Antibacterial Poly(D,L-Lactic Acid) Coating of Medical Implants

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Journal of Antimicrobial Chemotherapy (2003) 51, 585591

DOI: 10.1093/jac/dkg105

Advance Access publication 28 January 2003

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2003 The British Society for Antimicrobial Chemotherapy

Antibacterial poly(D,L-lactic acid) coating of medical implants using a

biodegradable drug delivery technology

Hans Gollwitzer1,2*, Karim Ibrahim1, Henriette Meyer1, Wolfram Mittelmeier2, Raymonde Busch3

and Axel Stemberger1

1Institut fr Experimentelle Onkologie und Therapieforschung; 2Klinik und Poliklinik fr Orthopdie und

Sportorthopdie; 3Institut fr Medizinische Statistik und Epidemiologie, der Technischen Universitt Mnchen,

Ismaninger Strasse 22, 81675 Munich, Germany

Received 4 September 2002; returned 16 October 2002; revised 26 November 2002; accepted 26 November 2002

Objectives: Biomaterial-associated bacterial infections present common and challenging com-plications with medical implants. The purpose of this study was to determine the antibacterialproperties of a low molecular weight biodegradable poly(D,L-lactic acid) coating with integratedantibiotics gentamicin and teicoplanin.

Methods: Coating of Kirschner-wires was carried out by a solvent casting technique underaseptic conditions with and without incorporated antibiotics. Release kinetics of gentamicin andteicoplanin were studied in phosphate-buffered saline. Initial bacterial adhesion of Staphylo-coccus epidermidison coated and bare implants was determined by radiolabelling and countsof detached viable organisms.

Results: The incorporated antibiotics showed a continuous release over a period of at least 96 hwith an initial peak of release in the first 6 h. Attachment of non-viable microorganisms, detectedby radiolabelled bacteria, was increased significantly by the polymer coatings (P< 0.05). Incontrast, the number of viable bacteria was reduced by the pure polymer (P< 0.01) and further bythe polymerantibiotic combinations (P< 0.05).

Conclusions: Poly(D,L-lactic acid) coating of implants could offer new perspectives in preventingbiomaterial-associated infections. Combinations with other drugs to formulate custom-tailoredimplant surfaces are feasible.

Keywords: polylactide, PDLLA, drug release, Staphylococcus, biomaterial

Introduction

Since the first applications of biomaterials in medicine,

infections and deficient tissue-integration represent the most

important complications, which still limit the unrestricted use

of biomaterials in humans. Implant-associated infections

account for nearly 50% of the estimated 2 million nosocomial

infections in the United States per year,1 and infection rates up

to 100% are reported for certain implants, like external fix-

ation pins.24 Treatment of these infections is associated with

high complication rates and places an enormous burden on

both the patient and healthcare providers; prolonged hospital

stay, increased morbidity and mortality, and serious economic

sequelae being common consequences.5

It is possible that the risk of infection can be reduced by an

antiseptic surface coating for medical implants. The purpose

of this study was therefore to determine the anti-infective

properties of a new biodegradable coating for medical

implants with regard to Staphylococcus epidermidis, one of the

most important pathogens in biomaterial-associated infec-

tions.6,7 This coating is based on a polymer of low molecular

weight poly(D,L-lactic acid) (PDLLA) and can be combined

with drugs like antibiotics or growth factors to establish a

locally acting drug-delivery system.

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*Corresponding author. Tel/Fax: +49-89-4140-7242; E-mail: [email protected]


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Materials and methods

The polymer coating

The commercially available Resomer R203 is a polymer of

PDLLA with a molecular weight of 29 000 Da and was pur-

chased from Boehringer Ingelheim (Ingelheim, Germany).The polymer is a racemic mixture of the D- and L-enantiomers

of lactic acid and serves as a biodegradable coating for med-

ical implants.

Implants were coated with PDLLA by a solvent casting

technique. In brief, the drug-carrier was dissolved in ethyl-

acetate (SigmaAldrich AG, Deisenhofen, Germany) at a

concentration of 133.3 mg/mL. The coating solution was main-

tained on dry ice to prevent evaporation of the organic solvent

and a subsequent increase in the polymer concentration.

To create a local drug delivery system, 5% (w/w) of the

antibiotics gentamicin sulphate (COM Pharma, Hamburg,

Germany) and/or teicoplanin (Aventis, Frankfurt, Germany)

were added to the polymer solution. The implants were coated

by two dip-coating procedures to achieve a dense and regular

polymer coating. All coating steps were carried out under

aseptic conditions in a laminar air-flow.

Medical implants

Commercially available Kirschner-wires (K-wires) of stain-

less steel (ISO 5832-1; Synthes GmbH & Co. KG, Umkirch,

Germany) and titanium alloy (TiAl6V4, ISO 5832-11 and ISO

5832-3; Synthes GmbH & Co. KG) were studied. The wireswere cut into lengths, cleaned and sterilized by autoclaving.

The wires used for microbiological and antibiotic release

studies and their abbreviations are shown in Table 1.

Bacterial strains

A clinical isolate of a biofilm-forming strain ofS.epidermidis

(strain SE 183) was used for the in vitro studies. The test strain

was susceptible to both gentamicin and teicoplanin (genta-

micin MIC 2 mg/L, teicoplanin MIC 0.5 mg/L). Biofilm for-

mation was demonstrated by qualitative assessment with the

tube assay previously described by Christensen et al.8 Stock

cultures of the isolate were lyophilized or stored at 70C.

Preparation of bacteria

S. epidermidis (SE 183) was cultured to late logarithmic

growth phase on blood agar plates at 37C for 18 h before test-ing. Bacterial cells were then resuspended in normal saline

and adjusted to 5 107 cfu/mL by visual comparison with a0.5 McFarland standard. This suspension was diluted with

normal saline to an inoculum of 2.5 105 cfu/mL.

Antibiotic release

Drug release from antibiotic- and polymer-coated K-wires

(n = 4; size 1.5, length 150 mm; stainless steel) was studied in

10 mL of phosphate-buffered saline (PBS) at 37C (pH 7.4).Samples were coated with the PDLLA polymer (133.3 mg/mL

ethylacetate) including gentamicin or teicoplanin (5% w/w).

PDLLA-coated samples without antibiotics served as a con-

trol. To assess antibiotic stability during the test period, PBS

of the control groups was supplemented with the gentamicin

and teicoplanin. Aliquots of 500 L were taken at the timepoints 10 min, 1, 6, 24 and 96 h from the PBS and assayed for

gentamicin and teicoplanin. Antibiotic concentrations were

determined by the fluorescence polarization immunoassays

TDx/TDxFLx Gentamicin assay (Abbott Laboratories, Abbott

Park, IL, USA) and Innofluor Teicoplanin assay system

(Opus Diagnostics, Fort Lee, NJ, USA).

Total bacterial adhesion

The total amount of live and dead adhering bacteria was

studied in a radiolabelling experiment, as described by Chris-

tensen et al.9S. epidermidis was grown in MuellerHinton

broth (Oxoid GmbH, Wesel, Germany) substituted with

[3H]thymidine. After 24 h of incubation on a rotary shaker

(100 rpm), inoculum suspensions were prepared as described

earlier. Coated and bare K-wires of stainless steel and

titanium alloy (n = 9; size 1.8, length 12 mm) were incubated

with the radiolabelled bacterial cell suspension for 2 h at 37Cunder static conditions. Individual wires were removed with

sterile forceps, washed three times with sterile normal saline

Table 1. Sample groups and surface coating of stainless steel (S) and titanium alloy (T) for the

in vitro tests

Surface coating Stainless steel Titanium alloy

Bare K-wires without coating (control group) S1 T1

K-wires coated with PDLLA S2 T2K-wires coated with PDLLA and gentamicin (5.0% w/w) S3 T3K-wires coated with PDLLA and teicoplanin (5.0% w/w) S4 T4

K-wires coated with PDLLA and gentamicin (1.7% w/w)

and teicoplanin (3.3% w/w)

S5 T5


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587

for 15 s and air-dried. After transfer to scintillation vials con-

taining scintillation fluid, counts per minute were measured in

a-counter (1219 Rackbeta; LKB Wallac, Bromma, Sweden).

Adhesion of viable bacteria

Adhesion of viable bacteria was evaluated in a bacterialadhesion assay. Coated and bare K-wires of stainless steel and

titanium alloy (n = 10; size 1.8, length 12 mm) were immersed

in 2 mL of the bacterial suspension (2.5 105 cfu/mL normalsaline) and incubated for 2 h at 37C under static conditions.After washing in normal saline, the K-wires were placed in

vials containing 2 mL of trypsin solution (1% w/w) and

sonicated (Sonorex RK255H, 50 kHz; Bandelin Electronic,

Berlin, Germany) for 15 min to remove the adhering micro-

organisms. Serial dilutions of each trypsin solution were

plated on blood agar plates for quantification of viable organ-

isms. Blood agar plates were incubated for 48 h at 37C, and

the cfu were counted visually.Complete detachment of the adhering microorganisms by

sonication was verified through scanning electron micro-

scopy (SEM).

Morphological analysisSEM

Representative K-wire specimens from the in vitro studies

were prepared for SEM as follows. Specimens were fixed

in an aqueous solution of 1% (v/v) glutaraldehyde (Serva,

Heidelberg, Germany) in a buffer of sodium phosphate

(dibasic) monohydrate (Merck KGaA, Darmstadt, Germany)

and sodium hydroxide (Merck KGaA) for a minimum of 16 h.The buffer contained 18.8 g/L sodium phosphate (dibasic)

monohydrate and 4.3 g/L sodium hydroxide. Dehydration

was carried out in an ascending ethanol series (50708090

100%) and samples were dried in a critical point dryer (CPD

030; BAL-TEC AG, Balzers, Liechtenstein) with carbon di-

oxide. Once coated with gold palladium (Polaron Autocoating

Unit SEM 5200; Quorum Technologies, Newhaven, UK)

each specimen was examined through a low vacuum SEM

(JEOL 5900; JEOL Germany GmbH, Eching, Germany).

Calculations and statistical methods

Data from bacterial adherence studies were compared for

statistical significance using non-parametric methods and the

method of closed testing procedure,10 with P < 0.05 consid-

ered significant (KruskalWallis and MannWhitney tests).

Results

Morphological analysis

After the dip-coating procedure, a stable and regular PDLLA

coating could be observed through SEM. Complete detach-

ment of adhering bacteria was observed after sonication.

Antibiotic release

The concentrations of the released antibiotics gentamicin and

teicoplanin in the elution buffer are shown in Figure 1. Acontinuous drug release could be demonstrated for at least

96 h for both antibiotics. After an initial peak of release in the

first few hours, a slow and continuous drug release could be

observed for the rest of the testing interval. Gentamicin

showed a pronounced initial peak of release in the first hour.

Teicoplanin release, in contrast, was delayed compared with

gentamicin, the initial peak of release lasted 6 h and theremaining teicoplanin was released to the PBS with kinetics

similar to that of gentamicin. Both gentamicin and teicoplanin

showed a marked variation of initial release between indi-

vidual wires (between 3.87 and 10.58 mg/L for gentamicin

and between 2.71 and 15.11 mg/L for teicoplanin after 6 h).

Antibiotic concentrations of the control groups remained

constant throughout the entire study interval and gentamicin

and teicoplanin were stable in PBS in control experiments for

at least 96 h.

Total bacterial adhesion

The total biomass of bacteria adhering to the wires was

measured using the radioactively labelled bacteria method. In

Figure 1. Elution profiles of gentamicin (left-hand panel) and teicoplanin (right-hand panel) into PBS from individual stainless steel K-wires with

PDLLA coatings containing either gentamicin or teicoplanin. In each panel the different lines show the release kinetics of the four samples tested.


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this method, the number of counts per minute is directly pro-

portional to the number of implant-adhering microorganisms,

and independent of their viability (data not shown). The

median counts and inter-quartile ranges (along with outliers)

obtained for the different wires are shown in Figure 2. The

titanium alloy used showed a slightly lower biomass ofS. epi-

dermidis adhering to the wire than stainless steel alloys,

although the results are not significantly different (P > 0.05).

However, for both types of wire a marked increase in bacterial

adhesion can be observed for all PDLLA-coated samples

compared with the bare specimens. For all of the titanium

alloy wires this increase was significant at the P < 0.05 level,

whereas for the stainless steel wires the increases were

statistically significant (P < 0.05) for all except the PDLLA/

gentamicin-coated (S3) wire.

With regard to the effect of antibiotic integration, genta-

micin showed a tendency (P = 0.050.1) to reduce bacterial

biomass after 2 h compared with pure PDLLA. Integration of

teicoplanin on the other hand significantly increased thebiomass of S. epidermidis compared with implants coated

with pure PDLLA and the PDLLA/gentamicin combination

(P < 0.05).

Adhesion of viable bacteria

The number of viable bacteria adhering to the stainless steel

and titanium alloy implants was evaluated in a quantitative

adhesion assay. Results are expressed in colony forming units

per agar dilution plate (Figure 3), this measurement being a

direct indicator of the number of viable counts adhering to the

implants.

Figure 2. Total biomass (median and inter-quartile ranges) ofS. epidermidis adhering to either stainless steel and PDLLA-coated K-wires (n = 9;

left-hand panel) or titanium alloy and PDLLA-coated K-wires (n = 9; right-hand panel), as assessed by the binding of radiolabelled bacteria.

*, extremes (cases with values more than 3 box lengths from the upper or lower edge of the box); H, outliers (cases with values between 1.5 and 3 box

lengths from the upper or lower edge of the box).

Figure 3. Median viable counts ofS. epidermidis adhering to either stainless steel and PDLLA-coated K-wires (n = 10; groups S1S5 according to

Table 1; left-hand panel) or titanium alloy and PDLLA-coated K-wires (n = 10; groups T1T5 according to Table 1; right-hand panel). *, extremes

(cases with values more than 3 box lengths from the upper or lower edge of the box);H, outliers (cases with values between 1.5 and 3 box lengths from

the upper or lower edge of the box).


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The PDLLA-coated implants significantly reduced adhe-

sion of viable staphylococci compared with bare K-wires

made from either titanium or stainless steel alloy (P < 0.01).

Combination of PDLLA with either gentamicin or teico-

planin or both antibiotics on the implant together reduced

viable counts to almost undetectable levels (P < 0.05).

Although there were no significant differences between

gentamicin and teicoplanin (P > 0.05), there was a slight

tendency towards less bacterial growth with gentamicin-

containing coatings, and also between the titanium alloy and

stainless steel (Figure 3).

Discussion

Bacterial adhesion to biomaterials and the capability of many

microorganisms to form biofilms on foreign bodies are well-

established steps in the pathogenesis of implant-associatedinfections.1113 In a biofilm, bacteria are protected from the

host immune defence11,14 and exhibit a marked but reversible

increase in antibiotic resistance.11,15,16 Even high local drug

concentrations beyond the MBC for planktonic microorganisms

do not completely eradicate bacteria located in biofilms.15,17

As a result, prevention of bacterial colonization and bio-

film formation is an important consideration and may be sup-

ported by the use of antiseptic surface coatings. In the present

work, a new antibacterial surface coating using a biodegrad-

able drug-delivery system was studied.

The chosen PDLLA coating enables the possibility of

covering medical implants with a biodegradable and biocom-patible surface coating. This polymer is applied to implants by

a solvent casting technique that allows coating of alloys and

plastics with polished, irregular or porous surface materials.

Breakdown of the polylactic acids is based on hydrolytic

splitting of the polymer backbone over several months to

oligomers, and release of lactic acid, which is metabolized in

the citric acid cycle of the organism.1820

Incorporation of antibacterials into this coating to give a

local drug-delivery system ensures high concentrations around

the implant for long periods of time and the risks and side-

effects for the host organism are minimized compared with

systemic drug application.21

In this study, elution fluids were not changed, and an elu-

tion model that is broadly applied in the literature, especially

with antibiotic loaded bone cement, was used.22,23 Powdered

teicoplanin and gentamicin do not dissolve very well in the

volatile organic solvents used, and the final coating on the

implants consisted of antibiotic particles in the polymer. This

leads to an initial peak of release and high variability between

experiments, caused by fine drug particles located at, and

washed out of the polymer coating surface, a process that is

not well-defined. On the other hand, after the initial peak,

subsequent release is very predictable and comparable for all

samples, being primarily the result of diffusion of incorpor-

ated antibiotics from deeper levels of the coating.

In this study, we observed significant differences between

the results of adhesion studies depending on whether total or

viable bacteria were assessed. The coating increased the total

amount of attached microorganisms but at the same time

significantly reduced the number of viable bacteria even with-

out antibiotics, suggesting that the bare PDLLA coating has

bactericidal action against adhering S. epidermidis SE 183.

Since most of the bacterial adhesion studies only employ one

type of test, either counting the total number of attached

microorganisms2426 or counting only viable bacteria,2729

results from these studies may be misleading. Since genta-

micin and teicoplanin are integrated into the polymer as fine

particles, drugs located at the surface can be washed out to

leave small irregularities in the polymer. As one of the most

important features in primary bacterial adherence is the

topography texture,30,31 this may promote the bacterial adhe-

sion seen in this study. Apart from morphological features

of the polymer, physiochemical characteristics like surface

charge influence bacterial adhesion as well. In the present

study, teicoplanin increased the total number of adhering micro-

organisms compared with gentamicin. Gallardo-Moreno et al.32

and Wilcox et al.33,34 have previously described promotion of

bacterial adhesion to implant surfaces by subinhibitory concen-

trations of glycopeptide antibiotics.3234 Our results would sug-

gest that this effect may also occur at inhibitory concentrations

and highlight the importance of undertaking total as well as

viable bacterial counts.

PDLLA has excellent features with respect to implant coat-ing, with high mechanical stability,35 good osteoinductive

potential36 and excellent biocompatibility in vivo.36 The

material also shows good anti-thrombogenic characteris-

tics.37 In this study we have been able to demonstrate that the

antibacterials gentamicin and teicoplanin can be incorporated

into the polymer to give local drug-delivery systems that

reduce bacterial adhesion in vitro. We conclude that a com-

bination of the antibioticpolymer coating with other drugs to

create a multifunctional and custom-tailored surface is there-

fore possible and could offer new opportunities in the use of

established biomaterials.

Acknowledgements

We gratefully acknowledge the contributions of I. Kappstein

(Associate Professor of the Department of Hospital Epidemi-

ology, University Hospital, Technical University Munich,

Germany) for inspiring discussions, constructive micro-

biological advice and for reading the manuscript. We would

also like to thank V. Vatou and M. Chihaja for their assistance

in microbiological testing. Furthermore, we acknowledge the

contributions of D. Hall of the 1st Medical Clinic of the Tech-

nical University Munich for assistance in preparing the manu-


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script. This study was supported by a grant from the Bavarian

Research Cooperation for Biomaterials (FORBIOMAT).

The authors did not receive any payments or benefits from any

other research fund, foundation, educational institution or

other non-profit organization. No benefits in any form have

been received or will be received from a commercial party

related directly or indirectly to the subject of this article.

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Antibacterial Poly(D,L-Lactic Acid) Coating of Medical Implants