Vol 14, Issue 1 - International Academy of Periodontology

Transcription

I
Journal of the International
AP
Academy of Periodontology
The official journal of the International Academy of Periodontology
Volume 14 Number 1 January 2012
Published by
The International Academy of Periodontology
I
AP
Volume 14
Number 1
January 2012
Journal
of the
International Academy of
Periodontology
ISSN 1466-2094
EDITORIAL BOARD
Mark R Patters
Editor
Memphis, TN, USA
Andrea B Patters
Associate Editor
Sultan Al Mubarak
Riyadh, Saudi Arabia
P Mark Bartold
Adelaide, SA, Australia
Michael Bral
New York, NY, USA
Effect of Local Nifedipine Administration on Rat Gingiva
Davide Bencivenni, Mirdza E. Neiders, Sebastiano Andreana,
Michelle L. Moffitt and Robert E. Cohen
1
Histological Evaluation of Osseous Defects Combined with
Orthodontic Tooth Movement
Mai Shafik Attia, Eatemad A. Shoreibah, Samir A. Ibrahim and
Hamdy A. Nassar
7
Regenerative Therapy of Osseous Defects Combined with
Orthodontic Tooth Movement
Mai Shafik Attia, Eatemad A. Shoreibah, Samir A. Ibrahim and
Hamdy A. Nassar
17
Nadine Brodala
Chapel Hill, NC, USA
Cai-Fang Cao
Beijing, People's Republic of China
Daniel Etienne
Paris, France
Ahmed Gamal
Cairo, Egypt
Vincent J Iacono
Stony Brook, NY, USA
Isao Ishikawa
Tokyo, Japan
Georges Krygier
Paris, France
Francis Mora
Paris, France
Hamdy Nassar
Cairo, Egypt
David Paquette
Chapel Hill, NC, USA
Rok Schara
Ljubljana, Slovenia
Uros Skaleric
Ljubljana, Slovenia
Shogo Takashiba
Okayama, Japan
Thomas E Van Dyke
Boston, MA, USA
Warwick Duncan
Dunedin, New Zealand
Nicola Zitzmann
Basel, Switzerland
The Journal of the International Academy of Periodontology is the official journal of the International Academy of Periodontology
and is published quarterly (January, April, July and October) by The International Academy of Periodontology, Boston, MA, USA and
printed by Hasti Digital Prints, Mumbai, India.
Manuscripts, prepared in accordance with the Information for Authors, should be submitted electronically in Microsoft Word to the
Editor at the [email protected] Editorial Office can be contacted by addressing the editor, Dr. Mark R.Patters, University of
Tennessee, College of Dentistry, 875 Union Avenue, Memphis, TN 38163, USA.
All enquiries concerning advertising, subscriptions, inspection copies and back issues should be addressed to Ms. Alecha
Pantaleon, Forsyth Institute, 245 First Street, Suite 1755, Cambridge, MA, USA 02142, Telephone: +1 617-892-8536, Fax: +1 617-2624021, E-mail: [email protected]. Whilst every effort is made by the publishers and Editorial Board to see that no inaccurate or
misleading opinion or statement appears in this Journal, they wish to make clear that the opinions expressed in the articles,
correspondence, advertisements etc., herein are the responsibility of the contributor or advertiser concerned. Accordingly, the
publishers and Editorial Board and their respective employees, offices and agents accept no liability whatsoever for the consequences of
any such inaccurate or misleading opinion or statement.
©2012 International Academy of Periodontology.
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, photocopying, or otherwise, without permission of the Academy.
Printed in India by Hasti Digital Prints, Mumbai
Journal of the International Academy of Periodontology 2012 14/1:1-6
Effect of Local Nifedipine Administration
on Rat Gingiva
1
2
3
Davide Bencivenni , Mirdza E. Neiders , Sebastiano Andreana ,
Michelle L. Moffitt4 and Robert E. Cohen4
1
School of Dental Medicine, University of Modena and Reggio
2
Emilia, Modena, Italy; Departments of Oral Diagnostic Sciences,
3
Restorative Dentistry, and 4Periodontics and Endodontics,
University at Buffalo, The State University of New York, School of
Dental Medicine, Buffalo, NY
Abstract
Background: Nifedipine, a calcium channel-blocking agent, has been associated with
gingival enlargement in humans. This enlargement has also been successfully established
in animal models. Previous investigators have administered nifedipine through a systemic
route, most commonly by oral intake. The aim of the present study was to measure the
effects of nifedipine administered directly into rat gingival interproximal papillae.
Methods: Twenty-four adult female rats were assigned to three groups. Each animal
received a series of three injections, one week apart; each injection was placed directly
into the interdental papilla of the maxillary and mandibular central incisors. Group 1
(control) received only saline. Group 2 received a low (10 µg/ml) concentration of
nifedipine, while Group 3 received a higher concentration (500 µg/ml). One week after
the last series of injections, gingival specimens were harvested from the injection site and
prepared for histological and immunocytochemical analyses. Results: Specimens from
Group 3 displayed a significantly greater number of ED2-positive cells compared to the
other two groups. Specimens from Group 2 showed a significantly higher mean count of
positive cells compared to Group 1. Collectively, our data suggest that repeated local
injections of 10 µg/ml and 500 µg/ml nifedipine each elicit an inflammatory response in
the gingival connective tissue. Conclusions: Immunocytochemical analysis revealed
dose-dependent increases of resident tissue macrophages in rats receiving nifedipine (p <
0.005). An increased inflammatory infiltrate also was observed via routine histology.
Gross macroscopic changes consistent with gingival enlargement were not observed.
Key words: Nifedipine, calcium channel blocking agents, immunosuppressant,
anticonvulsants, gingival enlargement
Introduction
Drug-induced gingival enlargement is typically
characterized by a thickening of the spinous layer of the
gingival epithelium and increase in collagen deposition
(Bulut et al., 2006). Although the mechanism of drugrelated gingival enlargement has not been completely
elucidated, it involves an interaction between a drug and
resident gingival fibroblasts, resulting in a lower rate of
collagen phagocytosis (Shimizu et al., 2002). Additional
factors also might contribute to gingival enlargement,
including age, sex, plaque, genetic predisposition and
local tissue characteristics (Guncu et al., 2007).
Correspondence to: Mirdza E. Neiders, Department of Oral
Diagnostic Sciences, University at Buffalo, The State
University of New York, School of Dental Medicine, 250
Squire Hall, Buffalo NY 14214. E-mail: [email protected].
© International Academy of Periodontology
Drug-induced gingival enlargement was first
associated with phenytoin in 1939, but since has been
associated with three different therapeutic classes of
drugs: calcium channel blockers, immunosuppressants,
and anticonvulsants (Lin et al., 2007). Although those
classes are unrelated to one another, they are all known
to affect intracellular calcium levels by inhibiting its
entry into the cell (Gelfand et al., 1986; Messing et al.,
1985). The decreased intracellular Ca2+ level seems to
play a crucial role in the pathogenesis of gingival
overgrowth. Intracellular calcium is required by
fibroblasts for the synthesis and secretion of enzymes
involved in collagen degradation, such as matrix
metalloproteinases (MMPs). The upregulation of
MMP gene expression is specifically triggered by
protein kinase C, via induction of the binding of the
Effect of Local Nifedipine Administration on Rat Gingiva 2
transcription factor AP-1 to a specific promoter
sequence of the MMP gene (Birkedal-Hansen, 1993).
Activation of protein kinase C is a calcium-dependent
process (Hardie et al., 1990; Shimizu et al., 2002). This
pathway may explain, at the transcriptional level, how
each of the drugs associated with gingival overgrowth
alters the sequence of events that lead to MMP
expression and release from gingival fibroblasts. The
diminished collagen breakdown results in a shift of the
homeostatic balance and a net accumulation of
extracellular matrix becomes clinically evident as
gingival overgrowth. This hypothesis has been
confirmed by reduced MMP-1 staining in drug-induced
gingival overgrowth tissue samples (Walters, 1993).
Fujii (1990) also showed the ability of nifedipine to
inhibit cellular calcium uptake in gingival fibroblasts.
Numerous studies have demonstrated that clinical
manifestations of drug-induced gingival overgrowth
are similar in animals and humans (Nishikawa et al.,
1996; Nishikawa et al., 1991). The gingival papilla and
the gingival margin typically become enlarged, and are
characterized by firm, red, nodular tissue. Sibling
animals offer the advantage of minimizing the
interpatient variability due to genetics or other
individual patient characteristics. Availability, ease of
handling and cost make rats one of the most suitable
animal models. The rat bears close resemblance to
humans with respect to periodontal anatomy,
development and composition of oral plaque, and
histopathology of periodontal disease (Nishikawa et al.,
1996; Nishikawa et al., 1991; Shaker et al., 2011).
Experimental induction of drug-related gingival
overgrowth in rats has been obtained by many
investigators (Kataoka et al., 2001; Nishikawa et al.,
1996; Nishikawa et al., 1991). Nishikawa et al. (1996)
administered nifedipine (250 μg/g diet) to rats and
observed macroscopic enlargement as early as 20 days
after oral ingestion. Longer treatment periods of up to
70 days did not result in increased severity of the
overgrowth. It also was observed that when the drug
was removed from the animals' diet, the gingival
macroscopic morphology returned to control levels
within 30 days (Nishikawa et al., 1991). The authors
concluded that several clinical features were common
to gingival overgrowth induced by calcium channel
blockers, phenytoin or cyclosporin A in rats: 1) a more
conspicuous enlargement of the buccal rather than the
lingual gingival; 2) less severe enlargement of the
maxilla than the mandible; 3) accumulation of dental
plaque influences the severity, but is not essential for the
onset of overgrowth; and 4) more severe overgrowth is
observed in young rats. It was determined that severity
is dependent upon drug blood levels, as well as the
duration of drug administration, with maximum
overgrowth developing between 30 and 40 days
(Nishikawa et al., 1991). Since those same factors have
been hypothesized as being important in drug-induced
gingival overgrowth in humans, the rat model may be a
valuable model for investigating drug-induced gingival
enlargement.
Histological analysis of nifedipine-induced,
enlarged gingival tissue reveals that both the epithelium
and the connective tissues are affected. The lamina
propria typically displays an increased vascularity and a
chronic inflammatory cell infiltrate. Bundles of
immature collagen fibres are densely packed or loosely
textured. The epithelium is thickened and acantotic
(Nishikawa et al., 1996; Nishikawa et al., 1991; Shaker et
al., 2011). Histomorphometric analysis also indicates
that responder rats have a 2.5-fold increase in mean
cross sectional gingival area (Nishikawa et al., 1991).
Previous human or animal studies generally have
been performed to investigate the incidence of and the
mechanisms of systemically administered druginduced gingival overgrowth. To date, there are no
studies that attempt to exploit this pharmacologically
induced tissue change in order to purposely regenerate
gingival tissue. To the best of our knowledge, there are
no animal studies where drugs have been delivered
locally or topically to specific sites. Consequently, the
aim of this study was to measure the effects of local
nifedipine delivery on gingival tissues using
macroscopic observation, as well as through
quantitative immunocytochemical analysis of a
macrophage subset that may serve as a marker of
fibroblast activation.
Materials and methods
This study was reviewed and approved by the
Institutional Animal Care and Use Committee,
University at Buffalo, The State University of New
York. Twenty-four adult female rats (Harlan Sprague
Dawley, Inc., Indianapolis, IN) weighing approximately
220 g were maintained on food pellets and water ad
libitum and housed in an air-conditioned, humiditycontrolled facility. A pure powder form of nifedipine
(Sigma Chemical, St. Louis, MO) and sodium chloride
(Fischer Scientific, Fair Lawn, NJ) were combined for
the preparation of the nifedipine slurry. The injections
were performed using a 10 ml syringe with 30 gauge
needles, delivering a volume of 50 µl at each injection.
Twenty-four rats were assigned to three groups of
eight rats each. Rats in Group 1 were given only saline
(control group), rats in Group 2 received nifedipine (10
µg/ml) and rats in Group 3 received nifedipine (500
µg/ml). Those doses were chosen because they were
comparable to gingival crevicular fluid concentrations
observed in humans taking therapeutic doses of
nifedipine (Ellis, et al., 1993). Each animal received
three similar injections one week apart. The injections
were located at the maxillary and mandibular right side
of the gingival papilla, distal to the upper and lower
incisors. The animals were euthanized one week after
the third and last injection via an intraperitoneal dose of
phenobarbital (150-200 mg/kg). Tissue samples from
the injection sites were obtained for routine
3 Journal of the International Academy of Periodontology 2012 14/1
Mean number of cells per field positive for ED2 primary antibody
Mean number of ED2-postive cells per field
Group 1
4.67 ± 1.68
Group 2
19.72 ± 13.28
Group 3
74.56 ± 28.23
hematoxylin & eosin staining and for immunocytochemistry.
Specimens for routine histology were fixed in 10%
formalin at room temperature and slides 10 µm in
t h i ck n e s s we r e o b t a i n e d . S p e c i m e n s f o r
immunocytochemistry were frozen in liquid nitrogen
o
and stored at -70 C. Four-micron cryostat sections were
o b t a i n e d f r o m e a ch f r o z e n s a m p l e. Fo r
immunostaining, the frozen slides were brought to
room temperature for 30 minutes and then fixed in
acetone for 10 minutes. After fixation, the sections were
dried for 3 minutes, then washed for 5 minutes in Trisbuffered saline (TBS, 20 mM Tris-HCL, 500 mM NaCl,
pH 7.5), followed by incubation for 20 minutes in
normal rabbit serum diluted 1:5 in TBS for blocking of
non-specific background. The normal serum was
successively removed by tapping, and the sections were
treated with the primary monoclonal antibody (ED2)
diluted 1:500 in TBS, as described by Kataoka et al.
(2001) and as modified by us (Cohen et al., 1991). This
monoclonal antibody is specific for tissue-resident
macrophages. All sections were washed in TBS for 5
minutes and then exposed for 30 minutes to
biotinylated rabbit anti-mouse immunoglobulins
(Dako Corp. Carpintera, CA) diluted 1:400 in TBS.
After washing in TBS for 5 minutes slides were treated
with avidin-biotin complex-alkaline phosphatase
(Dako Corp. Carpintera, CA) for 30 minutes, then
rinsed with TBS for 5 minutes. Localization of antigens
was achieved by color development with a solution
prepared from 2 mg naphthol AS-MX phosphate free
acid (Sigma Chemical), 0.2ml N, N-dimethyl
formamide (Sigma Chemical) 9.8 ml of 0.1 M Tris
buffer pH 8.2 and 10 mg of Fast-Red TR salt (Sigma
Chemical). The sections were analyzed by light
microscopy after coverslipping with a water-based
mounting medium (Dako Corp. Carpintera, CA).
Quantification of phagocyte subsets was
performed in accordance with the methodology
described by Honda et al. (1990) and as modified by us
(Cohen et al., 1991). Ten randomly selected fields on
each specimen were analyzed by counting the number
of cells labelled with the monoclonal antibody, at 200x
magnification, from at least two duplicate connective
tissue sections (i.e., two adjacent sections from the same
specimen). The average number of cells per field
positive for each antibody was obtained for each
specimen. The values were computed and differences
between the control group and the two treatment
groups were determined by one-way analysis of
variance (ANOVA) cor rected for multiple
comparisons.
Results
Macroscopic findings
Macroscopic examination did not reveal any alteration
in the gross appearance of the rats' gingiva at the
injection sites. The gingival tissue maintained its normal
appearance with characteristic consistency, size and
color, and without any sign of tissue enlargement. This
observation was consistent irrespective of the group
and the observational time.
Group 1: control Microscopic examination of the
eight mandibular and eight maxillary specimens was
consistent with normal microscopic anatomy. The rat
incisor is characterized by continuous enamel
formation that occurs on the buccal aspect of the tooth
with the lingual aspect remaining exposed dentin
(Graner et al., 1995). No inflammatory infiltrate was
detected in any of the control sections obtained from
the mandibular arch.
Figure 1 is representative of the soft tissue
attachment to dentin. The gingival sulcus is adjacent to
the dentinal surface and the sulcular epithelium is
keratinized with the epithelial layer ranging between 15
and 30 cells in thickness. Orthokeratinization was
observed in each control section involving both oral
and sulcular epithelium, and is consistent with the
normal anatomy of the dento-gingival unit in rats
(Graner et al., 1995). The interface between the root and
the periodontal soft tissues was characterized by a thin
Figure 1. Photomicrograph of a gingival papillae from
the maxillary arch of a rat from Group 1 (control). The
gingival sulcus is adjacent to the dentinal surface and
the sulcular epithelium is keratinized. (20X, H&E).
Figure 2. Photomicrograph of the buccal papilla from
the mandibular incisor of a rat receiving high-dose
nifedipine (Group 3). Sections from this group
generally displayed an epithelial layer that
maintained its integrity and was comparable to
control sections. This section shows low cellularity
and scattered lymphocytes. (100X, H&E).
Figure 3. Photomicrograph of gingival connective tissue obtained from a rat receiving high-dose
nifedipine (Group 3). The section was processed for immunocytochemistry using ED2-specific
monoclonal antibodies with an avidin-biotin-alkaline phosphatase technique, and color
developed with naphthol AS-MX phosphate/Fast-Red TR. (200X).
epithelial layer, 2 to 4 cells thick, representing the
junctional epithelium. The connective tissue layer, or
lamina propria, was without pathology in terms of
collagen content, size and cellularity.
Group 2: low dose nifedipine. Specimens from Group 2
displayed a sulcus lined by orthokeratinized epithelium,
10-15 cell layers thick. The connective tissue exhibited
normal extracellular matrix accumulation. Six out of
eight specimens showed mild inflammatory infiltration,
consisting of scattered polymorphonuclear neutrophils
and a few lymphocytes.
Group 3: high dose nifedipine. Sections from this group
displayed an epithelial layer that maintained its integrity
and was comparable to control specimens. Its thickness
ranged between 15 and 20 cell layers. The connective
tissue was comparable to control sections in terms of
collagen content (Figure 2). Mild inflammatory
infiltration characterized by dispersed granulocytes and
lymphocytes were detected in five of eight specimens.
No frank alteration of papilla size was observed at the
injection sites of any test specimens.
Immunocytochemical findings
Cells recognized by ED2 monoclonal antibody had a
dendritic appearance, with small nuclei, little cytoplasm
and multiple elongated processes as described in
previous studies (Cohen et al., 1991; Kataoka et al., 2001).
The mean values of ED2-positive cells from the three
groups are summarized in Table 1. ED2-positive
macrophages were observed in specimens from all three
groups, but the mean count of ED2-positive cells was
significantly higher in Group 3 specimens (Figure 3),
compared to the other two groups (p < 0.0005) and
Group 2 compared to Group I (p < 0.0005). Differences
between maxillary and mandibular samples were not
evident, so those specimens were pooled for
immunocytochemistry. A dose-dependent mean
number of ED2-positive cells were noted in specimens
from animals treated with nifedipine. The average
number of ED2-positive cells was 4.6, 19.7 and 74.5
cells/field in Group 1, Group 2, and Group 3,
respectively.
Discussion
Unfavorable gingival architecture may adversely affect
plaque control and be unesthetic. Prosthetic
rehabilitation of edentulous areas and interproximal
spaces may be required to eliminate elongation of the
clinical crown and/or appearance of black spaces (black
triangles) between teeth caused by the loss of interdental
papilla, which may be of concern for both patients and
dental practitioners. Loss of interdental papilla may also
have biological consequences, such as food impaction,
increased dentinal sensitivity, and increased caries
incidence.
At this time, soft tissue regeneration only can be
achieved through surgical procedures such as gingival or
connective tissue grafting to increase the volume and
extent of soft tissue (Carnio, 2004). Results may be
technique-sensitive and variable; patients may decline
treatment due to cost, time or procedural factors (Ellis et
al., 1995). Although a less invasive and more costeffective approach might result in greater patient
acceptance, such procedures currently are not available.
This study was performed to measure local gingival
enlargement following repeated administration of
nifedipine in the interdental papilla. Although
significant changes in ED2-positive macrophages were
noted immunocytochemically, gross alterations in
gingival morphology were not observed.
Macrophages may play a role in the initial stages of
fibroblast activation. Bellon et al. (2011) have found that
macrophages may participate in human peritoneal
fibrosis through the stimulation of fibroblast cell
growth and cytokine production. Other studies have
demonstrated the expression of fibroblast growth
factors and receptors on macrophages and mast cells
with an increase in collagen production (Akimoto et al.,
1999; Barron and Wynn, 2011; Prasse et al., 2006; Takei et
al., 1989). Consequently, it is possible that macrophages
may participate in intracellular communication during
the first stages of nifedipine-induced gingival fibrosis.
Although a decrease in gingival enlargement might be
associated with discontinuing the use of nifedipine,
further studies examining additional macrophage and
fibroblast subsets would be indicated to more fully
elucidate the precise mechanisms of inflammatory cell
activation and to assess the permanence of druginduced enlargement in this model.
To the best of our knowledge this study is the first
attempt to induce drug-related gingival enlargement
through local drug delivery. The presence of nifedipine
in the gingival tissue may be required for production of
gingival enlargement (Ellis et al., 1992; Ellis et al., 1995).
However, it is possible that local administration under
the conditions used in this study did not produce an
effective concentration of nifedipine at the level of the
dentogingival unit (Ellis et al., 1995; Thomason et al.,
1998). The concentration of nifedipine utilized for each
injection was derived from the concentration typically
found in the gingiva of responder patients (i.e., patients
taking nifedipine and developing gingival enlargement),
but this may be more effective in humans compared to
rats.
Two different concentrations were used (10 and
500 µg/ml) to analyze dose response. It is possible that
a greater macroscopic effect might have been achieved
by using higher nifedipine concentrations and
increasing the frequency of drug delivery (daily or
continuously instead of weekly), and/or by extending
the length of the study.
We were able to demonstrate an inflammatory
response through immunocytochemical analysis.
Specimens from Group 3, the group injected with the
highest nifedipine concentration, displayed a
significantly greater number of ED2-positive cells
compared to the other two groups. Specimens from
Group 2 showed a significantly higher mean count of
positive cells compared to Group 1. Collectively, our
data suggest that repeated local injections of nifedipine
with concentrations of 10 µg/ml and 500 µg/ml elicit
an inflammatory response in the gingival connective
tissue. Fibrosis, with collagen deposition, may occur as
a result of prolonged inflammation (Ishida et al., 1995).
The duration of this study was only three weeks; a
longer experimental period could lead to a more
pronounced fibrosis, provided that inflammation
persists for an extended period. More subtle changes in
mononuclear cell subsets also might have been detected
using a panel of monoclonal antibody probes specific
for other macrophage subsets, as well as for T- and Blymphocytes. Future studies will be directed towards
further exploration of those variables.
Acknowledgment
There were no significant forms of support associated
with this research. This study was supported in part by
the Department of Periodontics and Endodontics,
University at Buffalo, The State University of New
York, School of Dental Medicine. There is no financial
relationship between any author and a commercial firm
that would pose a conflict of interest.
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Journal of the International Academy of Periodontology 2012 14/1:7-16
Histological Evaluation of Osseous Defects
Combined with Orthodontic Tooth Movement
Mai Shafik Attia1, Eatemad A. Shoreibah1, Samir A. Ibrahim2 and
1
Hamdy A. Nassar
1
Department of Oral Medicine, Periodontology, Oral Diagnosis
2
and Radiology, and Department of Orthodontics, Faculty of
Dental Medicine – Girls' Branch, Al Azhar University, Cairo, Egypt
Abstract
Background: The aim of this study was to histologically evaluate the effectiveness of
different times of initiating orthodontic tooth movement on enhancement of bone
formation in surgical bony defects. Methods: In 18 male guinea pigs, 3-4 months of age, a
bony defect was created in the alveolar process midway between the central incisor and
mandibular 1st molar. These bony defects were implanted with bioactive glass particles
and collagen membrane. According to the application of the orthodontic tooth
movement, the animals were divided into three groups, each comprised of six guinea
pigs. In Group I, the orthodontic tooth movement was initiated immediately after the
surgical procedure. In Group II, the orthodontic tooth movement was applied 2 weeks
after the surgical procedure, while in Group III no orthodontic tooth movement was
applied. Section blocks for histology were made at 1, 3 and 6 weeks after the surgical
procedure. Results: All experimental sites showed active bone formation with plump
osteoblast and osteoid matrix deposition in the treated area. In Groups I and II a dense
fibrous tissue formation and highly cellular coarse bone were seen at six weeks. The
histomorphometric analysis showed that Group I revealed the greatest number of newly
formed trabeculae: 2.4, 6.4 and 8.6 at 1, 3 and 6 weeks, respectively. In addition, Group I
defects revealed a greater total surface area of newly formed bone than Groups I and III:
2.96 mm 2 at the end of the study period. Conclusion: The combined
orthodontic/regenerative therapy seemed to enhance the process of bone formation.
Bone formation was histologically observed in all test groups. Defects treated with
immediate application of orthodontic tooth movement showed a statistically significant
increase in trabecular count and total surface area of newly formed bone than the other
experimental groups.
Key words: Surgical bony defects, combined orthodontic / regenerative
therapy, bone grafts, membranes, follow-up studies
Introduction
Bone is a connective tissue and guarantees protection
and support to the organ function. Bone is a dynamic
tissue that constantly undergoes turnover (Proff and
Romer, 2009).
In orthodontic tooth movement, sites of tension
display osteogenesis over an extensive surface area, a
framework consistent with modeling. However, sites of
compression undergo phases of remodeling cycles
(King et al., 1991b). The tooth movement will occur
only if the hard tissue around the tooth can undergo
proper breakdown and build-up. Such remodeling
Correspondence to: Mai Shafik Attia Department of Oral
Medicine, Periodontology, Oral Diagnosis and Radiology,
Faculty of Dental Medicine – Girls' Branch, Al Azhar
University, Cairo, Egypt.
Telephone: (02) 24112135/(02) 24053623
E-mail: [email protected]
requires the presence of cells able to resorb
(osteoclastogenesis) and cells able to form bone
(osteogenesis) (Diès et al., 1996; Skoglund et al., 1997).
One important consideration is how remodeling
cycles are initiated. Much experimental evidence has
linked bone remodeling to microdamage and to
subsequent increased cellular activity. Microcracks in
bone caused by fatigue or trauma may play an important
role in the initiation of remodeling cycles (Galley et al.,
2006). Crack displacements are capable of tearing
osteocyte cell processes, which may directly secrete
bioactive molecules into the extracellular matrix,
triggering a remodeling response (Hazenberg et al.,
2006). The increased prevalence of microcracks at
compression sites in orthodontic tooth movement
further suggests that they are important in initiating
orthodontic bone remodeling (Verna et al., 2004).
Much like tooth eruption, osteogenesis associated
Attia et al: Effects of Orthodontics on Osseous Defects in Guinea Pigs 8
with orthodontics is mediated by various
osteoinductive molecules. In general, most of these
molecules are regulated by tensile strains and act by
stimulating osteoblast progenitor cell proliferation in
the periodontal ligament, with subsequent bone
formation and inhibition of bone resorption.
Molecules that have been linked in this way to
orthodontic tooth movement include transforming
growth factor-beta (TGF-β) (Brady et al., 1998), various
bone morphogenic proteins (BMPs) (Mitsui et al.,
2006), bone sialoprotein (BSP) (Domon et al., 2001) and
epidermal growth factor (EGF) (Guajardo et al., 2000;
Gao et al., 2002).
Enhanced periodontal and bone regeneration by
orthodontic tooth movement towards a bony defect
(Geraci et al., 1990; Nevins and Wise, 1990; Liou and
Huang, 1998) and intrusive movement (Melsen, 1986;
Cardaropoli et al., 2001) have been reported. However,
others did not find this effect (Polson et al., 1984;
Wennstrom et al., 1993). Moreover, the type of bone
towards which the tooth moves also plays an important
role. Tooth movement into cortical bone results in
fenestration or dehiscence, i.e., loss of cortical bone
plate integrity (Steiner et al., 1981 and Steigman et al.,
1993), while tooth movement in the alveolar trough
results in subsiding of injury/repair cycles (Steigman et
al., 1993).
Recently, bioactive glass (BG), a ceramic material,
has gained much attention because of its unique silica
component compared to other bioceramic alloplastic
graft materials. The formation of a silica-gel layer on
the surface of the graft particles is thought to be
responsible for the bioactivity and osteoconductivity of
the material (Nishida et al., 2006).
Bioabsorbable collagen membranes have been
tested for their ability to promote regeneration in
intrabony defects (Mattson et al., 1995; Benque et al.,
1997; Mattson et al., 1999). The collagen materials
possess additional advantages over other bioabsorbable
membranes (Locci et al., 1997) with no specific immune
reaction (Schlegel et al., 1997). In an attempt to achieve
periodontal regeneration, the present study included
the use of bioactive glass with a bioabsorbable collagen
membrane.
The rationale behind this study was to utilize the
potential enhancing effect of orthodontic tooth
movement on bone formation. To the best of our
knowledge, the influence of the timing of the
application of force has not been previously
investigated. This study evaluated bony tissue
responses at different times of initiation of
orthodontic tooth movement.
eighteen male guinea pigs, aged 3-4 months, weighing
from 250-450 gram. Pre-operatively, the animals were
anaesthetized with 120 mg/kg intramuscular ketamine
hydrochloride (Vardimon et al., 2001).
The submental region was shaved and then
scrubbed with a disinfectant solution. Using a facial
approach, a submental incision was made using a BardParker scalpel with a #15 blade. A mucoperiosteal
elevator was then used to reflect the tissues and expose
the bone. By using the mental foramen as a reference
point two bony defects were created using surgical bur
number 2 attached to a low speed motor with an
irrigation system, each midway between the mental
foramen central incisor. The size of bony defects was
fixed by using a sterile standard fabricated stent, which
was 4 mm in depth x 4 mm in width. The surgical bony
defects were thoroughly irrigated, then bioactive glass
(Bio-Glass = surface activated resorbable bioactive
glass, Excellence Pharma, Inc., Egypt) was compressed
in the defects until the level of the material was flush
with the labial cortical bone of the mandible (Figure 1).
The collagen membrane (Biocollagen, Bioteck S.r.l
Fermi, Arcugraro VI, Italy) was placed over the
bioactive glass (Figure 2) and stabilized. The wound
edges were then approximated using a tissue forceps
and an atraumatic needle.
Figure 1. The bony cavity was filled with bioglass.
The research protocol for this study was approved by
the institutional Animal Care and Use Committee of
Al-Azhar University. The study was conducted on
Figure 2. The collagen membrane was added to
cover the graft material.
Figure 3. An orthodontic spring was applied to
allow tipping movement.
Figure 4. Separation of the teeth.
The mandible was dissected from the soft tissues. The
mandible was then placed in jars labeled by animal
number and investigation duration. Fixation of the
tissue was done using 10% formalin for 3 weeks.
Decalcification of the specimens were done using
ethylene diamine-tetra acetic acid (EDTA) 125 g/L
distilled water and sodium hydroxide as a buffer for 3
weeks. The samples were then dehydrated in ascending
grades of ethyl alcohol starting with 70% up to 100%
absolute alcohol followed by methyl benzoate for one
day followed by paraffin benzol for two hours. To
remove the alcohol residue the samples were bathed
three times in paraffin wax and placed in wax blocks of
suitable size to be ready for cutting. Cutting of the
samples was done using a Leitz Wetzlar microtome that
obtained serial sections at 5-8 µ thick, and about 10
sections were taken from each sample.
Image analysis
The microscopic fields were randomly selected and
those containing the highest number of newly formed
bone were selected. Four different microscopic fields at
the magnification x100 were photomicrographed for
each microscopic slide. Images were captured with the
aid of a digital video camera (5.1 megapixel, Olympus,
Japan) mounted on a light microscope (BX60,
Olympus, Japan). Captured digital images were
imported to a computer system and were displaced for
image analysis.
The brightness and contrast of the captured
images were automatically processed prior to
calculation using software (Photoshop 7.0, Adobe
Corp.). Using the image analysis software (Soft Imaging
System, Sis-5, Germany), the image was transformed
into 8-bite for automated grey scale level of the newly
formed bone trabeculae. Edges of bone trabeculae
were traced, and then color-code thresholding was
carried out. Finally, the color-coded bone trabeculae
were cut as desired areas and other structures such as
bone marrow were excluded from the field prior to
calculation.
Count of the number of newly formed bone
trabeculae and the total surface area of bone trabeculae
was automatically calculated using Excel software
(Office 2003, Microsoft Corp.®). Data were tabulated
for further statistical analysis.
The orthodontic tooth movement was applied
immediately after the surgical procedure in Group I,
and the animals were sacrificed at 1, 3 and 6 weeks. The
animals in Group II were subjected to the surgical
procedure, after 2 weeks the orthodontic tooth
movement was applied, and the animals were sacrificed
at 1, 3 and 6 weeks. Group III animals were sacrificed at
1, 3 and 6 weeks after implantation of bone graft and
collagen membrane in the bony cavities.
The orthodontic tooth movement was directed
towards the treated defect. The orthodontic appliance
consisted of standard tubes (0.018 x 0.015) placed on
the central incisors and attached together by a spring to
allow tipping movement of teeth (Figure 3). The spring
was activated once per week (Figure 4). After sacrificing
the animals, their mandibles were separated for the
preparation of paraffin blocks and histological
evaluation.
The histological results revealed that during the six
weeks period of bony cavity preparation and insertion
of regenerative materials with or without orthodontic
force application, several tissue changes took place
among the surgically created cavities.
Histological preparation
After each period of investigation the guinea pigs were
sacrificed and the site of surgery was carefully removed.
Group I
At one week, the histological sections of the surgical
bony cavities, using H&E stain, revealed that the
Results
Figure 5a
Figure 5b
Figure 5c
Figure 5d
Figure 5e
Figure 5. A photomicrograph of the surgical bony cavity of Group I: a) at one week showing the
presence of bone spicules (black arrow) interspersed with fibrin meshwork (blue arrow) (H&E
stain x100); b) at one week showing fibrillar fibrous tissues (blue arrow) intermingled with
spicules of newly formed bone (black arrow, Mallory's stain x100); c) at three weeks showing
coalescence of large areas of bone trabeculae (black arrows, Mallory's stain x100); d) at six
weeks showing mature mineralized bone trabeculae with osteocytes inside lacunae (black
arrow). The bone trabeculae had haphazard arrangement with marrow spaces in between (blue
arrow, H&E stain x100); 5 e) at six weeks period showing closely intertwining and condensing
collagen fibrils (yellow arrow) and numerous dilated vascular channels (Mallory's stain x100).
original periphery of cortical plates consists of dense
compact lamellar bone with the presence of
irregularities. These irregularities have been rounded
off by shallow depressions (Howship`s lacunae) that
identify regions of bone with osteoclastic resorption.
Inside the cavities, the presence of red blood cells
entrapped in the fibrin meshwork in addition to a
homogeneous degenerative tissue indicating necrosis
and spicules of bones were seen (Figure 5a).
Mallory`s stain revealed no difference between the
aforementioned structures inside the cavities
(Figure 5b).
At three weeks, H&E stain revealed that large areas
of bone trabeculae had been formed with a scanty
fibrin meshwork in between. The newly formed bone
trabeculae were numerous and intermingled with each
other. These features were well demarcated using
Mallory`s stain (Figure 5c).
After six weeks, a dense fibrous tissue formation
and highly cellular coarse (woven) bone were seen. The
Figure 6a
Figure 6b
Figure 6c
Figure 6d
Figure 6. A photomicrograph of the surgical bony cavity of Group II: a) at one week showing
bone irregularities that have been rounded off by shallow depressions (Howship's lacunae,
black arrow, Mallory's stain x100); b) at three weeks showing a communication of newly
formed bone with the periphery of the bony cavity (black arrow, Mallory's stain x100); c) at six
weeks showing a highly cellular formed bone (black arrow) nearly filling the entire cavity (H&E
stain x100); d) at six weeks period showing newly formed bone interspersed by fibrous tissue
(blue arrow, Mallory's stain x100);
newly formed bone trabeculae were numerous and
intermingled with each other. In addition, small
numbers of flattened, quiescent osteoblasts lining the
bone trabeculae were seen. The latter showed haphazard
arrangement (Chinese-letter pattern) with marrow
spaces in between (Figure 5d). Mallory`s stain revealed
that collagen in some areas showed closely interwining
and condensing fibrils, which are seen by virtue of a
deep blue color. Fibrous tissues interspersed with bone
trabeculae with less fibrin meshwork were also seen
(Figure 5e).
Group II
At one week, H&E stain revealed large islands of
osteoid tissue that were dispersed in a fibrous tissue.
These features were apparent with numerous marrow
spaces when utilizing Mallory`s stain. A difference in
staining with variability of color indicated the presence
of new and old bone. The latter showed irregularities
that were rounded off by shallow depressions
(Howship`s lacunae) that identify regions of bone with
osteoclastic resorption (Figure 6a).
After three weeks, H&E stain revealed the
presence of numerous fragments filling the cavities
intermingled with fibrin meshwork. A communication
of the newly formed bone with the periphery of the
bony cavity was seen. These features were distinguished
when Mallory`s stain was utilized (Figure 6b).
At the end of six weeks, H&E stain revealed that
the fibrin meshwork showed shrinkage and
degeneration. The cavities were almost filled with zones
of new bone, which enveloped the periphery of the
cavities. In addition to the presence of coarse woven
bone, formation of trabeculae of lamellar bones was
seen (Figure 6c). Mallory`s stain revealed the same
features (Figure 6d).
Group III
At one week, H&E stain revealed the presence of blood
clots inside the cavities with formation of fibrin
meshwork. Red blood cells were entrapped in the fibrin
meshwork surrounded with dense fibrous tissue
(Figure 7a). Mallory`s stain revealed that a fibrous
component of granulation tissue surrounded the cavity
Figure 7a
Figure 7b
Figure 7c
Figure 7d
Figure 7e
Figure 7f
Figure 7. A photomicrograph of the surgical bony cavity of Group III: a) at one week showing fibrin
meshwork all over the surgically created cavity (H&E stain x100); b) at one week showing fibrous
component of granulation tissue surrounds the bone graft particles (yellow arrow, Mallory's stain
x100); c) at three weeks showing bony spicules (black arrow) within the bony cavity (H&E stain
x100); d) at three weeks showing mature fibrous tissue (blue arrow) with newly formed bone (black
arrow) within the surgically created cavity (Mallory's stain x100); e) at six weeks showing
immature woven bone (black arrow) with presence of fibrous tissue in between (blue arrow, H&E
stain x100); f) at six weeks showing mature collagen fibrils interspersed with fragments of newly
formed bone (yellow arrow) with marrow spaces in between (blue arrow, Mallory' stain x100).
elements, including the bone graft materials (Figure 7b).
At three weeks, H&E stain revealed that bony
fragments inside the cavities were dense and mature,
replacing areas of the fibrin meshwork. They were
similar to that at the original periphery (Figure 7c).
Mallory`s stain revealed the same features (Figure 7d).
After six weeks, H&E stain revealed that areas of
the bony cavities were filled with zones of new bone,
which enveloped the periphery. In addition to the
presence of coarse woven bone, formation of
trabeculae of lamellar bones with marrow spaces were
seen. Interestingly, the lamellar bones showed
maturation and pattern of the Haversian canal systems
(Figure 7e). Mallory`s stain revealed immature collagen
fibrils interspersed with fragments of newly formed
bone (Figure 7f).
Statistical analysis
Data were analyzed by computer with the Statistical
®
Package for Social Science (SPSS) ver 16.0. Analysis of
Table 1. The trabecular count and the total surface area of newly formed bone
among the three groups.
2
Total surface area (mm )
Trabecular count
Group
1 week
3 weeks
6 weeks
1 week
3 weeks
6 weeks
I
2.4
6.4
8.6
0.17
2.72
2.96
II
2.2
5.2
8
0.95
1.64
2.33
III
0.2
2
5.4
0.011
0.49
1.90
Table 2. The mean difference values for comparison of trabecular count
among the three groups (ANOVA). p < 0.05 was considered statistically significant.
Mean
difference p -value
1 week
Group
Mean
difference p -value
3 weeks
Mean
difference p -value
6 weeks
I
II
-2
0.009
-3.2
0.009
-2.6
0.008
I
III
-2.2
0.005
-4.4
0.001
-3.2
0.002
II
III
-30.2
1.0
1.2
0.56
-0.6
1
Table 3. The mean difference values for comparison of total trabecular surface area
among the three groups (ANOVA). p < 0.05 was considered statistically significant
Group
Mean
difference
1 week
p -value
Mean
difference p -value
3 weeks
Mean
difference
6 weeks
p -value
I
II
-0.94
0.002
-1.15
0.0001
- 0.43
0.92
I
III
-0.84
0.001
-2.23
0.0001
- 1.05
0.06
II
III
0.775
0.01
-1.08
0.001
- 0.63
0.43
variance (ANOVA) was used to compare the trabecular
count and total surface of newly formed bone among
the means of the three groups. Table 1 shows the
changes in the trabecular count and total surface area of
newly formed bone among the three groups at 1, 3 and
6 weeks. Tables 2 and 3 show the mean difference values
for comparison of trabecular count and total trabecular
surface area among the three groups.
Discussion
The present study was employed to histomorphometrically evaluate the osseous defects treated with
combined orthodontic/regenerative therapy at
different times of initiation of the orthodontic tooth
movement. The concept of combining orthodontic
tooth movement with regenerative therapy is based on
the assumption that regenerative procedures could be
enhanced by orthodontic tooth movement
(Nemcovsky et al., 1996; Diedrich, 1997; Stefania et al.,
2000; Vardimon et al., 2001; Ogihara and Marks, 2002;
Stefania et al., 2002; Ogihara and Marks, 2006; Maeda et
al., 2007). Previously, several authors have reported the
use of combined orthodontic regenerative therapy in
the treatment of osseous defects. The determination of
the effect of the best time to initiate the orthodontic
tooth movement has not been discussed. Therefore, to
our knowledge, it appears that this is the first report to
evaluate the effect of different times of initiating the
orthodontic tooth movement. Immediate application
of orthodontic tooth movement with regenerative
surgery in treatment of intraosseous defect was for the
first time selected. In this study, the selection of the
period of two months (60 days) for initiation of
orthodontic tooth movement in Group II was based
upon several studies that evaluated periodontal
regeneration from 60 to 90 days after regenerative
therapy and noted advanced healing of the periodontal
tissues (Caffesse et al., 1993; Araujo et al., 2001; Ogihara
and Marks, 2006).
This study apparently showed that orthodontic
tooth movement, when applied within a certain time
period and of known magnitude, could be used as an
adjunct factor for bone regeneration. It was also
noticed that the presence of an extrinsic mechanical
stimulus causes bone repair. The osteoclastic –
osteoblastic coupling mechanism required for bone
apposition/resorption response corresponds with the
results of this study. Osteoclastic recruitment is most
likely to occur not only as a sign for increased
resorption activity, but also it could act as a signal for
bone deposition at a site in close proximity to the
resorption activity (Vardimon et al., 2001). The rapid
disappearance of graft particles in Groups I and II may
be explained by the rapid recruitment of osteoclasts to
the bony cavity that accompanied the orthodontic
tooth movement.
In the present study, bioactive glass was used as the
grafting material because a number of in vivo and in vitro
studies have highlighted the potential for bioactive glass
as an effective synthetic regenerative scaffold (Sculean
et al., 2002; Sculean et al., 2005; Keles et al., 2006). In
addition, the collagen membrane was specially selected
in this study owing to the following properties: it is
chemotactic to fibrolasts, it provides a scaffold for
periodontal ligament cell migration, it is a weak
immunogen, and it can be easily manipulated and
adapted (Yaffe et al., 1984; Mattson et al., 1999; Michele,
2002).
Regulation of bone formation by mechanical
loading force seems to play a significant role in new
tissue formation. The integrin-mediated signal
transduction cascade is the main mechanism of
mechanotransduction in cells and is associated with
osteogenesis (Tang et al., 2003). Cell multiplication is
the first reaction in the beginning of tooth movement.
Indeed, fibroblast numbers were doubled in the three
days after the commencement of tooth movement
(Meikle, 2006). Orthodontic tooth movement can
stimulate preosteoblasts and mesenchymal cells to
differentiate into osteoblasts (Faber et al., 2005).
Moreover, growing numbers of cytokines are known to
be related to bone formation. Intense production of
TGF-β1 mRNA and the translated protein contributes
to angiogenesis and coincides with osteoblast
migration, differentiation and the formation of
extracellular matrix (Mehrara et al., 1999). Expression
of collagen type IV is increased in the basement
membrane of newly formed blood vessels and lamin is
diffusely distributed in the matrix undergoing
mineralization (Campisi et al., 2003). Concurrently,
expression of BMP2, 4 and 7 in the connective tissue is
also increased (Mehrara et al., 1999).
In this animal study, the amount and type of bone
formation was in agreement with the overall process
previously described. The histomorphometric findings
showed that cell differentiation, cell multiplication,
bone formation and blood capillary hyperplasia were
active with the orthodontic tooth movement.
Moreover, osteogenesis can be considered as a hallmark
of Groups I and II, in which the combined orthodontic
regenerative therapy was used. This was previously
proved to be mediated by various osteoinductive
molecules that act by stimulating osteoblast
osteoprogenitor cells in the periodontal ligament with
subsequent bone formation and inhibition of bone
resorption (Guajardo et al., 2000; Domon et al., 2001;
Gao et al., 2002; Mitsui et al., 2006). In this study, bone
regeneration by periodontal regenerative materials in
Group II (in which the active orthodontic treatment
started two weeks after regenerative surgery) showed
the remodeling process as a function of orthodontic
tooth movement in the presence of newly formed bone
with activation and recruitment of osteoclasts followed
by formation of new bone by osteoblasts at the
surgically created cavity.
Groups I and II, in which combined
orthodontic/regenerative therapy was used showed
higher trabecular count and greater total surface area of
newly formed bone than the group in which
periodontal regenerative treatment alone was used
during the study period. The trabecular count of newly
formed bone and the total surface area were 1.5 fold
greater in the bony cavities treated by combined
orthodontic/regenerative surgery than bony cavity
defects treated by regenerative surgery alone.
Moreover, Group I (in which orthodontic tooth
movement started immediately after finishing the
regenerative surgery) showed a higher trabecular count
and more total surface area of newly formed bone than
the other groups at 1, 3 and 6 weeks.
The histomorphometric results of our study were
in agreement with Vardimon et al. (2001); Araujo et al.
(2001); Silva et al. (2006) and Nemcovsky et al. (2007).
They reported that the orthodontic movement was not
pre-requisite to the results obtained with the
regenerative periodontal treatment. Moreover, bone
formation in the bony cavities was greater in quantity in
groups that received combined orthodontic/
regenerative therapy than groups that received
regenerative therapy alone.
In conclusion, the combined orthodontic
regenerative therapy resulted in favorable histological
outcomes. The surgical bony defects treated with
combined orthodontic regenerative therapy with
immediate application of orthodontic tooth movement
showed greater trabecular count and more total surface
area of newly formed bone than those treated with
regenerative surgery followed by delayed application of
orthodontic tooth movement two weeks later.
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Journal of the International Academy of Periodontology 2012 14/1: 17-25
Regenerative Therapy of Osseous Defects
Combined with Orthodontic Tooth Movement
1
1
2
Mai Shafik Attia , Eatemad A. Shoreibah , Samir A. Ibrahim and
Hamdy A. Nassar1
1
Department of Oral Medicine, Periodontology, Oral Diagnosis
2
and Radiology, and Department of Orthodontics, Faculty of
Dental Medicine – Girls' Branch, Al Azhar University, Cairo,
Egypt
Abstract
Background: Combined orthodontic/regenerative therapy can resolve complex clinical
problems and enhance bone formation. The purpose of this study was to evaluate the
effectiveness of different times of initiating the active orthodontic tooth movement on
the regenerative potential of the intrabony defects. Methods: Fifteen adult patients with
at least three intrabony defects and malocclusion were included. A total of 45 defects
were divided into three groups and treated in a split mouth design. The defects were
treated with combined orthodontic regenerative therapy with immediate application of
orthodontic tooth movement or delayed application of orthodontic tooth movement
(test groups) or with regenerative therapy alone (control group). The following hard and
soft tissue measurements were recorded prior to initial surgery and after six months and
one year: probing depth (DP), clinical attachment level (CAL), bone density (BD) and
bone fill (BF). Results: The most significant results were greater for the group treated
with combined orthodontic/regenerative therapy with immediate orthodontic tooth
movement. The measures for PD reduction and clinical attachment level gain were 4 ±
0.8 and 5.1 ± 1.4 mm respectively for immediate application of orthodontic tooth
movement, and 3.7 ± 0.9 and 4.3 ± 0.6 mm with delayed application of orthodontic
movement two months later. Moreover, immediate orthodontic tooth movement
showed the most significant increase in bone density and bone fill, which reached
74.2 ± 14.2 and 3.7 ± 1.1 mm respectively at the end of the study period. Conclusion:
This study evaluated the effect of orthodontic tooth movement on enhancement of
periodontal regenerative outcomes. The results demonstrated that a significant
improvement in clinical and radiographic parameters was observed. When comparing
the different groups, a statistically significant difference was found with immediate
application of orthodontic tooth movement.
Key words: Combined orthodontic regenerative therapy, bone grafts, alloplasts,
membranes, bioabsorbable, periodontal regeneration, intrabony defects, follow-up
studies
Introduction
Periodontitis is an inflammatory disease affecting the
composition and integrity of periodontal structures,
causing the destruction of connective tissue matrix and
cells, the loss of fibrous attachment and the resorption
of alveolar bone (American Academy of
Periodontology, 1986; Grenstein and Lamster, 1997;
Papapanou et al., 1997; Caffesse et al., 2002). The
objective of periodontal therapy continues to be
preservation of the dentition, maintaining health and
comfort during the patient's lifetime. In addition,
Corresponding author: Mai Shafik Attia
Department of Oral Medicine, Periodontology, Oral
Diagnosis and Radiology Faculty of Dental Medicine – Girls'
BranchAl Azhar University, Cairo, Egypt
Telephone:(02)24112135/(02) 24053623
E-mail: [email protected]
periodontal therapy includes not only the arrest of
progressive periodontal disease, but also the restitution
of those parts of the supporting apparatus that have
been destroyed by disease (Allen, 1988; Cortellini and
Bowers, 1995; Laurell et al., 1998).
Many events required for regeneration of
periodontal tissues are similar to those required for
formation of periodontal tissues. For example, in both
situations, while not necessarily in this order,
appropriate cells must be attracted to and attach at the
site. An appropriate matrix must be secreted by cells in
order to provide an environment conductive to cell
proliferation and differentiation, resulting in cells
having the capacity to function as periodontal ligament
fibroblasts, cementoblasts or osteoblasts (Macneil and
Cobb, 1999).
The boundaries of orthodontic tooth movement
Attia et al. :Effects of Orthodontics on Infrabony Defects in Humans 18
have broadened to include treatment of patients of all
ages. Adult patients now represent a significant
percentage of the population in almost every
orthodontic practice. The treatment of those patients
often raises some difficult issues (Basadra et al., 1995).
Many adult orthodontic patients have underlying
periodontal defects that need to be resolved (Kokich,
2002).
Studies have shown that teeth with reduced but
healthy periodontium can be orthodontically moved
with no enhancement of periodontal destruction. In
this case, orthodontic tooth movement acts as a
stimulating factor of bone apposition when forces are
maintained within physiological limits. However,
enhanced periodontal destruction and connective
tissue attachment loss were observed when teeth were
moved into inflamed defects (Eliasson et al., 1982; Reed
et al., 1985; Lindskog-Stokland et al., 1993; Wennstrom
et al., 1993; Diedrich and Wehrbein, 1997; Vardimon et
al., 2001; Nemocovsky et al., 2007).
studies have highlighted the potential for bioactive glass
as an effective synthetic regenerative scaffold (Sculean
et al., 2002; Sculean et al., 2005; Keles et al., 2006).
Moreover, bioabsorbable barrier membranes were
selected to avoid several drawbacks that have been
documented with non-absorbable barriers or with no
barriers, including the risk of bacterial contamination
on exposure of the membrane, and the need for a
second reentry procedure, which may disrupt healing
and create further bone and attachment loss (Eicholz et
al., 2000).
Measurements
All participants received an intraoral clinical status
assessment, study casts and a complete radiographic
evaluation (intraoral periapical, panoramic and
cephalometric using digital radiology). All baseline
clinical parameters were recorded the day of surgery.
Measurements were made with a William's probe and
recorded to the nearest millimeter at mid-facial, midlingual, mesial and distal line angles from the free
gingival margin (FGM) to the base of the pocket to
evaluate probing depth (PD) changes, and from the
cementoenamel junction (CEJ) to the base of the
defect (BD) to evaluate attachment level changes. Hard
tissue measurements were obtained as follows: bone
density (BD) was assessed using the DBS-Win
software, which is a part of the recently introduced
Vista scan system. The mean gray value in each region
of interest was calculated (256 gray levels of color
resolution) by assigning the gray value (0) to black, and
the value 256 to white (Yokota et al., 1994). To measure
bone density, three successive parallel lines were plotted
to cover the surface area of the defect. Then the gray
levels at certain points on the lines were recorded. The
mean values of those measurements represent the
defect (Figure 1). To measure bone fill, we plotted a line
from the crest of the alveolar ridge (as a reference
point) to the base of defect (Figure 2).
Fifteen adult patients 25 to 48 years of age (10 female
and 5 male) were included in this study. Each patient
presented with a form of chronic periodontitis with
malocclusion and evidence of at least three
radiographic intrabony defects with associated probing
depth of > 5.0 mm following initial non-surgical
therapy. All the patients were non-smokers,
systemically healthy and had no contraindications for
periodontal therapy (Abramson, 1996). Pregnant
females, as well as breast-feeding mothers and patients
with history of periodontal surgery or antimicrobial
therapy for the previous six months were excluded
from this study. All patients were given information
about the proposed treatment and were asked to sign a
surgical consent form approved by the local ethics
committee.
Initial periodontal therapy consisted of full mouth
scaling and root planing utilizing both hand and
ultrasonic instruments under local anesthesia. Four to
six weeks following the initial phase of treatment, a
reevaluation was performed to assess probing depth
and clinical attachment level.
Figure 1. Densiometric radiographic measurement.
Figure 2. Linear radiographic measurement.
Figure 3. Periodontal probe showing 3 mm
intrabony defect
Figure 4. Flap reflection and removal of
granulation tissue from the defect.
Figure 5. Bioglass was compressed into the
intrabony defects.
Figure 6. Biocollagen membrane was placed over
the defect and sutured with a sling suture.
Figure 7. The flap was sutured by using OOO silk
suture to cover both bone graft and membrane.
Surgical procedures
Local anaesthesia (2% lidocaine containing 1:100,000
epinephrine) was utilized followed by intrasulcular
incision. Full thickness flaps were elevated from both
the buccal and the lingual aspects. All granulation
tissues were removed from the defects. The 3- or 2-wall
defects included in the study were those that were
determined after flap reflection. The root surfaces were
thoroughly debrided with hand and ultrasonic
instruments (Figure 3 and 4). Root conditioning was
applied using tetracycline solution for 3 minutes
(Darhous et al., 1995). The wound was rinsed several
times with sterile saline solution. In all groups, the
defects were filled with a bioactive glass (Bio-Glass:
surface activated resorbable bioactive glass,
manufactured in Egypt and distributed by Excellence
Pharma Inc). The bone graft was mixed with sterile
saline solution to obtain a sandy consistency. The
resultant coagulum was transferred to the bone defects
with a sterile amalgam carrier and compressed by
manual pressure with a sterile condenser (Figure 5).
The collagen membrane (Biocollagen, Bioteck S.r.l
Fermi, Arcugraro VI, Italy) was trimmed and adjusted
to cover the defect and at least 2-3 mm of the
surrounding bone. The coronal portion of the barrier
was tightened and sutured on the root with resorbable
sling suture (Figure 6). The flap was placed at the original
presurgical levels and was sutured using vertical
mattress sutures (Figure 7).
The patients were treated using the segmented arch
technique to change the inclination of extruded,
malaligned and migrated teeth. The forces used were
Before
After
A
B
Before
After
C
D
Figure 8. Clinical case presentation. A: Patients with periodontitis and crowding of
upper and lower teeth with maxillary protrusion. B: After 12 months of treatment
realignment of anterior teeth with correctable overjet. C: Patient with periodontitis,
spacing between maxillary teeth and maxillary protrusion. D: After 12 months of
treatment with correctable overjet and overbite and treatment of spacing.
light and continuous, about 10 to 15 g per tooth,
depending on the amount of the periodontal support.
The anchorage consisted of labial arch and two
stainless-steel segments connecting posterior teeth.
Titanium arch wires number 7 were used in the
treatment protocol.
The orthodontic tooth movement was applied
towards all the treated defects. The tooth movement in
Group I was initiated immediately after finishing the
periodontal surgery. In Group II, tooth movement was
applied two months after periodontal treatment.
Orthodontic tooth movement was not applied to
Group III.
All patients included in the study were instructed to
rinse twice daily for two minutes for two weeks after
surgery with 0.12% chlorhexidine gluconate
(Antiseptal, Kahira Co. for Pharm. And Chem., IND
Cairo-ARE) and not to brush the treated area for the
first two weeks. Systemic antibiotic therapy was
prescribed: doxycycline hyclate, 100 mg every 12 hours
for 10 days (Doxymycin, Nile Co. for Pharm and Chem.
IND. Cairo-ARE) (Yukna et al., 2001, 2002; Ogihara
and Marks, 2002; and Cortellini and Tonetti, 2005).
Recall appointments were carried out every week for
the first month and then monthly for professional
prophylaxis and oral hygiene reinforcement.
Statistical analysis
Data were presented as means and standard deviation
(SD) values. Student's t-test was used to compare
between means of the two groups. Paired t-tests were
used to study the changes by time in each group. The
significance level was set as p ≤ 0.05. Standard analysis
®
was performed with SPSS 16.0 (statistical package for
scientific studies, SPSS Inc. Chicago, IL) for Windows.
Results
All 15 patients completed treatment and had no adverse
reactions to therapy. Healing was uneventful in the 45
sites involved in this study.
Clinical parameters - probing depth
As shown in Table 1 and Figure 10, the mean PD
reduction in the three groups was recorded. With
immediate application of orthodontic tooth movement
(Group I), there was a reduction in PD measurements
of 43.7% and 69.7% at 6 and 12 months, respectively.
The mean percent change in PD between 6 and 12
months was 46%. With delayed orthodontic tooth
movement (Group II), there was a decrease in PD by
40.8% and 62.4% at 6 and 12 months, respectively,
compared to the baseline measurements. The mean
C
A
B
Figure 9. Radiographic case presentation. (A) Preoperative periapical radiograph showing 5 mm
intrabony defect on the mesial surface of the upper left lateral incisior. (B) Six months postoperatively
showing the same case with 3 mm defect fill record. (C) Twelve months postoperatively showing the
same case with complete defect fill and adjustment of upper left lateral incisor.
Table 1. The means, standard deviations (SD), results of ANOVA and Duncan's tests for
comparison of percentage decrease in PD among the three groups
Group I
Group
Group II
Group III
p -value
Mean
SD
Mean
SD
Mean
SD
43.7
9.9
40.8
9.8
45.3
12.9
NS
Base line – 12
months
69.7
9.8
62.4
10.8
61.4
10.8
NS
6 months – 12
months
46
14.4
37.1
12.1
33.8
15.8
NS
Period
Base line
–
6
months
Table 2. The means, standard deviations (SD), results of ANOVA and Duncan's tests for
comparison of CAL among the three groups
Group
Group I
Group II
Group III
p-value
Period
Mean
SD
Mean
SD
Mean
SD
Baseline –
6
months
42.7
13.2
43.3
23.9
47.9
13.2
NS
Baseline – 12
months
67.8
13.5
64.7
18.7
73.7
11.2
NS
6 – 12 months
44.8
14.5
37.8
17.7
51.1
18.3
NS
Group I
Group II
Group I
Group III
Percent Changes in Clinical Attachment
Percent Changes in Probing Depth
90
80
70
60
50
40
30
20
10
0
Group III
Group II
80
70
60
50
40
30
20
10
Base line – 6 months
Figure 10. The mean percent changes in probing
depth in Groups I, II and III during the study period.
Group I
90
0
Base line – 6 months Base line – 12 months 6 months – 12 months
Base line – 12 months 6 months – 12 months
Figure 11. The mean percent changes in clinical
attachment level in Groups I, II and III during
the study period.
Group III
60
120
50
100
Percent Changes in Bone Fill
Percent Changes in Bone Density
Group II
100
40
30
20
10
Group I
Group II
Group III
80
60
40
20
0
0
6 months – 12 months
Figure 12. The mean percent change in bone density in
Groups I, II and III.
percent decrease in PD between 6 and 12 months was
37.1%. With no orthodontic tooth movement (Group
III), there was a reduction in PD by 45.3% and 61.4% at
6 and 12 months. Statistical analysis regarding mean
percent change in PD showed that there was no
statistically significant difference among the three
groups through all periods. However, the groups in
which orthodontic tooth movement were combined
with periodontal regenerative therapy (Groups I and II)
showed greater reduction in PD than the group treated
with periodontal regenerative therapy alone (Group
III). In addition, immediate application of orthodontic
tooth movement (Group I) showed the maximum
reduction in mean PD measurements.
Table 2 and Figure 11 show the mean percent change
in clinical attachment level from baseline with
immediate application tooth movement (Group I) was
42.7% and 67.8% at 6 and 12 months. With delayed
application of orthodontic tooth movement (Group
II), the mean percent change in CAL was 43.3% and
6 months – 12 months
Figure 13. The mean percent change in bone
fill in Groups I, II and III.
64.7% at 6 and 12 months, respectively. With no
orthodontic tooth movement (Group III), the baseline
Table 3. The means, standard deviations (SD), results of ANOVA and Duncan’s tests for
comparison of percentage increase in bone density among the three groups. )
Group
a
Group I
Group II
Group III
p-value
Period
Baseline – 6
months
Baseline – 12
months
Mean
SD
Mean
SD
Mean
SD
17.1
8.1
18.4
6.8
18
4.1
NS
41 a
14.8
33.6 b
10.5
33.5 b
7.6
HS
6 – 12 months
24.9 a
5.8
12.7 b
3.2
13.1 b
4
HS
Greatest percent change; blower percent change
Table 4. The means, standard deviations (SD), results of ANOVA and Duncan’s tests for
comparison of bone fill among the three groups
Group
Group I
Group II
Group III
p-value
Mean
SD
Mean
SD
Mean
SD
Baseline – 6
months
49
12.1
50.5
8
52.8
15.1
NS
Baseline – 12
months
82.7
12.4
77.6
11.7
82.4
12.5
NS
6 – 12 months
69.2
20.9
55.8
18.6
64.4
23.5
NS
Period
Discussion
The present study was employed to evaluate osseous
defects clinically and radiographically when treated
with combined orthodontic/regenerative therapy at
different times of initiation of orthodontic tooth
movement. The concept of combining orthodontic
tooth movement with regenerative periodontal therapy
is based on the assumption that regenerative
procedures could be enhanced by orthodontic tooth
movement (Nemcovsky et al., 1996; Diedrich, 1997;
Stefania et al., 2000; Vardimon et al., 2001; Ogihara and
Marks, 2002; Stefania et al., 2002; Ogihara and Marks,
2006; Maeda et al., 2007).
Previously, several authors have reported the use
of combined orthodontic regenerative therapy in the
treatment of osseous defects. Determination of the
best time to initiate the orthodontic tooth movement
has not been discussed. Therefore, to our knowledge, it
appears that this is the first report to evaluate the effect
of different times of initiation of orthodontic tooth
movement. Immediate application of orthodontic
tooth movement with regenerative surgery in the
treatment of intraosseous defects was for the first time
selected. In this study, selection of the period of two
months (60 days) for initiation of orthodontic tooth
movement in Group II was based upon several studies
that evaluated periodontal regeneration from 60 to 90
days after regenerative therapy and noted advanced
healing of the periodontal tissues (Caffesse et al., 1993;
Araujo et al., 2001; Ogihara and Marks, 2006).
This study shows that orthodontic tooth
movement, when applied with a certain duration and
magnitude, could be used as an adjunct factor for
periodontal regeneration. It was also noticed that the
presence of an extrinsic mechanical stimulus causes
bone repair. The osteoclastic – osteoblastic coupling
mechanism required for the bone apposition/
resorption response corresponds with the results of
this study. Osteoclastic recruitment is most likely to
occur not only as a sign for increased resorption activity,
but also it could act as a signal for bone deposition at a
site in close proximity to the resorption activity
(Vardimon et al., 2001).
studies have highlighted its potential as an effective
synthetic regenerative scaffold (Sculean et al., 2002;
Sculean et al., 2005; Keles et al., 2006). In addition, the
collagen membrane was especially selected in this study
owing to the following properties: it is chemotactic to
fibroblasts, it provides a scaffold for periodontal
ligament cell migration, it is a weak immunogen, and it
can be easily manipulated and adapted (Yaffe et al.,
1984; Mattson et al., 1999; Michele, 2002).
In this study, the groups in which orthodontic
regenerative therapy was applied showed greater
reduction in probing depth and more gain in clinical
attachment level. These results were in agreement with
Liou and Huang (1998), Cardaropoli et al. (2001),
Nemcovsky et al. (2004) and Nemcovsky et al. (2007).
However, others did not find this effect (Polson et al.,
1984; Wennstrom et al., 1993). This may be attributed to
failure of elimination of plaque-induced lesions prior
to initiation of orthodontic therapy. Moreover, other
factors responsible for this effect might be the lack of
using periodontal regenerative surgeries and the
improper oral hygiene measures during the course of
orthodontic treatment.
The Vista scan system using the DBS-win software
with a phosphor-image plate can produce images
following exposure. This requires lower radiation dose
levels and has the capability of computer image
analysis. DBS-win software provides a special software
program that enables accurate linear bone height
measurements and bone density measurements around
each tooth involved (Yalcinkaya et al., 2006).
Regarding the amount of bone fill, it was
significantly increased in all groups through the study
period. Upon comparing the three groups, there was a
greater increase in bone fill in Group I (in which
immediate orthodontic tooth movement was applied)
during the study period than in the other groups.
There was a significant increase in bone density in
the three groups at the end of the study period. The
groups in which the combined orthodontic
regenerative therapy was used demonstrated more
increase in bone density than the group in which
periodontal regenerative surgery was used alone. In
addition, Group I showed the highest increase in bone
density at the end of the study period.
In conclusion, the combined orthodontic
regenerative therapy resulted in favorable clinical and
radiographic outcomes. It suggests that combined
orthodontic/regenerative therapy might be a good
option for achieving periodontal regeneration. Defects
in which immediate orthodontic tooth movement was
applied (Group I) demonstrated superior results than
defects in which delayed orthodontic tooth movement
was applied (Group II). Both groups showed more
favorable results than Group III (defects treated with
regenerative surgery alone).
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Vol 14, Issue 1 - International Academy of Periodontology

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