Dental pulp regeneration aided by blood and

blood substitutes after experimentally

induced periapical infection
William C. Myers, D.D.X., M.X., and Stuart B. Fountain, D.D.S.,
M.Sc.(Dent.), Chapel Hill, N. C.
UNIVERSITY OF NORTH CAROLINA

This study was based on the hypothesis that if, after periapical infection, the pulp
canal is opened maximally, then filled with whole blood and/or blood substitutes in
contact with the periapical tissues, and dental pulp may regenerate. Results showed
that regeneration or ingrowth of connective tissue into the root canals of these
monkey teeth was not enhanced by blood and/or blood substitutes. The tissue
ingrowth measured between 0.10 and 1.00 mm. and fell within this range regardless
of the filling material, including those teeth left void. Overextension of the instrumen-
tation of the root canals in this ‘study caused widespread inflammation and, in some
cases, cyst formation. The majority of the teeth developed root resorption which
correlated with the presence of chronically inflamed granulation tissue. Of the teeth
exhibiting root resorption, only incompletely developed cuspids showed any type
of repair of the resorptive defect during the time period of this study. These cuspids,
which had incomplete root formation, continued to deposit additional root length, or
bridging by a calcified material was attempted, even after seven of these teeth
required removal of the coronal seal in order to drain acute abscesses.

I t is widely believed that it is preferable to maintain the vitality of the dental

pulp whenever possible. If inflammation of the pulp occurs, then an attempt
should be made to retain viable pulp in the root canals. These treatments range
from the use of pulp-capping procedures to pulpotomies and partial pulpec-
tomies in order to retain a portion of vital pulp tissue within the root canal.
However, information from the past several years indicates that the pulp has a
potential to regenerate subsequent to its degenertaion or removal.
Several studies involving replanted or transplanted teeth have demonstrated
that the pulp tissue may be replaced by ingrowing vital tissue. Clark, Tam, and
Mitchell,l Myers and F1anagan,2 A gnew and Fong,3 Costich and assoeiates,4 and
Miller5 have demonstrated pulpal regeneration of varying degrees into the pulp
441
 Oral Surg.
March, 1974

canals of autogenously transplanted teeth. It is thought that the pulpal tissue

remaining in the tooth after the blood supply was severed could act as a matrix
for the ingrowth of new granulation tissue. In this respect, the remaining pulpal
tissue would function similar to a blood clot, which forms the scaffold for wound
repair.
In other histologic studies it has been shown that, when a blood clot was
allowed to form in the canals after biomechanical preparation, there was an in-
growth of capillaries and fibroblasts. Vidair and Butcher,6 Erausquin and Maru-
zabal,’ and Ostbysl 9 have demonstrated this phenomenon after removal of the
pulp. For this ingrowth of tissue to occur, two factors seem to be pertinent:
(1) the diameter of the canal and (2) the presence of a matrix. ijstby’ also
mentioned the factor of desiccation of the fibrin clot, followed by its degenera-
tion. The problem of infection of the blood clot must also be considered.
The present study was designed to examine the potential for regeneration of
dental pulp after infection within the canal and periapical tissues. It is based
on the hypothesis that if, after periapical infection, the pulp canal is opened
maximally, sterilized, then filled with whole blood and/or blood substitutes in
contact with the periapical tissues, the dental pulp may regenerate.

MATERIALS AND METHODS

This study utilized the twelve anterior teeth of each of four CIebuSapellae
monkeys. The incisor teeth of these animals compared in size to fully formed
human deciduous teeth. The cuspids were much larger in size and, in two
monkeys, had incomplete root formation. The four permanent first premolars of
each monkey were used as controls.
At the initial treatment the monkeys were first anesthetized with phencycli-
dine hydrochloride.” Access was made to all twelve anterior teeth, and the lengths
of the teeth were determined radiographically with files in place. The pulps
were extirpated with a broach, and the canals were minimally instrumented to
length in order to remove all of the pulp tissue. The canals were contaminated
with the monkey’s own saliva on an endodontic reamer and broach. These teeth
were left open to the oral environment for a minimum period of 14 days. This
period of time was considered to be adequate for chronic and acute infections
to be entrenched in the teeth and periapical tissues.
After this designated period of infection, radiographs were taken with an
endodontic file set to the instrumented length and placed in each tooth. This
was done in order to demonstrate the correct establishment of length and com-
plete removal of the pulpal tissue. If at this time the teeth contained any viable
tissue, as evidenced by bleeding upon instrumentation to length, they were then
instrumented larger and evaluated 7 days later. This procedure was continued
until there was no evidence of remaining hemorrhaging tissue within the root
canals. When this was established, the canals were cleaned biomechanically under
the rubber dam, with the use of 5.25 per cent sodium hypochlorite. The instru-

‘Sernylan, Bio-Ceutic Laboratories, Inc., St. Joseph, MO.

Volume 3i Dental pulp regeneration 443
Number 3

ments and solution were confined within the boundaries of the pulp canal space,
and the apical constriction was maintained during this phase.
The next treatment ranged between 4 and 28 days after biomechanical
instrumentation and irrigation of the canals. At that time, each canal was
cultured. Subsequent to the culture, a solution of 5.25 per cent sodium
hypochlorite was agitated in the canals with a file and allowed to remain there
for approximately 5 minutes. The purpose of this was to irrigate any debris out
of the canals and to establish a sterile environment ‘within the canals. The canals
were then irrigated with normal saline solution. An endodontic file that was the
size of the last file used in the canal was lengthened 2 mm. past the measured
length of the tooth and was then inserted to the new length, thereby enlarging
the apical constriction. This provided a wide-open foramen contacting the peri-
apical area in order to aid pulpal regeneration. The canals were then dried and
the bleeding was stopped with sterile paper points.
The pulp canal space in the experimental teeth was then prepared in one
of the following ways :
(a) Filled with titrated whole blood from the same animal clotted with
thrombin in situ.
(b) Filled with Gelfoam* and monkey blood from the same animal.
(c) Filled with isotonic saline (control teeth).
(d) Left void and sealed at the access opening (control teeth).
The filling sequence was the same for each monkey and was arranged to give a
representative sample of the different materials chosen. After placement of the
material into the canal space, the coronal portion of the canal was cleaned to
the cervical line and the tooth was sea1ed.t The animals were killed at intervals
designed to give observation periods of 2 weeks, 3 months, and 6 months.
RESULTS
Thirty-six of the original forty-eight teeth were examined histologically for
tissue regeneration, Nine of these were filled with blood, seven were filled with
blood and Gelfoam mixture, four were filled with isotonic saline solution, and
sixteen were left void. The animal designated as the l-month animal died 4 days
after biomechanical preparation and sealing of the twelve experimental teeth.
These teeth were not filled with the experimental materials; nor was the apical
constriction purposely enlarged. Histologic examination of these twelve teeth
revealed that nine had been instrumented beyond the apex, thereby enlarging
the apical constriction. These nine teeth were included under the results of canals
left void with a 0.5 week postoperative period.
Fig. 1 presents a graphic summary of results of tissue growth versus time.
Tissue regeneration occurred in twenty-eight of the thirty-six teeth, or 77.8 per
cent. This growth ranged from 0.10 to 5.10 mm. The tooth with the largest
amount of tissue ingrowth (5.10 mm.) was a cuspid with an incompletely formed
root. The amount of tissue regeneration was approximately the same regardless

*Gelfoam Powder, Upjohn Co., Kalamazoo, Mich.

tCavit, Premier Dental Products Co., Philadelphia, Pa.
 444 Myers and Fountain Oral Surg.
March, 1974

:
55

50 I L

Ezo-
E
z
E 15.

3
0
I IO-

n
05.

O-
II
b 2 12 24 0 2 12 24
WEEKS WEEKS

GELFOAM AND BLOOD

20

1
I.5

; IO-
:
z 1

i O:-L 0 2
WEEKS
I2 24 6 5
WEEKS
2 I2 20 24

Fig. 2. Summary of results. Each vertical bar represents the results of growth in one
tooth.

of the canal treatment, with the 2-week specimens exhibiting the greatest amount
of ingrowth. Tissue which grew into the canals at all time periods consisted
predominantly of granulation tissue. The 2-week specimens contained a moderate
inflammatory cell infiltrate of acute and chronic cells (Fig. 2)) whereas the
3-month and g-month specimens contained a heavy infiltrate of predominantly
chronic inflammatory cells (Fig. 3). The ingrowing tissue did not resorb the
blood clot placed in the canal. Instead, the clot appeared to serve as a matrix
for the ingrowth of tissue.
In the majority of cases the tissue ingrowth was accompanied by periapical
inflammation and root resorption. Of the nine teeth with a J-day postoperative
time which were instrumented beyond the apex, six contained no tissue ingrowth.
The periapical tissue contained acute inflammatory exudate which extended into
the surrounding bone and along the periodontal ligament. In the longer post-
operative periods the periapical tissue consisted of granulation tissue, with the
amount of inflammatory cell infiltrate increasing in the a-month and 6-month
specimens. It was difficult to classify the periapical lesions as granuloma, abscess,
Volume 37 De&al pulp regeneration 445
Number 3

Fig. d. Short-term growth into root canal. Postoperative period-2 weeks. Mandibular left
lateral incisor filled with blood. D, Dentin; C, canal; N, granulation tissue ingrowth. (Masson’s
trichrome stain. Magnification, x25.)

or cyst. Most were combinations of the elements comprising these lesions, but
the majority could be called granulomas and several were abscesses. Inflamma-
tory epithelial hyperplasia, or proliferation, was found in eleven teeth of the
study. Actual formation of a cystic cavity was found in only two instances
(Fig. 4). In the 2-week specimens, one tooth exhibited epithelial hyperplasia;
in the 3-month specimens, four teeth; and in the g-month specimens, six teeth.
The root resorption correlated very closely with the presence of chronic peri-
apical inflammation. Root resorption was not found in the 4-day specimens but
was quite extensive in the 2-week specimens. The amount of root resorption
increased progressively in the a-month and 6-month specimens (Fig. 3). No
evidence of repair of the resorptive defects was found, except in incompletely
developed cuspids.
The canal space contained necrotic pulpal debris, acute inflammatory exu-
date, and colonies of microorganisms. Extensive colonies of microorganisms were
found in a large number of canals of the 3-month and 6-month specimens. These
were in contact with the ingrowing tissue, and in several specimens appeared
to prevent tissue ingrowth (Figs. 5 and 6).
The eight cuspids in the two long-term monkeys had underdeveloped roots
and reacted differently than the other teeth in the study. Seven of these cuspids
required removal of the occlusal seal in order to establish drainage so that ful-
minating abscesses could be treated. Subsequent attempts to reseal these teeth
were successful in only one case; therefore, six were allowed to remain open to
446 Myers and Fountain Oral Surg.
March, 1974

Fig. 4

Fig. J. Long-term growth into the root canal. Resorption associated with chronic inflam-
mation. Postoperative period-3 months. Mandibular incisors. C, Canal; I, interradicular area ;
R, resorption; G, granulation tissue. (Hematoxylin and eosin stain. Magnification, x25.)
Fig. 4. Epithelial proliferation. Postoperative period-6 months. Maxillary left lateral
incisor. D, Dentin; C, canal filled with debris; E, epithelium; F, fibrous connective tissue
capsule; P, inflammatory cell exudate. (Hematoxylin and eosm stain. Magnification, x25.)

the oral environment for the duration of the experimental period. The results
showed no difference between those cuspids that were closed and those that were
left open. The periapical areas were completely healed in six specimens, and only
a slight area of inflammation remained in the other two specimens. All of these
teeth exhibited continued root formation, with attempts at apical closure or
apical bridging by a cementoid-osteoid type of tissue. The walls of three teeth
appeared to have been perforated during instrumentation, but a hard-tissue
bridge was forming to the intact wall. The ingrown tissue consisted of areas of
inflamed granulation tissue and areas of healthy fibrous connective tissue. In
three of these cuspids, odontoblasts were present in the ingrown tissue and were
Volume 37 Dental pulp regeneration 447
Number 3

Fig. 5. Colonies of microorganisms inhibiting tissue ingrowth. Postoperative period-3

months. Mandibular right central incisor. M, Colonies of microorganisms; D, dentin; C, canal;
A, abscess. (Hematoxylin and eosin stain. Magnification, x25.)
Pig. 6. Close-up view of apex shown in Fig. 4. M! Colonies of microorganisms; H, dentin;
C, canal; E, inflammatory cell exudate. (Hematoxylm and eosin stain. Magnification, x100.)

actively depositing regular dentin (Figs. 7 and 8). The ingrown tissue had pro-
liferating epithelium covering the superior aspect in three instances.

DISCUSSION
The results of this study indicate that a limited amount of soft-tissue in-
growth did occur in most cases. The extent of the growth and the type of tissue
which invaginated into the canals were very similar, regardless of the canal
treatment. In the large majority of cases, this tissue was granulation tissue con-
taining an inflammatory cell infiltrate. This finding indicates that the whole
blood, or a paste of Gelfoam and blood, failed to enhance the amount of tissue
growth into these previously infected canals.
Previous studies which used vital uninfected teethe-l0 showed that healthy
448 Myers and Fountain Oral Surg.
March, 1974

Fig. 7. Results of apical perforation in underdeveloped cuspid. Postoperative period-6

months. Maxillary right cuspid. C, Canal; E, epithelium, . P, fibrous connective tissue; I, inflam-
matory cell exudate; B, bone. (Hematoxylin and eosin stain. Magnification, x2.2.)
Fig. 8. Ingrowth in cuspid wit,h underdeveloped root. Postoperative period-3 months.
Ma.ndibular right cuspid. F, Fibrous connective tissue; H, dentin; G, granulation tissue;
E, inflammatory cell exudate; R, resorption. (Hematoxylin and eosin stain. Magnification,
x2.2.)

connective tissue grew into the canals, replacing the blood clot. 6stby,8 in his
original study, thought that this would occur in necrotic teeth as well as in vital
uninfected teeth. iistby and HjortdalQ found in a later study that only one of
twelve teeth that originally contained necrotic pulps exhibited tissue ingrowth.
The periapical tissues in these cases contained granulomas and cysts. These re-
sults compare very favorably with the results of the present study. Vidair and
Butcher6 also had similar results with infected teeth. They found that ingrowth
of connective tissue into previously infected canals would not occur regardless
of sterilization of the canal. In the present study, four canals gave nega-
tive cultures prior to filling, but all four exhibited periapical inflamma-
tory tissue.
These results can be explained by residual infection in the dentinal tubules,
necrotic tissue remaining in the dentinal tubules, or changes in the dentin which
made it toxic to tissue growth. In the present study, 5.25 per cent sodium hypo-
chlorite was used to irrigate the canals and was allowed to remain in the canals
for a period of 5 minutes. Colonies of microorganisms were nevertheless found
in the canals. This would seem to indicate that infected dentin is very difficult
to sterilize, and this may be the explanation for the large amount of inflammation
associated with these teeth.
Volume 3i Dental pulp regeneration 449
Number 3

The postoperative time with the over-all largest amount of tissue ingrowth
was the 2-week period. This was a somewhat unexpected finding. The explanation
for this could be that in the longer postoperative periods the microorganisms
had more time to colonize the canal and infect the clot or regenerating tissue,
thus inhibiting any further tissue ingrowth. Another explanation could be that,
as granulation tissue matured from the apex, the large amount of newly formed
collagen and its shrinkage led to contraction of this fibrous scar and, conse-
quently, a diminished vascularization. I1 It is possible that the collagen which
formed at or near the apical foramen diminished the blood supply to the tissue
more coronally in the canal.
The finding of nine overinstrumented teeth out of twelve in the J-day speci-
mens indicates that, with our present clinical techniques, we must be instru-
menting beyond the apex in a large percentage of cases. This overinstrumentation
created a severe inflammatory reaction, with subsequent root resorption. There
appeared to be a definite correlation between the extent of root resorption and
the chronicity of the inflammation present. There also appeared to be a definite
correlation between the amount of inflammatory epithelial hyperplasia and the
chronicity of the inflammation present. Six of the eleven teeth associated with
epithelial hyperplasia were in the 6-month specimens.
One tooth lost the occlusal seal after 2 months and was allowed to remain
open to the oral environment for the remaining 1 month of its experimental
period. This tooth exhibited the largest amount of tissue ingrowth of any tooth,
other than the underdeveloped cuspids, and was one of the few teeth that did not
have any periapical inflammation. This finding indicates that opening infected
teeth to the oral environment may have little, if any, detrimental effects,
because it tended to resolve extensive areas of inflammation and appeared to
initiate the repair process.
This was also found to be true in the underdeveloped cuspids. The eight
eight cuspids with incomplete root formation continued to exhibit apical deposi-
tion of calcified tissue. Although this was interrupted with development of ful-
minating abscesses in seven of these teeth, it continued after opening of the pulp
chamber to the oral environment. This indicated that the potential for complete
root formation remains after periapical infection and inflammation. Leaving
these teeth open to the oral environment did not impede the apexification process.
The persistence of periapical infection in this study demonstrates that a
previously infected root canal should receive further treatment other than en-
largement and cleaning if complete healing is to ensue. This supports the work
of Seltzer and his associates,12 who have stated that, if the canal is properly
obturated, the periapical inflammation will resolve.

SUMMARY AND CONCLUSIONS

Forty-eight anterior teeth in four monkeys were studied in order to determine
whether whole blood or blood substitutes would enhance regeneration of pulp
tissue into necrotic root canals.
The pulps of the teeth were extirpated and the canals were left open to the
oral environment for 2 weeks. One monkey died during the first week, which
caused the loss of twelve experimental teeth. The remaining thirty-six teeth were
450 Myers and Fountain Oral Surg.
March, 1974

treated endodontically and filled with either whole blood, Gelfoam and whole
blood, or isotonic saline solution, or were left void. The pulp chambers were
sealed with Cavit. The animals were killed after various time intervals, and the
teeth were studied histologically.
The results showed that ingrowth of healthy viable tissue into previously
infected root canals cannot be expected under the conditions of this study. Over-
extension of the instrumentation of the root canals caused widespread inflamma-
tion and contributed to some cyst formation. Most of the teeth developed root
resorption which correlated with the presence of chronically inflamed granula-
tion tissue. Of the teeth exhibiting root resorption, only incompletely developed
cuspids showed any type of repair of the resorptive defect during the time period
of this study. These cuspids, which had incomplete root formation, continued to
deposit additional root length, or bridging by a calcified material was attempted,
even after seven of these teeth required removal of the coronal seal in order to
drain acute abscesses.
The conclusion is that regeneration or ingrowth of connective tissue into the
root canals of previously infected monkey teeth is not enhanced by blood and/or
blood substitutes.
REFERENCES
1. Clark, H. B., Tam, J. C., and Mitchell, D. F.: Transplantation of Developing Teeth,
J. Dent. Res. 54: 322-328, 1955.
2. Myers, H. I., and Flanagan, V. D.: A Comparison of Results Obtained From Transplan-
tation and Replantation Experiments Using Syrian Hamster Teeth, Anat. Rec. 130: 497-
507. 1958.
3. Agnew, R. G., and Fong, C. C.: Histologic Studies on Experimental Transplantation of
Teeth. ORAL S~JRC. 9: 18-39. 1956.
4. Cost&h, E. R., Avery, J. K., MacKenzie, R. S., and Haley, E. W.: Freezing and In Vitro
Culture of Hamster Teeth Before Transplantation and Replantation, J. Oral Surg. 24:
500-516, 1966.
5. Miller, H. : Transplantation and Reimplantation of Teeth, ORAL SURG. 9: 84-95, 1956.
6. Vidair, R. V., and Butcher, E.: Regeneration of Tissue Into the Pulp Canal of Monkey’s
Teeth, J. Dent. Med. 10: 163-166, 1955.
7. Erausquin, J., and Maruzabal, M.: Evolution of Blood Clot After Root Canal Treat-
ment in Rat Molars, J. Dent. Res. 47: 34-40, 1968.
8. ii&y, B.: The Role of the Blood Clot in Endodontic Therapy.L” Actn Odontol. &and.
19: ii3-353, 1961.
9. iistby, B., and Hjortdal, 0.: Tissue Formation in the Root Canal Following Pulp Removal,
&and. J. Dent. Res. 79: 333-349, 1971.
10. Davis, M. S., Joseph, S. W., and Bucher, J. F.: Periapical and Intracanal Healing
Following Incomplete Root Canal Fillings in Dogs, ORAL SURG. 31: 662-675, 1971.
11. Robbins, S.: Pathology, ed. 3, Philadelphia, 1967, W. B. Saunders Co., pp. 63-72.
12. Seltzer, S., Soltanoff, W., Sinai, I., Goldenberg, A., and Bender, I. B.: Biologic Aspects
of Endodontics. IIT. Periapical Tissue Reactions to Root Canal Inst.rumentation, ORAL
SURo. 26: 534-546, 694-705, 1968.

Reprint requests to:

Dr. William C. Myers
2007 Randolph Rd.
Charlotte, N. C. 28207