Visualization of the A detailed study was made by Wilderman, Wentz and

4
Orban, to determine the origin of granulation tissue
Microvascularization of the which forms the new gingiva when bone is exposed.
Within a period of from six to ten days, granulation tis-
Healing Periodontal Wound sue covered the osseous surface; further differentiation
continued in the process of healing; the gingiva was
IV. Mucogingival Surgery: apparently normal histologically at 28 days.
7
Seven patients were treated by Pfeifer using the api-
Full Thickness Flap cal repositioned flap and split flap techniques. He noted
that there was much more osteoclastic activity when the
mucoperiosteal flap technique was applied. If periosteum
by
and gingival connective tissue were permitted to remain
SIMAO K O N * (split flap), the extent of bone resorption was less.
ARTHUR B. NOVAES** Osteoclasts were practically absent on the periodontal
M . P. R U B E N * * * ligament side of the osseous septa. The healing and
maturation of the soft tissue were similar in both pro-
H . M . GOLDMAN****
cedures.
D E S P I T E T H E FACT that gingival flaps have been used
1, 2
In order to study the healing of the split thickness
for several decades, the importance of this technique 8
flap, Staffileno et al conducted an investigation on four
has been emphasized recently by the more frequent need
adult dogs. The gingiva was split in such way that the
for access to bone for correction of advanced osseous
3-13
epithelial attachment was preserved; after the dissection,
lesions. In consequence many wound healing studies
the gingival flap was repositioned to the original position.
have been designed to evaluate not only the initial tissue
Histologically the authors were able to describe an epi-
damage and reaction, but also the sources and mode of
thelial bridging of the exposed connective tissue which
attachment of regenerative soft tissue and bone. Most of
started within 48 hours, and was complete in six days.
the information derived from those investigations give
At the same time marked cellular activity was taking
little attention to microvascularization of the wound.
place in the connective tissue. "Budding capillaries were
3
Linghorne and O'Connel, in 1950, studied the re- seen in the clot and also projecting from around the
attachment of the soft tissue after mucoperiosteal flaps. severed collagen bundles along the wound edge. New
They created an osseous lesion by removing radicular collagen fibers were being laid down as the fibrin threads
bone thus exposing the tooth; the flap was subsequently were resorbed." Bone resorption reached a peak at six
repositioned and sutured. Histologic sections were ob- days and gradually waned thereafter. Osteoblastic ac-
tained which documented healing for periods ranging tivity was more intense at 21 days. The final result was
from five days to one year. They concluded that: (1) a functional repair without significant anatomical de-
9

soft tissue reattachment can occur; (2) new cementum formity. Staffileno et al modified the above described
is laid down either on old cementum or on resorbed root experiment by excising the gingival side of the flap in
structure; (3) the reattached connective tissue fibers are order to compare the cellular activity in hard and soft
aligned parallel to the tooth; (4) epithelium migrates tissues. Investigation at 21 days revealed a "slight ana-
downward onto the root when connective tissue reattach- tomical deformity of the dentogingival junction." This
ment fails; (5) alveolar bone regeneration occurred in deformity was characterized by: (a) a more shallow
some degree. sulcus, (b) a dentogingival junction slightly apical to its
preoperative position.
This project represents a portion of the requirements for the
Master of Science in Dentistry Degree, awarded by Boston Uni- Mucoperiosteal flaps in humans were carried out by
versity, School of Graduate Dentistry, May 19, 1968. 10
This investigation was supported in part by a grant f r o m the Kohler and Ramfjord. Full flaps were raised; bone and
United States Public Health Service, National Institute of Dental cementum were preserved and protected from inadvert-
Research, Grant No. DE-02569-01, -02. ent damage. The flaps were then repositioned and su-
*O.A.S. fellowship; School of Dentistry, Sao Paulo University,
Sao Paulo, Brazil; graduate student, Boston University, School of tured. The clinical and histological observations showed
Graduate Dentistry. Present address: Faculdade de Odontologia, complete healing without any significant loss of perio-
Universidade de Sao Paulo, R. Tres Rios 363, Sao Paulo, S.P., dontal structure.
Brazil.
**C.A.P.E.S. (Brazil) fellowship, School of Dentistry Ribeirao
Preto, Sao Paulo, Brazil; graduate student, Boston University,
Differences in the healing process following the flap
11
School of Graduate Dentistry. technique were observed by Grant when grooves
***Professor of Stomatology, Boston University, School of were made in the roots. Six notched teeth had an api-
Graduate Dentistry.
****Dean and Professor of Stomatology, Boston University,
cally directed epithelial proliferation; all of the other
School of Graduate Dentistry. specimens presented an unaltered epithelial attachment.
Page 5/441
Page 6/442 K O N , NOVAES, R U B E N , G O L D M A N

11
Grant demonstrated, in humans, sequestration of M A T E R I A L S AND METHODS
alveolar bone following full flap operations. Sequestra-
tion could occur even months after the operation; the This investigation was performed on eight young mon-
patient or dentist may not necessarily be aware of it. This grel adult dogs weighing an average of 12 kilograms.
necrotic bone may be expelled, resorbed or encapsu- Full thickness mucoperiosteal flaps were raised which
lated by connective tissue fibers. extended from the mesial of the first to the distal of the
third upper premolar, where relaxing vertical incisions
were made (Bard Parker blades #15). Each flap was
lifted with a periosteal elevator (#7 wax spatula);
their extent included at least one millimeter of alveolar
mucosa. Notwithstanding all of the care which had been
taken to remove the connective tissue from the bone and
teeth, some remained and was removed with Gracey
curettes. The flap was returned to its original position,
and sutured vertically and interproximally with Ethicon
exodontia sutures (000, Atraloc needles). After one week
the animals were anesthetized with 0.5 cc of pentobarbi-
tal sodium (Diabutol) per kilogram of body weight,
and the sutures were removed.

Immediately after surgery and on the following day,

600,000 units of penicillin were given by intramuscular
injection to each of the animals. Once daily, each dog
was fed Purina ration with full vitamin and mineral
(Wesson salt mixture) supplementation, having water
for consumption ad libitum. For the first three weeks
after surgery, the animals were fed a soft diet, later
being returned to a standard laboratory chow (hard)
diet in order to inhibit debris and plaque accumulation
15, 1 6
at the gingiva.

Perfusion of India ink, a filtered carbon black sus-

FIGURE 1. Control specimen. High and heavy muscu-
pension (Gunther Wagner), was performed following
lar insertion. (Mallory, X100). the technique which was described in detail in the first
17
article of this series. A silicone rubber material (Micro-
fil) was utilized in one of the animals to permit compari-
In addition to studying tissue regeneration after flap son of perfusion media.
12
surgery, Hiatt, Stallard, Butler and Badgett, measured
the strength, and evaluated the condition, of the flap After sacrifice with an overdose of pentobarbitol so-
attachment. Forces required to separate the flap from dium the jaws were removed, cut in blocks and kept for
the underlying tissues were measured by a tensiometer two weeks in a solution of 10 percent buffered formalin.
at varying periods after flap replacement. They con- Generally the time required for the subsequent decalci-
cluded that "the initial attachment of the flap was fication in five percent nitric acid ranged from three to
through the epithelium and the fibrin did not appear to five weeks. The blocks were embedded in paraffin and
contribute significantly to the retention of the flap." cut into sections 8 microns thick. The sections were
When the applied forces separated the flaps, the split stained by hematoxylin-eosin and Mallory techniques
was always in the soft tissue, leaving a portion of the (Goldman-Bloom modification). Some of the specimens
new epithelial tissue on the root surface. It would appear were not decalcified so that they could be studied as
that a large accumulation of fibrin and complete elimina- cleared sections (400 microns thick) and will be the
tion of cementum delays healing. subject of a forthcoming report.

The microvascularization has not received a thorough RESULTS

evaluation in the studies on wound healing. This article
Clinical Findings
is part of an investigation which intends to observe
changes, alterations and behavior of vessels in areas Flaps comprising the epithelium, connective tiissue
where gingivectomy, curettage and mucogingival sur- and periosteum, from the mesial of the first to the distal
geries (periosteum retention and full thickness flap) of the third premolars, and encompassing half of the
were performed. 13, 1 4
papillae and the entire attached gingiva, were raised be-
VASCULARIZATION OF THE H E A L I N G MUCOPERIOSTEAL FLAP Page 7/443

FIGURE 2. Control specimen, (a) The epithelial

adherence is on enamel its apical termination is
at C.E.J. (Mallory, X35). (b) Higher magnifica-
tion of (a). Long rete pegs and keratinizing sur-
face of epithelium; capillary loops labelled by
carbon black (Mallory, X100). (c) Group III
fibers covering the tip of buccal septum (Mallory,
X35).
Page 8/444 K O N , NOVAES, R U B E N , G O L D M A N

yond the mucogingival line (Fig. 4a, b). The bone was but the bone was not exposed. The 36-hour animal
exposed. However, in a few of the animals some connec- demonstrated a well-adapted flap, with the interradicular
tive tissue remained, primarily at the alveolar crest and area being covered by edematous gingival tissue. The
at the site of strong muscular insertions. Theflapwas margin of the gingiva of the two-day specimen did not
returned to its former position and secured by inter- follow the anatomy of the tooth in the interradicular
proximal and lateral sutures (Fig. 4c). There was little area, where some necrotic soft tissue could be observed
bleeding from the interproximal and vertical incisions. (Fig. 8a).
In two of the animals the depression which indicates It was interesting to note that the interradicular aspect
the beginning of the interradicular zone could be seen, of the 36-hour specimen was again exposed at four days.
This was due possibly to gingival shrinkage as the in-
flammatory process subsided, or possibly because of in-
advertent positioning of the flap apically.
At seven days, when no irregular factors had inter-
fered, the gingival margin was thin and followed the

FIGURE 3. Control specimen, (a) Inflamed subsulcular area,

(b) Higher magnification of (a); vessels labelled by carbon
black. (c) Higher magnification of (a). Increase in number of FIGURE 4. (a) Upper premolars, area where full thickness
vessels perfused with carbon black, a characteristic of the flap was performed. (b) Flap is lifted. (c) Flap is returned to
inflammatory process (Hematoxylin-eosin. Original magnifi- its former position and secured by interproximal and lateral
cations X100). sutures.
 VASCULARIZATION OF T H E H E A L I N G MUCOPERIOSTEAL F L A P Page 9/445

anatomy of the tooth, the "sulcular parallel" lines could indicating patency of vessels in these areas. After 85
be observed, and the interproximal granulation tissue days, healing and the tissue coloration produced by per-
appeared clinically epithelized. The sutures were loose fusion were similar to the control animals (Fig. 16a).
or missing in some specimens, with a consequent forma-
tion of hyperplastic granulation tissue beneath the flap
RESULTS
at this location.
Histological Findings
In two weeks the wound was clinically healed in the
first and second premolar areas; in the third premolar, Control. In the overall picture, the anatomy of the
where the muscle attachment interfered, the gingival area showed a thin buccal bone septum with a very
margin was still rolled. One of the animals had inflam- heavy muscular insertion at the crest. Consequently, the
mation and retarded healing associated with adverse zone of the attached gingiva was very narrow. The mus-
environmental factors such as: pull of musculature on cle described above is characteristic of the third pre-
flap or marginal gingiva, loss of sutures and the presence molar area of the dog (Fig. 1 ). No apparent interference
of muscles in the wound healing is manifested in the sec-
tions taken from the first and second premolars.

The gingival stratified squamous epithelium is well

keratinized, with a large number of long, thick rete pegs
(Fig. 2a, b). There is much variation in the depth of the
sulcus, depending upon the area from which the sections
were taken. Some of the animals had a long sulcular epi-
thelium reflecting cellular desquamation. The bottom of
the epithelial attachment is at the cementoenamel junc-
tion (Fig. 2a). The subsulcular area is inflamed, and
the perfusion indicates increase in vascularization (Fig.
3a, b, c). The cut ends of the circular fibers comprise a
large part of the corium.

The long rete pegs and keratin layers are easily dis-
cernible with high magnification, which permits observa-
tion of the perfused capillaries between the rete pegs
(Fig. 2b). Dense connective tissue, well collagenated with
some perfused vessels, underlies the epithelium.

Small vessels, labelled by carbon black, are present in

a dense and organized periodontal ligament. The crestal
aspect of the ligament and the tip of the buccal bone are
covered by heavy bundles of Group III fibers which
emanate from the dentogingival junction (Fig. 2c). It is
interesting to note the arrangement of the Group III and
periosteal fibers, which increase in thickness at the point
FIGURE 5. Zero hour specimen. Separation of the soft wher the heavy muscle is inserted. The periosteum as-
tissue from the bone during histologic preparation sumes its normal shape and thickness more apically to
(Mallory, X35). the insertion. With higher magnification the connection
of hairs in the gingiva. Great improvement in this area, of muscular and connective tissue bundles forms a well
and the reduction of inflammation could be observed delineated area. The muscle joins the thick connective
concomitant with the change from a soft to a hard diet tissue in an oblique fashion.
at three weeks. Although healing progressed uneventfully
Zero Hour. A separation of the soft tissue from the
for 31 days, the incisional lines were still noticeable. The
bone, which occurred inadvertently during histologic
perfused tissues showed red spots in the interproximal
preparation, makes the incision clearly visible (Fig. 5).
areas for two days, suggesting that some vascular ob-
The flap was detached apically for quite some length
struction had occurred impairing blood supply to these
beyond the zone of attached gingiva in order to expose
zones. When the marginal area was not damaged, the
a reasonable amount of bone, since one of the purposes
sulcular parallel lines were clearly visible.
of the study was to investigate the relationship between
The seven-day healing perfusion showed that the red soft tissue and bone in the healing process and possible
spots described above were replaced by carbon black, changes in vascularization.
Page 10/446 K O N , NOVAES, R U B E N , G O L D M A N

FIGURE 6. Zero hour specimen, (a) Sulcular epithelium was preserved (H-E, X35). (b) Higher magnification of (a). Vessels of
the flap labelled by carbon black (H-E, X100). (c) Higher magnification of (a). Marginal gingiva with perfused vessels (H-E,
X100).

The flap was raised in a manner to preserve the sul- irregular surface topography, frequently all of the con-
cular epithelium (Fig. 6a). The area of the dentogingival nective tissue was not removed.
junction was severed in order to reach and expose the
bone. A very thin blood clot covered the exposed bone Two Days. At this time the attachment of theflapto
and connective tissue surface. The vessels of the flap subjacent structures seems to be histologically weak.
which were not disturbed when the soft tissue was raised Shrinkage during histologic preparation resulted in de-
were labelled with carbon black (Fig. 6b). With higher tachment of the soft tissue, and the blood clot which
magnification it is possible to observe the shape of the occupies the space between the flap and the bone gen-
rete pegs, as well as that of the keratin layer, the remain- erally remains with one or the other. No extensive
ing sulcular epithelium, and particularly the perfused changes took place in the gingival tissue in this section.
vessels in the gingival margin (Fig. 6c). These vessels There is an increase in flap vascularization with some
extend parallel to the nonkeratinized epithelium and in vasodilation (Fig. 7a, b). The shape of the rete pegs
at the tip of the gingiva is different from that of the
a capillary loop pathway, between rete pegs beneath the
attached gingiva, tending to beflattenedor absent. Con-
oral keratinizing epithelium.
sequently, the small capillary loops are not seen in-
In areas where muscle attachments predominated it between the shallow rete pegs. Immediately below the
was difficult to separate the soft tissue from the bone. sulcular epithelium where the instruments had to be
Histologically, it was evident that where bone had an forced to detach the flap, there is some damage and
 VASCULARIZATION OF THE H E A L I N G MUCOPERIOSTEAL F L A P Page 11/447

FIGURE 7. Two-day specimen, (a) Marginal area. Increase in vascularization (H-E, X35). (b) Higher magnification of (a). Vessels
perfused with carbon black (H-E, X100). Note in Figure (a) that a blood clotfillsthe slit in the flap; tissueflapinadvertently lac-
erated during surgery.

hemorrhage. The adherence mediated by the blood clot flap. For attachment to occur, the blood clot would have
between the soft and hard tissue is not substantial enough to be resorbed and new connective tissue be synthesized
to hold them together. The periodontal ligament is not to reform the attachment.
altered. Apparently there was no visible change in the
Six and Seven Days. A n inflammatory reaction and an
function of the vessels which remain in the soft tissue.
increase in vascularization of the remaining connective
The blood clot which formed between the soft and the
tissue and flap can be clearly observed.
hard tissue, does not seem to have a tendency to remain
on either one. In histologic preparation, when the flap At this time the flap still seems to be prone to separa-
had been lifted unintentionally the clot sometimes re- tion from the subjacent tissues when tension is applied.
mained on the bone (Fig. 8b, c), or on the soft tissue. This suggests that at six-seven days postoperatively,
The thickness of the clot varied according to the damage even though connective tissue synthesis has commenced,
caused to the soft tissue and the degree of adaptation of the annexation of the corium of the flap to bone and re-
the flap to the bone. Hemorrhage, an inflammatory cell maining gingival corium is a tenuous one. The blood clot
infiltrate, and an increase in vascularization are found between the soft and the hard tissue has been replaced
close to the surface of the wound. by young connective tissue being formed from the con-
nective tissue cells of the bone and flap. Inasmuch as
Four Days. As the adherence between the bone and the separation occurred in the blood clot which remained
the connective tissue is now apparently stronger, the flap in the middle of the wound, it is reasonable to assume
remains in place during histologic preparation (Fig. 9a, that this is the weakest point (Fig. 10). New collagen
b). No formation of new sulcus can be observed at this fibers emanating from the older connective tissue are
time. The shape of the gingival margin is modified and forming a phase of the granulation tissue interposed be-
appears flat and thick. In this specimen, the flap is tween the flap and subjacent hard and soft tissues. Perio-
maintained in place more securely. Possibly this is due dontal ligament is intact; there is some resorption of the
to the blood clot, since there is no epithelial attachment. bone on the periosteal side of buccal bone septum where
A small artifact present between the clot and the bone some narrow spaces are open and perfused vessels are
suggests that at this time the clot has a more adherent clearly observed (Fig. 1 la, b, c).
quality in relation to the soft tissue than to the bone.
Twelve Days. The flap is reattached to the bone and
There is no change in the buccal bone plate either on its
tooth, with the sulcular wall on enamel. There is mild
buccal or its periodontal ligament side. Some vessels,
inflammation of the marginal gingiva and some desqua-
close to the blood clot, are visible due to the carbon
mation (possibly indicative of cell turnover) of the new,
black perfusion.
thin sulcular epithelium. The oral epithelium is keratin-
There were instances in which connective tissue fibers ized and has rete pegs which are essentially normal in
were left inserted in the bone. During the initial phase of shape, although somewhat rounded at their tips. The
healing they had not been reattached to the flap due to a bulk of the gingiva is comprised of a very dense connec-
thick blood clot having formed between bone and the tive tissue (Fig. 12).
Page 12/448 K O N , NOVAES, R U B E N , G O L D M A N

FIGURE 8. Two-day specimen, (a) Clinical aspect, (b) Clot remains on

bone (H-E, X35). (c) Higher magnification of (b). Perfused vessels where
theflapwas lifted (H-E, X430).

Sixteen Days. Bone apposition predominates on the

buccal bone plate. Islands and projections of new bone
are present. Although a small area close to the crest is
still undergoing resorption, the periodontal ligament side
of alveolar bone is not affected.
When adverse factors interfered with the apposition
of the flap to bone, excessive granulation tissue was
formed between the flap and the tooth. Sulcular epithe-
lium proliferated to cover this new connective tissue.
This epithelium with an irregular morphology formed
the new inner sulcular wall.

Twenty-three and Thirty-one Days. Healing appeared

to progress normally, with reformation of the bone
which had been lost from the crest and buccal aspect of
the osseous septum. The flap is reattached to the tooth
by dense, organized, connective tissue. The muscle is in
a more apical position and is attached via connective
tissue to the outer portion of the septum. At the point
where the muscle fibers join the connective tissue, the
The pathway and shape of the large vessels which ex- bundle offibersare thick and dense.
tend from the mucobuccal fold and are contained in the
flap, apparently were not affected (Fig. 13a, b, c). A The nick on tooth surface, made unintentionally dur-
dense and regenerated connective tissue covers the crest ing the surgical procedure, contains epithelium in close
of the bone. A t the same time the buccal bone plate pre- adherence to the tooth surface. The marginal portion of
sents both osteoblastic and osteoclastic activity. The re- the gingiva manifests an inflammatory response. There
sorption takes place only at the buccal side of the crestal is an increase in vascularization and epithelial prolifera-
area. There is an alignment of osteoblasts on the perio- tion at this area. Carbon black particles within the vascu-
dontal ligament side of the septum, in the marrow lar lumina identify more small vessels in the inflamed
spaces, and on the buccal aspect of the alveolus, where area than in the remainder of the corium (Fig. 14a, b).
the resorption had previously occurred. The dentogingival junction and the periosteal side of the
 VASCULARIZATION OF THE H E A L I N G MUCOPERIOSTEAL F L A P Page 13/449

buccal bone are comprised of completely healed, dense

connective tissue. The facial aspect of the buccal cortical
plate is being rebuilt by new bone which has been formed
around the vessels and marrow spaces.

Thirty-eight Days. The tissue which was affected by

surgery has been regenerated. The sulcular wall is in-
flamed, and there is some apical migration of the epi-
thelial attachment. A t this location, an artifact suggests
that the epithelial and connective tissue attachments are
still imperfect. A n increase in number of labelled vessels
in the marginal gingiva could be observed, thus identify-
ing a phase of an inflammatory reaction (Fig. 15). Buc-
cal cortical bone has been reconstructed by intense
osteoblastic activity.

Fifty-five Days. At this time no major differences are

observed. The periodontal tissues have been recon-
structed, the appearance of which is similar to the 85-
day specimen.

Eighty-five Days. The presence of a secondary, ac-

quired cuticle identifies the bottom of the sulcus on
enamel. One specimen has a long, thin epithelial attach-
ment extending to the cementoenamel junction and com-
prised of few layers of epithelial cells (Fig. 16b, c, d). A
mild inflammatory state in another specimen is contained
at the tip of the gingival margin; and some epithelial pro-
FIGURE 9. Four-day specimen, (a) Thick blood clot between liferation is present with increase in number of vessels
hard and soft tissue (H-E, original magnification X35). (b) labelled by carbon black (Fig. 17a, b).
Higher magnification of (a) (H-E, original magnification
X100). The clot also contains many extravasated leucocytes. The dentogingival junction has been effectively re-
newed. The buccal bone plate which had reflected a re-
sorptive lesion after surgery is now rebuilt, resulting in a
smooth, homogenous topography of buccal bone plate
(Fig. 18a, b).

DISCUSSION

The blood clot observed interposed between the flap

and subjacent tissues, in the two- and four-day speci-
mens was thick and increased in width from the crestal
to the apical areas. This signified an incomplete or im-
perfect adaptation of the gingiva to the underlying bone
and gingival corium. This clot was resorbed and con-
comitantly replaced by young connective tissue derived
from marrow spaces of bone, the crestal aspect of perio-
dontal ligament and the connective tissue of the mucosa
at apical aspect of flap. Intensive fibroblastic activity
was first observed in the six-day specimen. Many new
capillaries containing carbon black could be seen in
an area of much cellular activity. It should be noted that
endothelial proliferation from preexisting capillaries is
18
marked at this time, continuing until the 16th to 18th
19
day postoperatively. According to Toto et al, the new
fibroblastic population and new capillaries are derived
FIGURE 10. Six-day specimen. Blood clot that was not
from the undifferentiated reserve connective cells. A thin
replaced by young connective tissue is the weakest
point (Mallory, X35). blood clot remained in the middle of the young connec-
 Page 14/450 K O N , NOVAES, R U B E N , G O L D M A N

FIGURE 11. Seven-day specimen, (a) Buccal bone plate (H-E,

X35). (b) Higher magnification of (a). Periodontal ligament
(H-E, X100). (c) Higher magnification of (a). Some marrow
spaces are open and perfused vessels are clearly observed
(H-E, X100). Some resorptive activity is evident and is medi-
ated by osteoclasts. An osteolytic type of resorption may FIGURE 12. Twelve-day specimen. Flap is reattached;
occur at same time produced by lysosomal enzymes of osteo- mild inflammatory reaction; vessels perfused by carbon
cytes. black (Mallory, X35).
VASCULARIZATION OF THE H E A L I N G MUCOPERIOSTEAL FLAP Page 15/451

FIGURE 13. Twelve-day specimen, (a) Perfused ves-

sels extending parallel to periosteum (H-E, X35).
(b) Higher magnification of (a) (H-E, X100). (c)
Higher magnification of (a) (H-E, X100).
Page 16/452 K O N , NOVAES, R U B E N , G O L D M A N

FIGURE 14. Thirty-one day specimen, (a) The margin of the gingiva manifests an inflammatory response
(H-E, X35). (h) Higher magnification of (a). Labelled vessel beneath sulcular epithelium (H-E, X100). Col-
lagen resorption is also evident.

tive tissue; when a separation (flap detachment) oc-

12
curred it did so at that location. Hiatt et a l pointed out
that the weakest part of the attachment between the flap
and the bone was in the area of the fibrin clot. They
reported that during the first days of the healing process
the flap could be easily detached from the tooth. In this
experiment, when detachment of the flap occurred dur-
ing histologic preparation, it was in the zero hour, two-
or four-day specimens. This fact also demonstrates that
the clot contributes very little to maintaining theflapin
position. A t seven days the flap was partially reattached
by new connective tissue fibers in the form of reticulum
and collagen. The 12-day specimen showed well organ-
ized and regenerated connective tissue at the dentogin-
gival area, as well as on the surface of the bone.

The presence of carbon black solution within the ves-

sels demonstrated little inhibition offlapvascularization
when the flap was detached, thus the viability of the
cellular components and the hydration of the flap were
maintained. A t two days the vessels which were con-
tained within the flap showed some dilation. The vessels
labelled by the perfused material increased in number
until the sixth day postoperatively, when budding and
FIGURE 15. Thirty-eight day specimen. Increase in sprouting capillaries were identified within the granula-
number of labelled vessels in the marginal area. Imper-
tion tissue at the interface between flap and bone. The
fect attachment (H-E, X35). The pocket epithelium is
hyperplastic. number of labelled vessels diminished gradually over a
 VASCULARIZATION OF THE H E A L I N G MUCOPERIOSTEAL FLAP Page 17/453

FIGURE 16. Eighty-five day specimen, (a) Healing and perfusion were similar to the control specimen, (b) Long epithelial attach-
ment (H-E, X35). (c) Bottom of epithelial attachment is at the cementoenamel junction (H-E, X35). (d) Higher magnification of
(b). Labelled vessels beneath the thin epithelial attachment (H-E, X100). Vessels extend parallel to epithelium; a normal vascular
characteristic of the area.
Page 18/454 K O N , NOVAES, R U B E N , G O L D M A N

FIGURE 17. Eighty-five day specimen. (a) Mild inflammation (Mallory, X35). (b) Higher magnification of (a).
Secondary cuticle (Mallory, X100). When new epithelial adherence forms postoperatively, there is no evi-
dence of keratinization; the new epithelial "attachment", though derived from oral epithelium assumes quali-
ties comparable to those of the original sulcular epithelium.

period of 12 days, during which period the connective In the earlier specimens, debris, blood and a sugges-
tissue achieved a more normal appearance. tion of mechanical retraction of the flap impeded the
epithelization of the crestal aspect of the flap and the
The osteoclastic activity observed on the facial aspect
formation of a new epithelial attachment and sulcular
of the buccal septum in the six-day specimen reached its
epithelium. A t 12 days, complete regeneration of the
peak a day later, but bone resorption at the crest of the
dentogingival junction which was comprised of tooth,
bone (buccal side) could be observed for periods rang-
dense collagen fibers, and thin sulcular epithelium and
ing to 16 days. A t 12 days the presence of osteoblasts epithelial attachment had occurred. A mild inflamma-
was predominant. The bone apposition became more tory reaction existed within the marginal gingiva.
intense so that in the 38-day specimen the buccal bone
septum was anatomically reestablished. A bone resorp- It was found that the correct positioning of the flap,
tive phase was dominant at first; however, in later speci- together with the necessary means to maintain it in place
mens bone formation was seen to coincide with resorp- during thefirstweek of healing were two of the most im-
tion. Later the process was purely appositional. portant requirements for success in a flap operation.
When the sutures are loose, or when muscle pull creates
These findings were similar to those described by a space between the flap and the tooth, the space is filled
12
Hiatt et al, who reported the presence of osteoclasts by a fibrin clot. This situation could be detrimental to
in the marginal bone through the third postoperative 12
the reattachment. Hiatt et a l reported that in studying
week, but ". . . in all instances this bone loss had been the strength of the flap attachment during healing, "The
recovered by one month. The earliest bone repair was healing was delayed by a large accumulation of fibrin."
observed at two weeks."
Pressure applied to the flap is another means of ob-
4
In an experiment on bone which was denuded, it was taining better adaptation of the soft to the hard tissue. In
found that extensive bone loss resulting in an anatomical one of the animals, a large space having the appearance
deformity took place. Correlation of thesefindingswith of a "pseudocyst" was created between the flap and the
those of this experiment suggested that a gingival cover tooth. This could have been avoided had adaptive pres-
inhibits resorptive activity and permits an appositional sure been applied immediately after surgery and main-
response to occur. tained postoperatively.
 VASCULARIZATION OF THE H E A L I N G MUCOPERIOSTEAL F L A P Page 19/455

FIGURE 18. Eighty-five day specimen, (a) Buccal bone plate is rebuilt (H-E, X35). (b) Buccal bone plate has
smooth and homogenous topography (Mallory, XI00).

The "sulcular parallel lines," which apparently are the The intravascular instillation of filtered carbon black
capillaries at the tip of the gingival margin, were re- solution was a valid mean of demonstrating the presence,
vealed through close examination of the kodachromes distribution and patency of vessels and the modifications
taken during the experiment. Although these lines dis- which occurred in them immediately after the incision
appeared just after surgery, they were observed again in and during the healing process. The presence of the per-
approximately two days. This finding was seen more fused material made it simpler to follow, to localize and
clearly when carbon black was perfused for the purpose to identify the vessels. The solution was useful for de-
of labelling the capillaries of the gingival margin. lineating vasodilation, variances in vasopermeability
during the healing process, severance of vessels at the
S U M M A R Y AND CONCLUSIONS
wound surface, the relationship between the vessels and
the blood clot, the increase in the number of vessels after
A study was carried out for the purpose of observing injury, and the return of the vessels to normalcy with
the wound healing process and the behavior of the blood progressive healing.
vessels when full thickness (muco-periosteal) flap tech-
1. The greatest degree of cellular activity in the area
niques were performed. Pelikan carbon black suspension
took place at six days, which was responsible for the
(Gunther Wagner) was filtered and injected into the
formation of new connective tissue (reparative tissue)
common carotid arteries of young adult mongrel dogs.
which, in turn, replaced the existing blood clot.
The perfusion technique was performed shortly before
the animals were sacrificed; that is, immediately after 2. Osteoclastic and osteoblastic activities were pre-
and at 2, 4, 6, 7, 12, 16, 23, 31, 38, 55 and 85 days after dominant respectively at 7 and 12 days although there
the surgery. Thus perfusion was performed in vivo to was some coincidence of these processes. The bone was
delineate the then patent, living circulation. completely rebuilt at 31 days.
The macroscopic aspect of the wound was evaluated 3. The vasculature was labelled with the carbon black
clinically and by means of kodachromes. Histologic sec- suspension, thereby demonstrating changes in vasculari-
tions (8 microns in thickness) were prepared and stained zation. The vessels within the flap were not significantly
by the Mallory technique (Goldman-Bloom modifica- damaged when it was raised and retracted. Dilation of
tion) and hematoxylin-eosin. the vessels constituted the initial response; this vasculari-
Page 20/456 K O N , NOVAES, R U B E N , G O L D M A N

zation increased during the following six days. The num­ 9. Staffileno, H . , Levy, S. and Gargiulo, Α . : Histologic
ber of patent blood vessels and their dilatation dimin­ Study of Cellular Mobilization and Repair Following a Peri­
ished gradually and achieved a better appearance at 12 osteal Retention Operation via Split Thickness Mucogingival
Flap Surgery. J. Periodont, 37:117-131, 1966.
days.
10. Kohler, C . A . and R a m f j o r d , S. P.: Healing of Gin­
gival Mucoperiosteal Flaps. Oral Surg., Oral Med., and Oral
4. Sulcular parallel lines could be identified through Path., 13:89-103, 1960.
close examination of the kodachromes. When carbon 11. Grant, D . Α . : Experimental Periodontal Surgery:
black was injected these lines appeared darker, suggest­ Sequestration of Alveolar Bone. J. Periodont., 34:409-425,
ing that they are capillaries. The tip of the gingival mar­ 1967.
gin is thin, and its transparency reveals the presence of 12. Hiatt, W. H , Stallard, R. E . , Butler, E . D . and Bad-
gett, B. : Repair Following Mucoperiosteal Flap Surgery with
these small vessels. The sulcular parallel lines which Full Gingival Retention. J. Periodont, 39:11-16, 1968.
were not evident immediately after surgery could be 13. Kon, S. : Visualization of the Microvascularization of
seen again in the two-day and later specimens. the Healing Periodontal Wound: I. Curettage; II. Mucogin­
gival Surgery—Full Thickness Flap. Thesis for M.Sc.D.,
Boston University, School of Graduate Dentistry, 1968.
REFERENCES 14. Novaes, A . B.: Visualization of the Microvasculari­
zation of the Healing Periodontal Wound: I. Gingivectomy;
1. W i d m a n , L . : The Operative Treatment of Alveolar II. Mucogingival Surgery—Periosteum Retention Technique.
Pyorrhea. Brit. Dent. J., 37:105, 1917. Thesis for M.Sc.D., Boston University, School of Graduate
2. G o l d m a n , H . M . : Periodontia, ed. 3. St. Louis, C . V . Dentistry, 1968.
Mosby Co., 1953. 15. Ruben, M . P., McCoy, J., Person, P. and Cohen, D .
3. Linghorne, W. J. and O'Connel, D . C : Studies in the W. : Effects of Soft Dietary Consistency and Protein Depriva­
Regeneration and Reattachment of Supporting Structures of tion on the Periodontium of the Dog. Oral Surg., Oral Med.,
the Teeth. I. Soft Tissue Reattachment. J. Dent. Res., 29: and Oral Path., 15:1061, 1962.
419-428, 1950. 16. Egelberg, J. : Local Effect of Diet on Plaque F o r m a ­
4. W i l d e r m a n , M . N . , Wentz, F . M . and Orban, B. J . : tion and Development of Gingivitis in Dogs. I. Effect of
Histogenesis of Repair After Mucogingival Surgery. J. Perio­ Hard and Soft Diets. Odontologisk Revy, 16:31, 1965.
dont, 31:283-299, 1960. 17. Kon, S i m a o , Novaes, A . B., Ruben, M . P. and Tur­
5. W i l d e r m a n , M . N . : Repair After a Periosteal Reten­ ner, H : Visualization of the Microvascularization of the
tion Procedure. J. Periodont, 34:487-503, 1963. Healing Periodontal Wound. I. Technique. Periodontics,
6. G l i c k m a n , I., S m u l o w , J. B., O'Brien, T . and Tannen, 6:257-263, 1968.
R.: Healing of the Periodontium Following Mucogingival 18. Majno, G . : Wound Healing, Advances in Biology of
Surgery. Oral Surg., Oral Med., and Oral Path., 16:530-538, Skin, Vol. 5, Montagna, W. and Billingham, R. E . New York,
1963. M a c m i l l a n Co., 1964.
7. Pfeifer, J. S. : The Reaction of Alveolar Bone to Flap 19. Toto, P. D . and Abati, A . : The Histogenesis of Gran­
Procedures in Man. Periodontics, 3:135-140, 1965. ulation Tissue. Oral Surg., Oral Med., and Oral Path., 16:
8. Staffileno, H . , Wentz, F . and Orban, B.: Histologic 218, 1963.
Study of Healing of Split Thickness Flap Surgery in Dogs. 20. W i l d e r m a n , M . N . : Exposure of Bone in Periodontal
J. Periodont, 33:56-69, 1962. Surgery. D . Clin. N . A m e r i c a , p. 23-26, March, 1964.

Announcements
T H E AMERICAN A C A D E M Y OF ple University School of Dentistry, Post-Graduate Division.
ORAL MEDICINE "Wound Healing as Related to Electro-Surgical Procedures."

The A m e r i c a n A c a d e m y of Oral Medicine will hold its S e m i - Edgar A . Tonna, B.S., M.S., Ph.D., Professor of Histology,
Annual Meeting on December 5, 6 and 7, 1969 at the Park New York University Dental Center. "Wound Healing in Bone."
Sheraton Hotel, 7th Avenue and 55th Street, New York City. Leo Zach, A.B., D.D.S., Professor of Pathology, New York
The Scientific Session and Luncheon (9:30 to 4:30) on Sunday, University Dental Center. "Wound Healing in Soft Tissues."
December 7, 1969 will have as its t h e m e : Wound Healing: Nor­
mal and Abnormal. For further information write to: Dr. Bernard T u c h m a n , 200
Central Park South, New York, New York 10019.
The following clinicians will participate:
T H E UNIVERSITY OF M A R Y L A N D
Merwyn A . Landay, D.D.S., Associate Professor and Chair­
SCHOOL OF DENTISTRY
m a n , Department of Periodontology, T e m p l e University, School
of Dentistry. "Periodontal Aspects of Wound Healing." The University of Maryland School of Dentistry announces
the following courses in the Continuing Education Division.
Leopold P. Lustig, D.M.D., Assistant Clinical Professor,
Crown and Bridge Prosthodontics, Tufts University, School of Modifications of Periodontal Techniques as Indicated by Local
Dental Medicine. "Periodontal Prostheses." Environment. January 21 and 22, 1970, 8:30 a . m . to 5:00 p . m .
Lecturer: Henry M. G o l d m a n , D.M.D., Dean, Boston University
Maurice Oringer, D.D.S., Instructor of Electro-Surgery, T e m ­ School of Graduate Dentistry. Tuition: $100.