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
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Biomaterials
1992. Vol. 13 No. 9
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of carbon
particles:
A. Brandwood
et a/.
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