Concentration-dependent cytotoxicity of copper ions on mousefibroblasts in vitro: effects of copper ion release from TCu380Avs TCu220C intra-uterine devices

Bianmei Cao & Yudong Zheng & Tingfei Xi &Chuanchuan Zhang & Wenhui Song &

Krishna Burugapalli & Huai Yang & Yanxuan Ma

Published online: 24 April 2012# Springer Science+Business Media, LLC 2012

Abstract Sustained release of copper (Cu) ions from Cu-containing intrauterine devices (CuIUD) is quite efficient forcontraception. However, the tissue surrounding the CuIUDis exposed to toxic Cu ion levels. The objective for thisstudy was to quantify the concentration dependent cytotoxiceffects of Cu ions and correlate the toxicity due to Cu ionburst release for two popular T-shaped IUDs - TCu380A andTCu220C on L929 mouse fibroblasts. Fibroblasts were cul-tured in 98 well tissue culture plates and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphehyltetrazolium bromide (MTT) assaywas used to determine their viability and proliferation as afunction of time. For cell seeding numbers ranging from10,000 to 100,000, a maximum culture time of 48 h wasidentified for fibroblasts without significant reduction in cell

proliferation due to contact inhibition. Thus, for Cu cytotox-icity assays, a cell seeding density of 50,000 and a maxi-mum culture time of 48 h in 96 well plates were used. 24 hafter cell seeding, culture media were replaced with Cu ioncontaining media solutions of different concentrations, in-cluding 24 and 72 h extracts from TCuIUDs and incubatedfor a further 24 h. Cell viability decreased with increasingCu ion concentration, with 30 % and 100 % reduction for40 μg/ml and 100 μg/ml respectively at 24 h. The cytotoxiceffects were further evaluated using light microscopy, apo-ptosis and cell cycle analysis assays. Fibroblasts becamerounded and eventually detached from TCP surface due toCu ion toxicity. A linear increase in apoptotic cell populationwith increasing Cu ion concentration was observed in thetested range of 0 to 50 μg/ml. Cell cycle analysis indicatedthe arrest of cell division for the tested 25 to 50 μg/ml Cu iontreatments. Among the TCuIUDs, TCu220C having 265 mm2

Cu surface area released 9.08±0.16 and 26.02±0.25 μg/ml,while TCu380A having 400 mm2 released 96.7±0.11 and159.3±0.15 μg/ml respectively following 24 and 72 h extrac-tions. The effects of TCuIUD extracts on viability, morphol-ogy, apoptosis and cell cycle assay on L929 mouse fibroblastscells, were appropriate for their respective Cu ion concen-trations. Thus, a concentration of about 46 μg/ml(~29 μM) was identified as the LD50 dose for L929mouse fibroblasts when exposed for 24 h based on ourMTT cell viability assay. The burst release of lethal con-centration of Cu ions from TCu380A, especially at theimplant site, is a cause of concern, and it is advisable touse TCuIUD designs that release Cu ions within cytotoxiclimits yet therapeutic, similar to TCu220C.

Keywords Intrauterine devices . Copper . Cell behavior .

Material-cell interactions

B. Cao :Y. Zheng (*) : T. Xi :H. Yang :Y. MaSchool of Materials Science and Engineering,University of Science and Technology Beijing,Beijing 100083, People’s Republic of Chinae-mail: zhengyudong@mater.ustb.edu.cn

T. Xi (*) : C. ZhangNational Institute for the Controlof Pharmaceutical & Biological Products,Beijing 100050, People’s Republic of Chinae-mail: xitingfei@tom.com

W. SongWolfson Center for Materials Processing,School of Engineering and Design, Brunel University,West London UB8 3PH, UK

K. BurugapalliBrunel Institute for Bioengineering, Brunel University,Uxbridge, Middlesex UB8 3PH, UK

B. CaoBeijing Institute of Medical Device Testing,Beijing 100120, People’s Republic of China

Biomed Microdevices (2012) 14:709–720DOI 10.1007/s10544-012-9651-x

1 Introduction

Since the first reporting of the contraceptive efficacy of Cuin IUDs by Zipper et al., Cu metal has been widely used inthe form of wires, tubes and beads for contraception (Zipperet al. 1969). CuIUDs gained popularity because, they arenon-hormonal, highly effective, inexpensive, long-lasting(use of single device reported for 20 years), rapidly revers-ible and safe contraceptives (Hov et al. 2007; Hubacher etal. 2001; Sivin 2007; Thiery et al. 1985). Currently, there areover 150 million IUD users (about 15 % of the world’swomen of reproductive age) around the world (Hubacheret al. 2006, WHO 2002).

CuIUDs are usually made of Cu wires or tubes woundaround T or U shaped flexible plastic material and theirnames typically contain a number indicating the total avail-able Cu surface area in mm2. Depending on the available Cusurface area, currently manufactured TCuIUDs, are indicat-ed for short-term usage of 3 years (200, 220, 250 mm2) orfor long-term of 5, 8 or 10 years (300, 375, & 380 mm2)(Kulier et al. 2007). However, in practice, effective contra-ception was reported for TCu220C and TCu380A for atleast 10 and 20 years respectively (Sivin 2007; Thiery etal. 1985).

The high effectiveness of Cu for contraception, unlikemetals such as Pt and Ag, is due to Cu metal’s high solu-bility and contraceptive ability even in the presence ofchloride ions (Chang et al. 1970). The corrosion products,primarily Cu2+ ions, released from the CuIUD cause therelease of uterine inflammatory reaction products - leukocytes and prostaglandins - by the endometrium in responseto the inserted CuIUD; together, they form a hostile envi-ronment in the uterus that reduces not only the viability ofsperms and eggs, but also the receptivity of endometrium toimplantation of embryos (Arancibia et al. 2003; Araya et al.2003; Beltran-Garcia et al. 2000; Hagenfeldt 1972; Kaplanet al. 1998; Ortiz et al. 1996; Roblero et al. 1996; Shimizu etal. 1991; Toder et al. 1988; Yin et al. 1993). However, likeany therapeutic drug, CuIUDs also induce side effects(pain,bleeding and pelvic inflammatory disease), the causes forwhich are not yet fully understood, but are reported todecrease to some extent over time (Hubacher et al. 2009;Stanback and Grimes 1998).

Two aspects of Cu ion toxicity need particular attention:one, the exposure of tissues in intimate contact with thedevice to (often lethal) Cu ion concentrations, and two, thechronic systemic exposure of rest of the body (Arancibia etal. 2003; Cai et al. 2005; Cao et al. 2008; Grillo et al. 2009;Hefnawi et al. 1974). In either case, cells take up Cu ions,and when the uptake crosses a threshold limit, the cellsbecome apoptotic and die (Araya et al. 2003; Aston et al.2000; Grillo et al. 2009; Hayashi et al. 2006; Obata et al.1996; Prasad et al. 1996; Singh et al. 2006). The entire

genital tract is reported to be exposed to 25–80 μg/day ofCu2+ ions released from an inserted CuIUD (Arancibia et al.2003; Kjaer et al. 1993). The resulting damaged uterinetissue might largely be removed by menstruation, oftenincreasing the severity and time of menses (Beltran-Garciaet al. 2000). Furthermore, the sustained daily release of Cu2+

ions also causes chronic systemic exposure (De la Cruz et al.2005; Okereke et al. 1972; Shubber et al. 1998). For in-stance, irrespective of the age of the user or the length oftime of using TCu380A, De la Cruz et al., reported thesustenance of nearly double the normal values for Cu con-centration in blood (De la Cruz et al. 2005).

The elevated plasma Cu levels due to TCu380A arereported to cause time-dependant increases in clinical oxi-dative stress biomarkers such as thiobarbituric acid reactivesubstances, protein carbonyls, glutathione, nitrates, nitrites,metallothioneins, ceruloplasmin, and the hepatic enzymes -lactate dehydrogenase and transaminases. As a result, toprevent oxidative damage, Arnal et al. recommended avoid-ing the continuous use of TCu380A beyond 2 years (Arnalet al. 2010).

In addition, the sustained high concentration of Cu, if notexcreted fast enough, results in accumulation of Cu intissues (Cu overloading). The Cu uptake by cells is reportedto be mediated primarily by ceruloplasmin, histidine andother Cu containing proteins (DiDonato and Sarkar 1997).Within the cell, Cu is distributed in all components, includ-ing nucleus, mitochondria, lysosomes, endoplasmic reticu-lum and cytosol (Linder 1991; Prasad et al. 1996). ExcessCu in cells is thought to interact non-specifically with var-ious macromolecules, modify their conformation or causesite-specific damage (Burkitt 1994; Grillo et al. 2010; Kanget al. 2004; Letelier et al. 2005). The resulting disruption offundamental cellular processes triggers apoptosis (Araya etal. 2003; Aston et al. 2000; Grillo et al. 2009; Hayashi et al.2006; Obata et al. 1996; Prasad et al. 1996; Singh et al.2006). However, threshold limits of Cu accumulation be-yond which cellular damage is triggered, as demonstrated inWilson and Menkes disease models having abnormal Cuaccumulation, varies between different cell types (hepato-cytes, neurons, and kidney cells) (Aston et al. 2000; Hayashiet al. 2006). Thus, a major objective for this study was toidentify the threshold for Cu induced cytotoxicity to fibro-blasts, one of the important (connective tissue formation andremodeling) cell type, affected in the uterus.

The overall objectives for this study were to evaluate Cuion cytotoxicity on L929 fibroblasts as a function of con-centration and correlate with that due to Cu ions in the burstrelease extracts of TCu220C and TCu380A. Cells werecultured on tissue culture plastic and MTT cell viability,Annexin V—propidium iodide (PI) apoptosis, and cell cycleanalysis assays were used to assess the effects of Cu ions onthe viability and proliferation of L929 fibroblasts. To this

710 Biomed Microdevices (2012) 14:709–720

goal cells were treated with Cu ion solutions of concentra-tion 0.1, 0.5, 1, 5, 10, 25, 40, 50 and 100 μg/ml, as well as24 and 72 h extracts of TCu220C and TCu380A IUDs. Thecell culture experiments were performed similar to thatrecommended in ISO-10993-5 standard and the results dem-onstrated a Cu ion concentration-dependent (including thatin TCuIUD extracts) cytotoxicity (ISO10993-5 2009).

2 Materials and methods

2.1 Materials

The IUDs, TCu380A and TCu220C were supplied by Tian-jin Medical Instrument Factory, China. Both IUDs are madeof bare Cu wires or tubes wound on the two cross-arms andthe stem of T-shaped high-density polyethylene (HDPE).The TCu380A had a Cu wire of 0.24±0.01 mm diameterwound on the stem and Cu tubes on each side of the twocross-arms, while TCu220C had five Cu tubes on the stemand two Cu tubes on each side of the two cross-arms. TheCu tubes on both devices had outer and inner diameters of2.18±0.03 and 1.48±0.01 mm respectively. The purity ofCu in both IUDs was 99.99 %. The devices were producedabout 6 months before being used in the experiments.

The L929 mouse fibroblast cell line was provided byShanghai Institute for Biological Sciences, Chinese Acade-my of Sciences. MTT was purchased from AMRESCO-Inc;dimethyl sulfoxide (DMSO) and 0.25 % Trypsin (with0.02 % ethylenediaminetetraacetic acid (EDTA)) from Sig-ma–Aldrich; Pen-Strep, L-Glutamine, Dulbecco’s modifiedEagle’s medium (DMEM) without phenol red and Fetal bo-vine serum (FBS) from GIBCO®, Invitrogen; and Dulbecco’sphosphate-buffered saline (DPBS) (Ca/Mg free) and DMEMwith phenol red from Hyclone, Thermo Scientific Inc.

2.2 Cu ion extracts from the IUDs

Sterile TCu380A, TCu220C, and T-shaped HDPE stemwithout Cu wires or tubes were incubated in 10 % FBSsupplemented DMEM (FBS-DMEM) culture medium (1 mlper 0.2 g of Cu) at 37 °C for 24 and 72 h. HDPE extract wasused as a negative control and 5 % DMSO solution as apositive control. The total weight of Cu on TCu220C andTCu380A, devices used in this study, was 0.3250 and0.1845 g respectively, and total Cu volume was 70.57 and39.87 mm3 respectively. As a result, the weight to volumeratios for Cu TCu220C and TCu380A were similar(~0.00462 g/mm3) (Table 1). However, their total Cu sur-face areas were different: 265 and 400 mm2 respectively.Furthermore, the surface of the fine Cu wire wound onTCu380A appeared to have micro-cracks, compared to a

smoother surface on Cu tubes, on the tested TCuIUDs (Caoet al. 2008).

The Cu ion concentrations in Cu solution standards andextracts from the TCuIUDs were analyzed by atomic ab-sorption spectroscopy (FAAS Thermo Electron CorporationM6AA System) using a Cu hollow-cathode lamp with anair–acetylene flame (acetylene, 1.0 L/min). The measure-ment was performed at a wavelength of 324.8 nm. Cu ionconcentrations in extracts were determined against a standardcurve of absorbance at a wavelength 324.8 nm obtained usingCu ion standard solutions of concentrations 0.0, 0.1, 0.2,0.4, 0.8, 1.6, 2.0μg mL−1. The standard curve (A01.9902C(mg/ml)+0.0009, where A is the absorbance and C theconcentration of Cu ions) had R2 (linearity) of 0.9999.

2.3 Cell culture

The L929 mouse fibroblasts were cultured to about 80 %confluence in a T75 flask containing DMEM culture medi-um supplemented with 10 % FBS, 100 U/ml penicillin,100 mg/ml streptomycin, and 2 mmol/L L-Glutamine, at37 °C in humidified atmosphere of 5 % CO2 and 95 % air.The cells were washed with fresh DMEM, trypsinized,centrifuged, re-suspended and counted under an invertedmicroscope (ZEISS, Axiovent) using haemocytometer, andcell viability was determined using tryphan blue exclusionmethod. Cell seeding concentrations, for the in vitro bio-compatibility assays, were varied to suit the different testrequirements described below.

2.4 MTT cell viability assay

The viability of L929 fibroblasts on tissue culture plasticwas measured by monitoring their metabolic activity usingMTT assay. At the time of assay, cells cultured in 96 wellplates were washed with DPBS, and then incubated in 100 μlof DMEM (without phenol red) containing 5 mg/ml MTT for4 h at 37 °C in a 5 % CO2 humid atmosphere incubator. Afterincubation, the medium was removed and cells washed withDPBS. To develop the color 100μL of DMSO was added and

Table 1 Total geometric surface area and weight to volume ratio of Cuon TCuIUDs available for Cu extraction (in 10 % FBS supplementedDMEM culture medium at 37 °C) and the corresponding Cu ionconcentration in TCuIUD extracts

CuSurfacearea(mm2)

CuWeight toVolumeratio(g/mm3)

24 hExtract(μg/ml)

72 h Extract(μg/ml)

TCu220C-IUD 265 0.00462 9.08±0.16 26.02±0.25

TCu380A-IUD 400 0.00463 96.7±0.11 159.3±0.15

Biomed Microdevices (2012) 14:709–720 711

the plate shaken gently for 10 min. The absorbance wasmeasured on a microplate reader (SPECTRA MAX plus384, Molecular Devices) at a wavelength of 570 nm, with areference wavelength of 650 nm, against medium only blank.

2.5 Cell viability as function of number of seeded cellsand time

To assess the MTT cell viability as a function of cell numb-ers and time, cells were seeded at 1×104, 2×104, 4×104, 5×104, 6×104, 8×104 and 10×104 cells/well (100 μl/well) in96 well plates. Following cell seeding, MTT cell viabilityassay was done every day for 7 days to study the growth andproliferation of L929 mouse fibroblast cells.

2.6 Effect of Cu ions on cell viability

A series of Cu ion solutions, 0.1, 0.5, 1.0, 5.0, 10.0, 25.0,40.0, 50.0 and 100.0 μg/ml were prepared by serial dilutionof a commercial standard cupric ion solution using FBS-DMEM cell culture medium (the National Institute of Me-trology, China) and their effects on cell viability determinedusing MTT assay. L929 fibroblast cells were seeded at 5×104 cells/well in 96 well plates (100 μl/well). After 24 h cellattachment, cells were washed with DPBS and incubated inculture media containing the different Cu ion concentrationsfor 24 h at 37 °C in a CO2 incubator. Culture mediumwithout Cu ions was used as a negative control, while 5 %DMSO solution in distilled water as a positive control. After24 h treatment, the medium was removed and cells washedin DPBS followed by the MTT assay. The results wereexpressed as percentage relative growth rate (%RGR) cal-culated using the equation:

%RGR ¼ OD for Test Material=OD for Negative Controlð Þ � 100 :

Similarly, the effect of Cu ion concentration in extractsfrom TCu380A and TCu220C on cell viability was alsoevaluated by the MTT assay and the results expressedas %RGR.

2.7 Morphology of cells on tissue culture plastic underinverted light microscope

The effect of TCuIUD extracts on fibroblast morphologywas also studied using inverted light microscope equippedwith phase contrast accessories.

2.8 Annexin V apoptosis assay

For the Annexin V apoptosis assay, L929 fibroblasts wereseeded in 96-well plates at a density of 1×106 cells/well. Cuions solutions in FBS-DMEM culture medium making up to

25, 40 and 50 μg/well; and 24 and 72 h extracts for bothTCu380A and TCu220C were added to the wells seededwith fibroblasts. Culture medium without Cu ions was thenegative control. After 24 h of treatment, Annexin V assaywas performed using Annexin V-Fluorescein isothiocyanate(FITC)/propidium iodide (PI) apoptosis kit (BD Pharmin-gen, San Jose, CA) as described previously [25]. Briefly, thetest cells were trypsinized, washed in DPBS and re-suspended in binding buffer at a concentration of 1×106

cells/ml. To 100 μl of cell suspension, 5 μl each of AnnexinV-FITC and PI solutions were added and incubated in thedark for 15 min at room temperature. An additional 400 μlof binding buffer was added and the cells were analyzed byflow cytometry using BD FACSCalibur™ (BD, FranklinLake, NJ) equipped with CellQuest software. Cells werecategorized as viable (Annexin V-/PI-), early apoptotic(Annexin V+/PI-) or late apoptotic (Annexin V+/PI+), andexpressed as a percent of total gated cells.

2.9 Cell cycle analysis

CycleTEST™ PLUS DNA Reagent Kit (BD Biosciences,San Jose, CA) in combination with PI was used for cellcycle analysis. Cell seeding and treatments were similar tothat used for apoptosis assay. The cells were incubated in thedark with DNA staining solution at 4 °C for 10 min and thenanalyzed using BD FACSCalibur™ equipped with Modi-fit3.0 software. The number of gated cells in G0/G1, S, orG2/M phases was presented as % of total cells.

2.10 Statistical analysis

All cell cultures were performed in triplicate for each mea-surement at each time point. All statistical computationswere performed using SPSS Version 16.0. Data is expressedas mean±standard deviation. Differences of P<0.05 basedon Student’s t-test were considered significant.

3 Results

3.1 Cu extraction from TCuIUDs

Cu content in TCu380A extracts was substantially higherthan in TCu220C extracts at both 24 and 72 h of extractionin FBS-DMEM culture medium (Table 1). TCu380Ahaving ~1.5 times higher apparent geometric surface area(400 mm2) released ~10 and 6 times more Cu ions at 24 and72 h respectively compared to TCu220C (265 mm2). Thissignificantly higher Cu burst release from TCu380A isattributed to the physical form of Cu wound on the T-shaped HDPE. On the stem of TCu380A Cu wire is wound,which apparently releases Cu at much faster rate than Cu in

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the form of tubes. The two cross-arms on TCu380A andwhole of TCu220C have Cu tubes wound around (Cao et al.2008; Lu et al. 2008).

3.2 Cell viability as a function of number of seeded cellsand time

Initially, the L929 fibroblasts’ proliferation in 96 well plateswas tested. Cells were seeded at numbers of 1×104, 2×104,4×104, 5×104, 6×104, 8×104 and 10×104, each in tripli-cate. After 1, 2, 3, 4, 5, 6, and 7 day intervals, cells werewashed and assayed for MTT cell viability. Linear regres-sion curve fitting on the optical density results were linear at1 day (y00.000004x-0.0016; R200.9970) for the cell seed-ing range of 1×104 to 10×104, and thereafter the linearitydecreased with time (R2 values of 0.9970, 0.9787, 0.9567,0.8875, 0.8339, 0.6955 and 0.5829 respectively for the timepoints 1, 2, 3, 4, 5, 6 and 7 days).

3.3 Effect of Cu ion concentrations on viability of mouseL929 fibroblasts

We seeded cells at a density of 5×104 cells per well of a 96well plate, intending to maximize the number of cells ex-posed to the different Cu ion solutions (concentrations rang-ing from 0.1 to 100 μg/ml or 24 and 72 h extracts fromTCuIUDs containing up to 160 μg/ml). The time pointsrelevant for our Cu cytotoxicity assays were 24 and 48 h.Cells were allowed to attach to the tissue culture plastic for24 h, following which the non-attached cells washed off,before adding fresh medium with or without Cu ions forcytotoxicity testing. The Cu ion exposure was typically forup to 24 h, i.e., 2 days since initial cell seeding, unlessotherwise mentioned. At 24 h after cell seeding, the cellsappear to be in the logarithmic phase (R200.9970), and the

linearity between viability and cell seeding numbers for therange of 1×104 to 10×104 cell/well at 2 days (R200.9787),did not decrease significantly. Hence, we assumed theeffects of contact inhibition by the study end point of 2 days,interfering with Cu cytotoxicity assays, to be minimal forthe cell seeding density of 5×104 cells/well.

Dose-dependent toxic effects of Cu ions were observedon the mouse L929 fibroblasts (Fig. 1). The data isexpressed as %RGR with reference to negative controls.The viability of cells decreased with increasing Cu ionconcentration, reaching zero viability when exposed to100 μg/ml for 24 h.

3.4 Cytotoxic effect of Cu ions in TCuIUD extracts

The cytotoxicity results for Cu ions in TCuIUDs extracts arepresented in Fig. 2. The cells were seeded in 96 well plates,allowed to attach and spread on tissue culture plastic for24 h followed by exposure to the TCuIUD extracts in mediafor 24 h. The positive control, DMSO, also significantlydecreased the cell viability (%RGR of 5.58). Except for 24 h

%R

GR

0

10

20

30

40

50

60

70

80

90

100

110

0%

RG

R10 200 30

Cupric Ion Concentration (µµg/ml) 40 50 60 70

LD50: ~4

0 80

46 µg/ml (

90 100

(~29µM)

Fig. 1 Concentration-dependent Cu ion toxicity onmouse L929 fibroblastsrepresented as % RGR based onMTT cell viability assay.Negative control cells were notexposed to Cu ion solution.n03

Fig. 2 Cell viability assay results for mouse L929 fibroblasts exposedto extracts from TCu380A and TCu220C, expressed as % RGR versusnegative control. \ast indicates p<0.05 vs negative control

Biomed Microdevices (2012) 14:709–720 713

extracts of TCu220C, all the other groups significantlylowered %RGR compared to the negative control group.Effectively, the toxicity of the TCuIUDs is dependent onthe amount of Cu ions released into the surroundingmedium. The data expressed as % RGR (Fig. 2) showedthat the extracts decreased L929 fibroblast viability in linewith their Cu ion content, similar to that observed inFig. 1. A difference in the mean % RGR values for72 h extracts (77.61 %) containing 26.02 μg/ml Cu com-pared to that (85 %) observed for standard 25 μg/ml Cu ionstandard solution. However, this difference was not statisti-cally significant, due to the relatively high standard devia-tions observed for cell viability data for TCuIUD extracts.

3.5 Morphology of L929 fibroblasts treated with TCuIUDextracts

Figure 3 shows the light microscopic images of cells ontissue culture plastic after 24 h of exposure to test (TCuIUDextracts) and control media solutions. The cell exposed tonegative control (HDPE extract) show a confluent layer offibroblasts, with well spread cells (Fig. 3a). In contrast, thecells were rounded and mostly detached from the surface oftissue culture plastic for the positive control (Fig. 3b). Thecells exposed to extracts from TCu220C at both 24 and 48 hshowed near normal fibroblast morphology (Fig. 3c&d).However, there is a decrease in the density of cells with

increasing time of incubation with TCu220C extracts. Incontrast, the cells exposed to TCu380A extracts weredetached from surface of tissue culture plastic and rounded(Fig. 3e & f). Thus, both 24 and 72 h extracts fromTCu380A proved fatal (due to the high Cu ion concentra-tions ~96 and 159 μg/ml) to mouse L929 fibroblasts.

3.6 Apoptosis analysis

Flow cytometry plots showing the cell count events of L929fibroblasts labeled with Annexin V and PI are presented inFig. 4. The events in bottom left quadrant indicate AnnexinV-/PI- viable cells, bottom right—Annexin V+/PI- earlyapoptotic cells, and top right quadrant—Annexin V+/PI+late apoptotic cells. Dose dependent Cu ion-induced apo-ptosis of L929 mouse fibroblasts cells is evident from thegradual shift of the fluorescent events from viable to earlyapoptotic and late apoptotic cells for Cu ion concentrations0, 25, 40 through to 50 μg/ml (Fig. 4). The dose-dependenttoxic effects of Cu ions on L929 cells are also quantitativelyillustrated by a proportional and a significant (p<0.05)increase in late apoptotic cells vs Cu ion concentration inFig. 5. 25, 40 and 50 μg/ml Cu ion solutions caused 14.23,30.5 and 47.12 % apoptotic cells respectively. Thus nearly50 % cells were dead when exposed to 50 μg/ml.

The effects of 24 and 72 h Cu ion extracts from TCuIUDs220 C and 380A on L929 fibroblast apoptosis was also

Fig. 3 Inverted light microscope images showing the morphology ofmouse L-929 fibroblasts treated with, (a) negative control (0 % Cu);(b) positive control (DMSA, 0 % Cu); (c) TCu220C 24 h extract(9.08±0.16 μg/ml Cu); (d) TCu220C 72 h extract (26.02±0.25 μg/ml

Cu); (e) TCu380A 24 h extract (96.70±0.11 μg/ml Cu); and f) TCu380A72 h extract (159.30±0.15 μg/ml Cu). Magnification a–d 100x; and e–f200x

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evaluated (Figs. 6 & 7). The results further confirmed thedose-dependent effects of Cu ions on cells. TCu220C’s 24and 72 h extracts contained ~9 and 26 μg/ml Cu ionsrespectively. Accordingly the percentage of late apoptoticcells (7.65 and 11.96) was low (Figs. 6 & 7). In contrast, allcells were dead by 24 h of treatment with both extracts from

TCu380A. This can be attributed to the lethal Cu ion con-centrations of ~96 and 159 μg/ml. To obtain meaningfulintermediate stage cell apoptosis results, the treatment timeof cells with TCu380A extracts was reduced to 12 h. Theresults showed that majority of the cells lost their viabilityand are in early or late apoptotic stages (Fig. 6). About 33and 47 % of the total cells respectively for 24 and 72 hextract the TCu380A were in the late apoptotic cell stagewithin 12 h of exposure (Fig. 7).

3.7 Cell cycle analysis

Cell cycle analysis was used to further characterize the Cuion toxicity on L929 fibroblasts. Nuclear DNA was stainedwith propidium iodide and the fluorescence intensity of PIstained nuclei was measured using flow cytometry to clas-sify the cells into G1, S or G2/M phases of cell cycle. L929mouse fibroblasts cells were exposed to a series of Cu ionsolutions to evaluate Cu ion induced cell cycle perturba-tions. As shown in Fig. 8 and Table 2, after 24 h of Cu ionsolution exposure, the percentage of cells in S phase decreased

Fig. 4 Cu ion-induced apoptosis for 1×106 L929 mouse fibroblastsexposed to 100 μl of cell culture medium containing Cu ion concen-trations of (a) 0, b) 25 (c) 40 and (d) 50 μg/ml for 24 h. For the plots ofPI versus Annexin V-FITC in (a) to (d), early apoptotic cells are in the

bottom right; Late apoptotic cells in the right top; and live cells in theleft bottom quadrants. *p<0.05 for Cu ion solutions in (b), (c) and (d)vs negative control (a). n03

Fig. 5 Percentage of late apoptotic L929 fibroblasts as a function ofexposure to different Cu ion concentrations. 1×106 L929 fibroblasts wereexposed to 100 μl of cell culture medium with and without Cu ions. n03

Biomed Microdevices (2012) 14:709–720 715

significantly for all Cu ion concentrations compared withnegative control cells (p<0.05), while the percentage of thecells in G2/M phase increased (p<0.05). The decrease in S-

phase cells is indicative of inhibition of DNA replication,while the increase in G2/M phase cells of perturbation orarrest of cell division, the reduction in cell proliferation.Similar effects were observed when cells were treated withTCu220C extracts (results not shown), while all cells weredead by 24 h when treated with TCu380A extracts due towhich we couldn’t capture any meaningful cell cycle pertur-bation effects.

4 Discussion

Cu-containing IUDs are popular contraceptives becausethey are inexpensive, rapidly reversible, highly efficientand reliable. Moreover, a CuIUD once implanted can func-tion for a long-time (3 to 20 years) (Sivin 2007). Thecontraceptive effects are due to the Cu ions released fromthe Cu wires/tubes wound around a T shaped polymer.

Fig. 6 Cu ion-induced apoptosis for 1×106 L929 mouse fibroblastsexposed to 100 μl of (a) 24 h TCu220C, (b) 72 h TCu220C, (c) 24 hTCu380A, and (d) 72 h TCu380A extracts in cell culture medium. TheCu ion exposure time for (a) and (b) was 24 h and that for (c) and (d)

was 12 h. For the plots of PI versus Annexin V-FITC in (a) to (d), earlyapoptotic cells are in the bottom right; Late apoptotic cells in the righttop; and live cells in the left bottom quadrants. *p<0.05 for a) to d) vsnegative control (0 μg/ml Cu). n03

Fig. 7 Percentage of late apoptotic L929 fibroblasts exposed to thedifferent TCuIUD extracts. The Cu ion exposure time for the TCu220Cextracts was 24 h and that for TCu380A extracts was 12 h. n03

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Effectively the CuIUDs are biomaterials capable of sus-tained Cu ion release. In our earlier study we reported theinitial burst release of Cu in the first three days from twocommercial TCuIUDs followed by a sustained release insimulated uterine and body fluids. Further, we also showedCuIUDs get corroded faster when implanted in rat uterus (invivo) than in simulated fluids (in vitro) (Cao et al. 2008). Inthis study, we analyzed the Cu ion concentration dependentcytotoxicity to L929 mouse fibroblasts cells and correlated

with that due to the burst release extracts from two com-monly used CuIUDs - TCu220C and TCu380A.

The weight to volume ratio of total Cu on each of theTCu220C and the TCu380A IUDs was similar(~0.00462 g/mm3), but the total surface area availablediffered (265 and 400 mm2 respectively). Yet, an abnor-mally high (6 to 10 times higher) burst release of Cu ionsfrom Cu wires on TCu380A (0.24±0.01 mm diameter)was observed compared to TCu220C (Table 1). This couldbe due to a size dependent non-linear increase in corro-sion with decreasing Cu wire diameter, as reported by Luet al. They observed the formation of a diffusion layerhaving an optimum thickness of 0.56 mm on Cu wiresurface when immersed in a salt solution. If the radius ofthe immersed Cu wires was smaller than the diffusionlayer thickness, a size dependent non-linear accelerationin Cu corrosion was observed with decreasing Cu wireradius (Lu et al. 2008).

The mode of action for TCuIUDs is through the inhibi-tion of sperms’ motility and viability, thus preventingsperms from fertilizing oocytes. However, like any

Fig. 8 Cell cycle assay results showing the cell population distribu-tions based on their stage in the cell cycle (G0/G1; S and G2/M), as afunction of exposure to (a) 0, (b) 25, (c) 40 and (d) 50 μg/ml Cu ion

concentrations. 1×106 L929 fibroblasts were treated with 100 μl ofdifferent Cu ion solutions for 24 h. n03

Table 2 Cell cycle perturbation induced by Cu ions—percentage ofcells in the different phases of the cell cycle (G0/G1; S and G2/M) aftertreatment of 1×106 L929 fibroblasts with 100 μl of different Cu ionsolutions for 24 h. n03

Cu ion Treatment (μg/ml) G0/G1 (%) S (%) G2/M (%)

0 54.44±0.4 30.49±1.0 15.08±1.1

25 55.93±0.7 21.72±0.7 22.35±0.8

40 50.58±1.3 29.45±0.5 19.96±0.6

50 53.81±1.5 24.55±0.8 21.64±0.4

Biomed Microdevices (2012) 14:709–720 717

therapeutic drug, Cu ions released from TCuIUDs alsocause cytotoxicity to other cells both in the vicinity of theimplant site (in the uterus) and in remote tissues (includingliver, kidney, spleen and lungs). Fibroblasts are one suchcells in the close vicinity of implant that are affected by theCu ion toxicity. They are crucial in the repair of the tissuedamaged by inflammatory responses due to the implants andnew tissue formation in uterus. Furthermore, L929 fibro-blasts are the cells recommended by the international stan-dard ISO-10993-5 for in vitro biocompatibility evaluations.Hence we chose L929 fibroblasts for our evaluation of Cuion concentration dependent cytotoxicity.

In culture, fibroblasts move (locomotion), multiply (pro-liferation) and grow (growth) until they cover all the surfacearea available on the tissue culture plastic forming a conflu-ent mono-layer. However, when a fibroblast comes in con-tact with its neighboring cell, the cell walls fuse at thecontact points triggering a decrease in growth and stopproliferating, which phenomenon is called contact inhibi-tion. To prevent the interference of contact inhibition withthe Cu ion toxicity assays, cell seeding density and culturetime were varied and MTT cell viability assessed. For a wellof a 96 well plate, a linear increase in cell viability wasobserved when cells were cultured for 24 h for the cellseeding numbers of 1×104 to 10×104. A gradual decreasein linearity of MTT cell viability data was observed on adaily basis up to 7 days of testing, which was indicative ofthe high cell numbers in the small space of a well in 96 wellplate causing contact inhibition (Meisler 1973a; Meisler1973b). For the MTT assay to test Cu cytotoxicity, a cellseeding density of 5×104 cells/well and a maximum culturetime of 48 h was chosen to ensure that the number of cellsexposed to the different Cu ion concentrations was maxi-mized without interference from contact inhibition.

The MTT cell viability assay revealed a concentrationdependent decrease in viability of L929 fibroblasts (Fig. 1).Exposure of about 50,000 fibroblasts to 100 μl of 40 μg/ml(25 μM) Cu ion solution caused about 30 % decrease in theviability of L929 mouse fibroblasts. Considering a 30 %decrease in cell viability to be cytotoxic, as recommendedby ISO 10993–5 standard, 40 μg/ml was identified as cyto-toxic for L929 fibroblasts (Fig. 2) (ISO10993-5 2009).Furthermore, the MTT cell viability results correlated wellwith apoptosis assays. We observed a decrease in cell via-bility and a matching raise in apoptotic cell population withincreasing Cu ion concentration. Cell cycle assay indicatedthe arrest of DNA replication and as a result, the arrest ofcell proliferation. Overall, our cell viability data suggeststhat the LD50 for L929 fibroblasts is about 46 μg/ml(~29 μM) and almost all cells were dead when exposed to100 μg/ml (~62 μM) of Cu ions (Fig. 1).

The cytotoxic effects due to Cu ions in burst releaseextracts from TCu200C and TCu380Awere consistent with

their respective Cu ion content. Our results, further reiteratethat, the burst release in the first few days, especially for theTCu380A, exposes cells in close proximity to the device tolethal concentration of Cu ions (Pereda et al. 2008). TCu380Ahad a burst release extract concentration of >95 μg/mlwhich was lethal for the L929 mouse fibroblasts, as demon-strated by our cell viability, apoptosis and cell cycle analysisassay. Clinical reports document that TCu380A required re-moval in 15 % of users due to pain and bleeding (side effects)within 1 year of insertion (Hubacher et al. 2009). TheTCu220C burst release extracts, on the other hand, had con-centrations that are not as lethal. However, in clinical practice,pregnancy and expulsion rates of 0.8 to 2.2 % and 0 to 6.4 %respectively for TCu220C, and 0 to 1 % and 2.4 to 8.2 % forTCu380Awere reported following use for one year (Kulier etal. 2007). Thus, the difference in performance between thetwo devices is only marginal.

The sustained high concentrations of Cu ions in the bodydue to TCuIUD cause an accumulation of Cu ions insidecells both in the vicinity of the implant and systemically(Arnal et al. 2010; Okereke et al. 1972). However, thedefinitive correlation of specific disease conditions, e.g.,cancer, oxidative stress and liver dysfunction, to the long-term use of TCuIUDs in humans has been difficult to provebecause the elevations in toxicity biomarkers are often sub-clinical (Arnal et al. 2010). The resulting clinical symptomsare often disregarded as harmless side effects. But there isgrowing evidence from a large number of in vitro and invivo studies, in literature, suggesting the ill-effects of Cu iontoxicity.

Apparently, elevation in Cu ion levels in healthy individ-uals cause an increase in proteins involved in Cu ion me-tabolism, which in turn increase the active accumulation ofCu ions in cells systemically in the body (Arnal et al. 2010).The threshold for accumulation of Cu ions in cells beyondwhich Cu ion toxicity manifests varies between differentcell types. Hayashi et al. demonstrated that 8.5 to 16 ppmof Cu produced single strand breaks in brain cells of LongEvans Cinnamon rats, an animal model for human WilsonDisease. However, higher concentrations (200 to 400 ppm)were needed to damage hepatic and renal cells (Hayashi etal. 2006). Furthermore, Aston et al. observed a time depen-dant linear accumulation of Cu in human hepatoma cells. At72 h of culture in the presence of 4 and 64 μM (0.64 and10.22 μg/ml) of Cu, they estimated a Cu content of 0.11 and1.22 pg/cell and observed ~2 to 4 and 18 % necrotic cellsrespectively (Aston et al. 2000). The LD50 of Cu for 72 hculture of peripheral blood mononuclear cells (PBMCs) wasreported by Singh et al. to be 115 μM (18.4 μg/ml), whichincreased to 710 μM (113.3 μg/ml) when the cells werepretreated with 200 μM of Zinc (Singh et al. 2006). ForChinese hamster ovary cells, Grillo et al. reported a signif-icant decrease in viability of cells treated with ≥7.42 μg/ml

718 Biomed Microdevices (2012) 14:709–720

(4.65 μM) Cu and ~90 % decrease when treated with10.85 μg/ml (6.8 μM) for 72 h. Cortizo et al. tested Cutoxicity to URM106 rat osteosarcoma and MC3T3E1 oste-oblast cell lines. They incubated the cells with Cu wireshaving 0.1 cm diameter and 0.314 cm2 total Cu surface areaimmersed in the culture medium. At 24 h of incubation, theyobserved a release of 104 μg/ml Cu ions into the culturemedium, with 40 to 50 % decrease in surviving URM106 orMC3T3E1 cells and all cells were dead by 48 h (225 μg/mlCu) (Cortizo et al. 2004). Cell necrosis was evident from asearly as 4 h of incubation with Cu wires. Wahata et al.demonstrated the cytotoxic effects of Cu ions on humanmonocytes that play an important role in the biologicalresponse to implanted biomaterials. They tested the effectsof Cu and other metals on the mitochondrial function andtotal cellular protein in THP-1 monocytes for 4 weeks. A Cuion concentration ≥20 μM/l (3 μg/ml) produced a 35 %increase in total protein content, while 60 μM/l (9.6 μg/ml)produced a 75 to 125 % and 75 to 150 % rise in total proteinand succinic dehydrogenase activity. The concentrationstested were said to be near lethal for the THP-1 mono-cytes by 24 h of cell culture (Wahata et al. 2002). Cuion toxicity to human vascular endothelial (HVE) andfibroblasts (HAIN-55) was reported by Kishimoto et al.It is interesting to note that HVE cells were moresusceptible to concentration-dependent Cu cytotoxicitythan HAIN-55 cells (Kishimoto et al. 1992). Overall,our results are also indicative of similar cytotoxiceffects on L929 mouse fibroblasts as that reported inliterature for a variety of cells lines. Differences in thenature of cells, culture time, and units for the presenteddata make it difficult to tabulate comparable data. How-ever, from the above discussion, it is evident that thethresholds for Cu accumulation vary for the differentcell types in the body.

In human use, elevated plasma concentrations of Cu inblood were shown to cause chromosomal aberrations inblood lymphocytes (Shubber et al. 1998), when CuIUDswere used for over one year. Such positive correlations forlong-term use of CuIUDs and DNA damage must be takenas a safety warning. At the same time, exposure to a mini-mum of 8×10−6 mol/L (0.5 μg/ml) of Cu ion concentrationfor 20 min is required to significantly reduce the motility ofspermatozoa (Araya et al. 2003). Thus a balance of Cu ionconcentration needs to be achieved in the uterus fluid,wherein the therapeutic effect is 100 %, yet the side effectsare minimal.

To conclude, our findings suggest a LD50 dose of about46 μg/ml (~29 μM) Cu ions for L929 mouse fibroblastsand >99 % cell death with 24 h exposure to 100 μg/ml(~62 μM). Cu ion concentration in burst release extracts (upto 160 μg/ml) for TCu380A is lethal to fibroblasts, andexposure of uterine tissues to such lethal concentrations

(>100 μg/ml) should be viewed as a cause of concern. Fur-thermore, there is growing clinical evidence to suggest thedeleterious cytotoxic and overloading effects due to TCu380A(Arnal et al. 2010; Aston et al. 2000; Beltran-Garcia et al.2000; De la Cruz et al. 2005; Grillo et al. 2010). As a result, itis advisable to encourage the use of alternative TCuIUDs thatrelease Cu ions well within the cytotoxic limit, yet highlyeffective for contraception.

Acknowledgements The authors acknowledge the financial supportfrom National Science and Technology Support Project of China(Grant No.2006BAI15B08), National Natural Science Foundation ofChina Project (Grant No. 51073024) and the Royal Society-NSFCinternational joint project grant (No. 5111130207).

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