In vivo degradation of processed dermal sheep collagen evaluated ...

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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

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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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