Volume 83 • Number 9

A Novel Approach to the Use

of Doxycycline-Loaded Biodegradable
Membrane and EDTA Root Surface
Etching in Chronic Periodontitis:
A Randomized Clinical Trial
Ahmed Y. Gamal* and Radi M. Kumper*

Background: The release profile of 25% doxycycline (DOX)

gel loaded on a biodegradable collagen membrane (COL)
after 24% EDTA root surface etching was evaluated.
Methods: Thirty systemically healthy patients, each with at
least one pair of contralateral interproximal intrabony defects
‡4 mm deep, along with an interproximal probing depth ‡6 mm
and clinical attachment loss ‡4 mm, were randomized into

I
t is well established that plaque con-
two groups. Group 1 consisted of sites treated with open-flap trol is a critical determinant of the
debridement followed by placement of DOX gel-loaded COL success or failure of various outcomes
(DOX–COL), whereas group 2 sites were treated with flap of periodontal therapy.1,2 Data indicate
surgery followed by the placement of DOX–COL after EDTA that decreasing the biofilm load may lead
etching of the exposed root surfaces (DOX–COL + EDTA). Sam- to improved clinical results and more
ples of gingival crevicular fluid were obtained 1, 3, 7, 14, and predictable periodontal healing.3,4 Placing
21 days after surgery. Separation was performed, and quantita- bone grafts and membranes in infected
tive measurements of DOX were taken with a high-performance sites may limit periodontal healing suc-
liquid chromatography. Clinical evaluation and follow-up for cess.5 It was found that, during the surgi-
6 months were performed. cal and postoperative healing phases,
Results: At 21 days, DOX–COL + EDTA group showed 5.3 both non-biodegradable and biodegrad-
mg/mL value. However, no DOX was detected in samples able membranes could serve as a nidus
of the DOX–COL group. DOX–COL + EDTA-treated group for the growth of the microorganisms
retained more DOX during the periods of 3, 7, 10, and 14 harbored in the oral cavity.6 In addition,
days than did the DOX–COL group. the clinical characteristics of the surgi-
Conclusion: EDTA root surface etching could enhance cal site may initially contribute to bac-
DOX availability in the gingival crevicular fluid after its release terial contamination and then lead to
from the collagen membrane. J Periodontol 2012;83:1086- additional complications in healing.7
1094. Because of the infective nature of peri-
odontal disease and the possibilities of
KEY WORDS
contamination of regenerative materials,
Doxycycline; guided tissue regeneration; edetic acid; gingival various approaches to the use of anti-
crevicular fluid; membranes; periodontitis. microbial agents in regenerative proce-
dures delivered by rinsing, irrigation,
* Department of Periodontology, Faculty of Dental Medicine, Al-Azhar University, Cairo, systemic administration, and local de-
Egypt.
livery systems have been discussed in
several studies.8-12 The use of controlled
drug delivery systems to administer anti-
infectives into periodontal pockets offers
the advantage of high and prolonged
anti-infective concentrations within the

doi: 10.1902/jop.2011.110476

1086
J Periodontol • September 2012
periodontal pocket, eliminates compliance concerns,
and may be associated with a lower risk of adverse sys-
temic effects and development of resistant bacteria.12,13
One such product is a locally administered con-
trolled-release doxycycline (DOX) that is effective
against Gram-positive and Gram-negative bacteria,
protozoa, and various anaerobes.14 DOX exerts the
antibacterial action by inhibiting the microbial protein
synthesis that requires access into the cell wall and
lipid solubility. DOX binds the ribosome to prevent
ribonucleic acid synthesis by avoiding addition of
more amino acid to the polypeptide.15 It is frequently
used in dental treatments for soft tissue and bone re-
generation after periodontal disease because of its
strong activity against periodontal pathogens, such
as Aggregatibacter actinomycetemcomitans, Porphyr-
omonas gingivalis (Pg), and Tannerella forsythia.16,17
DOX is also known to inhibit the matrix metalloprotei-
nases.18 It has been speculated that dentin etching
leaves the collagen fibrils unprotected and vulnerable
to degradation by endogenous metalloproteinases
(MMPs) capable of degrading all extracellular matrix
components. Human dentin contains collagenase
(MMP-8), gelatinases MMP-2 and MMP-9, and en-
amelysin MMP-20.19-22 Dentin collagenolytic and ge-
latinolytic activities can be suppressed by protease
inhibitors,23 indicating that MMP inhibition could be
beneficial in the preservation of collagen fibrils. Col-
lagen preservation was demonstrated in an in vivo
study in which the application of DOX was shown to
have a broad-spectrum MMP-inhibitory effect.24 The
antibiotic possesses a bone resorption inhibition
characteristic, mainly by inhibiting the osteoclastic
collagenase that is mediated by altered regulation of
cytoplasmic calcium.25 Anti-inflammatory properties
were also reported because of their ability to suppress
the polymorphonuclear leukocytes, particularly by
scavenging action or by blocking prostaglandin E2.26
In addition, it has the ability to promote the attach-
ment of fibroblasts to root surfaces,27 resulting in an in-
crease in the production of collagen and bone.24,28-30
Topical DOX application has the capability of con-
ditioning dentin and also of enabling fibrin linkage,
both of which favor the formation of a new attach-
ment.31,32 It has the ability to concentrate in the gin-
gival crevicular fluid (GCF) at levels substantially
greater than in serum,33-36 a property that enhances
their action against microbes.37 It exhibits a high de-
gree of substantivity by binding to the periodontally
diseased root cementum and dentin, acting as a res-
ervoir while slowly releasing the antibiotic in a bio-
logically active form above the minimum inhibitory
concentration (MIC) levels, into the adjacent environ-
ment for several days after its topical application,
thereby prolonging its therapeutic effects.38 Random-
ized controlled trials revealed that the direct applica-
Gamal, Kumper
tion of biodegradable DOX sustained-release devices
to the periodontal pockets or its delayed application
into the pockets after initial treatment resulted in a sig-
nificant gain in clinical attachment levels (CALs) and
a significant reduction in probing depths (PDs). Also,
clinical data demonstrated that DOX use can partially
counteract the negative effect of smoking on peri-
odontal healing after non-surgical therapy.34,39
One of the most important limitations of local deliv-
ery vehicles is that the main bulk of the delivery vehicle
or its acidic degradation products could interfere with
regenerating tissues.40 In addition, tissue-implanted
materials could be surface friendly to biofilm forming
bacteria; this could present a potential risk of retained
graft material acting as a surface for the reestablish-
ment of the original bacteria or for the initiation of an in-
fection by new bacteria.41 In their previous scanning
electron microscopic study,42 the authors reported that
EDTA gel could be used as a non-space-occupying
delivery vehicle that significantly improved chlorhex-
idine (CHX) substantivity to periodontally affected
root surfaces without compromising regenerating tis-
sues. In their recent study,43 the authors reported that
CHX could be maintained for up to 14 days at a level
higher than that of the MIC after EDTA root surface
etching. In the same study, the authors also reported
significant improvements in the clinical soft tissue pa-
rameters compared to open flap debridement (OFD).
Such improvement was attributed to the prolonged
availability of CHX secondary to EDTA root surface
etching. However, direct antimicrobial gel applica-
tion to the root surface could create a smear-like layer
effect, compromising clot and periodontal ligament
(PDL) cell adhesion to EDTA-exposed dentinal tu-
bules and dentin-associated collagen. Baker et al.44
reported that, after EDTA root surface removal of
the organic smear layer, additional conditioning of
the dentin surface with protein constructs produces
a surface morphology similar to that of the smear
layer with poor fibrin clot retention. Gamal45 reported
that EDTA root surface etching enhances clot-
blended small-sized particle b-tricalcium phosphate
graft adhesion to the periodontally exposed root
surfaces. Clot-blended graft adhesion to EDTA bio-
modulated root surface could also be compromised
by direct antimicrobial gel application to the root
surface. To the authors’ knowledge, quantification
of DOX loaded on collagen membrane (COL) after
EDTA root surface etching has not been reported
previously.
The objective of the present study is to evaluate
EDTA root surface etching as an indirect delivery ve-
hicle that could enhance DOX availability in the GCF
after its release from the collagen membrane. Clinical
evaluation and follow-up for 6 months were per-
formed to determine whether such clinical changes
1087
EDTA Enhanced Doxycycline GCF Availability
could be attributed to DOX level alterations after
EDTA root surface preconditioning.
MATERIALS AND METHODS
Participant Selection and Grouping
Thirty systemically healthy, non-smoking individ-
uals (19 males and 11 females aged 36 to 45 years;
mean age: 31.22 years) undergoing periodontal
therapy at the Department of Periodontics, Faculty
of Dental Medicine, Al-Azhar University, Cairo, Egypt,
from February 2010 to January 2011, were selected
for the study. Intraoral periapical radiographs (IOPRs)†
were taken to confirm the presence of suitable bony
defects for the selection of participants. The inclusion
criteria were the presence of paired, contralateral inter-
proximal intrabony defects (IBC) ‡ 4 mm deep (dis-
tance between alveolar crest and base of the defect
on IOPRs) along with an interproximal PD ‡ 6 mm
and CAL ‡ 4 mm after phase I therapy (scaling and root
planing [SRP]). All teeth were vital, asymptomatic
first and second molars without furcation involve-
ment. Patients with any systemic illness known to
affect the outcomes of periodontal therapy, those
pregnant and/or lactating, those smoking or using
other tobacco products, those taking drugs known to
interfere with wound healing, those with allergy or sen-
sitivity to tetracyclines, and those with unacceptable
oral hygiene (plaque index [PI]46 > 2) after the reeval-
uation of phase I therapy were excluded from the study.
In addition, teeth with gingival recession, endodontic
involvement, opened interproximal contact, or Grade
II or greater mobility47 were also excluded. Research
procedures were explained to all patients, and they
agreed to participate in the study and signed the
appropriate informed consent form of Al-Azhar Uni-
versity. Experimental protocol was approved by the
Local Ethical Committee of Al-Azhar University
(OMD 138-2010).
Presurgical Therapy
Before the surgery, each patient was given careful
instructions on proper oral hygiene measures. Full-
mouth supragingival and subgingival SRP procedures
were performed in quadrants under local anesthesia.
This procedure was performed using a combination
of hand curets‡ and an ultrasonic scaler using the
P10 tip§ until the surface was clean, hard, and smooth
as determined by the investigator (RK). Patients were
recalled every other day and received detailed me-
chanical plaque control instructions, which consisted
of their brushing using a soft toothbrush with a roll
technique and flossing. Supragingival plaque re-
moval was performed whenever necessary. Peri-
odontal reevaluation was performed 6 to 8 weeks
after phase I therapy to confirm the suitability of
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Volume 83 • Number 9
the sites for this study. Periodontal disease status was
determined by clinical periodontal assessments for
the selected sites, including PI,46 gingival index (GI),48
PD,49 and CAL.50 The clinical measurements were
obtained using a periodontal probei to the nearest 0.5
mm at baseline. Preoperative and postoperative non-
standardized radiographs were taken with the long-
cone parallel technique. Additionally, in all cases, the
precise position of the periodontal probe was recorded
using intraoral photographs taken at the same magni-
fication and from the same position. The deepest point
of baseline defects was included in the calculations.
For the patient to act as his or her own control, the
two contralateral sites were randomized at the split-
mouth level by the flip of a coin (which was flipped each
time by the same individual [Dr. M. El Destawy, Faculty
of Dentistry, Al-Azhar University, Cairo, Egypt], who
was not involved in the study in any other way) and
placed into two groups. Group 1 (G1) consisted of sites
treated with OFD, followed by placement of DOX gel-
loaded COL¶ (DOX–COL), whereas group 2 (G2) sites
were treated with flap surgery, followed by the place-
ment of DOX–COL after EDTA etching of the exposed
root surfaces (DOX–COL + EDTA).
DOX Gel Preparation and Loading
DOX solution was prepared by dissolving 25 mg DOX#
in 100 mL distilled water (25%). A weighed amount
of methylcellulose powder (4%)** was sprinkled
gently and stirred at 50 rpm using a magnetic stirrer.
Stirring was continued until a thin hazy dispersion
without lumps was formed. Leaving DOX solution
overnight in the refrigerator may be necessary for com-
plete gel dispersion in case the gel is not completely
formed.51 A fixed volume of 100 mL DOX gel was
applied to one side of the COL 3 hours before its use.
Surgical Procedures
After administration of local anesthesia, buccal and
lingual intrasulcular incisions were made, and muco-
periosteal flaps were elevated. Care was taken to pre-
serve as much interproximal soft tissue as possible.
For better access to the surgical site or to achieve
better closure, the flap was extended one or two teeth
mesially or distally in most cases. Vertical releasing
incisions were performed when deemed necessary.
The granulation tissue was removed from the defects,
and meticulous SRP was performed with the use of
ultrasonic instruments and curets. No osseous recon-
touring was done. One site received defect coverage
with DOX–COL (G1; 30 sites) with the inner surface
† Extraspeed, Eastman Kodak, Rochester, NY.
‡ Gracey curets 1/2 and 7/8, Hu-Friedy, Chicago, IL.
§ Cavitron, Long Island City, NY.
i University of Michigan O probe with Williams markings, Hu-Friedy.
¶ BioCollagen, Bioteck, Torino, Italy.
# Doxycycline, Pfizer Egypt under authority of Pfizer, New York, NY.
** Methylcellulose powder, Sigma Chemicals, Munich, Germany.
J Periodontol • September 2012 Gamal, Kumper

(DOX-coated surface) in contact with the defect and

the outer surface in contact with the gingival tissue.
Four weeks later, contralateral sites received DOX–
COL after EDTA gel†† was applied with a sterile sy-
ringe with a blunt-ended cannula into the exposed
and debrided root surfaces and allowed to stay for 3
minutes. The roots were then rinsed for 3 minutes with
a physiologic saline solution (G2; 30 sites). Finally,
the mucoperiosteal flaps were repositioned and se-
cured in place using 4-0 non-absorbable black silk
surgical suture,‡‡ using modified vertical mattress
and interrupted sutures. Participants were asked not
to use dental floss for 14 days at the selected sites
to avoid displacement of the membrane.
Fourteen days after surgery, sutures were removed Figure 1.
and normal oral hygiene procedures were allowed ex- Pure DOX experimental standard of HPLC (10 mg/mL).
cept for the use of chemotherapeutic mouthrinses and
brushing of the selected sites. One surgeon (AG) per-
formed all surgeries. Clinical and radiographic mea-
surements were reassessed at 3 and 6 months after
therapy to evaluate the quantitative changes in the
defect and to do critical comparisons with the DOX
availability. All clinical measurements were recorded
by one calibrated masked examiner (RK).
GCF Sampling
With the use of a capillary tube (5 mL), GCF samples
were collected52 by a single examiner (RK), who was
masked to the attribution of the sites to DOX–COL or
DOX–COL + EDTA. After isolation and drying of the
selected site with cotton rolls, disposable micro-
pipettes§§ were placed intrasulculary at the mesiofa-
cial line angle of the selected site to a maximum
depth of 2 mm below the gingival margin. Micro-
pipettes were left in place until 5 mL fluid was col-
lected. GCF samples were collected 1, 3, 7, 10, 14, Figure 2.
and 21 days after therapy and diluted in saline solu- Example of the GCF DOX level of 7 days DOX–COL + EDTA sample
tion (50 mL). Samples were labeled, carried in a dark using HPLC.
container, and kept at -80C until used.
Detection of DOX by High-Performance Liquid the changes by time within each group. The signifi-
Chromatography cance level was set at P £0.05. Statistical analysis
High-performance liquid chromatography (HPLC) was performed with statistical software.##
was equipped with a binary pumpii C18 column¶¶
(5 mm, 250 · 4.6 mm). Mobile phase was 34% aceto- RESULTS
nitrile/66% 50 mM acetate buffer (acidic pH 3.8) at 1 Postoperative healing was uneventful and revealed
mL/min flow rate. An ultraviolet detector (LC 1,210; good soft tissue response to both treatments in all
UV/Vis detector) was used to estimate and quantitate cases. No complications, such as allergic reactions,
DOX in samples at 257 nm according to Hebert et al.53 abscesses, or infections, were observed throughout
using an experimental standard (10 mg/mL) that the study period. All patients complied with the re-
was injected to HPLC separately to give a peak at a call appointments throughout the 6-month study pe-
retention time of 3.8 minutes (Figs. 1 and 2). riod, except for three patients from G1 who did not
†† EDTA gel, Biora, Malmo, Sweden.
Statistical Analysis ‡‡ Braided black silk suture 4-0, W. L. Gore, Flagstaff, AZ.
Data were presented as mean – SD values. A paired t §§ Fisherbrand disposable micropipettes, Fisher Scientific, Pittsburgh, PA.
ii LC-1110, GBC Scientific Equipment, Hampshire, IL.
test was used to compare control and experimental ¶¶ Hypersil Elite C18, Fisher Scientific.
sides within each group. It was also used to study ## SPSS, IBM, Chicago, IL.

1089
EDTA Enhanced Doxycycline GCF Availability Volume 83 • Number 9

continue their follow-ups for sample collections. It was
decided not to include their samples in the analysis of
data. As a result, 27 of 30 G1 sites completed the
study. The periodontal status of the two studied
groups was demonstrated using the mean – SD for
the appropriate clinical measurements: PI, GI, PD,
CAL, and depth of the IBC. At baseline, all GI and PI
scores were within the clinically healthy scores (1).
IBC measurement for the DOX–COL samples was
4.9 – 0.5 mm and for the DOX–COL + EDTA samples
was 5.2 – 0.4 mm. The differences were statistically
non-significant (P £0.05). Table 1 shows the mean –
SD baseline and follow-up clinical measurements
for the two study groups during different observation
periods. Six months after treatment, significant clini-
cal improvements were obtained in PD reduction and
clinical attachment gain in DOX–COL + EDTA- and
DOX–COL-treated sites (P £0.05). DOX–COL +
EDTA-treated sites showed more significant clinical
improvements when compared to DOX–COL-treated
sites (P £0.05). Significant improvement was also
reported in the reduction of the IBC depths in both
groups, with a significant difference between G1 when
compared with that of G2 in favor of the DOX–COL +
EDTA group.
Table 2 shows DOX concentrations in the G1
(DOX–COL) and G2 (DOX–COL + EDTA) treated
groups during different observation periods. GCF
samples obtained 1, 3, 7, 10, and 14 days in the
DOX–COL + EDTA group showed statistically sig-
nificant higher mean DOX concentration than that
of the non-etched G1 treated sites. At 21 days, the
DOX–COL + EDTA group showed 5.3 mg/mL values;
however, no DOX was detected in any of the col-
lected DOX–COL (G1) samples. The DOX–COL +
EDTA-treated group appeared to retain more DOX
during the period of 3, 7, 10, and 14 days compared
with the DOX–COL group.
DISCUSSION
In consideration of the technical aspects of perio-
dontal reconstructive procedures for intraosseous
defects, one major factor important for periodontal
regeneration is to eliminate, or reduce to a great extent,
the chances of postoperative infection and contami-
nation of the blood clot and, possibly, the implanted

Table 1.
Mean – SD Baseline and Follow-Up Clinical Measurements for Different Study Groups

DOX–COL DOX–COL + EDTA DOX–COL DOX–COL + EDTA DOX–COL DOX–COL + EDTA

Parameter (baseline) (baseline) P value (3 months) (3 months) P value (6 months) (6 months) P value

PI 0.3 – 0.2 0.5 – 0.2 NS 0.4 – 0.2 0.4 – 0.3 NS 0.4 – 0.2 0.5 – 0.2 NS

GI 0.4 – 02 0.4 – 0.2 NS 0.6 – 0.4 0.4 – 0.3 NS 0.4 – 0.2 0.3 – 0.3 NS

PD (mm) 6.6 – 0.9 5.2 – 0.4 NS 3.7 – 0.5 2.6 – 0.4 0.04 3.5 – 0.7 2.5 – 0.4 0.02

CAL (mm) 4.6 –1.07 4.3 – 0.8 NS 2.9 – 0.7 1.8 – 0.7 0.02 2.5 – 0.6 1.9 – 0.5 0.01

IBC (mm) 4.9 – 0.5 5.2 – 0.4 NS 2.9 – 0.5 1.4 – 0.4 0.01 2.6 – 0.4 1.4 – 0.4 0.01
NS = non-significant (P £0.05 is considered significant).

Table 2.
DOX Concentration in DOX–COL and DOX–COL + EDTA Groups

Observation Difference Release Release Release Release

Period (days) DOX–COL DOX–COL + EDTA P Value (G2 - G1) Period (days) Value (G1) SD Period (days) Value (G2) SD P Value

1 503.0 – 1.3 705.1 – 3.7 0.043* 202.1 – 2.1

3 209.2 – 2.52 365.5 – 3.3 0.043* 156.3 – 3.3 1 to 3 293.8 2.1 1 to 3 339.6 2.4 0.745

7 72.3 – 1.2 249.3 – 1.9 0.038* 171 – 1.7 3 to 7 136.9 3 3 to 7 22.2 2.3 0.036*

10 29.6 – 1.3 173.7 – 1.3 0.0023* 144.1 – 1.4 7 to 10 42.7 2.3 7 to 10 69.6 2.6 0.024*

14 14.7 – 2.2 31.9 – 1.3 0.317* 17.2 – 1.3 10 to 14 14.9 2.8 10 to 14 141.8 4.4 0.040*

21 0–0 5.3 – 1.2 0.043* 5.3 – 1.5 14 to 21 14.7 0 14 to 21 26.6 1.4 0.041*
* P £0.05.

1090
J Periodontol • September 2012
biomaterial or biologic agent, which would unavoid-
ably lead to impaired healing outcome. Antimicrobial
local delivery during the early stages of healing might
enhance the seal of the attachment apparatus, which
will augment protection of regenerating tissues, a fac-
tor that could improve periodontal regeneration. DOX
is one the most important antimicrobial agents that
has the ability to concentrate in the GCF.37 Many
studies reported that direct exposure of the acidic
media of tetracycline could either reduce adhesion of
PDL cells to periodontally involved root surfaces or
may be retained within the tubules and continue its
demineralizing action.54,55 In view of this, it appears
more reasonable to expose indirectly the defect area
to DOX by loading it to a delivery vehicle, such as
COL. In this way, the creation of a DOX gel smear-like
effect obscuring the positive effects of the EDTA-
exposed dentinal tubules and dentine-associated col-
lagen also can be avoided. Baker et al.44 reported
that, after EDTA root surface removal of the organic
smear layer, additional conditioning of the dentin sur-
face with protein constructs produced a surface mor-
phology similar to that of the smear layer with poor
fibrin clot retention. The hypothesis behind this pres-
ent work is to maximize the availability of DOX in the
GCF through its delivery on COL while at the same
time make use of the advantage of EDTA root surface
exposure of dentinal tubules and dentin-associated
collagen to enhance such availability without inter-
fering with root surface characteristics. In that way, it
is reasonable to surmise that DOX was delivered in
a double-delivery approach. The first is the COL as
a direct delivery vehicle, and the second is the EDTA
root surface etching as an indirect delivery vehicle
for the released DOX ingredients from the COL. This
double-delivery effect could prolong DOX availability
in the GCF, providing a biochemical reclogging
prevention factor for exposed dentinal tubules and
dentin-associated collagen by reducing bacterial
colonization. The collagen membrane by its me-
chanical root surface protection could also add a
mechanical reclogging prevention factor for the
exposed dentinal tubules. At the same time, avoid-
ing direct DOX coating to the root surface could
reduce any possible deleterious effects on PDL
fibroblast viability and clot adhesion.
For evaluating the released DOX at different time
periods, micropipette sample collection appears to
be ideal because it provides an undiluted sample of
‘‘native’’ GCF whose volume can be accurately as-
sessed. In addition, GCF flow by its physical protective
effects through flushing the pocket is considered an
excellent undispersed media. The selection of the
intrabony pocket type is another factor that helps
in maintaining DOX undispersed for accurate eval-
uation of its availability and release pattern. At base-
Gamal, Kumper
line, the plaque control was optimal, and GI scores
were £1 for both groups. There were no statistically
significant differences in PD, CAL, and IBC. Therefore,
the GCF flow rate was nearly consistent, and the DOX
containment and the subsequent release from the IBC
could be under the same circumstances. The authors
decided to start GCF sample collection 1 day after sur-
gery because GCF samples that were collected imme-
diately usually were contaminated with blood. The
HPLC method using ultraviolet detection for the deter-
mination of CHX in GCF was reported to be a sensitive
method with excellent selectivity and reproducibility,
and it has been applied to a bioequivalence clinical
study with great success.56
Analysis of the GCF in the present study reveals
that DOX concentrations in the test DOX–COL +
EDTA-treated sites are significantly higher than that
in the non-etched DOX–COL surfaces for £21-day ob-
servation periods. This period appeared to be more
suitable for availability of DOX because more pro-
longed subtherapeutic levels could increase the risk
of bacteria developing resistance to DOX. At 21 days,
DOX concentration in the DOX–COL + EDTA group
appeared to be available by a mean value of 5.3
mg/mL. This value was reported to be within the min-
imum antibacterial DOX inhibitory concentration.
Most of the subgingival microorganisms are suscepti-
ble to tetracycline at an MIC £1 to 2 mg/mL. However,
species, such as Eikenella corrodens, Prevotella ora-
lis, Selenomonas sputigena, and some strains of Cam-
pylobacter and Veillonella, are susceptible at MIC ‡16
mg/mL. Microorganisms associated with periodontitis
can also develop a resistance after exposure to sub-
inhibitory concentrations of antibiotics.57 Slots and
Rams58 reported that the MIC at which Pg, Prevotella
intermedia, Campylobacter rectus, and Fusobacte-
rium nucleatum are susceptible to DOX is at a concen-
tration £6.0 mg/mL. Laboratory studies on human
subgingival plaque showed that DOX at 125 mg/mL
can inhibit an average of 99% of the cultivable bacte-
ria.59 Larsen60 reported that DOX is effective against
putative periodontal pathogens at a concentration of
0.1 to 6.0 mg/mL. Expanded substantivity of DOX
after EDTA root surface etching could be explained
by the DOX mechanical retention into the opened
dentinal tubules and by an increase in the wettability
of the exposed dentin collagen of the EDTA-etched
exposed root surfaces. In the authors’ previous stud-
ies,42,43 they reported that EDTA gel root condition-
ing can result in exposure and widening of dentinal
tubule orifices that could add a mechanical retentive
effect by the impaction of silica particles carrying
CHX within the tubules. In addition, the same studies
noted that exposure of dentinal tubules, intertubular
dentin, and dentin-associated collagen increases
the amount of root surface areas onto which CHX
1091
EDTA Enhanced Doxycycline GCF Availability
gel can attach. Sole EDTA application that could en-
hance DOX availability for £21 days appears to favor
the cost–benefit value of such an approach when com-
pared with the costly single or frequent applications
of the currently available local delivery antimicrobials.
When the present release data are compared to the
release profile of the currently available DOX formu-
lation, the availability of DOX in the DOX–COL +
EDTA-treated group appears to be more extended
in a higher concentration than that which followed
the cellulose-acetate-loaded DOX formulation stud-
ied by Tonetti et al.61 The release of DOX from a com-
mercial 8.5% DOX*** formulation was reported to
maintain during the first 48 hours of application an
average of 1,500 to 1,700 mg/mL DOX in the peri-
odontal pocket34 and maintained a concentration
of 250 mg/mL DOX hyclate at the end of 7 days in
the GCF62 and 100 mg/mL at the end of 10 days.34
Kim et al.63 reported a mean GCF DOX concentration
of 1,085.30, 264.00, 273.94, and 258.00 mg/mL at 2,
5, 24, and 48 hours, respectively, after application
of 8.5% DOX.††† DOX hyclate at 10%‡‡‡ reported
250 mg/mL in the GCF during a period of 7 days.64
Deasy et al.65 used 25% tetracycline hydrochloride
in poly(hydroxybutyric acid) as a biodegradable poly-
mer matrix and showed sustained release over 4 to 5
days, with a significant burst effect at day 1. In the
present study, to avoid disturbance of the COL,
GCF samples were obtained from the most coronal
part of the subgingival space. This could explain the
lower initial and follow-up levels of DOX that was ap-
plied to the underside of the membrane compared
with other studies that quantify free DOX gel in the
pocket.
The clinical 6-month follow-up data of the present
study indicate that significant improvements could
be obtained for deep IBC after EDTA root surface
etching and subsequent placement of DOX gel-
loaded COL when compared with non-etched DOX–
COL-treated sites. This could be attributed to the as-
sociated prolonged and higher values of DOX levels
for the DOX–COL + EDTA-treated group. However,
the main target of the present work is to quantify levels
of DOX during the early stages of healing to discern
whether such clinical improvement could be attrib-
uted to prolonged and increased DOX levels after
EDTA root surface preconditioning.
CONCLUSIONS
The present results suggest that the DOX–COL +
EDTA guided tissue antibacterial regimen is a conve-
nient method of obtaining a prolonged drug release
without compromising the space that should be oc-
cupied by regenerating tissues or by inducing smear-
ing of the root surface by DOX gel. If the outcomes
of the currently available delivery vehicles were com-
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pared with those of the DOX–COL + EDTA delivery,
the negative effects of space-occupying factors of
such materials, liability of bacterial contamination,
cost–benefit value, the possibility of liberating toxic
degradation products, together with the mechanical
protective effect of COL, all favor DOX–COL + EDTA
delivery.
ACKNOWLEDGMENTS
This work was partially supported by Al-Azhar Uni-
versity, Cairo, Egypt. The authors thank Dr. Khaled
Kerra, Private Dental Clinic, Cairo, Egypt, for his as-
sistance in statistical interpretation of the results.
The authors report no conflicts of interest related to
this study.
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Correspondence: Dr. Ahmed Y. Gamal, Department of
Periodontology, Faculty of Dental Medicine, Al-Azhar
University, Cairo, Egypt. E-mail: hgamal1@hotmail.com,
ahmedygamal@gmail.com.
Submitted August 13, 2011; accepted for publication
November 22, 2011.