Repair of Critical-Size Bone Defects Using

Bone Marrow Stem Cells or Autogenous Bone

With or Without Collagen Membrane:
A Histomorphometric Study in Rabbit Calvaria
Antonio Carlos Aloise, DDS, PhD1/Andr Antonio Pelegrine, DDS, PhD2/
Allan Zimmermann, MD, MS1/Rafael de Mello e Oliveira, DDS, MS1/Lydia Masako Ferreira, MD, PhD1
Purpose: The aim of this study was to evaluate bone healing after the use of a xenograft scaffold enriched with
bone marrow mesenchymal stem cells (BM-MSCs), an autogenous bone graft, or the scaffold without BM-MSCs.
Materials and Methods: Eighteen rabbits were used for this study; bilateral 12-mm-diameter defects were
created in the animals parietal bones. The bilateral defects were filled with a xenograft enriched with BM-MSCs
(test group [TG]), with autogenous bone graft (positive control group [PCG]), or with a xenograft alone (negative
control group [NCG]). In all groups, randomly, one defect was covered with a collagen membrane. The rabbits were
sacrificed 8 weeks after surgery, and their parietal bones were harvested and analyzed histomorphometrically.
Results: Within the PCG and the NCG, the defects covered with the barrier membrane showed better bone
healing. In the TG, the defects covered with the barrier membrane did not show better bone healing (intragroup
comparisons by Wilcoxon and Friedman tests for paired data). TG showed percentage of mineralized tissue
(MT) of 56.03% 3.49% with membrane and 57.71% 5.31% without membrane. PCG showed MT of 55.13%
4.83% and 49.69% 3.81% with and without membrane, respectively, and NCG showed MT of 26.77%
7.29% and 19.67% 2.66% with and without membrane, respectively. Conclusion: Both autogenous bone
graft and a xenograft enriched with BM-MSCs were equally effective for bone reconstruction and better than
the xenograft alone. The use of a barrier membrane seemed to have a synergistic effect on bone healing in PCG
and NCG but not in TG. Int J Oral Maxillofac Implants 2015;30:208215. doi: 10.11607/jomi.4010
Key words: bone marrow, bone repair, cell transplantation, osteogenesis, stem cells

one repair of critical defects can be assisted with

therapies that involve bone marrow, because this
tissue possesses cell populations that play an important role in bone homeostasis, known as bone marrow stromal cells.1 However, there is no consensus
about the best methodology for obtaining and using
bone marrow stromal cells. The relevant literature has
reported three main alternatives: (1) fresh bone marrow (in natura)24; (2) bone marrow mononuclear cell
concentrate, also called bone marrow mononuclear
fraction (BMMF)1,5,6; and (3) cultivated bone marrow
mesenchymal stem cells (BM-MSCs).7,8
1Department

of Plastic Surgery, Paulista Medicine School,

Federal University of Sao Paulo, So Paulo, Brazil.
2Department of Implantology, So Leopoldo Mandic Dental
School, Campinas, So Paulo, Brazil.
Correspondence to: Dr Lydia Masako Ferreira, Rua Pedro de
Toledo 781, 11o andar CEP 04039-032, So Paulo, Brazil.
Email: aca.orto@uol.com.br
2015 by Quintessence Publishing Co Inc.

Bone marrow stromal cells were discovered in the

late 1970s by a group led by Alexander Friedenstein,
who showed that bone marrow contains a population of plastic-adherent, highly proliferative cells that
are able to form a colony of fibroblasts (hence the
name colony-forming unit fibroblasts).9,10 Following
implantation in diffusion chambers, colony-forming
unit fibroblasts spontaneously formed bone, cartilage,
and fibrous tissue in vivo.11 Whereas Friedenstein et al
termed them determined osteogenic progenitors,12
the subsequent findings of their multipotentiality toward other mesenchymal lineages led Caplan to coin
the term mesenchymal stem cells,13 in analogy to hematopoietic stem cells, which were the best described
adult stem cell type at that time.
Modern bone-engineering strategies based on
osteogenic cells, osteoinductive stimulators, and osteoconductive scaffolds are recognized as potential
ways to create biologic tissue substitutes for regenerating large bone defects.14 The choice of cell sources
that can efficiently differentiate into bone tissue is the
first important step of bone engineering. Several cell

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Aloise et al

types can potentially be used as cellular components

in bone engineering. These include osteoblast, embryonic, and adult stem cells. Among these candidates,
MSCs, as adult stem cells, possess some characteristics
that make them more appropriate for use in promoting bone regeneration.15
A number of in vitro assays can be used to assess
the multipotentiality of these cell preparations.16
Osteogenic differentiation of human MSCs can be
triggered by exposure to specific culture supplements, including dexamethasone, ascorbate-2-phosphate, and beta glycerolphosphate.17,18 Adipogenic
differentiation can also be induced through the use
of induction medium containing dexamethasone, indomethacin, isobutylmethylxanthine, and insulin, but
these cultures require a higher seeding density than
that used for osteogenic differentiation.16 Finally, human MSCs placed in aggregate or pellet cultures in a
defined medium containing dexamethasone, ascorbate-2-phosphate, insulin, selenious acid, transferrin,
sodium pyruvate, and transforming growth factorbeta will undergo chondrogenic differentiation.16,19,20
The ability of MSCs to differentiate along these lineages is strongly suggestive of their multipotency
and stem cell nature. However, human MSCs do not
maintain these characteristics indefinitely, and they
present senescence with extensive subcultivation in
vitro, whereby they lose their proliferation and differentiation potential.2123
In this study, the bone healing rate after the use
of a tissue-engineering technique of bone xenograft
scaffolds enriched with BM-MSCs was compared with
the gold standard autogenous bone graft in a rabbit
model.

MATERIALS AND METHODS

For this study ,18 adult, skeletally mature New Zealand
white rabbits from the animal colony of Federal University of So Paulo, Brazil (UNIFESP), weighing 3.5 to
4 kg each were used. The animals were divided into
three groups of six animals each: the test group (TG), in
which BM-MSCs were associated with a xenograft; the
positive control group (PCG), which were treated with
autogenous bone graft; and a negative control group
(NCG) treated with the xenograft alone. All animals
were operated by the same researcher, and all evaluations were done by the same researchers.
The surgical protocol for this study was analyzed
and approved by the UNIFESP Ethics Committee (no.
2139/2011).
The animals underwent a period of adaptation to
environmental conditions prior to being housed on

UNIFESP premises. Rooms with a controlled temperature ranging from 18C to 20C and individual cages
specifically designed for rabbits were used. The animals
were fed commercial pellets and water ad libitum.
Anesthesia was induced by ketamine (40 mg/kg),
midazolam (2mg/kg), and fentanyl citrate (0.8g/kg)
and maintained using a mixture of isoflurane/nitrous
oxide (1%:1.5%)andoxygen (2/3:1/3) using a pediatricsize laryngeal mask airway. The top of the head of each
of the 18 animals was shaved and the site disinfected
with a povidone-iodine solution. After administration
of a local anesthetic injection (2% lidocaine with epinephrine 1:100,000), a sagittal incision was made and
the skin and periosteum were retracted.

Bone Marrow Harvest and Culture

After general anesthesia was achieved, autologous

bone marrow was obtained by an aspiration technique. Two milliliters of bone marrow aspirates were
obtained from each tibia of the 18 rabbits using a disposable 40 10 needle (1.10 38 mm) with 20-mL
disposable syringes, previously heparinized to prevent blood clotting. Only the initial volume aspirated
of bone marrow was used, because a greater amount
could result in peripheral blood aspiration.24
The following procedure was performed to obtain
the stem cells. After the bone marrow was aspirated,
it was transferred into polypropylene tubes containing
preservative-free heparin (1,000 units/mL). The bone
marrow and heparin were mixed well. The BMMF was
then separated from the bone marrow tissue by density-gradient centrifugation with Ficoll Histopaque-1077
(Sigma-Aldrich) according to the manufacturers instructions. In essence, 3.5 mL of the bone marrow tissue was diluted in a 1:1 ratio with phosphate-buffered
saline (PBS), which was placed in a conical tube (15 mL)
containing the same volume of Ficoll Histopaque-1077.
The final solution was centrifuged at 400g for 30 minutes at 22C. The mononuclear cell layer was then pipetted and washed twice with PBS. A volume of 4 mL
was established, and another centrifugation was performed at 200g for 10 minutes at room temperature.
The supernatant was discarded, and the pellet was
resuspended in 5 mL of Dulbecco minimum essential
medium (DMEM) (Sigma) containing 10% fetal bovine
serum (Sigma), 1% penicillin, 100 U/streptomycin, and
100 mg/mL solution (designated the standard culture
medium) and transferred to a 75-cm2 culture flask
(Corning) that already contained 5 mL of culture medium. The cells were incubated at a temperature of 37C
in an atmosphere composed of 95% air and 5% carbon
dioxide. The culture medium was exchanged every 48
hours, and the culture was monitored daily using an inverted optical microscope.
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Aloise et al

Creation and Grafting of the Critical-Size

Bone Defects

Two defects were then created, one on each side of the

midline, with a 12-mm-diameter trephine bur. In TG, a
xenograft enriched with BM-MSCs was used to fill the
bilateral defects, restoring their original contour. In PCG,
autogenous bone graft was harvested from the created
defect area and then particulated (Neodent) to fill the
bilateral defects, restoring their original contour. In NCG,
a xenograft was used to restore the contour of the defects bilaterally. In all groups, one of the calvarial defects
(randomly chosen) was covered with a barrier membrane. Randomization was performed using specific
software.25 In all rabbits, the incision was closed in layers.
The animals were given an intramuscular injection
of benzylpenicillin (40,000 IU) and sodium dipyrone
(0.25 mg/kg) postoperatively. The animals received
food and water ad libitum for the remainder of the experimental period.
The xenograft used in this study was Bio-Oss
(Geistlich Biomaterials), which is a particulate xenograft
bone substitute. The form used in this study was small
particles, with granules ranging from 0.25 to 1 mm.
The barrier membrane used was Bio-Gide (Geistlich
Biomaterials), which is a resorbable bilaminar collagen
membrane of porcine origin that prevents unwanted
invasion of tissues adjacent to the bone defect, thus
optimizing bone healing.
All 18 animals were sacrificed 8 weeks after the initial surgery. The parietal bones were then harvested
and processed for histologic and histomorphometric
evaluation.

Assays and Characterization

Adhesion of MSCs. Cell adhesion was analyzed 3 days

after seeding the cells in the culture flask using an inverted optical microscope.
Adipogenic Differentiation. To evaluate adipogenic differentiation, the cells were cultivated in sixwell plates (TPP Techno Plastic Products) containing
2 mL of complete DMEM/F12 medium supplemented
with 10mol/L insulin and 1mol/L dexamethasone
(Sigma Aldrich) for 15 days (adipogenic medium). The
plates were kept in a humidified incubator (37C; 95%
oxygen and 5% carbon dioxide), and the culture media
was changed every 3 days. After this period, the medium was aspirated, and the cells were washed twice
with PBS. Then, 2 mL of paraformaldehyde (0.4% in
PBS, Electron Microscopy Sciences) were added to the
cells. The fixative solution was aspirated after 30 minutes, and the cells were washed three times with PBS
as follows: once with PBS containing glycine 0.1 mol/L
for 10 minutes and twice with PBS for 2 minutes.
The cells were then incubated with Oil Red O dye
(0.5%) at room temperature for 30 minutes. The dye

was carefully removed with a 2-mL disposable pipette,

and the plate was washed five times with 2 mL of water.
The fixed and dyed cells were observed with a Nikon
Ti-U optical microscope and photographed using
NIS-Elements version 3.2 software (Nikon Instruments).
Osteogenic Differentiation. To determine osteogenic differentiation, the cells were cultured in six-well
plates (TPP Techno Plastic Products) containing 2 mL
complete DMEM/F12 medium supplemented with
50 mol/L ascorbic acid (Sigma Aldrich), 0.1 mol/L
dexamethasone, and 10 2 mol/L beta-glycerophosphate (Mallinckrodt Baker) for 21 days (osteogenic medium). The plates were kept in a humidified incubator
(37C; 95% oxygen and 5% carbon dioxide), and the
culture media was changed every 3 days. After this
period, the medium was aspirated, and the cells were
washed twice with PBS. Then, a solution of 2 mL paraformaldehyde (0.4% in PBS, Electron Microscopy Sciences) was added to the cells. The fixative solution was
aspirated after 30 minutes, and the cells were washed
three times with PBS as follows: once with PBS containing glycine 0.1 mol/L for 10 minutes and twice with PBS
for 2 minutes.
The cells were then incubated in a solution of sodium alizarin (40 nM, pH 4.1; Sigma Aldrich) at room
temperature for 30 minutes. The dye was carefully removed with a 2-mL disposable pipette, and the plate
was washed five times with 2 mL of water. The fixed
and dyed cells were observed with a Nikon Ti-U optical microscope and photographed using NIS-Elements
version 3.2 software (Nikon Instruments).
Chondrogenic Differentiation. To assess chondrogenic differentiation, the cells were cultured in six-well
plates (TPP Techno Plastic Products) containing 2 mL
complete DMEM/F12 supplemented with 10 mol/L
insulin, 0.1 mol/L dexamethasone, 50 mol/L
ascorbic acid, and 10 ng/mL transforming growth
factor-beta1 (Cell Signaling Technology) for 21 days
(chondrogenic medium). The plates were kept in a humidified incubator (37C; 95% oxygen and 5% carbon
dioxide), and the culture media was changed every 3
days. After this period, the medium was aspirated and
the cells were washed twice with PBS. Then a solution
of 2 mL paraformaldehyde (0.4% in PBS, Electron Microscopy Sciences) was added to the cells. The fixative
solution was aspirated after 30 minutes, and the cells
were washed three times with PBS as follows: once
with PBS containing glycine 0.1 mol/L for 10 minutes
and twice with PBS for 2 minutes.
The cells were then incubated in toluidine blue
(0.1%; Sigma Aldrich) at room temperature for 30
minutes. The dye was carefully removed with a 2-mL
disposable pipette, and the plate was washed five
times with 2 mL of water. The fixed and dyed cells were
observed with a Nikon Ti-U optical microscope and

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Aloise et al

Fig 1Fragments of parietal bone after

removal (8 weeks postoperative).

Fig 2 Histologic views of NVMT (yellow), VMT (orange), and NMT (blue). (a) TG; (b) PCG; (c) NCG (Stevenel blue; magnification 100).

photographed using NIS-Elements version 3.2 software (Nikon Instruments).

Immunophenotypic Characterization. Stem cells
from the second passage were used for immunophenotypic characterization. The cells were tripsinized,
and the cellular suspension was centrifuged (300g,
4 minutes). The cells were stained with antibodies
conjugated with fluorescein isothiocyanate (FITC),
allophycocyanin (APC), or phycoerythrin (PE) to determine expression of CD16-PE, CD34-APC, CD45-FITC,
CD73-PE, CD90-APC, and CD105-PE (BD Biosciences).
A total of 5 105 cells were resuspended in 0.2 mL
PBS and incubated with FITC-, APC- or PE-conjugated
antibodies for 20 minutes at room temperature and
protected from light. The samples were analyzed by
flow cytometry (Guava easyCyte, Millipore) to identify
specific fluorescence channels of each antibody.

Histologic Preparation and Histomorphometric

Analysis

An area of approximately 20mm2 around each grafted

site on the parietal bone was removed 8 weeks postoperatively (Fig 1). It was then decalcified by submersion in 10% ethylenediaminetetraacetic acid for 8 to 12
weeks at room temperature. The bone removed during
creation of the bone defects (baseline) was also processed to enable histomorphometric examination of
native bone. Two millimeters of the middle portion of

each specimen were selected and embedded in paraffin blocks. Next, 7-m sections of each specimen were
obtained and evaluated according to a semiquantitative histologic scoring system, in which an initial section
was used and the following three were discarded, successively, until all sections had been evaluated. Thirtysix sections of each specimen were stained with Mallory
trichrome and Stevenel blue and then qualitatively examined under light microscopy. Six areas of each section were analyzed (top left, lower left, top center, lower
center, top right, and lower right) (Fig 2).
Digital images were captured with a digital camera
(RT Color, Diagnostic Instruments) attached to a light
microscope (1.25 magnification). The digital images
were merged to create a single image for each histologic section using Adobe Photoshop Elements 2.0
(Adobe Systems).
Two blinded examiners (AZ and RMO) were calibrated previously. In cases of disagreement, the specimen
was reevaluated and a consensus was reached. The examiners traced all the images to measure new bone formation using Image Pro Plus 4.5 Software for Windows
(Media Cybernetics). Mineralized tissue (MT) and nonmineralized tissue (NMT) were measured. In all groups,
the nonvital mineralized tissue (NVMT) and vital mineralized tissue (VMT) were measured inside the MT area.
All results were scored in square micrometers and then
expressed as a percentage of the total area.
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Aloise et al

Fig 3 BM-MSCs after 21 days in

culture with DMEM.

50 m

50 m

50 m

50 m

Fig 4 Differentiation of BM-MSCs into osteoblasts stained for (a) calcium phosphate with alizarin red, (b) adipocytes stained for
lipid with Oil Red O, and (c) chondrocytes, stained for sulfated proteoglycans with 1% toluidine blue in 0.1 N hydrochloric acid.

Statistical Analysis

All quantitative data were analyzed with SPSS software

(version 17, SPSS Inc). The Wilcoxon test was used to
compare the results obtained with and without membrane coverage in relation to the variables MT and NMT
in both groups. The Friedman test for paired data was
used to compare the MT and NMT results for the situations with and without membrane coverage in both
groups. The Kruskall-Wallis test was used to compare
the groups with respect to each variable (MT and NMT)
and also with respect to the results obtained with and
without membrane coverage. The Bartlett test was
used to evaluate the homoscedasticity. A P value less
than .05 indicated statistical significance. The Spearman correlation coefficient was used to assess inter
examiner agreement.

RESULTS
Isolation and Cell Culture

The isolation and culture of bone marrow cells were

performed uniformly for all six rabbits in the test
group. There were no complications associated with
harvesting bone marrow, and in all animals, it was
possible to collect the same amount of bone marrow
(a total of 4 mL, ie, 2 mL from each tibia). The BMMF
was obtained from each animal for subsequent cell
culturing procedures. The same amount of BMMF
was obtained from all animals (2 mL) and contained

1.46 106 cells per sample. After 3 days, the culture

medium was removed, and adherent cells were counted after trypsinization (0.25% trypsin + 5 mmol/L
ethylenediaminetetraacetic acid) using trypan blue.
A constant rate was observed of 15 adhered cells per
flask of culture (0.001%). There were two subcultures
until they reached confluence of 80% of the surface of
a 75-cm2 flask, which took an average of 35 days. Upon
confluence, the BM-MSCs were removed and counted; an average of 2 106 cells per culture flask was
obtained.

Cell Adhesion and Differentiation Assays

The cells were evaluated for their ability to adhere in

the culture flask before the first exchange of culture
medium. All flasks had a mean of 15 adherent cells
(0.001%) (Fig 3). For the three differentiation tests
(adipogenic, chondrogenic, and osteogenic) the
BM-MSCs were cultured for 21 days and then photographed to evaluate the presence of the respective
markers (oil red O, toluidine blue, and alizarin red).
There was positive staining for all BM-MSCs in all cell
cultures; the markers were uniformly distributed on
the surfaces of the culture flasks (Fig 4).

Immunophenotype of Cells

Flow cytometry analysis showed that these cells expressed CD14, CD16, CD73, CD90, CD105. There was
no expression of markers CD31, CD34, CD44, CD45,
CD117 in any of the samples analyzed (Fig 5).

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Aloise et al

CD14

CD16

CD31

CD34

CD44

CD45

CD73

CD90

CD105

CD117

Fig 5 Flow cytometry of BM-MSCs (21 days in culture).

Table 1 Intergroup Analysis: Statistical Comparison of Mean Values (%) for MT and
NMT Between Groups
With membrane

Tissue
type

TG

Without membrane

PCG

NCG

TG

PCG

NCG

MT

56.03 3.49

55.13 4.83

26.77 7.29

.2

57.71 5.31

49.69 3.81

19.67 2.66

.03

NMT

44.97 5.88

44.87 3.78

73.23 6.87

.6

42.29 4.81

50.31 5.01

80.33 2.67

.03

P.05 indicates statistical significance (Kruskal-Wallis test).

Table 2 Areas (%) of NVMT and VMT Within MT Areas

With membrane
Tissue type
NVMT

TG

Without membrane

PCG

27.7 2.72

NCG

TG

13.52 3.00

27.23 6.80

PCG
0

NCG
13.08 1.72

VMT

28.24 6.17

55.13 4.83

13.06 5.24

30.48 4.65

49.69 3.81

6.56 1.20

MT (NVMT + VMT)

56.03 3.49

55.13 4.83

26.77 7.29

57.71 5.31

49.69 3.81

19.67 2.66

Histomorphometric Observations

In the TG, the sides on which the defects were covered

with the barrier membrane did not show better bone
healing (P > .05) (Table 1). In contrast, in the PCG and
NCG, the sides on which the defects were covered
with the barrier membrane showed better bone healing (P < .05) (intragroup comparison by Wilcoxon and
Friedman tests for paired data). The test group showed
mean MT of 56.03% 3.49% and 57.71% 5.31%

with and without membrane coverage, respectively.

The PCG showed mean MT of 55.13% 4.83% and
49.69% 3.81% with and without membrane coverage, respectively. The NCG showed mean MT of
26.77% 7.29% and 19.67% 2.66% with and without membrane coverage, respectively (Table 1).
The PCG group showed no NVMT. The other groups
showed varying amounts of NVMT (Table 2).

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DISCUSSION
The definition of adult MSCs involves the observation
of several factors, such as the capacity to adhere to
the plastic of culture flasks; differentiation into three
mesenchymal cell types (osteoblasts, adipocytes, and
chondrocytes) through stimulation of extracellular
matrix; and the expression of specific cellular markers.
In this study, BMMF was used for the cell culture, rather than fresh bone marrow, because this procedure
avoids undesirable cell types.26 The mean number of
cells in this mononuclear cell fraction was 1.46 106
( 0.3). On the third day after seeding these cells and
immediately before the first culture medium change,
there were 15 ( 3) cells attached (0.001%), which coincides with the findings of Minguell et al.21 Assays for
differentiation of the adherent cells were performed
with supplementation of the culture medium for 21
days and stains specific to each type of cell. It was observed that all cells evaluated differed, which proves
the undifferentiated state of these cells before induction by culture medium.
The cells expressed markers CD14, CD16, CD73,
CD90, and CD105 and did not express markers CD31,
CD34, CD44, CD45, and CD107, showing that the cells
were BM-MSCs. These results are similar to those found
by Horwitz et al26 and Hashimoto et al.27 A previous
study1 that used the same critical-size defect in rabbits showed that the use of the scaffold without bone
marrow cells provided poorer results for all histomorphometric parameters assessed, which suggests that
cellular therapy increases bone regeneration, especially when BMMF or bone marrow stem cells are used.
For all groups evaluated by Pelegrine et al1 and for
the PCG and NCG of the present study, the use of a collagen membrane contributed to superior results, with
significantly higher levels of VMT. In guided bone regeneration, the membrane provides mechanical isolation of graft material from the overlying soft tissues.
However, in the present study, in the TG, no benefit was
conferred by the membrane (P > .05). The TG, in which
BM-MSCs were used, was the only group in which the
membrane did not provide any benefit. It may be that
an interaction between the autologous mesenchymal
stem cells and the bone marrow microenvironment
inhibits cell migration of the overlying soft tissues.
It is also possible that there is a greater induction of
angiogenesis and vascular endothelial permeability
(diapedesis) at the recipient site, as suggested by Blau
et al.28 The most probable hypothesis to explain the
similarity of action of stem cells with the technique
of guided tissue regeneration might be the barrier
action of the cells themselves; however, the MSCs do
not act as a physical barrier, as does a traditional membrane, but rather as some sort of physiologic barrier.

Further research is warranted to confirm or reject this

hypothesis.
The amount of VMT seen after the use of BM-MSCs
(TG) was similar to the BMMF in the study of Pelegrine
et al1 and higher than the PCG and NCG. This phenomenon can be explained by the presence of other populations in the bone marrow, in addition to the MSCs,
that play a role in bone regeneration.29
In the TG and the NCG, the level of mineralized
tissue was the sum of NVMT and VMT; on the other
hand, in the PCG, all the MT was VMT. This can be explained by the lack of NVMT in autogenous bone. In
the test group, the sum of NVMT (27.79% 2.72% and
27.23% 6.80% with and without membrane coverage, respectively) and VMT (28.24% 6.17% and
30.38% 4.65%), with and without membrane coverage, respectively) after 8 weeks was similar to the MT
observed in the PCG (P > .05). In the group in which
only the xenograft was used (NCG), this sum (NVMT
plus VMT) was lower. In the NCG, most of the MT was
NVMT. This can be explained by the great distance between the remaining bone walls, ie, the critical-size
bone defect. These results may also point to the incorporation of the MSCs into the Bio-Oss in the TG of the
present study to accelerate the healing process. Furthermore, the use of MSCs inhibited resorption of the
xenograft particles. However, regarding the presence
of a VMT specifically, the PCG showed a higher level,
and this might contribute to better osseointegration
of titanium implants.
The clinical use of BM-MSCs is associated with variations in the method by which stem cells are isolated
and expanded; methods are not the same around the
world, resulting in different outcomes. At present, the
routine clinical use of cell culture is not acceptable because of the absence of definitive established markers
for MSCs, which reflects a dangerous lack of standardization. Although Yamada et al30 demonstrated excellent results when using a culture of MSCs from bone
marrow associated with platelet-rich plasma for sinus
floor augmentation, it must be recognized that stem
cell culture has disadvantages versus other methodologies, such as the use of BMMF or fresh bone marrow,
the cost, the time between removal and transplantation of the tissue, the risk of contamination, and the lack
of agreement regarding the number of cells required.

CONCLUSIONS
The association of bone marrow mesenchymal stem
cells with a scaffold and the use of autogenous bone
graft, with the adjunct use of a barrier membrane, resulted in similar patterns of mineralized tissue, which
were better than the xenograft alone. However, the

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Aloise et al

use of autogenous bone graft resulted in higher levels of vital mineralized tissue. The use of a membrane
made no difference when bone marrow mesenchymal
stem cells were used.

ACKNOWLEDGMENTS
The authors wish to thank Geistlich Pharma, Brazil, for donating the xenogeneic biomaterials used in this study. The authors
have no relationships with the other manufacturers cited in this
article.

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Use of fresh bone marrow or bone marrow mononuclear fraction.
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