Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation: comparison with augmentation by autologous bone in adult sheep.

Abstract

Our aim was to compare the osteogenic potential of mononuclear cells harvested from the iliac crest combined with bovine bone mineral (BBM) (experimental group) with that of autogenous cancellous bone alone (control group). We studied bilateral augmentations of the sinus floor in 6 adult sheep. BBM and mononuclear cells (MNC) were mixed and placed into one side and autogenous bone in the other side. Animals were killed after 8 and 16 weeks. Sites of augmentation were analysed radiographically and histologically. The mean (SD) augmentation volume was 3.0 (1.0) cm(3) and 2.7 (0.3) cm(3) after 8 and 16 weeks in the test group, and 2.8 (0.3) cm(3) (8 weeks) and 2.8 (1.2) cm(3) (16 weeks) in the control group, respectively. After 8 weeks, histomorphometric analysis showed 24 (3)% BBM, and 19 (11)% of newly formed bone in the test group. The control group had 20 (13%) of newly formed bone. Specimens after 16 weeks showed 29 (12%) of newly formed bone and 19 (3%) BBM in the test group. The amount of newly formed bone in the control group was 16 (6%). The results show that mononuclear cells, including mesenchymal stem cells, in combination with BBM as the biomaterial, have the potential to form bone.

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Please cite this article in press as: Gutwald R, et al. Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation:
Comparison with augmentation by autologous bone in adult sheep. Br J Oral Maxillofac Surg (2009), doi:10.1016/j.bjoms.2009.06.226
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Mesenchymal stem cells and inorganic bovine bone mineral
in sinus augmentation: Comparison with augmentation by
autologous bone in adult sheep
Ralf Gutwalda,1,2, Jörg Haberstrohb,2, Jens Kuschnierza,1, Carola Kistera,1,
Dominikus A. Lysekc, Michele Maglioned, S.P. Xaviere,1,
Toshiyuki Oshimaa,1, Rainer Schmelzeisena,1, Sebastian Sauerbiera,∗
aUniversitätsklinik für Zahn-, Mund- und Kieferheilkunde, Abteilung Klinik und Poliklinik für Mund-, Kiefer- und Gesichtschirurgie,
Hugstetter Str. 55, D-79106 Freiburg, Germany
bDivision of Experimental Surgery, BioMed Center, University Hospital Freiburg i. Br., Hugstetter Str. 55, D-79106 Freiburg, Germany
cGeistlich Pharma AG, Division Biomaterials, Clinical Research Associate Bahnhofstr. 40, 6110 Wolhusen, Switzerland
dDepartment of Oral Surgery, University of Trieste, Via Stuparich n. 1, I 34125 Trieste, Italy
eDepartment of Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Sao Paulo, Av. Do Café, Campus USP, 14040-904,
Ribeirao Preto – SP, Brazil
Accepted 29 June 2009
Abstract
Our aim was to compare the osteogenic potential of mononuclear cells harvested from the iliac crest combined with bovine bone mineral
(BBM) (experimental group) with that of autogenous cancellous bone alone (control group). We studied bilateral augmentations of the sinus
floorin6adultsheep.BBMandmononuclearcells(MNC)weremixedandplacedintoonesideandautogenousboneintheotherside.Animals
were killed after 8 and 16 weeks. Sites of augmentation were analysed radiographically and histologically. The mean (SD) augmentation
volume was 3.0 (1.0)cm3and 2.7 (0.3)cm3after 8 and 16 weeks in the test group, and 2.8 (0.3)cm3(8 weeks) and 2.8 (1.2)cm3(16 weeks)
in the control group, respectively. After 8 weeks, histomorphometric analysis showed 24 (3)% BBM, and 19 (11)% of newly formed bone in
the test group. The control group had 20 (13%) of newly formed bone. Specimens after 16 weeks showed 29 (12%) of newly formed bone
and 19 (3%) BBM in the test group. The amount of newly formed bone in the control group was 16 (6%). The results show that mononuclear
cells, including mesenchymal stem cells, in combination with BBM as the biomaterial, have the potential to form bone.
© 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Animal trial; Biomaterial; Cell transplantation; Mesenchymal stem cells; Sinus augmentation
Introduction
Autogenous bone is the gold standard for augmentation of
bone.Variousmethodsoftissueengineeringhavebeentested
toimproveosteoconductivescaffolds,butthesearelimitedby
availability and the morbidity associated with harvesting.1,2
∗Corresponding author. Tel.: +49 761 270 4701; fax: +49 761 270 4800.
E-mail address: sebastian.sauerbier@uniklinik-freiburg.de
(S. Sauerbier).
1Tel.: +49 761 270 4701; fax: +49 761 270 4800.
2Both authors contributed equally.
Usually either cells or biologically active molecules are
combined with an osteoconductive scaffold.3,4The selection
ofthemostappropriatescaffoldsisanimportantsteptowards
the regeneration of hard tissue.
Bovine bone mineral (BBM) is one of the most widely
used scaffolds used in sinus augmentation.5–7Its physical
propertiesaresimilartothoseofhumancancellousbone,both
in its morphological structure and its mineral composition.
To improve the rate of formation of new bone using BBM
as a scaffold, several authors have studied the feasibility of
combiningitwithgrowthfactorssuchasbonemorphogenetic
0266-4356/$ – see front matter © 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2009.06.226

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Please cite this article in press as: Gutwald R, et al. Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation:
Comparison with augmentation by autologous bone in adult sheep. Br J Oral Maxillofac Surg (2009), doi:10.1016/j.bjoms.2009.06.226
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proteins BMP-2, BMP-7 (OP-1), or platelet-derived growth
factor (PDGF).8–12However, BMP are expensive. To form
bone in rodents and higher primates, up to several mg are
necessary,13in contrast to the naturally occurring concentra-
tions of BMP of 1?g/g of bone.14
The alternative idea, to combine a biomaterial with
osteogenic cells, has recently been gaining interest. One
option is the application of stem cells that originate from
bonemarrow.15Mesenchymalstemcells(MSC)orbonemar-
row stromal cells are defined as multipotent progenitor cells
with the ability to generate cartilage, bone, muscle, tendon,
ligament, and fat.16
Current publications in various fields have shown the
benefit of using a cocktail of mononuclear cells without
expanding them in vitro before reimplantation—such as
grafting of autogenous marrow cells in non-union of the
tibia or in peripheral arterial disease.17,18In maxillofacial
bone grafting, Smiler et al. have shown that the use of bone-
marrow-derived MSC is feasible but without concentrating
the cells.19
Wehaveinvestigatedtheapplicationofmononuclearcells
– among them mesenchymal and haematopoetic stem cells –
together with BBM.
Material and methods
Animals
Thistrialwasapprovedbytheanimaltrialcouncil.Themean
age of the 6 sheep was 3 years (range 2.5–3.3) and mean
weight 87.3kg (range 72–98). All animals were examined,
weighed, dewormed, and kept in quarantine for at least 2
weeks. Each animal was kept under the same conditions and
looked after by a veterinary specialist.
Study design
Three animals each were assigned to an observation period
of 8 and 16 weeks.
Operation
A bilateral sinus augmentation procedure under general
anaesthesia was done through an extraoral approach. The
anterior wall of the maxillary sinus was exposed inferiorly
to the lower orbital rim by mobilisation of the masseter
muscle and the adherent periosteum before preparation of
the lateral window. The left sinus (control) was augmented
with 3.5cm2of autogenous cancellous bone harvested from
the iliac crest. Bone marrow was aspirated from the iliac
crest. MSC were extracted by Ficoll separation. A total
of 3.5cm2MSC and BBM (Bio-Oss®; Geistlich, Wol-
husen, Switzerland) was transplanted into the right sinus
(experimental). The wounds were closed with resorbable
sutures.
Collection and processing of marrow cells
Bone marrow 60ml was aspirated with a heparinised biopsy
needle,pipettedontoFicoll(Sigma,StLouis,MO,USA)ina
ratioof1:1,andcentrifugedat2400rpmfor25min.Theinter-
face layer was removed, resuspended in phosphate-buffered
saline (PBS) and centrifuged again (2000rpm for 10min).
The supernatant was pipetted off for washing. This proce-
dure was repeated. The resulting cell pellet was resuspended
in PBS. Viable nucleated cells were counted by trypan blue
dye exclusion.
Assay of colony forming units
Cells were placed at four different densities (5000, 25,000,
50,000, 100,000 MNC/ml) in 96-well plates (Becton Dickin-
son, Los Angeles, CA) and cultured in MSCBM-medium
(Cambrex, USA) in a humidified atmosphere of 5% car-
bon dioxide at 37◦C for 7 days. The medium was initially
changed after 24h and then every second day. MSC were
selected by plastic adhesion and counted under a micro-
scope.
Fluorescence marking
Calcein 10mg/kg body weight was given during the second
and third weeks to the animals in the 8-week survival group
and during the tenth and eleventh weeks to the animals in the
16-week survival group. Xylenol-orange 90ml/kg of body
weight was given during the sixth and seventh weeks to the
animals in the 8-week survival group and during the four-
teenth and fifteenth weeks to the animals of the 16-week
survival group.
Volume rendering
Based on the data from computed tomograms (CT) the vol-
ume of the augmentation was calculated by the program
VoXim 4.3 (©IVS Solutions AG, Germany).
Histological and histomorphometric evaluation
After they had been fixed in formalin and dehydrated, the
samples were infiltrated with resin (Heareus Kulzer, Hanau,
Germany)andpolymerisedunderultraviolet(UV)light.Sec-
tions were cut with a diamante microsaw (Microslice, IBS,
Cambridge, GB), placed on an Acrylglas carrier (Maertin,
Freiburg, Germany) and reduced to a thickness of 100?m
on a rotating grinding plate (Struers, Ballerup, Denmark).
Eight slides of each augmentation side were selected and
evaluated for each animal. The fluorescent markings were
analysed before being stained with azur II and pararosanilin
(Axiovert 135, Zeiss, Kochern, Germany; AnalySISΛD Soft
Imaging system, Olympus Europa GmbH, Hamburg, Ger-
many).

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Comparison with augmentation by autologous bone in adult sheep. Br J Oral Maxillofac Surg (2009), doi:10.1016/j.bjoms.2009.06.226
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Results
Assay of colony forming units (CFU)
Ameanof40millionmononuclearcells(range22−84×106,
SD 26×106) were obtained. A mean (SD) of 86 (50)
CFU/million MNC were isolated.
Volume rendering
After 8 weeks we found volumes of 3.3 (0.9)cm3on the
experimentalsideand2.8(0.3)cm3onthecontrolside.After
16 weeks the corresponding volumes were 2.7 (0.3)cm3and
2.5 (1.2)cm3.
Histological results
After 8 weeks
Connective tissue was found in all specimens from the
control side. There were signs of extensive remodelling
of transplanted cancellous bone and active new bony for-
mation uniformly throughout the entire augmentation area
(Figs. 1A and 2A). Osteoclasts were resorbing cancel-
lous bone particles and neighbouring newly formed bone.
Haversian systems were detected in places where there was
remodellingofnewlyformedbone,buttherewasnolympho-
cytic infiltration.
On the experimental side there was uniform and substan-
tial new bone formation (Figs. 1B and 2B). The bone was
mature and compact with a structure containing organised
osteons. There were no gaps at the bone–particle interface,
and no inflammatory cells. In a few areas it was possible to
find small capillaries within the augmented tissue.
After 16 weeks
On the control side the autogenous bone showed a high rate
ofremodellingandgenerally,histologicalfindingsweresim-
ilar to those after 8 weeks. Histological features were also
similar at 16 weeks to those at 8 weeks. Osteoclasts were
in the process of resorbing BBM particles and neighbour-
ingnewlyformedbone.Therewasnoinflammatoryinfiltrate
at the interface of the particles, and there were capillaries
adjacent to the bone.
Histomorphometric results
After 8 weeks
On the control side 2.0 (0.6)% were transplanted bone, 20
(12)% newly formed bone, and 78 (9)% fibrous tissue. Of
the bone 8 (6)% was formed before week 3 and 12 (5)%
between weeks 3 and 8 (Fig. 3A). On the test side 24 (3)%
was composed of BBM particles, 19 (11)% of newly formed
bone, and 57 (9)% of soft tissue. Of the newly formed bone 3
(2)% was formed within the first 3 weeks and 15 (9)% after
8 weeks (3A).
After 16 weeks
On the control side the volume of bone graft was estimated
at 4%. The rate of newly formed bone decreased to 16 (6)%,
and the amount of fibrous tissue was 80 (6)%. Of the bone
formed before week 11, 8 (5)% was newly formed, and a
further 6 (1)% until week 16 (Fig. 3B). On the experimental
Fig. 1. Histological section from an animal killed after 8 weeks. Overview of the whole augmentation area of the control side (autogenous bone) (original
magnification ×10). (A) Newly formed tissue is stained red and the marrow space white/blue (pararosanilin azur II staining). The outstanding difference on the
test side is the rarified trabeculae, resulting in a large marrow space. (B) The fluorescence microscopy picture shows the formation of new bone (fluorescence
marking with calcein-green and xylenol-orange).

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Fig. 2. Fluorescence microscopy of a histological section from an animal killed after 8 weeks. Overview of the whole augmentation area of the experimental
side (BBM and bone marrow cells) (original magnification ×10). (A) The newly formed tissue is stained red and the marrow space white/blue (pararosanilin
azur II staining). Because of the biomaterial there is more hard tissue than in the control group. (B) The fluorescence microscopy picture shows the formation
of new bone (fluorescence marking with calcein-green and xylenol-orange).
Fig. 3. Histomorphometric analysis of the augmenation sites after (A) 8 and (B) 16 weeks for the experimental (test) and control groups. The results are
displayed for each individual animal and as a mean for each group. Calcein stained areas=white; xylenol-orange=light grey; autogenous bone/BBM=grey;
marrow space=dark grey.
side the rate of BBM stayed at 19 (3)%, and newly formed
boneincreasedto28(2)%.Atotalof53(12)%wassofttissue,
and 8 (7)% of bone was newly formed before week 11 and
20 (6)% between weeks 11 and 16 (Fig. 3B).
Discussion
Graftingofthesinusfloorisconsideredtohavebeensuccess-
ful if dental implants can be inserted, and if they are stable
overtime.Theintegrationofthetransplantandtheosseointe-
gration of inserted implants are faster when autogenous bone
is added than with pure biomaterials. Thorwarth et al. found
better bone forming kinetics in BBM with 25% autogenous
bone than with BBM alone.20In the present study the addi-
tion of mononuclear cells led to formation of bone similar
to that of autogenous bone. Both groups showed that a mean
of 19% of new bone had formed. The earlier bone formation
(within the first 3 weeks in the control group) indicated a
lag time for MSC. Four months after augmentation the rate
of formation was similar in the two groups. The resorption
rate in the control group was higher resulting in less newly
formedboneandasmallervolumeoftransplant.Thevolume
maintenance on the experimental side was probably caused
by BBM, which does protect bone grafts from resorption.21
Thevolumeoftheautogenousbonedecreasedfromtheinitial
3.5cm3to 2.5cm3.
The yield of MNC added to BBM was not directly related
to the formation of bone. For example, sheep 3 had by far the
largestnumberofcells,butlessbonyregenerationthansheep
1. Other factors too could influence the rate of formation of
new bone. The rate in this study was comparable to results
reported elsewhere when BBM was mixed with autogenous
bone, and was rather higher than that from augmentations
made purely with BBM.22
Our results support the data of Hernandez-Alfaro et al.,
who found accelerated bone formation induced by autolo-
gousbonemarrowcellsinaclinicalfeasibilitystudyinwhich

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Comparison with augmentation by autologous bone in adult sheep. Br J Oral Maxillofac Surg (2009), doi:10.1016/j.bjoms.2009.06.226
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5
they used an ex vivo cultured and autologous bone-marrow-
derived cell product.23Comparisons were made within a
randomised investigation in humans who had bilateral sinus
augmentation with either BBM alone or combined with so-
called “tissue repair cells” (“TRC”, Astronom Biosciences,
Inc.).Whentheradiographicimageswereevaluated,thebony
dimensions measured had increased by 3 months postopera-
tively on the TRC/BBM side compared with the side given
BBMalone.Thehistomorphometricanalysisshowedthatthe
fractional surface area of BBM, as a ratio to total bone sur-
face area, was smaller on the TRC/BBM side. A tendency to
increase the formation of bone and to reduce the fractional
surface area of BBM confirmed the results in the present
animal trial. Bone-marrow-derived cells with BBM seem to
support the formation of bone.
Some recent studies have reported a technique called
“injectable bone”.24This involves the morphogenesis of
new bone tissue from isolated cells associated with bio-
compatible scaffolds. One month before the operation,
MSC are isolated from marrow aspirated from the patient’s
iliac crest. After in vitro culture, the cells are trypsinised
and used for implantation into human maxillary sinus.3,25
Despite some encouraging results, the number of patients
operated on is still small and the follow up period
short.
Theproceduresofseparating,growing,anddifferentiating
thelimitednumberofstemcellsmaketheclinicalapplication
difficult and expensive. More studies using control groups,
and more animal or clinical trials, are necessary to evaluate
theeffectivenessandreliabilityofthismethodcomparedwith
thesimplemethodofaspirationandcentrifugationusedinthe
present study.
Acknowledgements
We thank Ute Hübner1, Heike Jahnke2, Annette Lindner2,
andDr.HeinerNagursky2fortechnicalassistance.1CellLab-
oratory, and2Hard Tissue Research Laboratory, Department
for Oral- and Maxillofacial Surgery, Head: Prof. Dr. Dr. R.
Schmelzeisen, University Hospital Freiburg, Germany.
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