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: anyska@bezeqint.net

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

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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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