J. Biosci., Vol. 14, Number 3, September 1989, pp. 289–299. © Printed in India.
In vivo biocompatibility of aliphatic segmented polyurethane in rabbit
M. JAYABALAN*, K. RATHINAM, T. V. KUMARY and
MIRA MOHANTY
Divisions for Technical Evaluation of Biomaterials, Toxicological Screening of Materials
and Pathophysiology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for
Medical Sciences and Technology, Satelmond Palace, Trivandrum 695 012, India.
MS received 24 April 1989; revised 24 July 1989
Abstract. An aliphatic segmented polyurethane with soft to hard segment ratio 3 was
synthesised using hexamethylene diisocyanate, polypropylene glycol 400 and 1,4-butane
diol.A stainless steel cage implant system has been used to study the in vivo
biocompatibility of this polyurethane. United States Pharmacopoeia negative control
polyethylene was used for the comparison. Three cages, one with polyurethane another
with United States Pharmacopoeia polyethylene and the third control empty cage were
implanted subcutaneously in the dorsal aspect of rabbits. The inflammatory exudate
surrounding the material was aspirated from the cages on 4, 7, 14 and 21 days after
implantation. The total protein content in the exudate aspirated from all the 3 cages was
significantly higher at 7 days than in the reported normal rabbit serum of New Zealand
white rabbit but equal to that of our rabbit colony. The albumin concentration was lower
in the initial period but increased at 21 days post implantation period in all the cages.
Concentration of α1, α2 and γ-globulin also decreased in all cages at 21 days. Neutrophils
were predominant in all the exudates aspirated from polyurethane, polyethylene and
empty control cages during whole implantation period. This is attributed to the profound
effect of the cages on the surrounding vasculature. Macrophage was found to be seen
during acute phase of inflammation due to the migration of macrophage along with
neutrophil towards the inflammatory lesion. The percentage of neutrophils showed a faster
decline in the cage containing polyethylene at 21 days. The extra cellular alkaline
phosphatase activity, though higher in exudate from cages containing polyurethane at 14
days post implantation, was same in all 3 cages at 21 days. Leucine amino peptidase
activity was found to be decreased at 21 days of post implantation time though the empty
control cage exhibited an increase at 14 days post implantation. The inflammatory
response at 21 days was similar in polyurethane and the control polyethylene.
Keywords. Cage implants; aliphatic segmented polyurethane; inflammatory exudate;
proteins; leukocytes; enzymes; biocompatibility.
Introduction
Polyurethane has been widely used for biomedical applications which result in
short term or long term contact between the material and tissues of the body. The
tissue compatibility of the polyurethane depends upon the response of the cells and
their enzymes on the material and vice versa. One of the most important factors
which affect the material in vivo biocompatibility is the cellular interactions which
occur at the material-tissue interface. Histopathological techniques have been
widely adopted to determine the tissue tolerance of material with subcutaneous
implants and intramuscular implants. These histopathological techniques offer the
* To whom all correspondence should be addressed.
Abbreviations used: HDI, Hexamethylene diisocyanate; PPG, polypropylene glycol; BD, 1, 4-butane diol;
US P, United States Pharmacopoeia.
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gross tissue evaluations but they provide less information regarding cellular
interactions at the material-tissue interface. It is difficult to investigate material/cell
interactions at the interface during different periods of implantation under in vivo
conditions with the histopathological techniques. Imai et al. (1979) have introduced
a cell culture biocompatibility test system to study the material and cell interaction. However, this in vitro technique does not simulate the dynamic nature
of the cellular interaction at the interface. Marchant et al. (1983) have adopted a
new technique 'cage implant system' in which the candidate material is allowed to
bathe in the inflammatory exudate under in vivo conditions. The technique enables
to assess the level of inflammatory response to an implanted material. The latest
report by Spilezewski et al. (1988) deals with in vivo biocompatibility of catheter
materials investigated by the cage system. In all these studies the rat has been
selected as the animal model. In our present studies we have selected rabbit as the
animal model for in vivo biocompatibility of the candidate aliphatic segmented
Polyurethane using cage implant system.
Experimental
Preparation and characterization of polyurethane
The candidate polyurethane is polyether based. Hexamethylene diisocyanate (HDI)
and polypropylene glycol (PPG, molecular weight 400), were used (Fluka A.G.).
1, 4-Butane diol (BD) (Merck Schuthardt) was used as the chain extender.
Dibutyltin dilaurate (0·05 pbw) from Fluka AQ was used as catalyst. Adequate
quantity of dimethyl acetamide was used for transferring the reactants to the
reaction vessel. All the reactants were degassed for 30 min before the reactions.
Initially a prepolymer was prepared under nitrogen atmosphere using HDI and
PPG 400. The reaction was conducted for 30 min under exothermic conditions
leading to the maximum temperature of 120-130°C. The reaction mixture was
cooled to 30°C. Then the prepolymer was chain extended with BD and allowed to
react exothermically leading to the maximum temperature of 110-120°C for 90 min.
The polymer liquid was poured in a preheated (60°C) glass plate and cured at 60°C
for 12 h, 80°C for 3 h and 100°C for 2 h. The cured polymer was immersed in
distilled water at 25°C for overnight. Then the polymer was subjected to soxhlet
extraction using carbon tetrachloride, methanol and diethyl ether for removing
unreacted components, catalysts etc. The purified polymer was dissolved with
dimethyl acetamide (HPLC grade) and cast into film on a clean silicone oil-coated
glass plate and dried in air oven at 60–70°C. The casted film was subjected to
physicochemical and mechanical testing.
The molecular weight of the polymer was determined by gel permeation
chromatography (Waters Associates, USA) using differential refractometer, Vacuum
distilled dimethyl acetamide with 0·05% lithium bromide was used as solvent. µStyragel columns with pore size 105, 104 and 103Å were used. Differential thermal
analyses were carried out using Dupont 990 thermal analyser. The samples were
heated from ambient to 400°C in air at the rate of 10°C/min. Tensile tests were
carried out using Instron Universal testing machine as per the ASTM standard D
882. The formulation and properties of this polyurethane are given in table 1.
Aliphatic segmented polyurethane in rabbit
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Table 1. Formulation and properties of candidate polyurethane.
Preparation and implantation of polyurethane cages
The stainless steel cages were fabricated using stainless steel wire mesh (austenitic
stainless steel supplied by M/s. Shieves India, Bombay. The stainless steel type 316
containing 71·9% α-Fe, 18% Cr, 8% Ni, 2% Mo and 0·1% C. The mesh size was 24
and the wire diameter was 0·015 in. The cage length was 3·5 cm and diameter was
1 cm. The cage was fabricated into cylindrical shape. All wire ends were directed
into the cage and then compressed along the inner surface seams. The finished cages
were cleaned with benzene in soxhlet extractor and then with soap water and
deionized water ultrasonically. The nonporous and smooth candidate polyurethane,
was cleaned ultrasonically in ether and cut into rectangular specimens of length
3 cm, width 0·5 cm and thickness 0·5 mm. The material was put into the cage and
cleaned once again. These cages were packed in clean room and sterilized with γradiation with 2·5 Μ rad dose. The cage with polyurethane was code named as K3
polyurethane. A second set of cage was prepared for biomedical grade polyethylene
supplied by United States Pharmacopoeia (USP) Inc., Rockville, Maryland, USA,
(G5) as negative control. The cage with this polyethylene was code named as K4
polyethylene. A third set of empty cage was also prepared to serve as control to
both Κ3 polyurethane and K4 polyethylene. This was code named as K7 empty
cage. Albino rabbits from the Institute colony were used. They were maintained on
Hind Lever Pellets and water ad libitum. The animals were anaesthetized and
prepared for surgery. Two cages, one with candidate polyurethane (K3) and another
with USP negative control polyethylene (K4) and a third empty cage (K7) control
were selected for implantation subcutaneously in the dorsal aspect of each animal.
A single incision, 2 cm in length was made on the midline. Blunt scissor dissection
was then used to create implant sites by tunneling subcutaneously in the lateral
position of cephaled and caudal direction. The cage implant K3 was then inserted,
the end of the cage being 2·5 cm from the incision and the incision was sutured.
Similarly K4 and K7 cages were implanted through two other subcutaneous
pockets. Three animals were used for these studies.
Analysis of exudate
The pale yellow inflammatory exudate that accumulated within the cages were
periodically aspirated at the post implantation period of 4, 7, 14 and 21 days under
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sterile conditions using a 22 gauge needle with a syringe. The exudate collected
each time was not more than 0·5 ml. All samples were tested for sterility. Two drops
of exudate collected asceptically were inoculated into 10 ml of fluid thioglycollate
medium (Hi-media Laboratories). The inoculated medium was incubated at 35°C.
This was observed daily for 7 days for bacterial growth, turbidity and change of pH
of the media. If the growth and turbidity increased with time, exudate was declared
as contaminated. The exudate was analysed for total leucocyte count, differential
cell count, lysosomal enzymes and total protein. Quantitative leucocyte count was
carried out using a haemocytometer and differential leucocyte count was
accomplished by staining the smears with Leishman stain. Alkaline phosphatase was
measured using disodium phenyl phosphate as substrate as per the method described
elsewhere (Varley, 1967). Total acid phosphatase, prostatic acid phosphatase and
leucine amino peptidase were determined using Sigma kit 104 and 251 respectively.
Total protein content was determined by the biuret method (Reinhold, 1953).
Protein distribution was estimated by cellulose acetate electrophoresis using
Cosmos (Japan) electrophoretic apparatus. At the end of 21 days of implantation
time the animals were sacrificed and the cages were removed, opened, and implanted
cage and surrounding tissues were examined macroscopically (figure 1).
Figure 1. K 3, K4 and K7 cages with polymer strip harvested from rabbit.
Results
Adsorption of proteins is the primary event when foreign implant surfaces are in
Aliphatic segmented polyurethane in rabbit
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contact with tissue (Baier and Dutton, 1969). The subsequent cellular interactions
are determined by these adsorped proteins (Neuman et al., 1983). Cell adhesion in a
more general way governs the total response of blood and tissue to the polymer
implants. The results of the protein analysis of the exudate are given in table 2. At 4
days post implantation time, it was not possible to aspirate the exudate except in
one of the K7 empty cages.
The extracellular total protein content in each exudate was determined and no
significant differences were observed in K3 polyurethane, K4 polyethylene and K7
empty cages at 7 days. After 21 days implantation period, the total protein content
decreased. The total protein content at 7 days was significantly higher in the exudate
than in the normal rabbit serum (5·2 ± 0·79 g/dl) according to the values obtained
by Kozma et al. (1967). However, the total protein content was equal to that in
normal rabbit serum of our rabbit colony (9 ± 0·5 g/dl). The electrophoretic distribution of the protein is also given in table 2. At 7 days the percentage concentration
of α2, β and γ-globulin in the exudate were higher than normal rabbit serum values
of New Zealand rabbit and our rabbit colony while the value for albumin was
lower. However the values for αl, α2, β and γ decreased appreciably from the
original value while the value for albumin increased at 21 days. At this period the
K3 polyurethane, K4 polyethylene and K7 empty cage have more or less same
albumin value.
The results on the white cell analysis of the exudate are given in table 3. The
differential cell counts are given in tables 4 and 5. The collection of exudate was
carried out at around 10 AM in the collection day to avoid the diurnal variation in
differential cell counts (Kozma et al., 1974). Neutrophil is the predominant cell in
the exudate at all periods in K3 polyurethane, K4 polyethylene and K7 empty
cages. However the absolute value does decrease appreciably at the completion of
21 days of implantation. Macrophages along with neutrophils were seen in one of
the exudates of the empty cages at the end of the period, 4 days. This is attributed
to the fact that macrophage initially migrate into the bacterial inflammatory sites
simultaneously with neutrophil in rabbit (Issekutz et al., 1981). The percentage of
macrophage started increasing in the latter period even in K4 polyethylene and K7
empty cages. This is due to the continued migration of macrophage into the inflamematory lesion long after the neutrophil have stopped (Issekutz et al., 1981). The
increase in the macrophage concentration in K3 polyurethane cages from 7 to 14
days indicates the resolution of acute inflammatory response and start mild chronic
response.
While the cellular interactions appeared to be different for all K 3 , K 4 and K 7
cages, significant differences were also observed in the extra cellular alkaline phosphatase activity (figure 2). The results indicate increased activity for K3 polyurethane at 14 days of post implantation time while the values for K 4 polyethylene
and K 7 empty cages decreased. Since the alkaline phosphatase is localized to the
specific granules of polymorphonuclear, leucocytes, activity of this enzyme is an
important parameter to judge acute phase of inflammation. The activities of total
acid phosphatase and 'prostatic' acid phosphatase reflect both the acute and chronic
phase of inflammation. The total acid phosphatase activity decreased in all cages
with implantation period (table 6). While the prostatic acid phosphatase activity
increased for K 3 polyurethane and K 4 polyethylene cages at 21 days, significant
differences were observed between K3 and K4 cages in the initial period of inflam-
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Aliphatic segmented polyurethane in rabbit
295
Table 3. Variation in total leucocyte concentration in the
exudate with implantation time.
Table 4. Variation in neutrophil and eosinophil concentration in the exudate with implantation time.
Unit:cells/mm3
Table 5. Variation in macrophage and lymphocyte concentration in the exudate with implantation
time.
Unit:cells/mm3
mation as observed from the values of leucocyte. Leucine amino peptidase activity
also decreased with implantation time for K3 polyurethane and K4 polyethylene
cages in comparison with that of K7 empty cage at 14 days of implantation (figure 3).
Discussion
The cage implant system offers an understanding of the acute and chronic phase of
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Jayabalan et al.
Figure 2. Variation of extracellular alkaline phosphatase activity in the exudate with
implantation time.
Table 6. Variation in extracellular total acid phosphatase and prostatic acid phosphatase activity on
the exudate with implantation time.
inflammation which gives insight to the material as well as tissue response. The
changes in the exudate during the implantation can be understood with the variation
in total protein, protein distribution, cells and enzymes concentration. During the
initial phase of inflammation albumin concentration decreased and α1- and α2globulin concentration increased in comparison with the reported values of New
Zealand white rabbit serum. The decrease in αl, α2 and γ-globulin concentration
and increase in albumin concentration in the latter period of implantation indicate
the resolution of acute phase of inflammation (Marchant et al., 1983).
The level of inflammatory reaction to an implant is used to assess the biocompatibility of the implant. The acute phase of inflammatory reaction induced by the
polymer implant in the cage is characterised by the predominance of polymorphonuclear leucocytes. The chronic phase of inflammation is characterized by the
predominance of mononuclear leucocytes including macrophages and lymphocytes.
Predominance of neutrophil in all K3 polyurethane, K4 polyethylene and K7 empty
Aliphatic segmented polyurethane in rabbit
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Figure 3. Variation in extracellular leucine aminopeptidase activity in the exudate with
implantation time.
cages during the whole implantation time is a noted feature. It has been observed
that the neutrophils stop migrating into the bacterial lesions after 8 h in rabbits
(Issekutz and Movat, 1980). Moreover the neutrophils in tissues have shorter life
and die at the site of inflammation (Issekutz et al., 1980). Therefore the continued
predominance of neutrophil in all the K3, K4 and K7 cages is due to the profound
effect by the stainless steel cages on the surrounding vasculature as observed in the
case of pure metals by McNamara and Williams (1981). The K3 polyurethane cage
exhibits lower concentration of leucocytes in 7 days. The K4 polyethylene and K7
empty cages exhibit lower concentration of leucocytes in the latter period of implanttation indicating the faster resolution of inflammatory response. The leucocyte value
remained more or less same for K3 polyurethane cage during the implantation
period.
Recently importance has been given to growth factors and inhibitors produced
by mononuclear phagocytes and their role in the chronic inflammatory reaction.
These growth factors produced by monocytes and macrophages and other inflammatory cells have implications in the implant associated wound healing responses
(Nathan, 1987). These growth factors could be released upon activation of the
macrophage and induce the proliferation of fibroblasts around the cage implant to
form a capsule.
The presence of extra cellular acid phosphatase and Leucine amino peptidase
indicates the cellular activation during the chronic phase of inflammation.
In this study the alkaline phosphatase activity at day 7 is more or less same for
K 3 polyurethane, K 4 polyethylene and K 7 empty cages. Though K 4 and K 7 have
higher number of neutrophils at day 7 compared to K 3, all the cells were not
activated. This may be due to the migration of cells to the wound site in response
to chemotactic stimuli as reported by Spilezewski et al. (1988). By day 14, K 3
polyurethane has higher value for extracellular alkaline phosphatase activity in
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Jayabalan et al.
comparison with K4 polyethylene and K7 empty cages. However at day 21, all the
cages exhibit more or less same value of alkaline phosphatase activity, eventhough
K3 has higher number of neutrophils.
It has been found that macrophages have more receptors than neutrophils and
adhere strongly to surfaces (Anderson and Miller, 1984; Matlaga and Salthouse,
1983) and spread on the surface. But the surface is never completely covered by the
spreading cells. However the process of releasing extracellular enzyme is carried out
by phagosome-lysosome fusion (frustrated phagocytosis). If the material leaches
cytotoxic material the extracellular enzyme release can occur either by exocytosis or
by cell lysis. It has been found by Allison et al. (1966) that the acid phosphatase
release was increased when the material ingested Bas cytotoxic. In this study both
total and prostatic acid phosphatase activity values do not appear to have direct
correlation with the leucocyte concentration.
Generally when the biological response is changing from acute phase of inflamemation to chronic phase of inflammation the leucine amino peptidase activity may
be relatively high in comparison with the original activity. The cages K3 polyurethane and K4 polyethylene exhibited decreasing trend for the value of extracellular leucine amino peptidase activity. The K7 empty cage showed higher activity
for the implantation time, 14 days. At this implantation time both the macrophage
and lymphocyte in K7 are found to be nil. Therefore it is inferred that the higher
release of extracellular leucine amino peptidase may be due to the interaction of
macrophage with lymphocyte. Lymphocyte is one of the predominant cells in the
peripheral blood of adult rabbits in the morning. Lymphocytes are capable of
influencing Chemotaxis and enhance macrophage activity through the extracellular
release of lymphokines (Marchant et al., 1984).
The trend of cellular response to material in the rabbit is similar to that seen in
the rat. The cage implant system in rabbit has been adopted in this study with a
view to study the effect of macrophage infiltration along with neutrophil in the
inflammatory response. Considering the leucocyte and protein interaction with the
candidate polyurethane and extracellular enzyme activity it is inferred that the
chronic inflammatory response is gradually reducing with implantation time and
the candidate polyurethane is biocompatible.
Acknowledgements
The authors acknowledge the fund provided by the Department of Science and
Technology, New Delhi under the SERC scheme to carry out the project on the
studies on material-tissue interface. Grateful acknowledgements are due to
Dr. M. S. Valiathan and Mr. A. V. Ramani, for providing the necessary facilities.
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