Long-term Local and Systemic Safetyof Poly(L-lactide-co-epsilon-caprolactone)after Subcutaneous and Intra-articularImplantation in Rats
Yuval Ramot1, Abraham Nyska2, Elana Markovitz3, Assaf Dekel3,Guy Klaiman4, Moran Haim Zada5, Abraham J. Domb5
and Robert R. Maronpot6
AbstractThe use of biodegradable materials is gaining popularity in medicine, especially in orthopedic applications. However, preclinicalevaluation of biodegradable materials can be challenging, since they are located in close contact with host tissues and might beimplanted for a long period of time. Evaluation of these compounds requires biodegradability and biocompatibility studies andmeticulous pathology examination. We describe 2 preclinical studies performed on Sprague-Dawley rats for 52 weeks, to evaluateclinical pathology, biocompatibility, biodegradability, and systemic toxicity after implantation of 2-layered films or saline-inflatedballoon-shaped implants of downsized InSpace™ devices (termed ‘‘test device’’). The test devices are made from a copolymer ofpoly-L-lactide-co-e-caprolactone in a 70:30 ratio, identical to the device used in humans, intended for the treatment of rotator cufftears. Intra-articular film implantation and subcutaneous implantation of the downsized device showed favorable local and systemictolerability. Although the implanted materials have no inherent toxic or tumorigenic properties, one animal developed a fibro-sarcoma at the implantation site, an event that is associated with a rodent-predilection response where solid materials causemesenchymal neoplasms. This effect is discussed in the context of biodegradable materials along with a detailed description ofexpected pathology for biodegradable materials in long-term rodent studies.
Keywordsbiodegradable materials, safety studies, rotator cuff syndrome, biocompatibility, biodegradability
Introduction
One of the most common musculoskeletal disorders is shoulder
pain, which can have a prominent negative effect on quality of
life (Whittle and Buchbinder 2015). The rotator cuff (RC)
encompasses a group of muscles that engulf the shoulder joint
and allows movement and stabilization of this joint (Whittle
and Buchbinder 2015). Tears of the RC are the most common
cause for shoulder pain (Lorbach et al. 2015), and they often
necessitate surgical reconstruction (Yamaguchi et al. 2006).
However, the proper way to treat RC tears is still debated, con-
sidering the high rate of treatment failure (DeHaan et al. 2012;
Duquin, Buyea, and Bisson 2010; Lorbach et al. 2015), and
there is growing interest in ways to improve tendon-bone heal-
ing (Anz et al. 2014; Beitzel et al. 2013), including use of bio-
degradable orthopedic devices.
Biodegradable devices are commonly composed of poly-
meric materials that degrade into products that can be elimi-
nated from the body or degrade into normal metabolites
(Nyska et al. 2014). They are especially useful for orthopedic
applications taking into consideration their slow degradation,
allowing the gradual transfer of load to healing bones and joints
(Nyska et al. 2014; Ramot et al. 2015). Indeed, their use has
also been advocated for the treatment of the RC syndrome
(Zhao et al. 2015; Zhao et al. 2014).
A novel addition to the biodegradable armamentarium for
RC syndrome includes a subacromial implantable balloon-
shaped spacer (InSpace™ system, Ortho-Space, Caesarea,
1 Hadassah—Hebrew University Medical Center, Jerusalem, Israel2 Tel Aviv University and Consultant in Toxicologic Pathology, Timrat, Israel3 Ortho-Space Ltd., Caesarea, Israel4 Harlan Biotech Israel Ltd., Rehovot, Israel5 Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The
Hebrew University of Jerusalem, Ein Kerem, Jerusalem, Israel6 Maronpot Consulting LLC, Raleigh, North Carolina, USA
Corresponding Author:
Abraham Nyska, Haharuv 18, P.O. Box 184, Timrat 36576, Israel.
Email: [email protected]
Toxicologic Pathology1-14ª The Author(s) 2015Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/0192623315600275tpx.sagepub.com
Israel), which, when implanted between the humeral head and
acromion, permits smooth, frictionless gliding, supporting
shoulder biomechanics (Anley, Chan, and Snow 2014; Gervasi,
Cautero, and Dekel 2014; Savarese and Romeo 2012; Szollosy
et al. 2014). This device, which is intended for arthroscopic or
mini-open insertion in humans, has proven to treat shoulder
pain and improve range of motion when implanted in patients
with massive irreparable RC tears (Senekovic et al. 2013). It is
composed of a copolymer of poly-L-lactide-co-e-caprolactone
(PLCL) in a 70:30 ratio and is a well-known poly(a-hydroxy
acid; Levy et al. 2009). PLCL is a flexible but durable material,
which mirrors the chemical properties of the comprising mono-
mers: the lactide provides the toughness and the caprolactone
provides the elasticity. It is a Food and Drug Administration–
approved material (Burks et al. 2006), which has been used
in a large number of medical applications, including scaffolds
for tissue engineering (Jeong et al. 2004) and drug-loaded films
(Cho et al. 2008).
The proper evaluation of the safety of biodegradable mate-
rials is of great importance, taking into consideration the fact
that they are implanted in humans for a long period of time and
are located in close contact with human tissues (Fournier et al.
2003; Nyska et al. 2014; Vaisman et al. 2010). They require
both biodegradability and biocompatibility studies, with eva-
luation of these studies by toxicologic pathologists (Nyska
et al. 2014).
The aim of this communication is to describe the clinical
pathology, biocompatibility, biodegradability, and systemic
toxicity findings after implantation of the downsized InSpace
system (the test device) or its degradation products as observed
in 2 preclinical studies performed on Sprague-Dawley rats for a
period of up to 52 weeks. The purpose of the first study was to
evaluate the local biocompatibility of the device material in the
shoulder joint of rats. The purpose of the second study was to
evaluate biodegradability profile and clinical pathology after
subcutaneous implantation of the test device in rats. The over-
all findings reported here can serve as case studies for the
pathological evaluation and expected results for biodegradable
materials, and PLCL in particular.
Animals, Materials, and Methods
Test Device
The InSpace spacer, supplied by Ortho-Space Ltd., is con-
structed of medical grade copolymer of PLCL in a 70:30 ratio.
Implanted test devices were produced using the manufacturing
process and full ethylene oxide sterilization cycles identical to
the device as used in humans, but downsized to accommodate
animal size. In humans, the device is available in 3 different
sizes: small (40 � 50 mm), medium (50 � 60 mm), and large
(60 � 70 mm; Savarese and Romeo 2012). For the shoulder
implantation (first study), 2-layer films, sized to 5 � 5 mm
to accommodate rat shoulder size, were used. This adjusted
device size represents similar size and amount of material as
being used in human shoulders (50 � 50 mm). For the
subcutaneous implantation study (second study), each animal
was implanted with 2 test devices downsized to fit the selected
rat animal model. Each test device was downsized to 20 � 30
mm and inflated with approximately 2 ml of physiological sal-
ine. The relative device material amount and implanted area in
the rats was approximately 80 times greater than the relative
area and amount of material used in human subjects. The size
of the downsized device was selected to provide a device that
can be properly inflated and measured and big enough to enable
chemical characteristic evaluation, but that can still fit the ani-
mal’s size and shape without interfering the animal’s welfare
and in accordance with Annexure B of ISO 10993-6 and ISO
10993-11.
Animals and Housing
Male Sprague-Dawley rats, 7 to 8 weeks of age, were obtained
from Harlan Laboratories (Rehovot, Israel) and maintained on
standard chow (Harlan Teklad diet 2018S, Madison, WI). They
were allowed free access to drinking water, supplied to each
cage via polyethylene bottles with stainless steel sipper tubes.
The water was filtered (0.2 mm filter), chlorinated, and acidi-
fied (pH 5.0–5.9). During acclimation and throughout the
entire study duration, animals were housed within a limited
access rodent facility and kept in groups of a maximum of 3
animals/cage, in cages fitted with solid bottoms and filled with
wood shavings as bedding material (7093 Harlan Teklad
Shredded Aspen, Harlan Laboratories, Madison, WI). The rats
were allowed a 5- to 6-day acclimation period to facility con-
ditions (20–24�C, 30–70% relative humidity, and a 12-hr
light/dark cycle) prior to inclusion in the study. Animal care
and the test device implantation were conducted at a good
laboratory practices (GLP)–certified site (Harlan Biotech Israel
Ltd., Rehovot, Israel) and after receiving the approval of the
National Council for Animal Experimentation.
Experimental Design—First Study
For the first study, the 30 rats were randomized into 2 groups:
the test group consisted of 21 animals, and the control group of
9 animals. Animals were sacrificed at predetermined time
points of 12, 26, and 52 weeks postimplantation (Table 1). The
animals underwent analgesia (buprenorphine at a dose of 0.05
mg/kg by subcutaneous injection) and anesthesia (isoflurane
2–3% in 100% O2 at the rate of 0.8–1 L/min, administered
by face mask). Later, each animal was placed in lateral recum-
bency, with the right leg upward. The forelimb was positioned
in external rotation. A 2-cm skin incision was made over the
scapulo-humeral (SH) joint, which was exposed by blunt dis-
section. The supraspinatus tendon was exposed in its passage
under the bony arch consisting of the acromion, coracoid pro-
cess, and clavicle, and was resected at this location. The suba-
cromial space was debrided in order to enable test device
implantation.
In the test group, the test device (i.e., 2 layers of 5 � 5 mm)
was implanted in the subacromial space and fixated by single
2 Toxicologic Pathology
suture using appropriately sized nonabsorbable suture material.
The musculature and subcutaneous tissues were closed with
3/0 Vicryl sutures. The skin was closed with stainless steel
surgical clips. In the control group, an identical procedure was
performed, including full exposure of the SH joint, resection of
the supraspinatus tendon, and placement of the nonabsorbable
anchoring Vicryl suture, without implantation of the test
device. At the end of the day following surgery and twice on
the following day, animals were administered buprenorphine
by subcutaneous injection at a dose of 0.05 mg/kg.
Experimental Design—Second Study
For the second study, 15 animals were divided into 4 test
groups and were sacrificed at 4, 12, 16 (unscheduled), 26, and
52 weeks postimplantation (Table 2). All test devices were
inflated with sterile physiological saline and were sealed before
implantation. The amount of saline filled in each test device
was between 1.8 and 2.7 ml (average of 2.2 ml), and the aver-
age initial device weight was ~2.3 g. The animals underwent
analgesia (buprenorphine at a dose of 0.05 mg/kg by subcuta-
neous injection) and anesthesia (isoflurane 2–3% in 100% O2
at the rate of 0.8–1 L/min. administered by face mask). Later,
the animals were placed in ventral recumbency, and a 2- to
3-cm skin incision was made over the dorsal midline in the
thoracic area. Subcutaneous tunnels were created from the skin
incision in a caudal direction to both sides of the spine overly-
ing the lumbar paravertebral muscles.
Two saline-filled, preweighed test devices were implanted
in each animal on either side of the spine in the lumbar region,
1 test device on each side of the spine. The test devices were
anchored to the underlying muscle using nonabsorbable suture
material, which was used to identify the implantation site at test
device explantation. At the end of the day following surgery
and twice daily for up to 3 days postsurgery, animals were
administered buprenorphine by subcutaneous injection at a
dose of 0.05 mg/kg.
Viability and Weight
For both studies, viability check of all animals was performed
at least once daily, and detailed clinical examinations were car-
ried out and recorded once weekly. Body weights were mea-
sured prior to implantation and weekly thereafter. The last
body weight determination was carried out prior to scheduled
termination.
Hematology and Biochemistry
For both studies, blood for hematology and biochemistry para-
meters was collected just prior to euthanasia. Blood samples
were obtained by retro-orbital sinus bleeding under CO2
anesthesia and following an overnight food deprivation. At
least 300 μl whole blood was collected into EDTA-coated
tubes for hematology, and at least 700 μl whole blood was col-
lected into commercial serum separation gel tubes and centri-
fuged for separation of serum for biochemistry. The samples
were assayed for hematology using the ADVIA 120 Hema-
tology System (Siemens, Erlangen, Germany) and for bio-
chemistry using the ROCHE-HITACHI/MODULAR P-800
& Roche Cobas1 6000 Analyzer.
Necropsy and Tissue Handling
In the first study, following animal sacrifice, the implantation
sites were examined macroscopically to assess any evident tis-
sue reactions to the implanted test device. In 3 animals sacri-
ficed at the 12-week time point and 3 animals sacrificed at
the 26-week time point, the test device was carefully removed
from the implantation sites from both shoulders immediately
following sacrifice, taking care to cause as little damage to the
Table 2. Experimental design for the second study.
Number ofanimals
TreatmentSacrifice Timepoints (weeks
postimplantation)Test materialImplantationprocedure
3 PLCL:DownsizedInSpace™
Bilateralsubcutaneousimplantation inthe dorsolumbararea (2 testdevices peranimal)
44 121 16a
3 264 52
Note: PLCL ¼ poly-L-lactide-co-e-caprolactone.aUnscheduled sacrifice time point.
Table 1. Experimental design for the first study.
Group Number of animals
TreatmentSacrifice time points (weeks
postimplantation)Test material Procedure
Control 3 Not applicable Bilateral sham operation of shoulder joint,including RC tear
123 263 52
Test 8 PLCL: 2-layer films of InSpace™ Bilateral subacromial implantation followingRC tear
128 265 52
Note: PLCL ¼ poly-L-lactide-co-e-caprolactone; RC ¼ rotator cuff.
Ramot et al. 3
surrounding tissues as possible. The local draining lymph
nodes, that is, brachial and axillary lymph nodes, were assessed
macroscopically. The test and control sites from all animals
and the draining lymph nodes from the animals in the 52-week
sacrifice point were resected and preserved in 10% neutral buf-
fered formalin (approximately 4% formaldehyde solution). Tis-
sues were trimmed when necessary and embedded in paraffin.
Five step sections approximately 5 microns thick, at 250-mm
intervals, were prepared from each test or control site using
the suture site as the middle section. Slides were stained with
hematoxylin and eosin (H&E).
In the second study, all animals were subjected to a full
detailed necropsy. The following tissues were collected from
all sacrificed animals: brain, heart, lungs, liver, and kidneys.
These tissues were weighed and preserved in 10% neutral
buffered formalin until testing and pathology evaluated. In
addition, the tissue surrounding each test device was also pre-
served in 10% neutral buffered formalin for histopathological
examination. Tissues were trimmed when necessary and
embedded in paraffin. One section approximately 5 microns
thick was prepared from each site. Slides were stained with
H&E.
Histopathology Examination
For both studies, the histological local response parameters that
were assessed and recorded were based on the international
standards ISO 10993-6 (2007) and included (but were not
limited to) the presence and extent of fibrosis/fibrous capsule;
the extent of inflammation based on the number and types
of inflammatory cells present; degeneration as determined by
changes in tissue morphology and differences in tinctorial
staining; presence, extent, and type of necrosis; tissue altera-
tions such as fragmentation and/or presence of debris, fatty
infiltration, granuloma formation, and bone formation; pres-
ence and form of test device remnants, material fragmentation,
and debris; and the nature and extent of tissue ingrowth, if
applicable. The scaling of the lesions was based on the semi-
quantitative criteria presented by Shackelford et al. (2002), in
which grade 1 corresponds to lesion barely noticeable and/or
up to 10% of the tissue is affected; grade 2—the lesion is
noticeable but is not a prominent feature of the tissue and/or
up to 20% of the tissue is affected; grade 3—the lesion is a pro-
minent feature of the tissue and/or up to 40% of the tissue is
affected; grade 4—the lesion is an overwhelming feature of the
tissue and/or more than 41% of the tissue is affected.
Test Device Examination
In the second study, following sacrifice of the animals, the test
devices (or their residues) were explanted from their implanta-
tion sites, cleaned as much as possible from surrounding tissues
and weighed, and then analyzed for degradation by mass loss
and molecular weight change. The weight loss was determined
by weighing the recovered isolated dry polymer implant
residues.
Molecular weight of the implant residues was conducted on
gel permeation chromatography (GPC) systems, consisting of
Waters 1515 Isocratic high-performance liquid chromatogra-
phy (HPLC) pump, with L-7490 refractive index detector
(RI; Hitachi, Tokyo, Japan) and a Rheodyne (Coatati, CA)
injection valve with a 20-μl loop. Samples were eluted with
chloroform (HPLC grade) through a linear styragel column
THF, HR5E (Waters, MA) at a flow rate of 1 ml/min. The
molecular weights were determined relative to polystyrene
standards (Polyscience, Warrington, PA), using the Empower
computer program.
Statistical Analysis
For both studies, results were analyzed using GraphPad Prism
(Graphpad Software Inc., CA), using unpaired t-test, Tukey–
Kramer multiple comparisons test, and one-way analysis of
variance with Bonferroni multiple comparison test.
Results
Survival, Clinical Observations, and Body Weight Gain
In the first study, 1 control animal was removed prematurely
from the study at 12 weeks due to humane reasons. This animal
(that had only the sham operation without test device) dis-
played decreased motor activity and dyspnea as well as a
10% decrease in body weight compared to previous weighing.
There were no other treatment-related effects on body
weight, and all remaining animals showed normal body weight
gain until sacrifice. No abnormal clinical signs were observed,
except for a transient and local crusting on the left cranial
shoulder overlying the implantation site in 1 animal from the
test device group.
In the second study, 2 animals were removed from the study
ahead of their scheduled sacrifice time point. One animal had
ulceration of the skin overlying the test devices (both migrated
to the right side), from the second week postimplantation and,
therefore, was included in the 4-week termination group
instead of the originally planned 26-week group. The second
animal was removed on week 16 due to protrusion of the left
test device plug through the skin. Otherwise, there were no
treatment-related effects on body weight, and all animals
showed body weight gain at sacrifice. Crusting of the skin over-
lying the implanted test devices was present at 4 weeks postim-
plantation in 8 animals and disappeared from all animals except
1 by 8 weeks postimplantation. This observation was consid-
ered as procedurally related with no systemic adverse impact.
The presence of solution in the test devices in the second
study was evaluated by palpation of the skin or by visual detec-
tion at the sacrifice time point. Fluid was palpated in all test
devices up to 19 weeks postimplantation, and by approximately
22 weeks postimplantation, fluid was no longer detected by
palpation in any of the remaining test devices.
At week 45, a mass on the midline of the back was observed
in 1 animal from the 52-week sacrifice group. It grew progressively
4 Toxicologic Pathology
and reached a size of approximately 4 � 4 mm 1 day prior to
the 52-week scheduled study termination. None of the other
animals had similar observations at any of the sacrifice time
points.
Hematology and Clinical Chemistry
In the first study, statistical differences were noted in red blood
cells, hematocrit, mean corpuscular hemoglobin concentration,
glucose, and total protein levels between the control and test
groups at 26 weeks postimplantation (Table 3). At 52 weeks
postimplantation, there was a statistical significant difference
in the white blood cell levels (Table 3). However, all changes
were without clinical significance and were within normal ref-
erence ranges for hematology and biochemistry parameters in
these rats.
In the second study, no control group was available for
comparison. However, 2 animals had abnormal values com-
pared to the other animals in their groups. One rat that was
removed from the study due to skin ulceration had an elevated
white blood cell count. The rat with a large subcutaneous
mass in the implantation site had increases in creatinine and
urea levels along with an elevated white blood cells count due
to neutrophilia and anemia characterized by low red blood
cells count, low hemoglobin levels, and low hematocrit but
without a change to the cell size and hemoglobin content (data
not shown). These findings are secondary to the mass on the
midline of the back and were not a direct response to the test
device.
Pathology—First Study
In the first study, macroscopic examination of the tissue sur-
rounding the implantation sites revealed a hematoma-like dark
red–colored test device in 1 of the animals at the 12-week
interim sacrifice point. No macroscopic abnormalities were
found at the 26-week interim sacrifice point. At the 52-week
interim sacrifice point, a hardened substance or tissue was
observed at the suture site in 6 out of 10 implantation sites.
On 3 other cases, a square form (approximately 4� 5 mm) was
identified. No abnormalities were found at the sham-operated
animals.
Macroscopic examination of the test device and implanta-
tion site at the 12-week interim sacrifice point revealed opaque
films measuring about 5 � 5 mm, that were closely adherent to
the surrounding tissue in 4 of the 6 devices. One device had
curled to a cylinder-like structure and was also closely adherent
to the surrounding tissue. One device was observed as a double-
layered opaque film measuring about 5� 5 mm that was easily
separated from the surrounding tissue. At the 26-week interim
sacrifice point, the test devices were fragmented into small
pieces, encapsulated and/or inseparable or adherent to the sur-
rounding tissues.
Histopathologically, no treatment-related changes were
seen in the sham-operated animals. In the InSpace-implanted
animals, the sites of implantation located at the shoulder were Tab
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identified by the presence of cavity formation surrounded by a
mature fibrotic capsule (Table 4). At the 12- and 26-week
interim sacrifice points, the fibrotic capsule had a minimal
severity grade. At the 52-week sacrifice point, severity ranged
from minimal to mild (Table 4). Occasionally, a single layer of
macrophages of minimal (12 and 26 weeks) or minimal to mild
(52 weeks) severity was present at the interface of the copoly-
mer and the fibrotic capsule. Minimal presence of lymphocytes
and rare polymorphonuclear cells were sometimes present
within the fibrotic capsule. No evidence for chronic active
inflammation, necrosis, chondral damage, articular bone dam-
age, or surrounding muscle degeneration was noted at any time
point. Remnants of the implanted device, occasionally seen
within the cavity formation, were surrounded by mature fibro-
tic capsule at all time points.
At the 12-week interim point, the implanted test device
was not fragmented; while at the 26-week interim point, the
implanted device was fragmented into several pieces in some
of the rats. At the 52-week point, the implanted device was
fragmented into a few to multiple small pieces in the majority
of the implantation sites. When examining the irritation scoring
using ISO-10993-6 criteria, a marginal slight irritation (score
of 3.0) was confined to the test device at the first time point
of 12 weeks versus the respective control sites, while at the
26- and 52-week time points, the test device could be consid-
ered as nonirritant (score of <3.0; Supplementary Table 1).
At all time points, no adverse reactions such as mineraliza-
tion, necrosis, or immune-mediated inflammation were noted,
indicating good long-term tolerability of the test compound.
Pathology—Second Study
In 7 animals, the test devices on the left side were displaced to
the right side of the animal. It is not uncommon for subcuta-
neous implanted items in rodents to be displaced from the
implantation site, especially considering their body activity and
the relative large amount of loose skin in rats.
Macroscopic examination of the test sites at 12 and 26 weeks
postimplantation revealed that all test devices were encapsu-
lated by a connective tissue with some yellowish discoloration
of varying degrees in the surrounding tissue.
Macroscopic examination of the explanted test device
revealed that up to 16 weeks postimplantation, the test devices
were intact and were still filled with clear fluid to varying
degrees (Figure 1). While inflated, the test devices were not
adherent to their surrounding tissue aside from 1 test device
in the animal with ulcerated skin. At 26 weeks postimplanta-
tion, the test devices disintegrated to multiple small fragments,
which were encapsulated by surrounding tissue, and by
52 weeks postimplantation, the test devices were fragmented
into very small (<1 mm in size) pieces, embedded in and inse-
parable from the surrounding tissue.
Histopathologically, at all time points up to 26 weeks, the
sites of implantation located subcutaneously at the dorsolum-
bar area were identified by the presence of a cavity surrounded
by a mature fibrotic capsule (Table 5; Figures 2 and 3).
Occasionally, a single layer of macrophages with occasional
giant cells at the interface of the copolymer and the fibrotic
capsule was seen. No evidence of chronic active inflammation
or necrosis was present. Minimal presence of lymphocytes and
rare polymorphonuclear cells were sometimes present within
the fibrotic capsule. Up to the 16-week interim sacrifice point,
the test device was not fragmented. At the 26-week interim
sacrifice in all but one of the test sites, the implanted device
was fragmented into multiple pieces (Figures 4 and 5).
At the 52-week interim sacrifice, in all of the test sites, the
implanted device was fragmented into multiple small pieces,
each piece surrounded by a fibrotic capsule (Figures 6–9) with
occasional macrophages and giant cells at the interface of the
fibrous capsule and the fragmented device material (Figure
9). Overall, the local changes indicated favorable host response
to the device.
No evidence of systemic toxicity was present in the liver,
lungs, kidneys, heart, and brain from all animals, irrespective
of time of sacrifice or the presence of local reaction. A small
range of spontaneous hepatic and renal age-related background
lesions commonly seen in untreated rats of the same age and
strain were present.
At the 52-week time point, 1 rat had a subcutaneous mass
approximately 3.5� 4 cm on the midline of the back. The center
of the mass was red/pink with firm consistency and its periphery
was white and less firm. Histopathological examination of the
midline mass revealed the presence of a solid fibrous mass, that
was relatively well circumscribed, with irregular areas of necrosis
at the center. The histology features of the mass included a stori-
form growth pattern of uniform plump spindle cells, presence of
small rounded cells, some highly pleomorphic spindle cells, and
tumor giant cells (Figures 10–12). Occasional mitotic figures
were noted. Based on these histological features, the mass was
consistent with a pleomorphic fibrosarcoma. Fragments of the test
device were embedded within the neoplastic tissue.
Time-related Changes in Test Device Mass andMolecular Weight
In the second study, the explanted test devices were subject
to weight loss determination and molecular weight analyses.
These analyses were performed on test devices explanted
from the animals sacrificed at 4, 12, 16, and 26 weeks, since
at 52 weeks postimplantation, the test device was almost
fully eliminated, fragmented into very small (<1 mm) frag-
ments that could not be separated from the surrounding tissue
for evaluation. While the dry weight of the test device was
relatively stable until 16 weeks postimplantation, it was sig-
nificantly decreased by 26 weeks postimplantation
(Figure 13A). Molecular weight of polymers showed gradual
decrease with the progression of the study (Figure 13B).
Discussion
Both studies described in this article utilized PLCL, an
FDA-approved polymeric material that has been
6 Toxicologic Pathology
Tab
le4.
His
topat
holo
gyfin
din
gsin
rats
from
the
first
study
afte
rim
pla
nta
tion
of2-lay
erfil
ms
ofpoly
-L-lac
tide
acid
and
epsi
lon-c
apro
lact
one
copoly
mer
inth
esu
bac
rom
ialjo
int.
Sham
oper
atio
n(c
ontr
ol)
Tes
tdev
ice
expla
nte
dT
est
dev
ice
pre
sent
His
tolo
gyse
ctio
ndis
tance
from
sutu
relin
ein
mic
rom
eter
a
�500
�250
Sutu
resi
teþ
250
þ500
�500
�250
Sutu
resi
teþ
250
þ500
�500
�250
Sutu
resi
teþ
250
þ500
12-w
eek
post
impla
nta
tion
Fibro
sis/
fibro
us
capsu
le0.0
b(0
/6)c
0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.3
(2/6
)0.3
(2/6
)0.3
(2/6
)0.3
(2/6
)0.3
(2/6
)1.0
(10/1
0)
1.0
(10/1
0)
1.0
(10/1
0)
1.0
(10/1
0)
1.0
(10/1
0)
Pre
sence
ofm
acro
phag
es0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.3
(2/6
)0.3
(2/6
)0.3
(2/6
)0.3
(2/6
)0.3
(2/6
)1.0
(10/1
0)
1.0
(10/1
0)
1.0
(10/1
0)
1.0
(10/1
0)
1.0
(10/1
0)
26-w
eek
post
impla
nta
tion
Fibro
sis/
fibro
us
capsu
le0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.2
(1/6
)0.2
(1/6
)0.2
(1/6
)0.2
(1/6
)0.2
(1/6
)0.7
(7/1
0)
0.7
(7/1
0)
0.3
(3/1
0)
0.3
(3/1
0)
0.4
(4/1
0)
Pre
sence
ofm
acro
phag
es0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.2
(1/6
)0.2
(1/6
)0.2
(1/6
)0.2
(1/6
)0.2
(1/6
)0.7
(7/1
0)
0.7
(7/1
0)
0.3
(3/1
0)
0.3
(3/1
0)
0.4
(4/1
0)
Tes
tdev
ice
deb
ris
and
frag
men
tation
0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.6
(5/1
0)
0.5
(5/1
0)
0.1
(1/1
0)
0.1
(1/1
0)
0.3
(3/1
0)
52-w
eek
post
impla
nta
tion
Fibro
sis/
fibro
us
capsu
le0.2
(1/6
)0.3
(2/6
)0.0
(0/6
)0.3
(2/6
)0.3
(2/6
)N
AN
AN
AN
AN
A1.5
(9/1
0)
1.5
(9/1
0)
1.3
(9/1
0)
0.9
(6/1
0)
0.8
(6/1
0)
Pre
sence
ofm
acro
phag
es0.0
(0/6
)0.2
(1/6
)0.0
(0/6
)0.3
(2/6
)0.0
(0/6
)N
AN
AN
AN
AN
A1.1
(8/1
0)
1.1
(8/1
0)
0.8
(5/1
0)
0.8
(6/1
0)
0.6
(4/1
0)
Tis
sue
alte
rations
(within
the
cavi
tyof
impla
nta
tion—
dev
ice
frag
men
tation)
0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)0.0
(0/6
)N
AN
AN
AN
AN
A1.1
(6/1
0)
1.2
(7/1
0)
0.9
(5/1
0)
1.1
(6/1
0)
1.1
(6/1
0)
Not
e:N
A¼
not
avai
lable
.a Fi
vehis
tolo
gyse
ctio
ns
take
nat
250-m
min
terv
als
from
each
test
or
contr
olsi
teusi
ng
the
sutu
resi
teas
the
mid
dle
sect
ion.
bM
ean
seve
rity
score
sofle
sions
bas
edon
the
follo
win
g:0¼
norm
al,1¼
min
imal
,2¼
mild
,3¼
moder
ate,
and
4¼
seve
re.
c Num
ber
ofaf
fect
edsi
tes/
num
ber
ofex
amin
edsi
tes.
7
successfully used in a large number of medical applications
(Burks et al. 2006; Jeong et al. 2004; Cho et al. 2008).
Therefore, it is known that this copolymer is not pyrogenic,
is without in vitro and in vivo genotoxicity, and is free of
leachable materials that might be carcinogenic.
The PLCL is a fully biodegradable polymer, degrading into
its starting components, lactic acid and caprilic acid. It is later
secreted from the body as hydroxy acids or is naturally meta-
bolized into CO2 and water. According to Woodruff and
Hutmacher (2010), PLCL degradation is a 2-step process: first,
Figure 1. Macroscopic appearance of a test device (implant) removed from rats at various time points following subcutaneous implantation (sec-ond study). (A) Implant removed from a rat sacrificed at week 4 postimplantation. (B) Implant removed from a rat sacrificed at week 12 afterimplantation. (C) Implant removed from a rat sacrificed at week 16 after implantation. (D) Macroscopic appearance of host fibrotic capsular reac-tion around the implant fragments (left) and test device fragments (right) extracted from the capsular reaction from a rat sacrificed at 26 weekspostimplantation. The test device in A, B, and C still contains saline and its size is *20 � 30 mm.
Table 5. Histopathological findings in rats in the second study after bilateral subcutaneous implantation of a poly-L-lactide and epsilon-caprolactone copolymer (downsized InSpace™ device) in the dorsolumbar area.
Sacrifice interval 4 Weeks (n ¼ 3)a 12 Weeks (n ¼ 4) 16 Weeks (n ¼ 2) 26 Weeks (n ¼ 5) 52 Weeks (n ¼ 8)
Fibrosis/Fibrous capsule 3.0b (3/3)c 2.0 (4/4) 2.0 (2/2) 2.0 (5/5) 2.0 (6/8)Inflammation within the capsule (lymphocytes) 1.3 (3/3) 0.3 (1/4) 1.5 (2/2) 0.0 (0/5) 0.0 (0/8)Histiocytes 1.0 (3/3) 0.0 (0/4) 1.5 (2/2) 0.4 (2/5) 1.0 (6/8)Test device debris and fragmentation 0.0 (0/3) 0.0 (0/4) 0.0 (0/2) 1.6 (4/5) 2.0 (8/8)
aNumber of sites examined.bMean severity score based on the following: 0 ¼ normal, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, and 4 ¼ severe.cNumber of affected sites/number of examined sites.
8 Toxicologic Pathology
Figure 2. Animal sacrificed 4 weeks postimplantation. A central cavity (asterisk) is the site of implantation of a removed downsized test device inthe dorsolumbar area. A mature fibrous capsule surrounds the central cavity with minimal sporadic presence of mononuclear cells (arrows)between fibroblasts and collagen deposition. Hematoxylin and eosin (H&E) stain. Figure 3.—Animal sacrificed 12 weeks postimplantation. Amature fibrotic capsule without an accompanying inflammatory reaction in the dorsolumbar area surrounds the cavity (asterisk) from a removeddownsized test device. Hematoxylin and eosin (H&E) stain. Figure 4.—Animal sacrificed 26 weeks postimplantation. Site of implantation of thedownsized test device in the dorsolumbar area. A large cavity (asterisk) is the original site of implantation. Remaining fragmented pieces of theimplanted device (arrows) are each surrounded by separate fibrotic capsules. Hematoxylin and eosin (H&E) stain. Figure 5.—Animal sacrificed26 weeks postimplantation. This higher magnification of an implantation site contains a fragmented piece of the downsized test device (asterisk) ina cavity surrounded by a mature fibrotic capsule. A discontinuous single layer of macrophages (arrow) and multinucleated giant cells (arrowheads)are present at the interface of the test device location and the fibrotic capsule. Hematoxylin and eosin (H&E) stain. Figure 6.—Animal sacrificed52 weeks postimplantation. Low magnification view of the site of implantation from the dorsolumbar area showing microscopic fragments of thebiodegradable test device encapsulated by mature fibrous connective tissue (arrows). Hematoxylin and eosin (H&E) stain.
Ramot et al. 9
Figure 7. Animal sacrificed 52 weeks postimplantation. Higher magnification view of the dorsolumbar site of implantation (shown in Figure 6)with microscopic fragments of test device embedded in a matrix of mature fibrous connective tissue. Hematoxylin and eosin (H&E) stain. Figure8.—Animal sacrificed 52 weeks postimplantation. Higher magnification of Figure 7 showing fragments of the implanted device surrounded by amature fibrotic response. Hematoxylin and eosin (H&E) stain. Figure 9.—Animal sacrificed 52 weeks postimplantation. High magnification showingthe host reaction to fragments of test device material (asterisks). A discontinuous layer of macrophages (arrow) is at the interface of the fibrotic capsuleand test device location. A giant cell (arrowhead) is adjacent to a test device fragment. Hematoxylin and eosin (H&E) stain. Figure 10.—Animalsacrificed 52 weeks postimplantation. Site of implantation with fragments of the implanted test device (asterisks) surrounded by mature connec-tive tissue on the right and fibrosarcoma (arrows) on the left. The fibrosarcoma cells are more densely cellular than the mature connective
10 Toxicologic Pathology
nonenzymatic hydrolytic cleavage of ester groups; and second,
when the polymer has a lower molecular weight and is more
highly crystalline, it is subject to intracellular degradation by
macrophages, giant cells, and fibroblasts.
With the exception of a single fibrosarcoma, the tissue
responses in both rat studies reported here had an expected
favorable host response to the test device implants. The test
device was identified at all time points up to 52 weeks. The
implantation sites were histologically characterized by cavity
formation surrounded by a mature fibrotic capsule without evi-
dence of any adverse acute or chronic active inflammation. The
intact as well as fragmented device was associated with a single
layer of macrophages with occasional giant cells at the inter-
face of the copolymer and the fibrotic capsule with presence
of lymphocytes and rare polymorphonuclear cells within the
fibrotic capsule. This represents a well-ordered foreign body
reaction that would ultimately lead to complete removal of the
implanted copolymer material. Copolymer fragmentation was
present at 26 weeks with subsequent progression to very small
fragments (<1 mm), associated with mass and molecular
weight loss of isolated implants in rats at 52 weeks.
In a single animal at the 52-week termination point, a pleo-
morphic fibrosarcoma was noted at the site of implantation. It
was grossly and histologically intimately associated with the
implant and was therefore considered related to treatment.
However, there is vast literature documenting the association
between the long-term close contact between inert materials
and the induction of sarcoma in rodents. For example, sarco-
mas were reported to develop at sites of plastic film implanta-
tion (unplasticized vinyl chloride vinyl acetate copolymer of
different sizes and shapes; Brand, Buoen, and Brand 1976) or
after implantation of radiotelemetric transmitters (Shoieb
et al. 2012).
A long-term subcutaneous and intramuscular implantation
experiment (up to 106 weeks) in 70 rats, using poly-L-lactide
(PLLA), the principal (70%) component of the current test
device, resulted in the development of 3 cases of fibrosarcoma
located close to the implanted material (Pistner et al. 1993).
According to the authors, this phenomenon of tumors occurring
in rodents following long-term implantation is related to the
‘‘Oppenheimer phenomenon’’ or ‘‘solid state’’ carcinogenesis
correlated with the induction of sarcomas in rodent animal
models implanted with solid materials (i.e., inert plastics,
metals, and other materials), even though the materials them-
selves have no inherent toxic or tumorigenic properties (Kirk-
patrick et al. 2000; Schoen 2013).
A thorough histopathological evaluation was performed on all
tissues extracted from the implantation sites in the 2 studies
described by us. This investigation included all capsular tissues
involving the implanted material. It was confirmed that no foci
indicative of mesenchymal cell hyperplasia, suggestive of preneo-
plasia, were detected. Furthermore, histopathology evaluation of
Figure 13. (A) Dry weight measurements of the explanted test devices. Mean mg+ standard error of the mean (SEM), n¼ 5 for 4 and 26 weeks, n¼8 for 12 weeks, and n¼ 2 for 16 weeks. ***p < .001. (B) Molecular weight measurements of the explanted test devices. Mean Daltons+ SEM, n¼ 6for 4 weeks, n¼ 8 for 12 weeks, n¼ 2 for 16 weeks, n¼ 11 for 52 weeks. **p < .01, ***p < .001. one-way analysis of variance, Bonferroni’s posttest.
Figure 10.—(Continued) tissue on the right. Hematoxylin and eosin (H&E) stain. Figure 11.—Animal sacrificed 52 weeks postimplantation.Higher magnification of Figure 10. A storiform growth pattern (arrows) is present in the fibrosarcoma adjacent to small device fragments (aster-isks). Hematoxylin and eosin (H&E) stain. Figure 12.—Animal sacrificed 52 weeks postimplantation. Same site presented in Figures 10 and 11showing pleomorphic round and spindle cells (arrows) present in the fibrosarcoma adjacent to a device fragment (asterisk). Hematoxylin andeosin (H&E) stain.
Ramot et al. 11
implantation sites from other animal studies with the same device
material performed in pigs, dogs, and rabbits (data not shown),
with up to 1-year follow-up, indicated the formation of capsular
reaction with minimal inflammatory cell infiltration, without any
local mesenchymal cell proliferation suggestive of preneoplasia.
The histopathology evaluation also showed that there was a
time-related effect on the loss of integrity of the implanted
devices, with their being broken into more pieces and sur-
rounded by a relatively more intensive mononuclear cell
reaction at the 52-week than at the 26-week time point. The
mononuclear cell reaction is the expected body response to the
presence of biodegradable material and indicative of progressive
absorption of the test device material (PLCL; Nyska et al. 2014).
In this regard, it is relevant to mention the pivotal work of
Kirkpatrick et al. (2000), in which histopathological evaluation
was performed on rats up to 2 years postimplantation of bioma-
terials (i.e., polymers, metals, and ceramic). The authors
reported an approximately 26% incidence of various types of
mesenchymal tumors as well as the presence of a spectrum
of changes defined as ‘‘focal proliferative lesions through pre-
neoplastic proliferation to incipient sarcoma.’’ The authors
concluded that it is unlikely to implicate chemical sarcomagen-
esis as the prime etiological factor, due to the vastly different
structures involved, each having entirely different chemical
composition. In addition, it is improbable that leachable resi-
dual monomers from the polymer synthesis and/or compounds
used in the plasticizing process or other leachables could play a
pathogenic role, due to the standard medical grades of the
materials used in this investigation.
On the other hand, there are several studies reporting the
clinical and histopathology safety aspects of PLCL PLLA.
None of these studies identified local preneoplastic or neoplas-
tic reactions at the site of implantation in humans. Pistner et al.
(1993) reported that the only local long-term (i.e., 2 to 3 years
of follow-up) reaction observed in humans implanted with
crystalline block polylactide consisted of an ‘‘intense resorp-
tive histiocytic reaction,’’ but without sarcoma development.
McCarty et al. (2013) reported on a large series of patients with
complications observed following utilization of PLLA
implants to treat either labral or RC pathology. The follow-
up time was from 2 to 106 months after implantation. Lesions
included giant cell reaction in 84%, presence of polarizing
crystalline material in 100%, papillary synovitis in 79%, and
arthroscopically documented outerbridge grade III or IV chon-
dral damage in 70%. No local tumor induction was noted.
Freehill et al. (2003) conducted a retrospective cohort study
to investigate the complications related to the use of PLLA
implants for arthroscopic shoulder stabilization procedures.
The follow-up period ranged from 8 to 33 months. Synovial
biopsies showed giant cells and histiocytes typical of a for-
eign-body reaction. Typically, also in this study, the crystalline
structures, remnants of the implanted material, were sur-
rounded by a granulomatous reaction. No carcinogenic find-
ings were mentioned or noted in this study.
The exact pathogenesis of local foreign body carcinogenesis
is still not entirely clear. A potential mechanism could involve
long-term local minor inflammation. It is postulated that local
inflammation, even transient, could lead to the release of
growth factors and reactive oxygen species from the inflamma-
tory cells, followed by expansion of stem cell pools and by
the transient activation of the Hh and Wnt signaling pathways.
This activation increases the likelihood of accumulation of
multiple additional mutations, and eventually to tumor forma-
tion (Gottschling et al. 2001; Shoieb et al. 2012; Upham and
Wagner 2001). According to Amini, Wallace, and Nukavarapu
(2011), there is increasing evidence that the cause of the sar-
coma formation observed at the site of implantation of biode-
gradable polymers is not due to the chemical contents but
rather due to the implant’s form and size, length of the implan-
tation, and degree of inflammation.
The fact that local carcinogenesis was detected in only a sin-
gle rat implanted with the test device, without the presence of
any preneoplastic changes (i.e., hyperplasia) in other animals
from these experiments, as well as the fact that the manufactur-
ing process is free of harmful reagents that may be carcino-
genic, it is reasonable to suggest that a nongenotoxic
mechanism was involved in the pathogenesis of the fibrosar-
coma, and eventually, a threshold for human risk may be
suggested. In this regard, Grasso, Sharratt, and Cohen (1991)
extensively reviewed the potential link between sustained tis-
sue damage induced by nongenotoxic test materials/agents in
rodents, as a predisposing factor to tumor development. Their
analysis highlights the fact that at various sites (i.e., connective
tissue, liver, bladder, and forestomach), there is a threshold
dose, below which neither sustained tissue damage nor tumor
induction occurs, but above this level both effects are
manifested.
In general, rodents are much more prone than humans to
develop tumors of mesodermal origin, such as sarcomas, in
reaction to foreign body implantation. This might stem from
the fact that murine pericytes, the cells that serve as the origin
for the foreign body sarcomas, have an exceptionally labile
genome (Brand 1987). Therefore, when these cells are forced
into proliferation, they are much more likely to develop spon-
taneous genome errors.
Conclusions
With respect to the tissue response in the 2 rat studies, it can be
concluded that the PLCL test compound did not have irritant
properties upon long-term exposure and was favorably toler-
ated without foreign body granulomatous reaction, mineraliza-
tion, necrosis, or immune-mediated reactions indicative of an
adverse effect (Northup 1999; Nyska et al. 2014; Onuki et al.
2008; Ramot et al. 2015; Schuh 2008). There was a well-
ordered host response to the implant material with temporal
degradation of the copolymer over the 52-week follow-up
period.Given the lack of significant local irritation/inflammation
and lack of preneoplastic change in any of the rats implanted
with the test device for 1 year, we conclude that the single case
of fibrosarcoma reflects the known species-specific rodent
12 Toxicologic Pathology
predilection to develop rare mesenchymal neoplasms at
implantation sites (Greaves et al. 2013; Schoen 2013). We con-
clude that the risk of implantation-associated cancer in humans
implanted with the InSpace device, which is composed of the
biodegradable PLLA acid and epsilon-caprolactone copoly-
mer, is negligible.
Author Contribution
Authors contributed to conception or design (AN, EM, AD); data
acquisition, analysis, or interpretation (YR, AN, EM, AD, AK, MZ,
AJD, RM); drafting the manuscript (YR, AN, EM, RM); and critically
revising the manuscript (YR, AN, EM, AD, AK, MZ, AJD, RM). All
authors gave final approval and agreed to be accountable for all
aspects of work in ensuring that questions relating to the accuracy
or integrity of any part of the work are appropriately investigated and
resolved.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest
with respect to the research, authorship, and/or publication of this arti-
cle: Abraham Nyska and Robert R. Maronpot serve as consultants for
Ortho-Space Ltd. Yuval Ramot has received an honorarium from
Ortho-Space Ltd. Elana Markovitz and Assaf Dekel are Ortho-Space’s
Directors. Abraham J. Domb is an inventor of the biodegradable bal-
loon technology and has been associated with Ortho-Space. His con-
tribution to this research was not supported and was for scientific
interest only.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: The study
was funded by Ortho-Space Ltd.
Supplemental Material
The online data supplements are available at http://tpx.sagepub.com/
supplemental.
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