Secondary repair of alveolar clefts using human mesenchymal
Hossein Behnia, DMD,aArash Khojasteh, DMD,bMasoud Soleimani, PhD,cAzita Tehranchi,d
Ahad Khoshzaban, DDS,eSaeed Hidari Keshel, PhD,fand Reza Atashi, PhD,gTehran, Iran
SHAHID BEHESHTI UNIVERSITY OF MEDICAL SCIENCES, TARBIAT MODARES UNIVERSITY, TEHRAN
UNIVERSITY OF MEDICAL SCIENCES, AND STEM CELL TECHNOLOGY CENTER
Recently tissue engineering has become available as a regenerative treatment for bone defects; however, little
has been reported on the application of tissue engineering for regeneration of cleft defect tissues. Mesenchymal-
derived stem cells were applied to different kinds of bone substitute and compared in different animal models, but
their usage in human critical defects remained unclear. In this study we report 2 patients with unilateral alveolar cleft,
treated with the composite scaffold of demineralized bone mineral and calcium sulphate (Osteoset) loaded with
mesenchymal stem cells (MSCs). Computed tomograms showed 34.5% regenerated bone, extending from the cleft
walls and bridging the cleft after 4 months in one case and in the other there was 25.6% presentation of bone
integrity. The available data revealed the conventional bone substitute was not a suitable scaffold for the MSC-induced
bone regeneration. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:e1-e6)
Repair of bony defects continues to remain a challeng-
ing part of many reconstructive procedures. Currently,
the gold standard for grafting of bone defects is the use
of autogenous bone. To avoid morbidity at the donor
site or if large amounts of autogenous bone are neces-
sary, bone substitution materials can be used.1The
reconstruction of alveolar cleft defects is well estab-
lished, with the most widely accepted approach being
secondary alveolar cleft osteoplasty in mixed dentition
phase.2In conventional methods, autogenous bone
grafting has become an essential step in treating pa-
tients with alveolar cleft, and allows the placement of
dental implants for missing teeth in the final stages of
treatment.3It also assists in preventing maxillary seg-
mental collapse, particularly in patients with bilateral
cleft. Secondary grafting consistently produces trabec-
ular bone to unify the maxilla and provides odontogenic
support. Its high success rate makes it the preferred
approach at most centers.4With the advent of tissue-
engineering techniques, alternatives to the traditional
iliac crest bone grafting techniques are available. Re-
duced morbidity and improved healing with bone mor-
phogenic protein-2 in older patients with alveolar cleft
defects was reported.5Mesenchymal stem cells (MSCs),
which can be isolated from the marrow cavity as well as
from the trabecular compartment, have been shown to
have the ability to form new bone when transplanted.3
Bone marrow aspirated with resorbable collagen matrix
reported to have reduced morbidity in repair of alveolar
cleft defects.6Bone substitution materials can be com-
bined with vital cells such as MSCs to increase bone
formation.7-10Both synthetic and allograft materials
allow adhesion and growth of osteoblastic cells, or
osteogenic differentiation of precursor cells in vitro.11-13
In this study the authors performed alveolar bone recon-
struction with Osteoset (Wright, Arlington, OH, USA)
seeded with the human-derived MSCs.
REPORT OF A CASE
A 14-year-old girl was referred to the Department of Oral
and Maxillofacial Surgery. She had unilateral cleft lip and
This study was supported in part by a grant-in-aid of Iranian Dental
Research Center, Beheshti University of Medical Sciences (Vice
aProfessor and Chairman, Department of Oral and Maxillofacial
Surgery, Taleghani Hospital, Dental Research Center, Shahid Be-
heshti University of Medical Sciences, Tehran, Iran.
bAssistant Professor of Oral and Maxillofacial Surgery, Taleghani
Hospital, Dental Research Center, Shahid Beheshti University of
Medical Sciences, Tehran, Iran.
cHematology Department, School of Medical Sciences, Tarbiat Mo-
dares University, Tehran, Iran.
dAssociate Professor, Department of Orthodontics, Dental Research
Center, Shahid Beheshti University of Medical Sciences, Tehran,
eLaboratory Head Manager, Iranian Tissue Bank Research and Prep-
aration Center, Imam Khomeini Hospital, Tehran University of Med-
ical Sciences, Tehran, Iran.
fDirector of Cellular & Molecular Biology of Stem Cell Preparation
Unit, Farabi Hospital, Imam Khomeini Medical Complex, Tehran
University Medical Sciences, Tehran, Iran.
gLaboratory Manager, Stem Cell Department, Stem Cell Technology
Center, Tehran, Iran.
Received for publication Dec 8, 2008; returned for revision Mar 21,
2009; accepted for publication Mar 24, 2009.
1079-2104/$ - see front matter
© 2009 Published by Mosby, Inc.
palate and underwent surgical correction of the cleft lip and
palate. There was no fistula or oronasal communication in the
palate. The lip adhesion and revision surgical correction of
the lip with Millard technique provided satisfactory facial
esthetic results. Discontinuity was present only in the alveolar
bone and a small fistula could be seen in the depth of the
labial vestibule. The radiographic feature of the patient de-
picted eruption of the canine tooth adjacent to the cleft (Fig.
1). Presurgical orthodontic treatment had begun with excep-
tion of the alignment of tooth No. 8 that needed alveolar bone
grafting. Clinically, the size of the bone defect was minimal
and confined to the anterior portion of the maxilla. The risks
and benefits of this procedure were explained to the patient.
The girl and her family agreed to the procedure despite of the
absence of well-documented data to support mesenchymal-
induced bone regeneration in cleft condition. All procedures
were approved by the institutional ethical committee and
informed consent was obtained from all donors. Isolation and
cultivation of the MSCs was performed without xenogenic
supplements such as fetal calf serum (FCS).
A 10-year-old boy with unilateral alveolar cleft in the right
side of the maxilla was selected for this study. All possibil-
ities of this study were explained for the patient’s parent. The
alveolar defect was larger in the second patient and also the
surgical correction of the palate was not as good as the prev-
ious patient. Presurgical orthodontic treatment included max-
illary expansion without alignment of the teeth adjacent to the
cleft (Fig. 2).
Isolation and cultivation of mesenchymal stem
Isolation and differentiation of the cells was performed
based on our previous project regarding the use of stem cells
in human sinus augmentation.14Two weeks before surgery,
bone marrow aspirate (10-15 mL) was obtained from the
posterior iliac crest. The aspirate was diluted at 1:3 in Dul-
becco’s modified Eagle’s medium (DMEM)/F12 (Gibco,
Paisley, UK). On day 1, nonadherent cells were discarded and
adherent cells were washed with phosphate-buffered saline
(PBS) (Gibco) and then cultured in DMEM/F12 medium with
antibiotics and 20% autologous serum.
Preparation of human serum
FCS was replaced by human serum because of the ethical
committee concerns. From each donor bone marrow, 20 mL
of whole blood was drained into blood bags (Baxter, Deer-
field, IL), quickly transferred to 10-mL vacutainer tubes with-
out anticoagulants (BD, Plymouth, UK), and allowed to clot
for 4 hours at 4°C to 8°C. Subsequently, the blood was
centrifuged at 1800g at 4°C for 15 minutes. Serum was
collected and filtered through a 0.2 mm membrane (Sarstedt,
Aliquots of the sterile serum were stored at 20°C. The lab
process and cultivation of the cells for each patient was
performed without significant salience. Then, 15 to 20 mL of
human serum was drained; this amount was less than adult
samples. Mesodermal lineage differentiation was also dem-
onstrated with the osteogenic medium15and staining of the
Fig. 1. A, Radiographic evaluation of the patient. The canine is erupted and there is no bone in the mesial side of the root. B and
C, Presurgical orthodontic arch coordination of the teeth.
Behnia et al.
medium by Alizarin Red (Sigma Aldrich, St. Louis, MO,
USA), pH 4.2, for 5 minutes (Fig. 3).
Osteoset DBM (Wright) was used in this study. This com-
bination product is a resorbable bone graft substitute that acts
as a scaffold for new bone formation. Demineralized bone
matrix has been reported to provide osteoinductive bone
morphogenetic proteins (BMPs) that signal precursor cells
and stimulate the formation of bone at a defect site. However,
combining with the calcium sulfate causes structural strength,
increasing the resorption time. In both cases, 1 day before
transplantation, implants were loaded by the cells obtained
from the third subculture of the patient bone marrow–derived
stem cells. The cylinders were first washed with PBS and then
loaded with MSCs by placing 5 ? 105cells in 0.2 mL DMEM
medium on top of it.
Scanning electron microscopy
The morphology of the DBM scaffolds, with and without
cells, was observed by means of scanning electron micros-
copy (SEM; Vega, Tescan, Philadelphia, PA, USA). Before
the observation, samples of the cell-scaffold constructs were
fixed in 2.5% glutaraldehyde, dehydrated through a graded
series of ethanol, and vacuum-dried. All samples were coated
with gold using a sputter coater (Fig. 4).
Following crestal incision at the level of the gingival
sulcus, dissections were made in the scar tissue to reach the
bony surface of the cleft walls. The tissue was then elevated
in the subperiosteal plane to the levels of the anterior nasal
spine anteriorly, the lateral piriform rim superiorly, and to the
alveolar ridges inferiorly. The flaps of the nasal floor and the
oral mucosa formed the ceiling and the floor of the cleft
cavity, respectively. The scaffold with cells was transferred to
the defect by microforceps. The wound was subsequently
closed in a water-tight manner (Fig. 5).
Fig. 3. Alizarin red staining; arrow shows module of in vitro
Fig. 2. A, The panoramic view demonstrated the larger alveolar defect in left side of the second patient. B and C, Preop
orthodontic treatment limited to the expansion of the maxillary arch without alignment of the teeth lateral to the defect.
Volume 108, Number 2Behnia et al. e3
Evaluation of bone healing
For alveolar ridge healing, examiners used intraoral in-
spection and palpation. A panoramic scan was taken after 2
months and computed tomographic evaluation was obtained
after 4 months. One-millimeter coronal sections of alveolar
defect regions were obtained 2 months after surgery. These
defects were outlined using the Image J program (National
Institutes of Health [NIH], Bethesda, MD).
SEM revealed cell adherence to Osteoset in all sam-
ples taken after the third subculture of MSCs. The
successful healing left no fistula or oronasal communi-
cation. Panoramic view depicted the integrity of nasal
floor in both cases after 2 months. The outline of 1-mm
coronal sections of the alveolar defect was used to
determine the preoperative defect, postoperative defect,
and volume of bone fill obtained by Image Pro software
(NIH). The mean postoperative defect of patient 1 was
34.5%, and in patient 2 measured 25.6 % (Fig. 6). The
patients were referred back to the department of orth-
odontics to start the orthodontic tooth movement.
Although autogenous bone grafting remains the gold
standard in the reconstruction of the bone defects, dis-
advantages may include limited amount of bone and
donor-site morbidity. Tissue engineering approaches
can potentially obviate these problems. Bone tissue
engineering requires at least living osteoprogenitor
cells or osteoblast-like cells in combination with suit-
able scaffolds. The use of MSCs for bone regeneration
is currently becoming a popular practice.11,13Their mul-
in terms of cell harvesting, and their capacity to un-
dergo extensive replication without loosing their mul-
tipotential capacity make them an attractive cell source
for cell-based therapeutic approaches.9,12Several ex-
periments have demonstrated that MSCs can be in-
duced to transform into osteoblasts13; however, direct-
ing these cells into osteogenic differentiation is still a
major obstacle.16Most groups use 10% to 20% FCS in
their expansion medium.17-19Kuznetsov et al.17dem-
onstrated that MSCs cultured continuously with 20%
FCS formed bone more extensively than MSCs cul-
tured in human serum when combined with HA/TCP
(hydroxy apatite/three calcium phosphate) particles and
fibrinogen/thrombin. FCS has been implicated as a po-
tential vector for porion transmission.19In this pilot
human study the risk of porion transmission, although
minimal, was strong enough to prompt us to culture
Fig. 4. Scanning electron microscope views of the Osteoset. A (1/500), B (1/200): The porosity of the scaffold seemed to be
enough for the housing of the cells. C (1/100), D (1/50) revealed the MSCs laying down into the porous Osteoset.
Behnia et al.
MSCs with human growth factor. The integrity of the
maxillary alveoli was established in this manner but
the quantitative measurement of tomographic scans
showed less than half (?50%) bone fill in both pa-
tients. One possibility is that the elimination of FCS in
our setting and its replacement by human serum de-
creased the ability of the MSCs to form bone ectopi-
cally. Another possibility is the difficulties in transfer-
ring the MSCs. Scanning with electron microscope
confirmed the lay down of the cells, but after putting
the Osteoset in the alveolar cleft defect, there is no
definite way to demonstrate the living cells in human
Fig. 5. A, C: Exposing of the entire cleft and suturing of the nasal floor. B, D: Both defects were overfilled with Osteoset-loaded
MSCs. Closure of the defect without releasing incision.
Fig. 6. A, B: Radiographic investigation 4 months after the surgery demonstrated reconstruction of the nasal floor and filling of
the alveolar defect.
Volume 108, Number 2Behnia et al. e5
subjects. In these particular cleft cases, creation of the Download full-text
bony continuity may be enough for the successful orth-
odontic tooth movement, but this limited amount of the
bone formation may prohibit the use of human-derived
MSCs for the alveolar cleft tissue-engineered bone recon-
struction. Reduced morbidity of alveolar cleft healing
when treated with bone marrow aspirate in resorbable
matrix was reported,6but no quantitative measurement
of the newly formed bone has been done by the authors.
The major criticism in this study was the lack of the
histological or histomorphometric analysis of the bone
formation and definite amount, trabeculation pattern,
and direction of the bone formation. The amount of the
bone formation was not great enough in computed
tomography evaluation, but the lack of the donor site
morbidity, decrease in the time of the hospitalization,
and good soft tissue healing may be good criteria for
future comprehensive research in MSC-based bone en-
Special thanks to Dr. Kalantar Motamed for his pro-
fessional editing of the article.
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Arash Khojasteh, DMD, MS
Department of Oral and Maxillofacial Surgery
Shahid Beheshti University if Medical Sciences
Tabnak Avenue Vlenjak
Behnia et al.