull Johns Hopkins Hosp 1931; 48: 69 73. PUCHTLER PD, SWEAT F, DOSS N . A one-hour phosphotungistic acid-hematoxylin stain. Am J Clin Pathol 1963; 40: 334—7. Statistical Analysis System. SAS Users Guide: Statistics, Version 5 edition. Cary NC: SAS Institute, 1985. DELIVANIS PD, FAN V S C . The localization of blood-borne bacteria in instrumented unfilled and overinstrumented CAnah.J Endod 1984; 10: 521-4. EiNDSKOG E, BLOMLOF E , HAMMARSTROM E . Mitoses and micro-organisms in the periodontal membrane after storage in milk or saliva. Scand J Dent Res 1983; 91: 465-72. BYSTEDT H . Antibiotics in oral surgery. Stockholm: Karolinska Institute Odontol Diss, 1979. JUNIPER RP. Penicillin - the duration of its activity in blood clots. Br J Oral Surg 1972; 9: 222-7. PATERSON JA, CARDO V A , STRATIGOS G T . An examination of antibiotic prophylaxis in oral and maxillofacial surgery. J Oral Surg 1970; 28: 753-9. CuRRAN JB, KENNETT S, YOUNG AR. An assessment of the use of prophylactic antibiotics in third molar surgery. Int J Oral Surg 1974; 3: 1-6. BYSTEDT H , NORD C-E, NORDENRAM A. Effect of azidocillin, erythromycin, clindamycin and doxycycline on postoperative complications after surgical removal of impacted third molars. Int J Oral Surg 1980; 9: 157-65. BARZA M , SAMUELSON T, WEINSTEIN E. Penetration of antibiotics into fibrin loci in vivo. IE Comparison of nine antibiotics: effect of dose and degree of protein binding. J Infect Dis 1974; 129: 66-72. BERGAN T. Pharmacokinetics of tissue penetration of antibiotics. Rev Infect Dis 1981; 3: 45-666. EAVOIE GE, BERGERON MG. Influence of four modes of administration on penetration of aztreonam, cefuroxime and ampicillin into interstitial fluid and fibrin clots and on in vivo efficiacy against Haemophilus influenze. Antimicrob Agents Chemother 1985; 28: 404-12. HAU T, NISHIKAWA R A , PHUANGSAB A. The effect of bacterial trapping by fibrin on the efliciacy of systemic antibiotics in experimental peritonitis. Surg Gynecol Obstet 1983; 157: 252-6. HAU T, JACOBS DE, HAWKINS N E . Antibiotics fail to prevent abscess formation secondary to bacteria trapped in fibrin clots. Arch Surg 1986; 121: 163-8. ARNT W.
References 1. NoRDENRAM A. Autotransplantation of teeth. A clinical and experimental investigation. Acta Odontol Scand 1963; 21: Suppl 33. 2. OHMAN A. Healing and sensitivity to pain in young replanted human teeth. An experimental, clinical and histological study. Odontol Tidskr 1965; 73: 168-227. 3. FoNG CH; MORRIS H , GRANT T H , BERGER J. Experimental tooth transplantation in the Rhesus monkey. J Dent Res 1967; 46: 492-6. 4. MoNSOUR FNT. Pulpal changes following the reimplantation of teeth in dogs: a histological study. Aust Dent J 1971; 16: 227-31. 5. SHEPARD PR, BuRiCH RL. Effects of extra-alveolar exposure and multiple avulsions on revascularization of reimplanted teeth in dogs. J Dent Res 1980; 59: 140. 6. ANDREASEN JO. Effect of extra-alveolar period and storage media upon periodontal and pulpal healing after replantation of mature permanent incisors in monkeys. Int J Oral Surg 1981; 10: 43-53. 7. SKOGLUND A, TRONSTAD L. Pulpal changes in replanted and autotransplanted immature teeth of dogs. J Fndod 1981; 7: 309-16. 8. SKOGLUND A. Vascular changes in replanted and autotransplanted apicoectomized mature teeth in dogs. Int J Oral Surg 1981; 10: 43-53. 9. BARRET A P , READ PC. Revascularization of mouse tooth isographs and allographs using autoradiography and carbon-profusion. Arch Oral Biol 1981; 26: 541-5. 10. MoNsouR FNT, ATKINS KF. Proliferation of pulpal tissue following early transplantation of developing teeth. J Oral Maxillofac Surg 1983; 41: 738-42. 11. KRISTERSON E , ANDREASEN J O . Influence of root development on periodontal and pulpal healing after replantation of incisors in monkeys. Int J Oral Surg 1984; 13: 313-23. 12. HEYERAAS KJ, MYKING AM. Pulpal blood flow in immature dog teeth after replantation. Scand J Dent Res 1985; 93: 227-38. 13. MoNsouR FNT, ATKINS KF. Aberrations in pulpal histology and dentinogenesis in transplanted erupting teeth. J Oral Maxillofac Surg 1985; 43: 8-13. 14. KLING M , CVEK M , MEJARE I. Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol 1986; 2: 83-9. 55. ANDREASEN J O . Traumatic injuries of the teeth. 2nd ed. Copenhagen: Munksgaard, 1981; 223-4, 232. !6. ANDREASEN JO, HJORTING-HANSEN E . Replantation of teeth.
23.
24.
25. 26. 27.
28.
29. 30. 31.
32.
33.
34.
35. 36.
37.
38.
169
In immature and occasionally in mature reimplanted teeth the pulp can be completely revascularized, usually within 30 days after reimplantation (1-14). It has therefore been recommended that, in reimplanted immature teeth, endodontic treatment be postponed until infiammatory changes and/or inflammatory root resorption are seen in the control radiographs (15). These changes, however, may progress very rapidly in immature teeth (16), worsening the prognosis of the tooth. Therefore, it would be desirable if, on the basis of clinically available variables, the prognosis of pulp revasculariza-
Miemir Cvek\ Peter Cieaten-Jenes^ Jehn Austin^ Jehn Lewnie^ Margareta K\m\ Paui Fatti^ ^Departmenf of Pedodontics, Eastman Institute, Stockholm, Sweden, ^Dental Research Institute, MRC/University of Witwatersrand, Johannesburg, ^MRC/Laboratory Animal Unite, Johannesburg, "Division of Maxillo-Facial and Oral Department of Surgery, ^Department of Statistics and Actuarial Science, University of the Witwatersrand, Johannesburg, Republic of South Africa
Key words: doxycycline; reimplantation; revascularization; pulpal revascularization; dental pulp. Miomir Cvek, Department of Pedodontics, Eastmaninstltutet, Dalagatan 11, S-113 24 Stockholm, Sweden. Accepted for publication January 31, 1990.
tion could be more distinctly predicted and, eventually, improved by some therapeutic measures. From experimental studies, it can be concluded that some factors, such as the width of apical foramen, i.e. the stage of root development (2, 7, U), duration of extra-alveolar time and storage conditions (5, 6), could indicate the occurrence of pulp revascularization. A recent study of clinically reimplanted incisors, however, could only confirm the predictability of these factors to a hmited extent (14). In this study, pulp revascularization did occur only in immature teeth, but in these teeth it was not related to the width 157
Cvek et ai. of apical foramen, that is, to the stage of root development. Regarding the extent of extra-alveolar time and dry or wet storage, a weak but statistically significant increase in frequency of revascularization was found only in the teeth reimplanted within 45 min after avulsion, irrespective of storage conditions. However, revascularization also occurred in teeth with an extra-alveolar time that exceeded 2 h and included periods of 60 min dry storage. Furthermore, the frequency of revascularization was low (18%), when compared with the results obtained in experimental studies. All teeth in which revascularization did not occur exhibited a periapical radiolucency or an external infiammatory root resorption, i.e. processes known to be closely related to the presence of micro-organisms in the pulpal lumen (17, 18). Micro-organisms have been discussed as a reason for the occurrence of abscesses between vital and necrotic tissue in the pulpal lumen of reimplanted teeth (2), but this has not been experimentally investigated nor the presence of micro-organisms in the pulpal lumen explained. Prophylactic treatment with antibiotics has been recommended after reimplantation of accidentally avulsed teeth (15). The effects of such treatment on pulpal and/or periodontal healing, however, are, controversial. In clinical studies, no difference in the frequency of subsequent infiammatory processes, related to the presence of micro-organisms, could be found between groups of patients that were or were not treated svstemicallv with antibiotics after reimplantation or transplantation of teeth (14, 19). In a recent experimental study, on the other hand, a complete absence of external infiammatory root resorption was reported in mature reimplanted monkey teeth, which were not treated endodontically but the animals received systemic treatment with antibiotics for 6 d after reimplantation (20). As far as we know, no experimental study dealing with the effect of antibiotics on pulpal healing has been recorded. A series of experiments designed to imitate clinical conditions were therefore performed, using maxillary incisors in monkeys, in order further to study healing of the pulp in young reimplanted teeth. The purpose of the present study was to evaluate the significance of width of apical foramen, extent of extra-alveolar time, dry or wet storage, presence of micro-organisms in the pulpal lumen and systemic treatment with doxycycline, on the occurrence of complete pulp revascularization, i.e., the only type of pulpal healing to be aimed for in therapeutically reimplanted teeth. iWateriai and metheds The material consisted of 128 maxillary incisors in 32 young vervet monkeys [Cercopithecus aethiopspyger158
ythrus), primarily used in the production of poliomyelitis vaccine. The animals selected had unerupted permanent maxillary canines or had canines erupted, but not more than about the half of the crown of adjacent lateral incisor. The animals were immobifized with ketamine hydrochloride 10 mg/kg (Parke Davis, Johannesburg, RSA) and then anesthetized with pentobarbitone sodium (Parke Davis). Two intraoral radiographs were taken, one for the left and one for the right incisors, after which the animals were weighed. Thereafter, the maxillary incisors were extracted with forceps and then, in randomly selected animals, reimplanted in the following manner. In one group, the incisors were extracted one after another and placed in a Petri dish. After the last incisor was extracted, the first was reimplanted and followed by the others in order of extraction. For each tooth, this procedure required approximately 60 s, i.e., it corresponded to what in clinical situations is normally regarded as an immediate reimplantation. In the other 2 groups of animals the incisors were extracted and reimplanted after 30 or 60 min extra-alveolar time, respectively. In these groups, all right incisors were kept wet, intraorally in saliva, while all left incisors were kept dry in a Petri dish, with the incisal edge resting at the bottom and the tip of the root leaning against the vertical wall of the dish. The alveolar sockets were not treated and no attempt was made to remove coagulated blood from the tooth socket before reimplantation. After reimplantation, the reimplanted teeth were joined with a band of a composite material, placed over the acid-etched buccal surfaces of the crowns. This type of splinting prevents the postoperative loss of a single tooth but permits a certain mobility of the whole block of teeth during mouth activities. The material was divided into 2 groups. Group I consisted of 17 animals, which received no other treatment than the one described above. Group II consisted of 15 animals that were given 4 mg/kg doxycycline (Vibramycin, Pfizer, Amboise, France), intravenously, at the time of anesthesia, approximately 20 min before extraction and, 2 mg/kg doxycycline (Milvet Ethicals, Pietermaritzburg, RSA) intramuscularly for 5 consecutive days after reimplantation. Doxycycline is a broad-spectrum antibiotic that, after a single therapeutic dose, was found in the fiuid of gingival pockets and in the alveolar serum after tooth extraction, in concentrations that exceeded the inhibitory level for most of the oral bacteria (21, 22). Two monkeys had only central incisors erupted and 7 teeth were lost during various experimental procedures. This left 117 teeth, 63 in the first and 54 in the second group, for evaluation. The distri-
Puip revascuiarizatien: systemic antibietics Table 1. Distribution of 63 reimplanted maxillary incisors (group I), according to the duration of extra-alveolar time, wet or dry storage and the width of the apical foramen Storage and extra-alveolar time (min) Width of apical foramen (mm) 2.1 Total
wet
dry
1
30
6 1 3 5
4 2 2 4 2
5 1 3 1
15
14
10
60
30
60
Total
4 3 2 4
4 2 5
8 20 8 19 8
13
11
63
bution of the material is presented in Tables 1 and 2. The width of the apical foramen was measured from the intraoral radiographs obtained before extraction (Fig. 1). The radiographs were magnified in a photographic enlarger 5 times and the enlarged image of the apical foramen was measured with a ruler graduated in mm to the nearest half or whole mm, so that the reading error was at most 0.25 mm. The values were then divided by the magnifying factor 5, so that the reading error was reduced to 0.05 mm. The width of apical foramen varied from less than 0.5 to 2.8 mm. Four size limits were used: 0.5, 1.0, 1.5 and 2.0 mm. The teeth with a width of apical foramen 0.5 mm or less were considered as mature and the others as immature. For reasons related to vaccine production, the observation time varied from 6 to 8 weeks. The animals were killed with an overdose of barbitone sodium and then the neck and head were retrogradely perfused with physiologic sahne, followed by 10% buffered formafin (23). The teeth were removed in tissue blocks and kept in 10% buffered formalin for 7 d, decalcified in EDTA at pH 6.8, embedded in celloidin-paraffin and then the blocks were serially sectioned at 5-6 |J,m. In each group, 4
blocks were sectioned transversely and the remaining blocks longitudinally to the long axis of the teeth. Each tenth slide, containing 2 or 3 sections, was stained with hematoxylin-eosin and each eleventh with a modified Gram stain, according to Brown & Brenn (24). In addition, the teeth in 9 blocks were stained with a modified Mallory stain according to Puchtler et al. (25). Histologically, the material was evaluated with regard to the presence and amount of vital tissue, occurrence of infiammatory changes and presence of bacteria in the pulpal lumen. The vital tissue in the pulpal lumen was recorded as absent (0) or present. When present, the amount of vital tissue was estimated to occupy: (1) one third of the pulpal lumen, when it was seen only in its apical portion; (2) one half, when it was seen close to but below the level of marginal bone; (3) two thirds, when it was over this level but the revascularization was not complete; and (CPR) the whole pulpal lumen, when mesenchymal cells and capillaries were seen adjacent to the coronal roof of the pulpal lumen in all slides, i.e., the pulpal lumen was completely revascularized (Fig. 2). In the sections cut perpendicularly to the long axis of the teeth, the amount of vital tissue was calculated from all the sections. The micro-organisms were recorded as present
Table 2. Distribution of 54 reimplanted maxillary incisors treated pre- and postoperatively with doxycycline (group II), according to the duration of extraalveolar time, wet or dry storage and the width of the apical foramen Storage and extra-alveolar time (min)
wet
Width of apical foramen (mm)
1
2.1 Total
12
30
dry
60
30
60
Total
6 2 2 1
3 6 1 1
5 3 1 2
7 1 1
4 15 21 9 5
11
11
11
9
54
Fig. 1. Preoperative radiograph of the right incisors. The width of apical foramen for central and lateral incisor was measured to 2.8 and 1.9 mm, respectively ( x 5).
159
Cvek et ai.
Fig. 2. A, B. Complete revascularization of the pulpal lumen in an immediately reimplanted incisor, cells and capillaries at the coronal roof of the pulpal lumen. Note the longitudinally arranged collagen fibers, ( x 15 and x 35).
when they were seen in the pulpal lumen and/or in the dentinal tubules adjacent to this cavity. Infiammatory changes were classified as slight (few, scattered infiammatory cells), moderate (accumulation of infiammatory cells) or severe (formation of abscess). The statistical evaluation of the material was performed using a log-linear model analysis via the Catmod procedure of SAS (26) by which the results in the experimental groups were compared. Then, the teeth of both groups were subdivided into those with and without complete pulp revascularization and the significance of investigated variables for the occurrence of complete revascularization was analysed. The significance level chosen was 0.05.
Table 3. Amount of vital tissue in the pulpal lumen of 117 reimplanted maxillary incisors, 63 not treated and 54 treated systemically with doxycyctlne, distributed according to the width of the apical foramen Amount of vital tissue*
Width of apical foramen (mm): 2.1
4 3
The statistical analyses of the material revealed no difference between the results obtained in group I, in which the animals were not treated, and in group II, in which the animals were treated systemically with doxycycline, either when whole groups or separate parameters were compared. The results were therefore pooled and are presented in Tables 3-6. In mature teeth, i.e., in the teeth with the width of apical foramen 0.5 mm or less, none or only an insignificant amount of vital tissue was present in the pulpal lumen (Table 3). The mature teeth were, therefore, excluded from the further statistical evaluation. In the remaining 105 immature teeth, complete pulp revascularization occurred in 19
Table 4. Amount of vital tissue in the pulpal lumen of 105 reimplanted immature maxillary incisors, 55 in not treated and 50 in animals treated systemically with doxycycline, distributed according to the duration of extraalveolar time and dry or wet storage
Total
18%** * 0 = none; 1 = only in the apical area; 2 = to the level of marginal bone; 3 = over the level of marginal bone; CPR = complete pulp revascularization. ** Frequency (%) of CPR in immature teeth.
160
Resuits
Storage and extra-alveolar time (min) Amount of vital tissue*
wet 1
0 1 2 3 CPR
1 4 5 1 12
Total
23
dry
30
60
30
60
Total
9
8 8 3
9 8 2
2
6 10 1 2 1
24 39 18 5 19
21
20
20
105
1 4 21
* For explanation, see Table 3.
Puip revascuiarizatien: systemic antibiotics Table 5. Presence of micro-organisms in the pulpal lumen of 105 reimplanted immature incisors, distributed according to the amount of vital tissue Amount of vital tissue* 2
3
CPR
24
1 38
18 1
CO CM CM OO
Total
1
CM CO
absent present
0
CM CD
Micro-organisms
24
39
18
5
19
105
Total
* For explanation, see Table 3.
teeth (18%). In these teeth the width of the apical foramen, i.e., the stage of root development, could not be shown to be statistically significant, neither for the amount of vital tissue present in the pulpal lumen nor for the occurrence of a complete revascularization. The amount of vital tissue in the pulpal lumen of immature teeth is distributed according to the extra-alveolar time and wet or dry storage in Table 4. No statistically significant difference in the amount of vital tissue or the frequency of complete revascularization could be found between the teeth stored dry or stored wet, either when the whole groups or the time intervals, i.e., 30 or 60 min, were compared. Regarding extra-alveolar time, the only statistically significant increase in the frequency of the complete revascularization (P = 0.05) found was for the group of immediately reimplanted teeth compared with the other groups. Micro-organisms were found in the pulpal lumen of all mature teeth. The presence of micro-organisms in immature teeth, distributed according to the amount of vital tissue in the pulpal lumen, is shown in Table 5. Micro-organisms were present in most of the teeth v/ith the exception of the 19 teeth with a completely revascularized pulp, in which micro-organisms were found only in one. The relationship between the presence of micro-organisms in the pulpal lumen and absence of the complete revascularization was statistically significant {P = 0.0001). No difference in occurrence of micro-organisms was found be-
Table 6. Presence of micro-organisms in the pulpal lumen of 105 reimplanted immature incisors, distributed according to the duration of extra-alveolar time and storage Storage Time (min)
wet 1
30
dry 60
30
60
Total
iVlicro-organisms absent present
13 10
4 17
3 18
2 18
1 19
CO CM CM OO
Total
23
21
21
20
20
105
tween the teeth stored wet or stored dry. Regarding the duration of extra-alveolar time, fewer microorganisms {P = 0.05) were found in the group of immediately reimplanted teeth compared with the other groups. There was no difference between the 30- and 60-min extra-alveolar time intervals (Table 6). The micro-organisms could be seen in all parts of the pulpal lumen. In 2 groups of immature teeth, however, the presence and localization of bacteria could be related to two particular sites. In the apical portion of the pulpal lumen of 27 teeth, 16 in group I and 11 in group II (Table 7), with no or only an insignificant amount of vital tissue present, heavy contaminated sites were seen between abscesses below the vital tissue and the necrotized pulp (Figs. 3, 4). These sites, identified as blood clots, contained tangled fibrin strands and platelets which in the modified Mallory stain (25) were stained blue. Around the bacterial colonies, the clots were either structureless, amorphous and stained darkly in the hematoxylin-eosin stain or the clot had lost its consistency. The microfiora was mixed, but the rods predominated in the superficial and the cocci in the deeper parts of the clots (Figs. 3, 4). The other bacterial site was seen in the cervical area of 65 teeth, at crushed cementum and dentin, damage that apparently was caused by forceps at the time of extraction. The crushed tissue was covered with plaque that contained micro-organisms, which were seen to penetrate from the plaque into the dentinal tubules of 46 teeth. Furthermore, this penetration of micro-organisms could be followed, through serial sections and via dentinal tubules, from the mechanical damage right into the pulpal
Table 7. Inflammatory changes in periapical tissues and in the pulpal lumen, presence of bacteria and findings of contaminated blood clots or contamination via mechanical damage cervically in 105 reimplanted immature incisors, distributed according to the amount of vital tissue in the pulpal lumen Amount of vital tissue in the pulpal lumen* 0
1
24
39
18
24
36
5
1 38
2 1 15
2 1 2
15 4
24
14 38
7 16
2 3
4 1
Demonstrable contamination through blood clot 13 7 mechanical damage**
14 13
12
1
1
Inflammation periapically Inflammation in the pulpal lumen none slight-moderate severe total necrosis dentin resorption Micro-organisms present
CPR
19
24
* For explanation see Table 3. ** Only the teeth in which micro-organisms could be followed from mechanical damage cervically to the pulpal lumen.
161
Cvei( et ai.
Fig. 3. A. An incisor stored 60 min wet before reimplantation. a: remnants of coagulated pulp tissue ( x8). B. Apical area of the tooth, a: periapical osteitis; b: abscess; c: blood clot ( x 15). C. a: leukocytes; b: blood clot, tangled strands and platelets; c: darkly stained area of the clot containing micro-organisms (x 140). D. The same site stained according to Brown & Brenn. a: leukocytes; b, c: micro-organisms in the blood clot, rods prevailing at periphery and cocci deeper in the clot ( x 140).
162
Pulp revascuiarizatien: systemic antibiotics
Fig. 4. A. Apical area of an incisor stored 30 min wet. a: abscess; b: blood clot; c: coagulated pulp tissue (x45). B. Appearance of coagulated pulp tissue ( x 145). C. a: accumulation of leukocytes; b: appearance of the blood clot ( x 145). D. The same site stained with Brown & Brenn stain, a: leukocytes; b and c: rods and cocci in the blood clot (x 170).
163
Cvek et al. lumen of 34 teeth (Fig. 5). Among these, 14 exhibited ingrowth of vital tissue to the half or more of the pulpal lumen, and one had a completely revascularized pulp (Fig. 6). There were no differences in the frequency of contaminated blood clots or contamination from cervical damage on the root surface between group I and group H. Histologic observations regarding inflammatory changes in the pulpal lumen and its relationship to the presence of micro-organisms are presented in Table 7. With the exception of the completely revascularized teeth, most of the inflammatory changes were severe, i.e., abscesses had formed between vital and necrotic tissue in the pulpal lumen. In these, polymorphonuclear leukocytes were the predominant cells, while other inflammatory cells were only occasionally seen. Of the completely revascularized teeth, 4 showed slight or moderate inflammation that, in one tooth, could be related to the presence of bacteria in the adjacent dentinal tubules (Fig. 6). With regard to inflammatory changes, internal resorption of dentin with adjacent multinucleated clast cells were noted in 25 teeth. The resorptions were limited and localized to the predentin and superficial layers of dentin (Fig. 7), with the exception of one tooth, in which the process was more extensive. Among 23 teeth, in which the vital tissue occupied from one half to two thirds of the pulpal lumen, 5 showed periapical inflammation. The remaining showed apical reparative processes, such as formation of hard tissue or ingrowth of bone, without inflammation periapically, although an abscess was present coronally in the pulpal lumen. In most of these teeth, the accumulation of leukocytes could be followed from a coronal abscess through vital tissue into the apical or periapical area of the tooth (Figs. 7, 8). It is also clear that inflammatory changes were closely related to the presence of micro-organisms (Table 7). Discussion The results were in accordance with previous clinical findings (16). In mature teeth complete pulp revascularization did not occur, whereas in immature teeth it occurred with the same frequency as in the clinically reimplanted teeth and, furthermore, it did not depend on the width of the apical foramen, i.e., the degree of root development, or storage conditions. A significantly increased frequency of complete pulp revascularization was only found in the group of immediately reimplanted teeth, but in these the frequency of bacteria found in the pulpal lumen was also significantly decreased. In the material as a whole, the occurrence of a complete pulp revascularization was highly dependent on the 164
absence of bacteria in the pulpal lumen {P = 0.0001). Such a dominant influence of bacteria, i.e., the interference of inflammatory changes with the process of revascularization, render any discussion about the significance of other factors academic. However, in most of incompletely revascularized immature teeth, vital tissue was present in a part of the pulpal lumen. In these teeth, the amount of vital tissue was not related to the width of apical foramen, extra-alveolar time or storage conditions but possibly was determined by the level at which ingrowing vital tissue was confronted with microorganisms in the pulpal lumen. One may, therefore, speculate that, in absence of micro-organisms, complete revascularization might have taken place in all immature teeth. When the teeth, for example, were experimentally transplanted in a way that diminished possibilities of contamination, complete revascularization also occurred in the mature teeth, although compared with immature teeth it required a longer time (9). Extra-alveolar time and storage conditions may not be critical for the occurrence of revascularization per se but, on the other hand, these factors could be decisive for the type of tissue that replaces necrotized pulp and thus for the type of hard tissue formed in the pulpal lumen after revascularization has been completed (14). These aspects will be discussed in a following article. The prophylactic, systemic treatment with doxycycline had no effect on the frequency of pulp revascularization nor on the occurrence of inflammatory changes. This is in agreement with clinical experience as well as with the results in retrospective reimplantation and transplantation studies (14, 19). In this study, the monkeys were given doxycycline shortly before extraction and for 5- consecutive days after reimplantation. The absence of effect on the frequency of bacteria present in the pulpal lumen seems best discussed in relation to the pathways for its contamination. One of these is anachoretic, by blood-borne bacteria. Such contamination of freshly damaged tissue could be demonstrated when bacteremia was created in experimental animals by intravenous injections of a bacterial suspension (27). Whether similar bacteremia occurred in some animals of the present material and the doxycycline treatment had prevented contamination appears unlikely but is a possibility that cannot be excluded. The root surface of extracted teeth may be contaminated during the extra-alveolar time (28), which in the present material is illustrated by the finding of contaminated blood clots, formed at the time of extraction or pushed into the pulpal lumen during the reimplantation. Regarding preoperative administration of doxycycline, it has been shown that, after a single therapeutic oral dose, the inhibi-
Puip revascuiarizatien: systemic antiiiietics
B
%^
Fig. 5. Penetration of micro-organisms from mechanical damage on the root surface cervically into the pulpal lumen ii m an incisor reimplanted after 30 min wet storage. Micro-organisms could be via dentinal tubules followed in serial sections (stained according to Brown & Brenn) from cervical damage right into the pulpal lumen. A, B. Mechanical damage on the root surface covered with plaque and penetration of bacteria into dentinal tubules ( x 30 and x 140). C. Micro-organisms in dentinal wall, close to the pulpal lumen ( x 150). D, E. Micro-organisms in the pulpal lumen ( x 25 and 180). F. Formation of bacterial colonies in the necrotized pulp (p) tissue (x 150).
tory concentration in the serum is reached after 3 h (29). This may indicate that after a dose given shortly before extraction, the concentration in tissues of the extracted teeth in the present material was not high enough to prevent bacterial establishment on the root surface. Thus, contaminated teeth were reimplanted into the alveoli, most of which were filled with coagulated blood, known as an
excellent substrate for the growth of micro-organisms. With respect to the postoperative treatment, it has been shown that systemic penicillin may penetrate into the blood clot formed in alveolar sockets after extraction (30), but opinions about its prophylactic effect, for example, after extraction of third molars, are not unanimous (31-33). As far as we know, the effect of systemic treatment with anti165
Cvek et ai.
Fig. 6. A. Cervical part of an immediately reimplanted and completely revascularized incisor, dilated blood vessels and resorption (arrows) of the dentinal wall ( x40). B. Clast cell in the resorption lesion and a few, scattered leukocytes (arrows) in the pulp tissue (x 145). C. Brown & Brenn stain of the resorption site, micro-organisms in the adjacent dentinal tubules (x 145). D. Mechanical damage on the root surface, penetration of micro-organisms which via dentinal tubules could be followed to the site of resorption. Brown & Brenn stain ( x 135).
166
Puip revascuiarizatien: systemic antibiotics
Fig. 7. A. Apical portion of an incisor reimplanted after 30 min dry storage, a: reparative hard tissue formation at the apex; b: ingrowing, inflamed vital tissue; c: abscess and resorption (arrow) of dentin ( x 15). Insert: clast cell at the resorption site ( x 140). B. Coronal portion of the tooth, mechanical damage on the root surface (arrows), a: colonies of micro-organisms in the necrotized pulp tissue ( X 15).
biotics on the bacteria already entrapped in a blood clot has not been investigated. However, an essential component of a blood clot is fibrin and it can, therefore, be compared with implanted fibrin clots. It has been shown that systemic antibiotics may diffuse into implanted fibrin clots but for most antibiotics, concentrations found in the clots were significantly lower than those in the serum (34—36). Thus, when the contaminated fibrin clots were implanted, systemic treatment with antibiotics prevented dissemination of the bacteria but not the formation of the local abscesses (37, 38). This may explain the finding of abscesses close to contaminated blood clots in the teeth of animals treated with doxycycline in this material. Another source of pulp contamination demonstrated as a new finding is the mechanical damage of cementum and dentin in the cervical area of the tooth. This damage, found in more than half of the teeth, was caused by fbrceps at the time of extraction. In accidentally luxated teeth similar damage can be caused by the fulcrum effect from the margin 01 alveolar bone. The crushed tissue was covered by plaque from which micro-organisms, probably attracted by the disintegration products from necrot d pulp, penetrated into the dentinal tubules.
This penetration could be followed in serial sections of 33 teeth, from damage to the pulpal lumen. It is probable that the same occurred in other teeth with cervical damage, though it could not be verified, as the histological sectioning and staining may have missed some of the micro-organisms present in the tissues. Regarding the effect of doxycycline, inhibitory concentrations of the drug were found in the alveolar serum after oral administration (21). It is, therefore, possible that, in this material, some preventive effect on the formation of plaque was exerted during the postoperative treatment but after this, plaque could form in the crushed tissues and from it, micro-organisms could penetrate to the necrotized pulp. It is difficult to know whether prolonged treatment with doxycycline or some other antibiotic would decrease the frequency of this contamination pattern. However, if any effect is to be expected, the treatment should be extended over an interval that includes the time needed for revascularization as well as for the subsequent apposition of hard tissue on the dentinal walls which, by sealing off the dentinal tubules, might prevent access of bacteria to the pulpal lumen. This study confirms previous clinical findings about frequency and predictability of pulp revascul-
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w.n> '• ir^f#^sj z^. 5. An incisor reimplanted after 30 min wet storage. A. Penetration of micrO-organisms to coronal roof of the pulpal lumen, colonies in the necrotized pulp (p) (Brown & Brenn stain, x 100). B. Coronal part of the pulpal lumen, p: necrotic, contaminated pulp; a: abscess formation apically. C-E. Progression of the abscess (a) through grown-in or original vital tissue in the pulpal lumen towards apical area of the tooth, (B-E x 15). F. Note the dentin formed after reimplantation: arrow points the line between the original and new formed dentin ( x 33).
arization in reimplanted immature teeth. The dominant factor preventing occurrence of a complete pulp revascularization was presence of micro-organisms in 166
the pulpal lumen and the main pathway for its contamination seem to have been from a mechanical damage on the root surface in the cervical area of
Puip revascuiarization: systemic antibiotics the tooth. Prophylactic treatment with doxycycline failed to prevent the contamination of the infarcted pulp tissue or to eliminate micro-organisms from the pulpal lumen and other prophylactic methods should, therefore, be tested. However, the present results should not exclude administration of antibiotics after reimplantation of teeth. This may not increase the frequency of pulp revascularization in reimplanted teeth, but it may be of help in a separate tooth and may also enhance the healing of other injuries, such as those in periodontal tissues. The process of revascularization and the following formation of hard tissues in the pulpal lumen of reimplanted immature teeth will be discussed in a subsequent article.
I. Radiographic and clinical study of 110 human teeth replanted after accidental loss. Acta Odontol Scand 1966; 24: 263-86. 17. SuNDqviST C. Bacteriological studies of necrotic dental pulps. Umea: Umea University Odontol Diss No. 7, 1976. 18. ANDREASEN JO. Relationship between surface and inflammatory resorption and changes in the pulp after replantation of permanent incisors in monkeys. J Endod 1981; 7: 294-301. 19. SCHWARTZ O , BERGMAN P, KLAUSEN B. Autotransplantation of human teeth. A life-table analysis of prognostic factors. Int J Oral Surg 1985; 14: 245-58. 20. HAMMARSTROM E , BLOMLOF E , FEIGLIN B, ANDERSSON E ,
EiNDSKOG S. Replantation of teeth and antibiotic treatment. Endod Dent Traumatol 1986; 2: 51-7. 21. BYSTEDT H , DAHLBAGK A, NORD C - E . Concentration of azidocillin, erythromycin, doxycycline and clindamycin in dental alveolar serum after single oral doses. Int J Oral Surg 1977; 6: 65-74. 22. PASCALE D, CORDON J,
EAMSTER I, MANN P, SEIGER
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Concentration of doxycycline in human gingival fluid. J Clin Periodontol 1986; 13: 841-4. RETIEF DH, AUSTIN J C . The vervet monkey {Cercopithecus aethiops) as an experimental model for pulpal studies. J Dent Assoc S Afr 1973; 28: 98-103. BROWN J H , BRENN E. Method for differential staining of gram-positive and gram-negative bacteria in tissue sections. Bull Johns Hopkins Hosp 1931; 48: 69 73. PUCHTLER PD, SWEAT F, DOSS N . A one-hour phosphotungistic acid-hematoxylin stain. Am J Clin Pathol 1963; 40: 334—7. Statistical Analysis System. SAS Users Guide: Statistics, Version 5 edition. Cary NC: SAS Institute, 1985. DELIVANIS PD, FAN V S C . The localization of blood-borne bacteria in instrumented unfilled and overinstrumented CAnah.J Endod 1984; 10: 521-4. EiNDSKOG E, BLOMLOF E , HAMMARSTROM E . Mitoses and micro-organisms in the periodontal membrane after storage in milk or saliva. Scand J Dent Res 1983; 91: 465-72. BYSTEDT H . Antibiotics in oral surgery. Stockholm: Karolinska Institute Odontol Diss, 1979. JUNIPER RP. Penicillin - the duration of its activity in blood clots. Br J Oral Surg 1972; 9: 222-7. PATERSON JA, CARDO V A , STRATIGOS G T . An examination of antibiotic prophylaxis in oral and maxillofacial surgery. J Oral Surg 1970; 28: 753-9. CuRRAN JB, KENNETT S, YOUNG AR. An assessment of the use of prophylactic antibiotics in third molar surgery. Int J Oral Surg 1974; 3: 1-6. BYSTEDT H , NORD C-E, NORDENRAM A. Effect of azidocillin, erythromycin, clindamycin and doxycycline on postoperative complications after surgical removal of impacted third molars. Int J Oral Surg 1980; 9: 157-65. BARZA M , SAMUELSON T, WEINSTEIN E. Penetration of antibiotics into fibrin loci in vivo. IE Comparison of nine antibiotics: effect of dose and degree of protein binding. J Infect Dis 1974; 129: 66-72. BERGAN T. Pharmacokinetics of tissue penetration of antibiotics. Rev Infect Dis 1981; 3: 45-666. EAVOIE GE, BERGERON MG. Influence of four modes of administration on penetration of aztreonam, cefuroxime and ampicillin into interstitial fluid and fibrin clots and on in vivo efficiacy against Haemophilus influenze. Antimicrob Agents Chemother 1985; 28: 404-12. HAU T, NISHIKAWA R A , PHUANGSAB A. The effect of bacterial trapping by fibrin on the efliciacy of systemic antibiotics in experimental peritonitis. Surg Gynecol Obstet 1983; 157: 252-6. HAU T, JACOBS DE, HAWKINS N E . Antibiotics fail to prevent abscess formation secondary to bacteria trapped in fibrin clots. Arch Surg 1986; 121: 163-8. ARNT W.
References 1. NoRDENRAM A. Autotransplantation of teeth. A clinical and experimental investigation. Acta Odontol Scand 1963; 21: Suppl 33. 2. OHMAN A. Healing and sensitivity to pain in young replanted human teeth. An experimental, clinical and histological study. Odontol Tidskr 1965; 73: 168-227. 3. FoNG CH; MORRIS H , GRANT T H , BERGER J. Experimental tooth transplantation in the Rhesus monkey. J Dent Res 1967; 46: 492-6. 4. MoNSOUR FNT. Pulpal changes following the reimplantation of teeth in dogs: a histological study. Aust Dent J 1971; 16: 227-31. 5. SHEPARD PR, BuRiCH RL. Effects of extra-alveolar exposure and multiple avulsions on revascularization of reimplanted teeth in dogs. J Dent Res 1980; 59: 140. 6. ANDREASEN JO. Effect of extra-alveolar period and storage media upon periodontal and pulpal healing after replantation of mature permanent incisors in monkeys. Int J Oral Surg 1981; 10: 43-53. 7. SKOGLUND A, TRONSTAD L. Pulpal changes in replanted and autotransplanted immature teeth of dogs. J Fndod 1981; 7: 309-16. 8. SKOGLUND A. Vascular changes in replanted and autotransplanted apicoectomized mature teeth in dogs. Int J Oral Surg 1981; 10: 43-53. 9. BARRET A P , READ PC. Revascularization of mouse tooth isographs and allographs using autoradiography and carbon-profusion. Arch Oral Biol 1981; 26: 541-5. 10. MoNsouR FNT, ATKINS KF. Proliferation of pulpal tissue following early transplantation of developing teeth. J Oral Maxillofac Surg 1983; 41: 738-42. 11. KRISTERSON E , ANDREASEN J O . Influence of root development on periodontal and pulpal healing after replantation of incisors in monkeys. Int J Oral Surg 1984; 13: 313-23. 12. HEYERAAS KJ, MYKING AM. Pulpal blood flow in immature dog teeth after replantation. Scand J Dent Res 1985; 93: 227-38. 13. MoNsouR FNT, ATKINS KF. Aberrations in pulpal histology and dentinogenesis in transplanted erupting teeth. J Oral Maxillofac Surg 1985; 43: 8-13. 14. KLING M , CVEK M , MEJARE I. Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol 1986; 2: 83-9. 55. ANDREASEN J O . Traumatic injuries of the teeth. 2nd ed. Copenhagen: Munksgaard, 1981; 223-4, 232. !6. ANDREASEN JO, HJORTING-HANSEN E . Replantation of teeth.
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