In vivo behaviour of a biodegradable poly(trimethylene carbonate) barrier membrane: a histological study in rats.

Abstract

The aim of the present study was to evaluate the response of surrounding tissues to newly developed poly(trimethylene carbonate) (PTMC) membranes. Furthermore, the tissue formation beneath and the space maintaining properties of the PTMC membrane were evaluated. Results were compared with a collagen membrane (Geistlich BioGide), which served as control. Single-sided standardized 5.0 mm circular bicortical defects were created in the mandibular angle of rats. Defects were covered with either the PTMC membrane or a collagen membrane. After 2, 4 and 12 weeks rats were sacrificed and histology was performed. The PTMC membranes induced a mild tissue reaction corresponding to a normal foreign body reaction. The PTMC membranes showed minimal cellular capsule formation and showed signs of a surface erosion process. Bone tissue formed beneath the PTMC membranes comparable to that beneath the collagen membranes. The space maintaining properties of the PTMC membranes were superior to those of the collagen membrane. Newly developed PTMC membranes can be used with success as barrier membranes in critical size rat mandibular defects.

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In vivo behaviour of a biodegradable poly(trimethylene
carbonate) barrier membrane: a histological study in rats
A. C. Van Leeuwen•T. G. Van Kooten•
D. W. Grijpma•R. R. M. Bos
Received: 13 December 2011/Accepted: 24 April 2012/Published online: 9 May 2012
? The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract
the response of surrounding tissues to newly developed
poly(trimethylene carbonate) (PTMC) membranes. Fur-
thermore, the tissue formation beneath and the space
maintaining properties of the PTMC membrane were
evaluated. Results were compared with a collagen mem-
brane (Geistlich BioGide), which served as control. Single-
sided standardized 5.0 mm circular bicortical defects were
created in the mandibular angle of rats. Defects were
covered with either the PTMC membrane or a collagen
membrane. After 2, 4 and 12 weeks rats were sacrificed
and histology was performed. The PTMC membranes
induced a mild tissue reaction corresponding to a normal
foreign body reaction. The PTMC membranes showed
minimal cellular capsule formation and showed signs of a
surface erosion process. Bone tissue formed beneath the
PTMC membranes comparable to that beneath the collagen
membranes. The space maintaining properties of the
PTMC membranes were superior to those of the collagen
membrane. Newly developed PTMC membranes can be
The aim of the present study was to evaluate
used with success as barrier membranes in critical size rat
mandibular defects.
1 Introduction
Guided bone regeneration (GBR) is a widely used modality
for restoring bone deficiencies. In GBR the use of barrier
membranes leads to predictable bone formation, by pre-
venting in-growth of fibroblasts and provision of space for
osteogenesis within a blood clot formed in the defect [1]. A
variety of biocompatible membranes have been used to
achieve this desired barrier effect. The optimal barrier
membrane should exert biocompatible, synthetic, degrad-
able and space maintaining properties [2, 3]. Currently,
non-resorbable membranes have better space maintaining
properties compared to resorbable membranes. However, a
major disadvantage of non-resorbable membranes is the
need for their removal in a second operation.
The majority of clinically used resorbable membranes
are based on collagen. As collagen is an animal derived
product, these membranes carry the risk of disease trans-
mission from animal to human [3–5]. Other available
resorbable barrier membranes are synthetic polymeric
membranes based on lactide and glycolide. However, due to
an extensive foreign body reaction, adverse effects like
postoperative swelling have been reported when using these
materials[6–13].Itisknownthatthesematerialscanproduce
significantamountsofacidiccompoundsduringdegradation
inthebody,andsincebonedissolvesinacidicenvironments,
it can be expected that these polymers will not be the most
suited materials for use in GBR [7, 12, 14, 15].
Recently, we have developed a flexible synthetic
biodegradable barrier membrane prepared from poly(tri-
methylene carbonate) (PTMC) for GBR [16]. PTMC is a
A. C. Van Leeuwen (&) ? R. R. M. Bos
Department of Oral and Maxillofacial Surgery,
University Medical Center Groningen, PO Box 30001,
9700 RB Groningen, The Netherlands
e-mail: a.van.leeuwen01@umcg.nl
T. G. Van Kooten ? D. W. Grijpma
Department of Biomedical Engineering, University Medical
Center Groningen and University of Groningen, PO Box 196,
9700 AD Groningen, The Netherlands
D. W. Grijpma
MIRA Institute for Biomedical Engineering and Technical
Medicine, and Department of Biomaterials Science and
Technology, Faculty of Science and Technology, University of
Twente, PO Box 217, 7500 AE Enschede, The Netherlands
123
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DOI 10.1007/s10856-012-4663-x

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flexible, rubber-like, amorphous and biodegradable poly-
mer. The monomer from which it is prepared, trimethylene
carbonate (TMC), has been used to prepare copolymers
for use in barrier membranes in the medical field before
[17–20]. By gamma irradiation under vacuum, form-stable
elastomeric networks can be formed [21]. In vitro and in
vivo research has shown that this polymer is both bio-
compatible and can be degraded by surface erosion without
the formation of acidic degradation products [22, 23].
In the aforementioned study [16] the PTMC membrane,
prepared from TMC only, was evaluated and compared
with a collagen (Geistlich BioGide) and an expanded-
polytetrafluoroethylene (e-PTFE, GoreTex) membrane and
a non-treated control site for its suitability as a barrier
membrane for use in GBR over bony defects in rats. Both
quantitative micro-radiographical and quantitative micro-
computedtomographicalanalysis
amounts of bone formed underneath the PTMC membrane
[16]. Evaluation after 12 weeks showed that comparable
amounts of bone had formed underneath the PTMC and
reference membranes. The mean percentages of regener-
ated bone were 74, 71, 83 and 33 %, for respectively
the collagen, PTMC e-PTFE and the non-treated control
group [16].
The purpose of this current study was to evaluate his-
tologically the response of the surrounding tissue to the
PTMC membrane, the tissue formation beneath the PTMC
membrane, and the space maintaining properties of the
PTMC membrane. Furthermore, the degradation of the
membrane was assessed. The collagen membrane served as
a reference.
showed excellent
2 Materials and methods
2.1 Membrane synthesis
Polymerization grade 1,3-trimethylene carbonate (TMC)
was obtained from Boehringer Ingelheim, Germany.
Stannous octoate (SnOct2from Sigma, USA) was used as
received. The used solvents were of analytical grade and
purchased from Biosolve, The Netherlands. PTMC was
synthesized by ring opening polymerization of TMC
monomer under vacuum at 130 ?C for a period of three
days using stannous octoate as a catalyst. The polymer was
purified by dissolution in chloroform and precipitation into
a fivefold excess of ethanol 100 %. The precipitated PTMC
was dried under vacuum at room temperature. Analysis of
the synthesized polymer by proton nuclear magnetic reso-
nance (1H NMR), gel permeation chromatography (GPC)
and differential scanning calorimetry (DSC) according to
standardized procedures [21] indicated that high molecular
weight polymer had been synthesized. GPC measurements
showed that Mw= 443000 and Mn= 332000 g/mol, while
NMR indicated that the monomer conversion was more
than 98 %. The glass transition temperature of this amor-
phous polymer was approximately -17 ?C, as thermal
analysis showed.
The purified polymer was then compression moulded at
140 ?C and a pressure of 3.0 MPa (31 kg/cm2) using a
Carver model 3851-0 laboratory press (Carver Inc, USA)
into films with a thickness of 0.3 mm and a diameter of
8 mm using stainless steel moulds. The PTMC membranes
were then sealed under vacuum and exposed to 25 kGy
gamma irradiation from a60Co source (Isotron BV, The
Netherlands). This sterilization method leads to simulta-
neous crosslinking of the polymer [21].
2.2 Study design
ThegammairradiatedPTMCmembraneswere evaluatedby
a subperiosteal implantation in rats. All procedures per-
formed on the animals were done according to international
standards on animal welfare and complied with the Animal
Research Committee of the University Medical Centre
Groningen. Twenty-four Sprague–Dawley rats from an
earlier study [16] were included in here. The surgical pro-
cedure consisted of drilling one bicortical critical size
(5.0 mm diameter) mandibular defect in the left mandibular
anglewithatrephine.Thecreateddefectswerecoveredwith
a barrier membrane on the buccal and lingual side using
either the PTMC membrane, or a collagen (Geistlich Bio-
Gide, Geistlich, Switzerland) membrane which served as
reference. (The procedures are described in the section
‘‘Implantation procedure’’.) At three different time periods
(2, 4 and 12 weeks) the rats were sacrificed. Per time period
eight rats were evaluated; four rats treated with the collagen
membrane and four rats treated with the PTMC membrane.
Theratswerehistologicallyevaluatedfortheresponseofthe
surrounding tissue to the membranes, and the tissue prolif-
eration at the defect site, covered by the membranes. Fur-
thermore, space maintaining properties and degradation of
the PTMC membranes were assessed.
(In a separate study, of which the results will be reported
in another communication, the bone sample that was
obtained with the trephine was transplanted to the contra-
lateral mandibular angle to evaluate the suitability of the
membranes on modelling and incorporation of autologous
bone grafts.)
2.3 Implantation procedure
The animals were anaesthetized with nitrous–oxygen–
isoflurane. After the mandibular area was shaved and
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disinfected, a peri angular incision was made and a
standardised circular 5.0 mm critical size bicortical defect
was drilled with a trephine in the left mandibular angle [24,
25]. In one group the PTMC membrane was used to cover
the defects. In the other group the defects were covered
with a collagen membrane (Geistlich BioGide, Geistlich,
Switzerland). The wound was closed in layers using
resorbable sutures (Vicryl Rapide 4-0, Ethicon, USA). A
single dose of Temgesic (0.05 mg/kg) was administered
perioperative for postoperative pain relief, and the diet was
composed of standard laboratory food.
At three different time intervals (2, 4 and 12 weeks), the
rats were anaesthetised by nitrous–oxygen–isoflurane
inhalation and sacrificed by an intracardially injected
overdose of pentobarbital. The mandibles were explanted
and fixed in 4 % phosphate buffered formaline solution.
2.4 Light microscopy: histological scoring analysis
After explantation, the specimens were decalcified and
dehydrated in a series of ethanol. The specimens were
embedded in glycidyl methacrylate. The tissue blocks were
cut perpendicularly to the defects with a microtome. The
resulting histological sections were 2 lm thick. From all
samples two sections were stained, one series with tolui-
dine blue and one with toluidine blue/basic fuchsin. The
histological sections were digitalized with a ScanScope GL
(Aperio Technology Inc., Vista, CA). Digital images were
then stored for further analysis. Histological sections were
qualitatively and semiquantitatively analyzed using a
modified histological scoring analysis (Table 1). Two his-
tological sections were evaluated for each animal. The
investigators were blinded for time interval and type of
material used during evaluation of the explanted samples.
During analysis each section was evaluated with respect to
the foreign body response (soft tissue response to PTMC
membrane), (the kind of) tissue proliferation at the defect
site and finally for the space maintaining properties of the
membrane. Each section received a single score. The
scores for each time interval were then averaged; mean and
standard deviation were reported.
2.5 Statistical analysis
The data sets were statistically evaluated with a Fisher’s
Exact Test using SPSS 17 (Statistical Package for the
Social Sciences, SPSS Inc., USA). The null hypothesis (the
means of each set are equal) was evaluated with 95 %
confidence level (a = 0.05).
3 Results
3.1 Clinical observations
None of the rats died or suffered from postoperative
complications. The animals started eating immediately
after the surgical procedure and did not loose weight.
3.2 Descriptive microscopic observations
A gross light microscopical inspection of all histological
sections was performed. Defects and the PTMC and col-
lagen barrier membranes could easily be identified after
2–4 weeks (Figs. 1, 2). The PTMC membrane appeared
white in the sections, the collagen barrier membrane
appeared similar to (collagen) connective tissue, although
it could be differentiated from host (collagen) connective
tissue.
At 12 weeks it was more difficult to identify the defects,
whereas in some animals the defect had closed. The col-
lagen membrane could not be identified anymore at
12 weeks because, besides extensive signs of degradation
of the membrane, the differentiation between membrane
and host collagen was virtually impossible. The PTMC
membrane showed extensive signs of degradation. Only
Table 1 Semiquantitative histological grading scale
Soft tissue response to membrane
Fibrous, mature, not dense, resembling connective
or at tissue in the noninjured regions
4
Shows blood vessels and young fibroblasts, few
macrophages and giant cells are present
3
Shows macrophages and other inflammatory cells in
abundance, but connective tissue components between
2
Dense and exclusively of inflammatory type1
Cannot be evaluated because of infection or other
factors not necessarily related to the material
0
Tissue proliferation in defect
Mature bone and differentiation of bone marrow4
Bone or osteoid formation3
Fibrous connective tissue: collagen fibers at defect site2
Fibrous connective tissue: cellular and vascular components1
Cannot be evaluated because of infection or other
factors not necessarily related to the material
0
Space maintaining properties of membrane
No contact between membranes at defect site, bone
formation in between
4
No contact between membranes at defect site, connective tissue
in between
3
Contact between membranes at defect site, bone formation
present
2
Contact between membranes at defect site, connective tissue in
between
1
Cannot be evaluated because of degradation or absence of the
material
0
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some remnants of the PTMC membrane could still be
identified after 12 weeks (Fig. 3).
After 2 weeks both the collagen membrane and the
PTMC membrane was surrounded by a thin fibrous cap-
sule. The thin fibrous capsule around the collagen mem-
branes was composed of loose connective tissue fibers with
the presence of very few macrophages, giant cells and
other inflammatory cells. The fibrous capsule around the
PTMC membrane was also composed of loose connective
tissue fibers though appeared thicker and showed more
invasion of macrophages and giant cells compared to the
collagen treated animals. The macrophages and giant cells
appeared to erode the surface of the PTMC membrane by
phagocytosis (Fig. 2b, d).
At 2 weeks high numbers of blood vessels were present
at the tissues surrounding the PTMC membranes and
especially located in the tissues filling the defects covered
with the PTMC membranes. By contrast the animals trea-
ted with the collagen membranes showed blood vessel
formation mainly localized in and throughout the mem-
branes, as well as in the surrounding tissues.
Osteoid (bone) formation was already observed after
2 weeks in both the collagen and PTMC membrane treated
groups. Interestingly at 2 and 4 weeks, two different pat-
terns of bone formation were identified. The animals
treated with the collagen membrane tended to form osteoid
and new bone throughout the defect by forming bony islets
(Figs. 2, 3). By contrast the PTMC membrane treated
animals showed osteoid and new bone formation origi-
nating from the defect borders. The pattern of new bone
formation was found similar for both groups after
12 weeks, since the majority of the sections by then
showed complete bridging of the defect with newly formed
mature bone.
3.3 Histological analysis
Figure 4a shows the data of the histological rating of the
soft tissue response to the collagen and PTMC membrane,
respectively. Statistical analysis of these data revealed that
after 2, 4 and 12 weeks significant differences existed
between the tissue response towards the used membranes
(p\0.001, p\0.001 and p = 0.006, respectively). The
tissue reaction to the PTMC membrane shows a higher
density of inflammatory cells after 2, 4 and 12 weeks
compared to the collagen membrane. For both membranes
Fig. 1 Lightmicrographsofratmandibulardefectscoveredwitheither
a collagen (a, c) or a PTMC (b, d) membrane after 2 weeks of
implantation. The collagen and PTMC membrane can be readily
distinguished. Degradation of the PTMC membrane can be observed.
Toluidin blue with basic fuchsin as counterstain was used for the
stainingofthehistologicalsections.(p)polymer,(ct)connectivetissue,
(b) bone, (fc) fibrous capsule, (asterisk) osteoid, (filled triangle)
collagen membrane, arrows indicate phagocytosed intracellular frag-
ments. Figures ‘c and d’ magnifications (920) of the marked regions
from respectively figures ‘a and b’, which are 92 magnifications
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the score increased at 12 weeks, which means that there
was a reduction in inflammatory cell density, and the
number of giant cells, and an increase in vascularization of
the surrounding tissue.
The data on tissue formation at the defect site, shown in
Fig. 4b, was similar for both membranes. After 2 and
4 weeks the majority of the sections showed amounts of
osteoid and bone formation in the defects for both mem-
brane treated groups. Between 4 and 12 weeks the newly
formed bone had matured and after 12 weeks extensive
amounts of mature bone were found for both membrane
groups. There were no statistical differences in the tissue
proliferation at the defect sites between the collagen and
PTMC treated animals (p = 0.467). Moreover, after
12 weeks mature bone with bone marrow had formed at the
defect sites, whereas after 4 weeks bone could be assessed
as immature.
Figure 4c presents the data on the space maintaining
properties of the two membranes. The PTMC membrane
showed a distinctively maintained space at the level of the
mandibular defects, between the buccal and lingual placed
membranes after 2 and 4 weeks (Figs. 1, 2). This space
was filled with either connective tissue, osteoid or newly
formed bone depending on the time of evaluation,
respectively 2, 4 or 12 weeks. The collagen membranes by
contrast showed no space maintaining at all after 2 and
4 weeks. After 12 weeks the space maintaining properties
of both membranes could not be assessed for two reasons:
firstly, because bone had bridged the majority of the
defects, and as a result there was no ‘space’ left to be
assessed, and secondly because of the extensive degrada-
tion of both membranes. The collagen membranes could
not be discerned anymore due to degradation and similarity
to the host connective tissue and for the PTMC membranes
only some phagocytosed particles were observed. As a
consequence the space maintaining properties could not be
assessed, and score ‘0’ was assigned to all specimens of the
12 weeks group.
4 Discussion
In the present study a rat model was used to investigate the
soft and hard tissue response to a barrier membrane com-
posed of solid PTMC. Furthermore the space maintaining
properties were assessed, as space maintaining is an
Fig. 2 Light micrographs of rat mandibular defects covered with
either a collagen (a, c) or a PTMC (b, d) membrane after 4 weeks of
implantation. The collagen membrane can be distinguished from the
rats connective tissue. Bone forms in and around the collagen
membrane, by contrats bone formation underneath the PTMC
membrane originates from the defect borders. Toluidin blue with
basic fuchsin as counterstain was used for the staining of the
histological sections. (p) polymer, (ct) connective tissue, (b) bone,
(fc) fibrous capsule, (asterisk) osteoid, (filled triangle) collagen
membrane, arrows indicate phagocytosed intracellular fragments.
(filled square) Indicates surface erosion degradation by macrophage
and giant cell activity. Figures ‘c and d’ magnifications (920) of the
marked regions from respectively figures ‘a and b’, which are 92
magnifications
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