Clin Oral Invest

DOI 10.1007/s00784-017-2050-1

ORIGINAL ARTICLE

Application of tissue-engineered bone grafts for alveolar cleft

osteoplasty in a rodent model
Paula Korn 1 & Maria Hauptstock 1 & Ursula Range 2 & Christiane Kunert-Keil 3 &
Winnie Pradel 1 & Günter Lauer 1 & Matthias C. Schulz 1

Received: 14 July 2016 / Accepted: 4 January 2017

# Springer-Verlag Berlin Heidelberg 2017

Abstract 46.2% which was a statistically significant difference

Objectives The clinical standard for alveolar cleft osteoplasty
is augmentation with autologous bone being available in lim-
ited amounts and might be associated with donor site morbid-
ity. The aim of the present study was the creation of tissue-
engineered bone grafts and their in vivo evaluation regarding
their potential to promote osteogenesis in an alveolar cleft
model.
Materials and methods Artificial bone defects with a diameter
of 3.3 mm were created surgically in the palate of 84 adult
Lewis rats. Four experimental groups (n = 21) were examined:
bovine hydroxyl apatite/collagen (bHA) without cells, bHA
with undifferentiated mesenchymal stromal cells (MSC), bHA
with osteogenically differentiated MSC. In a control group,
the defect remained empty. After 6, 9 and 12 weeks, the re-
maining defect volume was assessed by cone beam computed
tomography. Histologically, the remaining defect width and
percentage of bone formation was quantified.
Results After 12 weeks, the remaining defect width was
60.1% for bHA, 74.7% for bHA with undifferentiated MSC
and 81.8% for bHA with osteogenically differentiated MSC.
For the control group, the remaining defect width measured
* Paula Korn
paula.korn@uniklinikum-dresden.de
1
Department of Oral and Maxillofacial Surgery, Faculty of Medicine
BCarl Gustav Carus^, Technische Universität Dresden, Fetscherstr.
74, 01307 Dresden, Germany
2
Institute for Medical Informatics and Biometry, Faculty of Medicine
BCarl Gustav Carus^, Technische Universität Dresden, Fetscherstr.
74, 01307 Dresden, Germany
3
Department of Orthodontics, Faculty of Medicine BCarl Gustav
Carus^, Technische Universität Dresden, Fetscherstr. 74,
01307 Dresden, Germany
(p < 0.001).
Conclusions The study design was suitable to evaluate tissue-
engineered bone grafts prior to a clinical application. In this
experimental set-up with the described maxillary defect, no
promoting influence on bone formation of bone grafts con-
taining bHA could be confirmed.
Clinical relevance The creation of a sufficient tissue-
engineered bone graft for alveolar cleft osteoplasty could pre-
serve patients from donor site morbidity.
Keywords Alveolar cleft . Bovine bone substitute .
Histomorphometry . Mesenchymal stromal cells .
Tissue-engineered bone grafts
Introduction
One of the most common hereditary craniofacial anomalies in
humans is cleft lip and alveolar cleft with or without cleft
palate. In literature, the prevalence is stated with approximate-
ly 1 in 700 live births with variability depending on geograph-
ic origin, ethnic groups and environmental as well as socio-
economic factors [1]. An essential part of the surgical treat-
ment is the secondary alveolar cleft osteoplasty before the
eruption of the permanent canine teeth at a patient’s age of
nine to eleven years. The augmentation of the persisting alve-
olar bone defect closes the oronasal fistula, provides osseous
support for the dentition and stabilizes the dental arch as well
as the local soft tissue [2]. If the alveolar cleft osteoplasty is
not performed and the alveolar bone defect remains untreated,
a delayed or misguided tooth eruption might occur. This can
lead to functional and aesthetic limitations for the patient.
Currently, autologous bone grafts, e.g. from the iliac crest,
are the clinical standard for alveolar cleft osteoplasty [3].

The transplantation of autologous bone leads to another sur-
gically created wound in the donor site area which might be
associated with intra- and postoperative complications.
Furthermore, the availability of autologous bone is still limited
[4–6]. This led to an intensive search for alternative materials
to replace or supplement the autologous bone graft for alveo-
lar cleft osteoplasty.
In oral surgery, bone augmentation in non-critical size de-
fects is clinically established. Especially after cyst enuclation,
tooth extraction and before insertion of dental implants, bone
augmentation is applied routinely [7, 8]. There are different
groups of bone substitute materials which are all clinically
approved. According to their origin, they are classified into
autologous, allogenic, xenogenic and alloplastic materials.
However, the exclusive application of bone substitution ma-
terials in critical size defects is clinically not recommendable
[9]. For larger defects, the challenge is that the use of pure
substitute materials is not sufficient to induce a stable defect
bridging by formation of new bone. One reason is the poor
cellular ingrowth into substitute materials replacing extensive
bony defects. Furthermore, possible interferences of dental
eruption and/or jaw development limit the application of pure
materials.
The method of tissue engineering could be an option for the
preparation of bone grafts with the ability to promote the os-
seous healing of large defects [10]. Currently, in bone tissue
engineering, the main cell source are bone marrow-derived
mesenchymal stem cells [11, 12]. They have a high osteogenic
potential and were characterized in numerous studies.
Nevertheless, an in vitro cell expansions is always necessary
prior to in vivo application [12]. Besides the chosen cells, the
scaffold material is important for the creation of a tissue-
engineered bone graft. The scaffold has to enable cell adhe-
sion and proliferation before and after transplantation.
Furthermore, it should be biocompatible, osteoconductive,
mechanically stable and resorbable in an adequate time [13].
Gladysz et al. reviewed the current literature regarding stem
cell-based regenerative therapy for alveolar cleft reconstruc-
tion. Besides the required characteristics, there is no consen-
sus which scaffold materials is the best suitable for this appli-
cation. Ceramics, like calcium phosphate and HA, synthetic
and natural polymers are discussed and each group has spe-
cific advantages to improve the scaffold-cell interaction,
which seems to be a precondition for better clinical results
[14]. Against the background that sintered bovine hydroxyl
apatite (bHA) is a clinically widely used bone substitution
material, it is of clinical interest whether the material is also
suitable for the creation of tissue-engineered bone grafts to
promote the ossification in maxillary defects. In vitro studies
analysing bHA with osteoblast-like cells indicated a high
in vitro biocompatibility and a sufficient expression of char-
acteristic proteins and transcription factors [15, 16]. The
in vivo biocompatibility of bHA is well documented for
Clin Oral Invest
non-critical size defects, e.g. sinus floor augmentation [17].
There are only few studies analysing pure bHA in critical size
defects wherefore the suitability could not yet be confirmed
clearly [18]. Currently, to the best of our knowledge, no ex-
perimental study has evaluated tissue-engineered bone grafts
containing bHA in large defects like an artificial alveolar cleft.
The hypotheses of the present experimental study are as
follows: (1) It is possible to create bone grafts of bHA scaf-
folds and mesenchymal stromal cells in vitro. (2) The surgi-
cally created bone defect of 3.3 mm diameter in the maxilla of
adult Lewis rats will not reunite within the experimental peri-
od of 12 weeks. (3) The in vivo application of the different
tissue-engineered bone grafts containing undifferentiated or
osteogenically differentiated MSC leads to a significant im-
provement of defect ossification within the healing time of a
maximum of 12 weeks. The defect ossification was assessed
by radiological and histological analysis ex vivo.
Methods and materials
Animal model
The study design was approved by the Commission for
Animal Studies at the District Government Dresden,
Germany (AZ: 24(D)-9168.11-1/2013-7). Adult male Lewis
rats (Janvier Labs, Le Genest-Saint-Isle, France) with an av-
erage body weight of 460 g and an age of six to 8 months at
the beginning were chosen for this experimental study. The
animals (n = 84) were housed in a light- and temperature-
controlled environment. They had access to food and water
ad libitum.
Scaffolds
For this tissue engineering application bovine, deproteinized
hydroxyl apatite combined with 10% collagen (bHA,
BioOss® Collagen, Geistlich Biomaterials, Wolhusen,
Switzerland) was chosen. The commercially available
100 mg biomaterial blocks (5 mm × 5 mm × 10 mm; size of
the single hydroxyl apatite particle 0.25–1 mm) were cut into
slices with a diameter of 3.3 mm and height of about 1 mm
under sterile conditions. Due to the collagen content, the scaf-
fold material could be deformed in a moist environment and
adapted to the bony defect easily.
Cell culture and in vitro analysis
In general anaesthesia, initiated by intraperitoneal injection of
100 mg/kg body weight ketamine (Riemser Arzneimittel AG,
Greifswald, Germany) and 10 mg/kg xylazine (Pharma-
Partner Vertriebs-GmbH, Hamburg, Germany), the femur of
donor rats (adult, male Lewis rats) was removed and
Clin Oral Invest
subsequently, the animals were killed in a carbon dioxide bath.
For isolation of the mesenchymal stromal cells (MSC), the
femur was separated in the area of the epiphysis and the bone
marrow was aspirated under sterile conditions. Centrifugation
of the aspirate at 1200 rounds per minute (rpm) for 10 min as
well as aspiration of the supernatant and resuspension with
minimum essential medium (MEMα (Gibco®, Thermo
Fisher Scientific Inc., Waltham, USA)) followed.
Afterwards, the cells were transferred into culture flasks
(T-75 cell culture flask, Greiner Bio-One, Frickenhausen,
Germany) and placed into an incubator (Hera Cell, Heraeus
Kulzer GmbH, Hanau, Germany) at 37 °C and 5% CO2. The
haematopoietic stromal cells which were marked by a CD34
antibody (antibodies-online, Aachen, Germany) could be re-
moved from the eluate by negative selection using the
MiniMACS™ Separators (Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany). The remaining MSC were cultivated
in vitro with changing of the culture medium every 3 days
for a period of 7 to 14 days until a confluence of 95% was
achieved. Cells of the second passage were used for further
tissue engineering application.
For colonization, the portioned biomaterial was placed into
a 24-well plate and incubated with 2 ml culture medium for
24 h at 37 °C. In preparation of the cell colonization, the
scaffolds were washed with phosphate-buffered saline without
Ca 2+ and Mg 2+ (PBS, Life Technologies Cooperation,
Darmstadt, Germany). MSC of the second passage were
washed with 2 × 6 ml PBS too and then detached using 2 ml
of trypsin/EDTA. The mobilized cells were taken up in spe-
cific medium (undifferentiated MSC: MEMα; osteogenically
differentiated MSC: Opti-MEM® (Thermo Fisher Scientific
Inc.)) and passed over into a 1.5-ml falcon tube. After centri-
fugation at 8000 rpm for 5 min at room temperature, the re-
moval of the supernatant and resuspension with 1 ml medium
was performed. The measurement of the cell number was
carried out using a Casy® Cell Counter and an associated
analysis system (Roche Diagnostics GmbH, Mannheim,
Germany). Then, the substitute material was colonized with
cell-medium-suspension containing about 200,000 cells each.
Additional cell culture medium was added after 2 h so that the
initial adherence of the cells to the bone substitute material
was ensured. Depending on the experimental group, different
mediums were used on the colonized scaffolds for the final
cultivation for 3 days before in vivo insertion. In one group,
undifferentiated MSC were planned. Thus, the colonized scaf-
folds were cultivated applying MEMα. Another experimental
group should contain osteogenically differentiated MSC. The
differentiation of the cells into osteoblasts was performed
using the medium Opti-MEM® supplemented with 10% foe-
tal calf serum (Gibco®, Thermo Fisher Scientific Inc.), 50 μM
ascorbic acid (Sigma-Aldrich, St. Louis, USA), 10 mM β-
glycerophosphate (Sigma-Aldrich) and 10 μM dexametha-
sone (Sigma-Aldrich) [19]. In both groups, the tissue-
engineered bone grafts were stored in an incubator at 37 °C
with 5% CO2 and 95% humidity for 3 days.
The successful differentiation into osteoblasts was con-
firmed using an Alkaline Phosphatase Kit (Sigma-Aldrich,
Taufkirchen, Germany) according to the manufacturer’s in-
structions. Qualitative results were obtained by light micros-
copy. For further characterization of both cell lines, MSC as
well as osteogenically differentiated MSC, the gene expres-
sion for bone morphogenetic protein 4 (BMP-4) and the stem
cell factor runt-related transcription factor 2 (Runx2) were
quantified after 3, 7 and 12 days cultivation by quantitative
polymerase chain reaction (PCR). The isolation of the total
RNA from cells was done using the standard protocol of the
RNeasy Mini Kit (Qiagen, Hilden, Germany). After determi-
nation of the RNA concentration, an amount of 200 ng total
RNA was transcribed reversely using innuScript Reverse
Transcriptase, inNucleotide Mix and Random primer
(Analytik Jena AG, Jena, Germany). To quantify the expres-
sion of different genes, gene-specific TaqMan PCR primers
and probes (Bmp4: Rn00432087_m1; Runx2:
Rn01512296_m1; Rat ACTB (actin, beta) endogenous con-
trol: 4352931E) were obtained from PE Applied Biosystems
(Weiterstadt, Germany) and quantitative real-time PCRs were
performed using a TOptical cycler (Analytik Jena AG) as well
as the innuMix qPCR MasterMix Probe (Analytik Jena AG).
Absolute copy numbers of the studied genes and 18S cDNA
were determined using calibration curves generated with
cloned PCR fragment standards. Copy numbers of individual
transcripts are given in relation to those of 18S cDNA. Each
series of experiments was performed twice and Bno-template
controls^ with water were carried out parallely in all experi-
ments as described previously [20].
The analysis of the tissue-engineered bone grafts was com-
pleted by a scanning electron microscopy examination (XL-
30 ESEM; Environmental Scanning Electron Microscope,
Koningklijke Philips N.V., Eindhoven, Netherlands). The cel-
lular colonization of the scaffolds was visualized with a mag-
nification up to 500 under humid conditions.
In vivo application of the bone grafts
The rats were anaesthetized by intraperitoneal injection of
ketamine (100 mg/kg body weight) and xylazine (10 mg/kg
body weight) and fixed in a dorsal position. A simulated al-
veolar cleft was created surgically in the anterior maxilla of
each animal. First, a sagittal incision was made following the
mid-palatal suture. After elevation of a mucosal flap and re-
moval of the periosteum, a localized bone defect with 3.3 mm
in diameter was created using a diamond-coated cylindrical
shaped drill (DiT Dental-Instrumente GmbH, Oberlungwitz,
Germany). According to the randomized distribution, each rat
received one bone graft (Fig. 1):

Fig. 1 Overview of the study design. Each seven animals were sacrificed after 6, 9 and 12 weeks according to the schedule. Alizarin was applied 7 days
and calcein 3 days prior to sacrifice
Group I: Control, no bone graft (n = 21)
Group II: bHA without cells (n = 21)
Group III: bHA with undifferentiated MSC (n = 21)
Group IV: bHA with osteogenically differentiated MSC
(n = 21)
After insertion of the bone graft, the flap was repositioned
and wound closure was performed using 5-0 Ethilon suture
(Ethicon, Norderstedt, Germany). Postoperatively, the animals
received amoxicillin trihydrate (Fort Dodge Veterinär GmbH,
Würselen, Germany) 15 mg/kg body weight once and 4 mg/kg
body weight carprofen (Sigma-Aldrich) every 24 h for 4 days.
All drugs were injected subcutaneously. The animals were
fed a soft diet for the first 3 days and, subsequently, received a
regular diet. Postoperatively, the animals and their behaviour
were monitored and the body weight was measured every
2 weeks.
For the ex vivo assessment of the dynamic bone formation,
all rats received intraperitoneal injections of the fluorochrome
dyes alizarine (20 mg/kg body weight) and calcein (30 mg/kg
body weight) 7 and 3 days prior to sacrifice.
Sample preparation
The animals (seven per group) were sacrificed after 6, 9 and
12 weeks of healing time. After cone beam computed tomog-
raphy, the cranium was dissected and fixed in 4% formalde-
hyde for 48 h. As described previously after dehydration in a
graded series of ethanol, all samples were embedded in
methylmethacrylate (Technovit® 9100, Heraeus Kulzer,
Wehrheim, Germany) [21]. Coronal sections were produced
according to Donath’s sawing and grinding technique [22].
Thus, the 4 central sections of each specimen could be
achieved for evaluation. Subsequently, the sections measuring
60 μm in thickness were polished. After analysis of the fluo-
rochrome marker uptake, Masson-Goldner trichrome staining
followed.
Histological analysis
All samples were imaged by fluorescence microscopy and,
after staining, by light microscopy (Olympus BX 61,
Olympus Deutschland GmbH, Hamburg, Germany) and
cell ^ F Imaging Software for Life Science (Olympus).
Clin Oral Invest
Multiple image alignment was performed using an automatic
scanning table (Märzhäuser, Wetzlar, Germany). Thus, eight
images per sample were scanned with a 10 × 10-fold magni-
fication and manually fused to one image.
Fluorochrome marker uptake was analysed to assess the
dynamics of bone formation at the defect margins. The histo-
logical analysis focused on the structure of bone, the soft
tissue and the resorption of bone grafts in the defect area.
For histomorphometrical evaluation, the remaining width of
the defect was measured between the cranial and caudal defect
margins being located in the cortical bone. The remaining
defect width was related to the individual initial defect width
which could be clearly identified by differences in bone mor-
phology from the newly formed bone. Additionally, the con-
tent of newly formed bone in the whole defect area was mea-
sured and is displayed as a percentage value.
Formulas for histomorphometrical measurement:
Remaining defect width [%] = ((cranial distance of newly formed defect mar-
gins [mm] + caudal distance of newly formed defect margins [mm]) ÷ 2) ÷
(cranial distance of initial defect margins [mm] + caudal distance of initial defect
margins [mm]) ÷ 2)* 100
Bone formation [%] = (area of newly formed bone [μm2] ÷ area of the defect
[μm2]) * 100
All measurements were realized by one examiner who was
masked regarding to the experimental group.
Formulas for histomorphometrical measurement are as
follows:
Cone beam CT
Immediately after sacrifice, cone beam computed tomography
(3D Acciutomo, J. Morita MFG. Corp., Kyoto, Japan) of the
cranium was performed applying a tube voltage of 70 kV and
an amperage of 4 mA as described previously [23]. All data
was transferred to the workstation of a commercially available
navigation system (BrainLAB AG, Feldkirchen, Germany) as
standard digital imaging and communications in medicine
(DICOM) files. The artificial alveolar cleft was visualized on
the workstation monitor in multiplanar image reformations for
each animal using the image-data processing software of the
navigation system. The remaining defects were outlined on 16
Clin Oral Invest

slices per defect area (slice interval 0.25 mm) using drawing
tools. A three-dimensional reconstruction of the defect was
performed and the volumes of the remaining cleft defects were
calculated using a navigation software (iPlan 2.6 Cranial,
BrainLAB AG). To eliminate inter-operator variability and
to avoid mistakes in outlining the region of interest,
reformatting and measurements were performed by a single
examiner who measured all samples twice.

Statistical analysis

Statistical analysis was performed using SPSS Statistics

23.0.0.0 (IBM Cooperation, Armonk, USA). A mean value
was calculated for each defect resulting from the different
sections. For all experimental groups, mean and standard de-
viations were calculated. For data in all experimental groups
and for all healing times, a normal distribution could be as-
sumed. The influence of the fixed factors Bexperimental
group^ and Bhealing time^ was tested with an ANOVA with
additional consideration of the influences of the various indi-
viduals as a random factor. Bonferroni-adjusted multiple com-
parisons were done as post hoc tests.

Results

In vitro results

The removal and cultivation of the MSC was realizable with-
out limitations. The osteogenic differentiation of the MSC
could be demonstrated by alkaline phosphatase analysis
(Fig. 2a). Scanning electron microscopy visualized the surface
structure of the bone grafts where the attached cells could be
clearly identified (Fig. 2b). Both groups of cells were analysed
regarding the mRNA content for BMP-4 after 3, 7 and 12 days
of cultivation. Osteogenically differentiated MSC showed the
highest mRNA amount of BMP-4 after 7 days of cultivation
before a significant reduction to 79.9% (at experimental day
12) occurred. Additionally, the runt-related transcription fac-
tor 2 (Runx-2) was quantitatively determined. The mRNA
expression of Runx2 was 2.2-fold and 1.7-fold increased in
osteogenically differentiated MSC compared to undifferenti-
ated MSC. During the cultivation, the Runx2 mRNA de-
creases in differentiated cells continuously, whereas undiffer-
entiated cells do not show any differences in the mRNA ex-
pression of both, BMP-4 and Runx2. Results are depicted in
Fig. 3.
Clinical results
Eighty of 84 animals completed the study. This represents a
survival rate of 95.2%. Three animals of the following groups
died during surgery: bHA + osteogenically differentiated
Fig. 2 Alkaline phosphatase analysis of osteogenically differentiated
cells after 3 days of cultivation, magnification 100× (a). Scanning
electron microscopy of bovine hydroxyl apatite/collagen colonized with
undifferentiated mesenchymal stromal cells after 3 days of cultivation on
the scaffold, magnification 500× (b)
MSC (6 weeks healing time) and bHA + undifferentiated
MSC (9 and 12 weeks healing time). One animal died within
the healing time (bHA + osteogenically differentiated MSC;
12 weeks healing time). It was not necessary to kill animals
before the end of the study due to their health condition.
During the healing time, all animals showed no disturbance
regarding their behaviour and wound healing. The food and
water intake was not compromised. After an initial loss of
body weight postoperatively, the weight remained stable.
Radiological results
A cone beam CT of each defect area was made ex vivo and
analysed regarding the remaining defect volume. All experi-
mental groups showed a reduction of the defect volume after
12 weeks healing time compared to the first measurement
after 6 weeks (Table 1). This was found to be statistically
significant for the control group from 6 to 12 weeks
(p = 0.005) and bHA + osteogenically differentiated MSC
 Clin Oral Invest

from 6 to 9 weeks (p = 0.022). The comparison between the

experimental groups showed a significant smaller defect for
the control group than the bHA + ostegeonically differentiated
MSC after 12 weeks (p = 0.001). The application of pure bHA
led to a statistically significant smaller defect volume com-
pared to bHA + osteogenically differentiated MSC after
6 weeks (p = 0.044).

Descriptive histology

Polychrome fluorescence labelling The application of the

Fig. 3 Quantification of bone morphogenetic protein 4 (BMP-4) (a) and
runt-related transcription factor 2 (Runx2) (b) mRNA levels in undiffer-
entiated (grey columns) and differentiated rat mesenchymal stromal cells
(checked columns) by real-time PCR at different time points. The mRNA
levels of all tested genes are given in relation to that of actin, beta. Means
± standard deviation are given in all cases for n = 8–9 samples. Stars
indicating significant differences between differentiated and undifferenti-
ated cells at a fixed time: *p < 0.05, **p < 0.01, ***p < 0.005, and
significant differences between differentiated cells at different times:
#
p < 0.05, ##p < 0.01, ###p < 0.005; unpaired t test
fluorescence labels alizarin and calcein were performed 7
and 3 days prior to sacrifice of the animals. Alizarin was only
detectable in some ex vivo specimens whereas calcein led to a
sufficient labelling of the mineralized tissue being formed at
the time of application.
The control group without bone graft exposed a symmetri-
cal osteogenesis at both defect margins beginning at the for-
mer surgically created defect margins and continuing into the
direction of the defect centre. The distance between alizarin
and calcein labels was homogenous which can be assumed as
a consistent bone formation (Fig. 4a). At the caudal area of the
defect, appositional bone formation occurred unrelated to the
artificial defect due to a periosteal reaction. After 9 weeks, the
intensity of bone formation decreased, especially at the caudal
defect margins. The osteogenesis was focused on a cone-
shaped region in direction of the defect centre. With continu-
ing healing time, the defect margins became rounded and the
fluorescence labels occurred with less extent and intensity.
Sometimes, discontinuations of the labels were detectable
due to remodelling processes at the defect margins.

Table 1 Results of the cone

beam computed tomography: Experimental group Healing time (weeks) Number Remaining defect volume (mm3)
remaining defect volume (mm3)
Mean SD

control 6 7 13.43a 0.89

9 7 12.57 1.21
12 7 11.07a,c 2.32
bHA 6 7 12.43d 1.90
9 7 14.07 0.93
12 7 12.57 1.17
bHA + undiff. MSC 6 7 14.07 1.40
9 6 13.75 1.04
12 6 13.25 1.48
bHA + osteo. diff. MSC 6 6 14.50b,d 2.17
9 7 12.43b 0.89
12 6 14.08c 1.36

The analysis is based on the number of 80 animals and the values are displayed as mean and their standard
deviation
Between italic values marked with the same letter, a statistically significant difference was measurable (a p = 0.005,
b
p = 0.022, c p = 0.001, d p = 0.044)
Clin Oral Invest
Fig. 4 Polychrome fluorescence labelling (magnification 10 × 10,
multiple image alignment). Alizarin (red) was applied 7 days and
calcein (green) 3 days prior to sacrifice. The control group showed ho-
mogenous osteogenesis on both defect margins after 6 weeks of healing
time (a). After 9 weeks osseous integration of bovine hydroxyl apatite
granulate could be observed in a particular case (b)
The experimental group with pure bHA bone grafts
showed mainly the same characteristics as the control group
regarding the localization and quantity of the fluorescence
labels. Osteogenesis started at the surgically created sharp
defect margins and went into the direction of the defect centre,
likewise. The bHA granules did not expose mineralization
processes on their surface which could be detected with the
chosen magnification. After 9 weeks, an osseous integration
of the granules close to the defect margins occurred sporadi-
cally (Fig. 4b). The labels were generally reduced compared to
earlier time points after 12 weeks, indicating decreasing min-
eralization intensity with proceeding osseous healing.
Thinned fluorescence labels and smaller cone-shaped osteo-
genesis areas were the result of the application of bone grafts
containing MSC. Initial resorption occurred on the granules
after 12 weeks. Between undifferentiated and osteogenically
differentiated MSC, no differences regarding dynamics and
quantity of bone formation could be found by this evaluation
method.
Histomorphology
Four samples of each animal displaying the bone defect were
included into the analysis. The main result of this analysis was
that no complete bony reunion of the artificial defect occurred
in any experimental group. According to the details of the
local bone formation, differences depending on the applied
bone grafts were obvious. The control group showed a sym-
metrical formation of woven bone in cone-shaped bone
spiculae with a thin layer of osteoid on both defect margins,
whereas the defect itself was filled with fibrous tissue. The
fibrous tissue exposed a more and more organized structure
with ongoing healing time. With proceeding time, the bone
spiculae showed a centripetal growth. However, no bridging
of the defect occurred. On the basis of the cone adult lamellar
bone dominated. Woven bone and osteoid were located in the
middle and peak area of the cone. After 12 weeks, no osteoid
was detectable anymore and the cone became more rounded
(Fig. 5a). On the caudal defect margin periosteal bone forma-
tion occurred.
The application of pure bHA led to a similar cone-like
formation of bone spiculae with an increasing broad basis of
lamellar bone and woven bone in the peak area. In contrast to
the control group, this experimental group showed an earlier
maturation of the newly formed bone. The bHA granules were
embedded in fibrous tissue and mainly located in the centre of
the defect. In one particular case, the biomaterial was integrat-
ed into the bone. In general, the osteogenesis was oriented
cranially in the direction of the nasal septum (Fig. 5b).
The tissue-engineered bone grafts did not enhance the os-
teogenesis, irrespectively of which cells were used for the
colonization of the material (Fig. 5c, d). In comparison to
the control group, the size of the cone-like bone areas seemed
to be reduced at all three time points. After 12 weeks, the
content of osteoid was increased for bone grafts containing
undifferentiated MSC compared to pure bHA or scaffolds
colonized with osteogenically differentiated MSC indicating
a proceeding bone formation. In both groups, osteogenesis
occurred from the defect margin in the direction to the middle
of the defect, likewise. After 6 weeks, in areas of direct contact
between host bone and bone graft resorptions with formation
of Howship’s lacunae were visible. These were limited to the
host bone whereas the biomaterial showed no signs of resorp-
tion. During the healing time, the bHA granules were increas-
ingly integrated into fibrous tissue without any alteration of
their morphology. In some cases, several granules were em-
bedded in one capsule of multilayered fibrous tissue.
All experimental groups had in common that in particular
cases a destruction of the caudal part of the nose septum oc-
curred due to surgical preparation of the defect in the upper
jaw. However, no influence on the local bone formation in the
defect area was detectable. Furthermore, sometimes bHA
granules dislocated into the surrounding tissue. If this hap-
pened in an area of mechanical stress, e.g. onset of muscles,
local resorption on the particular bone resulted. A dislocation
into the nasal cavity did not cause histomophologically detect-
able alterations.
Histomophometry: remaining defect width
The control group exposed a remaining defect width of 63%
related to the individual initial defect width after 6 weeks with
a statistically significant reduction after 12 weeks (46%
p = 0.001) (Table 2). After 12 weeks, all experimental groups
containing bone grafts showed a statistically significant larger
 Clin Oral Invest

Fig. 5 Descriptive histology after 12 weeks of healing. Exemplary
images of all experimental groups: control group (a), pure bovine
hydroxyl apatite (b), bone grafts containing undifferentiated
mesenchymal stromal cells (c), bone grafts containing osteogenically
differentiated mesenchymal stromal cells (d). No complete osseous
healing of the defect occurred, but starting from the defect margin,
cone-like bone formation is detectable in all experimental groups. In b–
d, the formerly compact scaffold is reduced to single hydroxyl apatite
granulae, which are not osseointegrated. (Masson-Goldner trichrome
staining, magnification 10 × 10, multiple image alignment)

defect compared to the control (Table 3). At the end of the ex-
perimental study, the remaining defect width measured 60% for
pure bHA, 75% bHA + undifferentiated MSC and 82% for bHA
+ osteogenically differentiated MSC. Apart from the bHA +
osteogenically differentiated MSC, the other groups exhibited a
reduction of the defect width within the study period. For the
control group, this was statistically significant (p = 0.001). The
biomaterial bHA did not lead to a faster reduction of the defect
width, irrespective of MSC colonization, in comparison to the
control group. This was mostly statistically significant (Table 3).
The application of undifferentiated MSC was associated with a
smaller defect than the use of osteogenically differentiated MSC
at the end of the study but this difference was not statistically
significant. After 6 and 9 weeks, the group with osteogenically
differentiated MSC exposed a smaller defect than the group with
undifferentiated MSC which was statistically significant
(p = 0.044) after 9 weeks.
Histomophometry: percentage bone formation
After 6 weeks, the control group exposed 25% newly formed
bone in the defect area (Table 2). With proceeding healing

Table 2 Results of
histomorphometric analysis: Experimental group Healing time (weeks) Number Remaining defect width Bone formation
remaining defect width related to (%) (%)
the individual defect size after
surgery (%) and percentage of Mean SD Mean SD
newly formed bone in relation to
the defect size control 6 7 62.6a 17.4 24.5c,d 12.9
k
9 7 58.9 19.1 32.9c,e 14.6
12 7 46.2a,k 16.7 43.0d,e 13.3
bHA 6 7 63.3 19.7 22.8f,g 12.8
9 7 63.6 18.4 30.2f 14.0
12 7 60.1 14.5 30.8g 12.1
bHA + undiff. MSC 6 7 81.5 16.4 8.9h 7.3
9 6 82.7 10.5 10.1i 7.8
12 6 74.7 12.9 20.5h,i 10.9
bHA + osteo. diff. MSC 6 6 73.6 17.3 17.8 12.5
9 7 71.8b 11.9 22.0j 11.8
12 6 81.8b 9.7 11.5j 7.3

The analysis is based on the number of 80 animals and the values are displayed as means and their standard
deviation
Between italic values marked with the same letter a statistical significant difference was measurable within one
experimental group (a p = 0.001, b p = 0.047, c p = 0.018, d p < 0.001, e p = 0.003, f p = 0.038, g p = 0.018,
h
p < 0.001, i p = 0.002, j p = 0.002, k p = 0.006). For statistical inter-group analysis, see Tables 3 and 4
Clin Oral Invest

Table 3 Pair wise comparisons

between the values for remaining Experimental groups Healing time
defect width in relation to the
individual defect size after 6 weeks 9 weeks 12 weeks
surgery (%) with regard to the Sign. Sign. Sign.
different healing times. Between
italic values a statistically Control bHA 1.000 1.000 0.004
significant difference (p<0.05) bHA + undiff. MSC 0.000 0.000 0.000
was measurable bHA + osteo. diff. MSC 0.048 0.008 0.000
bHA Control 1.000 1.000 0.004
bHA + undiff. MSC 0.000 0.000 0.002
bHA + osteo. diff. MSC 0.060 0.257 0.000
bHA + undiff. MSC Control 0.000 0.000 0.000
bHA 0.000 0.000 0.002
bHA + osteo. diff. MSC 0.241 0.044 0.568
bHA + osteo. diff. MSC Control 0.048 0.008 0.000
bHA 0.060 0.257 0.000
bHA + undiff. MSC 0.241 0.044 0.568

time, the value increased statistically to 33% (p = 0.018) and Discussion

43% (p < 0.001). The application of pure bHA led to compa-
rable values after 6 (23%) and 9 weeks (30%), also with sta-
tistically significant increase (p = 0.038). However, after this
time, the osteogenesis seemed to reach a steady state and no
further bone formation occurred. In combination with MSC,
no additional effect of the local bone formation was measur-
able. In contrast, both groups with cells had a lower percent-
age of bone formation, which was statistically significant at
certain points (Table 4). The lowest rate of bone formation
with 11% was shown by the group of bHA + osteogenically
differentiated MSC after 12 weeks which represents a statisti-
cally significant reduction of bone formation from week 9 to
the end of the study (p = 0.002).
Children suffering from a congenital cleft alveolus need local
bone augmentation in the area of the cleft to stabilize the
dental arch and to enable the eruption of the permanent canine
[2]. Currently, autologous bone from the iliac crest is consid-
ered as golden standard for the augmentation. Due to reduced
availability and to possible donor site morbidity, there are
efforts to implement the current developments of bone tissue
engineering to create a sufficient alternative to autologous
bone grafts [14]. The present study analysed the application
of in vitro manufactured bone grafts containing bHA and
MSC in a model of alveolar cleft with regard to their potential
to promote local bone formation.

Table 4 Pair wise comparison

between the values for percentage Experimental groups Healing time
bone formation in relation to the
defect size (%) with regard to the 6 weeks 9 weeks 12 weeks
different healing times. Between Sign. Sign. Sign.
italic values a statistically
significant difference (p<0.05) Control bHA 1.000 1.000 0.000
was measurable bHA + undiff. MSC 0.000 0.000 0.000
bHA + osteo. diff. MSC 0.161 0.001 0.000
bHA Control 1.000 1.000 0.000
bHA + undiff. MSC 0.000 0.000 0.004
bHA + osteo. diff. MSC 0.528 0.034 0.000
bHA + undiff. MSC Control 0.000 0.000 0.000
bHA 0.000 0.000 0.004
bHA + osteo. diff. MSC 0.010 0.001 0.023
bHA + osteo. diff. MSC Control 0.161 0.001 0.000
bHA 0.528 0.034 0.000
bHA + undiff. MSC 0.010 0.001 0.023

Animal model
Currently, the complexity of a congenital human alveolar cleft
or palate cannot be displayed by any pre-clinical model.
Therefore, experimental studies are limited to artificial bone
defects in different models. The established small animal
model is the rat, because in its maxillary bone a cleft-like
defect can be prepared surgically [24, 25]. An alternative large
animal model would be the maxillary bone of dogs [26].
Genetically, it is also possible to create cleft models [27].
Due to a gene deletion, an incomplete development of the
palate and alveolus as well as other pathologies occurred in
a mice model. The gene defect led to perinatal lethality. This
model is similar to the sevoflurane-induced embryonic cleft
model, not suitable to evaluate new bone grafts [28]. In the
present study, the adult male Lewis rat was chosen due to the
possibility to transplant cells interindividually without immu-
nological disadvantages. Both analysing methods confirmed
that an initial bone defect of 3.3 mm led to a defect without
osseous bridging within the 12 weeks of healing observed in
the present study.
In vitro behaviour of the bHA bone graft
Tissue engineering is one approach to create bone grafts
in vitro and apply them in vivo to promote bone regeneration.
The basis is the assortment of suitable scaffold materials and
cells which were in accordance with the intended clinical re-
sult. The in vitro findings of the present study confirmed the
possibility to use bHA in the context of tissue engineering
applications [29, 30]. The biocompatibility and cytotoxity of
bHA were analysed by Liu et al. using human osteoblasts,
likewise [31]. The colonized biomaterial underwent cell vital-
ity staining and four different tests of biocompatibility. Thus,
the suitability of the material could be confirmed. Clinically,
these findings were completed by numerous studies evaluat-
ing bHA for various clinical applications [32–34]. The bone
marrow of adult mammals represents a sufficient source for
multipotent stem cells [35]. These cells have the potential to
differentiate into bone cells, cartilage cells, muscle cells, liga-
ment cells, tendon cells, adipose cells and stroma cells. In the
context of the study with the aim to promote bone formation in
a maxillary defect, osteogenic differentiation of the MSC was
chosen. The osteogenic differentiation could be realized suf-
ficiently and the following RNA analysis showed a higher
level of gene expression for BMP-4 in osteogenically differ-
entiated MSC compared to undifferentiated cells.
Physiologically, BMP-4 promotes the differentiation of MSC
as osteoprogenitor cells into osteoblasts [36]. The highest lev-
el of BMP-4 expression occurred in vitro after 7 days of cul-
tivation. Cells, like MSC, can be verified by stem cell markers
like Runx2. Runx2, an essential transcription factor in the
development of the skeletal system [37], is required for
Clin Oral Invest
osteoblast cell transition from proliferative to differentiated
cells [38–40]. Runx2 expression is followed by the upregula-
tion of genes expressed in osteoblasts like osteocalcin, Alpl
and collagen type I [41]. Runx2-deficient mice are devoid of
osteoblasts and, consequently, of bone tissue [42].
Mastrangelo et al. compared the expression of alkaline phos-
phatase, osteocalcin and Runx2 of osteogenically differentiat-
ed MSC cultivated on bHA or synthetic hydroxyl apatite [15].
The measured level of protein expression was higher on bHA
indicating its suitability for further tissue engineering
applications.
In vivo behaviour of the bHA bone graft
In conclusion of the measurements using cone beam CT, the
control defect exposed a statistically significantly smaller de-
fect volume than tissue-engineered bone grafts containing
osteogenically differentiated MSC. Between pure bHA, grafts
with undifferentiated MSC and the control group no statisti-
cally significantly differences occurred, likewise. The defect
volume decreased with proceeding healing time but statisti-
cally significantly only for the control group.
Histomorphometrically, the remaining defect width was eval-
uated. Again, the control defect showed the lowest values
compared to the other experimental groups for this parameter.
After 12 weeks, the control defect was statistically significant-
ly reduced compared to all other groups. It has to be highlight-
ed that control and pure bHA groups showed similar rates of
bone formation and defect width after 6 and 9 weeks.
However, during further healing time, a stagnation of bone
formation was detectable for pure bHA. Meanwhile, the bone
formation in the control group proceeded to grow statistically
significantly. Moreover, bone grafts containing undifferentiat-
ed MSC showed a continuing statistically significant osteo-
genesis after 12 weeks. In contrast, the application of
osteogenically differentiated MSC led to a significantly re-
duced bone formation rate with ongoing healing time. In the
context of the initial hypothesis of the study, it has to be con-
cluded that the application of tissue-engineered bone grafts
containing bHA and MSC did not enhance the bone formation
in this maxillary defect in a rodent model. The quantity of
bone formation and thus, the reduction of the defect size de-
creased in the given order at the end of the study period:
control group > pure bHA > bHA + undifferentiated MSC >
bHA + osteogenically differentiated MSC. An unexpected
finding of the study is that no enhancement of defect ossifica-
tion could be shown in the artificial defect. One possible rea-
son might be the application of a non-resorbable bone substi-
tute. After degradation of the collagen scaffolds, the remaining
particles might have been exposed to micromovements, thus
hampering a sufficient ossification of the defect. Furthermore,
the defect was located directly posterior of the maxillary inci-
sors in an area where chewing forces might occur.
Clin Oral Invest
Postoperatively, the animals were provided with soft diet to
avoid chewing pressure on the wound area. However, a total
wound rest might not have been achieved causing
micromovements, likewise, which is a drawback of the study.
In a clinical situation, nasal gastric tubes might have been
applied to avoid stress during the wound healing period.
However, this is not possible in an animal model. The appli-
cation of a thin biocompatible membrane to cover the bioma-
terial might avoid this in further animal studies [43].
Comparison of bHA bone grafts
The current state of research regarding tissue-engineered bone
grafts is extensive but just a few studies analysed these bone
grafts in alveolar cleft osteoplasty models. Mayer et al. eval-
uated a copolymer scaffold (poly(lactide-co-glycolide)) with
and without recombinant BMP-2 in a maxillary defect in a
dog model [44]. Also, collagen and hydroxyl apatite scaffolds
were combined with recombinant BMP and analysed regard-
ing their influence on local bone formation in a rat model [43,
45]. The mentioned studies had in common that the bone
grafts led to a positive effect on bone formation.
Nevertheless, the scaffolds were not colonized with cells.
Thus, a direct comparison with the present study is not rea-
sonable. Pourebrahim et al. applied hydroxyl apatite/
tricalciumphosphate scaffolds which were in vitro colonized
with osteogenically differentiated MSC in a cleft defect in
dogs [26]. The tissue-engineered bone grafts were compared
with autologous bone grafts. The mean percentage of bone
regeneration was measured after 15 and 60 days. Local bone
formation was significantly higher for autologous bone grafts.
Nevertheless, the analysed combination of hydroxyl apatite/
tricalciumphosphate and osteogenically differentiated MSC
was recommended as an alternative material for grafting of
this maxillary defect. The mentioned studies abstained from
testing the pure scaffold material or empty control defect like
other studies did. For example, BONITmatrix® is a clinically
established bone substitute material consisting of synthetic
hydroxyl apatite and tricalciumphosphate. In 2014, a study
analysing the effect of the colonization with either undifferen-
tiated or osteogenically differentiated MSC on local bone for-
mation in a maxillary defect of the rat was published. The
study included a control group as well as one group with the
pure scaffold for comparison [21]. The undifferentiated MSC
showed a higher potential to reduce the defect size by ossifi-
cation compared to the other bone grafts. In comparison with
the present study, undifferentiated MSC seemed to be more
efficient than osteogenically differentiated MSC, likewise.
Both studies led to oppositional results regarding the control
defect. In the cited study, the remaining defect volume and
width were the highest for the control group. In contrast, the
present study led to statistically significant reduced values for
this experimental group. Reasons for the contrary results
might be seen in the applied biomaterial (synthetic hydroxyl
apatite/tricalciumphosphate vs. bHA) and the chosen healing
times (up to 6 weeks vs. up to 12 weeks). Both biomaterials
differed regarding numerous material associated parameters.
Thus, differences in the experimental and clinical performance
are expectable. Synthetic and bHA are very stable and will be
resorbed slowly. It represents an initial placeholder for local
osteogenesis starting from the defect margins of the host bone.
Later, the material will be integrated into the newly formed
bone [16]. Due to its stability hydroxyl apatite is applied fre-
quently and with sufficient results. In particular cases, an
inhibiting effect of hydroxyl apatite on initial osseous healing
of extraction sockets could be found [46]. In the present study,
this inhibiting influence of pure bHA was detectable after
12 weeks because up to 9 weeks, the bone formation was
similar to the control and then a stagnation was measurable.
To what extent this observation in a large maxillary defect is
transferable to other defects, e.g. extraction sockets, is uncer-
tain. Synthetic tricalciumphosphate is less stable than hydrox-
yl apatite, therefore, degraded more rapidly than Thus, it is
often combined with hydroxyl apatite to improve its material
characteristics. In the context of the study, no promotion of
osteogenesis in a large maxillary defect was detectable and it
has to be concluded that the combination of synthetic hydrox-
yl apatite/tricalciumphosphate might be more efficient than
the application of bHA independently of the application of
cells.
Translation into clinical application
Due to the promising results for the implementation of tissue-
engineered bone grafts as an alternative to autologous bone
which were gained in several small and large animal studies,
first studies in humans were realized. Van Hout et al. reviewed
controlled, randomized clinical trials analysing tissue-
engineered bone grafts [47]. Just three studies fulfilled the
mentioned criteria until 2011 [48–50]. Different resorbable
scaffolds made of collagen where combined with BMP-2
and led to comparable values for volume and newly formed
bone. The authors recommended further studies to analyse the
quality of the newly formed bone in the defect area. The clin-
ical application of bHA for alveolar cleft osteoplasty was eval-
uated in a study with 23 patients by Benlidayi et al. [51]. There
was no statistically significant difference between defects
grafted with bHA or autologous bone. The clinical application
of pure bone substitute materials for large defects, e.g. con-
genital clefts, was discussed critically because the eruption of
the permanent canine can be disturbed [10]. Due to their re-
generative potential, MSC are one option for tissue engineer-
ing applications. Related to alveolar cleft osteoplasty, the suit-
ability of these cells was analysed by Pradel et al. and Behnia
et al. [23, 52]. The combination of the biphasic scaffold of
hydroxyl apatite/tricalciumphosphate, osteogenically

differentiated MSC and platelet-derived growth factor led to a
defect ossification of 51% after 6 months [23]. Nevertheless,
the study abstained from comparing the results with the clin-
ical standard, the autologous bone graft [52]. Pradel et al.
measured a defect ossification of 41% for bone grafts of
demineralized bone matrix and osteogenically differentiated
MSC after 6 months. The transplantation of autologous
spongious bone from the iliac crest resulted in 37% ossifica-
tion of the cleft [23]. The currently published studies regard-
ing the application of tissue-engineered bone grafts for alveo-
lar cleft osteoplasty have in common that the number of cases
is limited and all of them recommended further studies to
identify potential bone grafts. A future trend in bone tissue
engineering for cleft alveolar osteoplasty might be the imple-
mentation of 3D printing of individual scaffolds based on pre-
existing computed tomography scans of the particular patient.
Berger et al. demonstrated a successful preparation of individ-
ual tricalciumphosphate-polyhydroxybutyrate scaffolds colo-
nized with human MSC in vitro [53]. As a next step, these
kinds of bone grafts have to be analysed in vivo to evaluate
their potential to promote osteogenesis in maxillary defects.
Conclusion
For patients suffering from congenital alveolar cleft, the de-
velopment of bone grafts as an alternative to autologous bone
from the iliac crest would be advantageous. The method of
tissue engineering enables the creation of in vitro bone grafts
consisting of clinically established biomaterials and living
cells. Before these bone grafts can be analysed in humans,
pre-clinical studies are necessary to identify potential combi-
nations of biomaterials and cells leading to a high degree of
defect ossification. The present study examined tissue-
engineered bone grafts of bHA and MSC with regard to their
effect on the promotion of local osteogenesis in an animal
model of alveolar cleft. The following conclusions can be
drawn from the study: (I) The in vitro preparation of bone
grafts containing bHA and MSC, both undifferentiated and
osteogenically differentiated, was possible. (II) An artificial
bone defect of 3.3 mm represented a non-reuniting defect in
the maxilla of adult Lewis rats within the entire experimental
period of 12 weeks. (III) The study could not confirm that
bone grafts of bHA enhance ossification in a model of alveolar
cleft. This was independent of the application of MSC. (IV)
Undifferentiated MSC were more efficient compared to
osteogenically differentiated MSC regarding the enhancement
of defect ossification after 12 weeks of healing. (V) Future
studies should focus on resorbable scaffolds in the maxillary
defect model to allow osteoconduction in the defects. Also,
the implementation of new technologies like 3D printing of
defect-specific scaffolds should be considered to improve the
initial interaction between tissue and biomaterial.
Clin Oral Invest
Acknowledgements The authors want to thank Mrs. Diana Jünger for
her extensive assistance in preparing the bone grafts. Furthermore, the
authors are grateful to Dr. Roland Jung and the team of the Experimental
Centre of the Medical Faculty BCarl Gustav Carus^, Technische
Universität Dresden, for the care of the animals and assistance during
the surgical interventions. Dr. Heike Meißner is acknowledged for the
scanning electron microscope images. Moreover, the authors thank Mr.
Torsten Jannasch for the assistance in preparing the images.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Funding The study was financially supported by an internal research
grant BMeDDrive Projekt Start^ of the Faculty of Medicine BCarl Gustav
Carus^, Technische Universität Dresden, Germany. The bone substitute
was kindly provided by Geistlich Biomaterials, Wolhusen, Switzerland.
Ethical approval This article does not contain any studies with human
participants performed by any of the authors. All applicable international,
national and/or institutional guidelines for the care and use of animals
were followed.
Informed consent For this type of study, formal consent is not
required.
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