Page 1 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
1
Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions Anne Bernhardt, PhD1, Matthias Schumacher, Dipl. Ing.1, Michael Gelinsky, PhD1
Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital and Medical Faculty Carl Gustav Carus, Fetscher Str. 74, Technische Universität Dresden, 01307 Dresden, Germany
Dr. Anne Bernhardt (corresponding author) phone: +49 351 458 16690 fax: +49 351 458 7210 [email protected]
Matthias Schumacher phone: +49 351 458 6688 fax: +49 351 458 7210 [email protected]
Prof. Dr. Michael Gelinsky phone: +49 351 458 6694 fax: +49 351 458 7210 [email protected]
Page 2 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
2 Abstract Objectives: Peripheral blood mononuclear cells (PBMC) are an attractive source for the generation of osteoclasts in vitro which is an important prerequisite for the examination of resorption and remodeling of biomaterials. In the present study different preparation methods are used to obtain cell populations with rising content of CD14+ monocytes. We wanted to address the question whether there is a correlation between content of CD14+ cells in the preparation and functionality of formed osteoclasts. Materials and methods: PBMC obtained by density gradient centrifugation with and without further purification by plastic adherence or immunomagnetic separation of CD14+ cells were seeded on both cell culture polystyrene and a calcium phosphate bone cement (CPC) and cultivated under stimulation with M-CSF and RANKL. Cell cultures were characterized by histological and fluorescent staining of multinucleated cells positive for tartrate resistant acid phosphatase (TRAP) activity and the presence of actin rings, respectively. Furthermore, activities of osteoclast marker enzymes TRAP and carbonic anhydrase II (CA II) were quantified. For osteoclasts cultured on CPC resorption pits were visualized using scanning electron microscopy (SEM). Results: Monocytes of all preparations were successfully differentiated into multinucleated osteoclasts showing TRAP and CA II activity both on cell culture plastic and CPC. Preparations involving an additional plastic adherence step exhibited only minor increase of TRAP and CA II activity in the second week of cultivation. Furthermore, the number of resorption pits on CPC was reduced in these cultures compared to immunomagnetically enriched monocytes and preparations without additional plastic adherence steps. Optimal results concerning yield, number of multinucleated osteoclasts, activity of TRAP and CA II and resorption of CPC were obtained by simple density gradient centrifugation. Conclusion: All examined monocyte preparation protocols were suitable for the generation of osteoclasts both on polystyrene and calcium phosphate bone cement. Highly purified monocytes are not mandatory necessary to obtain functional osteoclasts for investigation of biomaterial resorption.
Page 3 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
3 1. Introduction During the last decade osteoclasts have gained more and more attention in the investigation of bone graft remodeling.1 The multinucleated cells derive from the hematopoetic stem cell line and are formed by the fusion of peripheral blood mononuclear cells (PBMC).2 Osteoclasts have become a useful tool for the in vitro examination of biomaterials resorbability.3 Furthermore, co-culture models of osteoblasts or their precursors with osteoclasts reveal insights into the complex cross-talk of bone cells during remodeling of bone graft materials.4-11 The isolation of primary osteoclasts has been established for bones of neonatal animals.9 Identification of RANKL and M-CSF as factors for differentiation of mononuclear blood monocytes enabled the generation of human osteoclasts in vitro.10,11 It has been shown that among the peripheral blood mononuclear cell (PBMC) fraction CD14 positive monocytes are the main precursor cells of osteoclasts.12,13 The success of resorbable bone implants depends strongly on their ability to be remodeled, ideally in a balanced manner, where the formation of new bone tissue occurs with a rate similar to the biomaterial resorption and thus ensures sufficient mechanical stability during the remodeling process. Final remodelling studies have to be performed in vivo, however preliminary resorption and remodeling tests in vitro may help to reduce animal experiments. Preparation of PBMC is usually performed by density gradient centrifugation of whole blood or leucocyteenriched blood fractions (“buffy coat”) with a density gradient medium of 1.077 g/ml density (Ficoll®-Paque, Histopaque®, Lymphoprep™). Isolated human PBMC comprise typically 1015 % CD14+ monocytes.12-14 Further purification of the CD14+ fraction can be achieved by several methods. Some researchers perform two-step density gradients with different densities of the gradient medium,8,15-17 or even combine two density centrifugation steps with the same medium
12
to further enrich the CD14 monocyte fraction of PBMC. A simple and
long-known method for monocyte enrichment from PBMC is to exploit the feature of monocytes to adhere to plastic surfaces.18 Higher yield and purity of CD14+ monocytes can be obtained by magnetic immune selection either by labelling of CD14+ cells (positive selection) or labelling of all other cell species occurring in the PBMC fraction to obtain
Page 4 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
4 untouched monocytes (negative selection). Furthermore, untouched monocytes can be enriched by cross-linking (rosetting) of unwanted cells to red blood cells by tetrameric antibody complexes. While the presence of highly purified, non-activated CD14+ monocytes is an ultimate requirement for immunological investigations, in vitro formation of osteoclasts for resorption and remodelling assays seems to work also with PBMC. However, the results on the potential of mononuclear cells derived from PBMC and purified CD14+ fractions to form osteoclasts are somewhat inconsistent. Nicholson and co-workers12 studied osteoclast formation from immunomagnetically enriched CD14+ monocytes in comparison to unfractionated PBMC cultured on bone slices. Number of multinucleated cells with positive staining for tartrate-resistant acid phosphatase (TRAP) and resorption area normalized to protein content were comparable in the two cultures. However, 10-fold seeding density of PBMC compared to purified CD14+ cells was applied. Another study also compares the formation of osteoclasts from PBMC and immunomagnetically purified CD14+ cells on bovine bone slices and on xenogenous bone mineral.19 According to the authors, the number of osteoclasts derived from the purified CD14+ fraction was higher, but the size of the multinucleated osteoclasts was smaller compared to those derived from PBMC. Botelho et al.
20
cultivated PBMC and immunomagnetically enriched CD14+ monocytes on
hydroxyapatite ceramics with different silica content. However, this study lacks a direct comparison between the two cell preparations, since different analyses were performed with PBMC and CD14+ monocytes. Comprehensive investigations on osteoclastogenesis of PBMC and their CD14+ and CD14- subpopulations were presented by Costa-Rodriguez and co-workers.14 It was demonstrated, that pure CD14+ monocytes gave rise to an osteoclast population with less pronounced osteoclastic phenotype compared to osteoclasts from the whole PBMC fraction. The authors postulate the ability of other monocyte subpopulations to positively modulate osteoclastogenesis. In the present study we prepare PBMC using different protocols and, accordingly, different content of CD14+ monocytes. We analyse the potential of the monocytes to differentiate into
Page 5 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
5 functional osteoclasts both on cell culture polystyrene and on calcium phosphate bone cement (CPC).
2. Materials and Methods 2.1. Preparation of calcium phosphate bone cement samples In this study, a CPC based on -tricalcium phosphate as originally described by Driessens et al.21 was used. Upon mixing with aqueous solution using a liquid to powder ratio of 500 µl/g the cement forms a mouldable paste that sets into Ca-deficient, carbonated hydroxyapatite. Samples for in vitro experiments were prepared as disks of 10 mm diameter and 1 mm height using silicone moulds and allowed to set for 4 days in a water-saturated atmosphere at 37°C. Set cement samples had a micro-porous surface and an average surface roughness of Ra = 0.48 ± 0.12 µm as described recently.22 Samples were sterilized by gammairradiation (25 kGy) and immersed in cell culture medium 24 h prior to cell seeding to allow medium equilibration. 2.2. Isolation of monocytes Human monocytes were isolated from buffy coats of healthy adult donors (German Red Cross, Dresden, Germany). Each buffy coat was sub-divided into equal parts that were used for the different preparation protocols. Thus the direct comparison between preparation protocols was not interfered by individual variances between cells of different donors. In total, buffy coats (75-91 ml, each obtained from 500 ml blood) of 9 donors were included into this study. Monocyte adhesion medium contained α-MEM containing 10 % heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin und 2 mM glutamine (all from Biochrom). For isolation of monocytes the following protocols were used: Protocol “RED” (whole PBMC fraction): The buffy coats were diluted 1:2 with phosphate buffered saline (PBS) containing 2 mM ethylendiamintetraacetate (EDTA) (Sigma-Aldrich) and 0.5 % bovine serum albumin (BSA) (both Sigma-Aldrich) (PBS E/B) and layered onto Ficoll Paque Plus (GE Healthcare) using Leucosep tubes (Greiner). Tubes were centrifuged
Page 6 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
6 for 10 min at 1000 g. PBMC fraction was re-suspended in ice-cold PBS E/B and centrifuged at 250 g and 4°C for 15 min. The washing step was repeated once. Protocol “YELLOW”: In addition to protocol “RED”, remaining erythrocytes were removed in between the washing steps. Therefore, the PBMC pellet was resuspended for 30 s in 2 ml of deionized water before adding 48 ml of ice-cold PBS (E/B) and centrifugated at 250 g for 15 min at 4°C. Protocol “RED ADH”: After performing protocol “RED”, monocytes were enriched by plastic adherence: Cells were re-suspended in monocyte adhesion medium, transferred into serum-coated T175 cell culture flasks (coated overnight with 10 ml heat-inactivated FCS at 4°C) and incubated for 2 h at 37°C and 5 % CO2. Medium was removed and cells were washed twice with medium to remove non-attached cells. Then, cells were incubated with ice-cold PBS E/B at 4°C on a shaker to detach the adherent cells. After 30-40 min, the cell suspension was pipetted out of the flasks and centrifuged for 15 min at 250 g. Protocol “YELLOW ADH”: After performing protocol “YELLOW”, monocytes were enriched by plastic adherence, as described in the protocol above. Protocol “CD14”: After performing protocol “RED”, CD14+ cells were magnetically labeled with CD14 microbeads (Miltenyi) and separated by magnetic activated cell sorting (MACS) technology according to manufacturer`s instructions. For all protocols, cell pellets were re-suspended in a suitable amount of PBS E/B for further analysis. 2.3. Evaluation of purity of monocyte preparations A first estimation of monocyte content of the different preparations was obtained by measurement of cell number and size distribution with an automated cell counter (scepter 2.0., Millipore) applying the Coulter principle. Relative frequency of monocytes was derived from the diameter histogram plot. Cells with a diameter between 7.6 and 12 µm were counted for the monocyte fraction. Purity of monocyte preparations was further analysed by fluorescence activated cell sorting (FACS). 100,000 cells were re-suspended in 100 µl PBS, stained with monoclonal anti
Page 7 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
7 human CD14-APC- or FITC conjugated antibodies (Miltenyi) and fixed with 4 % formaldehyde in PBS. FACS analyses were carried out using a LSR II system (BD). 2.4. Seeding and cultivation of monocytes on polystyrene and CPC CPC discs were incubated overnight in -MEM containing 10 % heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin und 2 mM glutamine before seeding with cells from different PBMC preparations to ensure pH equilibration.23 Monocyte content of the different preparations was estimated with the automated cell counter and seeding density was adapted with respect to the monocyte content of the preparations. 48-well polystyrene cell culture plates (Nunc) were seeded with 3*105 monocytes and CPC discs were seeded with 6*105 monocytes both in 500 µl medium per sample. Samples which were seeded with preparations “RED” and “YELLOW” were incubated overnight with monocyte adhesion medium (see 2.2). On the next day, the medium was changed into -MEM containing 5 % heat-inactivated FCS, 5 % human A/B serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 25 ng/ml M-CSF (antibodies-online). Samples seeded with further purified cells (plastic adherence or immunomagnetic sorting) were incubated with the M-CSF containing medium directly after seeding. After two days of cultivation, cells were additionally supplemented with 50 ng/mL RANKL (antibodies-online). Medium was changed twice weekly. Samples for biochemical measurements (d2, d9, d16) were frozen at -80°C after washing with PBS. After 16 days samples for microscopic investigations were fixed with 4 % formaldehyde in PBS. All cell culture experiments were performed with triplicate samples and repeated three times. 2.4. TRAP staining and light microscopy Samples for light and fluorescence microscopy were washed with pre-warmed PBS at the respective time points and fixed in 3.7 wt-% formaldehyde (Merck) in PBS for 15 min at room temperature. Excess fixation solution was removed by washing with PBS. TRAP staining was performed for 10 min at 37°C by immersion in 0.3 mg/ml Fast Red Violet LB (Sigma-Aldrich) in an aqueous staining buffer containing 0.05 M sodium acetate (Sigma-Aldrich), 0.05 M acetic acid (Sigma-Aldrich), 0.03 M sodium tartrate (Roth), 0.1 mg/ml naphthol AS-MX
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Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
8 phosphate disodium salt (Sigma-Aldrich) and 0.1 % Triton X100 (Sigma-Aldrich). After washing with PBS, cell nuclei were stained for 10 min using Mayers Haemalaun solution (AppliChem) followed by rinsing in tap water. Stained samples were photographed using a Zeiss Axiovert C40 microscope equipped with a Sony NEX-5 camera. 2.5. Fluorescence staining and CLSM For fluorescence staining, fixed cells (see 2.4) were permeabilised by incubation in 0.2 % Triton X100 in PBS for 3 min. Then, samples were washed in PBS again and blocking was performed using 1 % BSA in PBS for 45 min to prevent non-specific binding of fluorescence dyes. Simultaneous staining of cell nuclei and actin cytoskeleton was done using 20 ng/ml DAPI (4′,6-diamidin-2-phenylindol, Invitrogen) and 25 µl/ml AlexaFluor 488® phalloidin (Invitrogen), in 1 % BSA in PBS for 45 min in the dark. After washing in PBS, stained cells were imaged using confocal laser scanning microscopy (Leica TCS SP5, Leica Microsystems), located in the “MTZ imaging” light microscopy facility of the Medical Faculty of the Technical University Dresden. 2.6. SEM Cells were removed from the CPC surfaces by addition of 1 % Triton X-100 in PBS. To support cell lysis, the samples were sonicated for 10 min. Samples were washed with deionized water and dried at 60°C for 12 hours. After drying, samples were mounted on stubs, sputter coated with carbon and imaged using a Philips XL 30/ESEM with FEG (field emission gun), operated in SEM mode at 10 mm working distance and 6 kV using a SE detector. 2.7. Determination of DNA concentration, TRAP and CA II activity Cell lysis: Frozen cell-seeded samples were thawed for 20 min on ice followed by lysis with 1 % Triton X-100 in PBS for 50 min on ice. Cell lysis was supported by sonication for 10 min in an ice-cooled ultrasonic bath. Cell lysates were transferred to test tubes and stored on ice for subsequent analysis. DNA content: Cell lysates were mixed with Picogreen ds DNA quantitation reagent (Molecular Probes) diluted 1:800 in TE buffer (= 10 mM TRIS and 1 mM EDTA) and
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Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
9 incubated for 5 min in the dark. The intensity of fluorescence was measured with a multifunction microplate reader (Infinite® M200 Pro, Tecan) at an excitation and emission wavelength of 485/535 nm. DNA content was calculated from a calibration line of calf thymus DNA (Sigma). TRAP activity: According to Janckila et al.23, TRAP reaction buffer was composed of 2.5 mM naphthol-ASBI-phosphate (Sigma) in 100 mM sodium acetate (Roth) and 50 mM disodium tartrate pH 6.1 (both from Roth) containing 1 % ethylenglycolmonomethylether (Fluka) and 2 % Nonidet NP-40 (both from Fluka). Cell lysates (10 µl) were mixed with TRAP reaction buffer (50 µl) and incubated at 37°C for 30 min. Finally, 0.1 M NaOH (Roth) containing 0.05 % Nonidet P40 (150 µl) was added to stop the enzymatic reaction. The intensity of fluorescence was measured with the multifunction microplate reader at an excitation and emission wavelength of 405/520 nm. Calibrator solutions with different TRAP concentrations (0.6 to 12 U/L) taken from a commercially available ELISA kit (BoneTRAP, Medac, Germany) were used to correlate the fluorescence intensity with TRAP activity. TRAP activity was related to DNA content of the respective sample. CA II activity: Cell lysates (50 µl) were mixed with CA II reaction buffer containing 12.5 mM TRIS pH 7.5 (Roth), 75 mM NaCl (Sigma) and 2 mM 4-nitrophenylacetate (50 µl, Sigma). Absorbance was monitored at 400 nm for 5 min. Conversion to 4-nitrophenol was calculated from the slope of the absorbance plot using a calibration line from different dilutions of 1 mM 4-nitrophenol (Sigma). CA II activity was related to DNA content of the respective sample.
2.8. Statistics All cell culture experiments were performed using triplicates and the results are presented as means ± standard deviation. Analysis of variance (two way ANOVA, Origin 8.5.0G, OriginLab) for repeated measures was used to evaluate statistical significance at a level of p
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
1
Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions Anne Bernhardt, PhD1, Matthias Schumacher, Dipl. Ing.1, Michael Gelinsky, PhD1
Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital and Medical Faculty Carl Gustav Carus, Fetscher Str. 74, Technische Universität Dresden, 01307 Dresden, Germany
Dr. Anne Bernhardt (corresponding author) phone: +49 351 458 16690 fax: +49 351 458 7210 [email protected]
Matthias Schumacher phone: +49 351 458 6688 fax: +49 351 458 7210 [email protected]
Prof. Dr. Michael Gelinsky phone: +49 351 458 6694 fax: +49 351 458 7210 [email protected]
Page 2 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
2 Abstract Objectives: Peripheral blood mononuclear cells (PBMC) are an attractive source for the generation of osteoclasts in vitro which is an important prerequisite for the examination of resorption and remodeling of biomaterials. In the present study different preparation methods are used to obtain cell populations with rising content of CD14+ monocytes. We wanted to address the question whether there is a correlation between content of CD14+ cells in the preparation and functionality of formed osteoclasts. Materials and methods: PBMC obtained by density gradient centrifugation with and without further purification by plastic adherence or immunomagnetic separation of CD14+ cells were seeded on both cell culture polystyrene and a calcium phosphate bone cement (CPC) and cultivated under stimulation with M-CSF and RANKL. Cell cultures were characterized by histological and fluorescent staining of multinucleated cells positive for tartrate resistant acid phosphatase (TRAP) activity and the presence of actin rings, respectively. Furthermore, activities of osteoclast marker enzymes TRAP and carbonic anhydrase II (CA II) were quantified. For osteoclasts cultured on CPC resorption pits were visualized using scanning electron microscopy (SEM). Results: Monocytes of all preparations were successfully differentiated into multinucleated osteoclasts showing TRAP and CA II activity both on cell culture plastic and CPC. Preparations involving an additional plastic adherence step exhibited only minor increase of TRAP and CA II activity in the second week of cultivation. Furthermore, the number of resorption pits on CPC was reduced in these cultures compared to immunomagnetically enriched monocytes and preparations without additional plastic adherence steps. Optimal results concerning yield, number of multinucleated osteoclasts, activity of TRAP and CA II and resorption of CPC were obtained by simple density gradient centrifugation. Conclusion: All examined monocyte preparation protocols were suitable for the generation of osteoclasts both on polystyrene and calcium phosphate bone cement. Highly purified monocytes are not mandatory necessary to obtain functional osteoclasts for investigation of biomaterial resorption.
Page 3 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
3 1. Introduction During the last decade osteoclasts have gained more and more attention in the investigation of bone graft remodeling.1 The multinucleated cells derive from the hematopoetic stem cell line and are formed by the fusion of peripheral blood mononuclear cells (PBMC).2 Osteoclasts have become a useful tool for the in vitro examination of biomaterials resorbability.3 Furthermore, co-culture models of osteoblasts or their precursors with osteoclasts reveal insights into the complex cross-talk of bone cells during remodeling of bone graft materials.4-11 The isolation of primary osteoclasts has been established for bones of neonatal animals.9 Identification of RANKL and M-CSF as factors for differentiation of mononuclear blood monocytes enabled the generation of human osteoclasts in vitro.10,11 It has been shown that among the peripheral blood mononuclear cell (PBMC) fraction CD14 positive monocytes are the main precursor cells of osteoclasts.12,13 The success of resorbable bone implants depends strongly on their ability to be remodeled, ideally in a balanced manner, where the formation of new bone tissue occurs with a rate similar to the biomaterial resorption and thus ensures sufficient mechanical stability during the remodeling process. Final remodelling studies have to be performed in vivo, however preliminary resorption and remodeling tests in vitro may help to reduce animal experiments. Preparation of PBMC is usually performed by density gradient centrifugation of whole blood or leucocyteenriched blood fractions (“buffy coat”) with a density gradient medium of 1.077 g/ml density (Ficoll®-Paque, Histopaque®, Lymphoprep™). Isolated human PBMC comprise typically 1015 % CD14+ monocytes.12-14 Further purification of the CD14+ fraction can be achieved by several methods. Some researchers perform two-step density gradients with different densities of the gradient medium,8,15-17 or even combine two density centrifugation steps with the same medium
12
to further enrich the CD14 monocyte fraction of PBMC. A simple and
long-known method for monocyte enrichment from PBMC is to exploit the feature of monocytes to adhere to plastic surfaces.18 Higher yield and purity of CD14+ monocytes can be obtained by magnetic immune selection either by labelling of CD14+ cells (positive selection) or labelling of all other cell species occurring in the PBMC fraction to obtain
Page 4 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
4 untouched monocytes (negative selection). Furthermore, untouched monocytes can be enriched by cross-linking (rosetting) of unwanted cells to red blood cells by tetrameric antibody complexes. While the presence of highly purified, non-activated CD14+ monocytes is an ultimate requirement for immunological investigations, in vitro formation of osteoclasts for resorption and remodelling assays seems to work also with PBMC. However, the results on the potential of mononuclear cells derived from PBMC and purified CD14+ fractions to form osteoclasts are somewhat inconsistent. Nicholson and co-workers12 studied osteoclast formation from immunomagnetically enriched CD14+ monocytes in comparison to unfractionated PBMC cultured on bone slices. Number of multinucleated cells with positive staining for tartrate-resistant acid phosphatase (TRAP) and resorption area normalized to protein content were comparable in the two cultures. However, 10-fold seeding density of PBMC compared to purified CD14+ cells was applied. Another study also compares the formation of osteoclasts from PBMC and immunomagnetically purified CD14+ cells on bovine bone slices and on xenogenous bone mineral.19 According to the authors, the number of osteoclasts derived from the purified CD14+ fraction was higher, but the size of the multinucleated osteoclasts was smaller compared to those derived from PBMC. Botelho et al.
20
cultivated PBMC and immunomagnetically enriched CD14+ monocytes on
hydroxyapatite ceramics with different silica content. However, this study lacks a direct comparison between the two cell preparations, since different analyses were performed with PBMC and CD14+ monocytes. Comprehensive investigations on osteoclastogenesis of PBMC and their CD14+ and CD14- subpopulations were presented by Costa-Rodriguez and co-workers.14 It was demonstrated, that pure CD14+ monocytes gave rise to an osteoclast population with less pronounced osteoclastic phenotype compared to osteoclasts from the whole PBMC fraction. The authors postulate the ability of other monocyte subpopulations to positively modulate osteoclastogenesis. In the present study we prepare PBMC using different protocols and, accordingly, different content of CD14+ monocytes. We analyse the potential of the monocytes to differentiate into
Page 5 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
5 functional osteoclasts both on cell culture polystyrene and on calcium phosphate bone cement (CPC).
2. Materials and Methods 2.1. Preparation of calcium phosphate bone cement samples In this study, a CPC based on -tricalcium phosphate as originally described by Driessens et al.21 was used. Upon mixing with aqueous solution using a liquid to powder ratio of 500 µl/g the cement forms a mouldable paste that sets into Ca-deficient, carbonated hydroxyapatite. Samples for in vitro experiments were prepared as disks of 10 mm diameter and 1 mm height using silicone moulds and allowed to set for 4 days in a water-saturated atmosphere at 37°C. Set cement samples had a micro-porous surface and an average surface roughness of Ra = 0.48 ± 0.12 µm as described recently.22 Samples were sterilized by gammairradiation (25 kGy) and immersed in cell culture medium 24 h prior to cell seeding to allow medium equilibration. 2.2. Isolation of monocytes Human monocytes were isolated from buffy coats of healthy adult donors (German Red Cross, Dresden, Germany). Each buffy coat was sub-divided into equal parts that were used for the different preparation protocols. Thus the direct comparison between preparation protocols was not interfered by individual variances between cells of different donors. In total, buffy coats (75-91 ml, each obtained from 500 ml blood) of 9 donors were included into this study. Monocyte adhesion medium contained α-MEM containing 10 % heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin und 2 mM glutamine (all from Biochrom). For isolation of monocytes the following protocols were used: Protocol “RED” (whole PBMC fraction): The buffy coats were diluted 1:2 with phosphate buffered saline (PBS) containing 2 mM ethylendiamintetraacetate (EDTA) (Sigma-Aldrich) and 0.5 % bovine serum albumin (BSA) (both Sigma-Aldrich) (PBS E/B) and layered onto Ficoll Paque Plus (GE Healthcare) using Leucosep tubes (Greiner). Tubes were centrifuged
Page 6 of 34
Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
6 for 10 min at 1000 g. PBMC fraction was re-suspended in ice-cold PBS E/B and centrifuged at 250 g and 4°C for 15 min. The washing step was repeated once. Protocol “YELLOW”: In addition to protocol “RED”, remaining erythrocytes were removed in between the washing steps. Therefore, the PBMC pellet was resuspended for 30 s in 2 ml of deionized water before adding 48 ml of ice-cold PBS (E/B) and centrifugated at 250 g for 15 min at 4°C. Protocol “RED ADH”: After performing protocol “RED”, monocytes were enriched by plastic adherence: Cells were re-suspended in monocyte adhesion medium, transferred into serum-coated T175 cell culture flasks (coated overnight with 10 ml heat-inactivated FCS at 4°C) and incubated for 2 h at 37°C and 5 % CO2. Medium was removed and cells were washed twice with medium to remove non-attached cells. Then, cells were incubated with ice-cold PBS E/B at 4°C on a shaker to detach the adherent cells. After 30-40 min, the cell suspension was pipetted out of the flasks and centrifuged for 15 min at 250 g. Protocol “YELLOW ADH”: After performing protocol “YELLOW”, monocytes were enriched by plastic adherence, as described in the protocol above. Protocol “CD14”: After performing protocol “RED”, CD14+ cells were magnetically labeled with CD14 microbeads (Miltenyi) and separated by magnetic activated cell sorting (MACS) technology according to manufacturer`s instructions. For all protocols, cell pellets were re-suspended in a suitable amount of PBS E/B for further analysis. 2.3. Evaluation of purity of monocyte preparations A first estimation of monocyte content of the different preparations was obtained by measurement of cell number and size distribution with an automated cell counter (scepter 2.0., Millipore) applying the Coulter principle. Relative frequency of monocytes was derived from the diameter histogram plot. Cells with a diameter between 7.6 and 12 µm were counted for the monocyte fraction. Purity of monocyte preparations was further analysed by fluorescence activated cell sorting (FACS). 100,000 cells were re-suspended in 100 µl PBS, stained with monoclonal anti
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Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
7 human CD14-APC- or FITC conjugated antibodies (Miltenyi) and fixed with 4 % formaldehyde in PBS. FACS analyses were carried out using a LSR II system (BD). 2.4. Seeding and cultivation of monocytes on polystyrene and CPC CPC discs were incubated overnight in -MEM containing 10 % heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin und 2 mM glutamine before seeding with cells from different PBMC preparations to ensure pH equilibration.23 Monocyte content of the different preparations was estimated with the automated cell counter and seeding density was adapted with respect to the monocyte content of the preparations. 48-well polystyrene cell culture plates (Nunc) were seeded with 3*105 monocytes and CPC discs were seeded with 6*105 monocytes both in 500 µl medium per sample. Samples which were seeded with preparations “RED” and “YELLOW” were incubated overnight with monocyte adhesion medium (see 2.2). On the next day, the medium was changed into -MEM containing 5 % heat-inactivated FCS, 5 % human A/B serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 25 ng/ml M-CSF (antibodies-online). Samples seeded with further purified cells (plastic adherence or immunomagnetic sorting) were incubated with the M-CSF containing medium directly after seeding. After two days of cultivation, cells were additionally supplemented with 50 ng/mL RANKL (antibodies-online). Medium was changed twice weekly. Samples for biochemical measurements (d2, d9, d16) were frozen at -80°C after washing with PBS. After 16 days samples for microscopic investigations were fixed with 4 % formaldehyde in PBS. All cell culture experiments were performed with triplicate samples and repeated three times. 2.4. TRAP staining and light microscopy Samples for light and fluorescence microscopy were washed with pre-warmed PBS at the respective time points and fixed in 3.7 wt-% formaldehyde (Merck) in PBS for 15 min at room temperature. Excess fixation solution was removed by washing with PBS. TRAP staining was performed for 10 min at 37°C by immersion in 0.3 mg/ml Fast Red Violet LB (Sigma-Aldrich) in an aqueous staining buffer containing 0.05 M sodium acetate (Sigma-Aldrich), 0.05 M acetic acid (Sigma-Aldrich), 0.03 M sodium tartrate (Roth), 0.1 mg/ml naphthol AS-MX
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Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
8 phosphate disodium salt (Sigma-Aldrich) and 0.1 % Triton X100 (Sigma-Aldrich). After washing with PBS, cell nuclei were stained for 10 min using Mayers Haemalaun solution (AppliChem) followed by rinsing in tap water. Stained samples were photographed using a Zeiss Axiovert C40 microscope equipped with a Sony NEX-5 camera. 2.5. Fluorescence staining and CLSM For fluorescence staining, fixed cells (see 2.4) were permeabilised by incubation in 0.2 % Triton X100 in PBS for 3 min. Then, samples were washed in PBS again and blocking was performed using 1 % BSA in PBS for 45 min to prevent non-specific binding of fluorescence dyes. Simultaneous staining of cell nuclei and actin cytoskeleton was done using 20 ng/ml DAPI (4′,6-diamidin-2-phenylindol, Invitrogen) and 25 µl/ml AlexaFluor 488® phalloidin (Invitrogen), in 1 % BSA in PBS for 45 min in the dark. After washing in PBS, stained cells were imaged using confocal laser scanning microscopy (Leica TCS SP5, Leica Microsystems), located in the “MTZ imaging” light microscopy facility of the Medical Faculty of the Technical University Dresden. 2.6. SEM Cells were removed from the CPC surfaces by addition of 1 % Triton X-100 in PBS. To support cell lysis, the samples were sonicated for 10 min. Samples were washed with deionized water and dried at 60°C for 12 hours. After drying, samples were mounted on stubs, sputter coated with carbon and imaged using a Philips XL 30/ESEM with FEG (field emission gun), operated in SEM mode at 10 mm working distance and 6 kV using a SE detector. 2.7. Determination of DNA concentration, TRAP and CA II activity Cell lysis: Frozen cell-seeded samples were thawed for 20 min on ice followed by lysis with 1 % Triton X-100 in PBS for 50 min on ice. Cell lysis was supported by sonication for 10 min in an ice-cooled ultrasonic bath. Cell lysates were transferred to test tubes and stored on ice for subsequent analysis. DNA content: Cell lysates were mixed with Picogreen ds DNA quantitation reagent (Molecular Probes) diluted 1:800 in TE buffer (= 10 mM TRIS and 1 mM EDTA) and
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Tissue Engineering Part C: Methods Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions (doi: 10.1089/ten.TEC.2014.0187) This article 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.
9 incubated for 5 min in the dark. The intensity of fluorescence was measured with a multifunction microplate reader (Infinite® M200 Pro, Tecan) at an excitation and emission wavelength of 485/535 nm. DNA content was calculated from a calibration line of calf thymus DNA (Sigma). TRAP activity: According to Janckila et al.23, TRAP reaction buffer was composed of 2.5 mM naphthol-ASBI-phosphate (Sigma) in 100 mM sodium acetate (Roth) and 50 mM disodium tartrate pH 6.1 (both from Roth) containing 1 % ethylenglycolmonomethylether (Fluka) and 2 % Nonidet NP-40 (both from Fluka). Cell lysates (10 µl) were mixed with TRAP reaction buffer (50 µl) and incubated at 37°C for 30 min. Finally, 0.1 M NaOH (Roth) containing 0.05 % Nonidet P40 (150 µl) was added to stop the enzymatic reaction. The intensity of fluorescence was measured with the multifunction microplate reader at an excitation and emission wavelength of 405/520 nm. Calibrator solutions with different TRAP concentrations (0.6 to 12 U/L) taken from a commercially available ELISA kit (BoneTRAP, Medac, Germany) were used to correlate the fluorescence intensity with TRAP activity. TRAP activity was related to DNA content of the respective sample. CA II activity: Cell lysates (50 µl) were mixed with CA II reaction buffer containing 12.5 mM TRIS pH 7.5 (Roth), 75 mM NaCl (Sigma) and 2 mM 4-nitrophenylacetate (50 µl, Sigma). Absorbance was monitored at 400 nm for 5 min. Conversion to 4-nitrophenol was calculated from the slope of the absorbance plot using a calibration line from different dilutions of 1 mM 4-nitrophenol (Sigma). CA II activity was related to DNA content of the respective sample.
2.8. Statistics All cell culture experiments were performed using triplicates and the results are presented as means ± standard deviation. Analysis of variance (two way ANOVA, Origin 8.5.0G, OriginLab) for repeated measures was used to evaluate statistical significance at a level of p
