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On pores in fibrin gels
THROMBOSIS RESEARCH 26; 141-142, 1982 0049-3838/82/020141-02$03.00/O Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.
LETTER TO THE EDITORS-lN-CHIEF
ON PORES IN FIBRIN GELS
Birger BlombBck and Masahisa Okada The New York Blood Center, New York, 10021 New York
Accepted
(Received 24.2.1982. by Editor-in-Chief A-L. Copley)
We have in a recent article in this journal described experiments pertaining to the flow properties of fibringels (1). As a result of these studies we concluded that the size, shape and orientation of the pores in fibrin gels is, at a given pH and ionic strength, determined by the clotting time (Ct) of the gel forming system. Short clotting times produce tight gels with small pore sizes and long clotting times give gels having large pores. In the above study we used the Poiseulle equation for calculation of pore sizes (1,2,3). This equation applies to a capillary system with the capillaries parallel to the direction of liquid flow. It is, however, doubtfulwhether this simple model can be applied to the pores in fibringels. In order to obtain more information on the actual pore sizes in fibrin gels, we have performed permeation studies employing spherical latex particles of known particle size for calibration of the pores in the fibrin gels. Gels were prepared in special cups by adding varying amounts of thrombin to a series of solutions of human fibrinogen (experiments l-6 in Fig. 1). After completion of gelation the gels were mounted in a special flow device (1). The theoretical average pore size of the different gels was calculated on the basis of permeation experiments with water. A water suspension (0.02% w/v) of the latex particles was prepared. The suspension was percolated through the gel columns. Fig. 1 shows an experiment with one batch of latex particles. Particles in the effluent from the gel columns were determined by reading the turbidity (OD) of the effluent at 450 nm. As is shown in Fig. 1 the particles penetrated the gels having theoretical pore sizes between 4 and 3 pm, but they did not penetrate gels with pore sizes below 2.4 pm. It is evident that the transition from maximum to minimum penetration occurred in a narrow pore size range (2.4-2.7 r.lm). The theoretical pore size at 50% turbidity would represent the average size of pores and particles. From Fig. 1 we determined this value to 2.54 pm. Since the average diameter of the particles is 0.198 pm, the effective average pore size in the gels is about one order of magnitude less than the theoretically calculated pore size. The difference in pore size between minimum and maximum permeation is a measure of the variation in size of pores and particles. The hoKey-words: Fibrin, Gels, Gelpores.
141
142
ON PORES IN FIBRIN GELS
I
2 Theoretical
’
L
3
pore dm-neier,
Vol.26, No.2
urn
FIG. 1 Penetration
of latex particles
through fibrin gels.
Six (expts l-6) fibrin gel columns (total volume: 3.65 ml) were prepared as previously described (1). Gel formation was at 22-23OC in Tris-imidazol buffer, pH 7.4, containing 20 mM calcium. Fibrinogen: 2.08 g/l - 2.45 g/l. Ionic strength: 0.21 - 0.23. Thrombin: 0.04 0.17 NIH-units/ml.Gelation at room-temperature for 2 hrs. Permeation: Theoretical pore diameter was calculated as previously described (1). Suspensions in water of latex particles (Dow Chemicals, 0.198 pm ? 0.0036) were 0.02% (w/v). Of the suspensions lo-20 1 was percolated through the gels at a pressure of about 3-4 x 10x dyne/ cm . Turbidity was read at 450 nm. Maximum turbidity in effluent was recorded. Arrow: theoretical average pore and particle size. rizontal bar in Fig. 1 represents the variation in particle diameters (average particle size t 3 SD). It is evident that the variation in particle size almost completely accounts for the total variation observed. This then must mean that the variation in pore size is rather small. We have also performed experiments with latex particles of other dimensions. The results were similar to those described above. In addition,preliminary permeation experiments with Sendai virus suggest that the effective pore size determined from permeation experiments with latex particles is valid also for some viruses. AS summary we conclude: 1) the effective pores in fibrin gels are much smaller than theoretically calculated for a parallel capillary system and 2) the pores in fibrin gels appears to be remarkably uniform. ACKNOWLEDGEMENTS This work was supported by a grant from The National Institute Bethesda, Md (HI 27279-01).
of Health,
REFERENCES 1. 2. 3.
BLOMBACK, B. and OKADA, M. Fibrin gelstructure and clotting time. Thrombosis Research, 1_5, 51-70, 1982. FERRY, J.D. Ultrafilter membranes and ultrafiltraticn. Chem. !kv. 18, 373-455. 1936. WHITE, M.L. The permeability of an acrylamide polymer gel. J.Phys.Chem. 62, 1563-1565, 1960.
LETTER TO THE EDITORS-lN-CHIEF
ON PORES IN FIBRIN GELS
Birger BlombBck and Masahisa Okada The New York Blood Center, New York, 10021 New York
Accepted
(Received 24.2.1982. by Editor-in-Chief A-L. Copley)
We have in a recent article in this journal described experiments pertaining to the flow properties of fibringels (1). As a result of these studies we concluded that the size, shape and orientation of the pores in fibrin gels is, at a given pH and ionic strength, determined by the clotting time (Ct) of the gel forming system. Short clotting times produce tight gels with small pore sizes and long clotting times give gels having large pores. In the above study we used the Poiseulle equation for calculation of pore sizes (1,2,3). This equation applies to a capillary system with the capillaries parallel to the direction of liquid flow. It is, however, doubtfulwhether this simple model can be applied to the pores in fibringels. In order to obtain more information on the actual pore sizes in fibrin gels, we have performed permeation studies employing spherical latex particles of known particle size for calibration of the pores in the fibrin gels. Gels were prepared in special cups by adding varying amounts of thrombin to a series of solutions of human fibrinogen (experiments l-6 in Fig. 1). After completion of gelation the gels were mounted in a special flow device (1). The theoretical average pore size of the different gels was calculated on the basis of permeation experiments with water. A water suspension (0.02% w/v) of the latex particles was prepared. The suspension was percolated through the gel columns. Fig. 1 shows an experiment with one batch of latex particles. Particles in the effluent from the gel columns were determined by reading the turbidity (OD) of the effluent at 450 nm. As is shown in Fig. 1 the particles penetrated the gels having theoretical pore sizes between 4 and 3 pm, but they did not penetrate gels with pore sizes below 2.4 pm. It is evident that the transition from maximum to minimum penetration occurred in a narrow pore size range (2.4-2.7 r.lm). The theoretical pore size at 50% turbidity would represent the average size of pores and particles. From Fig. 1 we determined this value to 2.54 pm. Since the average diameter of the particles is 0.198 pm, the effective average pore size in the gels is about one order of magnitude less than the theoretically calculated pore size. The difference in pore size between minimum and maximum permeation is a measure of the variation in size of pores and particles. The hoKey-words: Fibrin, Gels, Gelpores.
141
142
ON PORES IN FIBRIN GELS
I
2 Theoretical
’
L
3
pore dm-neier,
Vol.26, No.2
urn
FIG. 1 Penetration
of latex particles
through fibrin gels.
Six (expts l-6) fibrin gel columns (total volume: 3.65 ml) were prepared as previously described (1). Gel formation was at 22-23OC in Tris-imidazol buffer, pH 7.4, containing 20 mM calcium. Fibrinogen: 2.08 g/l - 2.45 g/l. Ionic strength: 0.21 - 0.23. Thrombin: 0.04 0.17 NIH-units/ml.Gelation at room-temperature for 2 hrs. Permeation: Theoretical pore diameter was calculated as previously described (1). Suspensions in water of latex particles (Dow Chemicals, 0.198 pm ? 0.0036) were 0.02% (w/v). Of the suspensions lo-20 1 was percolated through the gels at a pressure of about 3-4 x 10x dyne/ cm . Turbidity was read at 450 nm. Maximum turbidity in effluent was recorded. Arrow: theoretical average pore and particle size. rizontal bar in Fig. 1 represents the variation in particle diameters (average particle size t 3 SD). It is evident that the variation in particle size almost completely accounts for the total variation observed. This then must mean that the variation in pore size is rather small. We have also performed experiments with latex particles of other dimensions. The results were similar to those described above. In addition,preliminary permeation experiments with Sendai virus suggest that the effective pore size determined from permeation experiments with latex particles is valid also for some viruses. AS summary we conclude: 1) the effective pores in fibrin gels are much smaller than theoretically calculated for a parallel capillary system and 2) the pores in fibrin gels appears to be remarkably uniform. ACKNOWLEDGEMENTS This work was supported by a grant from The National Institute Bethesda, Md (HI 27279-01).
of Health,
REFERENCES 1. 2. 3.
BLOMBACK, B. and OKADA, M. Fibrin gelstructure and clotting time. Thrombosis Research, 1_5, 51-70, 1982. FERRY, J.D. Ultrafilter membranes and ultrafiltraticn. Chem. !kv. 18, 373-455. 1936. WHITE, M.L. The permeability of an acrylamide polymer gel. J.Phys.Chem. 62, 1563-1565, 1960.