JOHN D. FERRY' AND PETER R. MORRISON H a r v a r d M e d i c a l S c h o o l , Boston, M a s s .
Fibrin film is prepared from human fibrinogen by clotting with thrombin under specified conditions so that the resulting clot can be compacted with very slight pressure. Treatment of the film with steam, after careful adjustment of the moisture content, sterilizes the film and modifies it$ physical properties as well as its susceptibility to enzymatic digestion. This treatment is also used to form shaped objects, such as tubes and cups. Fibrin film has been extensively employed in surgery. Plastics of fibrinogen and other plasma proteins may also be prepared by heat treatment with polyhydric alcohols.
T
H E separation of human plasma into a series of protein fractions (5) has made a number of products available for medical use (4). The separation of the different plasma proteins, and especially the isolation of fibrinogen, has also made it possible t o prepare plastic products for use in surgery (9). Thus, surgical devices for introduction into the human body may be constructed from human protein materials. The usefulness of these devices has depended upon their mechanical properties, which can be varied t o suit specifications, and upon the faculty of being eventually absorbed after implantation in the tissues, ' with almost noirritative reaction. Since fibrinogen, like the high polymers of which synthetic plastics are composed, is a n elongated molecule of high molecular weight and, furthermore, serves the physiological function of forming a solid structure i n the clotting of blood, it was used as a starting material in the preparation of plastic products. It was found possible t o prepare two types of Solid structures from 1
Present address, University ut Wisconsin, Madison, Wis.
fibrinogen: fibrin films, by a modification of the normal clotting process with bhrombin, and fibrinogen plastics, by treatment with heat in the presence of a plasticizer. Plastics may also be prepared from albumins and globulins by heat treatment. PREPARATION OF FIBRIN FILM
Fibrin film is prepared by clotting a solution of fibrinogen (usually fraction I of human plasma, 5) with thrombin (purified from fraction 111-2,6,18) and compacting the resulting clot under low pressure (7, 8). I n compaction the thickness of the clot is decreased to about one fiftieth of its original value, and a solution containing salts and the proteins other than fibrin is squeezed out. This process is entirely different from the usual methods of extrusion or casting films of high polymers, which cannot be applied to this system because the polymerization takes place at high dilution and the protein cannot be subjected t o elevated temperatures. It is made possible by the remarkable ease of syneresis of the fibrin clot when it is prepared under the proper conditions of concentration, ionic strength, and pH. Fraction I of human plasma may be employed immediately after its precipitation, or it may be stored in the frozen state a t -5" C. or lower for a few weeks without significant destruction of fibrinogen. Fraction I is dissolved in 4 volumes of sodium citrate buffer at p H 6.1 and ionic strength 0.3,and the solution is clarified by filtration. This solution may be used a t once for preparation of films, or i t may be dried from the frozen state, stored in vacuo for as long as two years, and reconstituted with distilled water. The fraction I solution is diluted with water and sodium chloride solution t o a fibrinogen concentration of 5 grams per liter and ionic strength 0.15, and the p H is adjusted, if necessary, t o 1217
INDUSTRIAL AND ENGINEERING CHEMISTRY
1218
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Vol. 38, No. 12
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Time in HoLtrs
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Figure 3.
in Hours
Figure 1. Effect of Fibrinogen Concentration on Clot Compaction
Effect of S a l t C o n c e n t r a t i o n on C l o ~ Compaction
10 grams fibrinogen per liter; 0.3 unit thrombin per 0.4 pound per square inch
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0.3 unit thrombin per cc.; 0.22 M sodium chloride: presaure. 0.4 pound per square inch
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STERILIZATION O F FIBRIN FILM
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tion is impossible. The compaction time may be somewhat shortened by increasing the pressure, but pressures much higher than 1 pound per square inch are likely to rupture the clot, Fibrin film thus prepared is a rubbery, white, opaque sheet cont)aining 30% fibrin and 7 0 7 , water. Its mechanical properties are somewhat similar to those of a cross-linked elastomer.
0.5
1.0
1.5
Time in Hours Figure 2.
Effect of Thrombin C o n c e n t r a t i o n on Clot Compaction
10 grams fibrinogen per liter; 0.22 M sodium chloride: pressure. 0.4 pound per square inch
6.2-6.3. Sufficient thrombin solution is added t o bring its concentration t o 0.3 unit per cc. (the unit of activity is defined by Edsall and Miller, 6), and the mixture is then pouied through lintfree cloth or stainless steel screen (to remove foam) into trays of aluminum or stainless steel, 8 X 24 X 1 inch. Formation of bubbles must be carefully avoided. Clotting takes place in a few minutes; the reaction is allowed to proceed for 1 hour t o ensure complete conversion of fibrinogen to fibrin. The clots are then turned out on sheets of fine muslin and pressed between pieces of plate glass '/Z-inch thick a t a pressure of about 0.4 pound per square inch to expel the syneretic fluid. 'VVrinkles in the cloth and air bubbles between the cloth and the clot must be avoided, because they produce imperfections embossed on the film surface. The effects of varying the conditions of preparation on the course of clot compaction are shown in Figures I to 4 (where lhickness of clot is plotted against time) for clots containing 16 mg. fibrin per sq. em., clotted with various concentrations of fibrinogen, thrombin, and salt, and pressed under different pressures. Under favorable conditions about 1 hour is required foi compaction. Varying the fibrinogen concentration does not have much effect; the more dilute clots are compacted more rapidly but the volume of liquid t o be expressed is greater. The thrombin concentration is critical; if it exceeds 0.3 unit per cc, the compaction time is prolonged, but if it is less than this value, the time required for conversion of fibrinogen t o fibrin (approximately inversely proportional to thrombin concentration) is delayed. If the salt concentration is too high, the compaction time is prolonged; but i t must be high enough t o keep the fibrinogen in solution before clotting. If the pH is too low, the time required for conversion of fibrinogen to fibrin may be greatly prolonged, whereas if i t is too high, the compaction time is prolonged. At p H 6.7 the compaction time is 20 hours, and above this value compar-
For surgical use fibrin film must be sterile. It can be prepared under aseptic conditions from solutions of fibrinogen and thrombin which have previously been sterilized by filtration (17, 20). This procedure is laborious, however. Alternatively, the film can be st,erilized after it is prepared by treating it with heat under carefully specified conditions. The two types of heat treatment rvhich have been employed alter the film and cause it to be absorbed more slowly when implanted in the tissues. The longer persistence in the body of the heat-treated products is an advantage in many surgical applications. Boiling or autoclaving the freshly prepared fibrin film results in a degraded product with very little tensile strength, and these conventional methods of sterilization cannot be employed. However, sterilization of the film by heat can be accomplished without degradation if a part or all of the water present is first removed. Of course, removal of water tends to protect from destruction not only the fibrin but also bacterial spores. It is necexsary t o select conditions such that all bacteria will be cgrtainly killed (as shown by bacteriological tests) but the degradation of fibrin will be minimal. Two sterilization procedures have been used successfully: (a)replacement of the water by glycerol, followed by heat treatment in hot glycerol, and ( b ) partial desiccation, followed b!. heat treatment in saturated steam. In the hot glycerol method the film is first soaked in a large volume of glycerol at room temperature; the glycerol replaces t h r water, and the fibrin content remains about 30%. The film i b then immersed for 1 minute in glycerol a t 155" C., blotted with aseptic precautions, and packaged aseptically in previously sterilized containers. This process is satisfactory on a small scale but is not adapted to large scale manufacture. The first fibril: urns used clinically as dural substitutes (3) were sterilized in glycerol by S. H. Armstrong, Jr., and E. A. Bering, Jr. In the st,eaming method the film is first partially desiccated so that the moisture content is reduced from 70% to about 20%. If the final value is too high, the subsequent steaming will cause degradation; if it is too low, the film will be too brittle to handit:. The film is then autoclaved in saturated steam a t 15 pounds per square inch for 20 minutes. During the steaming the moisture content of the film rises t o about 25%. The process is carried out in the final container; after sterilization, the moisture content of the film is reduced t o 11 * 3%, and the container is sealed. The films prepared under contract with the United States Kavy werr sterilized in this way.
INDUSTRIAL AND ENGINEERING CHEMISTRY
December, 1946
1219
Pressure, Ib/sq. in. qo.2
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-
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1
0
1.5
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Hours
Effect of Pressure on Clot Compaction
fibrinogen per liter; 0.3 unit t h r o m b i n per cr.: s o d i u m chloride
0.22 M
TUBES AND CUPS OF FIBRIN FILM
The steaming process used for sterilization can be applied simultaneously to form shaped objects intended for specific surgical applications. This procedure depends upon two mechanical effects of the heat treatment of the fibrin. Thin layers of film which have been pressed together in contact before steaming adhere to each other, and, if the film is stretched t o conform t o a new shape before steaming, the stresses introduced are relaxed and the new shape is fixed. I n making seamless fibrin tubing, the moisture content of a strip of fibrin film is adjusted to about 50% (at which point the mechanical properties are optimal for manipulation), and the strip is rolled with slight tension about a cylindrical form. Ordinarily a strip of sufficient length to make ten or more successive layers is employed. The film is then further desiccated t o a moisture content of 20% and autoclaved. The steam treatment forms a strong, seamless tube, which can be removed from the cylindrical form at any time by immersion in water or salt eolution. In making cupshaped objects, the fibrin film is desiccated to ? moisture content of about 50% and stretched over a spherical form, and the edges are tied. Several successive layers may be used to build up the thickness. The moisture content is further reduced t o 20% and the film is autoclaved. The steam releases the stresses and fixes the film in the shape of the form. Fibrin a m in the form of sheets, tubes, and cups is illustrated in the pictures on page 1217. PROPERTIES OF FIBRIN
FILM
0
20
40
60
80
100
120
140
160
Percent Elongation
Stress-Strain Curves of Water-Equilibrated Fibrin Film
Figure 5.
which scatter less light and do not permit passage of protein molecules. With respect t o mechanical properties the untreated and glycerol-sterilized films may be described as soft and rubbery, with fairly rapid retraction after extension, whereas steam-sterilized film is tough, with a higher modulus and delayedelastic recovery, Upon desiccation all the films become progressively tough, inextensible, brittle, and finally very fragile. This behavior ie identical with t h a t of many high polymer systems as the plasticizer content is varied from the maximum compatible value down to zero (15). The stress-strain curves of typical samples of the three kinds of film, obtained a t a loading rate of about 50 grams per minute, are shown in Figure 5. The curve for untreated film (fibrin content, 29%) is almost linear. The initial modulus is about 60 grams per sq. mm., which is of the order of magnitude observed for rubbers (11). The curve for glycerol-sterilized film (fibrin content, 43%) has only a slight curvature. The curve for steam-sterilized film (fibrin content, 57%) has a sharp curvature and is S-shaped; it shows a high initial modulus, a n easier extension from 30 t o loo%, and a firming-up before break. These differences are primarily due t o differences i n fibrin content, as shown by the fact that, when untreated film is partly desiccated t o a fibrin content of 59%, approaching the equilibrium value for steam-sterilized film, its stress-strain curve becomes almost identical with t h a t of the latter (10). The tensile strength of the glycerol-sterilized film is slightly lower, and that of the steam-sterilized film considerably higher, than t h a t of the untreatedfilm.
The properties of the freshly prepared fibrin film, which consists of native human fibrin, and of fibrin film modified by steam treatment are discussed in detail elsewhere (8, 10). From a practical standpoint the properties of the steam-sterilized film are more significant, since this is the material which has the widest use in surgery. Table I compares the properties of untreated, glycerol-sterilized, and steam-sterilized films. Most of the data refer t o film in a state of equilibrium with water. (This is nearly equivalent t o its state TABLE I. PROPERTIES OF FIBRIN FILM when implanted in the tissues, Equilibrated with Water -. where i t is in equilibrium with Swelling MlongaStressTensile tion a t Index. tissue fluids.) The waterFibrin strain strength, break, in 1 M Film equilibrated fibrin content is % content Opacity Permeability curve g./mm. AcOH Untreated 30 Opaque considerably greater for the' Permeable to Almost 160-220 210-260 Dissolves hemoglobin linear heat-treated films than for the Glycerol43 TransImpermeable to Slight 160-180 330-350 24 sterilized parent hemoglobin curvaunmodified film. The marked ture Steamateri67 TramI m ermeable to 5-shaped 600 230 3.9 differences in opacity and perlized parent femoglobin; meability observed may be permeable to diglycine attributed to the difference in 4 Weight of swollen gel divided by weight of fibrin. fibrin content, the denser strucb Films with 14 mg. per sq. om. of fibnn in a 1% solution of oommereial trypsin (Cenoo) a t 37' C. tures having smaller interstices ~
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26
INDUSTRIAL AND ENGINEERING CHEMISTRY
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I n steam-sterilized film, the greatly diminished sv.dliiig I x i r 11 in water and in acid may be a t least partly att,ributed to incre:isi.tl cross linking of the structure, since the tensile strengtli o f thir material is much higher than that of the untreated film. Glyverol-sterilized film, on the other hand, shows somewhat dimiiiished swelling in water and acid but has it lower tensile strength. Thus cross linking, if it occurs here, must be accompanicd t ) ~ ,some molecular breakdown as well. It is significant that tlic, steam treatmen't is carried out a t a fibrin content of 75 to 8OC),, whereas the glycerol treatment is carried out in a much mow dilute state of 30 t o 40y0 fibrin. The former conditions ill Cavoi, caress linking and the latter breakdown, if both processri talic, place concurrently ( 2 1 , 2 3 ) . Both sterilization treatments enormously increase the times TI'quired for digestion by proteolytic enzymes, which can be considered as rough gages of the time of persistence of the film in aliimal tissues (14). This effect may be partly due to inability of t l i i ' enzyme t o diffuse into the treated films. Several of the properties of steam-sterilized film given in T a b h 1, together with others-for example, the moisture content, iii the package, the film weight per unit area, and the affinity for a11 arid and a. basic dye under standard conditions as measured b). the optical densities of dye-stained films-have been measured routinely in the control of production of t'his material. Thc rehults for several preparations of film, made by Armour and Company, are given in Table I1 and indicate the uniformit!. which can be attained. The amount of fraction I required for one fibrin film of staridard weight (14mg. fibrin per sq. cm.) and area (100 sq. cm.) currently produced is approximately that fractionated from one liter ni' plasma (four blood donations).
sntisfiicLury in thi-. wpplici~tion
!(I!,
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\\-as previously available. I t m e prepared for this pur.po,wu11dclcoiltract with the U'nited States S a v y from plasma, fr:iiqiiiiib ilrrived from blood collected by the American Rcd Cross. Fibrin film was used clinically by Schellcli (19) in t r e a h g 111 I fiii,ation> o f the tympanic rriemhrarie and septal perfor:itir)11?. P LA s r r c s b'ilii~iiiogc~ii plastic
prepared I'IYJIT~fibriiu~gvilwhicll has bcc,i: t h i t ' d h r n the frozen state with a low salt content. Ordinsril) tr:Lc:ticin I of human plaems (J),containing 40 t o 607, of fibrii11,g i ~ n ,is employed. The dry protein is mixed with a plnsticizc,:. .rirIi :is water, glycerol, ethylene glycol, or propylene glyco!. '.I'hi: compatibility of the last compound is limited, but the mort1)i)I:ir plasticizers can be mixed over a wide range of proportion?. : i t ltwht from 25 t o 80% protein. The mixture is pressed i i i :d mold :ind t r e a t d with heat-for. t,xample, a t 100' C. Fov I.;, ir
BIOLOGICAL AND CLINICAL APPLICATIONS
Extensive animal experiments on the use of fibrin film L L Z :I dural substitute and observations of the histological sequences following implantation were made by Bailey ( I , 3,12). Rlorrison and Singer ( 1 7 ) ,studying the rate of absorption of implanted films, showed that the persistence of film in the tissues could be adjusted by varying the degree of heat treatment. The use of sheets and tubes of fibrin film in nerve sut,ure was explored i n animals by Singer (20) and by Ingraham, Cobb, and Bering ( I S ) . The use of fibrin film tubes in blood vessel anastomosis was investigated hy Swenson and Gross ( 2 2 ) . Steam-sterilized fibrin film in sheet form has had ext,ensivv ulinic~aluse as a dural substitute and for the prevention of meningocwebral adhesions ( l a ) , a n d hac thus far proved entirely
Figure 6.
Fihrinogeii Plastic
, INDUSTRIAL AND ENGINEERING CHEMISTRY
December, 1946
122 1
placed by water. A t final equilibrium the protein concentration is always less than the original, and the modulus of elasticity is correspondingly lower. The imbibition of water is accompanied by development of opacity. Plastics made with water may be sterilized by partial desiccation and autoclaving with steam, as in the case of fibrin film. A possible procedure for sterilizing other types of plastic, suggested by J. D. Porsche and R. L. Kutz of Armour and Company, consists in heating a t 120’ C. in a mixt‘ure of 80% propylenc glycol and 20%water, Extensive animal experiments on the implantation of plastics prepared from fibrinogen and other proteins, and observations of the histological sequences accompanying their absorption, were made by Bailey and Ford (8). The use of fibrinogen plastic as a. a substitute for cartilage in the Lempert operation for otosclerosis is being investigated in animals by Meltzer (16). Plastics were prepared from other fractions of plasma by the same procedure used with fraction I. In general, globulin (fractions I1 111) and albumin (fraction V, 6) yield products which are stiffer than those made with fibrinogen in the same proportions. The moduli of elasticity of several plastics made from these proteins with glycerol are compared in Figure 8. Mixtures of the different proteins may also be employed.
+
ACKNOWLEDGMENT
Figure 7. Modulus of ‘Elasticityof Fibrinogen Plastics Plotted Logarithmically against Percentage of Protein
minutes. l’hc plastics are transparent and range i n color from pale amber to light brown (Figure 6). They become doubly refracting under stress. Qualitatively, a plastic made with a phlyhydric alcohol plasticizer is rubbery at a protein concentration of 25%, leathery a t SO%, and horny a t 75%. The modulus of elasticity, determined by bending a rectangular block of material, is plotted logarithmically against protein concentration in Figure 7. The stiffness increases by a factor of 600 as the protein content is increased from 30 to 80%. The three polyhydric alcohols are roughly equivalent in plasticizing effectiveness. When a glycol or glycerol plastic is immersed in water (or imp h n t r d in animal tissue), the plasticizer diffuses out and is re-
3.0 Y)
O
3
2.0
U
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5 0 . 60 -1 o/o Protein in Plastic Figure 8. RIodulus of Elasticity of Plasma Protein Plastics with Glycerol 40
8
Fibronogen Globulin Albumin
This work was carried, out under contract, recommended by the Committee on Medical Research, between the Office of Scientific Research and Development and Harvard University. The authors are indebted to William D. Williams for many of the measurements of modulus of elastirity of plastic material., and to Alan R. Downey for technical assistance. LITERATURE CITED
(I) Bailey, 0. T., unpublished experiments. (2) Bailey, 0. T., and Ford, R., Arch. Path., in press. (3) Bailey, 0. T.,and Ingraham, F. D., J . Clin. Investigation, 23, 597 (1944). (4) Cohn, E. J., Science, 101,51 (1945). ( 5 ) Cohn, E. J., Strong, L.E., Hughes, W. L., Jr., Mulford, D. J.. Ashworth, J. N., Melin, M., and Taylor, H. L., J . A m . Chem. Soc., 68,459 (1946). (6) Edsall, J. T., and Miller, S. G., in preparation. (7) Ferry, J. D., and Morrison, P. R., J . A m . Chem. SOC.(submitted). (8) Ibid. (submitted). (9) Ferry, J. D., and Morrison, P. R., J . Clin. Inveutiyation, 23, 566 (1944). (10) Perry, J. D., Singer, M., Morrison, P. R., Porsche, J. D., and Kuts, R. L.,J . A m . Chem. SOC.,in press. (11) Guth, E., and James, H. M., IND.ENO.CHEM.,33,1624 (1941). (12) Ingraham, F. D., and Bailey, 0. T., J . A m . Med. ASSOC., 126, 680 (1944); J . Neurosurg., 1, 23 (1944); Ingraham, F. D., Bailey, 0. T., and Cobb, C. J., J . A m . Med. Assoc., 128, 1088 (1945). (13) Ingraham, F. D., Cobb, C. J., and Bering, E. A., Jr., unpublished experiments. (14) Jenkins, H. P., and Hrdina, L. S., Arch. Surg., 44, 984 (1942); Jenkins, H. P., Hrdina, L. S., Owens, F. M., and Swisher, F. M., Ibid., 45,74 (1942). (15) Mead, D. J., Tiohenor, R. L., and Fuoss, R. M., J . A m . Chem. Soc., 64,283(1942). (16) Meltzer, P. E., unpublished experiments. (17) Morrison, P. R., and Singer, M., J . Clin. Investigation, 23, 573 (1944). (18) Oncley, J. L., Melin, M . , Richert, D. A., Cameron, J. R., and Gross,?. M., in preparation. (19) Schenck, A. D., Calif. Western Medicine, 63,80 (1945). (20) Singer, M., J . Neurosurg., 2, 102 (1945). (21) Spence, D., and Ferry, J. D., J . A m . Chem. SOC.,59, 1648 (19371. (22) Swenson, O., and Gross, R. E., Surgery, in press. (23) Tobolsky, A. V I and Andrews, R. D., J . Chem. Phys., 13, 3 (1945). T H Ipaper ~ is Number 53 in the series “Studies on Plasma Proteins” from the Harvard Medical School, on products developed by the Department of Phy.Gcal Chemistry from blood collected by the Bmerican Red Cross.
