Gingival Flap Attachment Healing with Amnion-Chorion Allograft Membrane: A Controlled, Split Mouth Case Report Replication of the Classic 1968 Hiatt Study

Abstract

Background: Whereas laminin-5 is a known glycoprotein with significant involvement in epithelial cell motility and junctional epithelium attachment and BioXclude™ amnion-chorion membrane (ACM) is known to contain significant amounts of laminin-5, it was postulated that reproduction of the Hiatt study with the addition of ACM would possibly lead to faster gingival flap attachment.
Methods: A 54 year old white male with a longstanding history of generalized moderate chronic periodontal disease and a 38 pack year history of smoking consented to participation in this study. For the purposes of this case report, the maxilla was divided into right and left halves for simultaneous treatment involving minor osseous recontouring and gingival flap replacement. Prior to flap closure, one half of the maxilla had a layer
of ACM placed onto exposed bone while the control side had nothing placed onto exposed bone. Tensile flap strength was measured by pulling on sutures with a tensiometer at 72 hours, 1 week, 2 weeks, and 3 weeks after surgery.
Results: The control side of the maxilla had simple flap displacement at 72 hours and 1 week after surgery with tension less than 1,000 grams. At 2 weeks, 1,600 grams of tension minimally displaced the flap while
at 3 weeks, flap displacement did not occur and sutures pulled through the flap with 2,100 grams of tension. On the experimental ACM side of the maxilla, simple flap displacement occurred at 72 hours. At 1 week,
1,700 grams of tension minimally displaced the flap. At weeks 2 and 3, flap displacement did not occur and sutures pulled through the flap with 2,000 or more grams of tension.
Conclusion: This human case report with a split mouth controlled design demonstrated faster gingival flap attachment with addition of ACM.

Full text

Wilcko et al
Background: Whereas laminin-5 is a known gly-
coprotein with significant involvement in epithelial
cell motility and junctional epithelium attachment
and BioXclude™ amnion-chorion membrane
(ACM) is known to contain significant amounts
of laminin-5, it was postulated that reproduction
of the Hiatt study with the addition of ACM would
possibly lead to faster gingival flap attachment.
Methods: A 54 year old white male with a long-
standing history of generalized moderate chronic
periodontal disease and a 38 pack year history of
smoking consented to participation in this study.
For the purposes of this case report, the maxilla
was divided into right and left halves for simulta-
neous treatment involving minor osseous recon-
touring and gingival flap replacement. Prior to
flap closure, one half of the maxilla had a layer
of ACM placed onto exposed bone while the
control side had nothing placed onto exposed
bone. Tensile flap strength was measured by pull-
ing on sutures with a tensiometer at 72 hours,
1 week, 2 weeks, and 3 weeks after surgery.
Results: The control side of the maxilla had
simple flap displacement at 72 hours and
1 week after surgery with tension less than
1,000 grams. At 2 weeks, 1,600 grams of
tension minimally displaced the flap while
at 3 weeks, flap displacement did not occur
and sutures pulled through the flap with
2,100 grams of tension. On the experimen-
tal ACM side of the maxilla, simple flap dis-
placement occurred at 72 hours. At 1 week,
1,700 grams of tension minimally displaced
the flap. At weeks 2 and 3, flap displace-
ment did not occur and sutures pulled through
the flap with 2,000 or more grams of tension.
Conclusion: This human case report with a split
mouth controlled design demonstrated faster
gingival flap attachment with addition of ACM.
Gingival Flap Attachment Healing with
Amnion-Chorion Allograft Membrane:
A Controlled, Split Mouth Case Report Replication
of the Classic 1968 Hiatt Study
Dan Holtzclaw, DDS, MS1 • H. Fritz Hinze, DMD1 • Nicholas Toscano, DDS, MS2
1. Private practice limited to periodontics and dental implants. Austin, Texas, USA.
2. Private practice limited to periodontics and dental implants. New York, New York, USA.
Abstract
KEY WORDS: Periodontal surgery, wound healing, amnion-chorion, case report
The Journal of Implant & Advanced Clinical Dentistry • 19

20 • Vol. 4, No. 5 • November/December 2012
BACKGROUND
Although they are relatively new to the prac-
tice of dentistry having been recently introduced
in 2008, placental allografts have been used in
medicine for over 100 years with initial use in skin
wound applications in the early 1900’s.1 While
the first version of dental placental allograft was
composed of dehydrated amnion alone, second
generation dental placental allografts are com-
posed of dehydrated amnion-chorion laminate
(BioXclude™, Snoasis Medical, Denver, Colo-
rado, USA). Placental barriers such as amnion-
chorion membranes (ACM) demonstrate many
unique properties. First and foremost, ACM’s
possess a variety of proteins which provide a bio-
active matrix to facilitate wound healing includ-
ing collagen types I, III, IV, V, and VI,2 laminin-5,
platelet derived growth factors alpha (PDGF-α)
and beta (PDGF-β), FGF, and TGF-β.3 With
the current knowledge that ACM’s possess a
wide array of proteins, it is not surprising that
skin wound healing studies of the early 1900’s
noted improved healing with placental dressings.
In 1968, Hiatt and colleagues4 published a
study that evaluated gingival healing following
replaced flap surgery. This classic wound heal-
ing study utilized sixteen older mongrel dogs with
periodontal disease as test subjects. Each dog
had a mucoperiosteal flap raised bilaterally over
maxillary canines, alveolar bone adjusted, roots
planed, and the flaps replaced with sutures. The
animals were sacrificed at 48 hours, 72 hours,
1 week, 2 weeks, 3 weeks, 1 month, 4 months,
6 months, and 12 months after surgery. Imme-
diately prior to sacrifice, the strength of flap
attachment at each surgical site was tested by
pulling suture loops with a tensiometer. Fol-
lowing sacrifice, block sections of the maxillary
canines were histologically examined. Tensiom-
eter data and histologic findings from this study
noted that initial flap “reattachment” occurred
within 1 week following surgery and strength-
ened during the second week to the point where
retained epithelial cells were noted on the root
surfaces following mechanical flap separation.
Whereas laminin-5 is a known glycopro-
tein with significant involvement in epithelial cell
motility and junctional epithelium attachment5,6
and BioXclude™ is known to contain significant
amounts of laminin-5,7 it was postulated that
reproduction of the Hiatt study4 with the addi-
tion of ACM would possibly lead to faster gin-
gival flap attachment. As such, the purpose of
this investigative case report was to test the
effects of ACM on the strength of gingival flap
attachment following replaced flap surgery.
CASE REPORT
A 54 year old white male with a longstanding his-
tory of generalized moderate chronic periodontal
disease and a 38 pack year history of smoking
consented to participation in this study. The
study was conducted in accordance with the
Declaration of Helsinki as revised in 2008. For
the purposes of this case report, the maxilla was
divided into right and left halves for simultane-
ous treatment. Following the administration of
local anesthesia, full thickness mucoperiosteal
flaps were elevated on both sides of the maxilla.
Degranulation with ultrasonic and hand instru-
ments was followed by calculus removal and
root planing. Negative osseous architecture was
corrected with rotary and hand instrumentation.
Following copious irrigation with sterile saline,
the right half of the maxilla had flap replacement
with simple interrupted 4-0 PTFE sutures while
Holtzclaw et al

The Journal of Implant & Advanced Clinical Dentistry • 21
the left half of the maxilla had a single layer of
ACM (BioXclude) placed onto exposed bone
surfaces (figure 2) prior to flap replacement
in a similar fashion. The patient was provided
pain medications and 0.012% chlorhexidine
rinse following surgery. Follow up visits were
performed at 72 hours, 1 week, 2 weeks, and
3 weeks after surgery. At each post-surgical
visit, following oral hygiene instructions and
deplaquing of the teeth with hand instruments,
loops of the sutures retaining the gingival flaps
were attached to a hooked tensiometer (fig-
ure 3) (American Weight, Norcross, Geor-
gia, USA). Gentle tension was applied to the
Figure 1: Amnion-chorion membrane (BioXclude™, Snoasis Medical, Denver, Colorado, USA) trimmed prior to placement on
experimental side of the maxilla.
Figure 2: ACM membrane placed on exposed bone of
experimental side of the maxilla prior to gingival flap
replacement.
Figure 3: Suture pull with tensiometer at 1 week healing.
Holtzclaw et al

22 • Vol. 4, No. 5 • November/December 2012
suture loop until either the flap displaced from
the underlying bone or the suture was pulled
free from the gingival tissue. Following removal
of all sutures, the patient was placed in peri-
odontal maintenance at 3 month intervals.
RESULTS
The results of suture tensiometer testing are
listed in table 1. On the non-ACM side of the
maxilla, gingival flap displacement occurred
at 72 hours and 1 week following surgery. At
2 weeks following surgery, significant resis-
tance to suture pulling was encountered prior
to the suture pulling through the flap, but a
small amount of gingival flap displacement
was noted. No flap displacement occurred 3
weeks following surgery. On the ACM side of
the maxilla, gingival flap displacement occurred
at 72 hours after surgery. At one week after
surgery, significant resistance to suture pulling
was encountered, but a small amount of gin-
gival flap displacement was noted as sutures
pulled through the flap. No flap displace-
ment occurred 2 or 3 weeks following surgery.
DISCUSSION
Wound healing is commonly divided into three
stages: inflammation, granulation tissue forma-
tion, and matrix formation/remodeling.8 When
dealing with the healing of a periodontal flap,
phases have been classified as: adaptation,
proliferation, attachment, and maturation.9
Adaptation
The flap adaptation phase occurs between
days 0-4 following surgery.9 Within the first few
hours of surgery, this process begins with the
formation of a fibrin clot between the bone and
connective tissue of the overlying gingival flap.
During this time, early inflammation ensues with
the release of polymorphonucleosites (PMN’s),
macrophages, and mast cells. The inflamma-
tory process clears necrotic cells to provide an
avenue for epithelial cell migration over con-
nective tissue. Sabag et al.10 noted that epi-
thelial migration begins in gingival wounds 2
days after surgery. This may occur by epithelial
cells dissolving their hemidesmosomal attach-
ment to release from the basement membrane
and begin migration from wound edges.11 In
Table 1: Tensile strength (in grams) required to seperate
gingival ap from underlying structures
72 hours 1 week 2 weeks 3 weeks
Control side (No ACM) 200 350 1,600 2,100*
Experimental Side 325 1,700 2000* 2,200*
(ACM added)
*Denotes sutures that pulled through the ginival flap without displacing flap from underlying structures
Holtzclaw et al

The Journal of Implant & Advanced Clinical Dentistry • 23
examining healing of pedicle flaps, Wilderman
and Wentz9 noted that between 2 to 4 days
after surgery, epithelial cells were in contact
with the tooth. They also noted that epithe-
lial cells migrated from wound edges at a rate
of 0.5mm per day and that the fibrin clot was
absent at the sites of epithelial proliferation.
Proliferation
The proliferation phase of gingival flap heal-
ing occurs between 4 to 21 days after surgery
with the greatest mitotic activity occurring at
day 4.9 At this time, blood supply to the healing
wound improves as capillary loops begin anas-
tomosing with cut vessels of the overlying gin-
gival flap.12 Osteoclastic activity begins around
days 3 to 4, reaching peak activity 8 to 10 days
after surgery. This resorptive process creates
a surface in which collagen fibrils of the hard
tissue matrix become denuded, establishing a
suitable substrate for newly forming collagen
fibrils.13 During this phase, granulation tissue
invades the fibrin clot with fibroblasts appearing
at days 6 to 10. The proliferating tissue seen at
this time contains many capillaries, fibroblasts,
lymphocytes, and PMN’s.9 Collagen formation
begins at days 7 to 21. Ravanti et al.11 noted
that gingival fibroblasts release matrix metallo-
proteinase 13 (MMP-13) to break down granu-
lation tissues. Between days 10-14, significant
amounts of epithelial migration are seen along
the root creating an epithelial cuff. By this
time, epithelial attachment is strong enough
to resist flap displacement.4 Osteoblastic
activity also begins around this time as well.14
Attachment
The attachment phase of gingival flap heal-
ing occurs between days 21 to 28 after sur-
gery9 with continued collagen formation apical
to the newly formed epithelial cuff. The 1968
Hiatt study4 demonstrated that by this time,
Table 2: Immunohistochemical analysis of amnion-chorin membrane
and porcine collagen membrane
Laminin Laminin-5 TGF FGF PDGF PDGF
Amnion 4.4 ± 0.55 4.2 ± 0.45 1.4 ± 0.9 0.7 ± 0.45 2.4 ± 0.55 3.2 ± 1.1
Chorion (p < 0.001) (p < 0.001) (p < 0.05) (p < 0.05) (p < 0.005) (NS)
(BioXcludeTM)
Porcine
Collagen 0.0 0.0 0.0 0.0 0.0 3.0 ± 0.0
(BioGide®)
Holtzclaw et al

24 • Vol. 4, No. 5 • November/December 2012
the fibrin clot of early healing had been largely
replaced with immature collagen fibers. Api-
cal epithelial migration no longer occurs dur-
ing this phase and is thought to be limited by
recently formed cementoid on the root sur-
faces which is typically seen by week 3 of
healing.9 At this point of healing, fibroblasts
and collagen fibers are parallel to the root
and bone surfaces. By the end of the attach-
ment phase, healing of the epithelial cuff has
progressed to a point where flap separation
does not occur, even under severe tension.4
Maturation
The maturation phase of gingival flap healing
occurs between days 28 to 180 after surgery.9
During this time, increased cementum and bone
formation occurs along with maturation of con-
nective tissue and connective tissue fiber bun-
dles. By one month after surgery, osteoblastic
activity reaches its peak, although slight bone
apposition has been seen 6 months later.14 By
6 months onward, newly formed cementum
has matured, newly formed bone has devel-
oped periosteum, and collagen fiber bundles
are arranged and inserted at right angles, as
compared to the earlier parallel arrangement.
This case report sought to replicate the
1968 Hiatt study4 in a human clinical setting
with the addition of amnion-chorion allograft
membrane (ACM). Utilizing a controlled split
mouth design with simultaneous surgery in a
single individual allowed for direct comparison
between healing with and without the addition
of ACM. Gingival flap attachment, to the point
where displacement by pulling on suture loops
did not occur, appeared to be nearly twice as
fast on the ACM side of the maxilla and with-
stood significantly more tension pull at earlier
time frames. This may be due to the signifi-
cant amounts of laminin and laminin-5 in the
ACM product BioXclude™. In comparing BioX-
clude™ to a widely available porcine collagen
membrane (table 2), Xenoudi and Lucas7 found
substantial amounts of laminin and laminin-5 in
the ACM product and no traces of these gly-
coproteins in the porcine product. Laminin
and laminin-5 are glycoproteins with significant
involvement in epithelial cell motility and junc-
tional epithelium attachment.5,6 The addition
of ACM prior to closure of a gingival flap may
allow for faster epithelial migration and subse-
quent initial flap attachment. Epithelial migra-
tion starts at 2-4 days following surgery and is
greatest in an apical direction along the root
surface 10-14 days after surgery.9 Hiatt4 noted
sufficient epithelial cell attachment at this point
to resist gingival flap displacement under ten-
sion. It is important to note that all of these
studies were performed on dogs, a species
that is widely known to heal significantly faster
than humans. In the present case report, gin-
gival surgery with the addition of ACM resulted
in substantially reduced healing times in terms
of flap adaptation and attachment. On the
ACM side of the maxilla, gingival flap displace-
ment occurred at 72 hours after surgery. This
is understandable as during the first 72 hours,
clearance of necrotic debris, fibrin clot forma-
tion, and initial epithelial migration begins. At
one week after surgery, significant resistance
to suture pulling was encountered, but a small
amount of gingival flap displacement was noted
as sutures pulled through the flap. It is impor-
tant to note that while some minor flap displace-
ment occurred with suture pulling at one week
Holtzclaw et al

The Journal of Implant & Advanced Clinical Dentistry • 25
following surgery, flap attachment was strong
enough to require that the sutures be pulled
completely through the flap in order to facilitate
this displacement. In the 1968 Hiatt study,4
at one week following surgery, similar findings
were observed and microscopic examination of
the gingival flap “torn from the tooth” revealed
intraepithelial tears with no epithelial cells
remaining on the root surface. In the present
case repot, no flap displacement occurred 2 or
3 weeks following surgery with suture pulling.
These findings are significantly different from the
non-ACM side of the maxilla in this case report
which took nearly twice as long to achieve
similar results as the ACM side of the maxilla.
CONCLUSION
This human case report with a split mouth
controlled design demonstrated faster gin-
gival flap attachment with addition of
ACM. Additional controlled studies with
histologic analysis are recommended to
expand upon and confirm these findings. ●
Correspondence:
Dr. Dan Holtzclaw
711 W. 38th Street • Suite G5
Austin, TX 78705
USA
info@lonestarperio.com
Disclosure
Dr. Holtzclaw has a financial interest in Snoasis
Medical and is a member of its clinical advisory
board.
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