influence of manganese ions on cellular behavior of human osteoblasts in vitro

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Influence of manganese ions on cellular behavior of human

osteoblasts in vitro

Frank Luthen a, Ulrike Bulnheim a, Petra D. Muller a, Joachim Rychly a,Henrike Jesswein b, J.G. Barbara Nebe a,*

a University of Rostock, Department of Internal Medicine, Schillingallee 69, 18057 Rostock, Germanyb University of Rostock, Institute of Biosciences, Albert-Einstein-Str. 3, 18059 Rostock, Germany

Abstract

Divalent cations like Mn2+ are known to strongly influence the integrin affinity to ligands and – in consequence – cell adhesion to extracellular

matrix proteins. Therefore, divalent cation supplementation of biomaterials could be a promising approach to improve the ingrowth and the

integration of implants. We were interested, whether manganese ions affect cellular functions like spreading, proliferation as well as gene

expression in human osteoblasts. MG-63 osteoblastic cells were cultured in DMEM with 10% FCS. MnCl2 was added at a concentration range of

0.01–0.5 mM for 24 h and 48 h. Spreading (cell area in mm2) of PKH26-stained cells (cell membrane dye) was analyzed using confocal

microscopy. Cell proliferation was measured by flow cytometry. Quantification of the phosphorylation status of signaling proteins was estimated

using the Bio-Plex 200 system. Gene expression of osteogenic markers at the mRNA and protein level was analyzed by quantitative real time RT-

PCR and Western blot, respectively. The results demonstrated that at higher concentrations of Mn2+ cells revealed a spindle shaped morphology.

Further analyses indicated a reduced spreading, proliferation as well as phosphorylation of signaling proteins due to the influence of Mn2+ in a

concentration-dependent manner. Although expression of bone sialo protein (BSP) at the mRNA level increased both after 24 h and 48 h in the

presence of manganese, no increased expression of BSP was detected at the protein level. The expression of alkaline phosphatase (ALP) and

collagen 1 (Col 1) mRNA decreased at >0.1 mM MnCl2. We speculate that the effect of manganese cations on cell functions is strongly

concentration-dependent and the release of manganese when incorporated in a biomaterial surface has to be thoroughly adjusted.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Manganese ions; Human osteoblasts; Proliferation; Gene expression

1. Introduction

The biologically important metal manganese is an essential

key cofactor for metalloenzymes (oxidases and dehydro-

genases), DNA polymerases and kinases (Culotta et al., 2005).

Furthermore, this divalent cation is known to strongly influence

the integrin avidity and the integrin affinity to ligands and – in

consequence – cell adhesion to extracellular matrix proteins

(Byzova et al., 2000; Mould et al., 1995; Zreiqat et al., 2002). In

addition, the stimulating effect on the affinity maturation of

avb1-integrins is accompanied by focal adhesion organization

and actin stress fiber formation (Dormond et al., 2004) which is

accompanied by enhanced cell migration (Byzova et al., 2000).

Therefore, divalent cation supplementation of biomaterials

could be a promising approach to improve the ingrowth and the

integration of implants. We were interested, whether manga-

nese ions affect cellular functions like spreading, proliferation

as well as gene expression in human osteoblasts to get insights

about the effectiveness as well as the concentration necessary

for the immobilization of divalent cations in the process of

biomaterial surface functionalization.

2. Materials and methods

2.1. Cell culture

Human MG-63 osteoblastic cells (osteosarcoma cell line, ATCC, LGC

Promochem) were cultured in six-well chambers (Greiner) in DMEM supple-

mented with 10% fetal calf serum (FCS Gold, PAA) with 1% gentamicin

(Ratiopharm) at 37 8C and in a 5% CO2 atmosphere. In general, cells were

seeded with a density of 3 � 105 cells/well. MnCl2 were directly added to the

cell suspension at a concentration range of 0.01–0.5 mM. The cultivation time

of osteoblasts was 24 h and 48 h (Luthen and Nebe, 2005). Cell morphology

was investigated under the confocal microscope using brightfield-image modus

(LSM 410, Carl Zeiss).

www.elsevier.com/locate/geneanabioeng

Biomolecular Engineering 24 (2007) 531–536

* Corresponding author. Tel.: +49 381 494 7771; fax: +49 381 494 7778.

E-mail address: [email protected] (J.G.B. Nebe).

1389-0344/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.bioeng.2007.08.003


2.2. Spreading

Human MG-63 osteoblasts were cultured for 24 h, trypsinated, washed in

PBS and the cell membrane stained with the red fluorescent linker PKH26

(PKH26 General Cell Linker Kit, Sigma) for 5 min in suspension as already

described (Luthen et al., 2005). The cells were then seeded into the wells and

cultured for 3 h, 16 h, 24 h, and 40 h. After fixation with 4% paraformaldehyde

(PFA, Merck) the cells were embedded with a cover slip. Spreading (cell area in

mm2) of 40 cells/specimen was measured using the software ‘area measure-

ment’ of the confocal microscope LSM 410 (Carl Zeiss).

2.3. Actin cytoskeleton

MG-63 cells were cultured in the presence of MnCl2 (0.1 mM) for 24 h.

Cells were fixed with 4% PFA (10 min, room temperature RT). After washing

with PBS, cells were permeabilized with 0.1% TritonX-100 (10 min, RT)

(Merck), incubated with phalloidine-TRITC (diluted 1:100, Sigma) for

30 min in the dark at RT, washed again, embedded and examined at the

LSM 410 (exc. 488 nm, Carl Zeiss) using a 63� oil immersion objective

1.25 oil/0.17.

2.4. Proliferation

The cell monolayer was trypsinated after 24 h of cultivation with 0.05%

trypsin/0.02% EDTA for 5 min. Cells in suspension were washed in PBS and

fixed with 70% ethanol over night at �20 8C. After washing twice cells were

treated with RNase (1 mg/ml, Sigma) at 37 8C for 20 min and incubated with

propidium iodide (PI) (50 mg/ml, Sigma) for at least 3 h on ice. Up to 20,000

events per sample were acquired by the flow cytometer FACSCalibur (BD

Biosciences). For the analysis of cell proliferation the cell cycle phases G0/G1,

S and G2/M were calculated in percent using ModFIT LT 3.0 for Power Mac G4

(BD Biosciences). For statistical evaluation S- and G2/M-phase cells were

defined as proliferative cells.

2.5. Phosphorylation of signaling proteins

Quantification of the phosphorylation status of signaling proteins was

estimated using the Bio-Plex 200 system (Bio-Rad Laboratories GmbH). Cell

lysates were simultaneously quantitatively analyzed by the Bio-Plex suspension

array system, a flow-based 96-well fluorescent microplate assay reader. Five

hundred ml/ml of each cell lysate were incubated with antibody-conjugated

beads (Phospho-ERK1/2 Assay #171V22238, Phospho-Akt Assay

#171V221075, Bio-Rad) in a microplate well to react with the specific

phosphorylated proteins as described in the manual.

2.6. Real time RT-PCR

Gene expression of osteogenic markers at the mRNA level was analyzed by

quantitative real time RT-PCR (ABI Prism1 7000 Sequence Detection System,

Applied Biosystems). Cells were washed twice with PBS and total RNA was

isolated using the NucleoSpin RNA II Kit (Macherey-Nagel) with DNase

treatment.

Fig. 1. Morphology of MG-63 osteoblasts. In the monolayer culture the cells switch to a more spindle like morphology when incubated with 0.1 mM and 0.5 mM

Mn2+. The index indicates the quotient of the cell length and the cell width: index of 1 = rounded cell, index of >1 = grade of longitudinal spreading (mean � S.D.,

n = 40) (Bright-field image, magnification 10�, LSM 410, Carl Zeiss).

F. Luthen et al. / Biomolecular Engineering 24 (2007) 531–536532


Reverse transcription was carried out starting from 500 ng of total RNA

using 1� first strand buffer, 10 U/ml SuperscriptTM II, 2 U/ml RNaseOutTM,

10 mM DTT, 0.5 mM (each) dNTPs, 2.5 mM randomhexamer (all Invitrogen) in

40 ml reaction volume and temperature holdings at 25 8C/10 min; 42 8C/50 min

and 70 8C/15 min after an initial denaturation of RNA at 65 8C/5 min. Quanti-

tative real time PCR assays were performed for alkaline phosphatase (ALP),

bone sialo protein (BSP), collagen 1 (Col 1), and monitored in duplicate.

Reaction conditions were: 1� TaqMan1 Universal Master Mix, 1�Assays-on-

DemandTM Gene Expression Assay Mix (ALP, BSP, Col 1—all Applied

Biosystems), 2.5 ml cDNA in a reaction volume of 25 ml. Thermocycling

conditions were: 95 8C/10 min followed by 40 cycles at 95 8C/15 s and

60 8C/1 min. Gene expression values were calculated by the comparative

DDCT-method (separate tubes with GAPDH as reference housekeeping gene).

2.7. Immunoblotting

Using mouse monoclonal anti-PCNA (Santa Cruz Biotechnology) and

polyclonal anti-human bone sialo protein II (DPC Biermann) immunoblots

were performed. Cells were lysed with a buffer containing 62.5 mM Tris–

HCl, pH 6.8, 5 mM EDTA, 2% SDS, 10% glycerol and 2% b-mercaptoetha-

nol. Proteins in total cell lysate were quantified using a Bradford assay (Bio-

Rad). Equal amounts of total cellular protein were separated by SDS-PAGE

and then transblotted to the PVDF membranes. The membranes were

incubated with appropriate primary antibodies overnight at 4 8C followed

by AP-conjugated secondary antibody (DAKO). For protein detection,

membranes were incubated with chemiluminescence reagent CDP star

(Roche) and exposed against X-ray films. Immunoblotting for each detected

Fig. 2. The time-dependent cell spreading of osteoblasts (3 h, 16 h, 24 h, and 40 h) is significantly inhibited due to the concentration-dependent influence of MnCl2(at 0.1 mM) (a). Cell area measured by the software ‘area measurement’ of the LSM 410 (U-test, p < 0.01, n = 40). In microscopical investigations this decrease in the

cell area of PKH 26-stained osteoblasts can optically be recognized after 24 h manganese ions treatment (b) (LSM 410, Carl Zeiss).

F. Luthen et al. / Biomolecular Engineering 24 (2007) 531–536 533


Fig. 3. In general, the proliferation of osteoblasts is inhibited due to manganese ions from 0.1 mM to 1.0 mM. The proliferative phase of the cell cycle (S+G2/M) is

significantly reduced after 24 h (flow cytometry, U-test, p < 0.05, n = 8) (a), which is accompanied by a reduction of the PCNA expression (Western blot analysis, one

representative example of three independent experiments) (b) as well as by a down regulation of the signal protein phosphorylation of p-Akt and p-ERK1/2 (Bio-Plex

200 System, U-test, *p < 0.05, +p < 0.01, n = 6) (c).

Fig. 4. The actin cytoskeleton of osteoblasts is pronounced and stress fibers are in parallel at a concentration of 0.1 mM MnCl2 (LSM 410, Carl Zeiss).



protein was repeated three times using lysates from three independent

experiments.

3. Results

The results demonstrated that at higher concentrations of

Mn2+ cells revealed a spindle shaped morphology (Fig. 1).

Further analyses indicated a reduced cell spreading (Fig. 2).

Due to the influence of Mn2+ in a concentration-dependent

manner the significantly reduced cell cycle phases S- and G2/M

(Fig. 3a) were accompanied by both, a reduced PCNA

expression (Fig. 3b) as well as inhibition of the phosphorylation

of the signaling proteins p-ERK1/2 – which regulate cell

growth and differentiation – and p-Akt which has putative roles

in cell proliferation and survival (Fig. 3c). In contrast, the

signaling protein IkB as an inhibitor of the NFkB-translocation

to the nucleus for gene activation and GSK3a (Glycogen

Synthase Kinase 3alpha), responsible for the activation of the

energy metabolism of the cell were not effected (data not

shown).

Non-treated and with 0.01 mM MnCl2 incubated cells

exhibited a meshwork of actin filaments. At the concentration

of 0.1 mM MnCl2 both, the generation of actin filaments and

the parallel direction of cytoskeleton appeared to be

pronounced (Fig. 4).

The expression of ALP- and Col 1-mRNA as early

differentiation markers of osteoblasts were slightly decreased

in a concentration-dependent manner (Fig. 5). Although

expression of the osteogenic marker BSP at the mRNA level

increased both after 24 h and 48 h in the presence of manganese

(Fig. 5), no increased expression of BSP was detected at the

protein level (Fig. 6).

4. Discussion

Because divalent cations like Mn2+ are known to influence

the integrin affinity (Byzova et al., 2000) we were interested in

the question, if manganese ions influence important cellular

functions like spreading and proliferation which are a

precondition for the cells to occupy an implant surface after

the first contact of cells. But we could find out that in human

osteoblasts both were inhibited by Mn2+ in a concentration-

dependent manner. Because our osteoblasts reveal a spindle

shaped morphology at higher concentrations of Mn2+ and

proliferation was reduced, which could indicate a cell

differentiation process we investigated cell differentiation

markers like ALP, Col 1 and BSP. We revealed that only the

late-stage differentiation protein BSP was increased at the

mRNA-level at a concentration already found for integrin

activation (Byzova et al., 2000; Legler et al., 2001). But this

increase could not be confirmed by Western blot analysis at the

BSP translation level after 24 h of culture. Therefore, future

long-time experiments are necessary to recognize if manganese

ions promote cell differentiation at the protein level.

5. Conclusions

We suggest that the effect of manganese cations on cell

functions is strongly concentration-dependent and the release

of manganese when incorporated in a biomaterial surface has to

be thoroughly adjusted.

Acknowledgements

The investigations as well as FL and UB were gratefully

supported by the program TEAM of the state Mecklenburg/

Vorpommern (UR 04 022 10), by the Deutsche Forschungs-

gemeinschaft (SPP 1100: NE 560/3-4) and by the BMBF

project ‘Tissue engineering of the bone’ (0101-31P3240).

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