TISSUE ENGINEERING: Part C

Volume 16, Number 2, 2010

ª Mary Ann Liebert, Inc.
DOI: 10.1089=ten.tec.2009.0269

In Vivo Comparison of Hard Tissue Regeneration

with Human Mesenchymal Stem Cells Processed
with Either the FICOLL Method or the BMAC Method

Sebastian Sauerbier, M.D.,1,* Andres Stricker, M.D., D.D.S.,1,* Jens Kuschnierz, M.D., D.D.S.,1
Felicia Bühler, D.D.S.,1 Toshiyuki Oshima, D.M.D.,1,2 Samuel Porfirio Xavier, D.D.S.,3
Rainer Schmelzeisen, M.D., D.D.S.,1 and Ralf Gutwald, M.D., D.D.S.1

Objective: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial
associated with mesenchymal stem cells (MSCs) separated by two different isolation methods.
Background: In regenerative medicine open cell concentration systems are only allowed for clinical application
under good manufacturing practice conditions.
Methods: Mononuclear cells, including MSCs, were concentrated with either the synthetic poylsaccharid (FI-
COLL) method (classic open system—control group, n ¼ 6 sinus) or the bone marrow aspirate concentrate
(BMAC) method (closed system—test group, n ¼ 12 sinus) and transplanted in combination with biomaterial.
A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD
1.0) bone biopsies
were obtained and analyzed.
Results: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9–29), and in
the FICOLL group was 15.5% (90% CI, 8.6–22.4). The 4.4% difference was not significant (90% CI, 4.6–13.5;
p ¼ 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages.
Conclusion: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can
form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to
substitute the open FICOLL system in bone regeneration procedures.

Introduction These new techniques of tissue engineering for bone recon-

struction may emerge as an alternative to conventional

P reclinical and clinical studies have demonstrated

the ability of bone marrow–derived stem and progenitor
cells to regenerate many tissues, including bone.1,2 Current
literature in various fields of medicine shows the benefit of
using a cocktail of mononuclear cells (MNCs) without ex-
panding them in vitro before reimplantation. Hernigou et al.
have demonstrated the healing of tiba nonunions with
grafting of autogenous marrow cells.3 Esato et al. showed
the advantage of injecting MNCs from bone marrow into
peripheral arterial disease, leading to neovascularization
around the injection sites.4 In the field of maxillofacial bone
grafting, Smiler et al. have shown that the use of bone
marrow–derived mesenchymal stem cells (MSCs) is feasible,
but they did not concentrate the cells.5 Soltan et al. suggest
that the use of bone marrow aspirate (BMA) that contains
adult stem cells may become a new ‘‘platinum standard.’’6
grafts. Harvesting of MSCs involves little donor-site surgery,
whereas autogenous bone harvesting is costly and associated
with certain morbidity. It also requires general anesthesia
and hospitalization.7,8 The minimal invasiveness of bone
marrow aspiration may give the possibility to save the re-
generative advantages of autologous bone grafting but
eliminates general anesthesia, hospitalization, and patient’s
morbidity.
The reinfusion of enriched progenitor cells and infarct
remodeling in acute myocardial infarction (REPAIR-AMI)
and autologous stem cell transplantation in acute myocardial
infarction (ASTAMI) trials showed differences in contractile
recovery of left ventricular function after infusion of bone
marrow–derived cells in acute myocardial infarction.9–11
These outcomes evoked a discussion about the effect of dif-
ferent MNC-possessing methods.12 A better understanding

1
Department of Oral and Craniomaxillofacial Surgery, University Hospital Freiburg, Freiburg, Germany.
2
Faculty of Health Sciences, Institute for International Cooperation, Okayama, Japan.
3
Department of Oral Maxillofacial Surgery and Periodontology, Faculty of Dentistry of University of Sao Paulo, Ribeirao Preto, Brazil.
*These two authors contributed equally to this work.

215
216
of the mode of action is required to optimize tissue-
engineering procedures for clinical applications.13,14
As most of the previous clinical trials involved bone
marrow–derived MNCs isolation by synthetic poylsaccharid
(FICOLL), this technique is currently viewed as the gold
standard.9,10,15 The FICOLL method can be an effective
procedure for MNC concentration that may be useful mainly
in hospitals. Thus, the limitations of the FICOLL method are
the time needed for its procedure and that a good
manufacturing practice (GMP) laboratory is required. Closed
systems are therefore necessary for the clinical use in oper-
ating facilities without GMP possibilities.16,17
To evaluate if the open FICOLL method can be substituted
by a closed system the new bone formation resulting from
transplanted MNCs was compared in patients who received
cells concentrated either by the FICOLL method or by the
closed bone marrow aspirate concentrate (BMAC) system.
Materials and Methods
Study population and protocol
The protocol was approved by the ethics committee of the
University of Freiburg, and the study was conducted in ac-
cordance with the Declaration of Helsinki. Patients older
than 18 years with need of dental implant placement in the
posterior maxilla were eligible for the study if they had a
maximum of 3 mm residual height of the alveolar ridge. In
addition, they had to be able to comply with study-related
procedures as, for example, returning for follow-up exami-
nations, exercising good oral hygiene, and being able to
understand the nature of the proposed surgery. Informed
consent was obtained before surgery. Exclusion criteria were
smoking, history of malignancy, radiotherapy or chemo-
therapy, pregnancy or nursing, general contraindications for
dental or surgical treatment, medication, treatment or dis-
ease, which may have an effect on bone turnover, bone or
connective tissue metabolism, and allergy to collagen.
Six sinus from four patients at an average age of 59.5 years
(range, 50–69 years) were comprised in the FICOLL group.
Twelve sinus from seven patients at an average age of 55
years (range 47–68 years) were included in the BMAC group.
There were more sinus in the BMAC group because later the
FICOLL method was completely replaced by the BMAC
system. A total of 50 implants were placed in a second sur-
gical intervention (17 FICOLL=33 BMAC). Implant survival
was evaluated after 1 year.
Harvesting of stem cells
The pelvic bone was punctured about 2 cm latero caudally
from the superior posterior iliac spine with a bone marrow
biopsy needle. With three 20 mL syringes (B. Braun, Mel-
sungen, Germany) each containing 0.3 mL of heparin solu-
tion (Heparin-Natrium, 10.000 U=mL, diluted with NaCl to
1000 U=mL, both B. Braun), 60 mL of bone marrow was
collected. The aspirate was pooled and anticoagulated with
5 mL of heparin solution (Heparin-Natrium, 10.000 U=mL,
diluted with NaCl to 1000 U=mL, both B. Braun). The BMA
was given to the laboratory to separate the mononucleated
cells with the FICOLL method. According to the instructions
of the manufacturer bone marrow cells were isolated directly
in the operating room using the BMAC system (Bone Mar-
SAUERBIER ET AL.
row Procedure Pack; Harvest Technologies, Plymouth, MA).
For the FICOLL method the aspirate was immediately pi-
petted on Ficoll (Sigma, St. Louis, MO) at a 1:1 ratio and
centrifuged at 2400 rpm for 25 min. The interface layer was
removed, resuspended in phosphate-buffered saline (pH
7.4), and centrifuged (2000 rpm, 10 min). For washing, the
supernatant was pipetted off. This procedure was repeated
once more. The resulting cell pellet was resuspended in
10 mL phosphate-buffered saline. The number of viable nu-
cleated cells was counted by the trypan blue dye exclusion
method. The procedure of selecting mononucleated cells out
of the BMAs took about 50 min.
Colony forming unit assay
To evaluate the number of MSCs out of the number of
mononucleated cells, a probate amount was kept and used for
continuous growth. The cells were plated in 96-well plastic
plates (Becton Dickinson, Los Angeles, CA) and cultured in
MSCBM Medium (Cambrex Corporation, East Rutherford,
NJ) in a humidified atmosphere of 95% air and 5% CO2 at 378C
for 7 days. Each sample was plated at a density of 100,000
nucleated cells=mL. The medium was initially changed after
24 h and then every second day. Nonadherent cells were
washed from the culture. MSCs were selected on the basis of
adhesion and proliferation on tissue culture plastic substrate.
Cells were counted under an inverted phase-contrast micro-
scope at a final magnification of 200.
Proof of pluripotency
The cells from the bone marrow concentrate were amplified
and differentiated into three cell lineages according to the
methods of Pittenger and coworkers.18 The lines consisted of
the chondrogenic, adipogenic, and osteogenic phenotype,
proofing that the transplanted cells were actually pluripotent
stem cells. Adipocytes were stained with oil red O, a lipophilic
red dye. Chondrogenic potential was confirmed by im-
munostaining with mouse anti-human aggrecan antibodies.
Osteogenic cells were characterized histologically by their
expression of high levels of alkaline phosphatase, collagen
type I, and calcification marked with the van Kossa staining.
Sinus augmentation procedure
A pedicled mucoperiosteal flap was raised to expose the
lateral wall of the maxillary sinus. A bone window of ap-
proximately 15–20 mm was outlined by a round burr at ap-
proximately 800 rpm with constant saline irrigation. The
Schneiderian membrane was detached from the bony surface
of the sinus floor. The site was augmented with bovine bone
mineral (BBM, Bio-Oss
0.25–1 mm; Geistlich Pharma AG,
Wolhusen, Switzerland) and enriched with mononucleated
cells in thrombin (Fig. 3). In the FICOLL group the thrombin
component of commercially available fibrin glue (Tissue Col;
Baxter Immuno, Heidelberg, Germany) and in the BMAC
group autologous thrombin produced from venous blood
(Thrombin Kit; Harvest Technologies) were used. A collagen
membrane (Bio-Gide
; Geistlich Pharma AG) was used to
cover the facial sinus wall defect on the surface of grafted
site. The mucoperiosteal flap was replaced, and wound clo-
sure was performed using resorbable suture material Vicryl
4.0 (Ethicon, Norderstedt, Germany). In the evening of the
COMPARISON OF FICOLL AND BMAC IN HUMANS 217

operating day the patients were discharged from the hospital
according to the outpatient protocol. After a 3-month healing
time of the augmented site, implant placement was per-
formed in a second-stage procedure under local anesthesia.
Cylindrical bone biopsies from the augmented maxillary
sinus were taken with a trephine burr (Gebr. Brasseler GmbH
KG, Lemgo, Germany), with 600 rpm. Then, after drilling to
the right diameter, screw-type implants were inserted.
Three months after implant insertion the implants were
exposed under local anesthesia and healing screws were in-
serted. Prosthetic attachment followed 4 weeks after implant
impression
signs of inflammation. The newly formed osseous lamellae
appeared as vital bone tissue containing osteocytes inside the
bone lacunae. In comparison to lamellar bone the biomaterial
could be easily identified by its size, shape, and color. In the
Azur II Pararosanilin staining the newly formed bone ap-
peared darker red than the BBM particles. The newly formed
bone lamellae connected the biomaterial particles and stabi-
lized the grafted complex. Bone formation appeared in the
macro pores of the biomaterial as well. Blood vessels could
be detected in the biopsy. The biomaterial with the newly
formed bone was integrated well in the surrounding host
bone (Fig. 1).

Histological evaluation Histomorphometric analysis

The burrs with the bone biopsies were fixed in formalin An overview of the estimated values and the 90% CIs of
for 48 h, rinsed in water, and dehydrated in serial steps of new bone formation, biomaterial, and marrow space are
alcohol (70%, 80%, 90%, and 100%) remaining for 3 days in displayed in Figure 2. The values for each histological slide
each concentration. After dehydration the samples were in- are given in Table 1.
filtrated with resin (Technovit 7200 VLC; Heareus Kulzer,
Hanau, Germany) for 2 weeks. The resin was polymerized in New bone formation
a UV light chamber for 10 h. After hardening, two sections of The estimated value of new bone formation for FICOLL,
300–400 mm thickness and parallel to the axis of the burr measured in percent, was 15.5%, with a 90% CI from 8.6% to
were cut with a diamante micro saw (Microslice; IBS, Cam-
bridge, UK). The sections were placed on an acrylic slide
(Maertin, Freiburg, Germany) and reduced to a thickness of
approximately 100 mm on a rotating grinding plate (Struers,
Ballerup, Denmark). The specimens were stained with Azur
II and Pararosanilin.
The histomorphometric examination was done with a
light microscope (Axiovert 135; Zeiss, Kochern, Germany).
The differentiation between BBM particles and the new
formed bone tissue was possible. The BBM particles were
marked and the new formed bone around the particles was
measured. The marking and measurements were performed
with the computer software AnalySISD Soft Imaging system
(Olympus Europa GmbH, Hamburg, Germany).

Statistical analysis
For the parameters NewBone (new bone formation), BioOss
(Biomaterial), and Marrows (marrow space), values were ex-
pressed in % of the evaluated area. A linear mixed mode (lme
of package nlme. under R) was used for analysis; 90% CIs
were computed from the contrast tables of the model.19,20

Results
All patients participated through the end of the study. No
patient dropped out. All patients recovered well from the
surgical procedure. Postoperatively, there were no com-
plaints about pain, hematoma, or infection at any time after
bone marrow aspiration and sinus floor augmentation.

Healing time
Average healing time was 143
39 days for group
FICOLL and 112
17 days for BMAC. The difference was
not statistically significant ( p ¼ 0.22).
Histological analysis
Histological specimens of both FICOLL and BMAC
showed similar results. The histological sections showed no
FIG. 1. A histological specimen after a healing time of 3.3
months. In the Azur II–Pararosanilin staining newly formed
bone is stained magenta. Connective tissue is marked blue
(A). For histomorphometrical evaluation residual bone is
marked yellow and bovine bone mineral is marked green
(B). It is visible that the new bone formation does not only
originate from the residual bone but also occurs in the aug-
mented area around the bovine bone mineral particles.
218 SAUERBIER ET AL.

FIG. 2. Above: Estimated values and 90% confidence intervals (CIs) of absolute values for new bone, biomaterial, and
marrow space. Below: Differences for bone marrow aspirate concentrate synthetic poylsaccharid (BMAC-FICOLL), with CIs
for the differences. Note that differences show significance even when CIs of absolute values overlap because of the crossed
experimental design. The difference of bone formation is not significant ( p ¼ 0.39). With 95% probability, new bone formation
of BMAC is not more than 4.58% points below the estimated FICOLL new bone formation of 15.5%. Biomaterial is signifi-
cantly higher for BMAC ( p ¼ 0.019). With 95% probability, biomaterial is higher for BMAC by at least 4.32% points. Both
limits of the CI for the difference are negative, so marrow space is significantly less for BMAC ( p ¼ 0.01). With 95%
probability, marrow space is less for BMAC by at least 7.48% points.

22.4% (Fig. 2, Table 1). This means that for FICOLL with 5%
probability the expected value is less than 8.6% and with 5%
probability it is greater than 22.4%. The estimated value of
BMAC is 19.9%, with a 90% CI from 10.9% to 29%. The
estimated value of the difference in new bone formation
between BMAC and FICOLL, the reference, was 4.4%. The
90% CI of the difference ranged from 4.6% to 13.5%. With
5% probability the expected difference of new bone forma-
tion between BMAC and FICOLL is therefore less than
4.6% and with a 5% probability greater than 13.5%. As the
signs of the lower and upper limits of new born formation
are different, the CI of the difference covers the value of 0.
This indicates that the difference in new born formation is
not significant ( p ¼ 0.39). With 95% probability, new bone
formation of BMAC is not more than 4.6% below the esti-
mated FICOLL new bone formation of 15.5%.
Biomaterial
The estimated value of biomaterial for FICOLL measured
in percent is 19.7%, with a 90% CI from 13.7% to 25.8%. The
estimated value of BMAC is 19.7 þ 12.2% ¼ 31.9%, with a
COMPARISON OF FICOLL AND BMAC IN HUMANS 219

Table 1. Histomorphometrical Values in % of Area for Each Histological Slide

Pat Sinus Slide New bone Biomaterial Marrow space Healing time Treatment

1 1 1 24.3 0.2 75.5 193 FICOLL

1 1 2 24.6 0.0 75.4 193 FICOLL
1 1 3 16.5 2.3 81.1 193 FICOLL
1 1 4 20.0 2.0 78.0 193 FICOLL
2 2 1 18.5 20.4 61.1 98 FICOLL
2 2 2 3.4 13.5 83.1 98 FICOLL
2 3 1 14.4 34.4 51.2 98 FICOLL
2 3 2 18.9 29.4 51.7 98 FICOLL
3 4 1 10.8 21.0 68.2 141 FICOLL
3 5 1 14.5 20.2 65.4 141 FICOLL
4 6 1 14.9 32.3 52.8 139 FICOLL
5 7 1 31.5 34.2 34.3 102 BMAC
5 7 2 29.6 35.8 34.6 102 BMAC
5 7 3 39.2 33.1 27.7 102 BMAC
5 8 1 34.1 26.0 39.9 102 BMAC
5 8 2 26.2 35.8 38.0 102 BMAC
6 9 1 26.5 23.8 49.7 130 BMAC
6 9 2 22.6 30.7 46.7 130 BMAC
6 10 1 29.5 27.4 43.1 130 BMAC
6 10 2 21.9 35.7 42.4 130 BMAC
7 11 1 19.0 32.4 48.6 137 BMAC
7 11 2 16.7 21.1 62.2 137 BMAC
7 11 3 20.6 34.9 44.5 137 BMAC
8 12 1 12.5 39.5 48.0 97 BMAC
8 12 2 11.8 37.3 50.9 97 BMAC
8 13 1 19.0 29.1 51.9 97 BMAC
8 13 2 15.9 32.1 52.0 97 BMAC
9 14 1 10.1 40.6 49.3 91 BMAC
9 14 2 8.2 45.3 46.5 91 BMAC
10 15 1 20.5 23.7 55.8 113 BMAC
10 15 2 17.7 18.8 63.5 113 BMAC
10 16 1 1.5 38.3 60.2 113 BMAC
10 16 2 1.0 41.7 57.3 113 BMAC
11 17 1 21.6 35.7 42.7 115 BMAC
11 17 2 24.9 31.1 44.0 115 BMAC
11 18 1 34.9 24.3 40.8 115 BMAC
11 18 2 26.3 25.3 48.4 115 BMAC

The clinical situation did not allow obtaining the same number of biopsies from each patient. The healing time is given in days. The raw
data are displayed to show the interindividually different regenerative potential and to facilitate metaanalysis in the future.
FICOLL, synthetic polysaccharid; BMAC, bone marrow aspirate concentrate.

90% CI from 24% to 39.7%. The estimated value of the dif-
ference in biomaterial between BMAC and the reference
FICOLL is 12.2%, with a 90% CI from 4.32% to 20%. This
means that with a 5% probability the expected Biomaterial
difference between BMAC and FICOLL is less than 4.32%
and with a 5% probability it is greater than 20%. Both limits
of the CI for the difference are positive, so biomaterial is
significantly higher for BMAC ( p ¼ 0.019). In other words,
with 95% probability, biomaterial is higher for BMAC by at
least 4.32% points.
Marrow space
The estimated value of marrow space for FICOLL, mea-
sured in percent, is 64.8%, with a 90% CI from 57.1% to
72.5%. The estimated value of the marrow space in the
BMAC group was 64.8 þ 17.4% ¼ 47.4%, with a 90% CI
from 37.6% to 57.3%. The estimated value of the difference in
marrow space between BMAC and the reference FICOLL is
17.4%, with a 90% CI from 27.2% to 7.48%. This means
that with 5% probability the expected difference of the
marrow space between the BMAC- and the FICOLL group is
less than 27.2% and with 5% probability it is greater than
7.48%. Both limits of the CI for the difference are negative,
so marrow space is significantly less for BMAC ( p ¼ 0.01).
With 95% probability, marrow space is less for BMAC by at
least 7.48% points.
Colony forming unit assay
The number of colony forming units (CFUs) did not
differ significantly. There were 28.7 (
14.1) CFUs=1106
bone marrow (BM)-cells in the FICOLL and 25.0 (
11.4)
CFUs=1106 BM-cells in the BMAC group.
Proof of pluripotency
The cultured MSCs could be differentiated successfully
into adipocytes as shown by oil red O staining, chondrocytes
as shown by aggrecan immuno staining, and osteoblasts as
220 SAUERBIER ET AL.

FIG. 3. Proof of Pluripotency. Plastic adherend cells from both groups could be differentiated as follows: Morphology of
cultured mesenchymal stem cells (MSCs) stained with methylene blue (A). Adipocyte differentiated from MSC. The adi-
pocyte is rounded and filled with lipid droplets that might fuse to form vacuoles that can be stained by Oil Red O, a lipophilic
red dye (B). Chondrocyte nodule cultivated by differentiation of MSCs. The extracellular protein aggrecan is specific for
chondrocytes. The immunohistochemical staining indicates the presence of aggrecan by its red fluorescence (C). Osteoblasts
differentiated from MSCs. Alkaline phosphatase is stained blue (D). Collagen type I is immunohistochemically indicated by
the brown staining (E). Calcification is revealed by a black color in the van Kossa staining (F).
COMPARISON OF FICOLL AND BMAC IN HUMANS
shown by calcification, alkaline phosphatase, and collagen
Type I activity (Fig. 3).
Follow-up and implant survival
Although no implant out of 17 was lost in the FICOLL
group, only one implant out of 33 failed in the BMAC group
before prosthetic loading. No implant was lost after loading.
All 49 osseointegrated implants were loaded and in function.
Up to now, all patients were subject to follow-up examina-
tions 2 years after implant placement.
Discussion
In the present study the bone-forming ability of cells iso-
lated with a point-of-care device for BMA was assessed in
comparison to bone marrow cells isolated by FICOLL. This
in vivo study was carried out in humans for two reasons. One
was to find out if the procedures enable bone growth in the
maxillary sinus to facilitate dental implant insertion. The
other reason was to see whether results from animal trials
with FICOLL can correlate to the outcome in humans when
the BMAC device is used.21
Sinus floor grafting is considered to be successful if dental
implants can be placed into a posterior maxilla that had
originally an insufficient bone height and if the implants are
stable over time. The one implant lost in the BMAC group is
of no significance as it is within the normal loss rate.22 There
are some advantages of autogenous bone for sinus aug-
mentation. For instance, the integration of the transplant and
the osseointegration of inserted implants are faster compared
to pure biomaterials. Thus, an earlier implant loading is
possible. Thorwarth et al. have shown that in the first 8
weeks BBM with 25% autogenous bone has superior bone-
forming kinetics compared to BBM alone.23 For longer
healing times this gap narrows and disappears after 3
months. The aim of the presented tissue engineering ap-
proach is to speed up initial bone formation and therefore
obtain earlier bone formation and osseointegration of the
implant. The present study has shown that adding MNCs to
BBM, among them MSCs, can lead to faster bone formation
compared to BBM alone. Both groups, FICOLL and BMAC,
were similarly effective. Yildirim et al. examined sinus ele-
vation with pure BBM in 15 sinus of 11 patients and found
14.7% (SD
5.0%) new bone formation after a healing time
of 6.8 months.24 Compared to the results, 15.5% new bone
formation after 4.8 months with FICOLL and 19.9% new
bone formation after 3.7 months with BMAC, this indicates
that adding MNCs containing MSCs is beneficial. The vol-
ume maintenance is most likely due to BBM, which has been
shown to protect bone grafts from resorption.25–27 The higher
BBM contents in the BMAC group may depend on the use of
autologous thrombin. It does not produce such a firm clot as
the commercial thrombin component. This is why in the
FICOLL group the BBM particles cannot be packed as dense
as in the BMAC group. The behavior of the marrow space is
reciprocal to the BBM value. Both methods, FICOLL and
BMAC, are suited to enhance new bone formation. The
number of mononuclear cells and mesenchymal stem added
to BBM was not directly related to the bone formation. This
indicates that there are other factors involved that determi-
nate the rate of new bone formation. Even without direct
relation between the number of added MSCs to BBM and the
221
rate of new bone formation, the growth rate is comparable to
the results described in literature, when BBM was mixed
with autogenous bone.26 The new bone formation in the
present study is higher when compared to histomorphome-
trical analysis of sinuses augmented purely with BBM.24 The
data presented here also support the data of Hernandez-Al-
faro, who found accelerated bone formation rates induced by
autologous bone marrow cells in a clinical feasibility
study, using an ex vivo cultured and autologous bone
marrow–derived cell product.28 Comparison took place
within a randomized investigation in humans undergoing a
bilateral sinus augmentation by using either BBM alone or in
combination with the so-called tissue repair cells (TRC;
Astronom Biosciences, Ann Arbor, MI). Compared to the
pure BBM side the bone dimensions evaluated by radio-
graphic imaging increased on the TRCþBBM side 3 months
after surgery. The histomorphometrical analysis revealed that
the fractional surface area of BBM, in relation to the total bone
surface area, was smaller on the TRCþBBM side. Shayesteh
successfully augmented severely atrophied sinus in six pa-
tients with tricalcium phosphate=hydroxyapatite scaffolds
that were vitalized with cultured MSCs.29 Although the bone
marrow cell–processing method differs from the one used in
the present investigation, it could be stated that the method is
feasible and that bone marrow–derived cells in maxillary si-
nus augmented with BBM seem to support bone formation.
Hermann et al. found 2.4 times more nucleated cells with
BMAC when comparing cells of 25 patients processed with
the BMAC or the FICOLL method in vitro.16 The same group
showed in an ischemic limb rodent model that the BMAC
group underwent faster regeneration than the FICOLL
group.16 This might be also because both separation methods
result in specifically different cell products. In a FICOLL
preparation 80–90% of the cells present are mononuclear, a
maximum of 5% are granulocytes, a maximum of 10% are
erythrocytes, and <0.5% are thrombocytes. The cells are
usually suspended in NaCl solution. The BMAC product is
suspended in autologous plasma and contains a maximum
of 54% granulocytes, a maximum of 34% MNC, and 17%
erythrocytes. The fact that the cells are not removed from
their natural plasma environment may help to sustain the
functionality of the cells.16 Another in vivo comparison of two
different BMA-processing methods (FICOLL vs. Lympho-
prep) was investigated in a murine model of hind limb is-
chemia by Seeger and coworkers.12 They concluded that cell
isolation protocols have a major impact on the functional
activity of bone marrow–derived progenitor cells and that
the assessment of cell number and viability may not entirely
reflect the functional capacity of cells in vivo.
Kasten et al. compared the FICOLL method in vitro with
two BMA-processing methods that use the closed SEPAX
system (Biosaye, Eysins, Switzerland).30 Compared to
FICOLL they found significantly higher numbers of CFUs
in the SEPAX-volume-reduction group followed by the
SEPAX-FICOLL group. As in the present study there, were
no significant differences in the differentiation abilities of
the cells. Sakai et al. introduced a simple centrifugal BMA-
processing method with a blood bag system that could be
potentially operated ‘‘chair side.’’ In 10 patients they found a
fivefold increase of nucleated and colony forming cells.31
The system has not been compared to others. With BMA
of 12 patients Horn et al. showed in vitro that red blood
222
cell-lyses with ammonium chloride could provide a more
effective alternative to the FICOLL method. The red blood
cell-lyses was faster, resulted in larger colonies (CFU-assay) 4.
and showed no differences in phenotype and differentiation
potential.32
The FICOLL method is an open system. The aspirate has
to leave the operating facility to be processed. This is costly, 5.
and transfusion regulations apply if the method is not used
in an experimental setting. A practical clinical approach
6.
therefore requires a closed system that can be operated chair-
side and does not require GMP facilities. The SEPAX system
is approved for the processing of umbilical cord blood. The
7.
Harvest BMAC system has the advantage that it is approved
for the processing of BMA for hard tissue regeneration.
Conclusion 8.
The FICOLL and the BMAC group showed comparable
new bone formation. When autologous thrombin is used, a
denser packing of biomaterial is possible than when cells are 9.
applied with the thrombin component of commercially
available fibrin glue. This leads to a higher proportion of
hard tissue in the BMAC group. The BMAC system is ef-
fective and suited for chair-side application. For a broad
10.
clinical application of this minimal invasive method a ran-
domized controlled trial with a closed chair-side system is
needed for further evaluation. In general, there is a lack of
in vivo comparisons of different BMA-processing methods.
Acknowledgments 11.
The authors are indebted to Ute Hübner,1 Heike Jahnke,2
Annette Lindner,2 Dr. Heiner Nagursky,2 Dr. Ali Al-Ahmad,3
and Dr. Rainer Schmelzeisen1,2 for excellent technical assistance.
1
Cell Laboratory, Department for Oral and Maxillofacial
Surgery, Head: Prof. Dr. Dr. R. Schmelzeisen, University
Hospital Freiburg, Germany.
2 12.
Hard Tissue Research Laboratory, Department for Oral
and Maxillofacial Surgery, Head: Prof. Dr. Dr. R. Schmel-
zeisen, University Hospital Freiburg, Germany.
3 cells used for cell therapy in patients with acute myocardial
Cell Laboratory, Department of Operative Dentistry, Head:
Prof. Dr. E. Hellwig, University Hospital Freiburg, Germany. 13.
Disclosure Statement
This study was technically supported by Harvest Tech-
nologies and Geistlich Biomaterials. Financial support for in
vitro work was provided by the Camlog Foundation. The 14.
laboratory work was financially supported by the Camlog
Foundation, Basel, Switzerland. Technical support was given
by Geistlich Biomaterials, Wolhusen, Switzerland, and Har-
vest Technologies, Munich, Germany.
15.
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Address correspondence to:
Sebastian Sauerbier, M.D.
Universitätsklinik für Zahn-
Mund- und Kieferheilkunde
Abteilung Klinik und Poliklinik für Mund-
Kiefer- und Gesichtschirurgie
Hugstetter Str. 55
D-79106 Freiburg
Germany
E-mail: sebastian.sauerbier@uniklinik-freiburg.de
Received: April 20, 2009
Accepted: May 27, 2009
Online Publication Date: July 7, 2009