282

The Internolionol Joumal o( Periodontics & Restorolive Dentishy


Ultrostructurol Observations
on the Wound Healing of Free
Gingival Connective Tissue
Autografts With and Without
Epithelium in Humans
Giorgio Colwa, MD, DOS'
CiusHniano Manani. MD'
Slefono Pomio-Benlertoti. MD. DOS. MSCD'
Sergro De Pooli. MD, DDS'
Cariólo Lucchesi, MD'
Paul A. Fugazzotio, DDS^
Both the free gingival epithelium-
connective tissue autogrcft and the
connective tissue outagraft have
been successfully employed in the
treatment of mucogingival problems.
The goals of both procedures include
the treatment of recession through the
establishment of on adequate zone
of attached gingiva'"' and the in-
creose af vestibular depth.
" Clinical Director, Deportment of Dentistry,
University of Ferrora, Ferraro, Italy.
' Assistont Professor of Human Anatomy,
University of Ferrara, Ferroro, Italy.
' Instructor of Period onto logy. University af
Ferrora, Ferrora, Itoly.
' Instructor of Period onto logy. University of
Ancono, Ancona, Italy.
* University of Ancona, Ancona, Italy.
" Reseorch Fellow, Boston University,
School of Groduote Dentistry, Boston,
Mossochu setts.
Correspondence oddress; Dr Poul A. Fu-
gazzotto, 25 FHigh Street, Milton, Mossa-
chusetts 02187.
283
The apparent superiority and the
predictability of one type of tech-
nique over the other is lorgely de-
pendent on fhe ability of the groft to
survive and an its potential for re-
pjjjp 10-13 Isjunnerous histologie studies
in different animal systems have
reparted on the heoling and revas-
culorizotion of the free gingival
graft.'^-^'
Sullivan and Atkins^'" demonstrat-
ed that the epithelial cells of the graft
tend to desquamate and re-epithe-
liaiization occurs from cells deeper
v/ithin the epithelial ridges. Oliver et
al'^ confirmed in rhesus monkeys that
the epithelium degenerates and des-
quomates by the fifth day and com-
plete necrosis occurs in some areos.
This study pointed out the contribution
to healing of proliferating epithelial
cells from the adjacent tissues, which
cavered the entire surface by the
eleventh day. Jansan and cawork-
ers," in on animal study, also re-
ported total sloughing of the epithe-
lium in fresh gingival graft tissues
within 48 hours. The remoining con-
nective tissue graft surface undergoes
re-epitheliolization from the periph-
eral mucosa ond gingiva. In 1969,
Staffileno and Levy" demonstroted
that the success af the graft depends
on the ability of the connective tissue
to withstand its tronsfer to a new lo-
cation. Lange and Bernimoulin^^ em-
phasized that the role of bosol epi-
theliol cells of free gingival grafts in
the re-epithelialization process pre-
vents Q complete degeneration of the
epithelium of the graft.
Parallels hove been drawn in other
studies of connective tissue gingival
autografts.'"•^•' There are indications
in the literature of o complete epithe-
liolization of a connective tissue graft
from the palote to alveolar mu-
l l . Number 4, 1991
 284
cosa.^^'" Donn^' demonstrated thot
the kerotinizotian of the epithelium is
directed by the underlying connective
tissue layer. The claim that the free
gingival graft is more predictable
thon the connective tissue autograft
in increasing the zone of attached
gingiva, from a cytologie point of
view, was not studied.
The object of the present investi-
gation was to evoluate, by means of
scanning electron microscopy, the ul-
trastructuraf modifications that occur
with the placement of epithelium-
connective tissue grafts, and connec-
tive tissue grafts.
Method and materials
The present study utilized two differ-
ent types of grafts; one composed
of epithelium and connective tissue
and the ather mode only of connec-
tive tissue. Forty areas in 28 patients,
aged 20 to 48, were treated at the
Clinica Odontoiotrica of the Univer-
sity of Ferrara, Italy, through the
plocement of free gingival grafts.
Twenty free gingivoi grafts, consisting
of both epithelium and connective tis-
sue and 20 grafts mode up only of
connective tissue, were ploced.
Orol hygiene instructions were giv-
en to the patients to ensure excellent
preoperotive and postoperative
plaque control in conjunction with
scaling and polishing procedures.
This regimen was repeated once a
week for o period of 2 weeks.
The Internalional Journal of Penodonlics & Restoroliue Dentistry
Following administratian of local
anesthesia, a horizontal sharp dis-
section was performed at the mu-
cogingival [unction to separóte the
alveolar mucosa, elastic, and muscle
fibers from the underlying periosteum.
Any remaining muscle fibers and
movable connective tissues were re-
moved, and every effort was made
to afford on immobile periosteal bed.
Laterally, the initiol incision was made
slightly coronal to the mucogingival
junction, to "overlap" the gingival
groft onto the attached gingiva co-
ronal to the mucagingivai junction.
After preparation of the recipient
site, measurements were taken with a
Michigan 0 periodontai probe to de-
termine the exact dimensions of the
free graft materai to be secured from
the hard palate. Masticatory mucoso
of the gingival zone of the hord pal-
ate was used as donor tissue. The
outline of the gingivoi graft was
scribed with o sharp scalpel. The do-
nor graft, composed of epithelium
and connective tissue, was then dis-
sected from the palate ¡Figs 1 to 3).
To procure the "connective tissue-
only" graft, the mucosal flap was
partiolly elevated from the hard pal-
ate and only the connective tissue
was removed and utilized os donor
tissue (Figs 4 to 9), The epithelium
was then replaced and sutured se-
curely.
The gingival graft material wos
then shaped to correspond to the
donor site and placed in position.
Two coronal marginal sutures were
placed to stabilize the graft. A cir-
cumferential suture was inserted into
the periosteal bed slightly opical to
the inferior margin of the graft,
looped around the cervical area of
the tooth, and tied to compress the
graff against the recipient bed. The
graft wos firmly compressed with a
moist gauze for two minutes and then
dressed with Coe-Pock periodontai
dressing (Coe Laborotories, Inc),
The ultra structura I ona lysis wos
conducted on 40 autogenous free
human graft biopsy specimens of tis-
sues ¡all less than 1 mm^), taken 30
days posto peratively with a punch
forceps [Aesculap OK 507). These
specimens were fixed immediately in
2% glutaraldehyde in 0.1 M caca-
dylate buffer (pH 7.3) followed by
postfixotion in 2% osmium tetroxide
buffered v/ith 0.1 M cocodylate (pfH
7,3), Tissues were then dehydrated in
increasing concentrations of ethonol,
and embedded in epoxy resin. Semi-
thin sections, 1 mm thick, were
stained with toluidine blue and ex-
amined vio light microscopy.
Ultrothin sections with interference
colours in the silver ronge ¡approxi-
mately 700 Â) were cut in Reichert
microtomes with glass knives. They
were mounted on copper grids,
stained with uronyl acetate and lead
citrate, and examined with a scan-
ning electron microscope (Zeiss EM-
9S2), Samples of attached gingiva
and hard palote, prepared for the
scanning electron microscope by the
same procedure, served as controls.
 285

fÄ, 1^* Al ¿j ^ ** í7(?

f CT
E

\
i / ^

Fig I The palatal components: (Bl Fig 2 Reflection of the partiol thickness epithelialized graft epithelium as the outer sur-
bone; (CTj connective tissue; ¡E) epithe- face of the flap. The incisions are located at a safe distance from the free gingival
lium. margin to prevent recession in the approximating area.

Figs 1 to 3 Schematic representation of

the free gingñ/ol graft (with epithelium)
procedure.

Fig 3 Removal of the free gingival graft

exposes a large orea of uncovered con-
nective tissue for a delayed healing
phase (healing by secondary intention).

Volume 11, Number 4, 1991

 286

Figs 4 ta 9 Schematic representation oí Fig 4 The palatal components: ¡B) Fig 5 Incision ond thin palatal flop
Ihe free gingival gran (wilhou! epithelium) bone. ¡CT) connective iissue; ¡El epithe- reflected, leaving underlying connective
procedure- lium. tissue.

Fig 7 Removal of connective tissue to

Fig 6 Further reflection of palatal ílap to gain easy access to connective tissue. use as a gingival graft.

The Iniemotioriol Joumol of Periodonlics & Restoralive Denhstiy


figs 8 and 9 Flap is sutured to palate. This procedure will facilitate the healing af the donor tissue ¡heoling by primory intentian).
Results
Attached gingiva
Under the light microscope, the ot-
toched gingivo appeared to be com-
posed of epithelium and lamina pro-
priQ, the lotter in close and direct re-
lationship with the periosteum, A
clearly delineated submucosa was
obsent. The lining epithelium was a
kerotiniztng stratified squomous epi-
thelium, while the lamina propria, with
na glands, was made of dense fi-
brous connective tissue, with only a
few elastic fibers. The bosement
membrane, which separotes the ep-
ithelium ond connective tissue, was
uneven and characterized by deep
287
epithelium ridges that surrounded the basal lamina, wos irregulor be-
fingerlike connective tissue papillae in cause of numerous short projections
which capillaries were abundant. All that extended into the underlying
of this is in o g ree ment with the results connective tissue, causing the for-
of previous studies.^''"^'' matian of introflections, in which the
Scanning electron microscopic basal lomina were found (Fig 10],
analysis showed that the oral epithe- Desmosomes joined adjacent epi-
lium wos subdivided into the char- thelial cells.
octeristic four loyers of cells: basal,
spinous, granular, and cornified. The
basal loyer wos made up of either
cylindrical or cuboidal cells, in a
single layer, immediately adjacent to
the connective tissue from which they
were seporoted by o basal lomina.
The cytoplosm contained differently
oriented bundles of microfilaments.
The cellular surface, in proximity to
Volume I I , Number 4, 1991
 The spinous cell layer consisted af
several loyers of large, polyhedral
cells. Numerous cytoplosmic pro-
cesses protruded from the cellular
periphery into o lorge intercellular
space, giving the cell surface a ¡ag-
g e d a p p e a r o n c e (Fig I I ] . The cyta-
plasmatic microfiloment population
was organized into bundles. Melan-
ocytes, with a b u n d a n t cytoplasmic
granules of pigments, were present
in the stratum spinosum.
In the granular layer, the cells be-
c a m e flattened parallel to the gingi-
val surface. Electron-dense keroto-
hyalin granules a n d microfilaments,
more prominent than in the stratum
spinosum, were found in the stratum
granulosum (Fig 12). Numerous des-
Fig 10 Epithelium of ottoched gingivo. Portions of bosal cefis show bundles of microfi-
loments. the basal surface extends Into the underlying connective tissue, mosomes joined the facing mem-
¡b! Microfilaments; ¡et) connective tissue; ¡nj nucleus; ¡bIJ basal lamina. fOriginol magni-
fication X 13,000.) branes of adjacent cells.
The stratum corneum was com-
posed of numerous layers of anu-
clear a n d flattened cells. The cyto-
plasm was densely packed with mi-
Fig 11 Cells of the spinous loyer of the gingivol epithelium. Note the numerous thin crofiloments, oriented along the long
cytoplasmic projections bridging Ihe intercelfufor spoces. ¡n) Nucleus; ¡is) intercellular axis of the cell ¡Fig 13).
space; ¡p) thin cytoplasmic proiections; ¡b) microfilaments. ¡Originol magnification x
5,000.1 The lamina propria was a layer of
dense connective tissue filled with a
network of collagen fibers, some of
which provided the firm attachment
of the lomina proprio to the perios-
teum of the alvealar pracess. Be-
tween bundles of collogen fibers, iso-
loted fibroblasts were present with
long a n d flattened cytoplasmic pra-
lections or lamellae. In the gingival
connective tissue, granular basophil
cells, choracferized by a cytoplasm
rich in electron-dense granules, were
found, while only occasionally were
single myelinated nerve fibers ob-
served. Numerous capillaries, char-
acterized by a small lumen and thin
wall, were present in the papillary
spaces between the projeclions of
the inner epithelial surface (Fig 14).

The Internationol Joumoi of Peiiodonhcs Ä Restorotiue Dentistry

 289

Fig 12 Epithelium covering attached gin-

giva. The cytapiosm of the gronulor layer
cells shows electrodense kerotohyalin
granules and bundles of microfilaments.
¡ni Nucleus; ¡k/ keratohyalin granules;
¡b) micrafilaments ¡Original magnification
X 14.000./

Fig 13 Kerotinized epithelium of ot-

toched gingivo. Ciosely packed, flattened
ceils of the cornified layer ¡stratum cor-
neum/ demonstrate numerous cytopiosmic
microfilaments oriented along the major
cellular axes ¡c/ Cells; ¡is/ intercellular
spoce; Ib/ microfilaments. ¡Original mag-
nification xll.OOO./

• 11, Number •!, 199t

290

Mucosa of the hard palate af the

danor site

Under the light microscope, the pal-

atal mucosa was composed of ker-
atinized stratified squamous epithe-
lium ond a lamina propria of con-
nective tissue, folding in irregular pa-
pillae. The lamina propria was firmly
bound to the periosteum of the al-
veolar process.
Examination under the scanning
electron microscope demonstrated
that the lining epithelium of the pal-
atal mucosa appeared to be sub-
divided into severol layers of celts:
basal, spinous, granular, and corni-
fied. The basal layer was made of
only one loyer of isoprismatic cells in
Fig 14 Lamina propnd ol attached gingivo. Capillaries in the connective tissue papillae contact with o continuous basal lam-
provide the nutritional supply to the gingival epithelium. Fibroblasts with thin projections
are also evident, (bv) Capillary; (II fibroblasfs; (be) basal cells. (Original magnification ina anchored to o loyer of microfibrils
X5.400.) (Fig 15). Interdigitating, adjacent cells
were connected to one another via
desmosomes. The cytoplasm con-
tained many microfilaments clustered
Fig 15 Basot orea of the epithelium of the hard palote of the donor site. Note ihe into bundles with various orientations.
lorge nucleus, numerous bundles ol cytophsmic filaments, the basot lamina, and the The large and ovoid nucleus was
anchoring fibnis of the underlying connective tissue, (bl) Basal lamina;
(n) nucleus; (is) intercellular space; (ml) microfibnis; (b) microfilaments. (Original magnifi- charocterized by the peripheral dis-
cation X 12.000.1 tribution of chromatin.

The stratum spinosum consisted of

severol layers of cells equipped with
short cytoplasmic processes, resem-
bling spines, attached to one anoth-
er. The intercellular spaces seemed
to be crossed by narrow intercellular
bridges at regular intervals, which
gave the cell surfaces a prickled op-
peorance. These consisted of des-
mosomic mechanisms, well devel-
oped along the interface of focing
plosmic membrane of adjacent cells.
In the cellular cytoplasm of the stra-
tum spinosum, bundles of microfila-
ments were present in clusters in
proximity to the intercellular bridges
¡Fig 1Ó). The granular layer consisted
of flattened cells with granules and

l of PenodonfrCï & Resforolive

 291

Pig 16 Epithelium of the hard palote al

the donor site. Spinous cells show narrow
intercellular bridges across the intercellular
spaces, ¡nj Nucleus; ¡b/ microfitoments;
¡pi thin cyloplasmic projections: I's) inter-
cellular space; ¡d) desmosome. ¡Original
magnification x ¡4,000J

Fig !7 Epithelium of Ihe hard palate at

the donor site. A portion of a ceil oí the
granular layer, which is rich in electro-
dense droplets and bundles of microfila-
ments. ¡bj Microfilaments; ¡k) keratohyolin
granules; ¡nJ nucleus. ¡Original mognifica-
iion x24,000.l

Volume H , Number <t, 1991

292

bundles of microfiloments in their cy-

toplosm (Fig 1 7).
The stratum corneum wos com-
posed of several layers of enucleat-
ed, flattened cells, surrounded by a
morkedly thickened cell membrane.
At the inferior border of the cornified
loyer, the cells were densely packed
together, while in their superficial por-
tions they tended to be forther opart,
but were still ottoched to one another
vio desmosomes ¡Fig 18).
The lamina propria of the palatal
mucoso was characterized by the
presence of large bundles of colla-
gen fibers with random orientations.
With the scanning electron micro-
scope, typicol fibroblasts and only
occasionally blood vessels were ob-
18 Epithelium of the hord polate at the donor site. Flattened cells of the stratum served. The connective tissue of the
carneum ore evident. The plasmatemma of adjacent ond more superficial cells show
desmosomes (c) Cell of the strotum comeum; (is) intercellulor space; fbl microfiloments; palatal mucoso was characterized
(dl desmosomes. (Original mognification x 10,000.)
by a predominance of collagen
fibers aver omorphous ond cellular
constituents (Fig 19),

Fig 19 Lamina propria of the mucosa of ths hard palote at the donor site. Large bun-
dles of coltogen fibers ore rondamly oriented, ¡cfj Collogen fibers. lOriqinol moanifica-
tian x24.000.¡

DI Penodonlics £• Re&ioraiive Denfisfiy

 293

Epithelium—connecfive tissue
autograft

Specimens of the free gingival epi-

thelium—connective tissue autograft,
taken 30 days postoperatively. were
abserved under the light microscope.
They consisted of a stratified squa-
mous epithelium, superficially cov-
ered by a keratinized cell layer, and
a lamina propria of dense connective
tissue. The border between the epi-
thelium and the underlying lamina
propria of the cannective tissue wos
uneven and characterized by deep
epithelial ridges that surrounded
fingerlike connective tissue papilloe.
Under the scanning electron mi-
croscope, the lining epithelium pre-
Fig 20 Epithelium of the epithelium-connective tissue outagraft. Bosot cells thot show o
sented v/ith the typicol strotified pat- welf-developed nucleus and a cytaplasmic membrane attached to basal lamina through
tern (ie, basal, spinous, granular, and hemidesmosomes are evident ¡n! Nucleus, ¡is) intercellular space; ¡bt) bosof famina; ¡b)
microfiloments; (h) hemidesmosomes. ¡Original magnificaban x 13,000.)
carnified layers). The shope and di-
mensians of the basal layer cells
were similar to those of "normal " tis-
sues, with connection of one cell to
another dependent on extensive in-
Fig 21 Epithelium of the epilhelium-conneclive tissue outograft. Spinous epithelial cells
terdigitattans and desmosamal at- show a highly vocuolized cytoplasm. Intercellulor spaces ore reduced, ana the desmo-
tachments. The nuclei were lorge, Somic junctions Ore fewer ¡n) Nucleus, (is) intercellular spoce, (d! desmosome; (v) vacu-
oli. (Originol magnificotion x 11,000.)
while cytoplasm was scarce, dem-
onstrating an abundance of microfi-
laments [Fig 20j. Hemidesmosomal
attachments were evident between
the basement membrane and the
cells.
in the spinous layer, there wos o
transition from rounded cells in the
deeper loyers to flattened cells in the
more superficial loyers. The intercel-
lular spoces were much narrower
than were the spaces between spi-
nous cells in the epithelium of the ot-
tached gingiva and in the mucoso of
the hard palate ¡Fig 21). The de-
mosomes joining intercellular bridges
were reduced in number, ond fewer
microfilaments were anchored to
them. Numerous vacuoli, containing

Volume n . Number 4, 1991

 294

mitochondrial remnants were present

¡Figs 21 and 22). Cellular organelles
were not detected and microfilamen-
tous structures were rare. The nuclei
demonstroted chromantin that wos
organized in large, high-electron-
dense granules. In short, pathog-
nomic signs of cellular degeneration
were evident.
The strotum granulosum consisted
of a few layers of flattened cells,
characterized by cytoplasmic gran-
ules and microfilaments, often in bun-
dles (Fig 23).
The strotum corneum was com-
posed of several layers of cells, with
fairly thickened plasmalemma, re-
sembling overlapping lamellae (Fig
24), Microfilomentous material in on
fig 22 Higher magnification of Fig 12. ¡n/ Nucleus; ¡v/ vacuoli. ¡Original magnification
electron-dense matrix was abundant
in the cytoplasm. Finely granular ma-
terials were found in the intercellular
space. Desmosomal attachments
were present between cells. Medan-
ocytic and cleor cells were also not-
ed.
Fig 23 Epithelium-connective tissue auiograft. Celts of the granular layer af the epithe- The lomina propria was mode of
lium are choracterized by eiectran-dense draplets and cytoplasmic microfiiament bun-
dles, ¡k/Electron-dense drapiets: ¡b I micro filaments ¡Original magnification x 28.500./ dense connective tissue, consisting of
iiTegularly oriented collagen fibers,
sometimes in bundles. The fibroblosts
of such fibrous connective tissue were
equipped with fanning pro[ections.
Delicate capillaries with walls com-
posed of three to four endothelial
cells were obsen/ed (Fig 25), These
cells demonstrated pyknolic nuclei
and an absence of cellular organ-
elles, once again indicating cellular
degeneration.

The IrlernaHonol Joumol of Penodontics & Restorative Denlistiy

 295

Fig 24 Epithelium of the epithelium-con-

nective tissue autograft. Thin parallel enu-
cleate sheets of the stratum corneurn ore
densely pocked with fitoment bundles.
The cell membranes have become thick-
ened and desmosomes are maintained,
(c) Cell; (d) desmosome; (is) intercellulor
space. (Original magnification x24.500.)

Fig 25 Lamina prapria of the epithelium-

connective tissue auiagraft. Nate the col-
lapsed walls of the capillaries and the
vacuolized cytoplosm of the endotheliol
cells. The nucleus is picnofic. (ec) Endoth-
eliol cell; (n) nucleus; (vl vacuoiL (Origi-
nal magnification x 12.000.1

• I I , Number-S, 1991
 296

Pree gingival connective tissue

outograft

Under the light microscope, the free

connective tissue autograft, taken 30
doys postoperatively, was histologi-
colly interchangeable with the epithe-
lium-connective tissue graft alreody
described. The connective tissue
outogrott w a s , therefore, compased
of o keratinized stratified squamous
epithelium ond lamina proprio, inter-
digitoting with the epithelial ridges.
Scanning electron microscopic o b -
servations demonstroted that the
basal layer consisted of one cylin-
drical cell layer; each cell was char-
octerized by thin cytoplasmic pro-
cesses on its basal surfoces (Fig 26).
Fig 26 Epithelium oí the connective tissue autogroft. Portion of a basal epithelial cell These fingerlike extensions of the cells
resting on bosal lamina Note the undulating microvilli following Ihe contours of the bas-
al lamina. The cells are attached vio hemiJesmosomes. ¡n) Nucleus; ¡bl¡ basol lamino; protruded into the underlying con-
(bl microfiloments; ¡h) hemidesmosomes; ¡cf¡ collagen fiber. ¡Original magnification nective tissue a n d were connected to
X 26,000.1
o continuous basal lamino via hem-
idesomosomal attachments. The
cytoplasm of the basal cells locked
cellular organelles, but was chorac-
Fig 27 Epithelium of Ihe connective tissue oulograit. Spinous epithelial cells. The inler- terized by numerous microfiloments in
ceiluiar ¡unctions are characterized by increased numbers of desmosomes.
¡n) Nucieus; ¡d) desmosome; ¡b) microfiioments; Inl) nucteotus. ¡Original magnification the basal zone.
X 12.000.)
The spinous cells showed ultra-
structurol characteristics thot resem-
bled those of cells in the strotum spi-
nosum of the attached gingiva ond
the hard palate, but not the chor-
acteristics of cells in the free epithe-
lium tissue outografts. Their cyto-
plasms were filled with a micrafilo-
mentous opporotus that mosked the
cellular orgonelles to a degree (Fig
27). The nuclei of the spinous cells
were round, with the chromotin moin-
ly locoted in proximity to the nuclear
membrane, and contained an evi-
dent nucleolus.

The granular layer consisted of

several layers of cells, whose cyto-
plasm was choracterized by mony
electron-dense droplets a n d micro-

The InternoKonol Journol o( Periodontics S Restoroüve Dentistry

 297

Fig 28 Epithelium of connective tissue

autaqraft. A portion of o cell of the gran-
ular layer is shown. Many electran-dense
droplets and microfilaments are present in
the cellular cytoplasm, (n) Nucleus; (b)
microfiloments; ¡k) electron-dense drop-
lets. (Origino! magnification x24,000.)

Fig 29 Epithelium of the connective tis-

sue outagrañ. The ceHs of the stratum
corneum shaw minimal structural detaHs
other than dense!y packed filament bun-
dles, (c) Cell; (is) intercellular spoce.
(Original magnification x24,000.)

• 11, Number 4, 1991

 298

Fig 30 Lamina propria of the connective

tissue autagraft. Wide-open waits of o
copilfory blood vessef ore evident The
junctions between adjacent endotheliol
cells show numerous cytaplosmic microfi-
faments in o horizontol cross section.
(n) Nucleus; (ec! endotheliol cell; ¡t! tight
junction, ¡f) lumen; ¡cf) coljagen fiber.
(Originof mognificotian x27,000.)

Fig 31 Lamina propna of the connective

tissue autogroft. A capillary with cof-
lopsed walls is demonstrated, ¡n) Nucfe-
us: ¡ec) endotheliof cell; ¡rbc) red blood
ceJI. ¡Originol mognification x 12,000.)

The International Joumol o( Penodorlics & Restorative Dentistry


filaments that were frequently aggre-
gated in bundles (Fig 28).
The stratum comeum was com-
posed of flattened, onucleor cells,
whose cytoplasm was filled almost
exclusively by microfilaments running
parallel to the long axes of the cells
1F¡9 29).
The underlying connective tissue
consisted of coarse collagen fibers
frequently aggregated in intermin-
gling bundles. Immediately below the
epithelium, the collogen fibers be-
come thinner, forming a band to
which the cellular projections of the
basal layer were anchored (see Fig
26). The prime cellular constituents of
the connective tissue were the fibro-
blasts, which showed evidence of
active production.
NumeroLiS capillaries with either
distended ¡Fig 30) or collapsed walls
were observed (Fig 31). The long,
flattened endothelial cells were par-
allel to the long axes of the vessels.
Tight [unctions were present belween
adjacent endothelial cells. The cyto-
plasm was filled with microfiloments
and vescicles of micropinocytosis.
The tuminal surfaces of the endo-
thelial cells were smooth in some
areas, while, in other regions, folding
had resulted in crypt formotion. The
external surfaces were lined with a
continuous basal membrane.
Discussion and conclusions
Numerous clinical studies have
shown a definitive biologic attach-
ment and blending of the free gin-
giva autogrofl inio the surrounding
periodontal tissues,^'"'^^'^' Attach-
ment of the groft materiol is accom-
plished by day 10 to 13.'^
Light microscopic studies hove
clorified the healing mechanisms, in-
cluding the cellular changes and vos-
cular development of the free gingi-
val graft placed in the donor
area."-'^''^
The ultra structura I observations via
scanning electron microscope have
dealt with numerous aspects of the
dentogingival interface of healthy hu-
man gingiva ond the normal human
periodontium.^"'^^ LJsing the transmis-
sion electron microscope, Listgarten^^
examined the gingival graft in mon-
keys.
However, an ultra structura I study
of free gingival epithelium-connec-
tive tissue ond connective tissue grafts
in humans has not been presented
before. Ultra structura I obsen/ctions of
epithelium-connective tissue grafts,
taken 30 days posto peratively, led to
the conclusion that the lining epithe-
lium was composed of four cellular
layers: basol, spinous, granular, and
cornified. Such cells presented a
morphologic picture resembling the
cells of the lining epithelium of the
attached gingiva and the mucosa of
the hord palate in the donor site, with
the exception of the spinous cell
layer. Here the trouma and degen-
eration were evidenced by vocuolo-
zation of the cytoplasm, the absence
of cellular organelles, a reduced
number of desmosomes, and nar-
rower intercellular spaces.
299
Both human ond animal studies
have previously reported degenera-
tive epithelial alterations of the graft
during the first week.'^'-*^ Histoiogic
studies have also reported a migra-
tion of epithelial cells from adjacent
wound margins between the second
and the fourth days."'"'" Oliver et
al'^ and Lange and Bernimaulin'^
mentioned a possible participation of
basal cells that remain in the graft in
re-epithelia!ization.
The presence of nonvital ar un-
healthy spinaus cells in the speci-
mens we examined could signify on
incomplete maturation of the epithe-
lium, because of a compromise of
nutritional supply from the blood ves-
sels of the lamina propria. This hy-
pothesis was reinforced by the fact
that the endothelial cells of the cap-
illories interspersed throughout the
bundles af collagen fibers in the graft
demonstrated ultra structura I charoc-
teristics of degeneration (ie, absence
of cellular organelles, vacuolization
of the cytoplasm, pyknotic nuclei).
Thus, the histoiogic alterations of the
epithelium were a consequence of in-
ten-uption of the nutrient supply to the
grafted material, whose survival must
initially depend on tissues of the sur-
rounding areas for nutrition.
Examination of the connective tis-
sue-only graft, 30 days postopera-
tively, revealed an epithelium similar
to that of both the control and the
epithelium-connective tissue graft.
VoiLime 11, Number^, 1991
 300
The morphologic picture of the spi-
nous cells resembled that of the at-
tached gingiva and mucosa of the
hard palate, but not that of the stra-
tum spinosum from the epithelium-
connective tissue graft. The epithelium
covering the Connective graft, 30
days posto peratively, appeared to
be more fully "recovered" from the
surgical trauma The stratum spinos-
um more fully resembled that of non-
insulted tissue of attached gingiva.
This was most likely related to the
larger number of capillaries present
in this tissue, which was taken from a
deeper level into the palate than was
the tissue used for an epithelium-
connective tissue graft. This would re-
sult not only in the presence of a
greater number of nutrients in the
graft at the time of removal, but olso
would provide more capillaries for re-
establishment of circulation within the
graft more quickly through connec-
tion with ingrowing capillaries from
the recipient site. This would result in
a more-rapid and less-froumotic ma-
turation of the graft. Regeneration of
the epithelium would be occom-
plished by migration of epithelial cells
from tissues surrounding the graft, os
il was with the epithelium-connective
tissue graft.
The connective tissue-only graft,
therefore, not only offers the advan-
tage of a more comfortable donor
site, because of replacement of the
"epithelial" flap, but also results in a
more-ropid, less-traumatic healing
and maturation of the graft in the re-
cipient site.
The frtlernationol Jo oí Penodonfics & Re&lorûhve DenlisHy
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Volume 11, Number 4, 19?1