99mtc-Methyl-Diphosphonate Binding To Mineral Deposits in Cultures of Mscs in Osteogenic Medium

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© Mary Ann Liebert, Inc.

DOI: 10.1089/ten.TEC.2018.0299
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TC‐METHYL‐DIPHOSPHONATE ( TC‐MDP) BINDING TO
MINERAL DEPOSITS IN CULTURES OF MARROW‐DERIVED
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
MESENCHYMAL STEM CELLS IN OSTEOGENIC MEDIUM
Tobias Grossner 1,2,3,; Tobias Gotterbarm 1,2,3; Victor H. Gerbaudo 4; Uwe Haberkorn
TC‐METHYL‐DIPHOSPHONATE BINDING TO MINERAL DEPOSITS IN CULTURES OF MSCs IN OSTEOGENIC MEDIUM (DOI: 10.1089/ten.TEC.2018.0299)
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;Myron Spector 2,3
1
University Hospital Heidelberg
Downloaded by STOCKHOLMS UNIVERSITET from www.liebertpub.com at 12/19/18. For personal use only.
Center for Orthopedics, Trauma surgery and Paraplegiology
Clinic for Orthopedics and Trauma surgery
Heidelberg, Germany
2
Tissue Engineering
Tissue Engineering
VA Boston Healthcare System
Boston, MA 02130
3
Department of Orthopedic Surgery
Brigham and Women's Hospital
Harvard Medical School
Boston, MA 02115

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4
Division of Nuclear Medicine and Molecular Imaging
Harvard Medical School
Boston, MA 02115
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Department of Nuclear Medicine
Heidelberg, Germany
CORESSPONDING AUTHOR
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Dr. med. Tobias Grossner M.D.
Center for Orthopedics, Trauma surgery and Paraplegiology
Clinic for Orthopedics and Trauma surgery
Schlierbacher Landstrasse 200 A, 69118 Heidelberg
Germany
Phone: +49 6221 56 35 443
Fax.: +49 6221 56 26 300
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Email: tobias.grossner@med.uni‐heidelberg.de

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CO‐AUTHORS
Prof. Dr. med. Tobias Gotterbarm
Kepler University Hospital
Department of Orthopedics
4021 Linz, Krankenhausstraße 9
AUSTRIA
Phone: +43 (0)5 7680 83 – 3199
Fax.: +43 (0)5 7680 83 – 743199
Email : Tobias.Gotterbarm@kepleruniklinikum.at

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Prof. Dr. med. Uwe Haberkorn
Department of Nuclear Medicine
INF 410, 69120 Heidelberg
Germany
Phone: +49 6221 56 7731
Fax.: +49 6221 56 5288
Email: uwe.haberkorn@med.uni‐heidelberg.de

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Victor H. Gerbaudo, PhD, MSHCA
Nuclear Medicine ‐ ASB1‐L1‐037A
75 Francis St , Boston MA 02115 USA
Phone: +1 (617) 732‐6290
Fax: +1 (617) 582‐6056
Email: gerbaudo@bwh.harvard.edu

Myron Spector, PhD
V.A. Medical Center ‐ Boston
Tissue Engr/Orthopedic Surgery, MS151
150 S Huntington Ave Jamaica Plain MA 02130 USA
Phone: +1 857/364‐6639
Fax: +1 857/364‐6791
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Email : mspector@bwh.harvard.edu

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ABSTRACT
Osteogenic differentiation of adult mesenchymal stem cells is a promising method for the
therapy of critical size bone defects and other applications, drawing attention to the
importance of in vitro assays for its evaluation. While there are standardized protocols to
induce osteogenic differentiation in monolayer cultures, there is a lack of validated
nondestructive procedures to quantify the osteogenic potential of mesenchymal stem cells
directly by accessing the mineral deposition. Here, we present a new method to
determine the osteogenic potential of mesenchymal stem cells using the radioactive
tracer, 99mTc‐MDP, to bind in vitro to newly produced mineral deposits from osteogenic‐
induced stem cells.
35mm petri dishes were seeded with goat and human MSCs and differentiated into the
osteogenic lineage. 99mTc‐MDP was applied to each dish, and acquisition of the bound
tracer was imaged with a gamma‐camera. The results revealed a significantly higher
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uptake in the osteogenic‐differentiated MSCs for both goat and human cells (p < 0,008
goat; 0,01 human), compared to controls grown in expansion medium. We were able to
show that 99mTc‐MDP labeling is a suitable method to quantify the amount of extracellular
mineral deposited by mesenchymal stem cells. Goat and human MSCs displayed similar
uptake characteristics (Spearman‐Rho Correlation coefficient r = 0,603). The radionuclide
method was validated by quantitative alizarin red staining, which showed a high
correlation (Spearman‐Rho Correlation coefficient r = 0,668). This radionuclide method
opens a new approach for evaluating osteogenic potential of mesenchymal stem cells and
for longitudinal studies of the process.

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IMPACT STATEMENT
The work is notable for describing a highly sensitive, quantitative and non‐destructive
method for evaluating the in vitro amount of mineral accompanying different types of
osteogenic differentiation of mesenchymal stem cells in a monolayer cell culture.
What is so unique and useful about the method is that it has the potential to be used to
define the kinetics of the differentiation process, reflected in the mineralization, without
destroying the monolayer. Therefore, it remains intact for further experiments.
KEY WORDS
Mesenchymal stem cells, osteogenic differentiation, mineral quantification, Technetium

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INTRODUCTION
Numerous in vitro studies have investigated the effects of osteogenic cell culture media on
bone marrow stromal cells (also referred to as mesenchymal stem cells, MSCs) [1‐3].
Methods to evaluate the formation of mineral deposits and expression of bone‐specific
proteins have been employed to track the differentiation of MSCs into osteoblast‐like cells
when the cells are grown in osteogenic medium. Mineralization of the extracellular matrix
in monolayer culture has been imaged by various histochemical staining techniques, in
which the applied dye reacts with the calcium content of the extracellular hydroxyapatite
(Ca5 (PO4)3(OH) or Ca10(PO4)6(OH)2. Von Kossa staining as well as Alizarin red staining is
generally accepted as a standard stain for imaging the mineral deposits [4‐9], but only the
Alizarin red stain has been accepted by the International Society for Cellular therapy as an
acceptable method to demonstrate osteogenic differentiation of MSCs [10].
There continues to be a need for a reliable method to quantify directly the amount of
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mineral deposited in the cultures and the time course of deposition. One of the most
common methods is to provide a semi‐quantitative assessment of the amount of alizarin
red staining by extracting the extracellular calcium‐bound dye in an extensive procedure
followed by photospectrography [9]. This process is time‐consuming, harmful to the cells
and the monolayer cell cultures are not available for further analysis. [11‐13]
Another qualitative and semi‐quantitative approach to demonstrate successful

differentiation into osteoblast‐like cells is to quantify and to assess the expression of bone
specific proteins, e.g., enzymes such as alkaline phosphatase, and to normalize the
obtained values to the DNA content of the monolayer cultures [3, 14].
Immunohistochemical staining for osteocalcin, osteopontin and osteonectin are also
applied methods [15]. All of these procedures, however, have the same disadvantage: they
are unable to show the hydroxyapatite content directly, as it is the most robust indicator
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of osteogenic differentiation. Furthermore, the prior methods require that the cell culture
be destroyed; therefore the cultures are not available for longitudinal studies. Other
methods which have been used, including scanning electron microscopy (SEM) [16, 17],
energy‐dispersive X‐ray microanalysis (EDX) [17, 18], transmission electron microscopy [13,
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17, 19, 20], x‐ray diffraction [17] and calcium radioisotope (45Ca) uptake [19] have this
same disadvantage.
For future clinical applications of osteogenic differentiated MSCs it is most important to
apply cells which are capable to produce a large amount of hydroxyapatite to maximize
the clinical effect, e.g., on bone healing. Therefore, there is a compelling need for a
standardized method able to directly quantify the amount of hydroxyapatite in the
extracellular matrix of MSC culture to identify cells potentially able to boost osteogenesis
in vivo in a clinical setting [21‐27]. Wang, et al., previously reported a method to directly
quantify the hydroxyapatite deposits produced by osteoblast‐like cells (viz., MC3T3 cells)
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cultured on polystyrene and Ti‐6Al‐4V alloy disks using a Technetium‐methylene
diphosphonate (Tc‐MDP) labeling method. 99mTc‐MDP is an established radionuclide, used
for decades in daily routine in nuclear medicine to detect bone lesions [28‐30]. Basis for
this innovative method is the ability of 99mTc‐MDP to bind to newly produced mineral in
vivo and in vitro. Here, radiotracer uptake is based on the phenomenon of chemisorption,
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with a typical binding energy of 800 kJ/mol, between the diphosphonate and the
hydroxyapatite crystals [31]. This novel approach was validated using inductively coupled
plasma to evaluate the calcium content of the cultures of mesenchymal stem cells
undergoing osteogenic differentiation[32].
The objective of the current study was to employ 99mTc‐MDP labeling to quantify the
mineralization of the extracellular matrix associated with the osteogenic differentiation of
mammalian MSCs (goat and human), when the cells were grown in osteogenic media for
differentiation. For comparison, cultures were also stained with von Kossa and alizarin red
stain for light microscopy and analyzed by energy dispersive x‐ray microanalysis (EDX) for
the presence of calcium and phosphorus in the deposits.
MATERIAL AND METHODS
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Experimental Design
Four groups of goat and human MSCs were employed in the study. All groups of cultures
were grown in expansion or osteogenic medium for 10 days, followed by a 21‐day culture
period in the same or alternate medium. In one group, MSCs were grown in expansion
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medium (EXP) for 10 days at which time the medium was changed to medium known to
stimulate the osteogenic differentiation of the cells. The MSCs were grown in this
osteogenic medium (OST) for 21 days. As a non‐differentiating control group, MSCs were
grown in EXP for the 21‐day period. In order to further investigate the effects of the type
of medium on the osteogenic differentiation of the cells, in a third group, MSCs were
initially expanded in the OST for 10 days and then grown in EXP for the ensuing 21‐day
period. In a fourth group, OST was used both during the 10‐day expansion period and the
21‐day differentiation period.
The radioactive labelling of the caprine cells was performed at the Division of Nuclear
Medicine and Molecular Imaging at the Brigham & Women’s Hospital, Boston MA USA;
and imaging was conducted using an E.CAM Variable gamma camera (Siemens Medical
Solutions, Hoffman Estates, IL, USA). The human cells were labelled at the Department of
Nuclear Medicine, University Hospital Heidelberg, Heidelberg, Germany, and imaging was
performed using a Siemens E.CAM+ gamma camera, (Siemens, Germany).
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Goat Bone Marrow Aspiration and Isolation of Marrow Stroma Cells
Bone marrow aspirates were obtained from the posterior iliac crests of 6 female Spanish
goats. After skin incision, penetration of the cortical bone was accomplished with a
Jamshidi‐Needle for bone marrow aspiration, 8‐gauge x 10 cm (Henry Schein Inc., Melville,
NY). From each iliac crest, 2 ml of bone marrow was aspirated into a 10ml syringe
containing 50U heparin (10U/ml, BD PosiFlush Syringe, Becton, Dickenson and Co., Franklin
Lakes, NJ). Isolation and primary culture of the MSCs were performed using a modification
of published protocols; cells from the 6 goats were grown separately. Briefly, the 2 ml of
anti‐coagulated bone marrow was diluted with 2 ml of phosphate‐buffered saline (PBS,
Invitrogen, San Diego, CA, USA), layered on 3 ml of Ficoll‐Paque™ (GE Healthcare,
Piscataway, NJ, USA) and centrifuged at 3000 rpm for 30 minutes. The resulting
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mononuclear cell layer was transferred from the interface, washed two times with PBS,
resuspended in EXP: low‐glucose Dulbecco’s modified Eagle’s medium (DMEM‐LG,
Invitrogen,) containing 10% heat inactivated (56ºC, 30 minutes) fetal bovine serum (FBS,
Invitrogen), 1% Penicillin/Streptomycin (Invitrogen). Cells were plated in 150 cm2 tissue
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culture flasks at a density of approximately 4×104 cells/cm2. The MSCs were selected
based on their ability to adhere to the tissue culture polystyrene. Non‐adherent cells were
removed with the first medium change after 48 hours. The culture medium was
subsequently changed 3 times per week. Upon reaching 80‐90% confluence, the caprine
cells were trypsinized and frozen at Passage 0 (P0) in liquid nitrogen in 0.5 ml aliquots
containing 5x105 cells in DMEM‐LG with 20% FBS and 10% dimethyl sulfoxide (DMSO,
Sigma).
Human Bone Marrow Aspiration and MSC Isolation
Bone marrow aspirates were obtained from the proximal femoral cavity of 6 healthy
donors under general anaesthesia during an elective surgical procedure for total hip
arthroplasty after informed consent and with ethics committee approval from the ethics
committee board, University Hospital Heidelberg, Heidelberg Germany.
During preparation of the proximal femoral bone cavity, 10‐15ml of bone marrow were
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collected into a 20ml syringe (BD) containing 1000 IU of heparin (5000 IU/ml, Braun).
Individual samples were diluted 1:1 with PBS and washed twice with phosphate buffered
saline (PBS). The mononuclear cell fraction was isolated by Ficoll‐gradient centrifugation
(Ficoll‐Paque™ PLUS; GE Healthcare). Mononuclear cells were plated in T‐150 polystyrene
tissue culture flasks (Falcon) at a density of 5*105/cm2 and cultured in a humidified 5% CO2
atmosphere at 37°C in EXP. After 48 hours, non‐adherent cells were removed and the
adherent cells were washed with PBS. Expansion medium was changed every 2 to 3 days.
At 90% of confluence cells were trypsinized. For further experiments, these P0 cells were
frozen in liquid nitrogen in 0.5 ml aliquots containing 5 x 105 cells in DMEM‐LG with 20%
FBS and 10% dimethyl sulfoxide (DMSO, Sigma).
Osteogenic Differentiation Assay
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P0 goat and human MSCs (n=6) were thawed and seeded in duplicates into T‐150 flask
(Falcon®) with 250,000 cells each, and cultured for 10 days to get enough cells for the
further experiments. While one flask of each donor was treated with EXP medium for 10
days, the other flask was filled and cultured with OST medium to differentiate the MSCs
towards the osteogenic lineage. For the goat cells EXP‐media supplemented with 100 nM
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dexamethasone (Sigma), 10 mM ß‐glycerol phosphate (Sigma) and 0.05 mM L‐ascorbic
acid‐2‐phosphate (Wako) was used. The human MSCs were treated with a slightly different
osteogenic media, using 100 nM dexamethasone (Sigma), 10 mM ß‐glycerol phosphate
(Sigma), and 0.170 mM L‐ascorbic acid‐2‐phosphate (Wako). Medium was changed every
two days. After 10 days, when the cultures reached about 80‐90% of confluence, cells
from each donor and group (EXP and OST) were trypsinized, resuspended and seeded at a
density of 30,000 cells/cm2 in duplicates into 35mm Petri dishes (Cornig). One dish from
each donor and expansion group was treated with EXP, and the other one with OST. The
cells were cultured at 37°C, 5% CO2 for 21 days, medium changed every two days.
In total we used four experimental groups for this study: 1) 10 days Expansion in EXP
media followed by 21 days cultivation in EXP media; 0 days of osteogenic differentiation.
2) 10 days Expansion in EXP media followed by 21 days cultivation in OST media; 21 days
osteogenic differentiation. 3) 10 days Expansion in OST media followed by 21 days
cultivation in OST media; 31 days osteogenic differentiation. 4) 10 days Expansion in OST
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media followed by 21 days cultivation in EXP media; 10 days osteogenic differentiation.
Duplicate cultures were prepared, with one dish from each donor and group analysed by
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Tc‐MDP Labelling followed by histology staining for mineral (von Kossa or alizarin red),
and the other identical dish employed only for SEM/EDX.
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Tc‐MDP Labelling of MSC Cultures
After 21 days of cell culture in OST or EXP, medium was removed from the cultures and the
3.5 cm diameter dishes were carefully washed twice with phosphate‐buffered saline (PBS,
Invitrogen). An aliquot of 5,5 MBq 99mTc‐MDP in 2 ml of NaCl 0,9% was added to each dish.
The activity of the Technetium was assayed with a dose calibrator (Boston: CRC‐15R,
Capintec, Ramsey, NJ, USA / Heidelberg: Activimeter ISOMED 1010, Nuklear‐
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Medizintechnik Dresden GmbH, Dresden, Germany). The dishes were then incubated at
room temperature for 2 hours. The remaining liquid 99mTc‐MDP was removed and the
dishes were washed three times (for approximately 30 minutes) in PBS to remove the
unbound radiotracer. The dishes (n=24 goat, 24 human) were simultaneously placed
under a gamma camera (Boston: E.CAM Variable, Siemens / Heidelberg: E.CAM+,
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Siemens). The dishes containing the goat cells were placed directly on the lower detector,
the second detector was placed above with a distance of 15cm and the counts were
acquired using both detectors. For analysing the human cells, only the detector over which
the cells were placed on (one‐detector setting) was used for counting and imaging. The
radionuclide counts were acquired for 3 minutes and then the images were analyzed using
Siemens software (Boston: E.SOFT 2.0, Siemens / Heidelberg: Xeleris, GE Healthcare). The
total number of counts for each culture dish was determined by placing a circular region of
interest (ROI) around each dish. ROIs had the same pixel size and were chosen a bit smaller
than the true diameter of the dish so that the very outer parts were excluded to eliminate
the edge effect, as the tracer accumulates here. The software calculated the total gamma
counts emitted from each ROI over the 3 minutes. For the goat cells, the counts from the 2
detectors were found to be within 10% of each other. The average from the 2 detectors
was used for statistical analysis.
One blank dish was labelled in parallel each time to detect the amount of background
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radionuclide binding. The amount of the blank binding was subtracted from the results
obtained from the dishes containing cells. After imaging, the dishes were stored at room
temperature for 72 hours to allow 99mTc‐MDP to decay to background levels.
Scanning Electron Microscopy (SEM) and Energy Dispersive X‐Ray Microanalysis (EDX) of
MSC Cultures
Goat MSC‐seeded dishes were fixed in 1 ml of 2.5% glutaraldehyde for 1 h, air dried. Goat
MSC cultures were observed with an environmental SEM (ESEM, XL30, FEI/Philips,
Hillsboro, OR), and EDX performed using the attached X‐ray analyzer on the XL30 ESEM.
Human MSCs culture dishes were carbon sputtered and investigated using a LEO 440 SEM
(LEO 440 REM, Zeiss, Oberkochen, Germany). The backscattered mode was used for
element weighted images and element analysis was performed with the Inca EDX‐System
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(Oxford Instruments, Oxfordshire, UK).
Qualitative Von Kossa Staining (caprine cells)
For mineral staining, the same cell culture dishes were used which were prior investigated
by 99mTc‐MDP labelling. The 35 mm‐diameter dishes of the caprine cultures were washed
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with distilled water and 2 ml of a 5% silver nitrate (Sigma) solution was added to the
dishes. The wells were placed under an ultraviolet lamp for 30 minutes. The unbound
silver ions were removed by adding 2 ml of a 5% sodium thiosulfate solution (Sigma) for
two minutes followed by washing with aqua dest.
Quantitative Alizarin Red Staining (human cells)
For osteogenic semi‐quantification of monolayer cell cultures the same cell culture dishes
were used which were prior investigated by 99mTc‐MDP labelling. The 35mm dishes were
fixed with 2,5ml 70% Ethanol (‐20°C) for 4 minutes followed by washing with aqua dest.
For the preparation of the staining solution, to 100ml aqua dest, 0,5g Alizarin Red S
powder (Sigma‐Aldrich A5533) was added. pH was measured, using a pH‐meter and by
adding ammonium‐hydroxide (Sigma 221228). pH was adjusted to 7,2. Subsequently, 1ml
of the staining solution was added to each dish and were incubated at room temperature
for 10 minutes followed by 5x washing with 1ml aqua dest. Then 1 ml of a 10% (w/v)
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cetylpyridiniumchloride solution dissolved in 10mM sodium phosphate was added to each
dish. After incubation for 10 minutes at room temperature on a shaker, 20µl from each
sample was put in a 96‐well plate and 180µl of the 10% (w/v) cetylpyridiniumchloride
solution was added. In parallel, standard dilution with Alizarin‐Red concentration between
62,5 µg/ml – 0,01 µg/ml was prepared in the 96‐well plate. Photometric absorption was
determined at 570nm in a 96‐well plate reader, followed by plotting a standard curve with
the results of the standard dilution. The absorption values of the samples were transferred
into alizarin red concentration by referring to the standard curve.
Statistics
One‐factor Analysis of variance (ANOVA with post hoc Test Bonferroni) and Fisher’s
protected least square differences (PLSD) post hoc testing was performed using Statview
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(SAS Institute, Cary, NC). Statistical significance was set to p < 0,05. Correlation analysis
was performed by IBM SPSS Statistics 20.

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RESULTS
Qualitative Light Microscopy
Von Kossa staining of the goat MSC cultures showed none to minimal positive staining in
the control group (EXP/EXP). In contrast, there was clear positive von Kossa staining in the
OST groups (EXP/OST and OST/OST), reflecting an increase in mineralized matrix over time.
The group treated with OST media during cell expansion showed a slightly positive stain
for mineral deposition.
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Tc‐MDP Labeling
Goat and human MSCs showed a noticeable effect of the OST for the 21‐day and 31‐day
differentiation period (EXP/OST, OST/OST) in culture with respect to the amount of binding
of the radiotracer (Fig. 1A and B). The highest uptake was generally seen in islands at
various locations in the dishes. There was variability in the uptake among goat and human
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MSC cultures. The mean gamma counts for the EXP/OST group was 11,879 (goat), 24,614
(human) and for the OST/OST group 9,314 (goat), 29,935 (human) while the mean gamma
counts for the EXP/EXP group was 5,159 (goat), 4268 (human) and for the OST/EXP group
5,630 (goat), and 4,208 (human)
Quantification of the Technetium counts (Fig. 2 A and B) reflected the qualitative uptake
findings. The mean values for the goat cell cultures in which the OST was used for the 21‐
day differentiation period were up to 2‐fold higher than the samples culture in EXP/EXP
(Fig. 2A), and for the human samples many‐fold higher (Fig. 2B).
One factor ANOVA with Bonferroni post hoc testing of the goat cell cultures demonstrated
a significant effect of medium on the radiotracer uptake for the EXP/EXP group compared
to the EXP/OST group (p = 0,03) and for the OST/EXP group compared to the EXP/OST
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group (p = 0,049).
One factor ANOVA with Bonferroni post hoc testing of the human cell cultures
demonstrated a significant effect of medium on the uptake for the EXP/EXP group
compared to the EXP/OST group (p = 0,005) and the OST/OST (p = 0,001). Also, there was a
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statistical significant difference for the gamma counts of group OST/EXP compared with
EXP/OST (p = 0,005 and OST/OST p=0,001).
Given that samples from individual goats displayed substantially more uptake than others,
a separate analysis of the goat samples was conducted in which the high and low
Technetium count values were omitted (resulting in n=4). In this case, the one‐factor
ANOVA displayed a more pronounced effect of medium on the uptake (p<0.001;
power=0.99). Fisher’s PLSD post hoc testing for the goat data demonstrated that the
following group comparisons were statistically different: EXP/EXP versus EXP/OST and
OST/EXP versus EXP/OST. In the case of the human samples, the following group
comparisons were statistically different: EXP/EXP versus EXP/OST; EXP/EXP versus
OST/OST; OST/EXP versus EXP/OST; and OST/EXP versus OST/OST.
Quantitative Alizarin Red Staining
The EXP/EXP controls only showed a minimal mean alizarin red content with 0.30 µg/ml
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while the samples of the osteogenic group (EXP/OST) displayed a mean alizarin red
content of 5,01 µg/ml. The OST/EXP group had a minimal mineral content, reflected by a
mean alizarin red concentration of 0,637 µg/ml while the OST/OST group had a significant
mineral deposit with an alizarin red concentration of 5,164 µg/ml. One factor ANOVA with
Bonferroni post hoc testing of the alizarin red content of the human cell cultures
demonstrated a significant effect of osteogenic medium on the stain concentration for the
EXP/EXP group compared to the EXP/OST group (p = 0,034) and the OST/OST (p = 0,027).
In addition, there was a statistical significant difference for the gamma counts of group
OST/EXP compared with OST/OST (p = 0,044).
For validation of the new method, Spearman‐Rho correlation analysis of the gamma
counts versus alizarin red content demonstrated a highly significant correlation with a p
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value < 0.001, r = 0.603. (Fig. 3)
The light microscopy of the stained dishes with a 10x magnification showed a nodule like
formation within the cell cultures of the EXP/OST group (Fig. 4 A) as well as for the
OST/OST group stained clearly positive for Alizarin red, reflecting centers of
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mineralization. The EXP/EXP group (Fig. 4B) showed no nodule formation and only an
unspecific slight background stain.
SEM/EDX
SEM of representative goat and human MSC cultures demonstrated the presence of a
substantially greater number of nodular deposits scattered through the monolayer culture
in the samples grown in osteogenic medium (EXP/OST) (Fig. 5A), in comparison to the
cultures with expansion medium (EXP/EXP) (Fig. 5B). EDX analysis of the nodular deposits
in the cultures grown in osteogenic medium displayed the prominent presence of calcium
and phosphorus (Fig. 6A). In contrast, in controls grown with expansion medium there
was almost no detection of calcium and phosphorus. (Fig. 6B).
DISCUSSION
MSCs are an attractive cell source in regenerative medicine [33‐35]. MSCs are capable to
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differentiate into different cell lineages including osteoblast‐like cells [3], and have been
evaluated for clinical use to facilitate and stimulate bone repair in critical size defects [22,
23, 36, 37]. There is lack of a non‐destructive method to proof the formation of mineral
deposits and to track the MSC differentiation into osteoblast‐like cells. Currently available
methods such as von Kossa and Alizarin red staining or alkaline phosphatase activity assays
are time consuming, are destructive to the cells in culture and are not capable to directly
show the hydroxylapatite content [32].
In this study we could show, that the a novel technique of 99mTc –MDP labelling, is a
suitable, reliable, non‐destructive and easy‐to‐handle method to quantify the calcium
deposition, e.g., osteogenic potential, synthesised by osteogenic differentiated goat and
human mesenchymal stem cells. These cells produce an anorganic matrix which
mineralizes by forming hydroxylapatite (Ca10(PO4)6(OH)2). The radiotracer binds with high
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affinity to this newly produced mineral as seen in the osteogenic treated groups (EXP/OST,
OST/OST). As shown by EDX/SEM the nodular configuration seen in these groups of the
monolayer cultures had significant more Calcium / Phosphorus content than the control
group, assuming that the tracer was bound to this anorganic matrix. While validating our
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novel method to the gold standard method of quantitative alizarin red staining, it was
found that there was a high significant correlation (r = 0.668, p < 0,001).
Our data using 99mTc‐MDP clearly indicates that human and goat cells display a similar
differentiation behavior for both cell lines, because both cell lines are mammalian. The
gamma counts of the human and goat cell cultures correlated at a high significant level (p
= 0,002; r = 0,603 Spearman Rho). This method is thus recommended for use with
osteogenic cells from other species.
As already stated by Wang et al. the short half‐live of 99mTechnetium of only 6,01h calls for
accurate dosage, close attention to incubation time, washing procedure and scanning
time. As noted from the live‐surveillance imaging of the gamma‐camera it is very likely that
also within a shorter acquisition time significant tracer uptake can be detected. Moreover,
the incubation time with the radiopharmaceutical can probably be reduced as well. We do
not expect the Technetium, to be harmful to the cells as extensive studies have been
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performed in vitro, confirming that even after 18 hours of exposition to the tracer, there
was no significant difference regarding the viability of leucocyte cells [38].
To confirm that the method does not interfere with osteogenic differentiation of MSCs in
vitro, further experiments are planned. Within the upcoming study we will look deeper
into the differentiation of the cells by analyzing biochemical markers and correlating the
findings with the amount of produced mineral and the 99mTc‐MDP uptake.
As this method clearly works with monolayer cell cultures on polystyrene, it is to assume
that also a three‐dimensional construct seeded with osteogenic differentiated cells can be
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quantified with the Tc‐MDP binding method. Investigating the labeling of other
monolayer culture surfaces should be possible, as long as a blank probe is labeled in
parallel to detect the background uptake. Here, it might be possible to quantify the
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osteoinductive potential of promising new biomaterials. Regardless of the blank specimen
it will be difficult to label monolayer cultures which consist out of a certain amount of
calcium (e.g. hydroxylapatite), as parts of the radiotracer would probably bind onto the
contained calcium as well, because of the affinity of the bisphosphonate present in the
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Tc –MDP.
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This radionuclide method for evaluating osteogenic differentiation warrants further
implementation in a longitudinal study of the mineralization process and to assess the
osteogenic potential of stem cell populations for clinical use.
ETHICS APPROVAL
For the harvesting and use of the goat stem cells permission and approval for animal
studies was given by VA Boston Healthcare System.
Permission‐and Protocol number: IACUC #221‐J‐0706.
For the harvesting and use of the human stem cells permission and approval was given by
the Ethics committee of the University Hospital Heidelberg, Medical Faculty, University
Heidelberg.
Protocol number: S‐309/2007.
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ACKNOWLEDGMENTS
Funding was provided, in part, by the U.S. Department of Veterans Affairs.
DECLERATIONS
Competing interests
None of the authors of this manuscript have any competing interests to declare.
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Figure Legends

which the cells were obtained is shown at the top of the figure.

by the 21‐day differentiation period is shown to the right. The goat subject number from
counts over 180 seconds. The medium used during the 10 day expansion period followed
Figure 1 A: Gamma camera images of the (A) goat (n=6) MSCs after acquisition of gamma
23
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subject number from which the cells were obtained is shown next to each dish.
by the 21‐day differentiation period is shown above or under the image. The human
counts over 180 seconds. The medium used during the 10 day expansion period followed
Figure 1B: Gamma camera images of the human MSCs (n=6) after acquisition of gamma
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MSC cultures grown in the various sequences of media are shown.

Figure 2: Mean values for 99mTc‐MDP counts/180 seconds ± SEM for (A) goat and (B) human
25
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Figure 3: Linear correlation analysis of the
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concentration (n = 12) showing a linear correlation of R2=0.77.

Tc‐MDP binding and the Alizarin Red

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nor a postive stain.

qualitative analysis. (A) Group EXP/OST showing the presence of positive stained
stained with alizarin red for the presence of calcium before extracting the dye for
nodules reflecting mineralisation centers while (B) Group EXP/EXP showed no nodules
Figure 4 A and B: 10x magnified light microscopy images of the human MSC cell cultures
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Page 29 of 30
500x magnification

control group (B). While there are many mineral deposits in the cultures grown in

Figure 5 A and B: SEM Images of the monolayer of the osteogenic group (A) and the
osteogenic medium, there are few in the control culture grown in expansion medium (B).
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Figure 6 A and B: EDX analysis of the monolayers in (A) and (B), respectively, showing the
definite presence of calcium and phosphorous in the osteogenic group (A). There is almost
none calcium and phosphorous in the expansion medium control group (B). The high
carbon amount in both analyses comes from the electrically conducting carbon layer which
was applied to the samples prior to SEM and EDX.
Tissue Engineering
99m

99mtc-Methyl-Diphosphonate Binding To Mineral Deposits in Cultures of Mscs in Osteogenic Medium

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99mtc-Methyl-Diphosphonate Binding To Mineral Deposits in Cultures of Mscs in Osteogenic Medium

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Another qualitative and semi‐quantitative approach to demonstrate successful

16. Boyde, A., et al., Scanning electron microscopy of bone cells in culture. Cell Tissue

34. Csaki, C., P.R. Schneider, and M. Shakibaei, Mesenchymal stem cells as a potential

Tc‐MDP binding and the Alizarin Red

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