In vivo biocompatibility of three different chemical

compositions of Ricinus communis polyurethane

Valdemar M. R. Barros,1 Adalberto L. Rosa,1 Marcio M. Beloti,1 Gilberto Chierice2
1
Department of Oral and Maxillofacial Surgery, School of Dentistry of Ribeirao Preto, University of Sao Paulo,
Av. do Cafe, s/n, 14040-904 Ribeirao Preto, SP, Brazil
2
Department of Analytical Chemistry and Polymer Technology, Chemistry Institute of Sao Carlos, University of Sao
Paulo, Av. do Trabalhador Sao-Carlense, 400, 13560-970 Sao Carlos, SP, Brazil
Received 27 August 2002; revised 11 February 2003; accepted 6 March 2003

Abstract: Alteration in the chemical composition of a biomaterial may be undertaken to improve its biological properties. The aim of this work was to evaluate the biocompatibility of three chemical compositions of Ricinus communis
polyurethane (RCP): RCPp (pure RCP), RCP CaCO3, and
RCP Ca3(PO4)2. RCP cylinders were surgically implanted
in rabbit femurs. After 8, 12, and 16 weeks, the femurs were
removed, xed, sectioned, ground, and stained by Stevenels
blue/Alizarin red S for light microscopy and histomorphometry. The osseointegration and osseoconductivity were
calculated by means of image analysis and the data were
submitted to the Kruskal-Wallis test followed by Dunns
test. Osseointegration was already completed after 8 weeks
on RCP Ca3(PO4)2 because similar values were found

INTRODUCTION
Reconstruction of local bone defects resulting from
trauma or bone tumors is a major problem in orthopedic and dental surgery. Currently, large amounts of
autogenous bone grafting are used to solve these clinical situations. Although autogenous bone grafts,
which combine osteoconductive, osteogenic, and osteoinductive properties, are the gold standard for
treating bone defects, only a few donors sites in the
skeletal system are appropriate for supplying the autogenous bone.1 Furthermore, morbidity is always associated with harvesting of autogenous bone.2 Because of these limitations, biomaterials are often used
as an alternative or supplement to autogenous bone.3
Polymers are the most versatile class of biomateriCorrespondence to: V. M. R. Barros; e-mail: vmbarros@
forp.usp.br
Contract grant sponsor: Fundacao de Amparo a` Pesquisa
do Estado de Sao Paulo (FAPESP); contract grant number:
00/2549-0
2003 Wiley Periodicals, Inc.

from week 8 to 16, whereas it showed a time-dependent

increase on RCPp and RCP CaCO3. The osseointegration
was greater on RCP Ca3(PO4)2 in all periods when compared with RCPp, and after 8 and 12 weeks when compared
with RCP CaCO3. None of the RCP samples presented
osseoconductivity. The present results showed that RCP
blended with calcium phosphate improved the biocompatibility by both enhancing and accelerating its osseointegration. Based on the absence of osseoconductivity, RCP was
considered to be a bioinert material. 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 235239, 2003
Key words: Ricinus communis polyurethane; osseointegration; osseoconductivity; polymer blend; biocompatibility

als, being extensively applied in medicine and biotechnology.4 Compared with other types of biomaterials, such as metals and ceramics, polymers offer the
advantage that they can be prepared in different compositions with a wide variety of structures and properties.5 Biodegradable polymers often have been combined with bioceramics to produce materials for bone
repair.6 These materials should maintain adequate
mechanical strength over critical phases of the tissue
healing process, should be osteoconductive, and
should degrade at a controlled rate to provide space
for the formation of new bone.6 Current research and
development is focused on tissue engineering, for
which such materials are considered to have a particularly signicant potential.5
Polymers used as biomaterials can be naturally occurring, synthetic, or a combination of both. A natural
polyurethane resin obtained by polymerization of the
polyester polyol, derived from Ricinus Communis, a
tropical castor bean, has been developed for bone
repair [Ricinus Communis polyurethane (RCP)]. This
polyurethane resin showed broblastic neoformation
progressively replaced by bone around and inside the

236

BARROS ET AL.

porosities of material with absence of late inammatory reaction, and no signs of systemic toxic effects,
when tested in rabbits.7 The biocompatibility of RCP
when implanted into rat alveolus was also observed.8
Histological examination of RCP implanted in rabbit
skulls showed bone neoformation after 6 weeks; however, the implant incorporation was not observed even
after 24 weeks.9
Alterations in the polymer chemical composition
may be undertaken to improve its biological properties. The addition of calcium carbonate to the RCP
composition produced a polymer blend that showed
better osseointegration than pure RCP (RCPp) when
implanted in rabbit tibia.10 Although the modication
of a polymers chemical composition by addition of
calcium carbonate can improve its biocompatibility,
the addition of calcium phosphate may represent a
more adequate choice. Both calcium carbonate and
calcium phosphate are known to be osteoconductive11;
however, studies using human osteoblasts showed
greater cell adhesion, proliferation, and differentiation
on calcium phosphate than on calcium carbonate.12
Previous study of the in vitro biocompatibility of
RCP showed that RCPs blended with calcium carbonate or, better yet, calcium phosphate, by favoring
events that promote matrix mineralization are more
biocompatible materials.13 In agreement with this, we
hypothesized that the addition of either calcium carbonate or calcium phosphate would improve the RCP
in vivo biocompatibility. To test this hypothesis, cylinders of RCP blended with calcium carbonate, and
blended with calcium phosphate were implanted in
rabbit femurs and their biocompatibility was evaluated by measuring the level of osseointegration and
osteoconductivity.

MATERIALS AND METHODS

nation of ketamine and xylazine (50 mg and 3 mg/kg, respectively). After a local anesthesia with 2% mepivacaine
plus adrenaline 1:100,000, the femoral shaft was aseptically
exposed and the periosteum was gently scraped from the
cortical bone. Two monocortical holes, 10 mm apart, were
drilled under copious irrigation with sterile saline using
burs at low speed. The RCP cylinder was gently tapped into
the hole until it was totally located beneath the external
surface of the cortical bone. The RCP samples were randomly distributed between distal and proximal holes in the
femoral shaft. The wound was closed with sutures. The
animals were maintained in cages and were permitted
weight bearing during the whole postimplantation period.
At 8, 12, and 16 weeks after implantation, the animals were
euthanized with a lethal dose of thiopental and the femurs
were harvested and processed for morphological and histomorphometric analyses. These periods of harvesting were
selected based on our previous work in which the biocompatibility of hydroxyapatite was evaluated by the same
method.14

Histological processing
The bone segments containing the RCP cylinders were
ground sectioned for light microscopy as previously described.15 Briey, immediately after harvesting the femurs,
the bone segments containing the RCP cylinders were immersed in 3% glutaraldehyde buffered with 0.1M sodium
cacodylate (pH 7.3) for 90 min. After xation, bone segments
were dehydrated in graded concentrations of alcohol and
embedded in resin (LR White Hard Grade, London, UK).
After polymerization, resin blocks were sectioned with a
diamond knife (Microslice 2; Ultra Tec Manufacturing Inc.,
Santa Ana, CA) to produce two samples per implant. Each
sample was polished and mounted on glass slides and the
resulting 40-m-thick mounted sections were further
ground and polished to a thickness of 20 m. Samples were
stained with Stevenels blue and Alizarin red S stain for light
microscopy.

RCP samples
RCP cylinders 2 mm in diameter and 4.5 mm high were
manufactured with three different chemical compositions:
RCPp (pure RCP), RCP CaCO3 (RCP blended with 30%
calcium carbonate), and RCP Ca3(PO4)2 (RCP blended
with 30% calcium phosphate). Before implantation procedures, all cylinders were cleaned in an ultrasonic bath and
autoclaved. Cylinders were kindly supplied by the Chemistry Institute of Sao Carlos at the University of Sao Paulo.

Implantation procedures
Twelve male rabbits (3 months old, 2.0 2.5 kg) received
two RCP cylinders in each femur. Animals were anesthetized with acepromazin (1 mg/kg), followed with a combi-

Histomorphometric analysis
Biocompatibility of the RCP samples was based on their
osseointegration and osseoconductivity capacity as showed
previously.14 For that, the perimeter of the transversal section of the samples was measured followed by the measure
of the length of this perimeter in close contact with the bone
matrix using a 10 original magnication. Osseointegration
was expressed as a percentage of the RCP cylinder surface in
close contact with the bone matrix of the cortical bone
whereas osseoconductivity was expressed as a percentage of
the RCP cylinder surface in close contact with the bone
matrix in the medullary canal (Fig. 1). Both of these parameters were determined using an image analyzer (Image Tool;
University of Texas Health Science Center, San Antonio,
TX).

IN VIVO BIOCOMPATIBILITY OF RCP

237

osseointegration was affected by the RCP chemical

composition after 8 weeks (H 8.43; df 2; p 0.01)
in the following order: RCPp RCP CaCO3
RCP Ca3(PO4)2; and after 12 weeks (H 7.61; df
2; p 0.02) in the following order: RCPp RCP
CaCO3 RCP Ca3(PO4)2. After 16 weeks (H 6.63;
df 2; p 0.03), there were no statistically signicant

Figure 1. Illustration of a transversal section showing the

RCP cylinder implanted in the femur. Solid line indicates
where osseoconductivity was measured and dashed line
indicates where osseointegration was measured.

Statistical analysis
The data presented in this work were subjected to the
Kruskal-Wallis test followed by Dunns test. Differences at
p 0.05 were considered statistically signicant.

RESULTS
Histological analysis
Histological examination revealed unresorbed RCP
cylinders localized in the implantation sites with no
persistent inammation or foreign body reaction. After 8 weeks of implantation, cortical bone regeneration
was observed around all RCP samples at the implantation sites [Fig. 2(A)]. New bone tissue showed close
interfacial contact with RCPs samples, and the mineralized bone matrix was characterized by mature bone
[Fig. 2(B)]. A layer of soft tissue was observed in close
contact to the RCP surface in the medullary canal [Fig.
2(C)]. This tissue was composed of a few elongated
cells and collagen bers located parallel to the RCP
surface. Morphological differences at the RCP bone
interface among the three compositions of RCP were
not observed.
Histomorphometric analysis
The percentage of osseointegration of the samples is
showed in Table I and Figure 3. The percentage of

Figure 2. Ground sections of RCP implanted in rabbit femurs stained with Stevenels blue/Alizarin red S. (A) Crosssection of RCP Ca3(PO4)2 cylinder after 8 weeks of implantation. Arrow indicates the direction of cylinder
implantation. Original magnication 5. (B) RCP CaCO3
tissue interface showing the cortical bone in close contact to
the implant after 12 weeks. Original magnication 10. (C)
RCPptissue interface showing soft tissue in close contact
with the implant, in the medullary canal after 8 weeks of
implantation. Original magnication 10.

238

BARROS ET AL.

TABLE I
Percentage of Osseointegration of the RCP Samplesa
Material
Period (weeks)

RCP

RCP CaCO3

RCP Ca3(PO4)2

8
12
16

10.42 11.12 (5)

26.73 1.47 (3)
37.50 8.25 (5)

23.23 20.28 (3)

33.36 13.03 (9)
47.15 12.87 (4)

55.62 20.80 (5)

61.68 16.97 (4)
60.14 15.17 (5)

All data are reported as means standard deviation. The number in parentheses represents the sample size.

differences between both RCPp and RCP CaCO3,

and RCP CaCO3 and RCP Ca3(PO4)2, but osseointegration was greater on RCP Ca3(PO4)2 when
compared with RCPp.
The percentage of osseointegration of the RCPp was
a time-dependent process (H 9.92; df 2; p 0.007)
in the following way: 8 weeks 12 weeks 16 weeks,
but RCP CaCO3 (H 3.63; df 2; p 0.16), and
RCP Ca3(PO4)2 (H 0.22; df 2; p 0.89) were not
affected by the implantation period.
None of the RCP samples presented osseoconductivity, even after 16 weeks of implantation.

DISCUSSION
The healing response after implants is modulated
by such factors as animal species, site of implantation,
and characteristics of the biomaterial.16,17 The biopolymers have been tested for application in orthopedic
and dental surgery.8,18 In this study, the biocompatibility of three different chemical compositions of RCP
was evaluated. The present results showed that all
implants, independent of the RCP composition, were
biocompatible, as demonstrated by the quality of the
RCPtissue interface and by the capacity for osseointegration.
The contact between a biomaterial and tissue may
show a range of responses, from induction of an inammatory response to the perception of the material

Figure 3. Degree of osseointegration of RCP samples. All

data are reported as means standard deviation.

as like-tissue invoking no reaction.19 The biocompatibility of a polymer depends on the specic adsorption of proteins to the polymer surface and the subsequent cellular interactions. Because of the nature of
these interactions, modications of the material have
been used to optimize cell response.5 In this study, a
polymer derived from Ricinus communis was blended
with calcium carbonate or calcium phosphate to improve its biocompatibility. Because all samples were
manufactured under the same conditions, the topography of all surfaces was the same for all evaluated
groups, with the exclusion of factors other than chemical composition that could inuence osseointegration.
The biocompatibility of RCP has been documented
both in vitro and in vivo. Carvalho et al.8 observed that
granules implanted into rat alveolus were encircled by
a brous capsule, which became thinner, and by the
sixth postoperative week, the tested area was almost
totally covered with mature bone. The presence of
RCP in the cervical third of rat alveolus led to a
retardation in the wound-healing process.20 The addition of calcium carbonate to the RCP composition
produced a polymer blend that showed better biocompatibility than RCPp when implanted in rabbit tibia.10
More recently, we showed in vitro that RCP blended
with calcium phosphate presented more bone-like formation than RCPp or RCP blended with calcium carbonate using rat bone marrow cells.13 However, this is
the rst in vivo study to compare the biocompatibility
of RCPp, RCP blended with calcium carbonate, and
RCP blended with calcium phosphate.
The RCPtissue interface exhibited areas in which
mineralized bone tissue was in very close contact to
the RCP surface, suggesting that bone was laid down
in direct contact to the RCP. In different experimental
systems, it has been shown that a mineralized bone
matrix can be produced in close contact to the RCP
surface.7,8,20 There were no morphological differences
at the RCP bone interface with respect to the differences in samples chemical composition, despite the
fact that osseointegration was affected by the RCP
chemical composition. Our results showed that osseointegration was increased by the presence of calcium carbonate at the 16th week and calcium phosphate after all evaluated periods. In addition, after 8
weeks, the osseointegration process was already com-

IN VIVO BIOCOMPATIBILITY OF RCP

pleted on RCP Ca3(PO4)2 because it did not increase

from 8 to 16 weeks. The osseointegration of RCPp and
RCP CaCO3 was considered to be a time-dependent
process because an increase was observed from week
8 to 16, even though this increase was not statistically
signicant for RCP CaCO3. In agreement with our in
vitro study of the biocompatibility of RCP,13 the
present results showed that the addition of calcium
phosphate improved the biocompatibility of the RCP
by both enhancing and accelerating its osseointegration. Because of this, further studies will be made to
establish a more adequate percentage of calcium phosphate to improve the RCP biocompatibility.
Recently, we have determined the biocompatibility
of hydroxyapatite evaluating the same parameters, in
the same animal model after 8 and 12 weeks of implantation.14 Hydroxyapatite presented a higher degree of osseointegration than RCP and also presented
osseoconductivity whereas RCP did not. This different
behavior could be attributed to the difference in their
biocompatibility spectrum. Considering that hydroxyapatite is a well-known bioactive material,21,22 it is
possible to conclude that RCP is a bioinert material.

239

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