Post on 08-May-2023
Characterization of biomimetic calcium phosphate labeled with fluorescent dextran for
quantification of osteoclastic activity.
Salwa M. Maria1,2, Christiane Prukner3, Zeeshan Sheikh1, Frank Mueller3, Svetlana V.
Komarova1,2, Jake. E. Barralet1,4
1Faculty of Dentistry, McGill University, Canada, 2Shriners Hospital for Children-Canada,
Montreal, Canada, 3Institute of Materials Science and Technology, Friedrich Schiller
University, Jena, Germany, 4Orthopaedics Division, Department of Surgery, Faculty of
Medicine, Montreal General Hospital, McGill University
Corresponding author: Jake. E. Barralet, McGill University, Montreal, Quebec, Canada H3G IA6, Telephone: 514-398-3908
Abstract:
Bone resorbing osteoclasts represent an important therapeutic target for diseases associated
with bone and joint destruction, such as rheumatoid arthritis, periodontitis, and osteoporosis.
The quantification of osteoclast resorptive activity in vitro is widely used for screening new
anti-resorptive medications. The aim of this paper was to develop a simplified semi-
automated method for the quantification of osteoclastic resorption using fluorescently labeled
biomimetic mineral layers with which can replace time intensive, often subjective and clearly
non-sustainable use of translucent slices of tusks from vulnerable or endangered species such
as the elephant.. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using
the pro-resorptive cytokine receptor activator of nuclear factor kappa-B ligand (RANKL). We
confirmed that fluorescent labeling did not interfere with the biomimetic features of
hydroxyapatite, and developed an automated method for quantifying osteoclastic resorption.
Correlation between our assay and traditional manual measurement techniques was found to
be very strong (R2 = 99). In addition, we modified the technique to provide depth and volume
data of the resorption pits by confocal imaging at defined depths. Thus, our method allows
automatic quantification of total osteoclastic resorption as well as additional data not
obtainable by the current tusk slice technique offering a better alternative for high throughput
screening of potential antiresorptives.
Introduction:
Osteoclasts are bone cells responsible for the resorption of the mineralized tissues in the normal
bone remodeling process. The abnormal resorptive activity of osteoclasts occurring in serious
disorders, such as osteoporosis [1], rheumatoid arthritis [2]; [3]; [4]; [5], periodontitis [6] and
cancer metastasis to bone [7]. Therefore, targeting osteoclastic resorption is an important
therapeutic strategy. While a number of therapies including bisphosphonates [8] and anti-
RANKL denosumab [9] are now successfully used to limit osteoclastic activity, intolerant and
resistant cases necessitate the development of novel treatments. For this, the ability to rapidly
screen the candidate chemical compounds is important.
We have previously validated a method of precipitating a thin layer of hydroxyapatite on
tissue culture plates and glass coverslips [10] to study osteoclast resorptive activity [11]. Calcium
phosphate substrates, including hydroxyapatite, can be fluorescently labeled, for example with
calcein, which was used to quantify osteoblast mineralization [12], or fluoresceinamine labeled
chondroitin polysulfate or calcein to study osteoclastic resorption [13].
Dextran is a nontoxic, water-soluble polysaccharide of a complex branched glucan,
composed of many glucose molecules with variable chain lengths [14]. Dextran can be
conjugated to a variety of fluorophores which expand the possibility of using these substrates for
the fluorescence studies. Dextran has the ability to chemically bind to the hydroxyapatite [15];
[16]. Dextran can also be internalized by a number of cells capable of endocytosis, including
osteoclasts [17].
In this study we used dextran (10,000 Da), conjugated to Alexa Fluor488 or Rhodamine
B to fluorescently label hydroxyapatite, and validated that the new fluorescent substrate is
supportive of osteoclast formation and resorption and amenable for automated quantification.
Materials and Methods:
Reagents: Chemicals used for mineral coating were purchased from Sigma and Fisher Scientific.
Dulbecco’s modification of eagle’s medium 1x, (DMEM), fetal bovine serum (FBS, 08050),
penicillin-streptomycin (450-202-EL), sodium pyruvate (600110-EL) and 0.25% trypsin with
0.1% EDTA, (325-043-EL) were from Wisent Inc, Quebec, Canada. Fluorescein isothiocyanate-
labeled-phalloidin (Phalloidin conjugate FITC) P5282, alendronate (A4978) were from Sigma-
Aldrich, Ontario, Canada. Recombinant glutathione S-transferase-soluble RANKL was purified
from the clones kindly provided by Dr. MF Manolson (University of Toronto, Canada) and
freshly reconstituted before each experiment. Dextran Alexa Fluor® 488 (D-22910) and
Rhodamine B (D-1824) of 10000 Da were from Invitrogen, New York, USA. Lactate
dehydrogenase activity assay kit plus (LDH) was from Roche Applied Science, UK.
Hydroxyapatite coating: The following solutions were prepared freshly before each coating as
described in detail in [10], Briefly, 2.5-fold simulated body fluid (SBF) was prepared by
mixing 50% Tris buffer (50 mM Tris base, pH=7.4 with 1M HC1), 25% calcium stock solution
(25 mM CaCl2·H2O, 1.37 M NaCl, 15 mM MgCl2·6H2O in Tris buffer, pH 7.4), and 25%
phosphate stock solution (11.1 mM Na2HPO4·H2O, 42 mM NaHCO3 in Tris buffer, pH=7.4).
Calcium phosphate solution (CPS) was prepared by first adding 41 ml HCl (1M) to 800 ml
MilliQ water, then dissolving 2.25 mM Na2HPO4·H2O, 4 mM CaCl2·2H2O, 0.14 M NaCl and
50 mM Tris, then bringing pH to 7.4 and the volume to 1 litre. The solutions were sterilized by
filtration with a 0.22 µm MillexGV, Millipore. Glass coverslips or tissue culture plates were
incubated with SBF (0.5 ml/well) for 3 days at room temperature. SBF was aspirated and CPS
(0.5 ml/well) was added for 1 day at room temperature, aspirated and 70% ethanol was added
and evaporated to sterilize the surface. The hydroxyapatite coated coverslips and plates washed
twice with distilled water and dried overnight at 37oC, were either used immediately or kept dry
at room temperature for up to 1 month. Prior to cell plating, coated plates were incubated with
FBS for 1 hour at 37oC.
Labeling hydroxyapatite with fluorescent dextran: The 25 nM solution of Alexa Fluor 488- or
Rhodamine B-conjugated dextran in distilled water was prepared and filtered. Sterile
hydroxyapatite-coated glass coverslips and tissue culture plates were incubated with 200 µl of
the dextran solution at 37oC for 48 h, washed twice with deionized distilled water (Milli-Q
water) and kept sterile and protected from light.
Characterization of fluorescent-hydroxyapatite layers: Low angle X-ray diffraction (XRD) data
was collected with Ni filtered CuKα radiation (λ = 1.54A) with 2 dimensional VANTEC area
detector at 40 kV and 40 mA using Bruker Discover D8 diffractometer. Step size of 0.02° was
used to measure from 10 to 50° over 2 frames with a count time of 400 s per frame. Scanning
electron microscopy (SEM) was performed using Hitachi S-4700. Scanning Electron Microscope
(Tokyo, Japan) with field emission gun (FEG) at an accelerating voltage of 10 kV and current of
10 amps. The sample stage was tilted to an angle of 35 degrees. Images were acquired using
secondary electron detector. For SEM the samples were coated Gold/Palladium using Hummer
VI sputter system with Argon gas under 80 millitorr vacuum, 10 milliamps: plasma discharge
current and voltage of 2.5 V as described previously [11].
Osteoclast cultures, transfer and identification: To generate osteoclasts, murine monocytic
cells RAW 264.7 (ATCC) were plated at 5,000 cells per cm2 in DMEM supplemented with
10% FBS, 1% penicillin-streptomycin, 1% sodium pyruvate and 50 ng/ml GST-RANKL as
described previously [18]; [19] on plastic tissue culture plates, on hydroxyapatite, or on
fluorescent hydroxyapatite. The medium was replaced every 2 days, until formation of
multinucleated osteoclasts (on day 5–7 day) was observed. To transfer mature osteoclasts onto
unlabeled or fluorescent hydroxyapatite, we first generated osteoclast-like cells from RAW
264.7 cells in 100 mm tissue culture plates. Then the osteoclast-like cells were purified, re-
suspended, and replated on the substrates at 20-200 cells/cm2 using the transfer protocol
described previously [11]. Tissue culture plates were returned to the incubator carefully and
maintained for 24-48 h. For fluorescence imaging, samples were fixed in 3.7%
paraformaldehyde stained with FITC-conjugated phalloidin for actin, and examined under
inverted fluorescence microscope (Nikon ECLIPSE TE 2000-U, USA).
LDH cytotoxicity assay: Cytotoxicity was assessed by the leakage of LDH into the culture
medium using [20] the LDH activity kit from Roche Applied Science (LDL50). Cells were
incubated on plastic surface or on the fluorescent hydroxyapatite for 24 h in 96-well plates
(Fisher), or on plastic surface with lysis solution, and the absorbance was measured using the
micro-plate readers (Infinite F200 TECAN).
Characterization of osteoclastic resorption: In the end of the culture period, the osteoclast-like
cells were washed twice with PBS and removed by incubation with 0.2% Triton-X-100 in
sodium chloride (1M) for 1-2 min at room temperature, then washed twice with distilled water
[10]. Unlabeled hydroxyapatite was examined using bright field microscopy. The fluorescent
hydroxyapatite was examined using the inverted fluorescence microscope (Nikon, ECLIPSE
TE 2000-U, USA). Three-five images per condition were collected and analyzed using ImageJ.
For manual quantification, each resorption pit was individually outlined and the areas were
quantified. For automatic quantitation the color images were first adjusted to obtain the best
brightness/contrast for the visual inspection of the resorption areas, and converted to binary
images, which were visually confirmed to correspond to the original images. The dark areas
were quantified. Using the training set of images, we determined that areas smaller than 100
pixel could not be reliably associated with resorption activity and therefore the particles below
100 pixels were removed by filtering from all the images. The areas of the remaining particles
were analyzed. The amount of the fluorescent dextran (Alexa Fluor 488 'green' and Rhodamine
B 'red') in the media was measured using the microplate reader (Infinite F200, TECAN). The
resorption volume was assessed using the confocal microscopy (Zeiss LSM510-META and
Olympus Fluoview, FV10-ASW1.7). A z-stack was obtained, and the volume was calculated as
sum of resorption areas in each level multiplied by the distance between the levels. To estimate
the resorption depth using fluorescence microscopy without confocal capabilities, images were
obtained at three levels for the same area of the substrates - superficial layer, at the glass level
and at the middle (3-4 µm depth).The volume of the resorption was calculated as sum of
resorption areas in each level multiplied by the estimated distance between the levels.
Statistical analysis: Data are expressed as mean ± standard error of the mean or standard
deviation, with n indicating the number of independent experiments or replicates recpectively.
Statistical difference was evaluated by Student t-test, or ANOVA followed by Tukey post-test
where appropriate and were considered to be significant at p < 0.05. Coefficient and significance
of correlation was assessed using Vassar Statistical Recourse page.
Results:
Characterization of the fluorescent hydroxyapatite: We used dextran conjugated to fluorescent
label Alexa Fluor 488 or Rhodamine B to improve the visualization of the thin film of
hydroxyapatite precipitated on the surface of glass coverslips or tissue culture plates as described
previously [11]. We have analyzed the degree of labeling of hydroxyapatite with fluorescent
dextran in the concentration range of 12.5-500 nM and found that sufficient fluorescence was
achieved even at low dextran levels (Fig. 1A). The labeled substrates were then incubated in
phenol red-free media for 48 h, and media fluorescence was measured. No significant
fluorescence was observed in media after incubation with hydroxyapatite labeled with 12.5-500
nM dextran, indicating stability of labeling. Low angle XRD patterns indicated the layer was
phase pure hydroxyapatite (Fig.1B). SEM demonstrated homogeneous and complete coating
prior to cell culture (Fig.1C) and after incubation with media without cells (Fig.1D). After
culture with RAW 264.7 cells differentiated with 50ng/ml into osteoclast-like cells for 5 days
characteristic resorption lacunae were evident (Fig.1E).
Fluorescent hydroxyapatite is not toxic and supports osteoclastogenesis: To examine if the
fluorescent hydroxyapatite have toxic effects on cultured cells, LDH cytotoxicity assay was
performed. RAW 264.7 cells were incubated on the fluorescent hydroxyapatite or the tissue
culture plates (control) for 24 h, and LDH release to the media was assessed. As a positive
control, cells were incubated with lysis solution. No cell toxicity of the fluorescent
hydroxyapatite was found (Fig.2A). To examine if fluorescent hydroxyapatite supports
osteoclastogenesis, were plated and cultured either untreated or treated with RANKL (50 ng/ml)
for 5 days to induce osteoclastogenesis. The fluorescent hydroxyapatite was stable after the
incubation with culture media alone (Fig.2B) or RAW 264.7 cells (Fig.2C). Multinucleated
osteoclast-like cells successfully formed on the fluorescent hydroxyapatite and were surrounded
by resorption zone free from coating (Fig.2D, white arrowhead). Both undifferentiated
RAW264.7 and osteoclasts internalized fluorescent dextran (Fig.2C,D yellow arrows). The
osteoclast-like cells formed on fluorescent hydroxyapatite exhibited actin ring normally
associated with resorption [21] (Fig.2E). We directly examined osteoclast resorptive activity on
the parallel samples of hydroxyapatite either unlabeled or labeled with fluorescent dextran, and
observed a significant correlation between the average resorption areas resorbed by osteoclasts
incubated on labeled and unlabeled hydroxyapatite, R2= 0.99, p <0.0001 (Fig.2F).
Automated quantification of osteoclastic resorption: Fluorescent labeling of the resorbable
substrates facilitated visualizing the substrates-free-areas resulted from osteoclastic resorption
(Fig.3A). Mature osteoclasts were transferred onto fluorescent hydroxyapatite as described
previously [11] and incubated with RANKL (0, 12.5, 25, 50 ng/ml), or with alendronate (100
µM) for 24-48 h. The cells were removed the images were obtained using the fluorescence
microscopy and quantified with ImageJ either manually by individually outlining each resorption
pit, or automatically by first adjusting the color image to obtain the best contrast, then converting
to binary image, then filtering the particles below 100 pixels and finally analyzing the size of the
remaining particles. We have found that the total resorption area increased significantly with the
increase in RANKL concentration (Fig.3B, left), and decreased significantly with osteoclast
inhibitor alendronate (Fig.3B, right). The correlation between the values obtained using manual
and automated quantification was significant at R2 = 0.82 (Fig.3C).
To examine if the amount of the fluorescent dextran in the media can be used as an
indicator of resorption, we measured the fluorescence intensity of the conditioned media
collected after 24 h of incubation of fluorescent hydroxyapatite with osteoclasts treated with
RANKL (0, 12.5, 25, 50 ng/ml) or alendronate (100 µM), or with untreated RAW 264.7 cells, or
with media only. No correlation between the resorptive activity in the sample and the amount of
dextran in the media was observed (Fig.4), likely indicating that internalization of dextran
interferes with its release into the incubation media.
Fluorescent hydroxyapatite facilitates measuring resorption volume: To obtain more detailed
3-dimentional information of the resorptive activity, we examined the osteoclastic resorption pits
formed on the fluorescent hydroxyapatite using the confocal microscopy. The z-stack was
obtained at 1 µm interval (Fig.5A). To facilitate visualization of the resorption depth, a color-
coded height stack with the color scale indicating the actual height was constructed (Fig. 5B).
The resorption pits of different depths were evident: while in some areas the fluorescent
hydroxyapatite was removed to the glass level (Fig. 5B, white outlined black arrow), in other
areas the resorption pit was more shallow (Fig.5B, white arrow). We have estimated the
resorption volume, and have found that it although there was a trend of increased volume with
increasing RANKL concentration it was not significant (P<0.05, ANOVA). (Fig. 5C).
We next developed a simplified method to determine the osteoclast-like cells’ resorption
volume using the fluorescent hydroxyapatite. Using fluorescence microscope without confocal
capability, the images were obtained focusing as the surface of the substrate, at the surface of the
glass and at the middle position (Fig.6A-C). Similar to confocal images, we were able to
distinguish between the resorption pits of different depth (Fig. 6D, white arrow and white
outlined black arrow). We have found significant correlation between the resorption volume of
the same samples estimated using fluorescence and confocal microscopy, R² = 0.93 (Fig.6E).
Discussion:
In this study, we developed an assay for an automated analysis of the osteoclastic resorption.
Fluorescent dextran labeling of the resorbable substrates facilitated visualizing the substrates-free
areas resulted from osteoclast resorption. We have shown that the data obtained using an
automated method to estimate the osteoclastic resorption significantly correlated with the manual
method of the resorption estimation. In addition, we demonstrated that fluorescently labeled
hydroxyapatite allows obtaining additional information regarding the resorption volume and
developed a simplified method to analyze resorption volume.
We have established that fluorescent labeling with dextran did not affect the basic
properties of the thin hydroxyapatite layers characterized for their stability and lack of non-
specific dissolution in the previous studies [10, 11]. In a similar approach, Miyazaki and
colleagues examined the fluorescent labeling of a carbonated calcium phosphate with three
different fluorescent polyanions: fluoresceinamine-labeled chondroitin polysulfate, Hoechst
33258-labeled deoxyribonucleic acid and calcein [13]. While demonstrating very promising
capabilities for quick assessment of the resorption index with fluoresceinamine-labeled
chondroitin polysulfate or Hoechst 33258-labeled deoxyribonucleic acid calcium phosphate, this
study had certain drawbacks, first in the evidence of a non-specific dissolution due to higher
solubility of the carbonated calcium phosphate, and second in a nonlinear correlation of
fluorophore release with the resorption index, suggesting certain lack of stability in fluorophore-
calcium phosphate binding. In contrast, the protocol used in this study allows for the
development of fluorescently labeled hydroxyapatite coatings with superior properties.
We have found that labeling the resorbable substrates with the fluorescent dextran was
not toxic to the cells and did not affect osteoclast differentiation or resorptive activity. Osteoclast
exhibited normal morphology and formed the actin ring structure that is associated with
resorption. Although osteoclasts and their precursors were to internalize dextran, their resorptive
activity was not affected by dextran internalization, as evident by a significant correlation and
similar range of the resorption areas resorbed by osteoclasts on labeled and unlabeled
hydroxyapatite, R2= 0.99, p <0.0001. These data confirm that this layer is suitable for analyzing
osteoclast resorptive activity.
We have used labeling of hydroxyapatite with the fluorescent dextran, which has the
capacity to bind effectively to hydroxyapatite [15]; [16], to facilitated visualizing and automate
the analysis of the substrates-free areas resulted from osteoclast resorption. In general, the
automated evaluation of the substrate-free areas on calcium phosphate substrates is complicated
by the crystallinity of the substrate that results in uneven illumination of the surface and poses
difficulties for automated thresholding. The automated quantification is commonly performed
without the demonstration of substrate appearance or validation of the quantification protocol
[13], and the when the substrates are shown it is often clear that automated quantification
represents only a rough estimate of osteoclastic activity [22]. The stability of hydroxyapatite
substrate and of dextran binding allowed for a high correlation between the manual and
automated quantification demonstrated in this study. Another approach to estimate a rapid
resorption index in live cultures, is to measure the release of fluorescent label into the media
[13]. However, we have found that level of the fluorescent dextran in the incubation media did
not correlate with the osteoclast activity likely due to it endocytosis by monocytes and
osteoclasts [23]; [17]. Similarly, it was previously demonstrated that when calcium phosphate
was labeled with calcein no correlation between the amount of fluorescence in the supernatant
and resorptive activity was observed [13]. Thus, labeling with different fluorescent indicators can
fine-tune the presented methodology to the specific experimental needs.
Measurement of the resorption volume represents a true measure of osteoclast resorptive
activity, however it is difficult to achieve in practice. Numerous studies proposed the use of
scanning electron microscopy [24], [25]; [26], confocal microscopy [27] and vertical scanning
profilometry [28], however most techniques are either time consuming or require specialized
instrumentation. In this study, we demonstrated that the measurements of the resorption volume
per pit can be simplified on the fluorescently labeled hydroxyapatite substrates, and developed a
convenient protocol to estimate the resorption volume using the fluorescence microscopy
without the confocal capability.
Thus, the method developed in this study allows simplifying and automating the analysis
of osteoclast resorptive activity, and provides additional measures of resorption volume that can
facilitate the development of novel osteoclast-targeting therapies.
Acknowledgment:
We thank Dr. MF Manolson (University of Toronto) for providing reagents used in this study.
This study was supported by the Canadian Institutes for Health Research (SVK) and Natural
Sciences and Engineering Research Council of Canada (JEB). SM was supported by the Faculty
of Dentistry, McGill University. JEB held Canada Research Chair in Osteoinductive
References:
[1] Hughes DE, Dai A, Tiffee JC, Li HH, Mundy GR, Boyce BF. Estrogen promotes apoptosis of
murine osteoclasts mediated by TGF-beta. Nat Med 1996;2:1132-6.
[2] Ritchlin CT, Haas-Smith SA, Li P, Hicks DG, Schwarz EM. Mechanisms of TNF-alpha- and
RANKL-mediated osteoclastogenesis and bone resorption in psoriatic arthritis. J Clin
Invest 2003;111:821-31.
[3] Amft N, Curnow SJ, Scheel-Toellner D, Devadas A, Oates J, Crocker J, et al. Ectopic
expression of the B cell-attracting chemokine BCA-1 (CXCL13) on endothelial cells and
within lymphoid follicles contributes to the establishment of germinal center-like
structures in Sjogren's syndrome. Arthritis and Rheumatism 2001;44:2633-41.
[4] Burman A, Haworth O, Hardie DL, Amft EN, Siewert C, Jackson DG, et al. A chemokine-
dependent stromal induction mechanism for aberrant lymphocyte accumulation and
compromised lymphatic return in rheumatoid arthritis. Journal of Immunology
2005;174:1693-700.
[5] Page G, Lebecque S, Miossec P. Anatomic localization of immature and mature dendritic
cells in an ectopic lymphoid organ: correlation with selective chemokine expression in
rheumatoid synovium. J Immunol 2002;168:5333-41.
[6] Bartold PM, Cantley MD, Haynes DR. Mechanisms and control of pathologic bone loss in
periodontitis. Periodontology 2000 2010;53:55-69.
[7] Halvorson KG, Sevcik MA, Ghilardi JR, Rosol TJ, Mantyh PW. Similarities and differences
in tumor growth, skeletal remodeling and pain in an osteolytic and osteoblastic model of
bone cancer. Clin J Pain 2006;22:587-600.
[8] Hussein O, Tiedemann K, Komarova SV. Breast cancer cells inhibit spontaneous and
bisphosphonate-induced osteoclast apoptosis. Bone 2011;48:202-11.
[9] Iqbal J, Sun L, Mechanick JI, Zaidi M. Anti-cancer actions of denosumab. Curr Osteoporos
Rep 2011;9:173-6.
[10] Patntirapong S, Habibovic P, Hauschka PV. Effects of soluble cobalt and cobalt
incorporated into calcium phosphate layers on osteoclast differentiation and activation.
Biomaterials 2009;30:548-55.
[11] Maria SM, Prukner C, Sheikh Z, Mueller F, Barralet JE, Komarova SV. Reproducible
quantification of osteoclastic activity: Characterization of a biomimetic calcium
phosphate assay. J Biomed Mater Res B Appl Biomater 2013.
[12] Hale LV, Ma YF, Santerre RF. Semi-quantitative fluorescence analysis of calcein binding as
a measurement of in vitro mineralization. Calcified Tissue International 2000;67:80-4.
[13] Miyazaki T, Miyauchi S, Anada T, Imaizumi H, Suzuki O. Evaluation of osteoclastic
resorption activity using calcium phosphate coating combined with labeled polyanion. J
Anal Biochem 2011;410:7-12.
[14] Hoorfar M, Kurz MA, Policova Z, Hair ML, Neumann AW. Do polysaccharides such as
dextran and their monomers really increase the surface tension of water? Langmuir : the
ACS journal of surfaces and colloids 2006;22:52-6.
[15] Fricain JC, Schlaubitz S, Le Visage C, Arnault I, Derkaoui SM, Siadous R, et al. A nano-
hydroxyapatite--pullulan/dextran polysaccharide composite macroporous material for
bone tissue engineering. Biomaterials 2013;34:2947-59.
[16] Niwa M, Li W, Sato T, Daisaku T, Aoki H. The adsorptive properties of hydroxyapatite to
albumin, dextran and lipids. Biomed Mater Eng 1999;9:163-9.
[17] Stenbeck G, Horton MA. Endocytic trafficking in actively resorbing osteoclasts. J Cell Sci
2004;117:827-36.
[18] Akchurin T, Aissiou T, Kemeny N, Prosk E, Nigam N, Komarova SV. Complex Dynamics
of Osteoclast Formation and Death in Long-Term Cultures. PLoS ONE 2008;3.
[19] Le Nihouannen D, Hacking SA, Gbureck U, Komarova SV, Barralet JE. The use of
RANKL-coated brushite cement to stimulate bone remodelling. Biomaterials
2008;29:3253-9.
[20] Fotakis G, Timbrell JA. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT
and protein assay in hepatoma cell lines following exposure to cadmium chloride.
Toxicol Lett 2006;160:171-7.
[21] Lakkakorpi PT, Väänänen KH. Kinetics of the osteoclast cytoskeleton during the resorption
cycle in vitro. Journal of Bone and Mineral Research 1991;6:817-26.
[22] Kartner N, Yao Y, Li K, Crasto GJ, Datti A, Manolson MF. Inhibition of Osteoclast Bone
Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction. Journal of
Biological Chemistry 2010;285:37476-90.
[23] Seto H, Kawakita H, Ohto K, Harada H, Inoue K. Membrane porosity control with dextran
produced by immobilized dextransucrase. Journal of Chemical Technology and
Biotechnology 2007;82:248-52.
[24] Boyde A, Ali NN, Jones SJ. Resorption of dentine by isolated osteoclasts in vitro. British
dental journal 1984;156:216-20.
[25] Fuller K, Thong JT, Breton BC, Chambers TJ. Automated three-dimensional
characterization of osteoclastic resorption lacunae by stereoscopic scanning electron
microscopy. Journal of bone and mineral research : the official journal of the American
Society for Bone and Mineral Research 1994;9:17-23.
[26] Grimandi G, Soueidan A, Anjrini AA, Badran Z, Pilet P, Daculsi G, et al. Quantitative and
reliable in vitro method combining scanning electron microscopy and image analysis for
the screening of osteotropic modulators. Microscopy research and technique
2006;69:606-12.
[27] Yamada Y, Ito A, Sakane M, Miyakawa S, Uemura T. Laser microscopic measurement of
osteoclastic resorption pits on biomaterials. Materials Science and Engineering: C
2007;27:762-6.
[28] Pascaretti-Grizon F, Mabilleau G, Baslé M, Chappard D. Measurement by vertical scanning
profilometry of resorption volume and lacunae depth caused by osteoclasts on dentine
slices. Journal of microscopy 2011;241:147-52.
Figure legends:
Figure 1: Characterization of the fluorescent hydroxyapatite. A: Representative images of
hydroxyapatite surfaces labeled with 500, 125, 50, and 12.5 nM fluorescent dextran. Same
exposure was used for all the images. B: XRD patterns of the fluorescent dextran-labeled-
calcium phosphate layer, crosses indicate standard hydroxyapatite peaks. C-E: SEM images of
fluorescent hydroxyapatite before and after culture, top and bottom rows are different
magnifications of the same image. The fluorescent hydroxyapatite untreated; dry (C), the
incubated fluorescent hydroxyapatite for 24 h with media with no cells (D), and with osteoclasts
(E), then cells were removed, and resorption pits formed underneath osteoclasts were imaged.
Figure 2: Fluorescent dextran labeling does not affect osteoclast response to hydroxyapatite. A:
RAW 264.7 cells were incubated on tissue culture plastic or fluorescent hydroxyapatite for 24 h,
and LDH levels in the media were assessed. As a positive control, lysis solution was added to
RAW 264.7 incubated on plastic. Data are means ± SD, n = 3 replicates, no significant difference
between plastic and fluorescent hydroxyapatite. B-E: Fluorescent hydroxyapatite (dextran
Rhodamine B 'red') was incubated for 24 h with media with no cells (B), with RAW 264.7 cells
(C) or with osteoclasts (D, E). Yellow arrows point at RAW 264.7 cells and an osteoclast
containing dextran intracellularly; white arrowhead points at a resorption area free from the
substrates next to an osteoclast. E: Osteoclast cultures on fluorescent hydroxyapatite (red, left)
were fixed and actin was labeled with FITC-conjugated phalloidin (green, middle). The overlay
(right) demonstrates an osteoclast with an actin ring that is surrounded by a resorption area free
of fluorescent substrates. F: Differentiated osteoclasts were transferred to the parallel samples of
unlabeled and fluorescent hydroxyapatite and incubated for 24-48 h with RANKL (0, 12.5, 25 or
50 ng/ml). Correlation of the resorption areas resorbed by osteoclasts on labeled and unlabeled
hydroxyapatite, R2= 0.99, p <0.0001.
Figure 3: Automated quantification of the osteoclast resorption on the fluorescent
hydroxyapatite. The fluorescent hydroxyapatite (dextran alexa Fluor 488) was incubated with
osteoclasts with media containing RANKL (0, 12.5, 25, 50 ng/ml), or media with alendronate
(100 µM) and no RANKL, then cells were removed and the substrates were imaged using
fluorescence microscopy. A: Representative images of fluorescent hydroxyapatite that was
incubated with osteoclasts without RANKL (left), with RANKL (middle), or with osteoclast
inhibitor, alendronate (100 µM) (right) for 24-48 h. B: Automated quantification of the
resorption areas per 111210 µm2. Data are mean ± SE, n = 4, *p < 0.0001 indicate statistical
significance assessed using ANOVA for the RANKL dependence and t-test for the effect of
alendronate. C: The resorption areas on fluorescent hydroxyapatite were quantified manually and
automatically and then correlation was assessed. The manual and automated quantification
methods of resorption exhibited significant correlation R2= 0.82, p <0.0001.
Figure 4: Media release of fluorescent dextran cannot be used as an indicator for the osteoclastic
resorption. Osteoclasts were incubated on fluorescent hydroxyapatite for 24 h with RANKL (0,
12.5, 25, 50 ng/ml, labeled as OC, R12.5, R25 and R50 respectively), or with alendronate
(Alend). As control, fluorescent hydroxyapatite was incubated with media only without cells (M)
or with RAW 264.7 cells in media without RANKL. Conditioned media was collected and
fluorescence was measured (black bars). In a selected experiment, cells were lysed, intracellular
fluorescence was measured and added to the measurements in conditioned medium (white bars).
Data are means ± SE, normalized to media only n = 4, no significant difference.
Figure 5: Assessment of the osteoclast resorption volume using the fluorescent hydroxyapatite.
Osteoclasts were incubated with RANKL (0, 12.5, 25 or 50 ng/ml) for 24-48 h on the fluorescent
hydroxyapatite (alexa-488, green), cells were removed and then images were taken using
confocal microscopy. A,1-9: The z-stack was obtained at 1 µm interval. The depth of the calcium
phosphate layers was 9 ± 3 µm. B: A color-coded height stack with the color scale indicating the
actual height above the bottom slice. White outlined black arrow indicates the resorption pit that
almost reached the glass level, white arrow indicates the resorption pit that did not reach the
glass level. C: The resorption volume increased with the increase in RANKL concentration. Box-
plots indicate the minimal values and 90th percentile (whiskers), the 25th and 75th percentiles
(the limits of the box), and the median values (the line within the box), n = 10-24.
Figure 6: A simplified method to determine the osteoclast resorption volume with the
fluorescent hydroxyapatite. Osteoclasts were incubated in media with RANKL (0, 12.5, 25, 50
ng/ml) or with alendronate (100 µM) for 24-48 h, the cells were removed and the substrates were
imaged at three levels: surface layer, 4 µm and 8 µm in depth and pseudo-colored red, green and
blue respectively. A-C: representative images of the substrate appearance following incubation
of osteoclasts with RANKL 50 ng/ml at 3 different levels: at the surface (A), 4 µm deep (B) and
8 µm deep (C). D: merge of A, B and C. White outlined black arrow indicates the resorption pit
that reached the glass level, white arrow indicates the resorption pit that did not reach the glass
level. E: The average resorption volume in the same samples was estimated using fluorescence
and confocal microscopy. Data are means ± SE, n = 4, the correlation R2= 0.93, *p <0.001.