In vitro ion adsorption and cytocompatibility of dicalcium phosphate ceramics
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In vitro cell testing of degradable bioceramics such as brushite or monetite is often challenging due to the ion release into or adsorption from the culture medium. These ionic changes are then mostly responsible for cell proliferation and activity, which prohibits the investigation of effects originating from surface topography or further material modifications.
Here, we aimed to solve this problem by developing a pre-conditioning regime following the repeated immersion of brushite and monetite samples in various Ca2+, Mg2+ and PO4 3− containing electrolytes, followed by studying ion adsorption / release as well as changes in phase composition and in vitro cytocompatibility with MG63 cells.
The results demonstrated that by using DMEM cell culture medium in a ratio of 10 ml/sample was sufficient to minimize changes of ionic composition after 7 d with a daily change of the medium. This leads to changes of the surface composition with dissolution of the brushite phase. In turn, this also positively influences the in vitro cytocompatibility with a 2–3 fold higher cell number and cell activity on the DMEM pretreated surfaces.
Controlled sample washing prior to cell testing using DMEM medium seems to be a valuable procedure not only to stabilize the pH during cell culture but also to maintain ion concentrations within a cell friendly range.
KeywordsBrushite Monetite Cell culture Ion adsorption
Dulbecco’s modified eagle’s medium
Inductively coupled plasma – mass spectroscopy
Joint committee of powder diffraction standards
Monocalcium phosphate anhydrous
Phosphate buffered saline
Calcium phosphate cements (CPC) are of high clinical interest for bone replacement due to their well-known biocompatibility in vivo [1, 2, 3]. Although a diversity of formulations are possible there are only two main products of the cement dissolution–precipitation reaction. Under neutral conditions hydroxyapatite (Ca10(PO4)6(OH)2, HA) is formed and under acid conditions orthophosphate is protonated and secondary phosphates such as brushite (CaHPO4•2H2O) or monetite (CaHPO4) are the main products of cement setting [4, 5, 6]. Whilst brushite is commonly formed due to kinetic reasons, monetite is only precipitated under highly acidic pH conditions, a water-deficient environment or by adding metal ions disrupting brushite crystal growth [7, 8]. Compared to HA cements, secondary phosphates have the advantage of a higher solubility under physiological conditions, which results in a faster resorption and bone remodeling in vivo . In comparison to brushite, monetite shows a lower solubility under physiological conditions, but resorbs faster in vivo since monetite does not transform into low soluble HA at physiological pH .
Although brushite and monetite based cements show promising results in vivo [11, 12, 13, 14], their in vitro characterization is challenging due to their metastable behavior under cell culture conditions. According to our experience this leads to a high release of phosphate ions into and an uptake of calcium and magnesium ions from the culture medium, whereas both effects have a detrimental effect on the cytocompatibility of the materials. This is problematic for the development of material modifications based on brushite or monetite (e.g. by adding bioactive metal ions), since it is then unclear whether observed effects during cell culture are correlated with the modification itself (e.g. by the released metal ion) or with the properties of the matrix and their capacity to change the ionic composition of the culture medium. The same would appear for an influence of surface topography on in vitro cellular behavior . Recently, we were able to show that silica modified brushite cements showed an improved cytocompatibility compared to pure brushite samples . However, this behavior was not correlated to the release of silicate ions, but to an altered dissolution profile of brushite during the in vitro experiments.
A solution to this problem might be a pre-conditioning of the samples prior to cell testing by immersion in calcium and magnesium containing solutions. This is thought to both remove unreacted, acidic cement raw materials, as well as to saturate the samples with calcium and magnesium ions. Here we studied in a systematic way the effect of such a washing regime of brushite and monetite samples prior to cell testing. Both changes of culture medium composition as well as the phase composition of the ceramic surface were correlated to the in vitro cytocompatibility determined with an osteoblastic cell line.
β-Tricalcium phosphate (ß-TCP) was prepared by sintering monetite powder (CaHPO4, Baker, Germany) and calcium carbonate (CaCO3, Merck, Germany) in a molar ratio of 2:1 for 5 h at 1050 °C. The sintered cakes were manually crushed with mortar and pestle and then sieved with 355 μm pore size-mesh prior to milling in a planetary ball mill (Retsch, Haan, Germany) for 60 min at 200 rpm. Cement powders were produced by mixing β-TCP powder in an equimolar ratio with monocalcium phosphate anhydrous (Ca(H2PO4)2, MCPA, Aldrich, Steinheim, Germany) in a coffee grinder for 30 s. Cement pastes were prepared by mixing the powder with water at powder to liquid ratios of 1.0, 2.0 and 3.0 g/ml. The pastes were transferred into silicone rubber molds (d = 15 mm, h = 2 mm) and set for 24 h at 37 °C at a humidity >90%. This resulted in a quantitative conversion of the cement powder into brushite according to XRD analysis. Monetite samples were prepared accordingly followed by autoclaving the samples at 121 °C for 20 min. All samples were sterilized prior to the following experiments by soaking in 70% ethanol followed by drying under sterile conditions.
Porosity characteristics of the samples were measured by mercury (Hg) porosimetry (PASCAL 140/440, Porotec GmbH, Hofheim, Germany). Specific surface area was determined by nitrogen adsorption (BET-method, Autosorb-iQ-AG, Quantachrome, Odelzhausen, Germany). The ionic composition of each medium was analyzed by inductively coupled plasma mass spectroscopy (ICP-MS, Varian, Australia) against standard solutions of 10 ppm Ca2+, Mg2+ or PO4 3− (Merck, Darmstadt, Germany). The phase composition of the samples was determined using X-ray diffraction (XRD) analysis with monochromatic CuKα radiation (D5005, Siemens, Karlsruhe, Germany) in a 2θ range from 20 to 40 ° with a step size of 0.02 °. This was performed on both finely ground samples (for changes of the bulk volume) as well as on intact samples to investigate the influence of the immersion regime on the surface composition. Qualitative assessment of the diffraction patterns occurred via JCPDS reference patterns for brushite (PDF Ref. 09–0077), monetite (PDF Ref. 09–0080) and β-TCP (PDF Ref. 09–0169).
In vitro cytocompatibility testing was performed using the osteoblastic cell line MG 63 (ATCC no. CRL-1427, Rockville, MD). Cells were cultured at 37 °C and 5% CO2 in DMEM medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Invitrogen Life Technologies). Cells were cultivated on polystyrene (PS) as well as on unwashed and pre-conditioned brushite and monetite specimen. Samples were placed in quadruplicate into the wells of a 24-well plate and covered with cell suspension. Cytocompatibility tests were performed by measuring cell proliferation as well as cell activity after 2 days in culture on all surfaces. Cell counting was performed using a CASY 1 TTC cell analyzer (Schärfe System, Reutlingen, Germany). Cell viability was analyzed using the cell proliferation reagent WST 1 (Roche Diagnostics, Mannheim, Germany), whereas after incubating the cells for 30 min with the a 1:10 dilution of the WST reagent in DMEM at 37 °C, the absorption of the supernatant was photometrically quantified (Tecan, Crailsheim, Germany) at 450 nm. For each method and sample four readings were recorded and the mean values and standard deviations were calculated.
Results and discussion
Porosity and specific surface area of brushite and monetite produced with a PLR of 1, 2 and 3 g/ml
Surface area [m2/g]
1 g/ml brushite
2.14 ± 0.03
2 g/ml brushite
1.95 ± 0.23
3 g/ml brushite
1.54 ± 0.14
1 g/ml monetite
1.86 ± 0.15
2 g/ml monetite
1.71 ± 0.24
3 g/ml monetite
1.62 ± 0.20
The results of the initial immersion study showed an even increasing release of phosphate, which raised the phosphate content by the factor of 20–35 (brushite) and the factor of 8–20 (monetite) compared to fresh medium. At the same time, the cements adsorbed most calcium (monetite >80%, brushite ~60–75% after more than 5 days) and magnesium ions (>95% for both matrices). Monetite generally adsorbed more Ca2+ and released less PO4 3−, whereas no clear difference was found for Mg2+.
In this study we analyzed the influence of the washing procedure of the metastable dicalcium phosphates brushite and monetite prior to in vitro studies. It is postulated, that monetite shows a better cell responses attributed to the lower solubility and thereby phosphate release of this phase . Due to the higher solubility of brushite it is completely washed out by PBS and DMEM, which resulted in a monetite surface and better cell response. Additionally, the saturation of magnesium and calcium had a beneficial effect for the cytocompatibility, as these essential nutrients are not adsorbed by the cement monolith anymore. The results clearly underline the need for controlled sample washing prior to cell testing. Here, the use of a large excess of serum free cell culture medium combined with multiple changing steps seems to be valuable to maintain ion concentrations within a cell friendly range. This will help to overcome the often observed discrepancy between the good in vivo results of calcium phosphate bioceramics (and their long term successful clinical use) and the strongly reduced cell growth on the ceramic surface under in vitro conditions.
We gratefully acknowledge financial support by the German Research Foundation (DFG) under the grant numbers GB1/20–1 and GR3232/3–1. This publication was funded by the German Research Foundation (DFG) and the University of Würzburg in the funding programme “Open Access Publishing”.
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All data sets supporting the results of this article are included within the article.
UG and MS designed the concept of the study and drafted the manuscript. All experiments regarding material synthesis and characterization were performed by MS. JEB and JG advised on the experimental design and modified the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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