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WO2000047148A1 - Procedes et compositions permettant de renforcer la migration des fibroblastes - Google Patents

Procedes et compositions permettant de renforcer la migration des fibroblastes Download PDF

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Publication number
WO2000047148A1
WO2000047148A1 PCT/US2000/003330 US0003330W WO0047148A1 WO 2000047148 A1 WO2000047148 A1 WO 2000047148A1 US 0003330 W US0003330 W US 0003330W WO 0047148 A1 WO0047148 A1 WO 0047148A1
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Prior art keywords
fibrinogen
solution
precipitate
produce
supernatant
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Inventor
Richard A. Clark
Dennis K. Galanakis
Azim Khan
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Research Foundation of the State University of New York
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Research Foundation of the State University of New York
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Priority to AU28756/00A priority Critical patent/AU2875600A/en
Priority to EP00907227A priority patent/EP1164992A4/fr
Publication of WO2000047148A1 publication Critical patent/WO2000047148A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/106—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen

Definitions

  • the subject invention is directed to methods and compositions for enhancing fibroblast migration and promoting wound healing.
  • U.S. Patent Nos. 4,950,483 and 5,024,841 each discuss the usefulness of collagen implants as wound healing matrices.
  • U.S. Patent No. 4,453,939 discusses a wound healing composition of collagen with a fibrinogen component and a thrombin component, and optionally fibronectin.
  • U.S. Patent No. 4,970,298 discusses the usefulness of a biodegradable collagen matrix (of collagen, hyaluronic acid, and fibronectin) for wound healing. Yamada et al .
  • 5,631,011 discloses a composition of HA and fibrin or fibrinogen.
  • Various other compositions have also been explored for their wound healing capabilities.
  • Kratz et al . (1997) used a gel of heparin ionically linked to chitosan.
  • Bartold and Raben studied platelet- derived growth factor ("PDGF").
  • Henke et al . (1996) disclosed that chondroitin sulfate proteoglycan mediated cell migration on fibrinogen and invasion into a fibrin matrix, while Nakamura et al . (1997) concluded that chondroitin sulfate did not affect wound closure in a corneal epithelial wound.
  • U.S. Patent No. 5,641,483 discloses topical gel and cream formulations containing human plasma fibronectin for healing cutaneous wounds.
  • Schultz et al . (1992) discloses a composition of epidermal growth factor ("EGF"), fibronectin, a synthetic collagenase inhibitor, and Aprotinin.
  • EGF epidermal growth factor
  • Fibrin matrices and components of fibrin matrices have been investigated for promoting wound healing. Besides being the ultimate plug of the hemostasis system, fibrin is part of a provisional matrix that provides tissue cells a scaffold for repopulation of a wound (Clark et al . (1982a)). Recently, however, it was discovered that fibroblasts, tissue mesenchymal cells, will not penetrate a pure fibrin clot (Greiling and Clark (1997) ) . Another plasma protein, fibronectin, normally found in blood clots must be present in the clot for fibroblast migration (Greiling and Clark (1997) ) .
  • Fibrin sealants are topical, biologically compatible, resorbable tissue adhesive that initiate the last phases of coagulation during wound healing.
  • the components of fibrin sealants typically consists of concentrated human fibrinogen in solution with various amounts of fibronectin and factor XIII, as well as other components.
  • the fibrin sealants are activated by addition of thrombin and calcium chloride and subsequently form a coagulum (clot) .
  • Methods of obtaining the concentrated fibrinogen include precipitation of plasma by cryroprecipitation, polyethylene glycol or ammonium sulphate (Brennan (1991) ) . Contradictory results are obtained, however, for these preparations.
  • the fibrinogen preparations improve healing (Gelich et al . (1995); Saclarides et al . (1992), in others no improvement in healing was found (Lasa et al . (1993); Byrne et al . (1992). Further, animal studies have not been predicative of clinical use.
  • the fibrin sealants utilized to date vary in purity levels. In view of the severity of the problem of chronic, nonhealing wounds, new and more effective matrices and methods for facilitating wound healing, and in particular, fibroblast migration are needed. The present invention is directed to meeting this need.
  • the present invention relates to a method for enhancing fibroblast migration at a wound site.
  • the method includes contacting the wound site with fibrinogen that is prepared by a process which includes precipitating plasma with glycine.
  • Another aspect of the present invention relates to a method for enhancing fibroblast migration at a wound site which includes contacting the wound site with a fibrinogen preparation which includes a lipid rich component .
  • Another aspect of the present invention relates to a composition which includes fibrinogen and a lipid rich component.
  • the present invention relates to a method and a composition for enhancing wound healing by enhancing fibroblast migration to the wound site.
  • enhancing fibroblast migration wounds that require the rapid formation of new tissue, such as chronic cutaneous ulcers and fresh surgical and traumatic wounds that cannot be closed, can be treated. Fibrinogen preparations that merely act as a blood clotting mechanism cannot achieve this objective.
  • Figure 1 is a schematic illustration of the three sub-fractions of one embodiment of the fibrinogen preparation of the present invention.
  • Figure 2 is a chromatogram illustrating the fibrinogen peak and lipoprotein peak of a preparation of the present invention.
  • Figure 3 is a comparison of the effect on fibroblast migration of fibrinogen isolates Cl (positive control) , C2 (negative control) , 2H, 2F, 2HL and 2FL.
  • Figure 4 illustrates the fibroblast migration activity of the lipoprotein peak of Figure 2.
  • the present invention relates to a method for enhancing fibroblast migration at a wound site.
  • the method includes contacting the wound site with fibrinogen that is prepared by a process which includes using glycine to precipitate fibrinogen from plasma.
  • Another aspect of the present invention relates to a method for enhancing fibroblast migration at a wound site which includes contacting the wound site with a fibrinogen preparation which includes a lipid rich component .
  • Another aspect of the present invention relates to a composition which includes fibrinogen and a lipid rich component .
  • enhancing fibroblast migration is meant to include any improvement or increase in the movement or mobility of fibroblast cells in a wound.
  • Fibroblast migration can be measured by a variety of methods. One particularly advantageous method is described in U.S. Patent No. 5,935,850 (the contents of which are hereby incorporated by reference herein) ; Greiling and Clark (1996) ; and Greiling and Clark (1997) . Briefly, organotypic dermal constructs consisting of human adult dermal fibroblasts embedded in floating type 1 collagen gels are pasted on 24 well tissue culture dishes coated with fibrin fibrils. Fibrin gels are then cast around the "dermal equivalent” forming an "inside- out” wound construct.
  • the number of fibroblast cells that migrate in the presence and absence of various soluble biologic response modifiers can then be quantified, for example using a Nikon inverted phase microscope, by visually counting identifiable fibroblast cell nuclei located outside of the construct.
  • the increase in fibroblast migration produced by contacting the wound with fibrinogen in accordance with the present invention e.g., (A-A 0 )/A 0 , where A and A 0 are the number of identifiable fibroblast cell nuclei located outside of the above-described construct in the presence and absence, respectively, of fibrinogen
  • a and A 0 are the number of identifiable fibroblast cell nuclei located outside of the above-described construct in the presence and absence, respectively, of fibrinogen
  • a "wound” and “wound site” are intended to include both acute and chronic dermal wounds including, for example, surgical incisional wounds, traumatic wounds, cancer extirpations, radiation wounds, venous leg ulcers, diabetic ulcers, and pressure ulcers .
  • the plasma employed in the present invention is collected by conventional methods and, in practice, can be from blood of a single individual, or, alternatively, it can be pooled from multiple individuals.
  • the plasma is from the same species of animal (e.g., human) as the wound being treated.
  • fibrinogen is isolated from the plasma by precipitation.
  • the method of precipitation is achieved with glycine and is carried out in a number of steps .
  • the plasma is precipitated with glycine to produce a precipitate and a supernatant in a manner known to those of ordinary skill in the art.
  • precipitation is carried out by adding glycine to the plasma in an amount such that the final concentration of glycine in the plasma/glycine mixture is from about 1.0 to about 2.1 M.
  • the glycine is added as dry glycine to the mixture.
  • precipitation is allowed to proceed during incubation. Incubation proceeds at temperatures below room temperature (e.g., refrigeration temperatures) , preferably from between about 2 °C and about 7 °C, more preferably about 5 °C.
  • Incubation occurs for from about 30 minutes to about 12 hours, preferably about 1 hour, until the precipitate is formed.
  • it is most convenient to conduct the precipitation by placing the plasma/glycine mixture in a standard refrigerator (i.e., at about 5 °C) .
  • a precipitate and a supernatant are produced, which can be separated by conventional methods, such as decanting or, preferably, centrifuging at temperatures from between about 2 °C and about 7 °C, preferably about 5 °C.
  • the precipitate will contain about 90% of the fibrinogen from the plasma.
  • the purity of the fibrinogen is above 50%. If a preparation having high purity content is desired, fibrinogen is further isolated from the precipitate of the first step.
  • a high purity content fibrinogen is defined as fibrinogen having a purity content of about or above 99%.
  • a second step is used to isolate fibrinogen from the precipitate of the first step.
  • second step generally refers to the process of adding buffer to a precipitate to produce a mixture, adding glycine to the mixture to produce a precipitate and a supernatant, and separating the precipitate and supernatant. This second step can be repeated as many times as desired.
  • the precipitate produced in the first step is dissolved in a suitable buffer to produce a solution.
  • the buffer has a pH of from about 6 to about 8, preferably from about 6.2 to about 7.6, most preferably about 6.4.
  • One suitable buffer for carrying out this process contains about 150 mM of sodium chloride, about 10 mM sodium phosphate, and 100 mM epsilon-aminocaproic acid in water, preferably in sterile water suitable for injection.
  • Other buffers suitable in the present invention include 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl .
  • the amount of buffer employed to effect the dissolution is preferably from about 30% to about 40% of the volume of the original plasma used in the first step.
  • the buffer used in this second step preferably has a volume of from about 0.3 V to about 0.4 V (i.e., between about 3/10 's of V to about 4/10 ' s of V) . More preferably, the volume of buffer employed to effect the dissolution of the precipitate is about 35% of the volume of the plasma used in the first step.
  • Glycine typically dry (as described above) , and at a suitable concentration is then added to the resulting solution in an amount such that the final concentration of glycine in the resulting mixture is from about 1.7 to about 2.2 M and, more preferably, about 2.1 M.
  • the resulting mixture is incubated, preferably at a temperature of from between about 2 °C to about 7 °C, most preferably at about 5 °C, for from about 30 minutes to about 2 hours, preferably for about one hour.
  • a precipitate and supernatant form which are separated, preferably by centrifugation at from between about 2 °C to about 7 °C, most preferably at about 5 °C.
  • This second step is advantageously repeated several times. Preferably, the second step is repeated at least twice.
  • the precipitate at the end of the second step will contain about 60% of the fibrinogen from the plasma.
  • the fibrinogen is at a purity of about 90%.
  • fibrinogen can be additionally or alternatively isolated from the supernatant (instead of the precipitate) of the original plasma/glycine mixture of the first step in a manner similar to that described above.
  • low purity fibrinogen means fibrinogen having a purity content of about or above 95%, but below 99%.
  • the low purity fibrinogen is produced by adding glycine, typically dry and at a suitable concentration, to the supernatant of the first step to produce a mixture.
  • the resulting mixture is incubated, preferably at refrigerator temperatures, for about 1 hour.
  • a precipitate and supernatant form which are separated, preferably by centrifugation at refrigerator temperatures.
  • the precipitate can then be dissolved in an appropriate buffer (e.g., the ones described above) , glycine added (preferably to a final glycine concentration of about 2.1 M) , the mixture incubated, and the resulting precipitate separated. This process can be repeated several times.
  • the precipitate can be advantageously further treated to purify the fibrinogen.
  • the further treatment includes dissolving the precipitate resulting from the glycine precipitation (s) described above in an appropriate buffer (e.g., as described above) to produce a solution where the precipitate is present in a volume of 1/2 to 1/3 of the original plasma, with 1/3 being especially preferred, and precipitating this solution.
  • the precipitation is achieved by adding a compound such as ammonium sulfate to the solution.
  • the ammonium sulfate is added as a saturated solution, and the amount of ammonium sulfate in the solution is about 25 percent of its saturation level.
  • the resulting solution is redissolved in a suitable buffer (as described above) and reprecipited, preferably with dialysis in 0.3 M NaCl .
  • a precipitate fraction of fibrinogen with a purity of greater than 95% is obtained.
  • fibrinogen having a purity of about or above 99% is obtained.
  • lipid rich fraction is obtained.
  • the lipid rich solids are found floating in the final supernatant produced by either method described above.
  • the lipid rich solids are separated from the final supernatant (typically by centrifugation) , added to a suitable buffer (as described above) and reprecipited, preferably with dialysis in 0.3 M NaCl, and treated with 25% saturated ammonium sulfate (as described above) .
  • the precipitate from this step is discarded and the resultant supernatant is rich in plasma lipids.
  • This supernatant which includes lipids is called the lipid rich component.
  • the lipids contained in the lipid rich component result in enhanced fibroblast migration when applied to a wound.
  • proteins bound to the lipids of the lipid rich layer result in enhanced fibroblast migration when applied to a wound.
  • the fibrinogen and the lipid rich component are stored, preferably at from about -50 °C to about -80 °C. Prior to use, they can be thawed, for example at 37 °C.
  • the fibrinogen prepared in accordance with the above-described methods enhances fibroblast migration during wound healing and, thus, enhances wound healing.
  • the lipid rich component enhances fibroblast migration during wound healing. Enhancement of wound healing refers to the traditional sense of wound healing where clean closure of the wound occurs. Since naturally occurring wound healing involves the movement of fibroblasts into the wound site, enhancement of wound healing can be assayed in vi tro using the model for cell transmigration provided in U.S. Patent No.
  • the model provides a contracted collagen gel containing fibroblasts surrounded by a fibrin gel.
  • fibrinogen prepared as described above is contacted with the fibrin gel, fibroblast movement from the collagen gel into the fibrin gel is enhanced.
  • the above described method of enhancing migration of fibroblasts at a wound site can advantageously further include contacting the wound site with other materials which promote wound healing.
  • Such contacting with other materials that promote wound healing can occur prior to, during, and/or after the wound site is contacted with fibrinogen.
  • the wound site can be contacted with a growth factor (such as platelet-derived growth factor ("PDGF") (described in, for example, Seppa et al . (1982) and in Senior et al . (1985)) or an extracellular matrix material (such as, fibronectin and hyaluronan) .
  • PDGF platelet-derived growth factor
  • an extracellular matrix material such as, fibronectin and hyaluronan
  • the wound site is contacted with fibronectin in addition to fibrinogen prepared by the process described above.
  • fibronectin is added to the fibrinogen by mixing the fibronectin with the fibrinogen.
  • the wound site can be contacted with more than one growth factor, more than one extracellular matrix material, or combinations of growth factor (s) and extracellular matrix material (s) .
  • the wound site is contacted with the lipid rich component.
  • the wound site can be contacted with the lipid rich component either alone or in combination with fibrinogen, one or more growth factors, one or more extracellular matrix materials or a combination thereof.
  • the fibrinogen prepared as described above can be contacted with the wound site by incorporating it in a fibrinogen preparation and then contacting the fibrinogen preparation with the wound site.
  • the fibrinogen preparation generally contains fibrinogen which enhances fibroblast migration.
  • inert additives may be incorporated into the fibrinogen preparation. These include preservatives, dispersants, diluents, and other physiologically compatible materials.
  • the fibrinogen is to be used concurrently with growth factor (s), extracellular material (s), or combinations thereof
  • the growth factor (s) and/or extracellular matrix material (s) can advantageously be incorporated in the fibrinogen preparation.
  • the fibrinogen, lipid rich component, growth factors, extracellular materials, or combinations thereof can be contacted with the wound site separately.
  • the fibrinogen preparation can further include thrombin so that the fibrinogen preparation, in addition to enhancing fibroblast migration, also promotes blood clotting.
  • the fibrinogen preparation of the present invention i.e., the fibrinogen preparation containing fibrinogen prepared as described above
  • wounds that are not bleeding are meant to include wounds that may be oozing blood but that would not be considered by the skilled clinician as requiring intervention to induce or promote clotting.
  • Fibrinogen preparations that are substantially free of thrombin are meant to include preparations in which the thrombin level is below the level which is generally viewed as being necessary to promote clotting when used in conjunction with fibrinogen.
  • fibrinogen preparations that are substantially free of thrombin include fibrinogen preparations in which the weight-to-weight ratio of thrombin to fibrinogen is less than 1, less that 0.8, less than 0.5, less than 0.3, or less than 0.1.
  • the fibrinogen or fibrinogen preparation of the present invention is contacted with the wound or wound site. Contacting can be carried out by any suitable method.
  • the fibrinogen or fibrinogen preparation can be delivered to the wound site via a syringe or pipet or by spraying or misting fibrinogen or fibrinogen preparation onto the wound.
  • Lyophilized fibrinogen or fibrinogen preparation can be used directly in powdered form, for example by sprinkling the powder on the wound.
  • the fibrinogen or fibrinogen preparation can be applied to the wound by incorporating it in a gauze pad, sponge, collagen or gel- type matrix and then applying the gauze pad, sponge, collagen or gel-type matrix to the wound.
  • the fibrinogen or fibrinogen preparation of the present application can be contacted with wounds that are bleeding or with wounds that are not bleeding.
  • the fibrinogen or fibrinogen preparation of the present application can be first contacted with the wound while the wound is bleeding and said contacting can be continued after bleeding ceases.
  • the contact which is maintained subsequent to cessation of bleeding is to be considered to be a contact with a non-bleeding wound.
  • the fibrinogen or fibrinogen preparation of the present application can be contacted with a wound only after bleeding from the wound stops .
  • the amount of fibrinogen or fibrinogen preparation to be applied to the wound depends on a variety of factors, such as the size of the treatment site, the nature and condition of the wound in need of treatment, and factors that might be unique to the patient being treated.
  • the optimal therapeutic amount for a specific wound can be determined by contacting the wound site with various concentrations of fibrinogen or fibrinogen preparation and observing the effect on wound healing.
  • the present invention is further illustrated by the following examples.
  • the fibrinogen preparations of the present invention were prepared from a IM glycine precipitation formed in the cold (4°C) that had been discarded in a previously described procedure (Galanakis (1995)).
  • glycine was dissolved in pooled human plasma to attain 1 M final concentration and the mixture was allowed to stand on ice for at least one hour. By subsequent centrifugation at 5°C, two fractions were obtained: the precipitate and the supernatant.
  • the precipitate obtained from plasma containing 1 M glycine, 5°C was redissolved (pH 6.4) and re-precipitated twice with 2.1 M glycine, 5°C.
  • the precipitate was then dissolved, precipitated with (NH 4 ) 2 S0 4 to 25% saturation, redissolved and exhaustively dialyzed vs. 0.3M NaCl.
  • the final product was stored at -80 °C. Greater than 95% purity was ascertained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • Sample 1 was isolated as previously described (Galanakis (1995) ) . It was the purest fibrinogen isolate. It had virtually all its alpha chains intact but lacked molecules with gamma chains that have an extended carboxy terminal which constitute approximately 15% of the fibrinogen in plasma.
  • the extended gamma chains are from a splicing mRNA variant . It extends the gamma chain by deleting the last two amino acids of the regular gamma chain and extending it with a 20 amino acid segment.
  • Sample 2 was isolated by a modification
  • Sample 2 was a highly pure fibrinogen isolate, but less pure than Sample 1, in that a minute contaminant of Factor XIII was detectable by biologic activity. It also had virtually all of its alpha chains intact and contained molecules with both extended and non-extended gamma chains.
  • Sample 3 was the same as Sample 2 , but was enriched with soluble fibrin.
  • Fibrin monomer was prepared as described in Galanakis et al. (1987). An amount of fibrin monomer (from a stock solution of 20 to 30 mg/ml, pH 4.5) was added to exceed 10% (mg/mg) of fibrinogen in solution, allowed to equilibrate at 37 °C and any clot that formed was removed. The resulting fibrinogen solution was termed fibrin-saturated and used. Care was taken to ascertain that the pH of the fibrinogen solution remained above 6 in storage and during clotting.
  • Sample 4 was isolated by a modification (Galanakis (1995)) of the procedure of Mosesson and Sherry (1966) .
  • Sample 4 had a purity similar to that of Sample 3, but contained a major population of molecules (approx. 20 to 30%) which were clottable but had degraded alpha chains .
  • Sample 5 was isolated by a modification (Galanakis (1995) ) of the procedure of Mosesson and Sherry (1966) .
  • Sample 5 was similar to Sample 4 in structure but less pure in that trace, but detectable, amounts of fibronectin and factor XIII were present.
  • Sample 6 was similar in all respects to Sample 5, with the exception of being enriched with soluble fibrin.
  • Fibrin monomer was prepared and added to Sample 5 as described above for Sample 3.
  • Sample 7 was isolated from the supernatant produced in Sample 1 above. It contained only minor amounts of fibrinogen, but was rich in plasma lipids.
  • DMEM Dulbecco ' s Modified Eagle's Medium
  • Fibroblast cultures at 80% confluence were harvested by treatment with a 0.05% trypsin/0.01% EDTA. Trypsin is inactivated by addition of soy bean trypsin inhibitor in PBS containing 0.2% BSA.
  • the cells are washed twice with DMEM + 2% BSA and resuspended at a concentration of 1 x 10 s cells/ml.
  • the fibroblasts are mixed with neutralized collagen (Vitrogen 100, Celtrix Labs., Santa Clara, CA.), 2% BSA, 30 ng/ml PDGF-BB, 30 ⁇ g/ml fibronectin , and concentrated DMEM so that the final concentration of DMEM and sodium bicarbonate is lx.
  • 600 ⁇ l of the cell mixture is added to the wells of a 24- well tissue culture plate, which has been precoated with 2% BSA.
  • the collagen is allowed to polymerize at 37° C.
  • the final concentration of collagen is 1.8 mg/ml and each gel contains 6 x 10" cells.
  • the gels are gently detached from the plastic surface to allow contraction with the addition of 0.5 ml DMEM + 2% BSA and 30 ng/ml PDGF-BB per well.
  • the gels are incubated overnight at 37° C in 100% humidity, 5% C0 2 and 95% air.
  • fibrinogen preparations described above were tested to determine if fibroblast migration was enhanced by the fibrinogen preparation of the present invention.
  • a relatively low solubility fraction can be harvested with IM glycine that is fibrin-rich and contains other proteins with low solubility. In typical fibrinogen purification schemes, however, this fraction is usually discarded because it is not readily soluble in physiologic buffer systems. It has been used, however, to isolate other proteins including fibronectin, Factor VIII and Factor XIII. Although there are alternative ways to obtain low solubility proteins from plasma, (e.g.
  • the present invention focuses on the fibrinogen isolate from the precipitate, since it is the only low solubility protein fraction from plasma which supports fibroblast migration.
  • the present invention is directed to the lipid rich component, which also supports fibroblast migration.
  • This matrix has potential for use in freshly debrided chronic cutaneous ulcers and fresh surgical and traumatic wounds that cannot be closed.
  • a fibrin clot is deposited in the wound space (Welch et al . (1990) ) .
  • the clot contains fibrin, fibronectin and vitronectin which together provide a provisional matrix scaffold for the movement of recruited cells into the wound space (Greiling and Clark (1997) ; Clark (1993a) ; Clark (1993b)).
  • platelets release a plethora of growth factors including the potent mesenchymal cell mitogen, platelet-derived growth factor (PDGF) (Heldin et al. (1996) ) .
  • PDGF platelet-derived growth factor
  • blood leukocytes especially neutrophils and monocytes
  • monocytes migrate into the fibrin-rich provisional matrix.
  • monocytes As monocytes mature into macrophages they begin to produce growth factors, including PDGF-BB, which are added to the wound space milieu (Rappolee et al . (1988); Shaw et al . (1990)) .
  • growth factors including PDGF-BB
  • Endothelial cells within blood vessels adjacent to the wound also proliferate, causing marked vessel hypertrophy (Clark et al (1982a); Clark et al . (1982b) ) .
  • a contracted collagen gel containing skin fibroblasts was pasted onto a surface of fibrin fibrils and surrounded by a fibrin clot (Greiling and Clark (1997)). This forms an "inside-out" wound environment.
  • 30 ng/ml PDGF was added to the fibrin clot. Fibroblast appearance in the translucent fibrin gel was quantified by cell counts. Cell accumulation in the fibrin gel was attributable to migration rather than mitogenesis as judged by the movement of nonproliferating, irradiated cells.
  • Fibronectin at a 1:10 molar ratio to fibrinogen and 30 to 100 ng/ml PDGF were added to the fibrinogen just prior to clotting with human thrombin as previously described to result in maximal cell migration (Greiling and Clark (1997)).
  • fibroblast migration enhancement FME
  • fibrinogen preparations that are of the highest possible purity, such as fibrinogen fraction 1-4, DEAEc peak 1 fraction, and others (produced by methods known to those skilled in the art) either possess low or no activity.
  • fibrinogen fraction 1-4 which can be prepared in bulk and whose moderate FME activity progressively decreases on repeat freeze thawing, thus enabling its use as a negative control.
  • positive and negative controls are termed Cl and C2 , respectively.
  • Fibrinogen isolates with high activity are isolated from plasma by procedures that yield two kinds of active isolates, one enriched with soluble fibrin and the other lacking fibrin enrichment.
  • the procedures also yield a lipid or lipoprotein rich (L) component which is enriched in FME activity.
  • Fibrin-rich fibrinogen preparation Glycine is dissolved in plasma to 1 M (or 1 molar) concentration and allowed to stand at 4° C overnight. The precipitate formed is dissolved in phosphate buffered saline, pH 6.4, subjected to reprecipitation with 2.1 M Glycine at 4° C, and this step is repeated. An additional precipitation step is performed with either 2.1 M Gly or 25% saturated Ammonium Sulfate. The fibrinogen isolate thus obtained is dialyzed vs 0.3 M NaCl at 4° C and termed 2F. As shown below, fibrinogen 2F may be further separated into sub-fractions . A large amount of insoluble fibrin gel forms during dialysis, and this is removed by centrifugation as described below.
  • Fibrinogen low in fibrin content preparation Fibrinogen low in fibrin content preparation .
  • An isolate is obtained from the plasma supernatant of the initial 1 M Gly step detailed in the paragraph above.
  • additional Glycine is added and dissolved to achieve 2.1 M Gly concentration and the precipitate obtained at 4° C is subjected to the same precipitation steps described above.
  • the final isolate is dialyzed as above and termed 2H.
  • fibrinogen 2H may be further separated into sub- fractions. During dialysis a small amount of insoluble fibrin forms and is removed by centrifugation as described in the paragraph below.
  • each of the fractions, 2F and 2H is subjected to centrifugation using at least 4000 XG for 30 or more minutes at 4° C.
  • the resulting three sub-fractions are illustrated in Fig. 1.
  • One sub-fraction is a lipid or lipoprotein component which contains insoluble material floating at the top of the solution. This subtraction is termed 2FL (from the 2F fibrin rich fraction) or 2HL (from the 2H low fibrin content fraction) .
  • 2FL from the 2F fibrin rich fraction
  • 2HL from the 2H low fibrin content fraction
  • the second sub-fraction is the bulk of isolated fibrinogen and is referred to herein as 2F sub- fraction (fibrin rich fibrinogen) or 2H sub-fraction (low fibrin content fibrinogen) .
  • the third sub-fraction is a pellet at the bottom of the centrifuged solution, which consists of insoluble fibrin gel and is discarded.
  • fibrinogen 2H invariably contains major amounts of sub-fraction 2HL, and fibrinogen 2F, by contrast, tends to contain lower amounts of sub-fraction 2FL. Such differences are demonstrable by dissolving the material and measuring its turbidity.
  • 2FL sub-fractions are of substantial amounts while others are of very small amounts compared to those in the 2HL counterparts from the same starting plasma.
  • a constellation of characteristics of the L isolates is their self-evident low density and coalescence into insoluble floating sheets or particles following centrifugation, their yellowish-white and opaque appearance on visual inspection (particularly marked in 2HL) , their capacity to be readily dispersed and redissolved in fibrinogen containing buffers, and their high turbidity when re-dissolved and assessed spectrophotometrically.
  • fibrin is formed in the assay from the same lot. Comparison can also be made with the same parent (or uncentrifuged fibrinogen preparation) .
  • fibrinogen can be removed so that the sub- fraction can be tested without its parent fibrinogen.
  • ammonium sulfate is added to 25% saturation and the precipitate is discarded.
  • the floating insoluble material on the top of the solution is then harvested and dialyzed.
  • An alternate method to sub-fractionate 2F or 2H isolates The fractions can be sub-fractionated by subjecting them to size exclusion chromatography, as shown in Fig. 2.
  • Absorbance values of the eluting fractions are obtained at 280 nm and at 350 nm.
  • Absorbance at 280 nm reflects the presence of protein and that at 350 nm reflects light scattering and, thus, the opacity caused by the lipid content of the fibrinogen solution.
  • This chromatographic procedure results in an early elution peak, labeled peak I. Fractions containing this peak appear white-opaque, show high absorbance values at 350 nm as expected, and show the presence of protein by their absorbance values at 280 nm.
  • this peak When dialyzed against water and freeze dried, this peak is insoluble in buffer but can be resolubilized at least in part in fibrinogen or other protein solutions.
  • the second peak, labeled peak II, consists of fibrinogen and soluble fibrin. This peak shows absorbance at 280 nm but little or negligible absorbance at 350 nm, as shown, and constitutes the bulk of protein applied to the column.
  • Fractions 2F and 2H have ample activity, with 2H showing moderately higher activity than 2F. Similarly, this activity remains high, as shown in Fig. 3, in both the L rich subfractions (2FL, 2HL) and fibrin rich sub- fractions (2H, 2F) . Moreover, these sub-fractions, 2FL and 2HL, show similarly high activity (See Fig. 3 insert) and this activity remains when fibrinogen is removed from 2HL and 2FL (not shown) . Because insoluble lipid in 2HL may induce formation of an abnormal fibrin matrix, care is taken to avoid insoluble lipid aggregates when using 2HL. This is not the case with 2FL, which contains a much lower lipid content and lacks such large insoluble aggregates .
  • Fibroblast enhancing (FME) activi ty Chromatographic sub fractions .
  • both peaks possess substantial fibroblast migration enhancing activity.
  • the fibroblast migration activity of the lipoprotein (L) peak of Fig. 2 is shown in the Fig. 4. This is consistent with the results from the post- centrifugation sub-fractions described above, indicating that the activity is present both in the L rich and the fibrinogen (L poor) peaks. Considering the small amount of L sub-fraction required to demonstrate the activity, this implies the lipid in these isolates contains relatively higher activity than does fibrinogen per se. That is to say, the activity is more lipophilic than fibrinogenophilic . Moreover, when fibrin-rich fibrinogen was isolated from the ascending limb of the chromatogram it too displayed substantial activity, not shown, consistent with the fact that fraction 2F is fibrin rich and shows activity comparable to 2H.
  • the fibroblast migration enhancement activity of fibrinogen isolated by the above procedures is associated with three components or sub-fractions : fibrin rich fibrinogen, lipid rich fibrinogen, and fibrinogen not enriched with either fibrin or lipid.
  • lipid rich fibrinogen (2FL or 2HL) displays somewhat higher activity, this activity remained when the lipid component was rendered free of fibrinogen by chromatography or other means.
  • fibrin rich fibrinogen which is also rendered lipid poor clearly retains its high activity.
  • this activity is progressively lost by freeze thawing of 1-4, and absent in DEAEc pure fibrinogen.
  • the lipid component can be isolated and re-introduced into fibrinogen of high purity or any other fibrinogen isolate for the purpose of further enriching its FME activity. This discovery makes it possible to monitor the amounts of this lipid component in any fibrinogen and/or soluble fibrin preparations.
  • Lamme E.N., et al . , J. Histochemistry and Cytochemistry, 44(11) -1311-1322 (1996).

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Abstract

L'invention se rapporte à des procédés et à des compositions permettant de renforcer la migration des fibroblastes au niveau d'une plaie. Ledit procédé consiste à mettre en contact la plaie avec du fibrinogène que l'on prépare selon un procédé consistant à faire précipiter du plasma avec de la glycine. Les compositions de cette invention contiennent un composant riche en lipides et du fibrinogène.
PCT/US2000/003330 1999-02-09 2000-02-09 Procedes et compositions permettant de renforcer la migration des fibroblastes Ceased WO2000047148A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7262174B2 (en) 2001-05-09 2007-08-28 Geron Corporation Treatment for wounds

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4453939A (en) * 1981-02-16 1984-06-12 Hormon-Chemie Munchen Gmbh Composition for sealing and healing wounds
US4950483A (en) * 1988-06-30 1990-08-21 Collagen Corporation Collagen wound healing matrices and process for their production
US5024841A (en) * 1988-06-30 1991-06-18 Collagen Corporation Collagen wound healing matrices and process for their production

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DD231006A1 (de) * 1984-08-28 1985-12-18 Bezirks Inst F Blutspende Und Verfahren zur herstellung gerinnungsaktiver plasmafraktionen aus humanblut

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4453939A (en) * 1981-02-16 1984-06-12 Hormon-Chemie Munchen Gmbh Composition for sealing and healing wounds
US4950483A (en) * 1988-06-30 1990-08-21 Collagen Corporation Collagen wound healing matrices and process for their production
US5024841A (en) * 1988-06-30 1991-06-18 Collagen Corporation Collagen wound healing matrices and process for their production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1164992A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7262174B2 (en) 2001-05-09 2007-08-28 Geron Corporation Treatment for wounds

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