Exodontia

The time sequenceof tissue regenerationin

human extraction wounds
Melvin H. Amler, M.S., D.D.S.,* New York, N. Y.

H ealing of normal extraction wounds has been studied experimentally

rats,lT * dogs,3-8 monkeys,gp lo sheep,ll and human beings with tissues obtained
in

(with certain exceptions12* I3 ) at necropsy or from diseased persons.14-16 These

studies, while yielding valuable information, can hardly be directly applicable
in a determination of the time sequence of tissue regeneration in healthy dental
patients and, therefore, must be consi,dered of only limited value. However,
notwithstanding this consideration, the time sequence developed on the basis of
these studies is universally accepted. It is used as the basis of discourses on
alveolar socket healing in various current textbooks and references17-1a and forms
the basis for definitive prosthetic, periodontal, and orthodontic clinical treat-
ment planning which frequently follows or is done in concurrence with exo-
dontia.
In view of this, a study of human tissues was arranged in which clinic pa-
tients were screened in order to eliminate, as far as possible, gross variables
which might interfere with normal tissue regeneration in extraction-wound
healing. This information has been organized to provide a more accurate and
detailed time sequence, which might more closely approximate normal tissue
regeneration in human cases than the information obtained by interpolation
from animal experimentation or from human autopsy material where the cause
of death was disease rather than trauma.

MATERIALS AND METHODS

In order to ascertain the physical condition of patients selected for post-
extraction biopsy, age was limited to between 30 and 50 years, the history was

This study was supported in part by Grant D-1065 from the United States Public
Health Service.
*Research Associate Professor, Department of Pathology, New York University College
of Dentistry; the Murray and Leonie Guggenheim Institute for Dental Research and the
Beth Israel Hospital.

309
310 Llmler O.S., OX. & 0.1’.
IvIadk. 19W

investigated, the diet, was checked for insnficicncies, and hospital records w(‘r(’
examined for obvious 1)athologic conditions. such as cndocrinc disorders, cardio-
vascular disturbances, and blood or blood-forming organ abnormalities. All dat,a
which could aflect soft-tissue or boric healing wcrc notjed and patients w~~r(:
screened accordinglv. ln this biopsy t,cchnique, alveolar bone was generally
avoided and tissues were taken only up to 50 days. The reasons for this were
twofold : (1) Avoidance oF removal of alveolar boric eliminated c\-en the slightest
possibility of patient disfigurement ; (2) up to the 50.day stage of bone healing,
only partial mineralization had taken place, and this permitted sections to be
cut without prior decalcification, thereby permitting the application of various
histochemical tests.
Tissues were fixed in Helly’s fluid and in 80 per cent ethanol at loo C. Ap-
proximately 185 biopsy specimens mere taken and stained for routine histologic
examination with hcmatosylin and eosin with 1 per cent phlosin B. For routine
histochemistry, sections wcrc stained as follows : acid polysaccharide ground sub-
stance, 0.5 per cent toluidine blue; glycogen and glycoprotein, Hotchkiss-
McRlannus-SchifP ; alkaline phosphatase, Gomori’s stainZ1; calcium, von Kossa
techniquez2 ; mast infiltration and basophilia, Giemsa staiP; connective tissue,
Mallory’s stain?“; silver impregnation and argyrophilia, a variant of the Biel-
chowsky-Mares& stainZ4 (with counterstain of van Gieson’s picrofuchsin)
The malt diastase was made up in a 0.1 per cent solution, by weight, and
tissues were incubated for 30 minutes at, 30° C. (The diastase, malt, Grade C
contained some proteinase activity.*)

OBSERVATIONS
Fig. 1 shows a photomicrograph of the fundic region of a ?-day-old healing
socket in which the organization of fibrin is apparent in large, concentric
whorls. Blood cells of the primary clot in approximately the same proportion
as the circulating blood can be observed between fibrin strands. Fig. 2 shows
a larger magnification of Fig. 1 through the central arca of the fibrin whorl.
Here the arrangement of red blood cells and fibrin organization can be noted.
The fibrin appears to intersperse the areas of red cells, forming small geometric
patterns. Fig. 3 shows a later stage of socket healing from a 4-day biopsy speci-
men. Blood cells and fibrin are present but are infiltrated and partially re-
placed by granulation tissue cells and some young connective tissue cells. Fig.
4 shows an S-day-old socket biopsy specimen stained for metachromasia with
0.5 per cent toluidine blue. The upper portion of the photomicrograph, showing
granulation tissue, represents the central area of the healing socket and is
negative for metachromasia, while the lower half of the figure reacted positively
For acid polysaccharides, indicating the development of young connective tissue.
Fig. ‘5 shows a lo-day specimen stained for alkaline phosphatase by the
Gomori technique. Here, too, the upper right portion of the specimen, which is
relatively unstained, represents the central immature area of the healing socket
where granulation tissue is still present. The central and lateral areas of the

*The diastase was obtained from Calbiochem Corp., California.

 Time sequence of tissue regeneration 311

Fig. 1 E

Fig. 3

Fig. 2. A S-day healing socket. Organization of fibrin is apparent in whorl formation.

Fig. 3. Larger magnification of central fibrin area of specimen shown in Fig. 1.
Fig. 3. A 4-day biopsy specimen, with granulation and some connective tissue.
Fig. 4. An g-day-old socket biopsy specimen stained with toluidine blue for acid polysac-
charides. Upper portion shows negative reaction, while lower area is positive, indicating de-
velopment of connective tissue.
 E

Fig. 5. A la-day biopsy specimen stained by the Gomori method for nIkaliur phosphatase.
Upper right portion is relatively unstained,. indicating granulation tissur., while central and
lateral areas are positive. This is eharacteristlc of connective tissue.
Fig. 6. A 21.day specimen stained for metachromasia. Heavy stain indicates positive re-
action.
Fig. 7. Same biopsy specimen as in Fig. 6, stained by P.A.S. method. Positive reaction in
bone indicates the presence of glycoproteiu.
Fig. 8. The same biopsy specimen as shown in Fig. 6 and 7 but stained for calcium by the
von Kossa technique. Central portion of bone spicule stains positively for calcium,
 Volume 27 Time sequence of tissue regeneration 313
Number 3

9 Fig. 10

11

Fig. 13

Fig. 12

Fig. 9. A 12.day biopsy specimen showing first proliferation of epithelium as downgrowth

before socket fundus has been replaced with connective tissue.
Fig. IO. Higher-power view of specimen in Fig. 9 showing epithelial downgrorvth toward
fundic region of healing socket.
Fig. 21. A 16-day specimen stained by the P.A.S. method, indicating glycogen, showing
the heaviest concentration of positive dark-staining area over the crest of the epithelium.
Fig. 1.8’. A 32.day specimen showing incomplete fusion of epithelium.
Fig. 1.3. Lower magnification of B-day specimen showing epithelization proceeding as a
thin linear growth over surface of healing socket. Fusion of epithelium in incomplete.
314 ilnlle7

healing socket, indicate a positive reaction to alkaline phosphatase staining,

characteristic of connective t&sac. Fig. 6 depicts a Pl-davy specimen with bone
stained for mctnchromasia, and it can 1~ observed that the bone development
commences with a heavy mctachromatic reaction indicating the presc~~ct of acid
polysaccharidc ground substance. As bone dcvelopmcnt proceeds, the bone loses
its metachromatic staining capacitor but reacts positively for glycoprotein
(darker arcas), as in Fig. 7 which shows a sprcimen from the same bone spiculc
of sock& as in Fig. 6. It can bc notctl that, in the specimen shown in Fig. 7,
stained by the PAS. mt~thod, the central, inner portion of spicule stains posi-
tively, indicating glycoprotein. (The prcsencc of glycoprotein rather than gly-
cogen was proved by the persistence of a positive stain to 1’A.S. following
incubation in malt diastase.) Fig. 8, another section of the same boric spicule
from socket shown in Figs. 6 and 7, is stainttd by the van Kossa method for
calcium. Here the central portion of bone spicule stains positively for calcium
presence indicating that. mincralizaiion occurs first within the central area of
the spicule and in this respect is similar to glycoprotein localization. Droplet
precipitation of calcium can be obscrvctl at the peripheral ends of the spiculr,
indicating the regenerating and most. a&\-c growth areas.
Fig. 9 shows the first proliferation of cpithelial regeneration as a down-
growth before the socket. fundus has been replaced with connective tissue, and
Fig. 10 is a higher-power vie\\- of Fig. 9, showing epithelial downgrowth toward
the same area of the socket.
A test stain with the PAS. method for glycogen (Fig. 11) indicate that
the heaviest concentration of the positive dark-staining area was present at the
crest of the epithelium over the alveolar ridge rather than at the downgrowth
portion of the regenerating cpithelium. This positive staining reaction was
proved to indicate the presence of glycogen rather than glycoprotein by its
removal following incubation in malt diastase. It appeared to be associated
with the most active region of cpithelial regeneration.
Fig. 12 intlicatcs a tissue section taken from a sock& 32 days following ex-
traction. TTerc WC can observe that complettt cpithelial closure has not yet taken
place. Fig. 13 shows a loaner magnification of a healing extraction socket of 21
days where cpithclixatitm can be seen to proceed as a thin linear growth over
the surface of the healing clot but has not, yet completely fused.

DISCUSSION
The use of histochtmical tests in studies of alveolar socket, healing has in-
troducctl a ncwcr concept in analysis of extraction wound healing in that not
only can anatomic structures be identified but information concerning the devel-
opment of biochemical factors, enzymes, and the presence of minerals can be
accumulated and demonstrated. In this way, a more detailed and accurate
study of the organization of tissue within the healing alveolar socket was
possible. (It was estimatctl that there was approximately a 5 per cent variabion
in experimental observations.) For instance, in other studiesl-I” where mention
was made of the progress of clot organization and development into connective
tissue, as well as of bone formation and mineralization, a certain amount of
 Time sequence of tissue regeneration 315

conjecture was involved. Fibers, when viewed in a section stained with hema-
toxylin and eosin, could not be distinguished or identified with great certainty
under all conditions. The same was true in the differential development of
granulation tissue into connective tissue, which has been a subject of much
speculation in previous studies of socket healing.6-8 With the application of his-
tochemical tests, however, this has become a procedure of much greater cer-
tainty. Collagen fibers staining blue with the Mallory stain and reticular fibers
staining brown or black with the application of the Bielchowsky test were more
readily identifiable. In differentiating granulation tissue from connective tissue,
for instance, the presence of acid polysaccharide ground substance and alkaline
phosphatase could be demonstrated histochemically. The presence of these
enzymes as well as of mature fibers indicated that more positive identification
and connective tissue could be demonstrated with greater assurance.
In studying bone formation, the presence of a glycoprotein by the periodic
acid-Schiff test indicated the maturation of the osteoid. The presence of calcium
deposits demonstrated by the von Kossa method indicated with reasonable ac-
curacy the degree of mineralization ; this was not possible by simply staining
with hematoxylin and eosin.
Table I indicates some of the significant investigations of typical studies of
extraction-wound healing with different species, based on purely morphologic
observations resulting from hematoxylin and eosin stains. The conclusions of
this present investigation are also listed for reference in Table I, on the last

Table I. Some of the significant investigations of typical studies of extraction

wound healing with different species
Begin-
Epithe- Epithe- “Organi- ning of Bone
Experi- lial pro- lial zation. of bone for- comple-
mental ““;emAn fusion clot” mattin tion
a&ma1 Investigator (says) (days) (says) (days)
Rat Huebsch (1952) 10-14 3 5
Smith (1g58) 14
Average : 13 3 5
“w Euler (1923) 8
Meyer ‘(:“gq 8
Schram 8 8
Claflin (1936) 3 7 3 5 31
Hubbel (1941) 3 to 7 3 to 4 4 to 5
Versnel (1953) 4 9 ii.; 22
Average 3.5 7.1 7.9 26.5
Sheep Harrison (1943) 12 6
Monkey Simpson {E] ; 14 7 28
Radden 15 to 16 6 to 7
Average 5 :i 15.5 6.8 i77.5
Man Deebach (1935) 17
Mangos 14 to 21 3 10
Christopher 14
Average 17.5 3 12
Man Amler (1968) 4 22 7 to 20 7 35
line, and are also noted diagrarlllnatically in +‘ig. I-1. They represent the time
sequence of tissue regeneration in alveolar sockets based on histologic studies
complemented by histochcmical tests.
In Table I, aside from Simpson’s” study, the commencement of epithelial
proliferation seems to follow a credible pattern : rat, 1 day; dogs, an average of
3.5 days ; monkeys, 3 days ; and my human studies, 4 days. However, when we
consider the fusion of cpithelium, which indicated the final closing of the socket
wound, the rat da.ta appear unusual since one would expect the rat epithelium to
fuse in less time than cpithelium of the dog or the monkey. In “organization of
clot,” the data in the t,able appear as follows: rat, 3 days; dog, an average of
Volume 27 Time sequence of tissue regeneration 317
Number 3

Table II. Summary of time sequence in normal human tissue regeneration based
on this study
Tissue 1 Days after extraction
Clot formation Same day
Replacement of clot by granulation tissue 7
Replacement of granulation tissue by connective tissue 20
Appearance of osteoid at base of socket
Filling of at least two thirds of socket fundus by trabeculae 3;
First evidence of epithelixation 4
Fusion of epithelium 24 to 35 and more

5.8 days ; sheep, 12 days; monkey, 15.5 days ; and man, 3 days. All data are
reasonable except in the case of the human study. Mangos,15who conducted this
study, noted an average of 3 days for “organization of clot.” This does
not appear credible, since it is the same healing time as seen in the rat and
more rapid rate than that found in the dog, sheep, and monkey. One would ex-
pect human tissues to heal at a slower rate than those of the lower mammals.
This could be expected particularly in Mangos’ investigationsI since the sub-
jects he studied consisted of two autopsy specimens from diseased persons, in
addition to seven biopsy specimens from hospital patients whose disorders were
so diverse as to include purpura, pulmonary tuberculosis, rheumatic fever,
and prostatic hyperplasia. It is not unreasonable to assume that this material
would indicate at least a degree of retarded healing and could not be con-
sidered to reflect the time sequence of tissue regeneration in extraction-wound
healing in the average, healthy dental patient. Claflin6 stated in 1936 : “. . . good
histological material from extraction wounds can be obtained only at necropsy
and since these patients have usually been very ill before death, a varying
degree of delayed healing can be reasonably expected.” In the present study
it was ascertained, on the basis of the histochemical tests, that a period of 20
days was required for all granulation tissue to be replaced by connective tissue.
The data in Table I, on bone healing and osteogenesis, have a credible se-
quence : rat, 5 days; dog, 6.7 days (average) ; sheep, 6 days ; monkey, 6.8 days;
and man (except for data from this investigation), an average of 12 days. In
this study it was noted that the first osteogenic fibers stained by histochemical
techniques appeared evident by the seventh day in isolated areas or attached
to old spicules of bone contiguous to the peripheral or apical alveolus. Dif-
ferences between the findings in this study and investigations of human casesby
others,14-la in which osteogenesis occurred in an average of 12 days, might be
explained by acknowledging that the debilitating illness of the subjects in-
volved could have caused varying degrees of delay in osteogenesis. Such differ-
ences might also be due to the use, in this study, of histochemical staining for
positive identification of osteogenic fibers and osteoid, which lessened the possi-
bility of error.
SUMMARY
1. A preliminary study of the time sequence of tissue regeneration in human
extraction wounds has been completed.
318 -1nder OS., O.M. &I0.1'.
March, 1969

2. In order to obviate some of the errors inherent in the studies of socket

healing from diseased human caws or by interpolation from animal experi-
mentation, biopsy specimens mere taken from apparently healthy persons who
wcrc screened to climinatc>, as far as possible, gross pathosis and xwiablcs which
might interfere with normal tissue regeneration.
3. Generally, the time sequence in normal human tissue regeneration based
on this study has been summarized diagrammatically in Fig. 14 and in Table II.
The author gratefully acknowledges the assistance of Dr. Irving Salman, Director and
Attending Oral Surgeon, Beth Israel Hospital, and Dr. Gunther G. Kronheim in obtaining
postextraction biopsies.

REFERENCES
1. Huebsch. R. F.. Coleman. R. D.. Frandson. M. M.. and Becks. II.: The Healing Process
Following Mola; Extract;ons, O&L SURG., URAL MED. & ORAL $ATH. 5: 864, 1955
2. Smith, R. L.: Role of Epithelium in the Healing of Experimental Extraction Wounds,
J. D. &es. 37: 187, 1958. -
3. Euler, H.: Die Heilung von Extraktionswunden, Deutsche Monatschr. Zahnh. 41: 655,
1923.
4. Meyer, W.: Die Heilung von Extraktionswunden unter abnormen Verhiiltnissen, Ztschr.
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