mechanism of antibiotic release from poly(methyl methacrylate) bone cement

C&Gxzl”Muterials 4 (1989) 109-122

Mechanism of Antibiotic Release from Poly(methy1 methacrylate) Bone

Sandra Downes

Institute of Orthopaedics, Stanmore, Middlesex HA7 4LP, UK

&

Peter A. Maughan

Teesside Polytechnic, Middlesborough, Cleveland TSl 3BA, UK

(Received 25 August 1988; sent for revision 21 October 1988; accepted 5 January 1989)

ABSTRACT

Antibiotic loaded poly(methy1 methacrylate) (PMMA) bone cement is commonly used in joint replacement surgery. The mechanism of antibiotic release was examined using a series of in-vitro experiments. An initial rapid rate of release was followed by a slower rate ouer 40 days. The quantity of antibiotic release was related to the surface area of the cement sample. Transfer of antibiotic across thin membranes of plain PMMA was almost negligible. The method of mixing the PMMA greatly affected the quantity of antibiotic released, the porosity and the surface features, whilst having no significant effect on the diffusion coej‘icient and the water equilibrium coeficient. It was established that the mechanism of antibiotic release is diffusion from the surface layers of the cement as opposed to capillary action via pores in the cement or diffusion through the entire matrix of the PMMA as previously proposed.

INTRODUCT%ON

Poly(methy1 methacrylate) (PMMA) has been used to deliver chemo- therapeutic agents for various applications: antibiotics in orthopaedics,’ germicides in optics2,3 and anti-fungal agents in dentistry.4T5 Although the pattern of drug release from PMMA is similar in all cases, the mechanism of release is not fully understood.

The mechanism by which antibiotics are released from bone cement 109

Clinical Materials 0267-6605/89/$03.50 0 1989 Eisevier Science Publishers Ltd, England. Printed in Northern Ireland

110 Sandra Dowries, Peter A. Maughan

has been investigated by several authors and several mechanisms have been proposed. Medcraft and Gardner6 and Elson et al.’ stated that the antibiotic diffuses through the entire matrix of the cement, while Marks et ~1.~ proposed that it is released via holes and pores in the cement by capillary action. Wroblewski” and Hughes et al. “’ suggested that diffusion through the cement is unlikely and the drug is simply washed off the surface layers of the cement.

The purpose of this study was to establish the mechanism by which antibiotic is released from PMMA bone cement.

MATERIALS AND METHODS

In-vitro release studies

The release of Gentamicin (Kirby-Warrick Pharmaceuticals Ltd) from blocks of PMMA was examined in vitro.

Preparation of bone cement PMMA is composed of a liquid monomer component and a polymer component. The liquid component was poured into a plastic mixing bowl and then the powder added to it. The two components were mixed using a plastic spatula at a rate of five beats per second for approximately 45 s until a dough-like paste was formed. The cement was shaped on to blocks in a stainless steel mould with dimensions 2cmx1.5cmxlcm.

E&ion of Gentamicin from loaded-PPMA. Ten blocks of cement were used. Each block was placed in an individual beaker in 20 ml phosphate buffered saline (PBS), pH 7.4. The beakers were kept at a constant temperature of 37 “C in a thermostatically controlled water bath. Twice daily, the beakers were gently mixed, a small aliquot (1 ml) of the saline was removed and stored at -20 “C for Gentamicin assay; the remaining saline was removed and replaced with 20 ml fresh PBS. At the time of assay, the samples were quickly thawed and assayed for Gentamicin by radio-immunoassay (Rianen, New England Nuclear, Southampton, UK).

The effect of changing the surface area on the release of Gentamicin from PMMA. One 40 g pack of Palaces-R (Kirby-Warrick Phar- maceuticals Ltd) with Gentamicin was mixed and then shaped into four blocks having the same volumes but different surface areas. The blocks

Antibiotic release from PMMA bone cement 111

fl PMMA cement membrane

Fig. 1. Schematic diagram of the cement holder. A, B indicate respective chambers.

were placed in 100 ml PBS in individual beakers at 37 “C. The beakers were gently mixed and IO ~1 aliquots removed for Gentamicin radio- immunoassay after 24 h.

Experiments to measure the transport of Gentarnicin across various thicknesses of plain PMMA cement. In order to run these experiments it was necessary to design 10 cement holders that comprised two chambers separated by a membrane of plain PMMA cement (Fig. 1). The cement thickness varied between O-21 and 1.00 cm. In chamber A was placed 20 ml PBS spiked with O-5 ml Gentamicin (40 mg/ml), i.e. 1 mg Gentamicin in 1 ml saline. In chamber B was placed 20 ml PBS. The chambers were placed on a Rolamix and maintained at 37°C. The saline in chamber B was sampled daily for 7 days and then weekly for 5.5 months. The samples were stored at -20 “C and quickly thawed at the time of assay for Gentamicin.

cement samples prepared with different porosities and surface es

Preparation of bone cement samples : method of mixing ne hundred blocks of PMMA (Palaces-R) bone cement were pre-

ared using five different mixing techniques, with 20 blocks in each group. Monomer (20 ml) was added to each 40 g pack of polymer and the two components were mixed at room temperature using a plastic mixing bowl and spatula. The same batch was used throughout. The mixing techniques were as follows:

Slow mix: Fast mix:

Centrifuged:

Pressurised:

Control mix:

Hand mix 1 beat per second for 45 s. Hand mix 4 beats per second for 45 s. Hand mix 2 beats per second for 45 s, followed by centrifugation at 2500 g for 120 s. Hand mix 2 beats per second for 45 s, followed by thumb pressure for 60 s. Hand mix 2 beats per second for 45 s.


Gentamicin release. The cement was allowed to set in stainless steel moulds with dimensions 2 cm x I-5 cm X 1 cm. Nineteen blocks of each mix of cement were used; each block was immersed in 20 ml PBS in individual beakers and maintained at 37 “C. After 24 h the PBS was assayed for Gentamicin.

Samples for the measurement of difusion coejlicient and equilibrium water content. The cement was mixed using the above techniques. Before curing, the cement was spread into a sheet of aluminium foil supported by a flat sheet of glass. Six samples were prepared for each cement mix (thickness 0.1-0.42 cm and diameter of approximately 4 cm).

The disc samples of the cement were placed in individual beakers containing deionised/distilled water, placed in a thermostatically controlled water bath at 37 “C. At 24-h time intervals the samples were removed from the beakers, any excess water removed by patting the sample with filter paper (Whatman No. 1) and the samples weighed. This was repeated until there was no further increase in the mass (approximately 100 days).

A plot of MJA4, versus t/21 was obtained for each sample, where t is time in seconds and MJh4, is the mass water uptake. For early stages of diffusion where t is small, the slope of the graph was found and used to calculate the diffusion coefficient (D):”

D= slope2 X z

16

The equilibrium water content is an important property, as it is a measure of the total water uptake at equilibrium. The equilibrium value (Co) is expressed in g/cm3. The value was calculated using

M = 2AlCo

where A is the surface area, 1 the half thickness and M, the mass of the water.

Electron microscopy of the differently prepared blocks of cement. One block of each mix of cement was used to study both the ‘as-cast’ and fractured surfaces of the bone cement. A scanning electron microscope, ‘ISI Super’, with a 15 kV accelerating voltage was used to examine .and photograph the cement samples. The fracture surfaces were prepared by polishing, using progressively finer silicon carbide papers. The ‘as-cast’ and the fracture surfaces were sputter-coated with gold prior to examination in the microscope.


The photographs were all taken with the microscope tilt angle at 30”. The error of orientation was calculated to be less than 20% using a stage micrometer with lo-’ mm divisions and at a magnification x 1000.

RESULTS

In-vitro release of Gentamicin

The release of Gentamicin from loaded PMMA was investigated. found that the release of Gentamicin was very rapid in the early stages and then fell off to a low level after about eight days (Fig. 2). Very low levels (below 1 pg/ml) of Gentamicin could be detected in the elution fluid for up to 40 days. The total quantity of Gentamicin released was between 0.OlOOOg and 0*01319g, which is between 10 and 13.2% (average 11.49%) of the original Gentamicin content of the cement.

The relationship between the release of Gentamicin and the surface area of the cement

locks of cement were prepared with various surface areas but similar volumes and loaded with the same quantity of Gentamicin per unit

120

60

40

20

0 0.25 1 2 3 4 5 6 7 8 40

Time(days)

W Gentamicln •.n SD.

Fig. 2. Release of Gentamicin from PMMA for 40 days. Each point represents the mean of 10 Gentamicin results.


TABLE 1 Gentamicin Released from Four Blocks of PMMA Bone Cement with Different

Surface Areas.

Block 1 Block 2 Block 3 Block 4

Mass Cd Volume (cm’) Surface area (cm*) Total concentration of

Gentamicin released after 24 h (pg/ml)

Total amount of Gentamicin released after 24 h (mg)

Total amount of Gentamicin released unit per area (mg/cm’)

12.75 13.14 13.38 12.94 6 6 6 6

22 23 31 32

19.34 24.21 31.10 29.50

1.934 2.421 3.110 2.750

0.087 0.105 0.100 0.080

weight. The surface area was measured as the dimensional surface area; it was assumed that the surface roughness was the same for all blocks as they were mixed and moulded in the same way. More Gentamicin was released from the blocks with greater surface areas. The quantity of antibiotic released was directly related to the surface area of the cement. In four blocks with surface area ranging from 22 to 32 cm* the total quantity of Gentamicin released per unit area ranged from 0.080 to 0.105 mg/cm* (average 0.93 mg/cm’) (Table 1). The correlation coefficient was 0.934, making the significance level 0.02.

Release of Gentamicin across various thickness of plain PMMA bone cement

Only a very small percentage of the Gentamicin crossed the cement membrane from chamber A to chamber B (less than 0.0125% even after 5.5 months). There was no direct relationship between cement thickness and passage (Table 2). It is interesting to note that in chambers 1, 2 and 4, with respective thicknesses 1.00, 0.98 and 0.60 cm, no Gentamicin crossed the cement even after 5.5 months.

Reiease of Gentamicin from differently prepared blocks of cement

The control method was mixed according to the manufacturer’s instructions. The mean level of Gentamicin released at 24 h was 106.32 f 4.6 pg/ml. The centrifuged cement gave an average Gen- tamicin level of 106.45 f 6.7 yglml. The slowly mixed cement gave an average Gentamicin level of 105.40 f 4.6 pg/ml, whereas the fast mix

TAB

LE

2 R

elea

se

of

Gen

tam

icin

@

g/m

l) ac

ross

Pl

ain

PMM

A

Cem

ent.

Tim

e B

lock

no.

1 da

y 2

days

3

days

4

days

5

days

6

days

7

days

8

days

2

wee

ks

3 w

eeks

4

wee

ks

3 m

onth

s 4

mon

ths

5;

mon

ths

Gen

tam

icin

re

leas

ed

afte

r 4

wee

ks

(%)

0.00

0.

00

0.00

0.

00

0.00

0.

00

0.00

0.

00

0.00

0.

00

0.00

0.

00 h

0~00

00

0~00

00

0~00

001

0.00

0 0.

0008

0.

0016

0.

0014

0*

0003

0~

0040

0.

0027

0.00

0.

00

0.00

0.

00

o-00

0.

00

o-00

0.

00

0.00

o-

00

0.00

0.

00

0.00

0.

00

o-00

0.

00

0.00

0.

00

0.13

0.

00

0.00

0.

00

0.00

0.

42

0.61

o-

00

0.00

0.

00

0.73

1.

34

o-00

0.

00

o-00

3.

61

4.70

0.

00

0.00

0.

00

4.69

9.

20

0.00

0.

00

o-00

11

.31

26.2

1 0.

00

0.14

0.

00

14.0

9 29

.41

0.00

0.

31

0.00

16

.10

32.6

4 0.

00

5.10

0.

00

-b

35.1

0 0.

00

5.74

0.

00

- 34

.90

0.00

6.

14

0.00

35

.62

o-00

o-

00

0.18

0.

00

0.61

0.

95

4.15

11

.36

19.2

1 24

.10

26.7

1 34

.90

b

o-00

6.

15

15.1

4 0.

01

12.4

1 16

.17

0.34

18

.91

18.4

0 0.

56

24.1

7 20

.80

0.51

31

.64

29.7

0 0.

89

39.0

4 34

.41

P-41

41

.40

36.2

9 1.

90

44.1

6 35

.80

2.71

51

.01

41.4

2 4.

16

71.3

0 50

.74

6.24

80

.41

54.1

0 11

.40

110.

14

59.0

1 19

.37

120.

19

59.5

9 29

.18

123.

61

58.7

1

u N

umbe

rs

in p

aren

thes

es

indi

cate

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men

t th

ickn

esse

s (c

m).

b C

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ber

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ed

and

Gen

tam

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w

as

unab

le

to

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mea

sure

d ac

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tely

. Ex

perim

ent

was

di

scon

tinue

d.

116 Sandra Dowries, Peter A. Vaughan

??Gentamicln l3 S.D.

Slow Fast Pressurised Centrifuged Control

MIX

Fig. 3. Gentamicin released from blocks of PMMA.

gave an average value of 90.26 f 4.8 pg/ml. The lowest release was observed from the pressurised cement with a mean Gentamicin level of 62.15 f 4.6 pg/ml. These results are illustrated in Fig. 3. The release is similar for the control, slow-mix, fast-mix and centrifuged cement. The release observed from the pressurised cement is significantly reduced when compared to the other mixes.

Effect of method of mixing on the diffusion coefficient of PMMA

The diffusion coefficient of a material is a measure of the uptake of water by unit mass in unit time. It is characteristic of the matrix of the cement and unaffected by the number of holes in the material. In this experimental work, the diffusion coefficient (3.59-14.6 x lop8 cm2/s) and the equilibrium water content (0.0453-0.063 g/cm3) were calculated for the differently mixed cements. Although the release of Gentamicin from the cement varied considerably according to the method of mixing, the quantity of antibiotic released from differently prepared cements were’ not related to the diffusion coefficient or the equilibrium water content (Figs 4 and 5).

Porosity of the samples of differently mixed cement

Porosity is expressed as the number of pores per mm’. Figure 6 shows the porosities of the differently prepared samples of PMMA bone


Control

Centrifuged

.iz ??Do.xld?

E Ej SD.

E d

Pressurised

i I 1 0 Do.em2/rt xltl? ’ O 20

Fig. 4. Diffusion coefficients (Do) for the cement samples. Each bar represents the average value for six discs of cement.

cement. The area of cement examined was 0.01 mm2 using magnification x 100; the results given are the average of five counts of five different areas of specimen. The error of orientation was calculate to be 20%.

The highest porosity was observed in the fast-mix cement (average porosity 5640 pores per mm’) and the lowest porosity was observed in the centrifuged cement (average porosity 1344 pores per mm*). The

Control

i I I I I 0.00 0.02 0.04 0.06 0.08 gram/cm3

Fig. 5. Equilibrium water content (Co) for cement samples. Each bar represents the average value for six discs of cement.


Centrifuged

H Porosity ! ??S.D. /

Fast

Porosity

Fig. 6. Porosity of differently prepared blocks of PMMA. Porosity expressed as number of pores per mm’.

porosities of the slow-mix, pressurised and control cements were similar.

Electron microscopy studies

The scanning electron micrographs of both the ‘as-cast’ surface and the fracture surfaces of the differently mixed cements were examined. The structure was that of tightly packed beads with pores scattered throughout the matrix. The ‘as-cast’ surface of the pressurised cement appeared to have a much smoother surface; the polymer beads were not as apparent as in the other cement mixes (Fig. 7). It can be assumed that the total surface area of the pressurised cement is less than the other cements due to the smoother surface of the cured cement.

DISCUSSION

Initially, there was a rapid rate of antibiotic release followed by a very slow or even negligible release. The cumulative amount of Gentamicin released was higher than could be accounted for by diffusion alone; this may have been due to some Gentamicin washing off the surface of the cement. The total quantity of Gentamicin released was only IO-13% of


the original content, over 87% of the antibiotic remaining locked in tbe cement. In agreement with Wahligl’ who reported that at the end of five years only 3-8% of the total Gentamicin had been released. Bayston and Milner13 suggested that the release of antibiotic is continuous but at a rate too slow to be detected by elution and serial transfer tests. The lower detection limit of the radio-immunoassay employed in these experiments is 0.1 ,ug/ml; therefore, if a continuous release is occuring it is negligible. There appears to be a high concentration of Gentamicin in the cement that is not liberated until the inner matrix of the cement is exposed to the elution fluid, supporting release of antibiotic from the surface of the cement.

The quantity of Gentamicin released from blocks of PMMA cement was directly related to the surface areas of the blocks (Table 1). Blocks with greater surface area exhibit greater antibiotic release, again indicating the importance of the surface layer of the cement in antibiotic release.

The transfer of Gentamicin across a thin membrane of plain PMMA is very low (<O.O125V ) o even after 5.5 months, indicating that PMMA is not particularly permeable to Gentamicin. If the mechanism of release of Gentamicin from PMMA is simple diffusion, one would expect the rate of diffusion to be directly proportional to the cement thickness and that eventually a state of equilibrium would be reached with the same concentration in both sides of the chamber. Whilst this was not the case, there was some transfer of Gentamicin across some of the thinner discs; this was probably due to leakage via small channels and cracks in the cement.

Cement samples with different porosities were prepared using five mixing techniques. Porosity was measured and expressed as the number of pores per unit area. Fast mixing increased porosity and centifuging the cement reduced the porosity, in comparison to slow and control mixes of cement. If the mechanism of drug release is via holes and pores in the cement mantle, as suggested by Marks et aZ.,3 one would expect the greatest antibiotic release from the most porous cement and the lowest release from the least porous cement. There was no direct relationship between the quantity of Gentamicin released and the porosity of the cement. Therefore, we suggest that release of antibiotic from PMMA bone cement is not via the pores in the matrix.

Scanning electron micrographs were made in order to establish whether there were any differences in the microstructure of the cement samples. It was found that the ‘as-cast’ and fracture surfaces of the cement samples were similar for the control, fast-mix, slow-mix and centrifuged cements. The surface of the pressurised cement was much


smoother with less surface detail than the other cements, indicatin that the total surface area of the pressurised cement was lower tha other cement samples. As a significantly lower Gentamicin released was observed from the pressurised cement, this is direct evidence that t rate of Gentamicin release is related to the total surface area of the cement.

CONCLUSIONS

It would appear, therefore, that drugs delivered from PMMA do not arise from within the cement matrix. Antibiotic is not released by either simple diffusion or via holes in the cement. The release of antibiotics from PMMA is a surface phenomenon and the drug is released from the outer surface layer of the cement alone.

REFERENCES

1. Buchholz, H. W., Prophylactic use of antibiotic-loaded cement: Long term results. In Progress in Cemented Total Hip Surgery and Revision, ed. R. K. Marki. Proceedings of a symposium, 16 October 1982 Amsterdam. Elsevier Science Publishers, Amsterdam. Excerpta Medica (1982) 36-42.

2. Mote, E. M., Scholessler, J. P. & Hill, R. M., Lens incorporated germicides. J. Amer. Optic. Assoc., 40 (1969) 291-3.

3. Mote, E. M. & Hill, R. M., Lens incorporated germicides I. J. Amer. Optic. Assoc., 41 (1970) 260-l.

4. Addy, M., In vitro studies into the use of denture base and soft liner materials as carriers for drugs in the mouth. J. Oral Rehabil., 8 (1981) 131-62.

5. Addy, M. & Handley, R., The effects of the incorporation of chlorohexi- dine acetate on same physical properties of polymerised and plasticised acrylic. J. Oral Rehabil., 8 (1981) 155-63.

6. Medcraft, J. W. & Gardner, A. D. H., The use of an antibiotic bone cement combination as a different approach to the elimination of infection in total hip replacement. Med. Lab. Technol., 31 (1974) 347-53.

7. Elson, R. A., Jephcott, A. E., McGechie, D. B. & Verettas, Antibiotic loaded acrylic cement. J. Bone Joint Surg., 59B (1977) 200-5.”

8. Marks, K. E., Nelson, C. L. & Lammenschlager, E. P., Antibiotic impregnated acrylic bone cement. J. Bone Joint Surg., 58A (1976) 358-63.

9. Wroblewski, B. M., Leaching out from acrylic bone cement: Experimental evaluation. Clin. Orthopaedics, 124 (1977) 311-12.

10. Hughes, S., Robertson, S., Want, S., Darrell, J., Kennedy, M. & Dash C. H., Cefluroxime in bone. Royal Society Medical International Congress Symposium, Series 38, 1980, pp. 163-71.


11. Braden, M., The absorption of water by acrylic resins and other materials. J. Prosth. Dent., 14 (1964) 307-16.

12. Wahlig, H., Specific properties of Gentamicin-Palaces. Paper Presented at 39th Congress of Nordisk Ortopaedisk, Forening, Odense, Denmark, 1978.

13. Bayston, R. & Milner, R. D. G., The sustained release of antimicrobial drugs from bone cement. J. Bone Joint Surg., 64B (1982) 460-4.

mechanism of antibiotic release from poly(methyl methacrylate) bone cement

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