- Email: [email protected]
Quantification of in vitro mineralisation using ion chromatography
Analytical Biochemistry 410 (2011) 244–247
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Quantification of in vitro mineralisation using ion chromatography Paul Souter ⇑, Alan Horner, Jim C. Cunningham Smith and Nephew Research Centre, Heslington, York YO23 1HG, United Kingdom
a r t i c l e
i n f o
Article history: Received 9 September 2010 Received in revised form 13 October 2010 Accepted 26 November 2010 Available online 3 December 2010 Keywords: Osteoblast Mineralisation Calcium Ion chromatography Citric acid
a b s t r a c t Analysis of in vitro mineralisation is an important tool in orthopedic research, allowing assessment of new therapeutic agents and devices; however, access to analytical equipment and accuracy of current methods can be a limiting factor. This current work investigated the use of calcium chelation with citric acid and subsequent analysis by ion chromatography as a method for accurately quantifying the extent of in vitro calcium deposition. Primary human osteoblasts were cultured on tissue culture plastic for 21 days under osteogenic conditions. At 3, 7, 14, and 21 days, alizarin red staining and citric acid calcium chelation of the cultures were performed. The use of alizarin red revealed increased calcium deposition over the culture period but was not sensitive enough to detect mineralisation at early time points after taking in to account background residual staining. The use of ion chromatography gave a limit of detection of 2 lg calcium, sensitive enough to detect mineralisation after 3 days, with no issues relating to background levels. We believe that the use of ion chromatography for quantifying in vitro mineralisation gives researchers an accurate, accessible, and cheap way of assessing novel technologies. Ó 2010 Elsevier Inc. All rights reserved.
The process of bone formation in higher vertebrates occurs via two routes: intramembranous ossification (direct bone formation) and endochondral ossification where bone is formed through an intermediate cartilaginous template [1]. Calcification of the organic extracellular matrix is initiated at the nucleation sites where it is believed that matrix vesicles rich in alkaline phosphatase, concentrated calcium, and inorganic phosphate lead to hydroxyapatite formation [2]. This apatite nucleation is the key process that causes the main constituent of bone to accumulate. In vitro analysis of this mineralisation process has become an important tool for assessing among other things, the osteogenic activity of anabolic agents, implant surfaces, and materials. Currently, a number of techniques are employed to examine this biological event, including investigating the regulation of genes such as osteocalcin and bone sialoprotein-1 [3,4], staining of in vitro cultures with histological chemicals (e.g., Von Kossa, alizarin red S, and Arsenazo III) which bind to the calcium aggregates [5–7], calcein incorporation [8], and labelling/quantifying calcium deposits with the radionuclide, Tc-99m-MDP [9]. Although the level of calcium deposition can be analysed by destaining the cultures followed by analysis of their optical density (e.g., Arsenazo III addition to culture extract [10]) or extracting the calcium (e.g., hydrochloric acid [4]), these methods can be inaccurate due to a low and narrow linear range (Arsenazo III analysis) or difficult to analyse.
⇑ Corresponding author. Fax: +44 (0)1904 824004. E-mail address: [email protected] (P. Souter). 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.11.041
Following on from a published method [9] that quantified calcium concentration by inductively coupled plasma optical emission spectroscopy (ICP-OES)1 following nitric acid extraction, an assay was developed to permit fast, sensitive and accurate assessment of in vitro mineralisation by chelation with citric acid and subsequent analysis by ion exchange chromatography, thereby negating the need for expensive ICP-OES equipment. Initial experiments allowed comparison to existing methods and analytical techniques, while secondary testing investigated the sensitivity of the method as well as the cell biology protocol for osteogenesis assays.
Materials and methods In vitro cell biology Primary human osteoblasts (PromoCell GmbH, Heidelberg, Germany) were resurrected and expanded in osteoblast growth media (PromoCell) and used at Passage 6. Osteoblasts were seeded into four 24-well plates (20 wells per plate) at a density of 40,000 cells/well in normal growth media (PromoCell) and incubated for 24 h at 37 °C, 5% CO2. The wells were equally divided between osteogenic and nonosteogenic culture conditions and analysis of mineralisation using alizarin red or ion chromatography. Following incubation, the media were exchanged on test wells for osteogenic media supplemented with 1 Abbreviations used: DI, deionised; ICP-OES, inductively coupled plasma optical emission spectroscopy; PBS, phosphate-buffered saline; %RSD, percentage relative standard deviation.
Quantification of in vitro mineralisation using ion chromatography / P. Souter et al. / Anal. Biochem. 410 (2011) 244–247
50 lg/ml ascorbic acid (Sigma, UK), 10 8 M dexamethasone (Sigma), and 3 mM b-glycerophosphate (Sigma). Controls were fed with growth media as before. Media were changed three times per week throughout the 21-day assay period. Time points of 3, 7, 14, and 21 days were studied.
245
Analytical Procedures document [11]. This involved an assessment of accuracy, precision, specificity, limit of detection, quantification limit, and linearity. Assay sensitivity – calcium quantification using citric acid and osteogenesis protocol
Existing calcium detection method – alizarin red A 1 mg/ml stock solution of alizarin red (Fluka, UK) was prepared in deionised water (DI H2O) and the pH adjusted to 5.5 using sodium hydroxide. The bottle was protected from the light and stored at room temperature. At 3, 7, 14, and 21 days, the allocated wells were stained with alizarin red. Briefly, the media were removed from the wells which were washed twice with sterile PBS, following which the cultures were fixed in 70% ethanol for 10 min. The wells were washed twice in excess DI water to remove all of the alcohol and 1 ml alizarin red solution was added to each well and incubated at room temperature for 30 min with agitation while protected from the light. After incubation, the stain was removed and the wells were washed thoroughly with tap water and left to dry. Following staining, images were captured using the Leica MZFIII Microscope. Once the cultures had been imaged, the wells were treated with 1 ml/well 5% perchloric acid (Riedel–de Haën) and rocked overnight at room temperature while protected from light. Calcium quantification using ion chromatography The ion chromatography system was manufactured by Metrohm and comprised of a Model 838 autosampler, Model 818 pump, Model 819 conductivity detector, and Model 837 degasser. A 50-ll injection of the test solution was made onto a cation exchange Metrosep C2 100 4 mm, 7 lm column. A 4 mM tartaric acid/ 0.75 mM dipicolinic acid eluent at a flow rate of 1 ml/min was employed with the analysis being undertaken at ambient temperature. The range and full scale settings for the conductivity detector were 1 mS/cm and 20 lS/cm, respectively. Column and eluent conditions employed were as recommended by Metrohm for the separation and quantification of alkali metals and alkaline earth metals. Under the conditions described these cations are retained by the column as a function of their specific charge to size ratio and anionic species unretained. The run time for the method was 12 min with calcium being eluted uniquely at approximately 7.5 min. The wells assigned for calcium quantification were washed twice with DI water and allowed to dry. Once all of the time points had been completed a 1-ml aliquot of a 10% citric acid solution was added to each well and the solutions were left to stand overnight at 4 °C to effect calcium chelation. Each 1 ml 10% citric acid solution was then transferred to a 13ml sample vial and the well washed with multiple aliquots of deionised water. The washings were then transferred to the 13ml sample vial and the solution was diluted to 10 ml to produce a 1% citric acid solution at a pH of approximately 2.5. The dilution step was crucial since the pH of a 10% citric acid solution is <2, which is outside of the tolerable pH range for the column (2–7). The system was calibrated with calcium standards ranging from 1 to 50 lg. This was achieved by pipetting a 1-ml aliquot of calcium chloride standards in deionised water ranging from 1 to 50 lg/ml of calcium into a sample vial along with a 1-ml aliquot of a 10% citric acid solution. The standard solution was then diluted with an 8-ml aliquot of deionised water to replicate the environment of the citric acid sample extracts, essential if an accurate value for the calcium content of sample solutions is to be obtained. The method was validated in general accordance with the International Conference on Harmonisation (ICH) Test for Validation of
The ion chromatographic procedure employed for the quantification of calcium deposition had a limit of quantification of 2 lg of calcium. In order to address concerns that 2 lg of calcium may represent a large amount of calcium for a culture to produce, the system was validated for sensitivity. Results Existing calcium detection method – alizarin red All osteogenic cultures stained positively with alizarin red with an increased intensity of staining apparent over the 21-day culture period, relating to increased calcium deposition. The control wells also showed some signs of increased staining over the 21 days despite growth in normal expansion media. Most obviously, there does not appear to be any mineralisation at the 3-day time point, with the control and osteogenic wells both showing baseline residual staining to the same degree of intensity; however, it could also be said that there is minimal visual difference between the 7 and the 14 days control and osteogenic cultures (Fig. 1). The increase in alizarin staining observed visually in control and osteogenic cultures over the 21 days was confirmed by the optical densities obtained from the destaining procedure (Fig. 2). Corrected absorbance data showed increases from 0.01 to approximately 0.03 (P < 0.05) and 0.01 to approximately 0.08 (P < 0.01) for the control and osteogenic cultures, respectively (Fig. 2). By this method it is not possible to determine if the culture time-dependent increase in alizarin red staining is calcium dependent or an artifact of the culture process and nonspecific accumulation. Subtraction of control (background) staining from the osteogenic data reveals the limited sensitivity of the alizarin red assay with appreciable difference (above 0) only being detected after 14 days (Fig. 3). Calcium quantification using ion chromatography The accuracy of the ion chromatography system employed for the work to assess calcium levels was found to be within 3% of the true value. Precision %RSD = 0.9. Calcium was not detected in a solution of 10% citric acid placed in a tissue culture plate overnight at 4 °C, showing that the method was suitably specific to calcium and the limits of detection and quantification were 1 and 2 lg, respectively. Linear regression coefficients for system calibrations were consistently in excess of 0.9995. The samples showed a clear trend of increasing calcium expression with time (Table 1 and Fig. 4), with the mineralisation from 7
Fig.1. Representative alizarin red staining of control and osteogenic cultures over 21 days.
246
Quantification of in vitro mineralisation using ion chromatography / P. Souter et al. / Anal. Biochem. 410 (2011) 244–247
0.100 0.080 0.070
35
Osteo Control
30
0.060
Calcium (µg)
OD (492-450nm)
40
Day 3 Day 7 Day 14 Day 21
0.090
0.050 0.040 0.030 0.020
25
R2 = 0.982
20 15 10
0.010
5
0.000 Control
Osteogenic
0 3
Fig.2. Alizarin red destain of osteoblast cultures. Error bars, standard deviation (error, SD). Downward diagonal bars, Day 3; chequered bars, Day 7; upward diagonal bars, Day 14; dotted bars, Day 21.
7
14
21
Time (days) Fig.4. Quantification of calcium expression by osteogenic cells at 4, 7, and 14 days. Error bars, SD. Downward diagonal bars, osteogenic cultures; upward diagonal bars, control cultures.
0.070 0.060
OD (492-450nm)
0.050
Day 3 Day 7 Day 14 Day 21
0.040 0.030 0.020 0.010 0.000
Fig.3. Corrected absorbance for alizarin red destained osteogenic cultures (error, SD). Downward diagonal bars, Day 3; chequered bars, Day 7; upward diagonal bars, Day 14; dotted bars, Day 21.
to 21 days exhibiting a linear trend (R2 = 0.9873). In addition, calcium expression levels could be determined after only 3 days of cell culture. Calcium was not detected in any of the control wells (limit of detection <2 lg). Discussion Alizarin red staining is a tried and trusted method for the evaluation of calcium deposition for in vitro bone forming studies. A significant limitation of the method is that results are sensitive
to the preparation and processing of samples during the staining procedure. For example, thoroughness of poststaining washes can be extremely influential on the results obtained (data not shown). Thus the results are only comparative within an assay and within a single user so limiting the ability to compare between operatives and experiments. Additional limitations of the alizarin red method highlighted by our data are that the assay has limited specificity and sensitivity. Time-dependent accumulation of staining in control cultures that was not seen with our ion chromatography method (described above) suggests non-calcium-dependent uptake and so lack of specificity in the binding of alizarin red to long-term cell cultures. The data suggest that the lack of detection of Ca by the ion chromatography method is not the lack of sensitivity of this method as in osteogenic cultures Ca accumulation was detected as early as Day 3 and increased almost linearly throughout the 21-day culture period The reasons for the culture time and potentially non-calciumdependent uptake of alizarin red have yet to be determined but may simply reflect entrapment with the monolayer as the cells become more densely packed due to cell division. A second limitation of the alizarin red method suggested by the data is the lack of sensitivity. At early time points 3 and 7 days alizarin red staining could not distinguish between control and test cultures and even when corrected at 7 days the corrected staining was almost zero. Whereas, as already noted, the ion chromatography method was able to detect increased Ca at Day 3 compared to the control cultures. The ability to analyse osteogenic data at earlier time points as seen with the ion chromatography method provides a valuable tool for the scientific community to accurately assess early miner-
Table 1 Calcium expression (lg) of human osteoblasts cultured on tissue culture plastic. Culture days
Calcium (lg) – replicates
Mean (lg)
3 7 14 21
5.93 11.33 19.76 32.70
7.22 10.41 20.45 33.50
5.99 11.88 15.30 25.43
6.71 8.48 14.39 25.68
7.13 7.76 17.98 27.24
6.60 9.97 17.58 28.91
3 7 14 21
N/D N/D N/D N/D
N/D N/D N/D N/D
N/D N/D N/D N/D
N/D N/D N/D N/D
N/D N/D N/D 2.05
<2 <2 <2 <2
Osteogenic
Control
N/D, none detected.
Quantification of in vitro mineralisation using ion chromatography / P. Souter et al. / Anal. Biochem. 410 (2011) 244–247
alisation responses to potential therapeutic agents and devices and so enable the selection of agents and surfaces that promote rapid bone deposition, increasing bone strength or bony integration of implanted devices. The sensitivity of the method to in vitro mineralisation has been proven to allow detection of 2 lg calcium within a culture and as shown in Fig. 4, this is more than adequate to assess osteogenic cultures at early time points when set up in the manner described here. Other ion chromatographic systems employing alternative column and eluent systems may be utilised to achieve the same end, but it is essential that the ability of any substitute system to cope with the acidic 1% citric acid sample environment and the accuracy of calcium content determination be evaluated rather than assumed. A potential limitation of the ion chromatography method described here is that when determining calcium levels at the low concentrations it is important to ensure that solutions are not exposed to glass, since this can result in calcium levels of around 0.1 lg/ml (data not shown).
Conclusions Ion chromatography provides a simple route to definitively evaluate calcium expression by osteogenic cell cultures, without the need for expensive ICP-OES analytical equipment.
247
References [1] W.A. Horton, C.R. Degnin, FGFs in endochondral skeletal development, Trends Endocrinol. Metab. 20 (7) (2009) 341–348. [2] H.C. Anderson, Vesicles associated with calcification in the matrix of epiphyseal cartilage, J. Cell Biol. 41 (1969) 59–72. [3] G.B. Schneider, H. Perinpanayagam, M. Clegg, R. Zaharias, D. Seabold, J. Keller, C. Stanford, Implant surface roughness affects osteoblast gene expression, J. Dental Res. 82 (5) (2003) 372–376. [4] D.M. Findlay, K. Welldon, G.J. Atkins, D.W. Howie, A.C.W. Zannettino, D. Bobyn, The proliferation and phenotypic expression of human osteoblasts on tantalum metal, Biomaterials 25 (2004) 2215–2227. [5] Y. Nakano, W.N. Addison, M.T. Kaartinen, ATP-mediated mineralization of MC3T3-E1 osteoblast cultures, Bone 41 (2007) 549–561. [6] J.E. Gough, J.R. Jones, L.L. Hench, Nodule formation and mineralisation of human primary osteoblasts on a porous bioactive glass scaffold, Biomaterials 25 (2004) 2039–2046. [7] Y.-D.C. Halvorsen, D. Franklin, A.L. Bond, D.C. Hitt, C. Auchter, A.L. Boskey, E.P. Paschalis, W.O. Wilkinson, J.M. Gimble, Extracellular matrix mineralisation and osteoblast gene expression by human adipose tissue-derived stromal cells, Tissue Eng. 7 (6) (2001) 729–741. [8] J.A.R. Gordon, C.E. Tye, A.V. Sampaio, T.M. Underhill, G.K. Hunter, H.A. Goldbery, Bone sialoprotein expression enhances osteoblast differentiation and matrix mineralization in vitro, Bone 41 (2007) 462–473. [9] H. Wang, V.H. Gerbaudo, L.W. Hobbs, M. Spector, Quantitation of osteoblast-like cell mineralization on tissue culture polystyrene and Ti6Al-4V alloy disks by Tc-99m-MDP labelling and imaging in vitro, Bone 36 (2005) 84–92. [10] C.A. Gregory, W.G. Gunn, A. Peister, D.J. Prockop, An alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction, Anal. Biochem. 329 (2004) 77–84. [11]
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Quantification of in vitro mineralisation using ion chromatography Paul Souter ⇑, Alan Horner, Jim C. Cunningham Smith and Nephew Research Centre, Heslington, York YO23 1HG, United Kingdom
a r t i c l e
i n f o
Article history: Received 9 September 2010 Received in revised form 13 October 2010 Accepted 26 November 2010 Available online 3 December 2010 Keywords: Osteoblast Mineralisation Calcium Ion chromatography Citric acid
a b s t r a c t Analysis of in vitro mineralisation is an important tool in orthopedic research, allowing assessment of new therapeutic agents and devices; however, access to analytical equipment and accuracy of current methods can be a limiting factor. This current work investigated the use of calcium chelation with citric acid and subsequent analysis by ion chromatography as a method for accurately quantifying the extent of in vitro calcium deposition. Primary human osteoblasts were cultured on tissue culture plastic for 21 days under osteogenic conditions. At 3, 7, 14, and 21 days, alizarin red staining and citric acid calcium chelation of the cultures were performed. The use of alizarin red revealed increased calcium deposition over the culture period but was not sensitive enough to detect mineralisation at early time points after taking in to account background residual staining. The use of ion chromatography gave a limit of detection of 2 lg calcium, sensitive enough to detect mineralisation after 3 days, with no issues relating to background levels. We believe that the use of ion chromatography for quantifying in vitro mineralisation gives researchers an accurate, accessible, and cheap way of assessing novel technologies. Ó 2010 Elsevier Inc. All rights reserved.
The process of bone formation in higher vertebrates occurs via two routes: intramembranous ossification (direct bone formation) and endochondral ossification where bone is formed through an intermediate cartilaginous template [1]. Calcification of the organic extracellular matrix is initiated at the nucleation sites where it is believed that matrix vesicles rich in alkaline phosphatase, concentrated calcium, and inorganic phosphate lead to hydroxyapatite formation [2]. This apatite nucleation is the key process that causes the main constituent of bone to accumulate. In vitro analysis of this mineralisation process has become an important tool for assessing among other things, the osteogenic activity of anabolic agents, implant surfaces, and materials. Currently, a number of techniques are employed to examine this biological event, including investigating the regulation of genes such as osteocalcin and bone sialoprotein-1 [3,4], staining of in vitro cultures with histological chemicals (e.g., Von Kossa, alizarin red S, and Arsenazo III) which bind to the calcium aggregates [5–7], calcein incorporation [8], and labelling/quantifying calcium deposits with the radionuclide, Tc-99m-MDP [9]. Although the level of calcium deposition can be analysed by destaining the cultures followed by analysis of their optical density (e.g., Arsenazo III addition to culture extract [10]) or extracting the calcium (e.g., hydrochloric acid [4]), these methods can be inaccurate due to a low and narrow linear range (Arsenazo III analysis) or difficult to analyse.
⇑ Corresponding author. Fax: +44 (0)1904 824004. E-mail address: [email protected] (P. Souter). 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.11.041
Following on from a published method [9] that quantified calcium concentration by inductively coupled plasma optical emission spectroscopy (ICP-OES)1 following nitric acid extraction, an assay was developed to permit fast, sensitive and accurate assessment of in vitro mineralisation by chelation with citric acid and subsequent analysis by ion exchange chromatography, thereby negating the need for expensive ICP-OES equipment. Initial experiments allowed comparison to existing methods and analytical techniques, while secondary testing investigated the sensitivity of the method as well as the cell biology protocol for osteogenesis assays.
Materials and methods In vitro cell biology Primary human osteoblasts (PromoCell GmbH, Heidelberg, Germany) were resurrected and expanded in osteoblast growth media (PromoCell) and used at Passage 6. Osteoblasts were seeded into four 24-well plates (20 wells per plate) at a density of 40,000 cells/well in normal growth media (PromoCell) and incubated for 24 h at 37 °C, 5% CO2. The wells were equally divided between osteogenic and nonosteogenic culture conditions and analysis of mineralisation using alizarin red or ion chromatography. Following incubation, the media were exchanged on test wells for osteogenic media supplemented with 1 Abbreviations used: DI, deionised; ICP-OES, inductively coupled plasma optical emission spectroscopy; PBS, phosphate-buffered saline; %RSD, percentage relative standard deviation.
Quantification of in vitro mineralisation using ion chromatography / P. Souter et al. / Anal. Biochem. 410 (2011) 244–247
50 lg/ml ascorbic acid (Sigma, UK), 10 8 M dexamethasone (Sigma), and 3 mM b-glycerophosphate (Sigma). Controls were fed with growth media as before. Media were changed three times per week throughout the 21-day assay period. Time points of 3, 7, 14, and 21 days were studied.
245
Analytical Procedures document [11]. This involved an assessment of accuracy, precision, specificity, limit of detection, quantification limit, and linearity. Assay sensitivity – calcium quantification using citric acid and osteogenesis protocol
Existing calcium detection method – alizarin red A 1 mg/ml stock solution of alizarin red (Fluka, UK) was prepared in deionised water (DI H2O) and the pH adjusted to 5.5 using sodium hydroxide. The bottle was protected from the light and stored at room temperature. At 3, 7, 14, and 21 days, the allocated wells were stained with alizarin red. Briefly, the media were removed from the wells which were washed twice with sterile PBS, following which the cultures were fixed in 70% ethanol for 10 min. The wells were washed twice in excess DI water to remove all of the alcohol and 1 ml alizarin red solution was added to each well and incubated at room temperature for 30 min with agitation while protected from the light. After incubation, the stain was removed and the wells were washed thoroughly with tap water and left to dry. Following staining, images were captured using the Leica MZFIII Microscope. Once the cultures had been imaged, the wells were treated with 1 ml/well 5% perchloric acid (Riedel–de Haën) and rocked overnight at room temperature while protected from light. Calcium quantification using ion chromatography The ion chromatography system was manufactured by Metrohm and comprised of a Model 838 autosampler, Model 818 pump, Model 819 conductivity detector, and Model 837 degasser. A 50-ll injection of the test solution was made onto a cation exchange Metrosep C2 100 4 mm, 7 lm column. A 4 mM tartaric acid/ 0.75 mM dipicolinic acid eluent at a flow rate of 1 ml/min was employed with the analysis being undertaken at ambient temperature. The range and full scale settings for the conductivity detector were 1 mS/cm and 20 lS/cm, respectively. Column and eluent conditions employed were as recommended by Metrohm for the separation and quantification of alkali metals and alkaline earth metals. Under the conditions described these cations are retained by the column as a function of their specific charge to size ratio and anionic species unretained. The run time for the method was 12 min with calcium being eluted uniquely at approximately 7.5 min. The wells assigned for calcium quantification were washed twice with DI water and allowed to dry. Once all of the time points had been completed a 1-ml aliquot of a 10% citric acid solution was added to each well and the solutions were left to stand overnight at 4 °C to effect calcium chelation. Each 1 ml 10% citric acid solution was then transferred to a 13ml sample vial and the well washed with multiple aliquots of deionised water. The washings were then transferred to the 13ml sample vial and the solution was diluted to 10 ml to produce a 1% citric acid solution at a pH of approximately 2.5. The dilution step was crucial since the pH of a 10% citric acid solution is <2, which is outside of the tolerable pH range for the column (2–7). The system was calibrated with calcium standards ranging from 1 to 50 lg. This was achieved by pipetting a 1-ml aliquot of calcium chloride standards in deionised water ranging from 1 to 50 lg/ml of calcium into a sample vial along with a 1-ml aliquot of a 10% citric acid solution. The standard solution was then diluted with an 8-ml aliquot of deionised water to replicate the environment of the citric acid sample extracts, essential if an accurate value for the calcium content of sample solutions is to be obtained. The method was validated in general accordance with the International Conference on Harmonisation (ICH) Test for Validation of
The ion chromatographic procedure employed for the quantification of calcium deposition had a limit of quantification of 2 lg of calcium. In order to address concerns that 2 lg of calcium may represent a large amount of calcium for a culture to produce, the system was validated for sensitivity. Results Existing calcium detection method – alizarin red All osteogenic cultures stained positively with alizarin red with an increased intensity of staining apparent over the 21-day culture period, relating to increased calcium deposition. The control wells also showed some signs of increased staining over the 21 days despite growth in normal expansion media. Most obviously, there does not appear to be any mineralisation at the 3-day time point, with the control and osteogenic wells both showing baseline residual staining to the same degree of intensity; however, it could also be said that there is minimal visual difference between the 7 and the 14 days control and osteogenic cultures (Fig. 1). The increase in alizarin staining observed visually in control and osteogenic cultures over the 21 days was confirmed by the optical densities obtained from the destaining procedure (Fig. 2). Corrected absorbance data showed increases from 0.01 to approximately 0.03 (P < 0.05) and 0.01 to approximately 0.08 (P < 0.01) for the control and osteogenic cultures, respectively (Fig. 2). By this method it is not possible to determine if the culture time-dependent increase in alizarin red staining is calcium dependent or an artifact of the culture process and nonspecific accumulation. Subtraction of control (background) staining from the osteogenic data reveals the limited sensitivity of the alizarin red assay with appreciable difference (above 0) only being detected after 14 days (Fig. 3). Calcium quantification using ion chromatography The accuracy of the ion chromatography system employed for the work to assess calcium levels was found to be within 3% of the true value. Precision %RSD = 0.9. Calcium was not detected in a solution of 10% citric acid placed in a tissue culture plate overnight at 4 °C, showing that the method was suitably specific to calcium and the limits of detection and quantification were 1 and 2 lg, respectively. Linear regression coefficients for system calibrations were consistently in excess of 0.9995. The samples showed a clear trend of increasing calcium expression with time (Table 1 and Fig. 4), with the mineralisation from 7
Fig.1. Representative alizarin red staining of control and osteogenic cultures over 21 days.
246
Quantification of in vitro mineralisation using ion chromatography / P. Souter et al. / Anal. Biochem. 410 (2011) 244–247
0.100 0.080 0.070
35
Osteo Control
30
0.060
Calcium (µg)
OD (492-450nm)
40
Day 3 Day 7 Day 14 Day 21
0.090
0.050 0.040 0.030 0.020
25
R2 = 0.982
20 15 10
0.010
5
0.000 Control
Osteogenic
0 3
Fig.2. Alizarin red destain of osteoblast cultures. Error bars, standard deviation (error, SD). Downward diagonal bars, Day 3; chequered bars, Day 7; upward diagonal bars, Day 14; dotted bars, Day 21.
7
14
21
Time (days) Fig.4. Quantification of calcium expression by osteogenic cells at 4, 7, and 14 days. Error bars, SD. Downward diagonal bars, osteogenic cultures; upward diagonal bars, control cultures.
0.070 0.060
OD (492-450nm)
0.050
Day 3 Day 7 Day 14 Day 21
0.040 0.030 0.020 0.010 0.000
Fig.3. Corrected absorbance for alizarin red destained osteogenic cultures (error, SD). Downward diagonal bars, Day 3; chequered bars, Day 7; upward diagonal bars, Day 14; dotted bars, Day 21.
to 21 days exhibiting a linear trend (R2 = 0.9873). In addition, calcium expression levels could be determined after only 3 days of cell culture. Calcium was not detected in any of the control wells (limit of detection <2 lg). Discussion Alizarin red staining is a tried and trusted method for the evaluation of calcium deposition for in vitro bone forming studies. A significant limitation of the method is that results are sensitive
to the preparation and processing of samples during the staining procedure. For example, thoroughness of poststaining washes can be extremely influential on the results obtained (data not shown). Thus the results are only comparative within an assay and within a single user so limiting the ability to compare between operatives and experiments. Additional limitations of the alizarin red method highlighted by our data are that the assay has limited specificity and sensitivity. Time-dependent accumulation of staining in control cultures that was not seen with our ion chromatography method (described above) suggests non-calcium-dependent uptake and so lack of specificity in the binding of alizarin red to long-term cell cultures. The data suggest that the lack of detection of Ca by the ion chromatography method is not the lack of sensitivity of this method as in osteogenic cultures Ca accumulation was detected as early as Day 3 and increased almost linearly throughout the 21-day culture period The reasons for the culture time and potentially non-calciumdependent uptake of alizarin red have yet to be determined but may simply reflect entrapment with the monolayer as the cells become more densely packed due to cell division. A second limitation of the alizarin red method suggested by the data is the lack of sensitivity. At early time points 3 and 7 days alizarin red staining could not distinguish between control and test cultures and even when corrected at 7 days the corrected staining was almost zero. Whereas, as already noted, the ion chromatography method was able to detect increased Ca at Day 3 compared to the control cultures. The ability to analyse osteogenic data at earlier time points as seen with the ion chromatography method provides a valuable tool for the scientific community to accurately assess early miner-
Table 1 Calcium expression (lg) of human osteoblasts cultured on tissue culture plastic. Culture days
Calcium (lg) – replicates
Mean (lg)
3 7 14 21
5.93 11.33 19.76 32.70
7.22 10.41 20.45 33.50
5.99 11.88 15.30 25.43
6.71 8.48 14.39 25.68
7.13 7.76 17.98 27.24
6.60 9.97 17.58 28.91
3 7 14 21
N/D N/D N/D N/D
N/D N/D N/D N/D
N/D N/D N/D N/D
N/D N/D N/D N/D
N/D N/D N/D 2.05
<2 <2 <2 <2
Osteogenic
Control
N/D, none detected.
Quantification of in vitro mineralisation using ion chromatography / P. Souter et al. / Anal. Biochem. 410 (2011) 244–247
alisation responses to potential therapeutic agents and devices and so enable the selection of agents and surfaces that promote rapid bone deposition, increasing bone strength or bony integration of implanted devices. The sensitivity of the method to in vitro mineralisation has been proven to allow detection of 2 lg calcium within a culture and as shown in Fig. 4, this is more than adequate to assess osteogenic cultures at early time points when set up in the manner described here. Other ion chromatographic systems employing alternative column and eluent systems may be utilised to achieve the same end, but it is essential that the ability of any substitute system to cope with the acidic 1% citric acid sample environment and the accuracy of calcium content determination be evaluated rather than assumed. A potential limitation of the ion chromatography method described here is that when determining calcium levels at the low concentrations it is important to ensure that solutions are not exposed to glass, since this can result in calcium levels of around 0.1 lg/ml (data not shown).
Conclusions Ion chromatography provides a simple route to definitively evaluate calcium expression by osteogenic cell cultures, without the need for expensive ICP-OES analytical equipment.
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