Effect of Autogenous Cortical BoneParticulate in Conjunction With EnamelMatrix Derivative in the Treatment ofPeriodontal Intraosseous DefectsLuigi Guida,* Marco Annunziata,* Salvatore Belardo,* Roberto Farina,† Alessandro Scabbia,†and Leonardo Trombelli†
Background: The aim of the present study was to assess the
additional clinical benefit of autogenous cortical bone particu-late (ACBP) when added to enamel matrix derivative (EMD),compared to EMD alone, in the treatment of deep periodontalintraosseous defects.
Methods: A total of 28 intraosseous lesions in 27 patients
with advanced periodontitis were included in this controlledclinical trial and randomly assigned to the EMD group (14 de-
fects) or to the EMD + ACBP group (14 defects). Immediately
before surgery (baseline) and after 6 and 12 months, probing
depth (PD), clinical attachment level (CAL), and gingival re-
cession (REC) were recorded. Radiographic depth of the de-
fect (DEPTH) was also measured at baseline and 12 months
Results: At 6 and 12 months, PD and CAL significantly im-
proved from baseline in both groups (P <0.000). No significant
differences in terms of CAL gain and PD reduction were de-
tected between groups. However, defect distribution according
to CAL gain was significantly different between groups
(P <0.05). DEPTH significantly decreased from baseline to 12
months in both groups (P <0.000); between-group differences
were not significant. At 12 months, a significantly greater REC
increase in the EMD group (1.1 – 0.7 mm) compared to the
EMD + ACBP group (0.3 – 0.8 mm) was observed (P <0.05).
Conclusions: Both EMD and EMD + ACBP treatments led
tial, which may potentially affect its re-
to a significant improvement in clinical and radiographic pa-
rameters at follow-up with respect to presurgery condition. The
combined approach resulted in reduced post-surgery recession
and increased proportion of defects with substantial CAL gain
(‡6 mm). J Periodontol 2007;78:231-238.
erative treatment is addressed to deep,non-contained intraosseous defects.14
Autograft; enamel matrix proteins; periodontitis; randomized
clinical trial; regeneration; treatment/surgery.
such as bovine porous bone mineraland demineralized freeze-dried bone al-lograft, has the potential to enhance the
* Department of Odontostomatological, Orthodontic and Surgical Disciplines, Second
University of Naples, Naples, Italy.
† Research Center for the Study of Periodontal Diseases, University of Ferrara, Ferrara, Italy.
EMD alone in terms of clinical attach-ment level gain15,16 or bone fill.17,18
Autogenous Bone Graft and EMD Versus EMD in Intraosseous Defects
Among the different available graft materials,
search Centre for the Study of Periodontal Diseases,
autogenous bone graft meets several ideal character-
University of Ferrara, Italy, from November 2003
istics, because it is potentially osteoinductive, bio-
to May 2004. Adult patients with advanced chronic
absorbable, low-cost, and easy to handle. It may be
or aggressive periodontitis21 were consecutively en-
speculated that the combined use of EMD and autog-
rolled for this study. Informed consent was obtained
enous bone graft could be advantageous, resulting in a
from the patients after explaining the nature of the
synergistic reconstructive effect. In this respect, the
investigation being conducted. Informed consent
autogenous bone graft may enhance the osteogenic
and research protocol were institutionally approved.
potential of the healing site, act as an effective space-
Exclusion criteria were: 1) systemic diseases that
maintaining scaffold for bone regeneration, and limit
contraindicated periodontal surgery; 2) medications
the collapse of supracrestal soft tissues into the defect.
affecting periodontal status; 3) pregnancy or lacta-
On the other hand, EMD may exert its biologic poten-
tion; and 4) full-mouth plaque score22 and full-mouth
tial at root level, inducing the biologic mechanisms
bleeding score >20% at the time of surgical procedure.
underlying the periodontal regenerative process. How-
Furthermore, third molars, teeth with Class III mobil-
ever, limited data are available about the use of EMD
ity, furcation involvement, inadequate endodontic treat-
and autogenous bone graft in the treatment of intraos-
Inclusion criteria were considered as follows: 1) at
A histologic study19 on non-human primates has
least one intraosseous defect in need of surgical treat-
investigated this combined approach, reporting en-
ment after initial periodontal treatment and reevalua-
couraging results in terms of amount of new cemen-
tion; 2) PD ‡6 mm; and 3) radiographic intraosseous
tum and bone. Recently, we reported a consecutive
series of cases where the effectiveness of a regenera-
Patients were given a cause-related treatment con-
tive procedure based on the preservation of supra-
sisting of oral hygiene instructions and multiple scal-
crestal soft tissue in association with an autogenous
ing and root planing sessions, using both hand and
cortical bone particulate (ACBP)-EMD combination
ultrasonic instruments. At least 4 weeks elapsed from
was investigated for the treatment of deep, 1- to 2-wall
the completion of the non-surgical therapy until
intraosseous defects.20 This approach resulted in sig-
nificant clinical attachment gain and probing depth
(PD) reduction and in a reduced marginal gingival re-
Radiographs were performed at baseline and 1 year
cession increase after 6 months of healing. To support
after surgery. Radiographic measurements included
the additional use of ACBP in conjunction with EMD,
1) the radiographic depth of the defect (DEPTH), mea-
we designed a 12-month prospective randomized
sured as the linear distance (in millimeters) from the
clinical trial where the two procedures (EMD alone
most coronal extension of the alveolar crest (as per-
versus EMD + ACBP) were compared in the treatment
pendicularly projected on the long axis of the tooth)
of deep intraosseous periodontal defects.
to the most apical extension of the defect (i.e., wherethe periodontal ligament space was considered hav-
ing a normal width);9 2) the radiographic defect fill
(percentage), calculated as follows: (baseline DEPTH
Two different approaches for the treatment of deep
- 12-month DEPTH)/baseline DEPTH · 100; and 3)
intraosseous defects were compared in a parallel
the radiographic defect angle (ANGLE) at baseline,
designed, randomized, controlled clinical trial. The
determined in degrees as the angle formed between
control group was treated by means of EMD‡ alone
the lines that represent the root surface of the involved
(EMD group), whereas the test group was treated with
tooth and the bone defect surface.23 All radiographic
EMD in association with an ACBP (EMD + ACBP
measurements were performed by a single trained
group). The same surgical procedures and the appli-
and calibrated examiner (AS), who was masked as
cation of EMD on the root surface were performed in
both control and test groups. Grafting with ACBP was
the only difference between the experimental groups.
The following clinical measurements were taken im-
Clinical outcomes were measured at baseline and 6
mediately before surgery (baseline) and 6 and 12
months post-surgery: local plaque score (LPS) and lo-
cal bleeding score (LBS), recorded dichotomously at
Patients were recruited among those seeking care for
surgical site as the presence or absence of supragin-
periodontal disease at the Department of Odontosto-
gival plaque and bleeding on probing, respectively;
matological, Orthodontic and Surgical Disciplines,Second University of Naples, Italy, and at the Re-
‡ Emdogain Gel, Straumann, Basel, Switzerland.
Guida, Annunziata, Belardo, Farina, Scabbia, Trombelli
clinical attachment level (CAL); PD; and marginal
After flap reflection, all soft tissue was removed
gingival recession (REC). Measurements were per-
from the defect, and the root surface was scaled and
formed at six sites (mesio-buccal, buccal, disto-buc-
planed with hand and ultrasonic instruments. In all
cal, mesio-lingual, lingual, and disto-lingual) around
cases, the exposed root surfaces were conditioned
the teeth presenting the defect; however, only the de-
with 24% EDTA geli for 2 minutes. The defect was then
fect-specific measurement presenting the highest
thoroughly rinsed with saline to remove gel remnants.
CAL at the time of presurgery recordings was consid-
For the EMD + ACBP group, an adequate amount of
ered for the analysis. Measurements were performed
cortical bone particulate was harvested from the buc-
by trained and calibrated examiners (SB and AS) with
cal cortical plate by means of a bone scraper.¶ The
a manual sensitive probe (at approximately 0.3 N
bone graft was collected from the surgical site adja-
force) with 1-mm increments§ and rounded up to
cent to the intraosseous defect. A first layer of EMD
the nearest millimeter. Examiners were not masked
was injected to condition the bone defect and the more
apical portion of the root surface. ACBP was posi-tioned to fill only the intrabony component of the
defect, avoiding any packing of the graft. Finally, a
Intrasurgical measurements, recorded immediately
second layer of EMD was injected to cover the grafted
after defect debridement, included probing bone level,
autogenous bone particles and to condition the por-
as the distance from the cemento-enamel junction
tion of the root surface coronal to the bone crest.
(CEJ) to the apical end of the defect; and intrabony
Therefore, a ‘‘sandwich’’ technique was adopted to
component of the defect, as the distance from the
treat the defect (i.e., apical layer of EMD, ACBP,
most coronal extension of the interproximal bone
crest to the apical end of the defect.
For the EMD group, the EDTA-treated root surface
and surrounding bony walls were conditioned with
the amelogenin gel according to the manufacturer’s
Two operators (LG and LT) performed all surgeries.
Each defect was randomly assigned to either EMD
Finally, flaps were positioned at the presurgery
group (14 defects) or EMD + ACBP group (14 defects).
level or slightly coronal to achieve primary closure
After local anesthesia, buccal and lingual sulcular in-
of the interdental area without any tension. Monofila-
cisions were made and mucoperiosteal flaps were ele-
ment non-resorbable 5-0 or 6-0# or polypropylene**
vated. Maximum care was exercised to preserve the
suturing material was used. Selection of the suturing
marginal and interdental tissues. Flap design in the in-
terdental area consisted of one of the following alterna-
tives: 1) sulcular incisions with the split of buccal and
The patients received systemic antibiotic therapy,††
lingual papilla;24 2) incision with the preservation of
2 g/day for 6 days, and 0.12% chlorhexidine mouth-
the buccal papilla, according to the simplified papilla
rinses,28 twice a day for 6 weeks. Mechanical tooth
preservation technique;25 3) incision with the preser-
cleaning was not allowed in the surgical area for the
vation of the buccal papilla, according to the modified
first 6 postoperative weeks. Sutures were removed
papilla preservation technique;26 or 4) incision with
at least 14 days following surgery. Patients were then
the preservation of the lingual-palatal papilla, accord-
placed on monthly recall visits, including professional
ing to interproximal tissue maintenance procedure.27
tooth cleaning, during the first 6 months, and every 3
Selection of flap design was based on: 1) width of the
months thereafter. Probing and subgingival instru-
interdental space, evaluated as the distance from
mentation was not performed £6-month reevaluation.
the CEJ of the tooth presenting the bone defect tothe CEJ of the adjacent tooth; 2) distance from the con-tact point or area to the bone crest, as radiographically
assessed; 3) apico-coronal width of interdental kerati-
Statistical software‡‡ was used for data analysis. Be-
nized tissue in the area of intraosseous defect; and 4)
cause no differences were observed whether the pa-
location and morphology of the bone defect, as deter-
tient or the defect was regarded as statistical unit,
mined with bone sounding. The gingival tissue was in-
we reported our data based on the number of defects.
cised at least one tooth mesial and distal to the defect
Measurements from each group were expressed as
site to provide access for visualization and instrumen-tation of the defect and, in the test group, for the follow-
ing phase of bone harvesting. Vertical incisions were
Micross, Meta C.G.M., Reggio Emilia, Italy.
placed mesial or distal to the treated defect, if they were
# Gore-Tex Suture, W.L. Gore & Associates, Flagstaff, AZ.
considered necessary for better access or primary clo-
** Perma Sharp Suture, Hu-Friedy. †† Augmentin, SmithKlineBeecham, Milan, Italy.
‡‡ NCSS-PASS, Number Cruncher Statistical Systems, Kaysville, UT.
Autogenous Bone Graft and EMD Versus EMD in Intraosseous Defects
mean values – SD. The presence of any randomiza-
All 27 patients completed the study, and complied
tion imbalance between the two experimental groups
with the 6- and 12-month reexaminations.
at baseline was tested by unpaired Student t test and
x2 analysis. Within-group comparisons for outcome
Incidence of LPS-positive defects was 21.4% at base-
variables were performed by paired Student t test,
line and remained unchanged (21.4%) at 12 months
whereas between-group differences were evaluated
in both groups (P >0.05). Incidence of LBS-positive
by unpaired Student t test at 6- and 12-month obser-
defects decreased from 71.4% at baseline to 7.1% at
vation intervals. A power analysis indicated that 26
12 months in the EMD group (P <0.000), and from
unpaired defects (13 in each group) would be suffi-
50% at baseline to 7.1% at 12 months in the EMD +
cient to demonstrate statistical significance at the
ACBP group (P <0.000). No statistically significant
P <0.05 level with a power of 0.85. In our study, 14
differences were detected between groups in terms
defects for each experimental group were treated.
of incidence of LPS- and LBS-positive defects at 6
Clinical and radiographic measurements at base-
Study Population and Defect Characteristics
line and 6 and 12 months are summarized in Table 2.
A total of 27 patients (13 men and 14 women, 30
At 6 months, CAL and PD significantly improved
to 65 years old; mean age: 46.3 – 8.7 years) with
from baseline (P <0.000) in both groups. The 12-month
28 intraosseous defects were selected and treated.
CAL and PD measurements did not significantly
Twenty-six patients contributed one defect, one pa-
change from the 6-month measurements, remain-
tient contributed two defects. Fourteen defects in
ing significantly improved with respect to baseline
14 patients were treated with EMD, 14 defects in 13
(P <0.000). In the EMD group, REC varied from
patients were treated with EMD + ACBP. All defects
1.7 – 0.7 mm at 6 months to 2.1 – 0.9 mm at 12
showed a predominant 1- to 2-wall component. Mean
months, whereas REC remained almost unchanged
age in the EMD + ACBP and EMD groups was 44.1 –
in the EMD + ACBP group. No significant differences
6.9 years and 48.4 – 9.9 years, respectively. There
in CAL gain and PD reduction were detected between
were seven females and two smokers in each group.
groups at 6 and 12 months. At 12 months, a signifi-
An analysis of defect characteristics at baseline
cantly greater REC increase in the EMD group com-
revealed no significant differences between groups
pared to the EMD + ACBP group was observed with
respect to baseline REC (P <0.05). A significantly dif-ferent distribution of defects according to 12-month
CAL gain was observed between groups (P <0.05)(Table 3): 50% of defects showed a CAL gain of ‡6
mm and 21% showed a CAL gain of 4 to 5 mm inthe EMD + ACBP group compared to 21% and 57%,
DEPTH significantly decreased from baseline to 12
months in both groups (P <0.000). DEPTH gain was
4.3 – 2.4 mm corresponding to a radiographic defectfill of 64.8% – 24.1% for the EMD group, and 4.3 –
1.3 mm corresponding to a radiographic defect fill
of 68% – 17.3% for the EMD + ACBP group. No signif-icant differences were detected between groups at 12
months (P >0.05). Defect distribution, according to
DEPTH gain, was similar between groups (P >0.05;Table 4).
The clinical effect of EMD to achieve the reconstruc-tion of lost periodontal tissues has been extensively
revised and confirmed. Available data from sys-
tematic reviews10,12,29 indicate that all EMD re-constructive treatment produces a more favorable
clinical improvement in hard and soft tissue param-
I = incisors; C = canine; P = premolars; M = molars; PBL = probing bone
eters of healing response (i.e., clinical attachment
level; IBD = intraosseous component of the defect; NS = not significant(P >0.05).
gain, pocket reduction, and bone fill) compared to
Guida, Annunziata, Belardo, Farina, Scabbia, Trombelli
Clinical and Radiographic Measurements at Baseline and 6 and 12 Months (mean – SD)
D0-6 = difference between baseline and 6 months; D0-12 = difference between baseline and 12 months; NS = not significant (P >0.05). * P <0.05. † P <0.01. ‡ P <0.000.
conventional open flap debridement procedure. How-
ever, although all studies generally showed an addi-
Defect Distribution According to CAL Gain
tional benefit with the use of EMD, a high degree of
variability in treatment outcomes (heterogeneity)was found in the included trials.29 This heterogeneity
may be partly explained by differences in the patientand defect selection among studies. For instance, the
use of a biomaterial with a gel-like consistency, such
as EMD, in a non–self-supporting intraosseous defectmay result in a limited clinical outcome due to the col-
lapse of the flap into the bone defect during the early
healing phase, particularly in deep, non-containingintraosseous defects.14 These observations seem to
* Significant difference between groups (P <0.05).
emphasize the clinical relevance to adapt the selec-tion of the reconstructive strategy to the anatomy ofthe treated area and the physical and biologic char-
acteristics of the regenerative materials used.29,30
In this perspective, the present study was under-
taken to evaluate whether and to what extent the ad-
ditional use of ACBP in conjunction with EMD mayimprove the clinical effect of EMD alone when used
in deep intraosseous defects, with a predominant1- to 2-wall component. The results indicate that both
the EMD + ACBP and EMD procedures led to a statis-
tically significant and clinically relevant CAL gain withrespect to presurgery condition, with more than 70%
of the defects presenting a CAL gain of at least 4 mm
for both treatment groups. No significant differences
* No significant difference between groups.
were detected between treatment groups in terms of
Autogenous Bone Graft and EMD Versus EMD in Intraosseous Defects
average CAL gain, PD reduction, and defect bone fill.
benefit derived from the additional use of ACBP. Pre-
However, EMD + ACBP treatment significantly in-
vious reports where a similar surgical approach was
creased the proportion of defects with substantial
associated to EMD treatment resulted in comparably
CAL gain (‡6 mm) and determined a smaller postop-
erative REC increase with respect to EMD alone.
A significantly smaller REC increase was found in
Recent studies indicated that the combination of
the EMD + ACBP group compared to the EMD group
EMD with bone substitutes, such as bovine porous
at the 12-month reevaluation. ACBP may efficiently
bone mineral and demineralized freeze-dried bone al-
have sustained the soft tissue healing, avoiding col-
lograft, has the potential to enhance the reconstruc-
lapse into the bony defect, during the tissue matura-
tive outcome compared to EMD alone in terms of
tion phase. This result compared to those stemming
clinical attachment level gain15,16 or bone fill.17,18
from clinical trials where EMD treatment was used
The choice to use ACBP in addition to EMD was sup-
in conjunction with slowly bioabsorbable biomate-
ported by data from a randomized controlled trial
rials, such as bovine porous bone mineral.16,17 These
showing a greater CAL gain after an access flap pro-
observations seem to suggest the additional use of
cedure with autogenous bone graft compared to an
ACBP in deep, non–self-supporting intraosseous de-
access flap procedure alone, for the reconstructive
fects, especially in areas of the dentition where the
treatment of deep intraosseous defects.31 Further-
esthetic outcome is considered of paramount im-
more, we previously showed that the adjunctive ap-
plication of ACBP with EMD resulted in clinically
Our results indicate that both EMD and EMD +
significant attachment gain and PD reduction in deep
ACBP reconstructive procedures result in comparable
intraosseous defects, with a predominant 1- to 2-wall
outcomes in terms of attachment gain and bone fill.
intraosseous component.20 In our study, the lack of
However, defect distribution according to CAL gain
substantial additional benefits observed in the EMD +
significantly differed between groups. In particular, a
ACBP group compared to EMD seems to confirm
higher prevalence of defects showing a CAL gain of
the biologic potential of EMD per se to support the
‡6 mm was observed in the EMD + ACBP group com-
clinical reconstruction of the lost attachment appara-
pared to the EMD group (Table 3). In contrast, defect
tus, and to limit the additional application of an autog-
distribution according to DEPTH gain was similar be-
enous bone particulate to enhance the EMD-induced
tween groups (Table 4). These data suggest that part
of the defects in the EMD + ACBP group showed a CAL
The observed values for CAL gain, PD reduction,
gain greater than the corresponding DEPTH gain,
and REC increase in the EMD group compared to
whereas in EMD-treated defects CAL gain substan-
some studies,9,14,16,32-34 whereas they differed from
tially paralleled DEPTH gain. The presence of a graft
others where less favorable outcomes had been ob-
supporting supracrestal tissues during the healing
served.35-39 The reason for these discrepancies may
phase could explain this observation. However,
be found in a wide range of predictors, including pa-
whether and to what extent the additional use of ACBP
tient selection, defect characteristics, maintenance
to EMD may affect the wound healing dynamics
phase, and surgical variables.39,40 In our material
of deep periodontal defects compared to mere EMD
the same surgical approach based on supracrestal
application needs be clinically and histologically con-
soft tissue preservation was used for both treatment
firmed. In this respect, several histologic studies41-44
groups; the only investigated variable was the appli-
in humans demonstrated the periodontal regenerative
cation of ACBP to fill the intraosseous component of
potential of the autogenous bone, leading to cemen-
the defect. In all cases flap design and suture tech-
tum and bone formation. In contrast, inconsistent his-
nique were adequately selected with respect to the
tologic results have been reported on the regenerative
morphologic characteristics of the defect to preserve
potential (i.e., presence and extent of newly formed
an adequate amount of supracrestal soft tissue and
bone and cementum) of EMD in the treatment of intra-
achieve primary closure in the interdental area.
Primary closure eliminates or, to a greater extent,
Two considerations about data analysis must be
reduces the chances of post-surgical infection and
made. First, among the 28 treated defects, four de-
contamination of the blood clot and, possibly, the bi-
fects were present in smoker patients. Due to the lim-
ologic agent or the graft. Incomplete primary closure
ited number of smokers in the study population,
may be of particular concern when a non-supportive
statistical analysis of treatment outcome between
material, such as EMD, is used to reconstruct in
smokers and non-smokers was not performed. Sec-
toto the attachment apparatus, including alveolar
ond, clinical examiners were not masked as to the
bone.7,15 In this respect, proper soft tissue manage-
surgical procedures, so that a potential bias in out-
ment, leading to optimal EMD-induced wound healing
come assessment cannot be completely excluded.
process, may partly account for the limited clinical
However, it must be considered that the examiners
Guida, Annunziata, Belardo, Farina, Scabbia, Trombelli
were expert and rigorous clinicians, trained and cali-
12. Venezia E, Goldstein M, Boyan BD, Schwartz Z. The
brated to ensure accuracy and reproducibility of clin-
use of enamel matrix derivative in the treatment of
periodontal defects: A literature review and meta-analysis. Crit Rev Oral Biol Med 2004;15:382-402.
13. Sanz M, Tonetti MS, Zabalegui I, et al. Treatment of
intrabony defects with enamel matrix proteins or bar-
With the limits of the present study, our data support
rier membranes: Results from a multicenter practice-
the clinical effectiveness of a regenerative procedure
based clinical trial. J Periodontol 2004;75:726-733.
based on EMD application, either alone or in combi-
14. Froum S, Lemler J, Horowitz R, Davidson B. The use
of enamel matrix derivative in the treatment of peri-
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This study was partly supported by a Research Grant
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of the Second University of Naples, Naples, Italy, and
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the Italian Ministry of Education, University and Re-
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of Periodontal Diseases, University of Ferrara, Fer-
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Correspondence: Dr. Luigi Guida, Department of Odon-
study of combined treatment with a collagen mem-
tostomatological, Orthodontic and Surgical Disciplines,
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Second University of Naples, Via L. De Crecchio 6, 80138
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Naples, Italy. Fax: 39-81-5665524; e-mail: luigi.guida@
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Accepted for publication September 8, 2006.
J. Am. Chem. Soc. 1997, 119, 10549 10550 Catalytic Pauson Khand Reaction in Super Critical the homogeneous reaction media after completion of thereaction. The reaction with water soluble ligand bound catalystsin organic/aqueous phase process8 and with perfluorinatedligands bound catalysts in the conventional organic solvent andNakcheol Jeong,*,† Sung Hee Hwang,† Youn Woo Lee,‡ an
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