biodegradation, transmission electron microscopy. Various collagen-based ... electron microscope operated at 40 kV. ..... Figure 6 Implantation interval: IO d.
In vivo degradationof processed dermal sheep collagenevaluatedwith transmissionelectronmicroscopy P.B.van Wachem,M.J.A.van Luyn and P. Nieuwenhuis Department
of Histology
9 7 13 EZ Groningen,
and
Cell
Biology,
Section
Biomaterials
Research,
University
of Gromngen,
Oosterwgel
69/Z,
The Netherlands
H.K.Koerten Laboratory
of Electron
Microscopy,
University
of Leiden,
Rijnsburgerweg
10, 2333
AA
Leiden,
The Netherlands
L. OldeDamink,H. Ten Hoopenand J. Feijen Depaflment Presented
of Chemical
Technology,
at Biointeractions
‘90,
Twente
Oxford,
University,
UK
2 l-23
PO Box 217,
August
7500
AE Enschede,
The Netherlands
1990
The in viva degradation of hexamethylenediisocyanate-tanned dermal sheep collagen was studied with transmission electron microscopy. Discs of hexamethylenediisocyanate-tanned dermal sheep collagen were subcutaneously distinguished.
implanted
in rats. Both an intra- and an extracellular
In addition to normal components
such as locally deviant neutrophil multinucleate phosphate
morphology,
infiltration
giant cells formed from different depositions,
were
observed.
hexamethylenediisocyanate-tanned
of basophil-like
cell types, aluminium
Foreign body multinucleate
dermal
route of degradation
of a typical foreign body reaction,
sheep
collagen
after
remarkable
cells, indications
silicate
could be
phenomena,
of foreign body
accumulations
and calcium
giant cells intracellularly internalization.
degraded
Both internalized
and
cellularly enveloped hexamethylenediisocyanate-tanned dermal sheep collagen degraded by the detachment of fibrils. Another extracellular route of degradation was characterized by calcium phosphate depositions in large bundles of hexamethylenediisocyanate-tanned dermal sheep collagen. From 6 wk. the hexamethylenediisocyanate-tanned dermal sheep collagen implant was replaced by rat connective tissue, which
was
subsequently
multinucleated phenomena
were
emphasize
also
degraded.
giant cells containing related
to the specific
the need for detailed
Keywords:
Collagen,
Various collagen-based in the biomedical
and tissue
However,
these
events
is needed
between
extensively
In this
of
the
study,
cyanate-tanned
dermal
subcutaneously
implanted
such
function
we
sheep
used
collagen
of
Butterworth-Heinemann
used and suggest level of newly
foreign
still persisted. cytotoxicity.
developed
body These They
biomaterials
microscopy
bio-
light micro-
understanding
of and
collagen-based
hexamethylenediiso(HDSC),
which
was
in rats. The material is successfully
Correspondence to Dr P.B. van Wachem @ 1991
electron
described4-6.
concern
for a better
of basophil-like
accumulations
of the material
have found applications
predominantly
presence
at the ultrastructural
In our opinion, more detailed knowledge
improvement
biomaterials.
been
the
applications.
transmission
Interactions
have
reports
scopy evaluations. further
biomaterials
fieldlm3.
materials
the cellular
biodegradation,
15 wk.
nature
evaluation
before they can be used for medical
After
aluminium/silicon-crystalline
used as a biological wound both after intramuscular after
intracutaneous
with
US Pharmacopeia
evaluation
injections XXI
were
It was tested in rabbits
observed
of small
of extracts9
(1985).
of the intramuscular
very slight toxic reaction. material
dressing’. implantation
and
The light microscopy
implantation
No adverse in the
pieces’
in accordance test showed
reactions
a
related to the
intracutaneous
injection
test. Referring
to the very slight toxic reaction
the intramuscular
implantation
study to evaluate
the degradation
transmission
electron
test,
microscopy
observed
in
it was the aim of this
of HDSC
in detail
using
(TEM).
Ltd. 0142-9612/91/020215-09 Biomatenals
199 1, Vol
12 March
215
Biodegradation of dermal sheep collagen: P.B. van Wachem et ol.
MATERIALS
AND
METHODS
Materials Hexamethylenediisocyanate(HMDIC)-tanned collagen
(batch
Nos.
335,
339
dermal
and
392),
sheep
subsequently
referred to as HDSC, was obtained from the Zuid Nederlandse Zeemlederfabriek, was
Oosterhout,
processed
immersed
from
in a lime-sodium
the epidermis then
machine
to obtain
tanned
split with
with
Germany). from
the
enzymes7.
band-knife
layer.
This
splitting
layer was
obtained
discs
then
from
of 8 mm were
of the
varied
of
The
Bayer,
punched
from
15
to
by gamma irradiation (2.5 Mrad,
Ede, The Netherlands).
Implantations.
A0
rats of approximately
were
used. The rats were
were
shaved
midline
depilated,
for removal
proteolytic
a diameter
weight
The material
was
solution
an industrial
30 mg. Discs were sterilized Gammaster,
which
(Desmodur@
Discs with The
with
dermal
HMDIC
HDSC.
skin,
sulphide
and purified
skin was
The Netherlands.
sheep
ether
3 month
anaesthesized,
and their skins disinfected
incisions
were
made.
with
of age
Si
their backs
I
ethanol.
Subcutaneous
Two
pockets
were
made with surgical scissors at the right and left sides of each incision.
Four
pockets
HDSC
discs
at a distance
Implants
per rat were
of about
implanted
in the
1 cm from the incisions.
with surrounding
tissue were harvested
2, 3, 5, 7 or 10 d and at every following
week
at 1,
from
2 to
15wk.
were
HDSC
fixed
phosphate
(non-implanted)
in 2%
(v/v)
buffered
and
HDSC
glutaraldehyde
saline (PBS).
(GA)
Specimens
implants in 0.1
were
mol
cut into
small blocks (2 X 2 X 2 mm) after at least 24 h of fixation at 4°C. Blocks dehydrated
were
post-fixed
in graded
alcohols
Semithin
sections
blue. Evaluation light
further
sections
were
for
microscope
X-ray
PBS,
with toluidine
For
which
so as to prevent
from the osmium the specimens. stained (TN) 400
signals
ultrathin
more difficult
7, discs, or what was left of them,
used to detect for
X-ray
geometry
with
a spot
of the instrument
signals
trace elements
within
were
microanalyser
scanning and transmission
at 80 kV and
of X-ray
sections,
uranyl acetate,
either unstained analysed
attached of
to a Philips
100
nm.
has been described
or
on a Tracer
electron microscope size
EM
operated
The
system
previously”.
Microscopic
HDSC
HDSC (non-implanted) and larger collagen the
typical
216
evaluation
original fibril structure, bundles
connective
Biomaterials
and a supple adhered
tissue.
199 1, Vol 12 March
The
intervals.
the was
From week
were sometimes
(Figure
la)
structure.
Upon
tothesurrounding HDSC
discs
were
Transversely
with
hard to
fibrils
elastin-like
sheet-like
lining collagen
in the
la). These particles contain aluminium X-ray
substances, microanalysis
were
crystalline particles,
(Figure
with
Ia).
lost the
of blood vessels
mainly
Si, observed
had
substances
show
(Figure
which
and remnants
elastin-like
cut bundles
collagen
substances,
observed. Some electron-dense
typical
structure
Localizing
mainly consists of a matrix of smaller
bundles.
gelatinous
were
also
present
Al and silicon
(Figure
16). and are
to as AI/Si-crystals. after
occurred.
morphnucleated
HDSC immediately
been
(TEM)
construction
Furthermore,
reaction
has a fibrous
subcutaneous
evaluation
Right
RESULTS
implantation,
after longer implantation
had
find.
referred
Macroscopic
discs
decreased.
and lead
had been implanted
from
Carbon-layered
only with
2000
with
capsule
days,
ln situ
by a rather thick fibrotic capsule. Thereafter, of the fibrotic
within
at 40 kV.
was
the interaction
the first month.
surrounded thickness
that,
during
with
10 d, 4 and 12 wk. Tissue blocks were fixed with GA but not with 0~0~
by palpation
showed
parts of blocks TEM,
easily localized inspection
with a Philips EM 201 operated
Figure 1 (a) Non-implanted HDSC with transversely cut HDSC bundles f.S) and elastin (EJ-like substances with sheet-like AI/Sicrystals (arrow) (original magnification X 17 000). (b) Spectrogram of crystals with aluminium (Al) and silicon (Si). The elements Cl, lJ and Co were derived from the preparative procedure.
both a
interactions
uranyl acetate
microanalysis
in specimens
in
b
in Epon 812.
those
TEM. with
were examined
electron
trace elements
of selecting
processing
Single-spot
0~0~
(1 pm) provided
of cellular
cut and stained
citrate. The sections transmission
sections
overview
HDSC as well as a means worth
1%
were cut and stained
of semithin
microscopical
with
and embedded
20 4m
KS.
PK?a Microscopy.
small
implantation After
neutrophils azurophilic
the
usual
1 d, dilated and
granules
were
tissue
Sometimes
either cell type had a deviant
surrounding
in detail for neutrophils
healing
vessels,
macrophages
connective
will be described
immediately
wound
blood
with
observed
polytheir in the
the HDSC disc.
morphology, below.
which
Biodegradation
Figure
2
Invading
~4685). HDSC
neutrophils
(c and d) Deviant which
had
Within
been
implanted
were
by the condensation organelles, intact
cytoplasm (Figures
of the
or, (2) locally
nucleus,
whilst
pronounced found,
deviant
but appeared After
by pseudopod These
nucleus
had
an
formation found
pattern
as part
pattern,
with
of
a still in the
developed
observed
1 or
and macrophages but they were
the implant.
morphology
After
and
shown
most 10 d
could still be as
swollen
tissue.
collagen:
interval:
mltochondria
P.B. van Wachem
2 d (orIginal
magnifrcatron
(M) and lipid formations
(L) in
in the number
collagen
of young fibroblasts
and
Fibroblasts
were
in the surrounding
identified
by their
connective
open
nucleus and by an abundance
of rough endoplasmic
(RER).
also
Macrophages
basophil-like cells,
were
and
granules, present.
further Mitotic
accumulation
were
many
blasts
referred figures,
3a and b) and extensive
cells showed
mitochondria. (FBM-G)
characteristics day
5 to day
of the different
were
and internalization
was observed. Furthermore,
Cells often
foreign
cells had developed. of different
cells, which will be described formation
fatty
young fibroblasts
cells and macrophages
by macrophages
giant
of cell fusion
intra- and extracellular
found at the edge of HDSC. Surrounding swollen
reticulum containing
observed.
basophil-like
of HDSC bundles
face-type
to as basophil-like signs
After 5 d, the first signs of ingrowing and infiltrating
multinucleate
et al.
X 15 360J.
of new
From encapsulated
(d)
sheep
(SJ (b). Implantation
by an increase
formation
contained
less intense.
2 and 3 d, HDSC discs became
(c) X3226
bundles
,n the cytoplasm.
(Figures
characterized
were
tissue, within
of a
2a and
and degeneration
phenomena
neutrophil
magnification
and organelles
HDSC
(0) of the glycogen
cells were
(or apoptosis),
in both neutrophils connective
(a) or between
and occasionally
and fatty accumulations
in the neutrophils
of implantation
(Figures
neutrophils
in a deviant
2c and d). These
2 d after implantation in the surrounding
The
the glycogen
disintegrated
10 d (d) (original
(1) in a normal
cell death
(F) network disintegration
disc, first formation
of glycogen.
in two ways:
of a programmed
with
occurred
indicated
and by concentrations
a fibrin
some macrophages observed.
morphology,
degenerating
HDSC
and cell infiltration
b). Primarily neutrophils, a lymphocyte
within
of neutrophils
for 5 d (c) or
the implanted
fibrin network
activated
entrapped
morphology
of dermal
body
FBM-G
types of precursor
in detail for the 10 d implant. 10,
infiltration,
types of FBM-G
Blomaterials
ingrowth
and
cells continued.
199 1, Vo’ol 12 March
217
Biodegradation of dermal sheep collagen: P.8. van Wachem et al.
Figure 3 Cell fusion in the connective tissue surrounding the HDSC implant after 3d of implantation (a) and detail shown (61 (original magRifi~ation (a] x 5 185 and {bj X23 2901.
By day 10, the great variety in cellular interactions compared with other implantation periods was remarkable: the inner interface showed complete ingrowth; the empty central part had a fibrin network, few neutrophits and locally extensive crystalline deposits in HDSC bundles, whereas at the outer interface high numbers of infiltrated neutrophits, sometimes locally showing deviant morphology as described previously, were present. The inner interface with complete ingrowth contained different ceil types, small bundles of newly formed rat collagen constructed of small calibre fibres and blood vessels (Figures 4a and b). At this interface, ingrowing macrophages, fibroblasts, basophil-like cells and a few capillary buds were present. Amongst the different cell types many FBM-G cells were found especially in the proximity of the fibrous capsule. These FBM-G cells had characteristics of either macrophages, fibroblasts or basophils. FBM-G cells surrounding and internalizing small parts of sometimes gelatinous HDSC bundles were always either
218
Biornaterials 199 1, Vat 12 March
Figure 4 [a and b) Different cell types. e.g. FBM-G cells /Gj. newly formed rat collagen (Rj and blood vessels IB) in the tiDSC ISI implant after 10 d ~origi~al mag~i~caiion {a) X3226 and lb) X4685j
macrophage-like (Figure !?a) or fibroblast-like (Figure 56) FBM-G cells. The macrophage-like FBM-Gcelts had azurophitic granules and usually many swollen mitochondria and myelin bodies. The fibrobtast-like FBM-G cells were characterized by their abundance of RER and the absence of azurophitic granules. Basophit-like FBM-G cells contained round or semiround granules and/or large muttiglobutar granules (Figure 64 ’ ‘-13. Within the round or semiround granules, sometimes membranous structures orwhorls, or inclusionsof glycogen, or densely packed particles (Figures 66 and c) were present. The large multiglobular granules sometimes contained fine parallel membranous (Figures 6d and e) or scroll-like structures (Figure 6f). Accumulations of AVSi crystals and
Biodegradarron of dermal sheep collagen: P B van Wachem et a/.
occasionally observed free in the cytoplasm (Figure 96) or within mitochondria of different types of cells. During these time intervals, detachment of fibrils was observed at the interface of both surrounded and internalized parts of HDSC. The presence of gelatinous substances in HDSC did not increase with time. From about 6 wk HDSC was replaced by rat tissue consisting of newly formed smaller and larger collagen bundles, a well-developed vasculature, FBM-G cells (which were sometimes degenerating), and some fibroblasts, eosinophils and lymphocytes. Newly formed rat collagen contained, especially in the larger bundles, membranous structures (remnants of degenerated cells) entrapped among the fibres (Figure 10). Such membranous structures were not present amongst collagen bundles of the surrounding connective tissue. Both de nova formation and degradation of rat collagen were observed. Degradation occurred by detachment of single fibres, followed by phagocytosis. as well as by obtaining gelatinous appearances. Extracellular Ca/P deposits were not found anymore. Blood vessels within the implant contained numerous pinocytotic vesicles in the endothelium and sometimes membranous structures in the lumen. With time, bundles of rat collagen with entrapped membranous structures appeared looser, interspersed with now somewhat smaller, mainly basophil-like, FBM-G cells (Figure 1 I). After about 10 wk, most basophil-like cells had almost normal sizes. Furthermore, locally degenerating fibroblasts and blood vessels next to fibroblasts and blood vessels with normal morphology were observed. After 15 wk, tissue with difficulty identified during explanation contained few rat collagen bundles not yet similar to collagen bundles in loose connective tissue, interspersed with mainly basophil-like FBM-G cells containing AL&i-crystalline accumulations in the granules,
/
DlSCUSSlON t ] I/
Figure 5 lmptanrarion tnferval: 70 d (a) Macrophage-like FBM-G cell surrounding HDSC (S) (original magnification X4685). (b) Fibroblast-like FBM-G cell mternalizmg HDSC. The fibroblast-like FBM-G cell contams an abundance of RER (arrows.- plasma membrane) (original magmfication x2 7 043).
elastin-like substances were particularly present in the granules of basophil-like FBM-G cells (Figure 6s). The empty central part of the 10 d implant contained scattered, sometimes rather extensive, deposits of electrondense needle-like crystals. These crystals were also observed in the 2. 3 and 4 wk implants. Both large bundles of uninternalized HDSC (Figures 7a andb) and small internalized parts of HDSC showed these crystals, in sharp contrast to newly formed rat collagen. X-ray microanalysis showed the presence of calcium (Ca) and phosphorous (P) in these crystals (Figure 8). The ratio between the Ca and P peaks in these deposits was 1.63 k 0.09, indicating the presence of hydroxyapatite. In the 3 and 4 wk implants, some of these deposits showed a clear central part containing only contours of fibrils but no trace elements (Figure 9a). From 10 d up to 6 wk. small deposits of needle-like crystals were
The in viva degradation of HDSC was studied. HDSC was degraded by both an intra- and an extracellular route and gradually replaced by rat tissue, which was thereafter also degraded. In addition to normal components of a typical foreign body reaction, remarkable phenomena, such as infiltrating neutrophils locally showing deviant morphologies, indications of FBM-G cells derived from different cell types, accumulations of AI/Si crystals and depositions of Ca/P crystals were observed. After 15 wk, the presence of FBM-G cells with Al/S crystals persisted. HDSC was degraded intracellularly after internalization of small parts by macrophage- and fibroblast-like FBM-G cells. One route of extracellular degradation occurred at the interface of HDSC and the surrounding cells. After some time, both internalized and surrounded HDSC degraded by detachment of fibrils, followed by phagocytosis, but not by obtaining gelatinous appearances. Tanning of HDSC may be a reason for detachment of fibrils taking some time and for not showing gelatin formation. Detachment of fibrils and phagocytosis have been reported before14, 15. Bat connective tissue replacing HDSC also degraded, possibly as part of a normal tissue-remodelling process. Detachment of fibrils from this tissue had already occurred, whilst at other sites collagen was still being formed de nova. Moreover, rat connective tissue obtained getatinous morphologies during degradation. However, Ca/P-crystalline depositions were not ob-
Biomaterials
199 7, Vol 12 March
219
3jodegradation of dermal sheep collagen: P.B. van Wachem et al.
Figure 6 Implantation interval: IO d. (a) Basophil-like FBM-G ceils containing round or semiround granules and/or large multiglobular granules (armws) (original magnification x 7 142J. (6) Granule with included glycogen (arrows) (original magnification X29 184. (c) Granules with densely-packed particles [arrows] (original magnification x ?5 360). (e) Detail of multiglobular granule (d) with fine parallel membranous structures farrows) (original magnification X48 450}. (f) Granule with scroll-like structures ~original magni~cation X48 450). (gj Accumulation of AILSi crystals, which has partly disappeared due to the preparative procedure {original magnification X 7 142).
served within the
case
another
this tissue,
of deposition
but only in HDSC; in large
route of extracellular
HDSC
this process
bundles,
degradation.
in
reflects
The peak ratio of
1.63 indicates that the deposits consist of hydroxyapatite’*. (Ca,*(PO~)~(OH)~).
The deposits
HDSC
changed
dense
to partially
contours Ca/P
their morphology
of the
crystals
composition. is associated
with
220
Biomaterials
at best
present,
by a substance
fixed
heart
valves)
199 I, Vol 72 March
i.e. the change central
parts
concept’*,
from and
HMDIC
tanning
completely
electron-dense
its proposed
fibres,
degradation
not
the of
show
explanation
evidence
Enzymes
to cleared form
a new
of
may finally
intra-
or
tissue
connective extracellular
play an essential
role in
of all types of collagen20-2’.
HDSC was tanned of investigators
and is cause
did
may also
of morphologies,
lg. In contrast to HDSC and rat connective
degradation.
of unknown
the major
that
tissue
bioprostheses
collagen
shows
only the deposited
or formaldehyde”,
study
which had replaced it, collagen of the surrounding
In general, the process of calcification well-cross-linked
This
result in calcification. The observed sequence
electron-
or phagocytosed
of collagen-based
being glutaraldehyde
(e.g. for implanted
Since were
solubilized
was replaced
Calcification
has been reported’6-‘8. cross-linker
fibrils
possibly
in internalized
from completely
electron-dense. original
were
and the collagen
and those
rg
failure.
reported
with HMDIC.
Only one other group
on the use of collagen
tanned
with
HMDIC4*22. These researchers did not light microscopically observe any morphological deviations using HMDIC-tanned burn
dressings
concluded
and
collagen
that toxicologically
sponges, MDIC
from
is a quite
which
they
acceptable
Biodegradatron of dermal sheep collagen: P.B van Wachem et a/
Ca
Ffgure 8 Spectrogram of needle-bke crystal on HDSC bundle showing phosphorus (P) and calcium (Ca). The elements U and Cu ware derived from the preparative procedure. The element Fe was present m the specimen nearby the crystalline deposit.
Figure 7 Electron-dense deposits of needle-like crystals present in HDSC bundles after 2 wk of implantation (original magnification (a) x 7 142 and (b) x 15 360)
tanning
agent.
suggest
cytotoxicity.
been
In contrast,
shown
(DAH),
which
to have cytotoxic
wound or more
of neutrophils,
healing3’, specific
morphology
implant.
that have
for cytotoxicity
cell-biomaterial cell-collagen
of neutrophils and by day
This clearly
product
of HMDIC,
may leak from HDSC was also
which
interactions
This devrant
soon after implant-
connective
tissue
still observed
primary
in normal
in genera131m3”
interactions4-6.
10 was
indicates
is the deviant
is not described
was observed
ation in both the surrounding the implant,
made
effects27-29.
One of the indications morphology
observations
effects of diisocyanates
and the hydrolysis
reported23-26
diaminohexane
we
Cytotoxic
cytotoxic
and within within effects
the of
HDSC. We define primary cytotoxic effects as effects resulting from direct releaseof contrast
to
cytotoxic
secondary
cytotoxic substances
substances
cytotoxic
effects
as a consequence
(possibly with
DAH) in
release
of
of HDSC degradation
Figure 9 (a) Daposits of Ca/P crystals with cleared central parts containing the contours of fib& but no trace elements 4 wk aver jmplantation (original magmfication x 13 360). (b) Ca/P crystals are present rn the cytoplasm of a FBM-G cell 5 wk after implantation (original magmfrcatmn X29 1841
%lomater~als 199 1. Vol 12 March
221
Biodegradation of dermal sheep collagen: P.B. van Wachem et al
Figure 10 Membranous structures, probably from degenerated cells, entrapped within a bundle of newly formed rat collagen (original magnification x29 184).
Figure 11 Rather loose rat collagen interspersed with smaller basophillike FBM-G cells. containing AI/.%-crystalline accumulations (original magnification X 7905).
by cells. Primary cytotoxicity can be removed by extensive washing of HDSC, preliminary results indicated that a washed HDSC implant did not show deviant morphology of neutrophils shortly after implantation. In contrast, secondary cytotoxic effects cannot be prevented by washing, as detected by us in an in vitro biocompatibility test34. In our study it is difficult to discriminate between primary and secondary cytotoxicity, with one changing gradually into the other. Apart from deviant neutrophil morphology, cytotoxicity may also be reflected by the extensive presence of both intraand extracellular lipid35, of cells with swollen mitochondria and of the high number of infiltrating basophil-like cells and basophil-like FBM-G cells. We characterized basophil-like cells by the morphology of the granules, which are typical for basophils, or cells of basophil origin, but not for macrophages”, “. Basophiloriginating cells are normally involved in inflammatory
222
Biomaterials
1991, Vol 12 March
responses related with infections and allergies; they have phagocytic capacity and can pinocytose”-‘3. The chemoattractant responsible for enhanced presence of basophillike cells is as yet unknown, although AI/S crystals may be candidates, since the FBM-G cells formed from basophil-like cells accumulated them. Formation of FBM-G cells from basophil-like cells has not been described before36-40. Basophil-like FBM-G cells sometimes contained both rather active and inactive nuclei which validates the hypothesis of formation by fusion. We cannot exclude the possibility of the multiglobular granules in basophil-like FBM-G cells consisting only of phagocytosed degenerated material, although this is unlikely since parallel membranous or scroll-like structures are not expected within the granule. Therefore, apart from granule morphology, other techniques have to provide further proof of the origin of basophil-like (FBM-G) cells and the possibility of extension of the current theory on FBM-G cells36-40. The formation of FBM-G cells from macrophages has often been reported in cell-biomaterial interactions. Current theory on FBM-G cells says that they are formed from macrophages by fusion36-40, although formation by nuclear division is not excluded. Also in this study, macrophages and macrophage-like FBM-G cells were observed. The attracting signal for macrophages and macrophage-like FBM-G cells may be the HDSC collagen itself. The development of fibroblast-like FBM-Gcells,whichwediscriminated from the macrophage-like FBM-G cells bytheir abundance of RER and the absence of small azurophilic granules, has been suggested previously37. Both macrophage-and fibroblast-like FBM-G ceils were possibly formed by fusion of cells, since, in some cases, cells simultaneously containing both rather active and inactive nuclei were observed. The presence of AVSi crystals is difficult to explain. Part of these structures was already present in HDSC before implantation and may have been introduced during some step in the fabrication procedure. But this cannot explain the extent of AI/Si crystals observed. So we believe that they were in part newly formed and further accumulated during degradation4’. Crystallization may occur since both silicon and aluminium are at hand. Si is an essential trace element present in all kinds of body fluids and connective tissues and is required for collagen biosynthesis42-45. Therefore, HDSC may function as a possible source of Si. Blood is the possible source of A146.47.Si and Al are known to readily combine and the formation of aluminium silicates at physiological pH has been described46. The presence of aluminium silicates has been reported for brain tissue and related with Alzheimer’s disease45-47. Detailed knowledge on collagen degradation was obtained from this study, which may in general be of use for a better understanding and further improvement of collagenbased biomaterials. Degradation of HDSC was compared with degradation of rat collagen from the replaced HDSC and with collagen of the fibrous capsule. Remarkable phenomena observed during HDSC degradation are related to this specific material and suggest cytotoxicity. Cytotoxicity may occur as a result of tanning. Tanning agents will always be released in some way, which remains a point of concern. Primary cytotoxic substances of HDSC may be removed by extensive washing, but secondary cytotoxic substances not. However, cytotoxic effects have also been reported for certain clinically applied collagen-based bioprostheses. For example, porcine heart valves leak toxic substances (GA), but it is claimed that this improves their function by prevention of cell ingrowth and therefore degradation48.
Biodegradarion
It is concluded that this study emphasizes the need for detailed evaluation at the ultrastructural level of newly developed biomaterials as signs of cytotoxicity can be detected to advantage using this observation method.
22
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