Ammonia Plasma Functionalized Polycarbonate Surfaces Improve Cell Migration Inside an Artificial 3D Cell Culture Module

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Ammonia Plasma FunctionalizedPolycarbonate Surfaces Improve Cell MigrationInside an Artificial 3D Cell Culture Module

Claudia Bergemann,* Antje Quade, Friederike Kunz, Stefan Ofe,Ernst-Dieter Klinkenberg, Michael Laue, Karsten Schroder,Volker Weissmann, Harald Hansmann, Klaus-Dieter Weltmann,Barbara Nebe

1. Introduction

The increasing demand for advanced, large-scale tissue

substitutes requires improved knowledge about tissue

interactions with scaffolds made from artificial materials.

Basic requirementsof suchmaterials areadequatemechan-

ical properties and interfacial biocompatibility. In parti-

cular, bone healing requires a mechanically resistant

matrix (scaffold) which is attractive to osteogenic cells

and allows revascularization for nutrient and oxygen

supply. A three-dimensional (3D) module for in vitro

investigations was developed to observe the behavior of

osteoblasts inside a porous scaffold. Special emphasis was

placed on the geometrical scaffold design regarding pore

size andpore design.[1] The systemwas composed of a stack

of titanium slices mounted on a clamp. This arrangement

enabled the separate monitoring of cell–growth patterns

on every single slice of the stack. This way, selective

information about the regulation of the cell physiology

was gained in the inner part of this 3D construct. This

knowledge could be used for the development of an

optimized scaffold design for orthopedic implants. In this

study, stacks of slices made of transparent polymer

materials were used to refine this 3D module for the

analysis of cell growth by light microscopy also in the

depth of the corpus.

The interaction between tissues and scaffolds is deter-

mined by the surface characteristics of the materials.

Surface chemistry, surface charge, bulkmaterial rigidity, as

Full Paper

C. Bergemann, F. Kunz, B. NebeBiomedical Research Center, Department of Cell Biology,University of Rostock, Schillingallee 69, 18057 Rostock, GermanyFax: þ49 381 4947778;E-mail: [email protected]. Quade, K. Schroder, K.-D. WeltmannLeibniz Institute for Plasma Science and Technology, Felix-Hausdorff-Str. 2, 17489 Greifswald, GermanyS. Ofe, V. Weissmann, H. HansmannInstitute for Polymer Technologies e.V., Alter Holzhafen 19, 23966Wismar, GermanyE.-D. KlinkenbergDOT GmbH, Charles-Darwin-Ring 1a, 18059 Rostock, GermanyM. LaueRobert-Koch-Institut, Postbox: 650261, 13302 Berlin, Germany

A three-dimensional (3D) cell culture module was used to test the requirements of scaffoldmaterial properties for improved bone cell ingrowth. For this purpose polycarbonate (PC)samples were treated with microwave-excited ammonia plasma in a defined time frame of1–300 s and the influence on initial attachment of osteoblast-like cells was determined. Perforated PC slices were treatedby 30 s of ammonia plasma and mounted on a clamp for 3Dcell ingrowth. Plasma treatment conditionswere ascertainedto stimulate cell adhesion and the migration of human bonecells into the depth of this artificial scaffold.

Plasma Process. Polym. 2012, 9, 261–272

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com DOI: 10.1002/ppap.201100059 261

well as morphological factors like holes, grooves, and

roughness determine the adsorption of proteins of the

extracellular matrix and thus significantly affect cell

adhesion to the material.[2] Optimal cell adhesion is in

turn a prerequisite for the proliferation and differentiation

of anchorage-dependent cells and for the stable integration

of an implant into the surrounding tissue.[3–5] In order to

obtain optimal cell growth conditions, the surface proper-

ties of polymer materials can be modified using several

techniques.[6] The functionalization of polymers with

N-containing groups is known to positively influence the

surface properties, like hydrophilicity, surface charge, and

free energy, which favor cell adhesion and spreading.[4,7]

Amino groups are widely used for application of polymers

in biochemistry, for example, for immobilization of

bioactive molecules. By the protonated amino groups

positive charges are introduced to the polymer surface

which probably attract negatively charged molecules like

proteins of the extra cellular matrix (ECM) in aqueous

solution.[8] Among several techniques, plasma treatments

are advantageous due to their high throughput and target-

oriented chemistry.[8] Other advantages of low pressure

plasma processes are chemical flexibility and avoidance of

hazardous and leachable substances. Plasma surface

functionalization can create chemical groups on the

polymer surfaces which can function as recognition sites

for protein adsorption and attachment of anchorage-

dependent cells in physiological cell culture media.[9]

Two plasma process strategies are available to equip

materials surfaceswithaminogroupsadvantageous for cell

attachment: deposition of thin plasma polymer layers by

plasma-enhanced chemical vapor deposition (PECVD) and

direct plasma functionalization.[10] In a depositing process,

an N-containing precursor is necessary to produce plasma

reactivespecieswhichareable to reactamongst themselves

on the top of the substrate, forming a thin film.[11] Direct

functionalization by low temperature plasmas sustained

in N2 or N-containing gases was used to generate

amino groups on different polymer surfaces, for example,

polystyrene (PS), polyethylene terephthalate (PET), poly-

carbonate (PC), and also on degradable polymers like

polyhydroxybutyrate (PHB) and polylactide (PLA).[12–17] For

the generation of amino groups microwave (MW) plasma

fed with ammonia gas is suitable.[18] MW-induced plasma

allows rapid functionalization without affecting the bulk

properties of the polymer. For a gas discharge at low

pressures (<1Torr) andhigh frequencies (>1MHz), it canbe

assumed that heavy particles such as gas molecules and

ions are still at the ambient temperature, typically at about

0.025 eV. Electrons under these conditions, however,

possess kinetic energies that are sufficiently high to cause

bond breakage and further ionization of the gas. Subse-

quently,highly reactivespeciesaswellasenergeticphotons

are produced by a gas discharge that can readily undergo

homogenous or heterogeneous reactions at solid surfaces

exposed to the plasma.[19] Wang et al. employed a low

pressure radio frequency (RF) discharge sustained in

ammonia for the modification of PLA scaffolds. Using the

methods of ink dyeing depth and scanning electron

microscopy (SEM) they observed an influence of the power

as well as treatment time on the modified depth and

degradation of the polymer.[20] Different plasma treatment

times generate different values of functional groups at

the surface of the polymer materials.[21] However, the

initial phase during plasma process is crucial and longer

treatment affects the number of amino groups at the

surface.[10,22]

Even though lowtemperatureplasmas sustained inN2or

N-containing gases were widely used to improve the

wettability of polymers the influence of plasma conditions

on initial cell attachment as a precondition for cell

proliferation and differentiation was not adequately

investigated. Therefore, to evaluate the effect of different

ammonia plasma treatment times on cell adhesion, the

biocompatible material PC was treated by ammonia

plasma for different duration and used as substrate for

the attachment of osteoblast-like MG-63 cells. The correla-

tion between plasma treatment time, numbers of amino

groups generated at the surface, and the initial cell

attachment was analyzed. Best plasma conditions for cell

colonization were used for the treatment of the perforated

PC slices of the 3D cell culture module to stimulate the cell

ingrowth into the depth of the artificial scaffold. Optimized

surface properties of the PC slices will be a key prerequisite

for cell colonization in the above-mentioned 3D cell culture

module.

2. Experimental Section

2.1. 3D Cell Culture Module

To gain insights into the cell behavior in a 3D environment, a novel

3D cell culture module was created.[1] To further develop this 3D

module we used a stack of slices made of transparent biocompa-

tible polymer materials, that is, PC. This newly generated 3D cell

culturemodule (Figure 1A) consists of a clamping ringwith outside

threading on which perforated PC slices (? 24mm and height

0.5mm) canbemountedandfixedwith afixing ringanda capwith

inside threading to create a porous 3D corpus (Figure 1B).[23] The

slices themselves were laser-cut and the number of slices and the

entire geometry is variable according to requirements. We used a

platform equipped with a set of five perforated slices. The holes of

adjacent slices are interconnected in a vertical direction. The

vertical pore sequence results in a meander-like single channel

configuration throughout the 3D construct (Figure 1C). The hole

sizes were deduced from literature where favorable pore sizes for

osteoblast ingrowth are reported to be around 500mm in

diameter.[24] Tongues in the clamping ring and grooves on the

slices were integrated for fixing and aligning of the PC slices in the

262Plasma Process. Polym. 2012, 9, 261–272

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.201100059

C. Bergemann et al.

https://www.researchgate.net/publication/229478090_Plasma_Processes_and_Polymers?el=1_x_8&enrichId=rgreq-450cb29a-6405-4061-89ac-155eebf6769e&enrichSource=Y292ZXJQYWdlOzIyMTY3ODkxNDtBUzoxODc3MjMxNTEzMjMxMzZAMTQyMTc2ODA5NTEyMQ==

stack. The screwed cap presses the fixing ring onto the stack and

ensures an exactly vertical position (Figure 1B). Clamping ring,

fixing ring, and capweremade of polyurethane casting compound

(MG 850, ebalta Kunststoff GmbH, Germany) and prepared in a

rapid tooling process.

2.2. Polycarbonate Samples

2.2.1. PC Disks

The PC disks used were made of commercially available PC

homopolymer (Bayer MaterialScience AG, Germany) which is

classified asmedical-gradematerial and has excellentmechanical,

thermal, and optical properties: tensile modulus 2400MPa

(1mm �min�1), equivalent fiber stress at maximum load 97MPa

(2mm �min�1), Vicat softening temperature 145 8C (50N;

50 8C �h�1), and haze< 0.8% (3mm). The necessary sterilization

of the samples by steam at 121 8C is possible because of the

synthetic polymermaterial’s heat deflection temperature of 125 8C(1.8MPa). Before the molding process, the thermoplastic basic

material was cleared of contamination in a specially staged

purification process. The PC has a water absorption of 0.12%

(equilibrium value: 23 8C; 50% relative huminity). Before further

processing, the residual moisture of the material was reduced to

below 0.01% by drying in a heating cabinet. A qualified hot-

pressing procedure with defined parameters (300 8C, 1.5 bar,

residence time: 3min, cooling time: 5min) was used to prepare

the substrate sample. The material was processed at 280 8C in a

compression mold. Reproducible substrate surface qualities

were achieved with specially processed tool surfaces (rough-

ness< 3mm). For the realization of small series (up to 300 samples),

coplanar material films with a dimension of 200mm�200mm�1mm were manufactured by a 200 T hydraulic laboratory press

(VOGT Maschinenbau GmbH, Germany). PC

diskswith a diameter of 11mmand a height of

1mmwere punched from this material with a

specially prepared mold.

2.2.2. Perforated PC Slices

The PC slices with the special fit for the 3D cell

culture module were made from PC precision

films (BayerMaterialScienceAG)withadimen-

sion of 210mm� 297mm� 0.5mm. Slices

were cut out by a laser procedure with an UV

laser (TruMicro5350,TrumpfGmbH,Germany).

Here, the uppermost (first) slice was chosen to

have round poreswith an initial size of 0.5mm

in diameter. The second and the fourth level

slices have oblong holes (0.5mm�1.5mm),

whereas, the third and fifth level slices possess

roundporesof? 0.5mm.Eachslicewas0.5mm

in height, and a stack of five slices generates

pore channels of an overall longitude of around

2.5mm (Figure 1C).

2.3. Surface Modification of PC Samples by

Plasma Treatment

Theplasma treatmentwasperformed inan industrial lowpressure

plasma processor (V55G, Plasma-Finish, Germany). A schematic

cross-section of the reactor is shown in Figure 2. The vacuum

chamber consists of aluminum and has the dimensions

40� 45�34 cm3 (width�depth�height). The MW source, with

a frequency of 2.45GHz, is located at the top of the reactor. It

generates large area, planar MW plasma just below the coupling

window.Aparabolic reflector above theMWwindowwas installed

inorder to facilitatehomogeneous treatmentof thesamples,which

Figure 2. Schematic drawing of the low pressure plasmaprocessorwith the MW source.

Figure 1. 3D cell culture module. (A) Exploded view (1: cap, 2: fixing ring, 3: stack of slices,and 4: clamping ring). (B) Sectional view. (C) Meander structures of the pores in thewhole stack with five slices.


� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plasma-polymers.org 263

Ammonia Plasma Functionalized Polycarbonate . . .

were placed on a metallic substrate holder approximately 5 cm

below the MW window. A detailed description is given in Steffen

et al. [25]

The plasma treatment process was performed solely with

ammonia (NH3) gas, using a flow rate of 40 sccm after purging the

chamber and stabilizing the gas for 1min. The applied MW power

was adjusted to 500W. The pressure during the process was 20 Pa.

The plasma conditions were chosen with respect to a minimal

thermal exposure but still sufficient density of reactive species.[25]

In order to generate homogenous plasma conditions for the

subsequent treatment procedure, NH3 plasma was carried out for

5min without samples. The treatment time of the PC samples

was varied between 1 and 300 s (1, 3, 5, 30, 60, 120, and 300 s)

using continuous wave (cw) mode. Perforated PC slices for the

3D cell culture module were treated separately by 30 s of

ammonia plasma. After the plasma treatment the reactor vessel

was flushed with dry nitrogen and vented. To avoid particles and

bacterial contaminations the plasma treated samples were

removed and packed in sterile under a laminar box. Time-related

changes on the PC surfaces after plasma treatment were

minimized by using the samples within 48h for cell biological

investigations.

2.4. Surface Characterization Using Physicochemical

Methods

2.4.1. X-ray Photoelectron Spectroscopy (XPS) Analysis

The chemical surface composition was determined by XPS. The

measurements were carried out with an AXIS Ultra DLD electron

spectrometer (Kratos Analytical, Manchester, UK). The XPS data

were recorded using monochromated Al Ka excitation (1 486 eV).

Spectra were acquired by setting the instrument to the medium

magnification (field of view 2) lens mode and by selecting the slot

mode, providing an analysis area of approximately 250mm in

diameter. Charge neutralization was implemented by low

energy electrons injected into the magnetic field of the lens

from a filament located directly atop the sample. Wide scans

and element spectra were recorded at a pass energy of 80 eV for

the estimation of the chemical element composition and at a

pass energy of 10 eV for the energetically highly resolved

measurements of the C 1s peak, respectively. Three spots in

different positions on each sample were analyzed and averaged.

Data acquisition and processing were carried out using CasaXPS

software, version 2.14.dev29 (Casa Software Ltd., UK). The

binding energy (BE) scale was corrected for charging using an

electron BE of 285.0 eV for the C–H/C–Caliph component in

the C 1s spectra. The full width at half maximum (FWHM) of

the C 1s components was �1.2 eV for the C 1s peak high energy

resolution measurements.

2.4.2. Chemical Derivatization for Quantification of

Amino Group Density

Gas-phase derivatization with 4-trifluoromethylbenzaldehyde

(TFBA; Sigma–Aldrich GmbH, Germany) was utilized to label

primary amines in order to evaluate their densities. The reaction of

the aldehyde group of TFBA with surface primary amine groups

yields a Schiff Base (Equation 1).

F3CO

HNH2 R OH2 F3C CH N R+ +C

(1)

Immediately after the plasma treatment the aminated PC

sampleswereplaced in a closedglass vial to be exposed tovapors of

0.5ml TFBA at a temperature of 37 8C for 2 h. To remove the

physisorbed products from the polymer surfaces the specimens

were transferred to the XPS chamber and pumped down overnight

to a pressure of 10�8mbar.

2.4.3. Contact Angle Measurements

Sessile drop contact anglemeasurements were carried out at 20 8Cusing the contact angle measuring system OCA 30 (Data Physics

Instruments GmbH, Germany). For the calculation of the

surface energy five contact angles each were measured with

double distilled water, ethylene glycol, and methylene iodide. The

dropsizechosenwas0.5ml. Thesurface freeenergywasdetermined

according to the theory of Owens–Wendt with polar and disperse

contributions.[26]

2.5. Cell Biological Investigations

2.5.1. Cell Culture of Human Osteoblasts

HumanMG-63 osteoblast-like cells (cell line, ATCC, No. CRL-1427TM,

LGC Promochem, Germany) were cultured in Dulbecco’s modified

Eagle medium (DMEM, Invitrogen, Germany) supplemented with

10% fetal calf serum (FCS, PAA Gold, PAA Laboratories GmbH,

Germany) and 1% gentamicin (Ratiopharm, Germany) at 37 8C in a

humidified atmosphere with 5% CO2. At subconfluency, cells were

detached with 0.05% trypsin/0.02% ethylenediaminetetraacetate

(EDTA) (PAA Laboratories GmbH) for 5min at 37 8C. After stoppingtrypsinization by the addition of complete cell culture medium, an

aliquot of 100ml was put into 10ml of CASY1 ton buffer solution

(Innovatis, Germany) and the cell number was measured in the

counterCASY1ModelDT(ScharfeSystem,Germany). Plasmatreated

PC disks were sterile per se and non-treated control disks were

sterilizedby steamat121 8C. PCdiskswere placed in a 48-well tissue

culture plate (Greiner BioOne, Germany) and coveredwith 200ml of

the complete cell culturemedium. The appropriate cell numberwas

seeded onto the disks as described for the following applications.

2.5.2. Investigation of Cell Adhesion

25ml of a cell suspension with 5� 104 cells were seeded onto the

plasma functionalized PC disks and non-treated controls in the 48-

well tissue culture plate (‘‘seeding’’ plate). Nine parallel experi-

mentsweredone, three sampleseach.After the cellshadadhered to

thematerial surface for10min, themediumwith thenon-adherent

cells was removed and transferred to a new 96-well tissue culture

plate (black, No: 655079, Greiner Bio One) (‘‘measurement’’ plate).

As a control for the total cell number, three samples of each cell

passage (5� 104 cells) were seeded on separate wells in the

measurement plate. The measurement plate was cultivated for



C. Bergemann et al.

30min at 37 8C in a humidified atmosphere with 5% CO2 and was

then processed for cell quantification by Hoechst 33342 fluores-

cence staining of DNA. Wells were washed with phosphate-

buffered saline (PBS) (PAA Laboratories GmbH), then dried and

frozen at �80 8C for 30min. After thawing, cells were incubated

with 200ml/well of Hoechst 33342 working solution containing

1� CyQuant Cell Lysis Buffer (Molecular Probes, Invitrogen,

Germany) and 10mg �ml�1 Hoechst 33342 (Sigma–Aldrich GmbH)

for 1 h. The fluorescence intensity per well was analyzed using a

microtiter plate reading fluorometer (Tecan Infinite M200, TECAN,

Germany) at 460nm (excitation 350nm). The percentage of

adherent cells was calculated by comparing the fluorescence from

the wells with non-adherent cells from the PC disks with the

fluorescence from wells with the total cell number.

2.5.3. Cell Spreading

To make the cells visible for microscopic investigations on the PC

surface, MG-63 cells were labeled with the fluorescent dye PKH26

for vital cells (PKH26-GL general cell linker kit, Sigma-Aldrich

GmbH, Germany) before seeding.[27] This fluorescent dye did not

influence the cell growthof culturedosteoblasts and the sensitivity

of the PKH26-stained cells to topographical and chemical features

of material surfaces was maintained, as seen in earlier experi-

ments.[1,28–30] The cell membranes of 1� 106 suspended cells (in

250ml diluentCaccording to thekit instructions)were stainedwith

red fluorescent linker PKH26 for 5min at 37 8C using a dilution of

2ml PKH26þ248ml diluentC.25ml ofa cell suspensionwith3�104

membrane-stained cells were seeded onto each of the plasma

functionalized PC disks and non-treated controls. After 15min,

when the cells had adhered to thematerials, the 48-well plate was

incubated at 37 8C in a humidified atmosphere with 5% CO2 for a

further 15min. After the whole cultivation time of 30min cells on

the materials were rinsed in PBS and fixed with 4% paraformalde-

hyde (PFA, Merck, Germany) for 10min at room temperature. The

cells on the PCdiskswere embeddedwithmountingmediumanda

cover slip.[1] Cells were examined under the confocal microscope

LSM 410 (exc. 543nm, Carl Zeiss, Germany) and the spreading (cell

area in mm2) of 40 cells/specimen was then analyzed using the

software ‘area measurement‘ of the confocal microscope LSM 410.

Two separate experiments with different cell passages were done

for each sample (n¼ 2, 40 cells each).

2.5.4. Scanning Electron Microscopy (SEM) and

Stereo Images

25ml of a cell suspensionwith3� 104 cellswere seededonto the PC

disks.After 15min,when thecells hadadhered to thematerials, the

48-well plate was incubated at 37 8C in a humidified atmosphere

with 5% CO2. After 24h cells were rinsed with PBS, fixed with 2.5%

glutaraldehyde (Merck), dehydrated through a graded series of

alcohol, and dried in a critical point dryer (K850, Emitech, UK). The

samples were characterized by the scanning electron microscope

DSM 960A (Carl Zeiss).

Cell height was estimated by using stereo images from

randomly selected sample regions. For this purpose, two images

ofeachregionwere takenatamagnificationof1 000timesandatilt

angle of�4 andþ48, respectively. Generation of stereo images and

height measurements were done using the stereo module of the

Scandium software (OSIS, Germany). The software generates a

height map of the imaged region using the position difference of

objects (parallax) in pictures obtained at different viewing (tilt)

angles. To estimate the cell height relative to the surface of the

substrate, apossible inclinationof the substrate relative to thebase

level of the height map had to be considered. The height of the cell

was estimated by drawing a line over the highest point of a cell

beginningandendingonacell-freeareaof thesubstrate. Extraction

of theheight profile provides all heights anddistances between the

points. By a simple trigonometric calculation cell height relative to

substrate could be calculated (Figure 3).

2.5.5. 3D Cell Culture

The components of the 3D cell culture module and non-treated PC

slices were sterilized by steam at 121 8C. Plasma treated PC slices

were sterile per se. MG-63 osteoblastic cells were seeded onto the

uppermost slice surface at a density of 3�105 cells/specimen

separately in a cell culture dish with complete culture medium.

After 30min of incubation at 37 8C in a humidified atmosphere

with 5% CO2, when the cells had adhered to the material, the slice

was mounted at the top of the 3D cell culture module with four

non-seeded slices as described above, placed in a cell culture dish

(? 60mm, Greiner Bio One) and covered with 22ml complete cell

culturemedium.Themediumwas changedevery secondday.After

14d of culture cells on every PC slice underwent a separate cell–

biological analysis. This could be easily realized by demounting

the module.

2.5.6. Vitality of Cells

MG-63 cells were cultured on the 3D module for 14d as described.

Vitalitywas analyzed by live/dead staining (L7013 LIVE/DEADCell

Viability Kit, Invitrogen) after demounting the slices from the

module and washing carefully with Hank’s Buffered Salt Solution

(HBSS, PAA Laboratories GmbH). Cellswere stainedwith a solution

of SYTO 10 and DEAD Red (5ml each in 2.5ml HBSS) for 15min

at 37 8C in the dark, washed again, and incubated with 4%

glutaraldehyde in HBSS for at least 1 h at 4 8C. Cells were examined

on the confocalmicroscope (LSM410,Carl Zeiss) usinganexcitation

wave length of 488 and 543nm, respectively. Living cells showed

green fluorescent nuclei and could, thereby, be distinguished from

dead cells identified by their red fluorescent nuclei. Images

Figure 3. Scheme of the trigonometric estimation of cell heightusing height values computed from stereo images, a¼ABS(a0 �a00), tana¼a/b, h0(a)¼b0 � tana, Cell height relative tosubstrate: h total¼ h�h0 �a00 (Scandium Software).




(512pixels�512pixels)were taken systematically from the center

to the edge of the top and central slices fromeach3Dmodule (slices

1 and 3) with a 10� air lens at zoom 1.

2.5.7. Statistics

Statistical analysis of data sets was performed using the software

SPSS 15.0 forWindows (SPSS Inc., USA). Data are expressed asmean

values� standard deviation and analyzed using the Mann–

Whitney U-test. Differences were considered statistically signifi-

cant at p<0.05 (�p<0.05, ��p<0.01, and ��p< 0.001).

3. Results and Discussion

The study presented here investigated the influence of

ammonia plasma on the surface properties of PC and the

effect onosteoblast behavior.MG-63 osteoblastic cellswere

used as a well-established line of human bone-like cells.[31]

Special interestwas focusedon theplasma time regimeand

its influence on surface properties as well as initial cell

reactions like adhesion and spreading and the effect on the

migration of the cells into the depth of the 3D cell culture

module equipped with perforated PC slices.

3.1. Surface Modification of PC by Plasma Treatment

3.1.1. Wetting Properties

Since unexposed PC does not contain any strongly polar

groups, its surface is hydrophobic and therefore water is

repelled from the surface. Subsequently, the measured

water contact angle (WCA) of non-treated PC is about 858due to its hydrophobic surface. As shown in Figure 4, short

ammonia plasma treatments over periods of 1–120 s at

500W, 20 Pa reduced the WCA. Initially, in the range of

5–60 s, theWCA reached its lowest value of around 458; thePC surface becomes more hydrophilic. After longer plasma

treatment times the wettability of the PC samples was

observed to decrease again and the WCA increased to 808.This observed trend of variation in wettability is related to

the incorporation of various functional groups at the

surface, cross-linking processes and chain scission. More-

over, spectroscopic measurements in ammonia plasma

revealed the presence of NHx radicals, atomic nitrogen, and

hydrogen.[32,33] Such active species can interact with each

other or with the surface of the substrate within very short

periods of time, a modification of the uppermost layer can

be achieved, resulting in reduced WCA values. However,

during the post plasma process or through exposure of the

activated surface to air, heterogeneous reactions, cross-

linking, and etching processes aswell as rearrangements of

polymer chains may occur.[10] These various reactions can

cause a negative effect on the surface functionalization

resulting in higher WCA. Our observed results suggest that

later processes become dominant at longer treatment

times, resulting in an increase in WCA values at t> 60 s.

The result of our investigations of the influence of

treatment duration on surface wettability of the plasma

treatedPC canbe correlated tomeasurements of the surface

energy as a function of plasma treatment times (Figure 5A).

For non-treated PC the observed surface energy was

39mN �m�1 with only a small number of polar groups

(3mN �m�1) but with a large amount of the dispersive

component (35mN �m�1). After short ammonia plasma

treatment times of 1–60 s at 500W, 20 Pa, the number of

polar groups increased strongly to a maximum value of

30mN �m�1 whichwas reached after 5 s and retained until

60 s. For clarification, the values for short treatment times

are shown in detail in Figure 5B. Longer plasma treatment

times of 120–300 s, however, were observed to reduce the

polar contribution (Figure 5A) which corresponds to the

obtained WCA values.

Theobservedtrend inWCAandsurfaceenergyvaluescan

be discussed in terms of the different processes occurring

during the plasma surface interaction but also of the post

plasma processes which lead to incorporation of polar

functional groups such as amino and hydroxyl, or carboxyl

groups, respectively. The functionalization processes are

competing with chain scission and cross-linking processes

which also take place simultaneously during exposure of

thepolymer surface to theplasma.At short treatment times

the functionalization process is predominant and the ratio

of polar functional groups at the surface increases. After

longer plasma process times, however, the competing

surface reactionsoccur, resulting inadecreaseof the ratioof

polar groups and subsequently in a decrease of surface

energy.

0 50 100 150 200 250 300

40

50

60

70

80

90

watercontactangle[°]

t [s]

non-treated PC (WCA:85°)

Figure 4. Effect of ammonia plasma treatment (500W, 20 Pa)on the wettability of PC surfaces dependent on the plasmatreatment duration. The error bars correspond to the standarddeviation estimated from five datasets for five measured points.



C. Bergemann et al.

3.1.2. Chemical Composition

Figure 6 shows the variation of the element ratios O/C and

N/C as a function of the ammonia plasma treatment

duration. A short treatment of 3 s (500W, 20 Pa) signifi-

cantly reduced the O/C ratio from�15% of the non-treated

surface to �12%. Up to 30 s of plasma treatment time, the

ratio remains around the same value with a further small

reductionof theO/Cratio to10%after the longest treatment

time of 300 s. The N/C ratio of the non-treated PC surface

was found to be very low and observed at the detection

limit. After only 1 s of treatment in ammonia plasma, N/C

was observed to sharply increase to a value of 2%, after 3 s

the N/C was about 5% and remained around at 5–6% until

30 s. When the treatment time was increased further, that

is, to 120 and 300 s, theN/C ratio decreased to a value of 4%.

In agreement with the trends in O/C and N/C element

ratios, the shape of the C 1s peak also changed due to the

shift of the C bindings dependent on the plasma treatment

time. Figure 7 displays the highly resolved measured C 1s

spectra of the non-treated PC surface compared to the

treated ones at three relevant times: 30, 60, and 300 s

(without TFBA-derivatization).

The C 1s peak of the non-treated PC has been

deconvoluted into five components corresponding to

C–Carom (BE: 284.5 eV), C–Caliph (BE: 285.0 eV), C–O (BE:

286.24 eV), OC(¼O)O (BE: 290.4 eV), and the shakeup (BE:

291.7 eV).[34] After ammonia plasma treatment two further

0 50 100 150 200 250 3000

2

4

6

8

10

12

14

16

18

XPS-elementratio[%]

t [s]

O/CN/C

Figure 6. Dependence of surface composition of PC samples onammonia plasma treatment times. The error bars correspond tothe standard deviation estimated from five datasets for threemeasured points.

Figure 7. Highly resolved measured C 1s spectra of non-treated(curve a) and ammonia plasma treated PC samples (b: 30 s, c: 60 s,and d: 300 s). Note that two further components C–N and C¼Ocan be detected after ammonia plasma treatment.

0 50 100 150 200 250 3000

10

20

30

40

50

60

70totaldispersepolar

surfaceenergy[mN/m]

t [s]

0 5 10 15 20 25 300

10

20

30

40

50

60

70totaldispersepolar

surfaceenergy[mN/m]

t [s]

A)

B)

Figure 5. Surface energy of PC after plasma treatment: (A) Depen-dence of the ammonia plasma treatment time up to 300 s. (B)Initial effect of ammonia plasma treatment up to 30 s (magni-fication of the time scale in A). The error bars correspond to thestandard deviation estimated from five datasets for fivemeasured points.




components C–N (BE: 285.8 eV) and C¼O (BE: 287.8 eV) can

be detected. With increasing plasma treatment time the

aromatic compound as well as the carboxylic/carbonate

groups decreased due to permanent damage of the PC

surface, with preference at the benzene ring and the

carbonate group (Figure 8). This is an indication that the

insertion of NHx into the PC surface takes place by breaking

the C¼O– and OC(¼O)O-bindings. The oxygen species

gained from the PC surface during this process can then

react with N and H and NHx forming various volatile

products. On the other hand, themeasured decrease inNHx

content could also be a result of simultaneously occurring

etching processes at the surface.

The measured trends in element ratios and C bindings

correlate well with our results for the surface energy data.

As discussed earlier, different processes occurring during

plasma surface interaction and post plasma oxidation

become dominant with increasing treatment times.

Additionally, chain scission and cross-linking processes

may also increasingly contribute to the observed surface

properties after long treatment times. Also etching

processes can take place simultaneously to surface

functionalization, as it is known to occur under certain

conditions such as long treatment times.

To summarize, at short plasma treatment times (up to

60 s) functionalization processes preferently occur at the

surface as suggested by the increase of the N/C element

ratio. During longer treatments (120–300 s), etching and

cross-linking or chain scission reaction prevail at the

aminated PC surface and the N/C and O/C ratios decrease.

This is supported by the observed values for the amine

group density shown in Figure 9. Short ammonia plasma

treatments in the range of 3–60 s generated 1–1.3% NH2

groups. Longer plasma treatments decreased thenumber of

NH2 groups on the surface of ammonia plasma treated PC

samples.

3.2. Cell Biological Investigations

The surface activation of PC samples by ammonia plasma

created additional functional groupswhich can function as

recognition sites for proteins and subsequently for bone

cells. For thestudypresentedhere, differences in thedensity

of functional groups as a function of the plasma time

interval were investigated concerning the influence on

initial cell attachment processes. Experimental results

demonstrated time-dependent differences in adhesion,

spreading, and morphology of MG-63 osteoblastic cells on

plasma treated PC disks as well as differences in the

migrationbehaviorof thecells in the3Dcell culturemodule.

3.2.1. Cell Adhesion

The cell adhesion capacity of ammonia plasma treated PC

disks is seen in Figure 10. Adherent MG-63 cells were

analyzed after 10min of cell culture on PC disks functio-

nalizedbyammoniaplasma for 1, 3, 5, 30, 60, 120, and300 s.

The values indicated that the treatment by ammonia

plasma even for 3 s has an impact on cell attachment. This

effect is enhanced by prolongation of plasma treatment

until timeintervalsof30 s. Forall timepointsbetween3and

120 s the number of adherent cells was significantly higher

than on non-treated surfaces, for example, for 30 s plasma

time the number of adherent cells was elevated about 24%

compared to non-treated PC. Further extension of the

Figure 8. Dependence of surface composition of PC samples onammonia plasma treatment times. Note that two further com-ponents C–N (see the black bar) and C¼O can be detected afterammonia plasma treatment and that aromatic components aswell as carboxylic/carbonate groups decrease with increasingplasma treatment time due to permanent damage of the PCsurface.

0 50 100 150 200 250 3000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

NH2/C

[%]

t [s]

Figure 9. Dependence of the number of the NH2 groups on the PCsurface on the ammonia plasma duration. The error bars corre-spond to the standard deviation estimated from five datasets forthree measured points.



C. Bergemann et al.

plasma time over 30 s did not show additional improve-

ment of cell adhesion but resulted in significantly higher

numbersofadherent cells for60and120 scompared tonon-

treated PC. Surfaces treatedwith ammonia plasma for 300 s

did not show better adhesion properties than pure PC

surfaces.

3.2.2. Cell Spreading and Morphology

Similar effects couldbeobserved for thespreadingofMG-63

cells on plasma treated PC surfaces. Spreading was

significantly enhanced on PC disks functionalized by

ammonia plasma for 3–300 s (Figure 11). A maximum cell

areawasdetected for cellsonPCafter60 splasmatreatment

(260mm2) compared to the area of cells grownonuntreated

PC (188mm2).

Differences in cellmorphology could be observed aswell.

MG-63 cells were well spread on all sample disks. But cells

culturedonammoniaplasmatreatedPCsurfaces seemtobe

more flattened and occupied a greater surface area than

cells on non-treated disks (Figure 12).

Measurement of the cell height of

MG-63 cells on plasma treated (30 and

300 s) and non-treated control PC disks

was realized by taking stereo SEM images

of each plate and subsequent analysis

using analySIS stereo software (Figure 3).

Calculation of the cell height confirmed

the optical impression of stronger

attached cells and the results for cell

adhesion on 30 s plasma treated PC disks,

which correlated with a reduced cell

height. This effect was significant for

30 s (Figure 13).

Our cell investigations showed best

results on PC samples treated by ammo-

nia plasma for 30 or 60 s. This is in

correlation with our results for the

analysis of surface energy properties

and chemical composition of PC surfaces,

where theelement ratioN/Cshowedmaximalvalues about

5–6%after ammoniaplasmatreatment for5–60 s (Figure6).

Thenumber ofNH2groupswas increased for this treatment

times (Figure9)and is reflected in the lowestvalues forWCA

around 458which results in increased cell attachment and

spreading and decreased cell height of MG-63 osteoblasts.

With elongated ammonia plasma treatment the number of

NH2 groups and the wettability of the surfaces decreased

again. As a consequence the attachment of osteoblast-like

cells was not promoted.

This is in line with earlier studies where optimal cell

attachment and growth for different cell types was shown

for polymer surfaceswithmoderatewettabilities.[35–37] Lee

et al.[7] investigated the effect of different surface

functional groups but similar wettabilities and find that

surfaces graftedwith amino groupswere best for adhesion

of Chinese hamster ovary cells. On PHB low pressure

ammonia plasmas were used by Nitschke et al.[16]. They

revealed that the number of generated amino groups was

not increased with elongated plasma treatment times and

Figure 10. Effect of ammonia plasma treatment of PC surfaces oncell adhesion (10min). (n¼9, 3 samples each, ��p<0.001).

Figure 11. Cell spreading of MG-63 cells (30min) on ammoniaplasma treated PC surfaces. (n¼ 2, 40 cells each, ��p<0.01,��p<0.001).

Figure 12. Effect of ammonia plasma treatment of PC surfaces on osteoblastmorphology(24 h cell culture). Note that cells seem to be more connected to the surface with theircell margins as well, and the cell area is enhanced on 30 s ammonia plasma treated PCdisks. SEM, magnification 1 000�, bar 20mm.




observed a saturating effect. Yang et al.[14] utilized

ammonia plasmas to improve fibroblast cell attachment

and growth on PLA surfaces. In all these investigations

adhesion of cells was determined after 90min or even

longer exceeding initial time frames. Our results agreewith

these earlier studies and extend our knowledge to

osteoblast cells and the influence of plasma generated

amino groups on the initial cell adhesion of MG-63 cells

within 10min, the cell spreading and cell height after

culture on plasma treated PC.

3.2.3. 3D Cell Culture

Our 3D cell culture module with five PC

slices was used to investigate the occupa-

tion of the depth of a scaffold by

osteoblast-like MG-63 cells. In conse-

quence of our results showing improved

cell culture properties on PC after short

time ammonia plasma, we used 30 s

ammonia plasma treatment for the opti-

mization of the surfaces of the perforated

PC slices for the 3D cell culture module.

The slices were treated by ammonia

plasma separately and the 3D module

was assembled just before 3D cell culture

experiments. As a control one module

with non-treated PC slices was utilized.

MG-63osteoblastic cellswere seededonto

the uppermost slice surface (slice 1) at a

density of 3� 105 cells/specimen sepa-

rately. After 30min of incubation, when

the cells had adhered to the material, the

seeded slicewasmountedat the topof the

3D cell culture module with four non-

seeded slices completing the 3D stack to

five slices. The 3D constructs (one with

slices treated by 30 s of ammonia plasma and one with

untreatedslices)werecultured for14 dandcell colonization

of the different slices was analyzed by live/dead staining

and confocal microscopy after demounting. To identify the

occupation of this artificial scaffold by osteoblast cells

especially toward the center the cell density on the top

(slice 1) and central slices (corresponding to slice 3) was

determined. Images were taken systematically from the

center to the edge of slices 1 and 3 from each 3D module.

Figure 14 shows representative images for these slices of

the plasma optimized and the non-treated 3D cell culture

module. Results showed that cells on both modules

occupied the top slices, but showed a higher cell density

on the plasma treated slice 1 than on the non-treated

control. Furthermore cells began to migrate into the depth

of the corpus in the 3D cell culturemodulewith the plasma

optimized surfaces visible by the occupation of the

channels (yellow dotted lines) on the central slice (slice

3). So we could show that the precondition of optimized

osteoblast cell attachment on ammonia plasma treated PC

slices resulted in increased cellmigration toward the center

of the 3D cell culture module and induced proliferation in

the channels.

In summary, the initial cell attachment and spreading

was significantly increased after ammonia plasma treat-

ment for 3–120 s on PC disks. Furthermore, cells showed a

significantly more flattened morphology on PC disks

Figure 13.Maximal cell height of MG-63 cells on ammonia plasmatreated PC disks (24 h), estimated from paired stereo SEM images.Note that cells have a reduced cell height on the 30 s plasmatreated PC due to distinct flattened phenotype (n¼ 10, �p<0.05)(see also Figure 12).

Figure 14. (A) Scheme of the 3D cell culture module and detailed drawing of themeander-like structure generated in a stack of five porous PC slices. (B) Confocalmicroscopic images of MG-63 cells (SYTO 10, green; DEAD Red, red) grown for 14 don the 3D module with perforated PC slices (pores 500mm in diameter; white dottedcircles), left: non-treated PC slices, right: PC slices treated with 30 s of ammonia plasma.The yellow dotted lines and circles on slice 3 represent the pore geometry from the slices1 and 2. Note that on plasma-functionalized PC slice 3 (right), cellular ingrowth towardthe scaffold center is enhanced (arrow), indicating a positive effect of ammonia plasmatreatment for the occupation of the artificial 3D scaffold by osteoblasts. (LSM 410; bar250mm).



C. Bergemann et al.

treated for 30 swith ammonia plasma. This is in correlation

with our results of the investigation of wetting properties

and PC surface chemistry, especially the number of NH2-

groups after ammonia plasma treatment which reached

maximal values after 3–60 s of plasma treatment. In line

with these observations, the best results for MG-63 cell

attachment and spreading were achieved on PC surfaces

treated with ammonia plasma for 30 and 60 s, suggesting

an influence of the density of amino groups on the cell–

material interaction. Finally, also migration and prolifera-

tion of osteoblastic cells into the 3D cell culture module

were improved by the application of plasma-optimized PC

slices.

This is probably due to the positive charge character of

amino functionalized surfaces under physiological pH

values and the resulting electrostatic interaction with

negatively charged cell surface components and with

serum proteins by hydrogen bonding. As it is known,

adhesive glycoproteins like vitronectin (VN) and fibronec-

tin (FN) adsorb onto plasma treated polymer surfaces in

competition with other serum proteins.[38–40] Anchorage-

dependent cells colonize artificial surfaces through binding

on this layer of glycoproteins by integrin receptors.[9] Steele

et al. investigated the influence of VNand FNadsorption on

the attachment of bone derived cells onto tissue culture PS

(TCPS). They showed that the attachment of cells is due to

the adsorption of VN from fetal bovine serum (FBS),

whereas, FN has to be preadsorbed onto TCPS.[38] Attach-

ment ofMG-63 osteoblastic cells was enhanced after 1 d on

oxygen plasmamodified PLA precoatedwith FN, VN, or FBS

by Alves et al. Nevertheless this effect was reversed after

prolonged culture.[40]

Electrostatic interactionseemstobeamajor factor for the

initial attachment of the cells and for subsequent cell

receptor guided adherence as well. Hyaluronan (HA), a

pericellularmatrix substance, playsakey role in initial cell–

biomaterial interactions.[41,42] Via HA, as a highly nega-

tively charged glycosaminoglycan, cells attract to posi-

tively charged surfaces; we were able to show this for

plasmapolymerizedallylamine (PPAAm)on titanium.[29] In

previous studies, positively charged amino groups from

PPAAm improved not only time-dependent cell adhesion

and proliferation of osteoblasts on titanium, but also the

organization of intracellular structures like the actin

cytoskeleton and the adaptor proteins paxillin and

vinculin.[27,43,44] On PPAAm surfaces cell migration was

significantly increased due to the mobility of vinculin

contacts.[45]

Here, we used ammonia plasma to generate amino-

functional groups directly on PC surfaces and we were

able to show similar effects on cell attachment, which

weremore or less distinctive dependingon theplasma time

intervals used. Following these results, it was proven that

the duration of the plasma treatment process has an

important influence on density and availability of amino

groups on PC surfaces and is beneficial for cell attachment

and the spreading of osteoblasts. For further improvement

of the 3D cell culture module we used PC slices which were

ammonia plasma treated for 30 s and, as expected, cell

colonization into the center of this artificial scaffold was

enhanced. Extension of this analysis to degradable poly-

mers and other cell types, for instance primary osteoblasts

or stem cells, will be interesting for further applications,

especially in tissue engineering.

With the knowledge that the time of the plasma

treatment can create customized adhesion-friendly PC

surfaces we have a tool for directing the cell migration into

thedepthofa scaffold.Further investigationsondynamic3D

cell cultures with stacks of more than five slices and longer

cultivation times will shed light on the question of whether

an effective occupation of the whole 3D corpus is possible.

4. Conclusion

Our study demonstrates that different ammonia plasma

treatment time isdecisive for the cell adhesionpropertiesof

PC surfaces. Modification of the plasma treatment time on

PC can, therefore, be used as a model to investigate the

influence of the density of functional groups on cell

attachment and morphology. Furthermore, plasma func-

tionalized PC surfaces are well suited as a substrate for

anchorage-dependent cells like osteoblasts. The newly

established 3D cell culture module can be used for in vitro

research and the development of an optimized scaffold

design for orthopedic implants.

Acknowledgements: The authors are grateful to the EuropeanUnion and to the Mecklenburg-Vorpommern State Ministry ofEconomic Affairs, Employment, and Tourism for financial supportwithin the joint project ‘‘Tissue regeneration’’ (GEREMA), sub-project VOCELL [EFRE: V220-630-08-TFMV-F-009, ESV: V220-630-08-TFMV-S-009], BIOPHIL [EFRE: V220-630-08-TFMV-F-010, ESV:V220-630-08-TFMV-S-010], and POLYLAY [EFRE: V220-630-08-TFMV-S-014, ESV: V220-630-08-TFMV-F-014]. The authorsacknowledge the excellent technical support of Roland Ihrke(INP Greifswald), Janine Wetzel (Cell Biology, University ofRostock), and Gerhard Fulda (Electron Microscopy Centre, Uni-versity of Rostock), and thank the SLV-MV GmbH Rostock forcutting the PC slices.

Received: March 25, 2011; Revised: September 12, 2011; Accepted:October 10, 2011; DOI: 10.1002/ppap.201100059

Keywords: ammonia plasma; cell adhesion; human osteoblasts;polycarbonate; surface modification

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Documents

Ammonia Plasma Functionalized Polycarbonate Surfaces Improve Cell Migration Inside an Artificial 3D Cell Culture Module