Journal of Oral Rehabilitation, 1993, Volume 20, pages 637-652

Periodontal cell migration into the apical pulp

during the repair process after pulpectomy in
immature teeth: an autoradiographic study
O. V O J I N O V I C and J. V O J I N O V I C Clinics for Children and Preventive Dentistry,
Faculty of Stomatology, University of Belgrade

Summary

The migration of dental papilla cells into the periodontium during the process of
root development may occur as part of the process involved in the formation of the
periodontal tissues. The question posed is whether such cells under pathological conditions could retromigrate from periodontium into dental pulp and together with other
apical pulp cells of immature teeth, take part in the production of additional dental
tissue, e.g. 1) the tertiary/dentine under deep carious lesion where odontoblasts had
been destroyed 2) the dentine bridge on an amputation wound and 3) calcified tissue
which closes an apex during the apexification process in immature teeth.
The migration of periodontal cells locally marked by H^ Thymidine immediately
after partial pulpectomy in immature dog's teeth* was analysed at observation periods
of 2, 24 and 50 h and also without H^ Thymidine labelling of periodontal cells 8 weeks
after pulpectomy. The marked cells were found in the early observation periods after
pulpectomy just in the places where the hard tissues were formed in the later observation period of 8 weeks. They were found in large numbers just around the coagulated
necrotic foci. The finding supports the assumption that firm necrotic masses are a very
important stimulative factor in the reparation process in pulp and periodontium. The
experiment also corroborated the existence of periodontal cell retromigration into
apical dental papilla of immature teeth. Future research should assess the possible
role of the pathological condition in the determination of undifferentiated odontogenic
ectomesenchymal periodontal cells into odontoblasts.
Introduction

The process of physiological cellular differentiation of odontogenic cells is determined

by a genetically conditioned succession of biological interactions (Ede, 1978; Wessels,
1977). Exogenous factors can influence this process, as well as the formative activity
of the cells, changing them both. Thus, dentine tissue produced by odontoblasts under
the conditions of defence and reparation are different in structure from that produced
under the normal circumstances of development i.e. tertiary dentine under deep
carious lesions, the dentine bridge under the amputation wound, the clacified conglomerate in the apex formed during the apexification process of immature teeth (Seltzer &
* All these experiments followed the guidelines proposed by the Ethical Committee of the LA.S.P.
Correspondence: Professor Dr O. Vojinovie, Stomatoloski Fakultet, Klinika Za Decju 1 Peventivnu
Stomatologiju, 11000 Beograd, Serbia.
. ...^ .
,; , ;

637

638

O. Vojinovie and J. Vojinovie

Bender, 1984; Ten Cate, 1985; Vojinovie, 1974, 1977, 1987; Vojinovie & Srnic, 1975.
There are two possible explanations of this phenomenon:
(i) pathological activity of the formative cells affect the structure of the tissues
produced by the cells;
(ii) incompletely differentiated newly mobilized cells which are to substitute the
destroyed ones, produce dental tissues incomplete in structure.
If the explanation under (ii) is acceptable, the first question to be posed is whether
the newly differentiated cells, which produce all the above mentioned pathological
dentine tissues, belong to the cellular clones strictly determined just for dentine or
whether the precursors genetically predetermined for the other dental tissues could
also be involved. The second question is whether these reparative cell precursors are
either localised in the neighbourhood of the destroyed odontoblasts or migrate out of
some other part of the dental germ or tooth, changing under new conditions, their
genetically predetermined differentiation course.
Numerous authors have pointed to the mutual ectomesenchymal origin of the cells
of the dental papilla, periodontium and the neighbouring alveolar one. Therefore, it
seems logical to assume that they also have the mutual cell precursors (Johnstone &
Listgarten, 1972; Thesleff & Hurmerinta, 1981; Ten Cate, 1969; Bernick & Grand,
1982).
During the process of root development, the migration of the cells of a dental
papilla into a dental follicle and biological interaction between these two kinds of cells
may occur. Yoshikawa & KoUar (1981) pointed to the possibility that the cells of the
dental papilla under physiological conditions, may take part in the formation of
periodontium. Osborn in 1984 and Osborn & Price 1988, concluded that the cells of
the dental papilla probably migrate into the dental follicle and that they, together with
the cells of the investing layer are responsible for the formation of the attachment
alveolar bone and gingival mesoderm. Others (Palmer & Lumsden, 1987; McCulloch
et al, 1987) have pointed to physiological migration of cells out of endosteal spaces
into periodontium and through vascular canals.
These kinds of cells influence the organization of the periodontal cellular population
and have effect on the cementum produced over the nearby root surface. The experiments described above indicate that the genetically harmonised interaction between
odontogenic precursors of dental papilla, periodontium and nearby bone, is necessary
for normal root development. Schroder (1973, 1985), investigated the effect of calcium
hydroxide ions upon the formation of a dentine bridge over an amputation would have
after pulpectomy. He concluded that the multilayer necrosis resulted in effect from
calcium hydroxide ions during the period up to 24h. The author thought that the layer
of firm necrosis closest to the pulp, mineralizing itself by attraction of salts, as well
as the layer of new odontoblasts just under the dentine bridge (this means under the
previously formed and mineralized firm necrosis layer), raises the question of the
odontoblast origin, as well as of their determination pathway.
There is little data available concerning cellular interactions in the pappiloperiodontal ligament interface under pathological conditions. For apexification and
apexogenesis after endodontic treatment of immature teeth the stimulative procedure
is very important to introduce the exact route of the reparatory cells. The possibility
of retromigration of the papilla cells (those which, according to Yoshikava and Kollar
(1981) and Osborn (1984) and Osborn and Price (1988), physiologically migrate from
the papilla into dental follicle) from periodontium into papilla, could be proof of the

Periodontal cell migration

639

existence of undetermined dentinogenic cells in periodontal ligament and their positive

role in the reparative process in the apical pulp of immature teeth.
The aim of our experiments was to investigate, by local application of cellular
markers in the already formed part of the apical periodontium in immature dog's
teeth, the following: 1) whether the periodontal cells retromigrate after pulpectomy
out of the periodontium into the dental papilla and 2) whether they take part in the
further process of dentinogenesis during the formation of the apex.
Materials and methods
Twelve two rooted premolars (24 roots) of beagle dogs aged 67 months were used in
the experiment (Table 1). The contralateral teeth of the same jaw were used as a
control. The experiment was performed right after the eruption of the teeth at the
time when radiologically no more than a third of the root was formed. The extirpation
of the pulp out of all the premolars was done under rubber dam using broaches
(0.4 mm wide) of a working length which corresponded to the formed part of a root
measured according to the radiograph. Attempts were made to remove the pulp out
of one canal up to the level of apical opening but out of the canal of the other root of
the same tooth up to the level 2 mm shorter than the apical opening (Scheme lA).
Bleeding was stopped by a dry cotton pellet. Rinsing was done only with sterile saline
solution and under slight pressure. The canals were dried carefully and filled with
Ca(OH)2 paste prepared immediately before the application and avoiding mechanical
injury of the tissue wound.
Sterile calcium hydroxide paste was prepared with distilled water without any
additional medicaments. After the canal had been filled, it was closed with phosphate
cement liner and contoured amalgam filling (Scheme lA). Just after the endodontic
treatment described above, one drop of H'' Thymidine was applied by a syringe with
0.45 mm lumen needle and shorter than the apical dentine edges. The length of needle
penetrating into the periodontium was controlled by X-ray (Scheme IB). In this way,
no more than 1 microcurie of H'^ Thymidine was injected. The injection was repeated
on the other pulp extirpated premolars of the same dog on the second premolars.
Table 1. The number of teeth used in the experiment
The kind of
teeth

I prem.

II prem.

III prem.

total

TH-^ applicated
observation
period
2h

3 teeth
(6 roots)

24 h
50 h
TH"^ nonapplicated
8 weeks
Total

3 teeth
(6 roots)
3 teeth
(6 roots)
1 tooth
(2 roots)
4 teeth
(8 roots)

1 tooth
(2 roots)
4 teeth
(8 roots)

1 tooth
(2 roots)
4 teeth
(8 roots)

3 teeth
(6 roots)
3 teeth
(6 roots)
3 teeth
(6 roots)
3 teeth
(6 roots)
12 teeth
(24 roots)

640

O. Vojinovic and J. Vojiriovic

after 22 h and on the third ones after 24 h. The dogs were sacrificed 2h after the last
experiment. The observation periods of 2, 24 and 50h were obtained on each animal
in this way.
On the three premolars of another dog the same experiment was performed without the application of H'^ Thymidine. This dog was sacrificed after 8 weeks (Table 1).
The experiments were done under general anaesthetic by intravenous barbiturate
injection. The dogs were sacrificed by giving large doses of anaesthetic and then 10%
formalin was injected by perfusion right into the heart.
After the preparation and removal of the complete jaws, fixation in 4% neutral
formalin was performed (formaldehyde solution 3740% 100 cc, aqua dist. 900 cc,
NaH2, PO4, H2O 4g., Na2HPO4 6.5 g). The jaws remained fixed for 7 days and then
out into small blocks. Each block included one tooth and the neighbouring bone. The
fixation of these blocks was performed in the following 7 days, the demineralization in
the solution of 24% EDTA, pH 7.4 lasting about 60 days.
Following demineralization and paraffin embedding, serial mesiodistally directed
buccolingual cutting was performed. Every 15th of the 5 mm sections was placed on
the plate together with the section of the control tooth, covered with gelatin, followed
by stripped emulsion (Kodak R-10), free of its gelatin liner. The plates coated in this
way were kept in the dark for 14 days at 4C. and developed with Kodak D-19. The
sections were stained with either H&E, Gomori or PAS technique.
Results
Pulpectomy could not be performed completely in either of the roots. In each, the
fragile buccal apical dentine edge was broken (Figs 1, 6, 10). For the sake of better
space orientation, during the anaylsis, the apex was divided into three regions (scheme
2A-transversal; buccolingual section, 2B-sagital: mesiodistal section). The region TI
includes the apex area on the level of the amputation would along the whole medisodistal
and buccolingual diameter. The region T2 encompassed the part of the root under the
region TI in the whole buccolingual diameter, but not in the whole mesiodistal one.
It incorporated only the part of the root where the marker was applied, which means
only the middle part of the root starting from the mesial towards the distal diameter
of the tooth (scheme 2A, 2B). The region T3 includes part of the root mesially and
distally from the region T2, that is the part which is furthest from the place of application of the marker (scheme 2B), but under the region TI.
Observation period of 2h
Region TI. Amputation wound: There were haemorrhagic foci with oedema and microphages. Subodontoblastic layer: Haemorrhagic foci were visible in all the specimens.
Region T2. Subodontoblastic layer: There were foci of haemorrhage in this region on
both buccal and lingual sides. The buccal dentine wall was interrupted in spots and
the apical immature pulp communicated, in these places, directly with periodontium
(Fig. Al). There were scarce macrophages localized in groups. The region of epithelium sheet: On the buccal side there were various sized areas of destruction. On
the lingual side, it was preserved along the whole mesiodistal diameter (Fig. 1 AH).
Region T3. Subodontoblastic layer: Haemorrhagic foci were noticeable in places, but
tnore rarely than in the region TI and T2. Epithelium sheet: It was well preserved on

Periodontal cell migration

641

B
Fig. 1. Observation period of 2h. Region T2, b, buceal side root; 1, lingual side of root; II, undamaged
side of epithelial sheet (H&E form 4%, EDTA 23%). (A) Transversal buceolingual seetion of the
root at the region T2. At this plaee, pulp direetly eommunieates with periodontium (21, 85x). (B)
Solitary marked cell found on the border of the damaged periodontium towards pulp. The places
where the marked cells were found are indicated by X and x on the part A (289x).

both lingual and buccal sides of the root.Localiszation of the marked cells: Only a few
marked cells were found in the periodontium, as well as on the pulp border near to it.
i.e. in places where dentine wall was broken (Fig.l A, x, X, IB).
Observation period of 24h
This period was characterized by the organization of the coagulum.
Region TI. On the amputation wound microphages were dominant. In comparison
with the period of 2h, macrophages were considerably more numerous. Coagulated
necrotic masses (CN Masses) were found in large quantites next to dentine walls
right under the amputation wound. They were found also in small quantities on the
amputation wound.
Region T2. Apical part of the pulp: Blood vessels gathered in masses especially
around the fractured dentine edge with initial formation of granulation tissue. The
layer of regular, undamaged dentine within this region both on the buccal and lingual side
was separated from CN masses by a darker line or space (Fig. 2, 1). Subodontoblastic
region: An eosinophilic band and even argyrophilic foci were located. In the middle
part of the pulp in this area monocytes were dominant. Lymphocytes were scarce.
Epithelium sheet: This was destroyed on the buccal while it was preserved on the
lingual.
Region T3. CN Masses were scarce and the odontoblast layer was mainly preserved.
Localization of the marked cells: Between macrophages on the amputation wound
itself in the region TI there were no marked cells. They were found next to or inside
CN masses located along dentine walls of the region T2 (Figs 2 and 3).

642

O. Vojinovic and J. Vojinovic

Fig. 2. Observation period of 24h. Region T2. Solitary makred cell inside CN masses, whieh are
separated from dentine by dark line. (H&E, form 4%, EDTA 24%) (578x) M, marked cell; k, CN
masses; 1, bordering line between CN masses and dentine; D, dentine.

Fig. 3. Observation period of 24h. Region T2. One of rare groups of marked cells next to CN masses
in subodontoblastic region. It is evident that odontoblasts are destroyed in this place. M, labelled cells;
D, dentine. K, coagulation necrotic masses (CN masses). 1, bordering line between predentine and
CN masses. (H&E. form 4%, EDTA 24%) (131x).

Observation period 50 h.
The beginning of the reparative process in this period was evident.
Region TI. On the amputation wound itself, there were thickly packed layers of cells
(Figs 4, M and 5, M) Macrophages were dominant, without lymphocytes. Cells were
numerous next to CN masses (Figs 4, 5, M), which were found next to dentine and
separated from it by a clear bordering line or space (Fig. 5).

Periodontal cell migration

643

Fig. 4. Observation period of 50h. Region TI, amputation wound. The labelled cells were condensed
just on amputation wound and in subodontoblastic region. They are evidently rarer in the middle
of pulp (H&E, form 4%, EDTA 24%) (131x) K, canal of root; D, dentine; P, apical pulp; M,
marked cells.

Fig. 5. Observation period of 50h. Region TI. Labelled cells next to dentine wall on amputation
wound. (H&E, form 4%, EDTA 24%, 433()53x). K, canal of root; M, marked cells; C, CN masses
next to dentine (D).

Region T2. On the lingual side the epithelial sheet was preserved and the apexogenesis
(the physiological formation of the apex) was evident (Fig. 6, N). On the buccal side
the dentine edge was fractured. Owing to dentine absence at this place, the pulp was
in direct contact with peridontium, in which CN masses could be found in areas (Fig.

644

O. Vojinovic and J. Vojinovic

1
Fig. 6. Observation period of 50 h. Region T2. Apical part of pulp (H&E, form 4%, EDTA 24%,
25-5X). p, apical pulp; o, bordering line of pulp towards damaged dentine apical edge; PR, periodontium;
D, dentine; K, CN masses; N, undamaged lingual side of apex. Firm neerotic masses squared under 1
represent the places on which new dentine tissue is going to be produeed in order to form new apieal
dentine wall in the later observation period presented in the Fig. lOA, B, and indicated with the letter
B; s, space between CN masses and dentine.

6, K). Along the border line between the pulp and periodontium, there were thickly
packed spindle-shaped cells (Fig. 7, M). The cells were densely grouped around and
inside the CN masses (Fig. 9). Next to the dentine wall on the undamaged lingual
side, there were also CN masses which were separated from dentine by a darker
border line or space (Figs 8 and 9B, C,D)
Region T3. The epithelial sheet was preserved on both sides. In the centre of the
apical pulp, there were no inflammatory cells in this region. The localisation of the
marked cells: they were found in large numbers in both the pulp and in periodontium.
In the region TI, they were found in the amputation wound, where the start of collagen formation could be seen in places (Figs 4 and 5). Next to CN masses, along the
dentine in this region, they were thickly grouped (Fig. 5). In the deeper regions,
towards the middle of pulp, the marked cells were more rarely found. The impression
was obtained that they migrate towards the wound (Fig. 4).
In the region T2, beside CN masses in the subodontoblastic region, on both the
buccal and lingual side, the marked cells were found mostly where the odontoblasts
had been destroyed. (Fig. 9B, C,D). They were distinguishable from the well preserved
odontoblasts by their shape and size. A thick layer of the marked cells was found on

Periodontal cell migration

645

Fig. 7. Observation period of 50 h. Region T2. Labelled eells on bordering pulp line towards damaged
dentine edge; pr, periodontium; M, marked cells; k, CN masses (H&E, form 4%, EDTA 24%, 289x).
Place location of cells is squared on Fig. 6 and indicated by No. 1. (D).

Fig. 8. Observation period of 50 h. Marked cells localized next to CN masses at dentine wall on
lingual undamaged side. Note dense marked cells just in subodontoblastic layer. They are extremely
rate in pulp centre (H&E, form 4%. EDTA 24%, 131 x). M, marked cells; K, CN masses; D, dentine.

the border between the pulp and periodontium on the buccal side, especially around
CN masses (Fig. 7). Generally, it should be emphasized that the marked cells were
found mainly around CN masses and always in groups (Figs 5,7,8 and 9). The localization of the marked cells around the epithelial sheet was attractive: They were
found next to a newly formed dentine wall on its pulp side. On the periodontal side,
they were only found apically. In the apical opening, next to the pulp limiting membrane, scarce groups of marked cells with small quantities of collagen were sporadically
observable. A few marked cells were recorded in the bone tissue only in the vicinity
of the place of the marker appHcation.
:
.
.,;

646

O. Vojinovic and J. Vojinovic

Fig. 9. Observation period of 50 h. Note marked eells localized around CN masses situated in
periodontium and next to dentine walls. Region T2. (H&E, form 4%, EDTA 24%). K, CN masses;
d, dentine; s, empty space between dentine and CN masses; m, marked cells; f, condensed eollagen;
p, periodontium; v. pulp; L, dark line between dentine and CN masses. (A) Marked cells inside
periodontium (m), around condensed collagen (f), on buccal side of root (289x). (B) Marked eells
inside pulp (m) next to and inside CN masses (k) loeated next to dentine (d) (234x). (C) Marked cells
(m) next to CN masses separated from dentine (d) with empty space (s). In the middle of pulp there
were no groups of marked cells (v) (131x). Buccal side of the root. (D) The lingual undamaged side.
Numerous marked cells (m) next to CN masses in odontoblasts. Bordering line (1) between dentine
and CN masses (k) is well noticeable (175x).

Observation period of 8 weeks

The apical part of the immature pulp did not show any inflammatory changes. Towards
the root canal, as well as towards the part of periodontium where the apical edge had
been broken, unspecific dentine tissues was formed. This tissue separated the pulp
from a new periodontium forming in this way, the buccal wall of a new apex (Figs 10
A, B, lOBB). The layers of this tissue which were formed first, were of an irregular
structure i.e. towards the periodontium (Figs 10, AR, 10, BR.). The closer to the pulp
the more regular the dentine structure became (Figs WA "T", lOB ' T " ) . The layer of
odontoblasts was also noticeable in the pulp by this tissue (Fig lOA, "O" lOB, "O").
A thin layer of predentine was also observed and therefore confirmed that regular
dentinogenesis was still taking place within the 8 week period (Fig. lOB "T").
At the side where the epithelial sheet was preserved, the root formation continued by the regular apexogenesis process. The root assumed an approximately
normal shape (Fig 10A, lOB). In the periodontium, which had the normal form on the

Periodontal cell migration

647

Fig. 10. Pathological dentine tissue was formed just on the place where marked cells had been
localized in the observation period of 50h (H&E, form 4%, EDTA 24%). D, regularly formed lingual
dentine wall; B, irregularly formed buccal dentine wall on the damaged side of root; C, dentine bridge
towarde canal lumen; O, odontoblasts; R, layer of atubullar dentine on damaged buccal dentine wall;
P, pulp; T, layer of tubular dentine on damaged bueeal dentine wall; TD, tertiary dentine; N, bone;
Ou canal lumen of root; pr, periodontium. (A) Reconstruction (25-5x). Observation period of 8
weeks. Third mandibular molar. It should be noticed that apex has almost regular shape during
apexification process of 8 weeks. Buccal damaged side of root (B) eonsists of two layers; primarily
formed irregular (R) and secondary formed, towards pulp, regular tubular dentine (T), with neighbouring
odonotoblasts (O). The first layer merges with the second one without a bordering line. (B) Observation
period of 8 weeks; second premolar of the same dog presented in Fig A. Odontoblastic layer to newly
formed apical wall is discernible (O). Process of apxification is identical to the one presented in Fig. A
(25-5X). (C) Observation period of 8 weeks. Dentine bridge on amputation wound. The same two
layers could be seen as on damaged buccal apical wall; irregular (R) and tubular dentine on pulpal
side (T) (117-5X) TD Tertiary dentine. (D) Tertiary dentine of the human permanent teeth, whose
formation was indueed by the trauma during the drilling. Note a layers of atubullar dentine between
dentine layers fomed before and after the trauma (175x).

undamaged lingual side, there were no pathological changes (Fig. lOA, D, lOB, D).
On the injured buccal side, the periodontium was larger and of irregular shape, but in
the observation period of 8 weeks, without any inflammatory cells (Fig. 10 Apr, 10
Bpr). It is important to point out that the new reparation hard tissue was formed,
after the observation period of 8 weeks, just in the places in which the labelled cells

648

O. Vojinovic and J. Vojinovic

were found accumulated in groups in the observation period of 50h. i.e. 1) On the
amputation wound a dentine bridge was formed after 8 weeks (Figs 4 and 5 in comparison with Fig 10A,C, 10 C). 2) Repair took place in the dentine root wall between
the pulp and periodontium in places where the dentine edge was broken (Figs 6 and 7
in comparison with Fig 10 A,B 10 B,B). 3) Tertiary dentine was formed on the
undamaged lingual side (Fig. lOA "TD" in comparison with Figs 8 and 9D). It was
interesting to note that the whole new dentine wall was formed on the damaged buccal
side after 8 weeks, which corresponed to the normal apical shape (Fig 10A B",
lOB "B"). The dentine tissue of this wall was very similar to the one which formed
the barrier on the amputation wound (Fig. IOC), as well as the tertiary dentine under
the deep cavities (Fig. lOD "TD". Both of them were of dentine type structure and
formed by odontoblasts.
Discussion
Immature teeth were used in the experiment because of the pronounced immaturity of
the cell population inside the apical part of the dental papilla. This situation offered
an opportunity to analyse the mode in which the genetically predetermined direction
of differentiation, migration and function of the periodontal cells was changed after
pulpectomy.
The marker was introduced locally (not intraperitoneally) in order to avoid the
simultaneous labelling of the dental papilla cells and to make the migration of essentially
periodontal cells observable. The observation periods of 2, 24 and 50h were supposed
to offer data on the migration of periodontal cells immediately after trauma and
before the moment the cells performed their ultimate determination.
The aim of the additional experiment on the dogs sacrificed after 8 weeks and
without labelling the periodontal cells, was to find the places in the apical immature
pulp in which the described dentine reparative tissue was definitively formed. It also
allowed comparison to be made with the places where the marked cells were found
in the earlier observation period of 50 h. The aim was to see whether the labelled
periodontal cells could take part in the formation of pathological dentine tissue. The
Tubilitec liner avoided the contamination of the amputation wound with the interposition of bacteria as an additional irritative factor in the determination process of
the marked cells. Calcium hydroxide for the canal filling was used because it has been
established that it does not bring about any immunological reactions (Schroder, 1973,
1985; Vojinovic, 1975).
The injuries of the fragile dentine edge, found always on the buccal side, could be
explained by the slope of the root, which directed the nerve broach towards the
buccal side. However, there is a slight possibility for the edge being broken from the
periodontal side, by the syringe needle during the marker application. The above
considerations arise from the fact that the bone was not injured in that place, whereas
a part of the apical dentine was missing (Figs 1,6), The position of fragments of the
injured dentine indicated that the injury occurred from the pulpal side and the pulp
tissue on the buccal side was injured, while the periodontium was spared (Fig. 6).
The identical injury was noticed in the teeth analysed 8 weeks after the experiment in
which Thymidine had not been applied (Fig. lOA, B). The identical injuries were
evident in all the other previous similar experiments (Vojinovic, 1974, 1975, 1986),
which were carried out without Thymidine.
The interruption of dentinogenesis in the places of the most severe trauma was

Periodontal cell migration

649

evident already after 2 and 24 h. This is confirmed by the absence of odontoblasts in

the area and haemorrhagic foci and spindle-shaped cells next to the dentine wall.
The findings of only a few labelled cells in the close vicinity of the application place
of the markers after 2h and their abundance after 50 h is in accord with the findings
by Gould et al. (1980). Following work on rats they suggested that proliferative
activity of the cells of the periodontium does not take place before 30 h. The rare
marked cells, found after 2h, were probably fibroblasts which, at the moment of the
application of the marker, were in the phase of physiological replication.
The most frequent localization of the marked periodontal cells next to CN masses,
no matter where they were located (in the pulp or periodontium) indicates that these
cells, together with the others, take part in the organization of the CN masses and
their later mineralization, as well as in the subsequent production of the mineralized
tissue around the same CN masses (Figs 2, 5, 7, 8 and 9).
The first layers of the dentine bridge on the amputation wound (Fig. lOA "C", lOB
"C", IOC), the first layers of the tertiary dentine in the places where odontoblasts
were destroyed (Fig. WA "CTD"), as well as the mineralized tissue which, in the
process of apexogenesis, separated the apical pulp stump from the periodontium (in
the areas of the broken dentine edge. Fig. lOA "B", WB "B"), were of the similar
irregular structure. This result points to possible identity of their formative cells. The
marked periodontium cells were grouped right in the places where the formation
of these tissues was to be expected (Figs IA, 4, 5 and 7) and already after 50h.
Doubtlessly, these cells are highly involved in the production of these tissues. At the
beginning of their activity, the cells (the CN foci having been organized) produced,
at first, an untypical dentine which, in the course of the process of cell differentiation,
becomes more and more similar to orthodentine (Fig. WA "B", WB "B", WC "TD",

WD "TD").
This fact points to the assumption that the marked cells in this experiment, disregarding their periodontal origin, could represent the precursors of odontoblasts
and, owing to pathological conditions, began their formative activity before they
reached their own histomorphologic perfection. Another possible role of these cells
could be some kind of 'helping clone' for the organized synthetic activity.
The marked cells next to the dentine wall were evidently different by their shape
and volume from normal odontoblasts (Fig. 9B, D). This was not the same with the
cells in the periodontium (Fig. 9A). This also could be evidence of possible periodontal
cell migration during the reparative process after pulpectomy in immature teeth.
The assumption that the marked cells inside the pulp tissue have not been marked
by the migration of cells but by the diffusion of the marker cannot be accepted for the
following reasons: 1) In the observation period of 2h, the presence of marked cells
was unnoticed in the apical opening, although this region was rich in cells in the division
stage at the time of apex formation. 2) Marked cells were found mostly in groups and
in the places where the formation of the dentine tissue was expected to take place
(Figs 4, 5, 7, 8 and 9); if the diffusion was to be the reason, the marked cells should be
dispersed all over the pulp. 3) The marked cells were found mostly round the CN
masses or inside them, and in the areas where the production of collagen was expected
to take place (the regions TI, T2, around the dentine fragments, on the border line of
the pulp towards the periodontium (Fig. 7). 4) They were rare on the periodontal side
of the apical edge, which would not have been logical if the cells had been marked by
the marker diffusion, because in that periodontal region there were abundant immature

650

O. Vojinovic and J. Vojinovic

cells. 5) In the centre of the pulp, there were a few marked cells, but they grew denser
and denser towards the amputation wound, which indicates the migration of the cells
rather than the marker diffusion (Figs 4, 8 and 9C). 6) In the periodontium, coronal to
the place of the marker application, there were no marked cells in comparison with the
number inside the pulp. The expectation would have been to find the cells in the periodontium rather than in the pulp since it would be easier for diffusion straight through
the periodontium than into the pulp around the dentine edge. In fact they were found
in the odontoblast layer both on the side of application (Fig. 9C) and on the opposite
side (Fig. 9D), but just a few could be seen in the middle of the pulp (Fig. 8), i.e.
between these two mentioned labelled layers.
Three questions arise from the reported experiments: 1) The meaning of the
differentiation of periodontal cells into odontoblasts, taking into the consideration
that they genetically have another formative task. 2) To explain the presence of the
odontoblast precursors in the periodontium. 3) To determine which inductive factors
provide the conditions for the process of differentiation of periodontal cells into
odontoblasts.
If the assumption is acceptable that the marked periodontal cells are the precursors
of the new odontoblasts, besides other pulp cells, then it should be supposed that in
the periodontium of immature teeth, there are such precursors which are formatively
multipotent. This means that their final determination depends not only on the genetic
information, but also on the outside conditions under whose influence the determination
takes place. The differentiation of these precursors into odontoblasts can happen only
if the cells have the appropriate pulp localization. Taking into consideration Yoshikawa
and KoUar's (1981) findings together with those of Osborn (1984) and Osborn and Price
(1988) that the dental papilla cells migrate into the dental follicle under physiological
conditions, it could be assumed that the same cells could retromigrate into the apical
pulp tissue under pathological conditions. The second question posed above could be
explained through an intercommunication of undifferentiated pulp cells and periodontal
cells which is possible through the wide apical opening.
The initiative factors for changing the direction of the cells which possible retromigrate from the periodontium, could be various including inflammation of amputation
wound, mechanical factors and broken dentine fragments. Within the ground substance,
special initiative factors could also exist, especially in the process of separating the
dental pulp from the periodontium (Fig. 6 and 10A) (Vojinovic et al., 1986). Having
in mind that the labelled cells were found mainly around or inside the CN masses,
they also could be one of initiative factors.
Naturally at present the explanation concerning the initiation factors which induced
the migration of cells can be accepted only as a supposition to be given further consideration. The reported experiments are clinically significant in that they recommend
that during the endodontic treatment of immature teeth, proper care should be given
to the periodontium so as to enable it to perform its activity as physiologically as
possible. Besides, the fragile apical dentine edges are often exposed to fracture by
pulpectomy. Therefore, in immature teeth, pulpectomy should be done with measured
needles a millimetre shorter than the dentine wall length. Conditions should be
provided in the apical part of the pulp and periodontium for the above mentioned
differentiation of reparatory cells and their undisturbed interactions in the pulpoperiodontal apical region.

Periodontal cell migration

651

Conclusions
:
The retromigration of the cells from the periodontium into the apical pulp stump after
pulpectomy in immature teeth is possible, provided that the cells have not fully
differentiated. These cells are probably the same ones which migrated under the
physiological conditions of dentine root development, from the dental papilla into the
periodontium in order to take part in the formation of the attachment apparatus. This
points to the multipotentiality of the pulpo-periodontal cells in the apical part of
immature teeth. Having come again into the dental papilla, under the pathological
conditions after pulpectomy, it is not impossible that they differentiate into odontoblasts. These experiments demonstrate that firm necrotic foci have some coordinating
effect on this cellular activity. This is supported by their localization in those places
where the formation of the reparation dentine as well as the multitude of marked
periodontal cells around them are expected (amputation wound, dentine wall, mineralized barrier between the apical pulp and periodontal ligament). Therefore, pulpectomy
in immature teeth should be done in a way which enables the intercommunication in
the pulpoperiodontal cellular population. This implies the preservation of the apical
pulp, with the utmost care given to the prevention of the injury or loading of the
apical part of the immature periodontium.
Acknowledgment

This research work has been supported by the Scientific Fund of The Republic of
Serbia No. 1315 h.
References
& GRAND, D . A . (1982) Development of periodontal ligament. In: Periodontal Lit^ament
in Health and Disease (eds B.K.B. Berkovitz, B.J. Moxham & N.N. Newman), p. 197. Pergammon Press, Oxford.
EDE, D . (1978) An Introduction to Developmental Biology, p. 39. Blackie, Glasgow-London.
GOULD, T.R.L., MELCHER, A . H . & BRUNTER, D.M. (1980) Migration and division of progenitor cell
population in periodontal ligament after wounding. .Journal of Periodontal Research, 15, 20.
JOHNSTON, M.C. & LISTGARTEN, M.A. (1972) Observation on the migration interaction and early
differentiation of orofacial tissue. In: Development Aspects of Oral Biology (eds H.C. Slavkin &
L.A. Baveta). p. 35, Academic Press, London Inc.
McCuLLOCH, C.A.G., NEMETH, E . , LOWENBURG, B . & MBLCHER, A.H. (1987) Pravascular Cells in
Endosteal Spaces of Alveolar Bone Contribute to Periontal Ligament Cell Population. Anatomical
Record, 219, 233.
OSBORN, J.W. (1984) From reptile to mammal: Evolutionary considerations of the dentition with
emphasis on tooth attachment. Symposium of Zoological Society, London (No. 52): 549.
OSBORN, J.W. & PRICE, D . G . (1988) An autoradiographic study of periodontal development in the
mouse. Journal of Dental Research, 67, 455.
PALMER, R.M. & LUMSDEN, A . S . (1987) Development of periodontal ligament and alveolar bone in
homografted recombinations of enamel organs and pappilary, pulpal and foUicular mesenchyme
in the mouse. Archives of Oral Biology. 32, 281.
SCHRODER, U . (1973) Reaction of human pulp to experimental pulpectomy and capping with calcium
hydroxide. Odontology Review, 24, Suppl. 25.
SCHRODER, U . (1985) Effect of calcium hydroxide-containing pulp capping agents on pulp cell migration,
proliferation and differentiation. Journal of Dental Research, 64 (Spec, issue), 54.
SELTZER, S. & BENDER, LB. (1984) The dental pulp (III edn), p. 1. J.B. Lippincroft Co., Philadelphia.
TEN CATE, A . R . (1969) The development of the periodontium. In: Biology of Periodontium (eds A.H.
Melcher & W.H. Bowen), p. 120. Academic Press, London.
TEN GATE, A.R. (1975) Formation of supporting bone in association with periodontal ligament
organization in mouse. Archives of Oral Biology, 20, 137.

BEKNICK, S.

652

O. Vojinovic and J. Vojinovic

A.R. (1985) Oral histology development, structure and function, p. 218. C.V Mosby
Co., St. Louis.
THESLEFF, I. & HuRMERiNTA, K. (1981) Tissuc interaction in tooth development. Differentiation,
18, 75.
VOJINOVIC, O . (1974) Induction of apical formation in immature teeth by different endodontic methods
of treatment. Journal of Oral Rehabilitation. 1, 85.
VOJINOVIC, O . & SRNIC, E . (1975) Induction of apical formation by the use of calcium hydroxide paste
in the endodontic treatment of immature pulpless teeth. Journal of British Endodontic Society,
8, 16.
VOJINOVIC, O . (1977) Influence of different endodontic methods of treatment upon the process of apical
closure of immature pulpless human teeth and the structure of the newly formed tissue in apical
opening. Journal of Oral Rehabilitation. 4, 335.
VOJINOVIC, J., VOJINOVIC, O . , MILIN, J. & TATIC, E . (1986) Biologia zuba (The biology of the teeth),
p. 185, 398, 251, Naucna Knjiga, Beograd.
VOJINOVIC, O . & STEVANOVIC, R. (1987) Irritative effect of drilling on deep dentine cavities and
chemical effect of zinc-oxide-cariophilorum paste and silver amalgam on the structure of tertiary
dentine. Stomatoloski glasnik srbije, Sept. 100.
WESSELS, N . K . (1977) Tissue interactions and development, p. 23. W.A. Benjamin INC, Menlo Park.
YOSHIKAWA, D . K . & KOLLAR, E . J . (1981) Recombination experiments on the odontogenic roles of
mouse dental papilla and dental sac tissues in ocular grafts. Archives of Oral Biology, 26, 303.

TEN CATE,