with United States Pharmacopoeia polyethylene and the third control empty cage .... pockets. Three animals were used for these studies. Analysis of exudate.
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.
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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
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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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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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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
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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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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. References Allison, A. C, Harington, J. S. and Birbeck, M. (1966) J. Exp. Med., 124, 141. Anderson, J. Μ. and Miller, K. Μ. (1984) Biomaterials. 5, 5. Baier, R. Ε. and Dutton, R. C. (1969) J. Biomed. Mater. Res., 3, 191. Imai, Y., Watanable, A. and Masuhara, E. (1979) Trans. Am. Soc. Artif. Intern. Organs. 25, 299. Issekutz, A. C, Movat, Κ. W. and Movat, Α. Ζ. (1980) Clin. Exp. Immunol., 41, 512. Issekutz, A. C. and Movat, H. Z. (1980) Lab. Invest., 42, 310. Issekutz, Τ. Β., Issekutz, Α. C. and Movat, Η. Ζ. (1981) Am. J. Pathol., 103, 47
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Kozma. C, Macklin, W., Cummins. L. M. and Maver. R. (1974) in The biology of the laboratory rabbit (eds S. A. Weisbroth, R. F.. Flat and A. C. Kraus) (New York: Academic Press) p. 50. Kozma. C. K., Pelas, A. and Salvador. R. A. (1967) J. Am. Vet. Med. Assoc, 151, 865. Marchant. R.. Hiltner, Α., Hamlin, C, Rabinovitch, Α., Slobodkin, R. and Anderson, J. Μ. (1983) J. Biomed. Mater. Res., 17. 301. Marchant. R., Miller, Κ. Μ. and Anderson, J. Μ. (1984) J. Biomed. Mater. Res., 18, 1169. Matlaga, Β. F. and Salthouse, Τ. Ν. (1983) J. Biomed. Mater. Res., 17, 185. McNamara, A. and Williams, D. F. (1981) Biomaterials, 2, 33. Nathan, C. F. (1987) J. Clin. Invest., 79, 319. Neuman. Α. W., Absolom, D R., Zingg, W., Van Oss, C. J. and Francis, D. W. (1983) in Biocompatible polymers. metals and composites (ed. Μ. Sycher) (Lancaster: Technomic Pub.) p. 53. Reinhold. J. G. (1953) in Standard Methods Clin. Chem. (ed. M. Reiner) (New York: Academic Press) vol. 1. p. 38. Spilczewski, K. L.. Anderson, J. M., Schaap. R. N. and Solomon, D. D. (1988) Biomaterials, 9, 253. Varley, H. (1967) Practical clinical biochemistry (London: William Heinemann Medical Books Ltd.) p. 455.
