An Assessment of 2,4 TDA Formation from Surgitek Polyurethane Foam under Simulated
Physiological Conditions MICHAEL
SZYCHER, PH.D.
AND
ARTHUR A.
SICILIANO, PH.D.
PolyMedica Industries, Inc. 2 Constitution Way Woburn, MA 01801 -
1. SUMMARY
polyurethane foam used in the manufacture of mammary prostheses were enzymatically treated for a total of thirty days. Papain (a plant thiol endopeptidase which has similar activity to the human lysosomal enzyme cathepsin B) was our enzyme of choice since it has both amidase as well as esterase activity. The experiment was conducted under physiological conditions closely simulating the microenvironment likely to be found around an implanted mammary pros-
samplers of
thesis. In our tests, 2,4 TDA was formed during enzymatic attack of this TDI-based polyurethane foam for the first four (4) days, reaching a maximum of 8.3 parts per million. After the initial burst, no further TDA was observed within the limits of detection of the experiment (10 parts per billion). Based on standard risk assessment, this amount of TDA translates into a risk of developing cancer of one in four hundred million. 2. FORMATION OF lBIDA FROM NIDI-R%SED POLYURETHANES
A 1978 article by Darby [1] at Travenol Laboratories was the first to alert the scientific community that a medical-grade polyurethane, 323
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324
Pellethane 2363-80A, contained an aromatic amine analog of 4,4’ diphenyl methane diisocyanate (MDI), namely 4,4’ diaminodiphenyl rriethane, commonly designated as MDA. This study identified MDA in aqueous extracts of the polyurethane, and also demonstrated the mutagenic potential of MDA using Salmonella typhimurium test strains. Darby and coworkers exposed the MDI-based polyurethane to steam sterilization conditions for several hours. Under these forcing conditions, the polyurethane linkages underwent thermolysis, leading to the formation of MDA. 4,4’ Methylene Dianiline (MDA) is a carcinogen [2-5], a mutagen [6], and a teratogen [7]. Other chemicals closely related to MDA have been tested in laboratory animals and also found to be carcinogenic. Such chemicals are 4,4’ methylene-bis[2-methyl-aniline] [8]; 4,4’ methy-
lene-bis[2-chloroaniline] [7,8]; 3,3’ dichloro-4,4’ diaminodiphenylether [9]; and its parent compound 4,4’ diaminoethylether [10]. Animal studies indicate that MDA is very toxic [11]. The rat oral and subcutaneous LDso values are 347 and 200 mg/kg, respectively, while the mouse intraperitoneal LDso is 74 mg/kg [12]. The lowest published dose
reported to produce systemic toxicity in man is 8.42 mg/kg orally; thus, NIOSH classifies MDA as a primary irritant [13]. MDA is a pomutagen, which is reactive in the Ames test in the concentration range of 5 to 150 pig. These data suggest that detectable migration of tent
MDA from implantable aromatic polyurethanes might be unacceptable in critical care clinical applications, even in parts per billion levels. MDA can be formed in aromatic-based polyurethanes by two independent mechanisms. The first mechanism involves the anhydrous thermal degradation of polyurethanes, with concomitant formation of MDA, carbon dioxide, and an olefin, as shown in Figure 1. The anhydrous thermal decomposition of aromatic polyurethanes undergoes chain cleavage only very slowly at 150 to 200°C; furthermore, the presence of other reactants and catalysts also influences the speed of this
decomposition reaction. The second mechanism involves a two-step thermohydrolytic reaction. In the first step, the aromatic polyurethane undergoes a dissociation to the corresponding aromatic diisocyanate, and the precursor
Figure 1. Anhydrous thermal degradation of aromatic-based polyurethanes, leading to formation
of methylene
dianiline (MDA).
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325
Figure 2. Postulated thermohydrolytic degradation of aromatic-based polyurethanes, leading to accelerated formation of methylene dianiline (MDA). The source of water can be twofold: (1) as chemisorbed moisture in improperly dried pellets, or (2) during prolonged steam sterilization.
alcohol. In the second step, and in the presence of water, the aromatic diisocyanate reacts with water to form aromatic amines such as MDA. The postulated two-step mechanism is presented in Figure 2. This
postulated thermohydrolytic decomposition reaction can only occur in the presence of water, and is much faster than the decomposition reaction discussed above. This reaction is thought to occur during extrusion, if the pellets of thermoplastic polyurethane are not properly dried prior to thermal processing. The presence of MDA contamination in biomedical-grade aromatic polyurethanes was detected and quantified in plasma storage packages fabricated from an aromatic thermoplastic polyurethane elastomer. Food and Drug Administration (FDA) investigators have reported 3 parts per billion of MDA in human plasma stored in aromatic polyurethane storage bags [13a,13b]. Although 3 parts per billion of MDA (in a blood bag containing about 10 grams of polyurethane) would represent an insignificant cancer risk, since the MDA would be dissolved in blood that would be infused in many debilitated patients it represented a clear danger. These and other considerations convinced the manufacturer to cease the use of aromatic polyurethanes in blood or plasma storage bags. Ulrich and collaborators [14] also studied the extent of the thermo-
hydrolytic generation of trace amounts of MDA in aromatic polyurethanes using an improved High-Pressure Liquid Chromatography (HPLC) method capable of detecting the aromatic diamine in the parts per billion range. Their investigations concluded MDA is indeed generated when the aromatic polyurethanes are subjected to one steam sterilization cycle at 120°C. Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
326
consistent with published data of Ernes [15] who detectable in trace amounts when an aromatic polyurethane specimen was boiled in water for 17 hours, or steam autoclaved for 1.5 hours at 125 ° C. However, it should be emphasized that polyurethane resin suppliers caution against the steam sterilization of polyurethanes. Subjecting polyurethanes to extreme conditions to obtain aromatic amines in vitro has been known for some time. In 1967 Mulder [16] obtained the hydrolysis of an ether-based aromatic polyurethane under the forcing conditions of 30% NaOH solution at 160°C using a pressure bomb. Reaction products in Mulder’s work included aromatic diamines and polyetherdiols. Marchant et al. [17] studied the effect of papain on an unstabilized polyurethaneurea for a period of one month in an in vitro model system. The authors state that &dquo;the presence of the enzyme papain accelerates the degradation of urethane and urea groups, leading to the formation of primary amine degradation products, which include methylene dianiline:’ Unfortunately the amount of MDA generated by the enzymatic treatment was not determined. Many highly successful prostheses are made from MDI-based polyurethanes with no adverse effects. So long as care is exercised by the manufacturer to avoid steam sterilization, or extrude wet polyurethane pellets [17a] no MDA has been detected in these prostheses. Only when these polyurethanes are subjected to extreme temperature conditions is MDA formed by thermohydrolytic degradation. The use of MDI-based polyurethanes in medical prostheses is extensive. Over the years, an enviable record of safety and efhcacy has been accumulated by polyurethane-based implantable prostheses; below is a partial list of some current biomedical applications which use MDI. These results
are
found that MDA
based
was
polyurethanes:
Artificial hearts Blood bags Pacemaker leads Percutaneous shunts Skin dressings Vascular grafts
Indwelling catheters Blood oxygenator tubing Heart valves
Hemodialysis tubing, Penile implants
membranes
Vascular stents
3. FORMATION OF TDA FROM TDI-I3~SED POLYURETHANE FOAM
If MDA-based polyurethanes form MDA under extreme conditions, could also expect that TDI-based polyurethane foam would form
we
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327 TDA under extreme conditions as well. It is well known that polyurethanes heated to extreme temperatures in the presence of water undergo an abnormal thermohydrolytic degradation reaction which releases toxic amines such as Toluene Diamine (TDA), as well as methylene dianiline [17b]. Polyurethanes are also known to degrade if subjected to high pH aqueous solutions. An experiment on the Surgitek polyurethane foam was performed by Batich et al. [18] under extreme laboratory conditions. The hydrolysis was carried out at 150°C in 3.0 N NaOH. Not surprisingly, 85 mg of TDA were released in a 500 mg sample of foam, or about 17%. Since the foam contains about 25% TDI, this meant that &dquo;an expected amount of TDA available for release would be about 410 mg from a 2 gram foam covert or 205 parts per million. [Author’s note: In fact, a typical mammary implant contains 1.35 grams of polyurethane foam.] Although based on extreme conditions, these investigators still concluded that: &dquo;Toluene diamine (TDA) is released from normal hydrolysis of the polyurethane foam cover which is known to degrade after implantation. TDA is highly toxic and materials which release it should not be used as implants&dquo; In late 1988, our laboratories were engaged by Surgitek to ascertain if TDA isomers were formed during simulated physiological conditions. Further, to determine the amount, and kinetic rate of any TDA released. The following paragraphs detail the results of our experiments. 4. EXPERIMENTAL PROTDCOL
The primary purpose of these experiments was to determine if TDA isomers are formed under the influence of the enzyme papain. Papain is known to decrease the physical properties of certain polyurethanes [18a]. The experiments were conducted under simulated physiological conditions to mimic the in vivo microenvironment around the foam implants. Analysis of enzymatically-formed TDA was carried out in accordance with the methods of Unger and Friedman [19]. Accurately weighed 150 mg samples of Surgitek foam lot number 1529 were exhaustively extracted in methylene chloride to remove any traces of leachable antioxidant, catalysts, surfactants, etc Evidence of purity was ascertained by assaying the extraction fluid in a Beckman DB UV spectrometer. Thus, in our nomenclature, test polyurethane foam means foam that was prepurified by means of the methylene chloride extraction. Pure samples of 2,4 and 2,6 TDA were purchased from Sigma ChemiDownloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
328
cal Co. Purity of the purchased TDA was ascertained by infrared analysis, as well as HPLC. The pure TDA samples were used to yield concentration standards for HPLC analyses. Papainase, 2X crystallized and lyophilized powder, lot number 78F8140 was purchased from Sigma Chemical Co. This lot had a specific amidase activity of 7 units per /4g of enzyme, and an esterase activity of 1 unit/yg of enzyme. The level of amidase activity in the papain suspension was measured periodically by the colorimetric method of Arnon [20], which involves the hydrolysis of N-alpha-benzoyl-DL-arginine-p-nitroaniline into benzoyl arginine and the colored compound p-nitroaniline. The absorbance of the p-nitroaniline product was measured at 410 nm. A unit of papain activity was defined as the amount of enzyme activity that could liberate 1 mole of p-nitroaniline in one minute at 25 ° C at pH = 7.5. The specific activity was expressed as the number of units of activity per microgram of protein. Since the specific activity of the enzyme is known to decrease linearly with time at a rate of 6.4% per day (Ref. [18], p. 2033), the treatment media was changed once every two days, to ensure a constant enzyme activity. In this fashion, the average specific activity of papain was measured at 12.5 units/pg. An enzyme activity of 75,000 u/ml was used ,
in
our
experiments.
The activating agent for papain was 0.05 M cysteine and 0.02 M EDTA in pH = 6.5, with 0.02% sodium azide to inhibit bacterial growth. The control foam was exposed to 0.05 M cysteine, 0.02 M EDTA in pH = 6.5, with 0.02% sodium azide, but lacking any papain. All solutions were changed every two days. The skived (machine-sliced) test polyurethane foam (1-1.5 mm thick) was manually cut into squares measuring 60 x 60 mm, yielding approximately 150 mg of foam. Extracted test polyurethane foams were immersed in the papain enzyme solution for a total of one month. No effort was made at this time to investigate if any potential high-molecular weight oligomers leached into the enzyme solution, although we recognize this may be important in assessing the overall degradation of the foam. After treatment, the foam samples were removed, and the enzyme solution extracted by partitioning into spectroscopic-grade methylene chloride at ambient temperature for 3 days. The methylene chloride was subsequently evaporated under vacuum at room temperature, and the residue redissolved in acetonitrile-water saturated chloroform elution solvent (8:2, v/v) for HPLC. Separation of components redissolved in acetonitrile and waterDownloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
329
saturated chloroform
carried out by normal-phase chromatogachieved with a silica adsorption column (10 tom particle size) at an elution rate of 3 ml/min, detected at 250 nm. Water saturated chloroform was prepared by adding 100 ml of water to 11 ml of chloroform shaking each for four minutes, and allowing the mixture to stand overnight. A ratio of 8 parts of acetonitrile to 2 parts of water-saturated chloroform was used, giving good resolution of 2,4 from 2,6 TDA, and adequate separation of TDA isomers from interfering peaks in the extract.
raphy. Separations
was
were
5. RESULTS
2,4 TDA was formed during the first 4 days of enzyme exposure. 2,4 TDA was formed to a maximum of 8.3 jig/gr foam (8.3 parts per million), as shown in Figure 3. After 4 days, the test foam reached steady-state equilibrium conditions, where no additional TDA was released.
Figure 3. Cumulative formation of TDA isomers following enzymatic exposure. Following enzymatic digestion, 2,4 TDA was formed from the polyurethane foam, to a maxi. level of 8.3 ~g TDAlgram of foam. After the fourth day, no further TDA was formed. Note: All test forms were exhaustively purified with methylene chloride prior to enzymum
matic
digestion. Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
330
Figure 4. Pictorial representation of monomers used
in the
synthesis of polyurethane
foam.
The Surgitek foam is synthesized from a 3,000 Mw ethylene adipate triol, and TDI, as depicted pictorially in Figure 4. This reaction results in a highly cross-linked polymer, as shown in Figure 5. Since TDI only composes about 25% of the formulation, the molecular interface is composed mainly of polyester linkages, and very few urethane linkages, as portrayed in Figure 6. We hypothesize that following implantation, both esterases and amidases reach the tissue/ prosthesis interface. The exposed urethane linkages are hydrolyzed by the amidase (shown in Figure 7), thus forming TDA. But, at the same time, esterases are also degrading the ester linkages, causing the formation of low molecular weight oligomers. We further hypothesize that these low molecular weight oligomers, shown in Figure 8, coat the surface of the polyurethane foam, preventing further enzymatic hydrolysis of the urethane linkages, which have retreated deeper into mobile oligomeric film. We base our hypothesis on the observation that ESCA analyses of retrieved mammary prostheses invariably lack the presence of urethane linkages within the first 50 Angstroms of surface [21]. An alternative explanation would be that chain cleavage occurs at the surface providing chain ends which swell and extend into the aqueous interface. These &dquo;spacer arms&dquo; could result in the observed phenomenon regarding the presence of nitrogen (i.e., urethane links) and their relative spatial distribution regarding the surface. Papain is both an esterase, as well as an amidase. In our tests, papain Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
Figure 5. Molecular depiction of the three-dimensional polyurethane foam structure. ester linkage; triangle = urethane linkage. Code: dot =
Figure 6. Outer nwlecular surface ofPU foam, showing mostly ester linkages, and a few linkages.
urethane
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331
Figure 7. Pictorial representation of TDA formation. (a) Urethane linkages intact. (b) Enzyme &dquo;cuts&dquo; both urethane linkages via amidase hydrolysis. (c) TDA molecule is formed.
Figure 8. Hypothesized oligomeric coating, protecting the underlying chain. Note: please see results section for alternative explanation.
332
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polyurethane
333
had an esterase activity of 1 u/tcg enzyme, and an amidase activity of 7 u/mg enzyme. Thus, the papain potentially hydrolyzed both urethane as well as ester linkages. TDA was only formed while the urethane linkages were still molecularly accessible to the enzyme; once the protective~oligomeric coating was formed, TDA ceased to be formed. The total amount of 2,4 TDA formed during our studies was 8.3 pig TDA/g foam. Using standard risk-assessment analysis we can calculate the risk of developing a malignancy due to this amount of TDA as follows : 8.3 tcg TDA Total g foam
^
2.24 x 101 ug TDA 2.7 g foam 2 in Total TDA Foam impl
For bolus events, risk is amount, thus:
2.24
10’ pig TDA 365 days
conventionally evaluated
6.14 x 10-2 ~g TDA
x
6.14
on an
x
annualized
10-5 mg TDA
~
-
day
day
Therefore, the lifetime risk associated with this
amount of TDA is:
(.21) [6.14 x 10-5 mg TDA/day (1 bolus yr)] x 10-9 - 2.54 ~~ ~ (65 kg body weight) (78 years life expectancy) or, 1 in
400,000,000. 6. DISCUSSION
The Darby study referenced in this communication (where the polyurethane was subjected to drastic steam sterilization) was the first of many examples in which the conditions used to degrade the polymer could be considered irrelevant and inappropriate. Accelerated testing is standard practice in many of the laboratory tests conducted on biomaterials. Since the rate of a chemical reaction doubles for every 10°C rise, increasing the temperature in accelerated tests is a favorite stratagem. However, it is known that polyurethane elastomers undergo several minor transition changes at temperatures as low as 50 °C. At temperatures of 50°C and above, polyurethane elastomers begin to lose their segmented structure, due to loss of intermolecular hydrogen bonding. This loss of hydrogen bonding, in turn, leads to increased molecular mobility, molecular rearrangement and abnormal susceptibility to (a) Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
334
chemical degradation such as oxidation, hydrolysis etc, and (b) loss of mechanical properties such as flexure endurance, abrasion resistance and tear strength. It is also known that extreme alkaline conditions rapidly hydrolyze polyurethane linkages, leading to the formation the corresponding amines. Extreme acidic conditions also degrade polyurethanes, but at a much slower rate. Neither high temperatures, nor high alkaline conditions can be envisioned to occur in vivo. Therefore, it is our contention that neither high temperature nor pH extremes are appropriate &dquo;accelerators&dquo; to predict long-term biodegradation or biostability of polyurethanes. Recent calls in the literature to ban the clinical use of TDI-based polyurethane foam mammary prostheses because of possible cancer induction by 2,4 TDA appear extreme. MDI-based polyurethane prostheses (all capable of producing cancer-causing MDA agents if degraded) are extensively used throughout the world; based on medical and scientific literature available to date, neither MDI-based nor TDIbased polyurethane implants have ever been linked to any cancers in humans. 7. CONCLUSIONS
All synthetic biomaterials can be forced to degrade by drastic chemical and/or physical treatments, with the concomitant release of toxic and/or carcinogenic constituents. Experimental degradation processes utilizing extreme destructive means have no bearing on the behavior and fate of biomaterials under physiological ar in vivo conditions. There is no scientific justification for discrediting a biomaterial on this basis. Other biomaterials are also known to degrade under extreme conditions : polytetrafluoroethylene (PTFE) if burned produces hydrofluoric acid, an extremely toxic substance; polyvinyl chloride (PVC) if overheated releases hydrochloric acid, another highly cytotoxic substance. To ascertain if TDA is a byproduct of enzymatic degradation of the polyurethane foam used to cover mammary prostheses, our laboratories performed an in vitro test, simulating physiological conditions as closely as possible. We selected to use the endopeptidase papain, which has broad specificity, and which has been utilized in vitro as an analogue of cathepsin B which is released by cells during acute and chronic
inflammatory response. In these tests [84], 2,4 TDA was formed for the first four (4) days, reaching a maximum of 8.3 parts per million. After the initial burst, no Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
335 further TDA was observed within the limits of detection of the experiment (10 parts per billion). Based on standard risk assessment, this amount of TDA translates into a risk of developing cancer of one in four hundred million. In standard risk analysis, a risk of 1 in a million (or less) is considered insignificant. Put in perspective, a risk of 1:400,000,000 is akin to the risk of dying as a result of being exposed to 3 hours’ worth of radiation in Denver; or the risk of dying as a result of ingestion of one teaspoon-
ful of peanut butter (which contains
a
powerful
toxin known
as
afla-
toxin). In 1991 the American Cancer Society estimates that 1 in 9 women risk the possibility of breast cancer during their lifetime. Considering that 1 in 9 normal women are at risk to develop breast cancer during their lifetime, the increased risk of developing a possible malignancy due to 2,4 TDA release following mammaplasty must be considered in-
significant. REFERENCES
Darby, T. D., H. J. Johnson and S. J. Northrop. 1978. "An Evaluation of a Polyurethane for Use as a Medical-Grade Plastic," Toxicology and Applied Pharmacology, 46:449. 2. Schoental, R. 1968. "Carcinogenic and Chronic Effects of 4,4’ Diaminodiphenyl Methane, an Epoxy Resin Hardener," Nature, London, 1.
219:1162-1163. Deichmann, W. B., et al. 1978. "Di(4 amino phenyl methane (MDA): 4-7 Year Dog Feeding Study," Toxicology, 11:185-188. 4. Munn, A. 1967. "Occupational Bladder Tumors and Carcinogens: Recent Developments in Britain," in Bladder Cancer, A Symposium Aesculapius. W. Deichmann and K. F. Lampe, eds., Birmingham, AL. 5. Schoental, R. 1968. "Pathological Lesions, Including Tumors in Rats after 4,4’ Diaminodiphenyl Methane and Butyrolactone," Science, 4:11461158. 6. Shimizur, H. and N. Takemura. 1976. "Mutagenicity of Some Aromatic Amino Compounds and Nitro Compounds, and Its Relation to Carcino3.
7.
8.
genicity," Sangyo Igaku, 18:138-139. McLaughlin, J., Jr. and P. P. Marliac, et al. 1963. "The Injection of Chemicals into the Yolk Sac of Fertile Eggs Prior to Incubation as a Toxicity Test," Tox. Appl. Pharmacol., 5:760-771. Stula, E. F. and H. Sherman, et al. 1971. "Experimental Neoplasia in
ChR-CD Rats with Oral Administration of 3,3’ Dichlorobenzidine, 4,4’ (2 MethylaniBis Methylene- and 4,4’ Methylene(2-Chloroaniline), Bis line)," Tox. Appl. Pharmacol., 19:380. 9. Stula, E. F., et al., ibid., 1975. Tox. Appl. Pharmacol., 31:159-176.
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336 10. Russfield, A. R., et al. 1975. ’The Carcinogenic Effect of 4,4’ MethyleneBis(2-Chloroaniline) in Mice and Rats," Tox. Appl. Pharmacol., 31:47-54. 11. Steinhoff, D. and E. Grundmann. 1970. "Cancerogene Wirkung von 3,3’ Dichloro, 4,4’ Diamino Diphenylather bei Ratten," Naturwiss, 58:676. 12. Steinhoff, D. 1977. "Carcerogene Wirkung von 4,4’ Diamino-Diphenylather," Naturwiss, 64:394. 13. 1976. NIOSH, Background Information on 4,4’ Diaminophenylamine (DDM). Technical Evaluation and Review Branch, Office of Extramural Coordination and Special Projects, Rockville, MD. 13a. Devices and Diagnostic Letter, 6(35):2 (August 31, 1979). 13b. Devices and Diagnostic Letter, 7(8):1 (January 2, 1980). 14. Ulrich, H. and H. W. Bonk. 1982. "Emerging Biomedical Applications of Polyurethane Elastomers," in Proceedings, SPI, 27th Annual Conference, Bal Harbour, FL, p. 143. 15. O’Mara, M. M., D. A. Ernes and D. T. Hanschumaker. 1984. "Determination of Extractable Methylene Bis(Aniline) in Polyurethane Films by Liquid Chromatography," in Polyurethanes in Biomedical Engineering. H. Planck et al., Elsevier Publishing Co., pp. 83-92. 16. Mulder, J. L. 1967. "Characterization of Linear Polyurethanes," Anal. Chim. Acta., 38:563-576. 17. Marchant, R. E., et al. 1987. "Degradation of a Polyether Urethane Urea Elastomer: Infrared and XPS Studies," Polymer, 28:2032. 17a. Pellethane CPR 2363-80A Technical Information Sheet, Upjohn Chemicals Plastics Research, Torrance CA, May (1979). Revision of June 1983. 17b. Szycher, M., V. L. Poirier and D. J. Dempsey. 1983. "Development of an Aliphatic Biomedical-Grade Polyurethane Elastomer," J. Elas. Plast., 15:87. 18. Batich, C., J. Williams and R. King. 1989. "Toxic Hydrolysis Product from a Biodegradable Foam Implant," J. Biomed. Mater. Res: Applied 18a.
19.
Biomaterials, 23(A3):311-319. Zhao, Q., R. E. Marchant, J. M. Anderson and A. Hiltner. 1987. "Long Term Biodegradation in vitro of Poly(Ether Urethane Urea): A Mechanical Property Study," Polymer, 28:2040-2046. Unger, P.D. and M. A. Friedman. 1979. "High Performance Liquid Chromatography of 2,6 and 2,4 Diaminotoluene, and Its Application to the Determination of 2,4 Diaminotoluene in Urine and Plasma," J. Chrom., 174:379-384.
20. 21.
Arnon, R. 1970. "Methods in Enzymology," New York, NY: Academic Press, 19:226. Szycher, M. and A. Siciliano. 1991. "Polyurethane-Covered Mammary Prosthesis: A Nine Year Follow-Up Assessment," Journal of Biomaterials
Applications, 5(4):282-322.
Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
Physiological Conditions MICHAEL
SZYCHER, PH.D.
AND
ARTHUR A.
SICILIANO, PH.D.
PolyMedica Industries, Inc. 2 Constitution Way Woburn, MA 01801 -
1. SUMMARY
polyurethane foam used in the manufacture of mammary prostheses were enzymatically treated for a total of thirty days. Papain (a plant thiol endopeptidase which has similar activity to the human lysosomal enzyme cathepsin B) was our enzyme of choice since it has both amidase as well as esterase activity. The experiment was conducted under physiological conditions closely simulating the microenvironment likely to be found around an implanted mammary pros-
samplers of
thesis. In our tests, 2,4 TDA was formed during enzymatic attack of this TDI-based polyurethane foam for the first four (4) days, reaching a maximum of 8.3 parts per million. After the initial burst, no further TDA was observed within the limits of detection of the experiment (10 parts per billion). Based on standard risk assessment, this amount of TDA translates into a risk of developing cancer of one in four hundred million. 2. FORMATION OF lBIDA FROM NIDI-R%SED POLYURETHANES
A 1978 article by Darby [1] at Travenol Laboratories was the first to alert the scientific community that a medical-grade polyurethane, 323
Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
324
Pellethane 2363-80A, contained an aromatic amine analog of 4,4’ diphenyl methane diisocyanate (MDI), namely 4,4’ diaminodiphenyl rriethane, commonly designated as MDA. This study identified MDA in aqueous extracts of the polyurethane, and also demonstrated the mutagenic potential of MDA using Salmonella typhimurium test strains. Darby and coworkers exposed the MDI-based polyurethane to steam sterilization conditions for several hours. Under these forcing conditions, the polyurethane linkages underwent thermolysis, leading to the formation of MDA. 4,4’ Methylene Dianiline (MDA) is a carcinogen [2-5], a mutagen [6], and a teratogen [7]. Other chemicals closely related to MDA have been tested in laboratory animals and also found to be carcinogenic. Such chemicals are 4,4’ methylene-bis[2-methyl-aniline] [8]; 4,4’ methy-
lene-bis[2-chloroaniline] [7,8]; 3,3’ dichloro-4,4’ diaminodiphenylether [9]; and its parent compound 4,4’ diaminoethylether [10]. Animal studies indicate that MDA is very toxic [11]. The rat oral and subcutaneous LDso values are 347 and 200 mg/kg, respectively, while the mouse intraperitoneal LDso is 74 mg/kg [12]. The lowest published dose
reported to produce systemic toxicity in man is 8.42 mg/kg orally; thus, NIOSH classifies MDA as a primary irritant [13]. MDA is a pomutagen, which is reactive in the Ames test in the concentration range of 5 to 150 pig. These data suggest that detectable migration of tent
MDA from implantable aromatic polyurethanes might be unacceptable in critical care clinical applications, even in parts per billion levels. MDA can be formed in aromatic-based polyurethanes by two independent mechanisms. The first mechanism involves the anhydrous thermal degradation of polyurethanes, with concomitant formation of MDA, carbon dioxide, and an olefin, as shown in Figure 1. The anhydrous thermal decomposition of aromatic polyurethanes undergoes chain cleavage only very slowly at 150 to 200°C; furthermore, the presence of other reactants and catalysts also influences the speed of this
decomposition reaction. The second mechanism involves a two-step thermohydrolytic reaction. In the first step, the aromatic polyurethane undergoes a dissociation to the corresponding aromatic diisocyanate, and the precursor
Figure 1. Anhydrous thermal degradation of aromatic-based polyurethanes, leading to formation
of methylene
dianiline (MDA).
Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
325
Figure 2. Postulated thermohydrolytic degradation of aromatic-based polyurethanes, leading to accelerated formation of methylene dianiline (MDA). The source of water can be twofold: (1) as chemisorbed moisture in improperly dried pellets, or (2) during prolonged steam sterilization.
alcohol. In the second step, and in the presence of water, the aromatic diisocyanate reacts with water to form aromatic amines such as MDA. The postulated two-step mechanism is presented in Figure 2. This
postulated thermohydrolytic decomposition reaction can only occur in the presence of water, and is much faster than the decomposition reaction discussed above. This reaction is thought to occur during extrusion, if the pellets of thermoplastic polyurethane are not properly dried prior to thermal processing. The presence of MDA contamination in biomedical-grade aromatic polyurethanes was detected and quantified in plasma storage packages fabricated from an aromatic thermoplastic polyurethane elastomer. Food and Drug Administration (FDA) investigators have reported 3 parts per billion of MDA in human plasma stored in aromatic polyurethane storage bags [13a,13b]. Although 3 parts per billion of MDA (in a blood bag containing about 10 grams of polyurethane) would represent an insignificant cancer risk, since the MDA would be dissolved in blood that would be infused in many debilitated patients it represented a clear danger. These and other considerations convinced the manufacturer to cease the use of aromatic polyurethanes in blood or plasma storage bags. Ulrich and collaborators [14] also studied the extent of the thermo-
hydrolytic generation of trace amounts of MDA in aromatic polyurethanes using an improved High-Pressure Liquid Chromatography (HPLC) method capable of detecting the aromatic diamine in the parts per billion range. Their investigations concluded MDA is indeed generated when the aromatic polyurethanes are subjected to one steam sterilization cycle at 120°C. Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
326
consistent with published data of Ernes [15] who detectable in trace amounts when an aromatic polyurethane specimen was boiled in water for 17 hours, or steam autoclaved for 1.5 hours at 125 ° C. However, it should be emphasized that polyurethane resin suppliers caution against the steam sterilization of polyurethanes. Subjecting polyurethanes to extreme conditions to obtain aromatic amines in vitro has been known for some time. In 1967 Mulder [16] obtained the hydrolysis of an ether-based aromatic polyurethane under the forcing conditions of 30% NaOH solution at 160°C using a pressure bomb. Reaction products in Mulder’s work included aromatic diamines and polyetherdiols. Marchant et al. [17] studied the effect of papain on an unstabilized polyurethaneurea for a period of one month in an in vitro model system. The authors state that &dquo;the presence of the enzyme papain accelerates the degradation of urethane and urea groups, leading to the formation of primary amine degradation products, which include methylene dianiline:’ Unfortunately the amount of MDA generated by the enzymatic treatment was not determined. Many highly successful prostheses are made from MDI-based polyurethanes with no adverse effects. So long as care is exercised by the manufacturer to avoid steam sterilization, or extrude wet polyurethane pellets [17a] no MDA has been detected in these prostheses. Only when these polyurethanes are subjected to extreme temperature conditions is MDA formed by thermohydrolytic degradation. The use of MDI-based polyurethanes in medical prostheses is extensive. Over the years, an enviable record of safety and efhcacy has been accumulated by polyurethane-based implantable prostheses; below is a partial list of some current biomedical applications which use MDI. These results
are
found that MDA
based
was
polyurethanes:
Artificial hearts Blood bags Pacemaker leads Percutaneous shunts Skin dressings Vascular grafts
Indwelling catheters Blood oxygenator tubing Heart valves
Hemodialysis tubing, Penile implants
membranes
Vascular stents
3. FORMATION OF TDA FROM TDI-I3~SED POLYURETHANE FOAM
If MDA-based polyurethanes form MDA under extreme conditions, could also expect that TDI-based polyurethane foam would form
we
Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
327 TDA under extreme conditions as well. It is well known that polyurethanes heated to extreme temperatures in the presence of water undergo an abnormal thermohydrolytic degradation reaction which releases toxic amines such as Toluene Diamine (TDA), as well as methylene dianiline [17b]. Polyurethanes are also known to degrade if subjected to high pH aqueous solutions. An experiment on the Surgitek polyurethane foam was performed by Batich et al. [18] under extreme laboratory conditions. The hydrolysis was carried out at 150°C in 3.0 N NaOH. Not surprisingly, 85 mg of TDA were released in a 500 mg sample of foam, or about 17%. Since the foam contains about 25% TDI, this meant that &dquo;an expected amount of TDA available for release would be about 410 mg from a 2 gram foam covert or 205 parts per million. [Author’s note: In fact, a typical mammary implant contains 1.35 grams of polyurethane foam.] Although based on extreme conditions, these investigators still concluded that: &dquo;Toluene diamine (TDA) is released from normal hydrolysis of the polyurethane foam cover which is known to degrade after implantation. TDA is highly toxic and materials which release it should not be used as implants&dquo; In late 1988, our laboratories were engaged by Surgitek to ascertain if TDA isomers were formed during simulated physiological conditions. Further, to determine the amount, and kinetic rate of any TDA released. The following paragraphs detail the results of our experiments. 4. EXPERIMENTAL PROTDCOL
The primary purpose of these experiments was to determine if TDA isomers are formed under the influence of the enzyme papain. Papain is known to decrease the physical properties of certain polyurethanes [18a]. The experiments were conducted under simulated physiological conditions to mimic the in vivo microenvironment around the foam implants. Analysis of enzymatically-formed TDA was carried out in accordance with the methods of Unger and Friedman [19]. Accurately weighed 150 mg samples of Surgitek foam lot number 1529 were exhaustively extracted in methylene chloride to remove any traces of leachable antioxidant, catalysts, surfactants, etc Evidence of purity was ascertained by assaying the extraction fluid in a Beckman DB UV spectrometer. Thus, in our nomenclature, test polyurethane foam means foam that was prepurified by means of the methylene chloride extraction. Pure samples of 2,4 and 2,6 TDA were purchased from Sigma ChemiDownloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
328
cal Co. Purity of the purchased TDA was ascertained by infrared analysis, as well as HPLC. The pure TDA samples were used to yield concentration standards for HPLC analyses. Papainase, 2X crystallized and lyophilized powder, lot number 78F8140 was purchased from Sigma Chemical Co. This lot had a specific amidase activity of 7 units per /4g of enzyme, and an esterase activity of 1 unit/yg of enzyme. The level of amidase activity in the papain suspension was measured periodically by the colorimetric method of Arnon [20], which involves the hydrolysis of N-alpha-benzoyl-DL-arginine-p-nitroaniline into benzoyl arginine and the colored compound p-nitroaniline. The absorbance of the p-nitroaniline product was measured at 410 nm. A unit of papain activity was defined as the amount of enzyme activity that could liberate 1 mole of p-nitroaniline in one minute at 25 ° C at pH = 7.5. The specific activity was expressed as the number of units of activity per microgram of protein. Since the specific activity of the enzyme is known to decrease linearly with time at a rate of 6.4% per day (Ref. [18], p. 2033), the treatment media was changed once every two days, to ensure a constant enzyme activity. In this fashion, the average specific activity of papain was measured at 12.5 units/pg. An enzyme activity of 75,000 u/ml was used ,
in
our
experiments.
The activating agent for papain was 0.05 M cysteine and 0.02 M EDTA in pH = 6.5, with 0.02% sodium azide to inhibit bacterial growth. The control foam was exposed to 0.05 M cysteine, 0.02 M EDTA in pH = 6.5, with 0.02% sodium azide, but lacking any papain. All solutions were changed every two days. The skived (machine-sliced) test polyurethane foam (1-1.5 mm thick) was manually cut into squares measuring 60 x 60 mm, yielding approximately 150 mg of foam. Extracted test polyurethane foams were immersed in the papain enzyme solution for a total of one month. No effort was made at this time to investigate if any potential high-molecular weight oligomers leached into the enzyme solution, although we recognize this may be important in assessing the overall degradation of the foam. After treatment, the foam samples were removed, and the enzyme solution extracted by partitioning into spectroscopic-grade methylene chloride at ambient temperature for 3 days. The methylene chloride was subsequently evaporated under vacuum at room temperature, and the residue redissolved in acetonitrile-water saturated chloroform elution solvent (8:2, v/v) for HPLC. Separation of components redissolved in acetonitrile and waterDownloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
329
saturated chloroform
carried out by normal-phase chromatogachieved with a silica adsorption column (10 tom particle size) at an elution rate of 3 ml/min, detected at 250 nm. Water saturated chloroform was prepared by adding 100 ml of water to 11 ml of chloroform shaking each for four minutes, and allowing the mixture to stand overnight. A ratio of 8 parts of acetonitrile to 2 parts of water-saturated chloroform was used, giving good resolution of 2,4 from 2,6 TDA, and adequate separation of TDA isomers from interfering peaks in the extract.
raphy. Separations
was
were
5. RESULTS
2,4 TDA was formed during the first 4 days of enzyme exposure. 2,4 TDA was formed to a maximum of 8.3 jig/gr foam (8.3 parts per million), as shown in Figure 3. After 4 days, the test foam reached steady-state equilibrium conditions, where no additional TDA was released.
Figure 3. Cumulative formation of TDA isomers following enzymatic exposure. Following enzymatic digestion, 2,4 TDA was formed from the polyurethane foam, to a maxi. level of 8.3 ~g TDAlgram of foam. After the fourth day, no further TDA was formed. Note: All test forms were exhaustively purified with methylene chloride prior to enzymum
matic
digestion. Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
330
Figure 4. Pictorial representation of monomers used
in the
synthesis of polyurethane
foam.
The Surgitek foam is synthesized from a 3,000 Mw ethylene adipate triol, and TDI, as depicted pictorially in Figure 4. This reaction results in a highly cross-linked polymer, as shown in Figure 5. Since TDI only composes about 25% of the formulation, the molecular interface is composed mainly of polyester linkages, and very few urethane linkages, as portrayed in Figure 6. We hypothesize that following implantation, both esterases and amidases reach the tissue/ prosthesis interface. The exposed urethane linkages are hydrolyzed by the amidase (shown in Figure 7), thus forming TDA. But, at the same time, esterases are also degrading the ester linkages, causing the formation of low molecular weight oligomers. We further hypothesize that these low molecular weight oligomers, shown in Figure 8, coat the surface of the polyurethane foam, preventing further enzymatic hydrolysis of the urethane linkages, which have retreated deeper into mobile oligomeric film. We base our hypothesis on the observation that ESCA analyses of retrieved mammary prostheses invariably lack the presence of urethane linkages within the first 50 Angstroms of surface [21]. An alternative explanation would be that chain cleavage occurs at the surface providing chain ends which swell and extend into the aqueous interface. These &dquo;spacer arms&dquo; could result in the observed phenomenon regarding the presence of nitrogen (i.e., urethane links) and their relative spatial distribution regarding the surface. Papain is both an esterase, as well as an amidase. In our tests, papain Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
Figure 5. Molecular depiction of the three-dimensional polyurethane foam structure. ester linkage; triangle = urethane linkage. Code: dot =
Figure 6. Outer nwlecular surface ofPU foam, showing mostly ester linkages, and a few linkages.
urethane
Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
331
Figure 7. Pictorial representation of TDA formation. (a) Urethane linkages intact. (b) Enzyme &dquo;cuts&dquo; both urethane linkages via amidase hydrolysis. (c) TDA molecule is formed.
Figure 8. Hypothesized oligomeric coating, protecting the underlying chain. Note: please see results section for alternative explanation.
332
Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
polyurethane
333
had an esterase activity of 1 u/tcg enzyme, and an amidase activity of 7 u/mg enzyme. Thus, the papain potentially hydrolyzed both urethane as well as ester linkages. TDA was only formed while the urethane linkages were still molecularly accessible to the enzyme; once the protective~oligomeric coating was formed, TDA ceased to be formed. The total amount of 2,4 TDA formed during our studies was 8.3 pig TDA/g foam. Using standard risk-assessment analysis we can calculate the risk of developing a malignancy due to this amount of TDA as follows : 8.3 tcg TDA Total g foam
^
2.24 x 101 ug TDA 2.7 g foam 2 in Total TDA Foam impl
For bolus events, risk is amount, thus:
2.24
10’ pig TDA 365 days
conventionally evaluated
6.14 x 10-2 ~g TDA
x
6.14
on an
x
annualized
10-5 mg TDA
~
-
day
day
Therefore, the lifetime risk associated with this
amount of TDA is:
(.21) [6.14 x 10-5 mg TDA/day (1 bolus yr)] x 10-9 - 2.54 ~~ ~ (65 kg body weight) (78 years life expectancy) or, 1 in
400,000,000. 6. DISCUSSION
The Darby study referenced in this communication (where the polyurethane was subjected to drastic steam sterilization) was the first of many examples in which the conditions used to degrade the polymer could be considered irrelevant and inappropriate. Accelerated testing is standard practice in many of the laboratory tests conducted on biomaterials. Since the rate of a chemical reaction doubles for every 10°C rise, increasing the temperature in accelerated tests is a favorite stratagem. However, it is known that polyurethane elastomers undergo several minor transition changes at temperatures as low as 50 °C. At temperatures of 50°C and above, polyurethane elastomers begin to lose their segmented structure, due to loss of intermolecular hydrogen bonding. This loss of hydrogen bonding, in turn, leads to increased molecular mobility, molecular rearrangement and abnormal susceptibility to (a) Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
334
chemical degradation such as oxidation, hydrolysis etc, and (b) loss of mechanical properties such as flexure endurance, abrasion resistance and tear strength. It is also known that extreme alkaline conditions rapidly hydrolyze polyurethane linkages, leading to the formation the corresponding amines. Extreme acidic conditions also degrade polyurethanes, but at a much slower rate. Neither high temperatures, nor high alkaline conditions can be envisioned to occur in vivo. Therefore, it is our contention that neither high temperature nor pH extremes are appropriate &dquo;accelerators&dquo; to predict long-term biodegradation or biostability of polyurethanes. Recent calls in the literature to ban the clinical use of TDI-based polyurethane foam mammary prostheses because of possible cancer induction by 2,4 TDA appear extreme. MDI-based polyurethane prostheses (all capable of producing cancer-causing MDA agents if degraded) are extensively used throughout the world; based on medical and scientific literature available to date, neither MDI-based nor TDIbased polyurethane implants have ever been linked to any cancers in humans. 7. CONCLUSIONS
All synthetic biomaterials can be forced to degrade by drastic chemical and/or physical treatments, with the concomitant release of toxic and/or carcinogenic constituents. Experimental degradation processes utilizing extreme destructive means have no bearing on the behavior and fate of biomaterials under physiological ar in vivo conditions. There is no scientific justification for discrediting a biomaterial on this basis. Other biomaterials are also known to degrade under extreme conditions : polytetrafluoroethylene (PTFE) if burned produces hydrofluoric acid, an extremely toxic substance; polyvinyl chloride (PVC) if overheated releases hydrochloric acid, another highly cytotoxic substance. To ascertain if TDA is a byproduct of enzymatic degradation of the polyurethane foam used to cover mammary prostheses, our laboratories performed an in vitro test, simulating physiological conditions as closely as possible. We selected to use the endopeptidase papain, which has broad specificity, and which has been utilized in vitro as an analogue of cathepsin B which is released by cells during acute and chronic
inflammatory response. In these tests [84], 2,4 TDA was formed for the first four (4) days, reaching a maximum of 8.3 parts per million. After the initial burst, no Downloaded from jba.sagepub.com at TEXAS A & M INTL UNIV on March 14, 2015
335 further TDA was observed within the limits of detection of the experiment (10 parts per billion). Based on standard risk assessment, this amount of TDA translates into a risk of developing cancer of one in four hundred million. In standard risk analysis, a risk of 1 in a million (or less) is considered insignificant. Put in perspective, a risk of 1:400,000,000 is akin to the risk of dying as a result of being exposed to 3 hours’ worth of radiation in Denver; or the risk of dying as a result of ingestion of one teaspoon-
ful of peanut butter (which contains
a
powerful
toxin known
as
afla-
toxin). In 1991 the American Cancer Society estimates that 1 in 9 women risk the possibility of breast cancer during their lifetime. Considering that 1 in 9 normal women are at risk to develop breast cancer during their lifetime, the increased risk of developing a possible malignancy due to 2,4 TDA release following mammaplasty must be considered in-
significant. REFERENCES
Darby, T. D., H. J. Johnson and S. J. Northrop. 1978. "An Evaluation of a Polyurethane for Use as a Medical-Grade Plastic," Toxicology and Applied Pharmacology, 46:449. 2. Schoental, R. 1968. "Carcinogenic and Chronic Effects of 4,4’ Diaminodiphenyl Methane, an Epoxy Resin Hardener," Nature, London, 1.
219:1162-1163. Deichmann, W. B., et al. 1978. "Di(4 amino phenyl methane (MDA): 4-7 Year Dog Feeding Study," Toxicology, 11:185-188. 4. Munn, A. 1967. "Occupational Bladder Tumors and Carcinogens: Recent Developments in Britain," in Bladder Cancer, A Symposium Aesculapius. W. Deichmann and K. F. Lampe, eds., Birmingham, AL. 5. Schoental, R. 1968. "Pathological Lesions, Including Tumors in Rats after 4,4’ Diaminodiphenyl Methane and Butyrolactone," Science, 4:11461158. 6. Shimizur, H. and N. Takemura. 1976. "Mutagenicity of Some Aromatic Amino Compounds and Nitro Compounds, and Its Relation to Carcino3.
7.
8.
genicity," Sangyo Igaku, 18:138-139. McLaughlin, J., Jr. and P. P. Marliac, et al. 1963. "The Injection of Chemicals into the Yolk Sac of Fertile Eggs Prior to Incubation as a Toxicity Test," Tox. Appl. Pharmacol., 5:760-771. Stula, E. F. and H. Sherman, et al. 1971. "Experimental Neoplasia in
ChR-CD Rats with Oral Administration of 3,3’ Dichlorobenzidine, 4,4’ (2 MethylaniBis Methylene- and 4,4’ Methylene(2-Chloroaniline), Bis line)," Tox. Appl. Pharmacol., 19:380. 9. Stula, E. F., et al., ibid., 1975. Tox. Appl. Pharmacol., 31:159-176.
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336 10. Russfield, A. R., et al. 1975. ’The Carcinogenic Effect of 4,4’ MethyleneBis(2-Chloroaniline) in Mice and Rats," Tox. Appl. Pharmacol., 31:47-54. 11. Steinhoff, D. and E. Grundmann. 1970. "Cancerogene Wirkung von 3,3’ Dichloro, 4,4’ Diamino Diphenylather bei Ratten," Naturwiss, 58:676. 12. Steinhoff, D. 1977. "Carcerogene Wirkung von 4,4’ Diamino-Diphenylather," Naturwiss, 64:394. 13. 1976. NIOSH, Background Information on 4,4’ Diaminophenylamine (DDM). Technical Evaluation and Review Branch, Office of Extramural Coordination and Special Projects, Rockville, MD. 13a. Devices and Diagnostic Letter, 6(35):2 (August 31, 1979). 13b. Devices and Diagnostic Letter, 7(8):1 (January 2, 1980). 14. Ulrich, H. and H. W. Bonk. 1982. "Emerging Biomedical Applications of Polyurethane Elastomers," in Proceedings, SPI, 27th Annual Conference, Bal Harbour, FL, p. 143. 15. O’Mara, M. M., D. A. Ernes and D. T. Hanschumaker. 1984. "Determination of Extractable Methylene Bis(Aniline) in Polyurethane Films by Liquid Chromatography," in Polyurethanes in Biomedical Engineering. H. Planck et al., Elsevier Publishing Co., pp. 83-92. 16. Mulder, J. L. 1967. "Characterization of Linear Polyurethanes," Anal. Chim. Acta., 38:563-576. 17. Marchant, R. E., et al. 1987. "Degradation of a Polyether Urethane Urea Elastomer: Infrared and XPS Studies," Polymer, 28:2032. 17a. Pellethane CPR 2363-80A Technical Information Sheet, Upjohn Chemicals Plastics Research, Torrance CA, May (1979). Revision of June 1983. 17b. Szycher, M., V. L. Poirier and D. J. Dempsey. 1983. "Development of an Aliphatic Biomedical-Grade Polyurethane Elastomer," J. Elas. Plast., 15:87. 18. Batich, C., J. Williams and R. King. 1989. "Toxic Hydrolysis Product from a Biodegradable Foam Implant," J. Biomed. Mater. Res: Applied 18a.
19.
Biomaterials, 23(A3):311-319. Zhao, Q., R. E. Marchant, J. M. Anderson and A. Hiltner. 1987. "Long Term Biodegradation in vitro of Poly(Ether Urethane Urea): A Mechanical Property Study," Polymer, 28:2040-2046. Unger, P.D. and M. A. Friedman. 1979. "High Performance Liquid Chromatography of 2,6 and 2,4 Diaminotoluene, and Its Application to the Determination of 2,4 Diaminotoluene in Urine and Plasma," J. Chrom., 174:379-384.
20. 21.
Arnon, R. 1970. "Methods in Enzymology," New York, NY: Academic Press, 19:226. Szycher, M. and A. Siciliano. 1991. "Polyurethane-Covered Mammary Prosthesis: A Nine Year Follow-Up Assessment," Journal of Biomaterials
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