646

COMMUNICATION Phagocytosis of carbon particles by macrophages in vitro A, Br~d~ood, Centre for Biomedical Australia Particles

of known

cultures

of murine

particles some

were

Kathyn

University

size ranges

of carbon

maGr0phage.S.

not phagocytosed

of which

migrated

particles

per hour were

rounded,

detached

findings argued

R. Noble and K. ~~~~e~

Engineering,

medical

devices,

Keywords:

Cells

surfaces.

presented and some from

particles

were

presented

were

phagocytosed.

by aggregations

Mean

lysis. The implications

implanted

medical

circulation

up to 2.5

of particles

underwent

in the size range

devices

8-20 pm,

by macrophages

became of these

is discussed. released

to lodge

It is

from

and transported

but large enough

Larger

of macrophages,

rates of phagocytosis

with a large excess

to be phagocytosed

to the vascular

from the implant

Carbon particles,

surrounded

released

exists where

are small enough remote

to in vi&o

carbon

of up to 20 pm diameter

on to the particle observed.

and subsequently

beds of tissues

fibre-reinforGed

but became

for the fate of particulates

lymphatics

NSW 2033,

Particles

from the substrate

that a mechanism

of New South Wales, PO Box 1, Kensington,

to the

in capillary

site.

macrophages,

phagocytosis

Received 22 January 1992; accepted 29 January 1992

The release of particulate carbon has been demonstrated for a number of prosthetic devices manufactured from carbon materials. These include wear debris from carbon fibs-reinfo~ed composites used in articular prostheses and particulate debris released, as a result of fibre disintegration*, from carbon fibre ligament prostheses. Acute tissue response to carbon implants is typified by a mild, short-lived inflammato~ response’. There is no conclusive evidence of any carcinogenic effects of carbon particulate debris2. Nonetheless, there is little knowledge of long-term effects of such particulate debris. Published animal studies have shown that particulate carbon can be phagocytosed by macrophages and may be transported from the implant site to remote tissues. Particles implanted in the medullary cavity of rabbit femurs were later detected in lung, liver, spleen and kidney3. Particles injected intravenously were found deposited in lung tissue and in lymphatic ducts4. Deposition of carbon particles in lymph nodes has been reported following intra-a~icular injection of carbon particles in rats4, intramuscular implantation of both carbon fibres and particles in rats4’ 5 and experimental replacement of achilles tendon with a carbon fibre prosthesis in rabbits’. Correspondence

Bio~aterials.1992,

to Dr A. Brandwood.

Vol. 13 No. 9

Histological evidence from these animal studies shows, not surprisingly, that the smaller particles are removed by lymphatic drainage, whereas larger debris remain close to the implant site. However, we are not aware of any published attempt to quantitate the range of particle sizes which may be phagocytosed by macrophages. In the present study we have attempted to characterize the size range of carbon particles which may be phagocytosed by macrophages in vitro and also the rate of phagocytosis and the maximum number of particles phagocytosed by individual cells.

EXPERIMENTAL Carbon particles were prepared from carbon fibrereinforced pyrolytic carbon (CFRC) (Isocarb, Science Applications International Corporation, Irvine, Ca, USA) by grinding in a ring grinder and sieved into size ranges GO, 20-30, 30-38, 38-53, 53-75 and >75gm. Suspensions of particles (0.1 g/ml in phosphatebuffered saline, pH 7.3) were prepared and sterilized by autoclaving, Murine peritoneal macrophages were obtained following procedures described by Conrad’ and Meltzer*. Briefly, 4 d before harvest of macrophages was 0 1992 Bu~e~orth-Heinemann 0142-§612/92/090646-03

Ltd

Phagocytosis

of carbon

particles:

A. Brandwood

required, 6-wk-old, outbred mice were injected with 1 ml thioglycollate broth (Difco) into the peritoneal cavity. Cells were harvested by peritoneal lavage with 10 ml phosphate-buffered saline, pH 7.3, containing 10 units/ ml heparin, via a 20 gauge needle. The lavage was carried out only once for each animal: approximately 8 ml lavage medium being recovered each time. Cells were sedimented at 250g for 10 min at 4°C then resuspended in 10 ml Eagle’s minimum essential’medium (EMEM) with Earle’s salts and non-essential amino acids (CSL, 73322010), supplemented with 10% (v/v) heatinactivated (56°C 30 min) fetal calf serum and 10 units/ml sodium heparin. Centrifugation was repeated and cells were resuspended in 6.0 ml EMEM. Cell suspensions were plated out into 24-well tissue culture plates (1.0 ml suspension per well) and incubated at 37°C overnight. The medium was then exchanged. This process removed any non-adherent cells present in the culture. Three separate experiments were carried out. In the first, suspensions (0.05 ml) of particles of different size ranges were added to individual cultures of macrophages. For the 8,~m may bypass the capillary bed via arteriovenous shunts. However, the importance of this route is uncertain’2-‘6. In summary, we have shown that CFRC particles of sizes up to 20 pm may be phagocytosed by macrophages in vitro. In vivo such particles may be deposited in lymph nodes and subsequently drain into the vascular circulation, Thus a mechanism exists where particles in the size range 8-20pm, released from medical devices, are small enough to be phagocytosed by macrophages and transported to the lymphatics and subsequently to the vascular ci~ulation but large enough to lodge in capillary beds of tissues remote from the implant site.

11

REFERENCES

12

Rushton, N., Dandy, D. J. and Naylor, C.P.E., The clinical, arthrnscopic and histological findings after replacement of the anterior cruciate ligament with carbon fibre, I. Bone ]t Surg. 1983,6SB, 308 Christel, P.S., The applications of carbon fiber-reinforced carbon composites (CFRC) in orthopedic surgery, CRC Crit. Rev. Biocomp. 1986, 2,184~218 Neugebauer, R., Helbing, G., Walter, D., Mohr, W. and Gistinger, G., The body reaction to carbon fibre particles implanted into the medullary space of rabbits, Biomaterials 1981, 2,182-184 Wolter, D., Biocompatibility of carbon fibre and carbon fibre microparticles, Aktuel. P&l. Chir. Orthop. 1983, 26,28-36 Tayton, K., Phillips, G. and Ralis, Z., Long term effects of carbon fibre on soft tissues, J. Bone Jt Surg. 1982, 64B, 112-114 Forster, I.W., Ralis, Z.A., McKibbin, B. and Jenkins, D.H.R., Biological reaction to carbon fibre implants: the

Biomaterials

1992. Vol. 13 No. 9

7

8

9

10

13

14

15

16

17

of carbon

particles:

A. Brandwood

et a/.

formation and structure of a carbon-induced ‘neotendon’, Clin. Urthop. Rel. Res. 1978, 131, 299-307 Conrad, R.E., Induction and collection of peritoneal exudate macrophages, in Manual of Macrophage Methodology: collection, characterisation and function (Eds H.B. Herscowitz, H.T. Holden, J.A. Bellanti and A. Ghaffar], Marcel Dekker, NY, USA, 1981, pp 5-11 Meltzer, MS., Peritoneal mononuclear phagocytes from small animals, in Methods for Studying Mononuclear ~hagocytes (Ed. D-0. Adams), Academic Press, London, UK, 1981, pp 63-67 Rae, T., A study on the effects of particulate metals of orthopaedic interest on murine macrophages in vitro, J. Bone Jt Surg. 1975,57B, 444-450 Nelson, D.S., Macropbages and Immunity, NorthHolland Publishing Co., Amsterdam, The Netherlands, 1969, pp 109-111 Madden, R.E., Paparo, M.S. and Schwartz, M.D., Limiting vascular diameters in various organs as determined by microspheres, Arch. Surg. 1968, 96, 130-137 Maxwell, L.C., Shepherd, A.P. and McMahan, C.A., Microsphere passage through intestinal circulation: via shunts or capillaries? Am. J. Physiol. 1985, 248, H217-H224 Ring, G.C., Blum, A.S., Kurbatov, T., Moss, W.G. and Smith, W., Size of microspheres passing through pulmonary circuit in the dog, Am. J. Physiol. 1961, 200, 1191-1196 Fan, F., Schuessler, G.B., Chen, R.Y.Z. and Chien, S., Dete~inations of blood flow and shunting of 9- and l&mm spheres in regional beds, Am. J. Physiol. 9979, 237, H25-H33 Maxwell, L.C., Shepherd, A.P., Riedel, G.L. and Morris, M.D., Effect of microsphere size on apparent intramural distribution of intestinal blood flow, Am. J. Physiol. 1981, 2417, H408-H414 Gelman, S., Granger, D.N., Fowler, K. and Smith, L.R., Clearance of g-mm spheres and rubidium in the intestinal circulation, Am. J. Fhysiol. 1984, 247, G13-G18 Sokal, R.F. and Rohlf, F. J., Biometry, 2nd Edn, Freeman, San Francisco, CA, USA

Phagocytosis of carbon particles by macrophages in vitro.

Particles of known size ranges of carbon fibre-reinforced carbon were presented to in vitro cultures of murine macrophages. Particles of up to 20 micr...
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