WO1995015763A1 - Process for the preparation of serum and platelet growth factors extract - Google Patents

Process for the preparation of serum and platelet growth factors extract Download PDF

Info

Publication number
WO1995015763A1
WO1995015763A1 PCT/CA1994/000601 CA9400601W WO9515763A1 WO 1995015763 A1 WO1995015763 A1 WO 1995015763A1 CA 9400601 W CA9400601 W CA 9400601W WO 9515763 A1 WO9515763 A1 WO 9515763A1
Authority
WO
WIPO (PCT)
Prior art keywords
platelet
serum
extract
plasma
concentration
Prior art date
Application number
PCT/CA1994/000601
Other languages
French (fr)
Inventor
Paul Brazeau
Thierry Abribat
Michel Ibea
Michèle BOUSHIRA
Original Assignee
Universite De Montreal
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universite De Montreal filed Critical Universite De Montreal
Priority to AU79885/94A priority Critical patent/AU7988594A/en
Publication of WO1995015763A1 publication Critical patent/WO1995015763A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum

Definitions

  • the invention relates to a process for the pro ⁇ duction of a whole blood derived serum and/or platelet extracts and their clinical uses in wound dressing,
  • the very first event that normally occurs in a wound is blood extravagation, that results in platelet aggregation and impregnation of the wound with platelet and serum constituents.
  • these constituents are polypeptide growth factors, which are known to play a major role in tissue regeneration.
  • Platelet ⁇ gran- ules which are released by aggregated platelets, are one of the richest physiological source of platelet- derived growth factor (PDGF) and transforming growth factor ⁇ (TGF ⁇ ), while serum contains high amounts of insulin-like growth factor I (IGF-I), IGF-II and their binding proteins (IGF-BPs) (Strovbant P & Waterfield MD, Embo.J., 1984, 2 :2963-2967; Assoian RK et al., J. Biol. Chem., 1983, 258:7155-7160: Sara VR et al., Physiol. Rev., 1990, 70:591-614) .
  • IGF-I insulin-like growth factor I
  • IGF-BPs binding proteins
  • PDGFs include PDGF, platelet derived angiogene- sis factor (PDAF), TGF ⁇ and platelet-factor-4 (PF-4), which is a chemoattractant for neutrophils (Knighton et al. , In Growth Factors and Other Aspects ofWound Healing: Biological and Clinical Implications, Alan R. Liss, Inc., pp. 319-329 (1988)).
  • PDGF is a mitogen and chemoattractant for fibroblasts and smooth muscle cells and is a stimulator of protein synthesis in cells of mesenchymal origin, including fibroblasts and smooth muscle cells.
  • PDGF is also a nonmitogenic chemoattractant for endothelial cells.
  • TGF ⁇ is a chemoattractant for macrophages and monocytes. Depending upon the presence or absence of other growth factors, TGF ⁇ increases the tensile strength of healing dermal wounds. TGF ⁇ also inhibits endothelial cell mitosis, and stimulates collagen and glycosaminoglycan synthesis by fibroblasts. Other growth factors, such as EGF, TGF ⁇ , the
  • HBGFs and osteogenin are also important in wound heal ⁇ ing.
  • EGF which is found in gastric secretions and saliva
  • TGF ⁇ which is made by both normal and transformed cells, are structurally related and may recognize the same receptors, which mediate cell pro ⁇ liferation on epithelial cells. Both factors acceler ⁇ ate re-epithelialization of skin wounds.
  • Surgical adhesives and tissue sealants which contain plasma proteins are known and are used for sealing internal and external wounds in order to reduce blood loss and maintain hemostasis.
  • Such sealants typically contain blood clotting factors and other blood proteins.
  • Stroetmann, U.S. Patent Nos. 4,427,650 and 4,427,651 describes the preparation of an enriched plasma derivative in the form of a powder that contains fibrinogen, thrombin and/or prothrombin, and a fibrinolysis inhibitor, and may also contain other ingredients, such as a platelet extract.
  • Fibrin glue which is also called fibrin sea ⁇ lant, is primarily formulated for clinical topical application and is used to control bleeding and promote wound healing.
  • the clinical uses of fibrin glue have recently been reviewed (Gibble et al., Transfusion, 1990, 3J):741-747; Lerner et al., J. Surg. Res., 1990, 48:165- 181).
  • Fibrin glues are commercially available.
  • IMMUNO AG Vienna, Austria
  • BEHRINGWERKE AG Germany
  • Fibrin glues are prepared from plasma. The precise components of each fibrin glue are a function of the particular plasma fraction used as a starting material. Typically fibrin glue contains a mixture of proteins that, upon mixing with thrombin, form a clot.
  • fibrin glue can be prepared from plasma by cryoprecipitation followed by fractionation, to yield a composition that forms a sealant or clot upon mixture with thrombin or an activator of thrombin.
  • Fractiona ⁇ tion of plasma components can be effected by standard protein purification methods, such as ethanol, polyeth ⁇ ylene glycol, and ammonium sulfate precipitation, and ion exchange, and gel filtration chromatography.
  • Fibrin glues generally include a fibrinogen concentrate, which contains fibronectin, Factor XIII, von Willebrand factor, and dried human or bovine throm ⁇ bin. It is prepared in lyophilized form and is mixed with a solution of calcium chloride immediately prior to use. Upon mixing, the components coagulate on the tissue surface and form a clot that includes cross- linked fibrin.
  • Factor XIII which is present in the fibrinogen concentrate, catalyzes the cross-linking, fibrin glue, by sealing tissues face to face, prevents air or fluid leaks, and thereby induces hemostasis.
  • fibrin glue promotes wound healing. It does no, however possess true wound healing properties. Because fibrin glue is suitable for both internal and external injuries and is useful to maintain hemostasis, it would be desirable to enhance its wound healing properties.
  • One aim of the present invention is to provide for an easy process for the isolation of whole blood derived serum/platelet extract containing non-denatured growth factors at a high concentration of about 54 to about 99.8% , which extract is suitable for use in a wound dressing composition.
  • Another aim of the present invention is to pro ⁇ vide for a process for the production of growth factors extracts nearly 100% identical to their human counter ⁇ parts.
  • Another aim of the present invention is to pro- vide a blood derived serum/platelet extract for use in a wound dressing composition being characterized by a total absence of potential viral transmission of human origin (Hepatitis, HIV, etc.).
  • a further aim of the present invention is to provide for pharmaceutical compositions for promoting wound healing.
  • the novel process in accordance with the present invention comprises the steps of: a) concentrating a platelet-rich-plasma prepared from whole blood by passing through a neutral support for adsorbing the platelets or by cen- trifuging whole blood at about lOOOg to about 3000g, preferably at 1500g; b) releasing the concentrated platelets of step a) by ultra-sound shock; c) precipitating the concentrated plasma of step a), a platelet-poor-plasma or a serum by adding an amount of acetone solution to obtain an ace ⁇ tone concentration of about 68 to about 95% at a temperature of about -15 to 5°C; thereby obtaining a solid plasma or serum precipitate; d) the plasma or serum precipitate of step c) is reconstituted to a concentration of about 0.5 to 6% of protein weight/volume and mixed to the released platelets of step b) to obtain a platelet-serum-extract of a concentration which substantially has a physiological concentration to give a maximum syner
  • the preferred wound dressing includes a combination of the main growth factors present at the wound site, i.e. PDGF, TGF ⁇ s and IGFs.
  • a process for the production of a whole blood derived serum/platelet extract is described along with its biological potencies as a wound healing enhan- cer when associated with a fibrin matrix and/or biode ⁇ gradable gelifying matrix.
  • the whole blood derived serum/platelet extract characterized by a total absence of potential viral transmission of human origin may be prepared from decontaminated human whole blood or whole blood of porcine or bovine origin or of other closely related species.
  • Fig. 1 is a reaction scheme of a process in accordance with one embodiment of the present inven ⁇ tion
  • Fig. 2 is a Western blot for the identification of IGFBPs in serum, platelet and serum/platelet extracts
  • Fig. 3 is a SDS-PAGE analysis of serum, plate ⁇ let and serum/platelet extracts
  • Fig. 4 illustrates the effect of a serum/plate- let extract (PSE) on a MTT test of human fibroblast survival
  • Fig. 5 illustrates the wound breaking strength of (A) control vs fibrin glue-treated rats (experiment 1) and of (B) control vs PSE-treated rats in presence or absence of fibrin glue (experiment 2);
  • Fig. 6 illustrates the dose-effect relationship of platelet/serum extracts (PSE) on wound breaking strength in rats
  • Fig. 7 illustrates the morphometric analysis of 14 day-old wounds treated with PSE alone, PSE in fibrin vs control wounds.
  • Fig. 1 illustrates the reaction scheme of a process in accordance with one embodiment of the pre ⁇ sent invention.
  • PRP is a platelet-rich-plasma, preferably of porcine origin prepared by low speed centrifugation in order to pellet and remove erythrocytes and leukocytes.
  • PE is a platelet extract.
  • NS is a mono- or poly-atomic neutral support, having an amorphous structure or a crystalline lattice (with or without defect) selected from the following polygons, but not limited to this list: hexagon, cubic, mono- or tri-clinic, romboedric and orthorhombic.
  • the support may be of carbon or heteroatomic matter. It may have a single or several anomeric phases and may include also in its network one or more of the following heteroa- tomes: cobalt, Nickel, sulfur, oxygen, tungsten, alu- minum, silicon, nitrogen, fluorine among others.
  • the preferred support is graphite powder or activated car ⁇ bon.
  • FM is a neutral filtering membrane especially designed using a non-soluble and hydrophobic organic polymer. This membrane offers a wide filtering surface which confers its almost non-saturable filtering capac ⁇ ity.
  • US is representing ultra-sound shock
  • ii n or " sp” is a platelet-poor-plasma resulting from a unique passage of support-adsorbed- platelet containing plasma through the filtering mem ⁇ brane.
  • PA-1 and PA-2 are each respectively pre ⁇ cipitating agent 1 and 2 of the following chemical for- mulas:
  • Q is a radical homo- or hetero-cyclic, alkyl, silyl, cycloalkyl, alkenyl, aryl, aralkyl including at least one functional group selected from the following, but not limited to this list, acid, alcohol, amine, amide, alde ⁇ hyde, ketone, enol, oxime, halide, imine, enamine, ester, ether, phosphite, phosphate, nitro, nitroso, sulfamide, sulfate, sulfite, sulfoxyde, sulfone, thiosulfone, thioether, and nitrile;
  • M is an ammonium or phosphonium ion or any positive ion resulting from the chemical ioni- sation of elements of groups 1 to 3 of the
  • Y is a carboxylate, a sulfate, a nitrate, a halide, a hexafluorophosphate, or a tetrafluoroborate ion, or any stable anion resulting from the Lewis acid-base interaction; and x and n are identical or different and are any number between 1 and 6.
  • the precipitating agents PA-1 and PA-2 may be used as such or combined together in a mixture with one or more agents, preferably in aqueous suspension or solution of a pH range from 1 to 12.
  • the preferred precipitating agent PA-1 is an amount of acetone solution where the resulting acetone concentration is about 68 to about 72%.
  • the preferred precipitating agent PA-2 is an amount of acetone solution where the resulting acetone concentration is about 84 to about 95%.
  • the process of the present invention excludes from the platelet-rich-plasma (PRP) step any manipula ⁇ tion or rinsing of the free platelets by successive centrifugation.
  • PRP platelet-rich-plasma
  • the first step of the process of the present invention consists mainly in the preparation of the platelet extract (PE) based on the use of ultrasound as a shock treatment for the release of the material of filtered platelet. In addition to its low cost, this step is simple and can be carried out in less than two hours, even using large quantities of material.
  • the second step of the process of the present invention consists mainly in the preparation of the serum extract (SE) while overcoming the drawbacks of the prior art procedures.
  • This step allows for the chemical transformation of a platelet-poor-plasma or of a serum in a hyperactive growth factor concentrate of molecular weight between 0 and 50 kDa.
  • SE may be prepared in advance or in parallel and may be stored and reconstituted in water or in any appro ⁇ priate physiological buffer such as saline, phospho- saline, Krebs or Hepes.
  • physiological buffer such as saline, phospho- saline, Krebs or Hepes.
  • porcine whole blood was used in accordance with the present invention due to its availability and its contamination-free charac- teristic.
  • young Landrace, Yorkshire, male cas ⁇ trated piglets (2 month-old, 15kg B.W. were purchased from a local breeder). They were housed for 2-3 days in our facilities with free access to water and food.
  • ketamine hydrochloride (10 mg/kg BW)
  • fluothane by intratracheal administration.
  • a cannula was aseptically inserted into one carotid artery and animals were exsanguinated.
  • ACD acid-citrate-dextrose
  • ACD acid- citrate-dextrose
  • the collected platelet-rich-plasma (PRP) was immediately mixed with synthetic activated carbon
  • Q is the amount of activated carbon to add (g)
  • k is an empirically determined constant (between 0.01 and 0.80, preferably 0.082)
  • [PT] is the platelet concen ⁇ tration per liter of PRP and V the PRP volume (L).
  • Activated carbon was used as a neutral support to selectively adsorb platelets.
  • Other supports such as silicon nitride, silicon carbide, or activated carbon G-60TM (Aldrich) or Carbon Decolorizing AlkalineTM (Norit A, Fischer Scientific, Nepean, Ont.) were also successfully used.
  • the mixture activated carbon-PRP was gently mixed for 2 min., then filtered through a Whatman folded filter paper 2VTM (Whatman International Ltd, England).
  • the filter containing platelets and acti ⁇ vated carbon, was washed twice with phosphosaline buffer (PBS, pH 7.0) in order to completely eliminate proteins from serum. The content of the filter was then transferred into a glass vial containing picopure distilled water.
  • PBS phosphosaline buffer
  • the vial was subjected to a shock treatment consisting of either heating at 100°C for one min. and then cooled at 25°C or of two ultra-sound treatment with a sonicator (KontesTM 40-watt; output control:60; monitor scale:20) for about 5 sec. to 10 min. with a pause of 30 sec. in between.
  • the mixture was finally centrifuged at 2,000 to
  • the activated carbon pellet was reconstituted in picopure water, centrifuged, and both supernatants were pooled and lyophilized.
  • the result ⁇ ing powder was the platelet extract (PE) .
  • the precipitate, corre ⁇ sponding to the platelets was resuspended in phosphate buffer saline (PBS) to obtain a final concentration of 10 ⁇ platelet equivalent per ml (pq/ml), which corre ⁇ sponds to the first washing step.
  • PBS phosphate buffer saline
  • the resuspended platelets were centrifuged for a second time at the conditions mentioned above.
  • the precipitated platelets were resuspended in picopure, distilled water to a final concentration of 10 ⁇ plate ⁇ let equivalent per ml (pq/ml).
  • the solution was cooled to about -5 to about 4°C in a ice bath and was subjected to a shock treatment consisting of two ultra-sound treatment with a sonica- tor (KontesTM 40-watt; output control:60; monitor scale:20) for 45 sec. with a pause of 30 sec. in between.
  • the treated solution was frozen using liquid nitrogen and lyophilized overnight.
  • the lyophilized platelets were reconstituted in picopure water, centri ⁇ fuged, and both supernatants were pooled and lyophilized.
  • the resulting powder was the platelet extract (PE).
  • a serum extract was prepared by double acetone protein precipitation in acidic conditions.
  • Whole serum was first acidified with formic acid (88% formic acid, Fisher Scientific, Neapan, Ontario) to pH 1.9- 2.0, then mixed with acetone (Anachemia, Montreal, Canada) to a final concentration of about 68% to about 72% acetone, the preferred acetone concentration being 70%.
  • the mixture was filtered through* a Whatman filter paper 2VTM.
  • the filtrate was collected and acetone was added to a final concentration of about 84% to about 95%, the preferred final concentration being 90%.
  • the mixture was stirred up and again filtered through a Whatman filter paper 2VTM.
  • the filter content was washed with pure acetone, then with ether in order to completely remove acetone.
  • Ether was air-evaporated, the final powder was reconstituted in picopure water (>18.2 megaohms) (1% w/v), filter-sterilized through 0.2 ⁇ m filters (Nalgene, Rochester, NY) and kept frozen at -75 to about -85°C.
  • the serum precipitate may be reconstituted to a concentration of about 0.5 to 6% of protein weight/volume, preferably to a concentration of about 1 to 2% of protein weight/volume (for example,
  • the PE was reconsti ⁇ tuted with the 1% SE to a final concentration of 10 10 platelet equivalent per ml.
  • the mixture was centri ⁇ fuged at 5000 g for 15 min. at +4°C, then filter ster ⁇ ilized through 0.2. ⁇ m filters and either stored at -75°C or lyophilized.
  • the final product was termed platelet-serum-extract (PSE).
  • PSE platelet-serum-extract
  • the PE was reconstituted in picopure water (>18.2 megaohms) (10 ⁇ platelet equivalent/ml).
  • PDGF antibody used in the kit. Results were therefore converted in porcine PDGF equivalent by dividing them by 0.38.
  • Transforming growth factor ⁇ l and ⁇ 2 were meas ⁇ ured in PSE using the Quantikine testTM commercially available (Amersham International, V.K.). Before meas ⁇ urement, extracts were mixed 1:2 either in distilled water or in trifluoroacetic acid (TFA) 1%, incubated for 1 hour at room temperature, lyophilized and recon ⁇ stituted with water to their initial volume. This pro ⁇ cedure was conducted in order to determine if TGF ⁇ was present in extracts as an active or latent, high molecular weight form.
  • TGF ⁇ was present in extracts as an active or latent, high molecular weight form.
  • Insulin-like growth factor-I and II were measured in SE by radioimmunoassay after for ⁇ mic acid-acetone extraction (Bowsher R.R. et al., Endocrinology, 1991, -128:805-814). Briefly, SE (100 ⁇ l) was acidified with formic acid 8.0 M, containing 0.5% Tween 20TM (Biorad Lab., Richmond, CA), then precipi ⁇ tated with acetone (350 ⁇ l).
  • the supernatant was then diluted in RIA buffer for IGFs measurements (1/60 for IGF-I and 1/300 for IGF-II, in a 30 mM phosphate buffer, pH 7.4, containing 0.02% protamine sulfate, grade II, 10 mM EDTA, 0.02% sodium azide and 0.25% bovine serum albumin RIA grade, all buffer components from Sigma Chemicals, St Louis, MO) .
  • IGF-I was then measured in neutralized extracts by double antibody RIA using recombinant human IGF-I (Bachem California) for iodination and standards, and polyclonal anti-hIGF-I antiserum kindly provided by Dr. Underwood (University of North Carolina) through the National and Pituitary Program of the NIDDK, as previ- ously described by Brazeau P. and Abribat T. (75" 1 Ann. Meet, of the Endocrine Soc., Las Vegas, NA, June 9-12, 1993, Abst. 1327) according to the following procedure. hIGF-I RIA PROCEDURE
  • IGF-II was also measured in neutralized extracts in the same RIA buffer by double antibody RIA (Brazeau P. and Abribat T., 75" 1 Ann.Meet, oftheEndocrineSoc., Las Vegas, NA, June 9-12, 1993, Abst 1327) using recom ⁇ binant human IGF-II (Bachem California) for iodination and standards, and monoclonal anti hIGF-II antibodies diluted 1/50 K (Amano International Enzymes Co) accord ⁇ ing to the following procedure.
  • IGF-II RIA 0.5% and cross reactivity of IGF-I in the IGF-II RIA was 3.5%.
  • ED50 from standard curves were typically 70 pg/tube. and 90 pg/tube for IGF-I and IGF-II RIAS, respectively.
  • IGFBP IGF-binding proteins
  • TBS Tris-buffered saline
  • NonidetTM P-40 Calbiochem Co, La Jolla, CA
  • Sheets were trans ⁇ ferred into sealed plastic bags containing 1.5. 10 ⁇ cpm 12 ⁇ I-IGF-I in 20 ml. After an overnight incubation with continual gentle agitation, sheets were washed twice in TBS-0.1% Tween 20TM, then 3 times in TBS, and finally autoradiographed at -70°C on X-ray films for 48 h. For quantitative analysis, bands of the nitrocellulose sheets corresponding to those on autoradiograms were cut and counted in a gamma counter.
  • PDGF levels averaged 3.5 ⁇ 0.5 ng/ml (mean ⁇ SEM of 7 measurements).
  • PDGF levels unexpectedly varied from 3.16 to 14.2 ⁇ g/1.
  • non extracted (water extracted) TGF ⁇ 2 content was 4145 ⁇ 542 pg/ml
  • acid extracted TGF ⁇ 2 content was identical (4169 ⁇ 489 pg/ml) sug- gesting that TGF ⁇ 2 was entirely present in PE in its active form of about 25 KD.
  • TGF ⁇ 1 levels unexpectedly varied from 0.873 to 3.9 ⁇ g/1.
  • TGF ⁇ 2 levels unexpectedly varied from 5.8 to 46.0 ng/1.
  • IGF-I and IGF-II concentrations averaged 200 ⁇ 16 ng/ml and 452 ⁇ 82 ng/ml respectively (mean ⁇ SEM of 7 measurements) .
  • IGFBPs could be detected both in SE and PSE but not in PE.
  • the major IGFBP in serum, IGFBP3 doublet migrating at 37 and 41 KD
  • IGFBP2 doublet migrating at 37 and 41 KD
  • IGFBP2 three other IGFBPs in serum (IGFBP2, 1 and 4, migrating at 31, 27, and 22 KD respectively) were found in SE and PSE, at a concentration of 15-25% that in serum.
  • IGF-I concentration unexpectedly varied from 16.9 to 88 ⁇ g/1 and IGF-II concentration varied from 47.9 to 356 ⁇ g/1.
  • TGF ⁇ 1 level unexpectedly varied from 1.98 to 21.5 ⁇ g/1 and TGF ⁇ 2 level varied from 0.027 to 0.290 ⁇ g/1.
  • Total protein content was 5.3 ⁇ 0.5 mg/ml in PE (mean ⁇ SEM of five determinations), 6.3 ⁇ 0.8 mg/ml in SE (mean ⁇ SEM of five determinations) and 12.2 ⁇ 0.8 mg/ml in PSE (mean ⁇ SEM of six determinations).
  • PE, SE and PSE were sub ⁇ jected to a SDS-PAGE electrophoresis on 12% polyacryla- mide gels, and silver-stained according to the method described by Morissey JH (Anal. Biochem., 1981, 117:307- 310).
  • a protein staining of typical PE, SE and PSE is shown in Fig. 3.
  • the major contaminant in PSE was identified as a 50-55 KD weighing group of proteins of seric origin.
  • PE porcine primary cultured fibroblasts
  • both PE and SE stimulated cell proliferation in a dose-dependent manner between 1 and 100 ⁇ l of extracts.
  • Combination of PE and SE was synergic on cell proliferation, that is the effect of PSE was more than the addition of the effects of PE and SE.
  • This synergy was observed at the doses of 1 and 10 ⁇ l/ml, but not 100 ⁇ l/ml. In fact, the 100 ⁇ l/ml dose was less potent than the 10 ⁇ l/ml dose in all PSE tested.
  • a dose response study from 0 to 200 ⁇ l/ml revealed that PSE induced a dose-related linear stimu ⁇ lation of cell proliferation from 0 to 10 ⁇ l/ml, the effect being progressively blunted between 50 and 200 ⁇ l/ml.
  • the maximal effect on cell proliferation was estimated to be at 10-50 ⁇ l/ml of PSE.
  • RPMI 1640 RPMI 1640 containing 10% FCS and 1% penicillin-streptomycin at a density of 10 ⁇ cells in one ml per well. Twenty four hour later, medium was replaced by a test medium (RPMI 1640TM, 0.5% FCS and 1% penicillin-streptomycin) containing test samples (0 to 200 ⁇ l/ml SE, PE or PSE; 0 to 100 ng/ml porcine PDGF-R & D system, MN, Cat.#125PD). Maximal stimulation of proliferation was evaluated by adding 10% FCS in control wells. Each dose of each test sample was assayed in triplicate.
  • both PE and SE stimulated cell proliferation in a dose-dependent manner between 1 and 100 ⁇ l of extracts.
  • Combination of PE and SE was synergic on cell proliferation, that is the effect of PSE was more than the addition of the effects of PE and SE.
  • This synergy was observed at the doses of 1 and 10 ⁇ l/ml, but not 100 ⁇ l/ml. In fact, the 100 ⁇ l/ml dose was less potent than the 10 ⁇ l/ml dose in all PSE tested.
  • a dose response study from 0 to 200 ⁇ l/ml revealed that PSE induced a dose-related linear stimu ⁇ lation of cell proliferation from 0 to 10 ⁇ l/ml, the effect being progressively blunted between 50 and 200 ⁇ l/ml.
  • the maximal effect on cell proliferation was estimated to be at 10-50 ⁇ l/ml of PSE. 3 .3 M. T. T . Test
  • a quantitative colorimetric assay for porcine primary cultured fibroblasts (PPCF) and human foreskin fibroblasts (HFF) survival was developed in order to assess potential cytotoxicity of the extracts.
  • the assay is dependent on the reduction of the tretrazolium bromide, from Sigma Chemical, St Louis, MO) by the mitrochondrial deshydrogenase of viable cells to form a blue formazan product.
  • the assay was run in 96 well plates in DMEM/Ham F12 medium (Gibco). Cells were plated at confluency (4.0 x 10 4 cells/well) and the procedure was conducted exactly as described by Hansen M.B. et al. (J. Immunol. Methods, 1989, 111:203-210). All test samples (0 to 100 ⁇ l/ml) were tested in tripli- cate.
  • MTT tests were performed on a dose range (0 to 100 ⁇ l/ml) of PSE. As shown in Fig. 4, even high concentrations of PSE (50, 75, 100 ⁇ l) had no negative effect on cell survival, indicating an absence of cytotoxicity. In fact, an increase in optical density was noted at concentrations higher than 50 ⁇ l/ml, that reflected a good cellular health.
  • Sprague-Dawley male rats (275-300g) were pur- chased from Charles River Canada Inc. (St Constant, Quebec) . They were housed in individual cages and fed ad libitum. Each experimental group contained 7 to 8 rats. Wounds were performed as previously described by Garrel DR et al. (J. Surg. Res., 1991, 5_l:297-302) . Briefly, one 6 cm-full-thickness cutaneous incision was made longitudinally on the dorsal skin of each rat under pentobarbital anesthesia. The incisions were immediately closed with Dermalon 4.0TM sutures placed 1 cm apart.
  • Serum and platelet extract were prepared as described above. Except otherwise specified, they were applied onto the wounds in combination with a fibrin matrix purchased from Haemacure Biotech Inc. (Pointe- Claire, Quebec). Platelet-Serum extracts were used to reconstitute a lyophilized thrombin powder (1 ml extract for 100 IU thrombin) in presence of calcium chloride (40 mM) . This mixture was applied directly onto the wound in combination with a bovine fibrinogen solution (40-50 mg/ml) in picopure water. For a 6 cm- long wound, 250 ⁇ l of each preparation was used (total volume: 500 ⁇ l) .
  • the dose-effect relationship of growth factor extract (27,83 and 250 ⁇ l of PSE, 10 10 platelet equiva ⁇ lent/ml in 1% SE) in 500 ⁇ l fibrin matrix, vs control (BSA, 60 mg/ml) was determined.
  • SE lyophilized and reconstituted in 250 ⁇ l sterile water) in 500 ⁇ l fibrin matrix, vs control (BSA, 60 mg/ml) were determined.
  • PSE as a wound healing enhancer was tested in a model of rat surgical wound. Measured parameters included wound breaking strength 14 days post wounding, as well as histological examination of the scars.
  • Morphological evaluation includes aspect of the epidermis and of the cells within the scar, whereas morphometric evaluation includes thickness of the epi ⁇ dermis surrounding the scar, width of the scar at the level of both superficial and reticular dermis and cel ⁇ lularity, by counting fibroblasts per microscopic field, magnification x 750.
  • PSE increased wound ' breaking strength in vivo when administered in a fibrin matrix. This effect was associated with increased scar width and cellular- ity. In all observed histological sections, no sign of inflammation, of chelo ⁇ d or of tumoral foci could be detected.
  • a seric and a platelet extract yielded a preparation containing at least sig ⁇ nificant amounts of PDGF, TGF ⁇ 2, IGF-I, II and IGFBPs.
  • growth factors were only characterized by immunoreactivity in accordance with one embodiment of the present invention, numerous reports confirm that platelets and serum are two major sources of growth factors. Platelets ⁇ granules are the richest in vivo source of TGF ⁇ and PDGF. Porcine platelets contain two isotypes, TGF ⁇ 1 and ⁇ 2. Human and porcine TGF ⁇ 1 have total sequence identity and TGF ⁇ 2 has approxi ⁇ mately 70% homology with TGF ⁇ 1.
  • TGF ⁇ s are natu- rally found as a latent, high molecular weight, inac ⁇ tive complexes in platelets.
  • the measurement of non-extracted and acid-extracted platelet extracts yielded the same amount of TGF ⁇ 1 and ⁇ 2, indicating that TGF ⁇ 1 and ⁇ 2 are present in our extracts in its active form of about 25 KD. Since activation of TGF ⁇ in the latent complex has been shown to occur by treatment with acid, urea or heat in vitro, it was concluded that the heating step of the PE preparation was responsible for this activa- tion.
  • PDGF is a disulfide-linked dimer with a molecu ⁇ lar weight 30-32 KD.
  • the subunits of the dimer are two related polypeptides designated the A and B chains.
  • human platelet PDGF has been shown to consist of PDGF-AB and PDGF-BB
  • porcine platelet PDGF consists primarily of PDGF-BB homodimers. Because of its higher affinity for the type B PDGF receptor, PDGF-BB is more potent than PDGF-AB in stimulating cell proliferation in vitro.
  • PDGF AB and BB have equal potency as wound healing enhancers.
  • Both PDGF and TGF ⁇ s are known as stimulators of wound healing. They both induce extracellular matrix synthesis, granulation tissue formation and increase wound breaking strength in a variety of animal models. Both recombinant TGF ⁇ 2 and PDGF-BB are currently tested in clinical trials as therapeutic agents for the healing of chronic ulcers with promising initial results.
  • platelet ⁇ gran- ules have been shown to contain a number of agents that might play a role in the process of wound healing.
  • these substances are Platelet Factor 4 (PF4) , a platelet-derived Endothelial Cell Growth Factor (pdECGF), an Epidermal Growth Factor-like protein (EGF), and traces of IGF-I, IGF-II and IGFBP3.
  • PF4 Platelet Factor 4
  • pdECGF platelet-derived Endothelial Cell Growth Factor
  • EGF Epidermal Growth Factor-like protein
  • Insulin-like growth factors I and II are two related peptides of approximately 7500 KD and of iden ⁇ tical sequence in human and pig. In serum, they circu ⁇ late in large amount, tightly bound to specific, high affinity binding proteins. IGF-I is the most important mediator of the biological actions of growth hormone and possesses strong systemic and local anabolic actions. Both IGFs and IGFBPs are present in wound fluid in the course of wound healing. When adminis- tered alone or in combination with IGFBPs, IGF-I has been shown to stimulate wound strength and granulation tissue formation in models of normal or impaired wound healing.
  • the analytical results showed that the process of extraction in accordance with the present invention successfully recovered IGFs and IGFBPs from serum, and PDGF and TGF ⁇ from platelets.
  • other bioactive substances such like PF4, pdECGF or EGF might also be present and might participate in the biological activity of PSE.
  • the process of the present invention for the manufacture of PSE is new, simple and inexpensive. It involves a few number of steps that can be performed in less than 24 hours to produce a mixture made of at least 5 important growth factors for wound healing (PDGF, TGF ⁇ , IGF-I, IGF-II, IGFBPs).
  • porcine origin of the blood in accordance with one embodiment of the present inven- tion, guaranties: 1) a total absence of potential transmission of human viral agents (hepatitis, HIV, etc.); 2) the production of growth factors nearly 100% identical to their human counterparts and, 3) a reli ⁇ able, largely available and highly reproducible source of raw material.
  • both the platelet and serum extracts of the present invention stimulated fibroblast proliferation in a dose dependent manner, and coadmin- istration of both extracts resulted in a synergic effect.
  • SE and PE contained IGFs and PDGF, respectively, it is suggested that the synergy was due to interactions between these two growth factors. This synergy was first reported in cultured 3T3 fibroblasts, then confirmed in vivo in animal models of wound heal- ing. It has been explained by the complementary roles of PDGF and IGFs in the cell cycle, PDGF being a compe ⁇ tence factor and IGFs being a progression factor.
  • PSE induced a dose-dependent increase of por ⁇ cine primary cultured fibroblasts (PPCF) or 3T3 up to 10 ⁇ l/ml, while the stimulatory effect was inhibited at doses equal or above 50 ⁇ l/ml.
  • PSE was not cytotoxic at doses up to 100 ⁇ l/ml and stimulated protein (collagen) synthesis in a dose dependent fash ⁇ ion between 0 and 100 ⁇ l/ml.
  • platelet/serum extract of the present invention is a powerful stimulator of granulation tissue formation. Its potential therapeutical applications include the treatment of chronic ulcers and surgical wounds, and the regeneration of injured soft tissues.
  • SE and PE of the present invention might be used alone or advantageously in combination (PSE) because they work synergically.
  • PSE in vivo, they have to be adminis ⁇ tered topically in a matrix slowly releasing them, such as fibrin glue, collagen, polymers among others.
  • the bioactive formulation is 50 to 1000 ⁇ l of a preparation made of 10 to 1000 IU/ml thrombin, 20 to 200 mg/ml fibrinogen, and 50 to 500 ⁇ l/ml SE (0.1 to 5%), PE (10 8 to 10 12 platelet equiva ⁇ lent/ml buffer) or PSE (10 8 to 10 12 platelet equiva- lent/ml SE 0.1 to 5%) in presence of CaCl2 (1 to 100 mM final) .
  • Q K.D X [PRP] . V. 10-9
  • K is an empirically determined constant of 0.082
  • D x is a ratio of the density of the neutral support with respect to the density of carbon (1 for graphite)
  • [PRP] is the platelet concentration per liter of PRP (464 X 10 9 )
  • V is the PRP volume in L (3)
  • the adsorbed platelet are washed twice with three liters of phos- phosaline buffer pH 7.4, then the washed filtrate is transferred in a beaker.
  • the membrane is rinsed with 1 L of distilled water and the temperature of the washed filtrate in the beaker is raised to 100°C for 1 min.
  • the washed filtrate in the beaker is filtered and/or centrifuged; the supernatant is lyophilized.
  • the lyophilisate constitutes the solid platelet extract (PE) which can be reconstituted.
  • PPCF control 75858 763 100 0.21 0.017 100
  • EXAMPLE II Preparation of Platelet Extract The procedure as described in Example I was carried out, except that carbure of silicium was used instead of graphite. This material has a density con ⁇ stant of 3.23, which resulted in a Q value of 335g of carbure of silicium used.
  • the precipitated carbon is kept and carefully mixed with the three liters of PRP. After a 2 min. mixing, the platelet-adsorbed-carbon mixture is fil- tered through a Whatman filter paper 2VTM. The fil ⁇ tered PPP is discarded.
  • the adsorbed platelet are washed twice with 450 ml of phosphosaline buffer pH 7.4, then the washed filtrate is transferred in a beaker.
  • the adsorbed platelet are resuspended in picopure water (>18.2 megaohms) to obtain a final con ⁇ centration of 10 pq/ml and the temperature of the beaker is cooled to about 0-4°C using an ice bath.
  • the adsorbed platelet are subjected to a first ultra-sound treatment with a sonicator (KontesTM 40- watt; output control:60; monitor scale:20) for about 45 sec, a pause of 30 sec. and a second ultra-sound treatment for about 45 sec.
  • a sonicator KontesTM 40- watt; output control:60; monitor scale:20
  • the washed filtrate in the beaker is centri- fuged for 5 min. at 5000g; the precipitate is resus- pended in a minimum quantity of picopure water and cen ⁇ trifuged again under the same conditions. Both super- natants are combined, frozen in liquid nitrogen and then lyophilized.
  • the lyophilisate constitutes the solid platelet extract (PE) which is reconstituted in picopure water (>18.2 megaohms) to obtain a ' final con ⁇ centration of 10 10 pq/ml, centrifuged for 30 min. at 10,000g.
  • the supernatant is filtered first through 0.8 ⁇ m and 0.2 ⁇ m filters (Nalgene, Rochester, NY). The preparation is aliquoted and kept at -80°C.
  • TGF ⁇ 2 46.0 ng/1
  • PPCF control 76982 1706 100 0.21 0.017 100
  • Example IV The procedure as described in Example IV is carried out, except that activated carbon DarcoTM G-60 (Aldrich) was used instead of carbon.
  • the resuspended platelets are centrifuged for a second time at the conditions mentioned above.
  • the precipitated platelets are resuspended in picopure water (>18.2 megaohms) to a final concentration of 10*" platelet equivalent per ml (pq/ml).
  • the solution is cooled to about 0 to about 4°C in a ice bath and is subjected to a shock treatment consisting of two ultra-sound treatment with a sonica- tor (KontesTM 40-watt; output control: 60; monitor scale: 20) for 45 sec. with a pause of 30 sec. in between.
  • a shock treatment consisting of two ultra-sound treatment with a sonica- tor (KontesTM 40-watt; output control: 60; monitor scale: 20) for 45 sec. with a pause of 30 sec. in between.
  • the treated solution is frozen using liquid nitrogen and lyophilized overnight.
  • the lyophilized platelets are reconstituted in picopure water to obtain a final concentration of 10 ⁇ pq/ml.
  • the membrane fragments are eliminated by ultracentrifugation or by filtration through 0.8 ⁇ m, 0.45 ⁇ m and 0.2 ⁇ m filters (Nalgene, Rochester, NY). The preparation is aliquoted and kept at -80°C.
  • a 300 ml of whole serum was 'first acidified with 18.2 ml of formic acid (88% formic acid, Fisher Scientific, Neapan, Ontario) at room temperature. After 5 min. of agitation at room temperature, 700 ml of pure acetone was added. The agitation was continued for another 5 min., then the suspension was filtered through a Whatman filter paper 2VTM.
  • the filtrate was collected and slowly trans ⁇ ferred in a beaker containing 1400 ml of pure acetone constantly agitated. The order in the addition is of extreme importance.
  • the precipitate is filtered through a Whatman filter paper 2VTM, washed three times with 500 ml of pure acetone, then three times with 500 ml of ether, then dried.
  • the solid mass is 1.7 g and is reconsti- tuted in 170 ml of picopure water (18.2 Ohm) (1% w/v), filter-sterilized through 0.22 ⁇ m filters (Nalgene, Rochester, NY) and aliquoted under laminar flux (Canadian Cabinets Co, Ltd., V6 MW99.C30TM). This solution of pH 6.5 and 1% of final concentration, con- stitutes the serum extract (SE).
  • the acidified serum is cooled at 4°C in a cold room. A quantity of 75% acetone is added and agitated magnetically for an hour to precipitate the proteins. The suspension is filtered through a Whatman filter paper 2VTM.
  • the filtrate was collected arid slowly trans ⁇ ferred in a beaker containing an amount of pure acetone to obtain a final concentration of 90% acetone. After an incubation of one hour, the suspension is filtered through a Whatman filter paper 2VTM.
  • the precipitate is washed twice with pure ace ⁇ tone, and then a third time with anhydride ether, then dried using a lyophilizer.
  • the solid mass is reconstituted in picopure water (>18.2 megaohms) to obtain a final concentration of 1%, filter-sterilized through 0.22 ⁇ m filters (Nalgene, Rochester, NY) and aliquoted under laminar flux (Canadian Cabinets Co, Ltd., V6 MW99.C30TM) and kept at -80°C.
  • PPCF control 74087 1761 100 0.21 0.017 100 1 ul/ml 0.20 0.006 93 10 ul/ml 0.17 0.007 84
  • Example VIII The procedure as described in Example VIII is carried out, except that the acidified serum is cooled at 0°C instead of 4°C. Also, the first precipitation using acetone is conducted such that the acetone is added dropwise until a final acetone concentration of 72% is achieved, the second acetone addition is effected under the same conditions until a final ace ⁇ tone concentration of 75% is achieved, and a third ace ⁇ tone addition is effected under the same conditions until a final acetone concentration of 90% is achieved.
  • Example I, II, III, IV, V or VI is reconstituted with SE of Example VII, VIII, IX, or X to obtain a final concentration of 10 ⁇ platelet per ml without neutralization.
  • the mixture is centrifuged at 10,000g for 15 min. at +4°C, then filter sterilized through 0.22 ⁇ m filters and aliquoted under laminar flux.
  • EXAMPLE XII
  • Example I, II, III, IV, V or VI is reconstituted with SE of Example VII, VIII, IX, or X to obtain a final concentration of 10 ⁇ platelet per ml.
  • the mixture is neutralized using a Amberlite IRA- 400(OH)TM resin, centrifuged at 10,000g for 15 min. at +4°C, then filter sterilized through 0.22 ⁇ m filters and aliquoted under laminar flux.

Abstract

The present invention relates to a process for the preparation of platelet-serum-extract, which comprises the steps of: a) concentrating a platelet-rich-plasma prepared from whole blood by passing through a neutral support for adsorbing the platelets or by centrifuging whole blood at about 1000g to about 3000g; b) releasing the concentrated platelets of step a) by ultrasound shock; c) precipitating the concentrated plasma of step a), a platelet-poor-plasma or a serum by adding an amount of acetone solution to obtain an acetone concentration of about 68 to about 95 % at a temperature of about -15 to 5 °C; thereby obtaining a solid plasma or serum precipitate; d) the plasma or serum precipitate of step c) is reconstituted to a concentration of about 0.5 to 6 % of protein weight/volume and mixed to the released platelets of step b) to obtain a platelet-serum-extract of a concentration which substantially has a physiological concentration to give a maximum synergy in promoting wound healing. The present invention also relates to pharmaceutical composition for promoting wound healing, which comprises an effective concentration of a platelet-extract, a serum-extract or a platelet-serum-extract prepared according to the process of the present invention in association with a pharmaceutically acceptable carrier.

Description

PROCESS FOR THE PREPARATION OF SERUM AND PLATELET GROWTH FACTORS EXTRACT
BACKGROUND OF THE INVENTION (a) Field of the Invention
The invention relates to a process for the pro¬ duction of a whole blood derived serum and/or platelet extracts and their clinical uses in wound dressing, (b) Description of Prior Art Wound healing is a complex cascade of cellular and biochemical events which lead to wound closure and repair of tissues- Three successive phases are classi¬ cally distinguished in this process:
1) the inflammatory phase, corresponding to increased vascular permeability and migration of leukocytes and macrophages;
2) the proliferative phase, characterized by fibroblast proliferation and collagen synthe¬ sis, resulting in granulation tissue formation; and
3) the remodeling phase, where collagen and granu¬ lation tissue rearrangements results in scar resorption.
The very first event that normally occurs in a wound is blood extravagation, that results in platelet aggregation and impregnation of the wound with platelet and serum constituents. Among these constituents are polypeptide growth factors, which are known to play a major role in tissue regeneration. Platelet α gran- ules, which are released by aggregated platelets, are one of the richest physiological source of platelet- derived growth factor (PDGF) and transforming growth factor β (TGFβ), while serum contains high amounts of insulin-like growth factor I (IGF-I), IGF-II and their binding proteins (IGF-BPs) (Strovbant P & Waterfield MD, Embo.J., 1984, 2 :2963-2967; Assoian RK et al., J. Biol. Chem., 1983, 258:7155-7160: Sara VR et al., Physiol. Rev., 1990, 70:591-614) .
PDGFs include PDGF, platelet derived angiogene- sis factor (PDAF), TGFβ and platelet-factor-4 (PF-4), which is a chemoattractant for neutrophils (Knighton et al. , In Growth Factors and Other Aspects ofWound Healing: Biological and Clinical Implications, Alan R. Liss, Inc., pp. 319-329 (1988)). PDGF is a mitogen and chemoattractant for fibroblasts and smooth muscle cells and is a stimulator of protein synthesis in cells of mesenchymal origin, including fibroblasts and smooth muscle cells. PDGF is also a nonmitogenic chemoattractant for endothelial cells.
TGFβ is a chemoattractant for macrophages and monocytes. Depending upon the presence or absence of other growth factors, TGFβ increases the tensile strength of healing dermal wounds. TGFβ also inhibits endothelial cell mitosis, and stimulates collagen and glycosaminoglycan synthesis by fibroblasts. Other growth factors, such as EGF, TGFα, the
HBGFs and osteogenin, are also important in wound heal¬ ing. EGF, which is found in gastric secretions and saliva, and TGFα, which is made by both normal and transformed cells, are structurally related and may recognize the same receptors, which mediate cell pro¬ liferation on epithelial cells. Both factors acceler¬ ate re-epithelialization of skin wounds.
The in vivo mode of action of these growth fac¬ tors involves chemoattraction at the wound site, cell proliferation and collagen synthesis. One very inter¬ esting feature of these products is that some of them, namely PDGF and IGFs, work synergistically in stimulat¬ ing wound repair (Lynch SE et al., J. Gin. Invest, 1989, £4:640-646; Greenhalgh DG et al., Wound. Rep. Reg., 1993, 1:54-62). Growth factors are, therefore, potentially use¬ ful for specifically promoting wound healing and tissue repair. The addition of exogenous growth factors to a wound has been shown to increase the rate at which the wound is closed, the number of cells in the healing area, the growth of blood vessels, the total rate of deposition of collagen, and strength of the scar (Carter et al. , in Growth Factors and Other Aspects ofWound Heal¬ ing: Biological and Clinical Implications, Alan R. Liss, Inc., pp. 303-317 (1988)). Platelet-derived wound healing for¬ mula (PDWHF), a platelet extract which is in the form of a salve or ointment for topical application, has been described by Knighton et al. (Ann. Surg., 1986, 204: 322-330). Knighton et al. (Ann. Surg., 1986, 204: 322-330 &
In GrowthFactors and Other Aspects ofWound Healing: Biological and Clinical Implications, Alan R. Liss, Inc., pp. 319-329 (1988)) describe a process for the preparation of a platelet- derived wound healing composition by centrifuging platelets and treating them with thrombin to stimulate the production of a releasate, which can be combined with collagen. Knighton et al. obtain a platelet- derived releasate having a concentration of growth fac¬ tors of about 10 to about 17%, which is not practically usable. Also, this platelet-derived releasate contains essentially denatured or non-mature growth factors due to the use of thrombin as a chemical shock to break apart the platelets and release their content.
Surgical adhesives and tissue sealants which contain plasma proteins are known and are used for sealing internal and external wounds in order to reduce blood loss and maintain hemostasis. Such sealants typically contain blood clotting factors and other blood proteins. Stroetmann, U.S. Patent Nos. 4,427,650 and 4,427,651, describes the preparation of an enriched plasma derivative in the form of a powder that contains fibrinogen, thrombin and/or prothrombin, and a fibrinolysis inhibitor, and may also contain other ingredients, such as a platelet extract.
Clinical evaluation of recombinant growth fac¬ tors are underway but it remains so far uncertain whether the therapeutic benefit of these drugs will pay for their high cost of production specially if used in combination.
Autologous human platelet derived wound healing formula, made of thrombin activated platelet α gran¬ ules, has also been shown to induce the healing of chronic ulcers, thus indicating that growth factor extracts may constitute an advantageous alternative to the use of recombinant growth factors if proven to be economically extractable.
In addition, improved fibrin glue delivery sys- terns have been disclosed (Miller et al., U.S. Patent No. 4,932,942; Morse et al., PCT Application WO 91/09641).
Fibrin glue, which is also called fibrin sea¬ lant, is primarily formulated for clinical topical application and is used to control bleeding and promote wound healing. The clinical uses of fibrin glue have recently been reviewed (Gibble et al., Transfusion, 1990, 3J):741-747; Lerner et al., J. Surg. Res., 1990, 48:165- 181). Fibrin glues are commercially available. For example, IMMUNO AG (Vienna, Austria) and BEHRINGWERKE AG (Germany) presently have such products on the market (U.S. Patent Nos. 4,377,572 and 4,298,598).
Fibrin glues are prepared from plasma. The precise components of each fibrin glue are a function of the particular plasma fraction used as a starting material. Typically fibrin glue contains a mixture of proteins that, upon mixing with thrombin, form a clot. For example, fibrin glue can be prepared from plasma by cryoprecipitation followed by fractionation, to yield a composition that forms a sealant or clot upon mixture with thrombin or an activator of thrombin. Fractiona¬ tion of plasma components can be effected by standard protein purification methods, such as ethanol, polyeth¬ ylene glycol, and ammonium sulfate precipitation, and ion exchange, and gel filtration chromatography.
Fibrin glues generally include a fibrinogen concentrate, which contains fibronectin, Factor XIII, von Willebrand factor, and dried human or bovine throm¬ bin. It is prepared in lyophilized form and is mixed with a solution of calcium chloride immediately prior to use. Upon mixing, the components coagulate on the tissue surface and form a clot that includes cross- linked fibrin. Factor XIII, which is present in the fibrinogen concentrate, catalyzes the cross-linking, fibrin glue, by sealing tissues face to face, prevents air or fluid leaks, and thereby induces hemostasis. By virtue of the ability to maintain hemostasis and reduce blood loss, fibrin glue promotes wound healing. It does no, however possess true wound healing properties. Because fibrin glue is suitable for both internal and external injuries and is useful to maintain hemostasis, it would be desirable to enhance its wound healing properties.
It would be highly desirable to be provided with a economical and easy to carry out process for the isolation of contamination-free blood derived serum/platelet extract for use in a wound dressing com¬ position. Such a blood derived serum/platelet extract should be absolutely free of Hepatitis B and HIV con- taminants. It would be highly desirable to be provided with a process for the isolation of whole blood derived serum/platelet extract containing non-denatured growth factors at a concentration of about 54 to about 99.8%, which extract is suitable for use in a wound dressing composition.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide for an easy process for the isolation of whole blood derived serum/platelet extract containing non-denatured growth factors at a high concentration of about 54 to about 99.8% , which extract is suitable for use in a wound dressing composition. Another aim of the present invention is to pro¬ vide for a process for the production of growth factors extracts nearly 100% identical to their human counter¬ parts.
Another aim of the present invention is to pro- vide a blood derived serum/platelet extract for use in a wound dressing composition being characterized by a total absence of potential viral transmission of human origin (Hepatitis, HIV, etc.).
A further aim of the present invention is to provide for pharmaceutical compositions for promoting wound healing.
Surprisingly and in accordance with the present invention there is provided a process for the prepara¬ tion of platelet-serum-extract, wherein the growth factors are substantially non-denatured and in high concentration essentially due to the new use of ultra¬ sound as a shock treatment. The novel process in accordance with the present invention comprises the steps of: a) concentrating a platelet-rich-plasma prepared from whole blood by passing through a neutral support for adsorbing the platelets or by cen- trifuging whole blood at about lOOOg to about 3000g, preferably at 1500g; b) releasing the concentrated platelets of step a) by ultra-sound shock; c) precipitating the concentrated plasma of step a), a platelet-poor-plasma or a serum by adding an amount of acetone solution to obtain an ace¬ tone concentration of about 68 to about 95% at a temperature of about -15 to 5°C; thereby obtaining a solid plasma or serum precipitate; d) the plasma or serum precipitate of step c) is reconstituted to a concentration of about 0.5 to 6% of protein weight/volume and mixed to the released platelets of step b) to obtain a platelet-serum-extract of a concentration which substantially has a physiological concentration to give a maximum synergy in promoting wound healing.
In accordance with the present invention, the preferred wound dressing includes a combination of the main growth factors present at the wound site, i.e. PDGF, TGFβs and IGFs.
In accordance with one embodiment of the pre¬ sent invention, a process for the production of a whole blood derived serum/platelet extract is described along with its biological potencies as a wound healing enhan- cer when associated with a fibrin matrix and/or biode¬ gradable gelifying matrix.
In accordance with one embodiment of the pre¬ sent invention, the whole blood derived serum/platelet extract characterized by a total absence of potential viral transmission of human origin (Hepatitis, HIV, etc. ) , may be prepared from decontaminated human whole blood or whole blood of porcine or bovine origin or of other closely related species.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a reaction scheme of a process in accordance with one embodiment of the present inven¬ tion;
Fig. 2 is a Western blot for the identification of IGFBPs in serum, platelet and serum/platelet extracts;
Fig. 3 is a SDS-PAGE analysis of serum, plate¬ let and serum/platelet extracts;
Fig. 4 illustrates the effect of a serum/plate- let extract (PSE) on a MTT test of human fibroblast survival;
Fig. 5 illustrates the wound breaking strength of (A) control vs fibrin glue-treated rats (experiment 1) and of (B) control vs PSE-treated rats in presence or absence of fibrin glue (experiment 2);
Fig. 6 illustrates the dose-effect relationship of platelet/serum extracts (PSE) on wound breaking strength in rats; and
Fig. 7 illustrates the morphometric analysis of 14 day-old wounds treated with PSE alone, PSE in fibrin vs control wounds.
DETAILED DESCRIPTION OF THE INVENTION
With reference to the drawings, it will be seen that Fig. 1, which illustrates the reaction scheme of a process in accordance with one embodiment of the pre¬ sent invention. The terms referred therein are defined as follows: "PRP" is a platelet-rich-plasma, preferably of porcine origin prepared by low speed centrifugation in order to pellet and remove erythrocytes and leukocytes. "PE" is a platelet extract. "NS" is a mono- or poly-atomic neutral support, having an amorphous structure or a crystalline lattice (with or without defect) selected from the following polygons, but not limited to this list: hexagon, cubic, mono- or tri-clinic, romboedric and orthorhombic. Depending on the exact procedure used, the support may be of carbon or heteroatomic matter. It may have a single or several anomeric phases and may include also in its network one or more of the following heteroa- tomes: cobalt, Nickel, sulfur, oxygen, tungsten, alu- minum, silicon, nitrogen, fluorine among others. The preferred support is graphite powder or activated car¬ bon.
"FM" is a neutral filtering membrane especially designed using a non-soluble and hydrophobic organic polymer. This membrane offers a wide filtering surface which confers its almost non-saturable filtering capac¬ ity.
"US" is representing ultra-sound shock, ii n or " sp" is a platelet-poor-plasma resulting from a unique passage of support-adsorbed- platelet containing plasma through the filtering mem¬ brane.
"PA-1" and "PA-2" are each respectively pre¬ cipitating agent 1 and 2 of the following chemical for- mulas:
Q-H or MxYn wherein:
Q is a radical homo- or hetero-cyclic, alkyl, silyl, cycloalkyl, alkenyl, aryl, aralkyl including at least one functional group selected from the following, but not limited to this list, acid, alcohol, amine, amide, alde¬ hyde, ketone, enol, oxime, halide, imine, enamine, ester, ether, phosphite, phosphate, nitro, nitroso, sulfamide, sulfate, sulfite, sulfoxyde, sulfone, thiosulfone, thioether, and nitrile;
M is an ammonium or phosphonium ion or any positive ion resulting from the chemical ioni- sation of elements of groups 1 to 3 of the
Periodical Table of Mendeleev;
Y is a carboxylate, a sulfate, a nitrate, a halide, a hexafluorophosphate, or a tetrafluoroborate ion, or any stable anion resulting from the Lewis acid-base interaction; and x and n are identical or different and are any number between 1 and 6.
The precipitating agents PA-1 and PA-2 may be used as such or combined together in a mixture with one or more agents, preferably in aqueous suspension or solution of a pH range from 1 to 12.
The preferred precipitating agent PA-1 is an amount of acetone solution where the resulting acetone concentration is about 68 to about 72%.
The preferred precipitating agent PA-2 is an amount of acetone solution where the resulting acetone concentration is about 84 to about 95%.
The process of the present invention excludes from the platelet-rich-plasma (PRP) step any manipula¬ tion or rinsing of the free platelets by successive centrifugation. In .accordance with the process of the present invention, there is no need for the expensive intermediates of the prior art, such as thrombin. The first step of the process of the present invention consists mainly in the preparation of the platelet extract (PE) based on the use of ultrasound as a shock treatment for the release of the material of filtered platelet. In addition to its low cost, this step is simple and can be carried out in less than two hours, even using large quantities of material.
The second step of the process of the present invention consists mainly in the preparation of the serum extract (SE) while overcoming the drawbacks of the prior art procedures. This step allows for the chemical transformation of a platelet-poor-plasma or of a serum in a hyperactive growth factor concentrate of molecular weight between 0 and 50 kDa. The platelet extract (PE) and the serum extract
(SE) may be prepared in advance or in parallel and may be stored and reconstituted in water or in any appro¬ priate physiological buffer such as saline, phospho- saline, Krebs or Hepes. The preferred embodiments of the process of the present invention are more specifically described as follows, which is intended to illustrate the invention rather than to limit its scope.
1. BLOOD EXTRACTS PREPARATION 1.1 Blood collection
For experimental purposes, porcine whole blood was used in accordance with the present invention due to its availability and its contamination-free charac- teristic. Thus, young Landrace, Yorkshire, male cas¬ trated piglets (2 month-old, 15kg B.W. were purchased from a local breeder). They were housed for 2-3 days in our facilities with free access to water and food. For blood collection, they were anaesthetized with ketamine hydrochloride (10 mg/kg BW), then with fluothane by intratracheal administration. A cannula was aseptically inserted into one carotid artery and animals were exsanguinated. Blood was collected in 20% acid-citrate-dextrose (ACD) buffer (8 g citric acid monohydrate; 22 g dextrose; 26 g sodium citrate dihy- drate in one liter of picopure distillated water, >18.2 megaohms). When serum was needed, blood was collected without any anticoagulant buffer.
1.2 Platelets extract (PE) 1.2.1 General procedure
Whole ACD blood was collected in 20% acid- citrate-dextrose (ACD) buffer and centrifuged for about 3 min. at 1500g (BRAKE=MAXIMUM; Rotor™ JA10; Beckman™ J2-21). The supernatant, platelet-rich-plasma (PRP), was collected, its volume was calculated and the plate¬ let's count was effected using a Coulter Counter ZM™ (Coulter Electronics of Canada, Ltd).
1.2.2 Procedure with solid support and shock treat¬ ment
The collected platelet-rich-plasma (PRP) was immediately mixed with synthetic activated carbon
(No. 24,227; Aldrich Chemical Co, MW) that had been previously soaked in picopure distilled water. The amount of graphite to add was determined using the following general formula I:
Q = k. [PT] . V. 10-9,
where Q is the amount of activated carbon to add (g), k is an empirically determined constant (between 0.01 and 0.80, preferably 0.082), [PT] is the platelet concen¬ tration per liter of PRP and V the PRP volume (L). Activated carbon was used as a neutral support to selectively adsorb platelets. Other supports, such as silicon nitride, silicon carbide, or activated carbon G-60™ (Aldrich) or Carbon Decolorizing Alkaline™ (Norit A, Fischer Scientific, Nepean, Ont.) were also successfully used. The mixture activated carbon-PRP was gently mixed for 2 min., then filtered through a Whatman folded filter paper 2V™ (Whatman International Ltd, England). The filter, containing platelets and acti¬ vated carbon, was washed twice with phosphosaline buffer (PBS, pH 7.0) in order to completely eliminate proteins from serum. The content of the filter was then transferred into a glass vial containing picopure distilled water.
The vial was subjected to a shock treatment consisting of either heating at 100°C for one min. and then cooled at 25°C or of two ultra-sound treatment with a sonicator (Kontes™ 40-watt; output control:60; monitor scale:20) for about 5 sec. to 10 min. with a pause of 30 sec. in between. The mixture was finally centrifuged at 2,000 to
10,000g for 5 min., the activated carbon pellet was reconstituted in picopure water, centrifuged, and both supernatants were pooled and lyophilized. The result¬ ing powder was the platelet extract (PE) .
1.2.3 Procedure with shock treatment and no solid support
The collected platelet-rich-plasma (PRP) was centrifuged for about 20 min. at 1500g (BRAKE=6; Rotor™ JA10; Beckman™ J2-21). The precipitate, corre¬ sponding to the platelets, was resuspended in phosphate buffer saline (PBS) to obtain a final concentration of 10^ platelet equivalent per ml (pq/ml), which corre¬ sponds to the first washing step. The resuspended platelets were centrifuged for a second time at the conditions mentioned above. The precipitated platelets were resuspended in picopure, distilled water to a final concentration of 10^ plate¬ let equivalent per ml (pq/ml).
The solution was cooled to about -5 to about 4°C in a ice bath and was subjected to a shock treatment consisting of two ultra-sound treatment with a sonica- tor (Kontes™ 40-watt; output control:60; monitor scale:20) for 45 sec. with a pause of 30 sec. in between. The treated solution was frozen using liquid nitrogen and lyophilized overnight. The lyophilized platelets were reconstituted in picopure water, centri¬ fuged, and both supernatants were pooled and lyophilized. The resulting powder was the platelet extract (PE).
1.3 Serum extract (SE)
A serum extract was prepared by double acetone protein precipitation in acidic conditions. Whole serum was first acidified with formic acid (88% formic acid, Fisher Scientific, Neapan, Ontario) to pH 1.9- 2.0, then mixed with acetone (Anachemia, Montreal, Canada) to a final concentration of about 68% to about 72% acetone, the preferred acetone concentration being 70%. The mixture was filtered through* a Whatman filter paper 2V™. The filtrate was collected and acetone was added to a final concentration of about 84% to about 95%, the preferred final concentration being 90%. The mixture was stirred up and again filtered through a Whatman filter paper 2V™. The filter content was washed with pure acetone, then with ether in order to completely remove acetone. Ether was air-evaporated, the final powder was reconstituted in picopure water (>18.2 megaohms) (1% w/v), filter-sterilized through 0.2 μm filters (Nalgene, Rochester, NY) and kept frozen at -75 to about -85°C.
The serum precipitate may be reconstituted to a concentration of about 0.5 to 6% of protein weight/volume, preferably to a concentration of about 1 to 2% of protein weight/volume (for example,
10 mg/ml=1.0%) .
1.4 Combination of platelet and serum extracts For all in vivo studies, the PE was reconsti¬ tuted with the 1% SE to a final concentration of 1010 platelet equivalent per ml. The mixture was centri¬ fuged at 5000 g for 15 min. at +4°C, then filter ster¬ ilized through 0.2. μm filters and either stored at -75°C or lyophilized. The final product was termed platelet-serum-extract (PSE). For the need of analytical and some in vitro studies, the PE was reconstituted in picopure water (>18.2 megaohms) (10^ platelet equivalent/ml).
2. ANALYTICAL PROCEDURES
Proteins were measured in PE, SE and PSE by a Lowry method (J. Biol. Chem., 1951, 193:265-275) using a commercial kit sold by Sigma Chemical (St Louis, MO). Platelet-derived growth factor (PDGF) was meas¬ ured in PSE by radioimmunoassay using a commercial kit (Amersha International, UK) according to the following procedure. PORCINE PDGF RIA PROCEDURE
Tube # Standard Buffer l125 -PDGF-2 A ! or Raised sample PP in goat
1- Trace — - 100 μl -
2- Background - 400 μl 100 μl -
3- Zero — 300 μl 100 μl 100 μl
4- 0.044 ng/tube 100 μl 200 μl 100 μl 100 μl
5- 0.137 ng/tube 100 μl 200 μl 100 μl 100 μl
6- 0.410 ng/tube 100 μl 200 μl 100 μl 100 μl
7- 1.230 ng/tube 100 μl 200 μl 100 μl 100 μl
8- 3.700 ng/tube 100 μl 200 μl 100 μl 100 μl
9- 11.000 ng/tube 100 μl 200 μl 100 μl 100 μl
10- 33.000 ng/tube 100 μl 200 μl 100 μl 100 μl
11-100.000 ng/tube 100 μl 200 μl 100 μl 100 μl
12- unknown ng/tube 100 μl 200 μl 100 μl 100 μl
Incubation: 20 to 24 hours at 22°C. Precipitation: 2nd antibody precipitation (100 μl) 50 μl 4% normal goat plasma and 50 μl donkey anti-goat gamma globu¬ lins (diluted 1 in 5 with buffer), vortex, leave 18 hrs or overnight at 22°C, centrifuge 35 miπs at 3200 rpm, decant and count Dilution of standard: Porcine PDGF 1 ng/ml frozen in aliquot of 500 ml (500 ng/500 μl) 1. 200 μl is added to 400 μl buffer = 33.3 ng/100 μl; 2. A serial dilution is made by 1/3 for standard from 100 ng/100 μl to
0.044 ng/100 μl. Dilution of Ab! (anti-hPDGF-BB, AB-220-NA):
1. Frozen in 40 μl aliquots of a dilution of 1/1 ;
2. 20 μl in 1980 ml of buffer make a dilution of 1/100. Take 100 μl of 1/100 Ab-] in 16 μl buffer, make a final dilution of 1/80K.
Tracer 25I-PDGF-] (Dupont) diluted in PDGF buffer so that 100 μl = 13 000 cpm.
According to the manufacturer's data, cross reactivity of porcine PDGF is 38% with the anti human
PDGF antibody used in the kit. Results were therefore converted in porcine PDGF equivalent by dividing them by 0.38. Transforming growth factor βl and β2 were meas¬ ured in PSE using the Quantikine test™ commercially available (Amersham International, V.K.). Before meas¬ urement, extracts were mixed 1:2 either in distilled water or in trifluoroacetic acid (TFA) 1%, incubated for 1 hour at room temperature, lyophilized and recon¬ stituted with water to their initial volume. This pro¬ cedure was conducted in order to determine if TGFβ was present in extracts as an active or latent, high molecular weight form.
Insulin-like growth factor-I and II (IGFI and II) were measured in SE by radioimmunoassay after for¬ mic acid-acetone extraction (Bowsher R.R. et al., Endocrinology, 1991, -128:805-814). Briefly, SE (100 μl) was acidified with formic acid 8.0 M, containing 0.5% Tween 20™ (Biorad Lab., Richmond, CA), then precipi¬ tated with acetone (350 μl). The supernatant was then diluted in RIA buffer for IGFs measurements (1/60 for IGF-I and 1/300 for IGF-II, in a 30 mM phosphate buffer, pH 7.4, containing 0.02% protamine sulfate, grade II, 10 mM EDTA, 0.02% sodium azide and 0.25% bovine serum albumin RIA grade, all buffer components from Sigma Chemicals, St Louis, MO) .
IGF-I was then measured in neutralized extracts by double antibody RIA using recombinant human IGF-I (Bachem California) for iodination and standards, and polyclonal anti-hIGF-I antiserum kindly provided by Dr. Underwood (University of North Carolina) through the National and Pituitary Program of the NIDDK, as previ- ously described by Brazeau P. and Abribat T. (75"1 Ann. Meet, of the Endocrine Soc., Las Vegas, NA, June 9-12, 1993, Abst. 1327) according to the following procedure. hIGF-I RIA PROCEDURE
Tube # Standard IGF-I ll25-RIGF-1 A ! or Buffer Raised sample in rabbit
1- Trace - - 100 μl -
2- Background - 400 μl 100 μl -
3- Zero - 300 μl 100 μl 100 μl
4- 15.6 pg/tube 100 μl 200 μl 100 μl 100 μl
5- 31.2 pg tube 100 μl 200 μl 100 μl 100 μl
6- 62.5 pg/tube 100 μl 200 μl 100 μl 100 μl
7- 125 pg/tube 100 μl 200 μl 100 μl 100 μl
8- 200 pg/tube 100 μl 200 μl 100 μl 100 μl
9- 500 pg/tube 100 μl 200 μl 100 μl 100 μl
10-1000 pg tube 100 μl 200 μl 100 μl 100 μl
11-2000 pg/tube 100 μl 200 μl 100 μl 100 μl
12- unknown pg/tube 100 μl 200 μl 100 μl 100 μl
Incubation: 20 to 24 hours at 4°C. Precipitation: 2nd antibody precipitation (100 μl) 50 μl 4% normal rabbit plasma and 50 μl goat anti-rabbit gamma globu¬ lins (diluted 1 in 5 with buffer), vortex, leave 18 hrs or overnight at 4°C, centrifuge 35 mins at 3200 rpm, decant and count. Dilution of standard: Human IGF-1 100 μg/ml frozen in aliquot of 10 μl (1 μg/10 μl) 1. 10 μl is added to 990 μl buffer = 100 000 pg/100 μl; 2. 50 μl is added to 950 μl buffer = 5000 pg/100 μl;
3. 400 μl is added to 600 μl buffer = 2000 pg/100 μl;
4. A serial dilution is made for standards from 2000 to 15.6 pg/100 μl. Dilution of Ab-j (Gropep):
1. Frozen in 50 μl aliquots of a 1/100 dilution; 2. 50 μl in 15 ml IGF-I buffer (final dilution for RIA 1/15 OK)
Tracer: 125I-RIGF-I diluted in IGF-I buffer so that 100 μl = 10 000 to 11 000 cpm.
IGF-II was also measured in neutralized extracts in the same RIA buffer by double antibody RIA (Brazeau P. and Abribat T., 75"1Ann.Meet, oftheEndocrineSoc., Las Vegas, NA, June 9-12, 1993, Abst 1327) using recom¬ binant human IGF-II (Bachem California) for iodination and standards, and monoclonal anti hIGF-II antibodies diluted 1/50 K (Amano International Enzymes Co) accord¬ ing to the following procedure. hIGF-II RIA PROCEDURE
Tube # Standard IGF-II |125. |GF-ll Ab} or Buffer Raised sample in rabbit
1- Trace — — 100 μl -
2- Background - 400 μl 100 μl —
3- Zero - 300 μl 100 μl 100 μl
4- 3.9 pg/tube 100 μl 200 μl 100 μl 100 μl
5- 7.8 pg/tube 100 μl 200 μl 100 μl 100 μl
6- 15.6 pg tube 100 μl 200 μl 100 μl 100 μl
7- 31.2 pg/tube 100 μl 200 μl 100 μl 100 μl
8- 62.5 pg/tube 100 μl 200 μl 100 μl 100 μl
9- 125 pg tube 100 μl 200 μl 100 μl 100 μl
10- 250 pg/tube 100 μl 200 μl 100 μl 100 μl
11- 500 pg/tube 100 μl 200 μl 100 μl 100 μl
12- unknowr ι pg/tube 100 μl 200 μl 100 μl 100 μl
Incubation: 20 to 24 hours at 4°C.
Precipitation: 2nd antibody precipitation (100 μl)
50 μl 4% normal mouse plasma and 50 μl goat anti-mouse gamma globulins (diluted 1 in 5 with buffer), vortex, leave 18 hrs or overnight at 4°C, centrifuge 35 mins at 3200 rpm, decant and count
Dilution of standard: Human IGF-I1 100 μg/ml frozen in aliquot of 10 μl (1 μg/10 μl)
1. 10 μl is added to 500 μl buffer = 200 000 pg/100 μl;
2. 50 μl is added to 1950 μl buffer = 5000 pg/100 μl;
3. 100 μl is added to 900 μl buffer = 500 pg/100 μl; 4. A serial dilution is made for standards from 500 to 3.9 pg/100 μl.
Dilution of Ab-| :
1. Frozen in 50 μl aliquots of a 1/10 dilution;
2. 50 μl in 50 ml IGF-II buffer (final dilution for RIA 1/50K)
Tracer: 125I-IGF-II diluted in IGF-II buffer so that 100 μl = 10000 to 11 000 cpm. Cross reactivity of IGF-II in the IGF-I RIA was
0. 2% and cross reactivity of IGF-I in the IGF-II RIA was 3.5%. ED50 from standard curves were typically 70 pg/tube. and 90 pg/tube for IGF-I and IGF-II RIAS, respectively.
IGF-binding proteins (IGFBP) were measured in PE, SE and PSE by a western ligand blotting methodol¬ ogy, using 125I-IGF-I as radioligand, adapted from the procedure originally described by Hossenlopp et al. (Anal. Biochem., 1986, 154:138-143) . Non reduced samples were subjected to SDS-PAGE electrophoresis on 12% poly- acrylamide gel slabs, then electrotransferred onto nitrocellulose. Air dried nitrocellulose sheets were sequentially incubated for 30 min. in Tris-buffered saline (TBS) containing 3% Nonidet™ P-40 (Calbiochem Co, La Jolla, CA), for 2h in TBS-1% BSA, and finally for 10 min. in TBS-0.1% Tween 20™. Sheets were trans¬ ferred into sealed plastic bags containing 1.5. 10^ cpm 12^I-IGF-I in 20 ml. After an overnight incubation with continual gentle agitation, sheets were washed twice in TBS-0.1% Tween 20™, then 3 times in TBS, and finally autoradiographed at -70°C on X-ray films for 48 h. For quantitative analysis, bands of the nitrocellulose sheets corresponding to those on autoradiograms were cut and counted in a gamma counter.
Growth factors and protein content were meas- ured in a series of platelet and serum extracts (PE, SE and PSE) .
In PE, PDGF levels averaged 3.5 ± 0.5 ng/ml (mean ± SEM of 7 measurements).
In PE, using the ultra-sound as a shock treat- ment, PDGF levels unexpectedly varied from 3.16 to 14.2 μg/1.
In four PE, non extracted (water extracted) TGF β 2 content was 4145 ± 542 pg/ml, and acid extracted TGFβ 2 content was identical (4169 ± 489 pg/ml) sug- gesting that TGFβ 2 was entirely present in PE in its active form of about 25 KD.
In PE, using the ultra-sound as a shock treat¬ ment, TGFβ 1 levels unexpectedly varied from 0.873 to 3.9 μg/1.
In PE, using the ultra-sound as a shock treat¬ ment, TGFβ 2 levels unexpectedly varied from 5.8 to 46.0 ng/1.
In SE, IGF-I and IGF-II concentrations averaged 200 ± 16 ng/ml and 452 ± 82 ng/ml respectively (mean ± SEM of 7 measurements) . As shown in Fig.2, IGFBPs could be detected both in SE and PSE but not in PE. The major IGFBP in serum, IGFBP3 (doublet migrating at 37 and 41 KD), was completely absent in serum extracts, while the three other IGFBPs in serum (IGFBP2, 1 and 4, migrating at 31, 27, and 22 KD respectively) were found in SE and PSE, at a concentration of 15-25% that in serum.
In SE, using the ultra-sound as a shock treat- ment, IGF-I concentration unexpectedly varied from 16.9 to 88 μg/1 and IGF-II concentration varied from 47.9 to 356 μg/1.
In SE, using the ultra-sound as a shock treat¬ ment, TGFβ 1 level unexpectedly varied from 1.98 to 21.5 μg/1 and TGFβ 2 level varied from 0.027 to 0.290 μg/1.
Total protein content was 5.3 ± 0.5 mg/ml in PE (mean ± SEM of five determinations), 6.3 ± 0.8 mg/ml in SE (mean ± SEM of five determinations) and 12.2 ± 0.8 mg/ml in PSE (mean ± SEM of six determinations).
Finally, a series of PE, SE and PSE were sub¬ jected to a SDS-PAGE electrophoresis on 12% polyacryla- mide gels, and silver-stained according to the method described by Morissey JH (Anal. Biochem., 1981, 117:307- 310). A protein staining of typical PE, SE and PSE is shown in Fig. 3. The major contaminant in PSE was identified as a 50-55 KD weighing group of proteins of seric origin.
3. IN VITRO ASSAYS
3.1 In vitro assays porcine primary cultured fibro¬ blasts
Jn vitro bioassays were performed using porcine primary cultured fibroblasts (PPCF) provided by our animal facilities (Centre de Recherche Louis Charles Simard, Hδpital Notre-Dame, Montreal, Canada). Cells were cultured in DMEM (Cat.#11885; Gibco BRL, Burling¬ ton, Ontario) containing 10% characterized Fetal Calf Serum (FCS, Hyclone Lab. Inc., Logan, UT) and 1% peni¬ cillin-streptomycin (Cat.#600-5140; Gibco BRL, Burling¬ ton, Ontario) for assays. All cell incubations were performed in temperature and air-controlled incubators (37°C, 100% humidity and 5% C02) -
3.1.1 Proliferation assay for porcine primary cul¬ tured fibroblasts
Cells were plated in 12-well-plates in contain¬ ing 10% FCS and 1% penicillin-streptomycin at a density of 80 x 103 cells in one ml per well. Twenty four hour later, medium was replaced by a test medium (DMEM, 0.5% FCS and 1% penicillin-streptomycin) containing test samples (0 to 200 μl/ml SE, PE or PSE; 0 to 100 ng/ml porcine PDGF-R & D system, MN, Cat.#125PD). Maximal stimulation of proliferation was evaluated by adding 10% FCS in control wells. Each dose of each test sample was assayed in triplicate.
Two days later, cells were washed with DMEM, then trypsinized with 1 ml Trypsine/EDTA 1 x (Gibco, Cat.#25-300-013) . Trypsinisation was stopped with 100 μl of pure FCS, cells in suspension were triturated, diluted. Finally, they were counted in a Coulter Counter ZM™ (Coulter Electronics of Canada, Ltd), equipped with a Coulter Channelyzer 256™, allowing for measurement of both cell number and cell average diame- ter.
The effect of PE, SE and PSE on cell prolifera¬ tion was determined using a porcine primary cultured fibroblasts (PPCF).
In this cell line, both PE and SE stimulated cell proliferation in a dose-dependent manner between 1 and 100 μl of extracts. Combination of PE and SE was synergic on cell proliferation, that is the effect of PSE was more than the addition of the effects of PE and SE. This synergy was observed at the doses of 1 and 10 μl/ml, but not 100 μl/ml. In fact, the 100 μl/ml dose was less potent than the 10 μl/ml dose in all PSE tested. A dose response study from 0 to 200 μl/ml revealed that PSE induced a dose-related linear stimu¬ lation of cell proliferation from 0 to 10 μl/ml, the effect being progressively blunted between 50 and 200 μl/ml. Thus, the maximal effect on cell proliferation was estimated to be at 10-50 μl/ml of PSE.
3.2 In vitro assays for 3T3-Swiss albino cells In vitro bioassays were performed using 3T3-
Swiss albino cells (ATCC No. CCL 92, American Culture Type Collection, Rockville, Maryland, U.S.A.). Cells were cultured in RPMI 1640™ (Cat.#430-3400EB, Gibco BRL, Burlington, Ontario) containing 10% characterized Fetal Calf Serum (FCS, Hyclone Lab. Inc., Logan, UT) and 1% penicillin-streptomycin (Cat.#600-5140; Gibco BRL, Burlington, Ontario) for assays. All cell incuba¬ tions were performed in temperature and air-controlled incubators (37°C, 100% humidity and 5% CO2). 3.2.1 Proliferation assay for 3T3-Swiss albino cells
Cells were plated in 12-well-plates in RPMI 1640 containing 10% FCS and 1% penicillin-streptomycin at a density of 10^ cells in one ml per well. Twenty four hour later, medium was replaced by a test medium (RPMI 1640™, 0.5% FCS and 1% penicillin-streptomycin) containing test samples (0 to 200 μl/ml SE, PE or PSE; 0 to 100 ng/ml porcine PDGF-R & D system, MN, Cat.#125PD). Maximal stimulation of proliferation was evaluated by adding 10% FCS in control wells. Each dose of each test sample was assayed in triplicate.
Two days later, cells were washed with RPMI 1640™, then trypsinized with 1 ml Trypsine/EDTA (Gibco, Cat.#25-300-013) . Trypsinisation was stopped with 100 μl of pure FCS, cells in suspension were tri¬ turated, diluted. Finally, they were counted in a Coulter Counter ZM™ (Coulter Electronics of Canada, Ltd), equipped with a Coulter Channelyzer 256™, allow¬ ing for measurement of both cell number and cell aver- age diameter.
In this cell line, both PE and SE stimulated cell proliferation in a dose-dependent manner between 1 and 100 μl of extracts. Combination of PE and SE was synergic on cell proliferation, that is the effect of PSE was more than the addition of the effects of PE and SE. This synergy was observed at the doses of 1 and 10 μl/ml, but not 100 μl/ml. In fact, the 100 μl/ml dose was less potent than the 10 μl/ml dose in all PSE tested. A dose response study from 0 to 200 μl/ml revealed that PSE induced a dose-related linear stimu¬ lation of cell proliferation from 0 to 10 μl/ml, the effect being progressively blunted between 50 and 200 μl/ml. Thus, the maximal effect on cell proliferation was estimated to be at 10-50 μl/ml of PSE. 3 .3 M. T. T . Test
A quantitative colorimetric assay for porcine primary cultured fibroblasts (PPCF) and human foreskin fibroblasts (HFF) survival was developed in order to assess potential cytotoxicity of the extracts. The assay is dependent on the reduction of the tretrazolium bromide, from Sigma Chemical, St Louis, MO) by the mitrochondrial deshydrogenase of viable cells to form a blue formazan product. The assay was run in 96 well plates in DMEM/Ham F12 medium (Gibco). Cells were plated at confluency (4.0 x 104 cells/well) and the procedure was conducted exactly as described by Hansen M.B. et al. (J. Immunol. Methods, 1989, 111:203-210). All test samples (0 to 100 μl/ml) were tested in tripli- cate.
In order to determine if the inhibition of the proliferative effect observed at doses higher than 50 μl/ml was due to a cytotoxic effect, MTT tests were performed on a dose range (0 to 100 μl/ml) of PSE. As shown in Fig. 4, even high concentrations of PSE (50, 75, 100 μl) had no negative effect on cell survival, indicating an absence of cytotoxicity. In fact, an increase in optical density was noted at concentrations higher than 50 μl/ml, that reflected a good cellular health.
4. IN VIVO ASSAYS 4.1 Animals and wounds
Sprague-Dawley male rats (275-300g) were pur- chased from Charles River Canada Inc. (St Constant, Quebec) . They were housed in individual cages and fed ad libitum. Each experimental group contained 7 to 8 rats. Wounds were performed as previously described by Garrel DR et al. (J. Surg. Res., 1991, 5_l:297-302) . Briefly, one 6 cm-full-thickness cutaneous incision was made longitudinally on the dorsal skin of each rat under pentobarbital anesthesia. The incisions were immediately closed with Dermalon 4.0™ sutures placed 1 cm apart.
4.2 Experimental treatments
Serum and platelet extract were prepared as described above. Except otherwise specified, they were applied onto the wounds in combination with a fibrin matrix purchased from Haemacure Biotech Inc. (Pointe- Claire, Quebec). Platelet-Serum extracts were used to reconstitute a lyophilized thrombin powder (1 ml extract for 100 IU thrombin) in presence of calcium chloride (40 mM) . This mixture was applied directly onto the wound in combination with a bovine fibrinogen solution (40-50 mg/ml) in picopure water. For a 6 cm- long wound, 250 μl of each preparation was used (total volume: 500 μl) . The contact of the fibrinogen with thrombin in presence of calcium chloride resulted in an immediate polymerisation of the fibrinogen to fibrin, a solid matrix entrapping growth factor extracts and slowly releasing them into the wound. Several experiments were conducted to establish the efficacy and the optimal dose of extracts. Experiment 1
The effect of fibrin matrix alone (500 μl) vs bovine serum albumine (BSA Sigma Chemical, St Louis, MO), 60 mg/ml as a control group was determined.
Experiment 2 The effect of growth factor extract (250 μl of
PE, 1010 platelet equivalent/ml in 1% SE) alone (diluted to 500 μl in sterile saline) or in 500 μl fibrin matrix were determined. Control rats were treated with a solution of BSA, 60 mg/ml. Experiment 3
The dose-effect relationship of growth factor extract (27,83 and 250 μl of PSE, 1010 platelet equiva¬ lent/ml in 1% SE) in 500 μl fibrin matrix, vs control (BSA, 60 mg/ml) was determined.
Experiment 4
The dose effect of growth factor extract (250,
500, 1000 μl of PSE, 1010 platelet equivalent/ml in 1%
SE, lyophilized and reconstituted in 250 μl sterile water) in 500 μl fibrin matrix, vs control (BSA, 60 mg/ml) were determined.
4.3 Wound breaking strength
In all four experiments, wound breaking strength was measured 14 days postinjury. Initial experiments showed that the maximal effect of extracts was seen at that time. Rats were sacrificed by decapi¬ tation, the dorsal skin of each rat was excised and cut into 4 strips, 1 cm wide by 5 cm long, with razor blades positioned onto a rigid support. Haemorrhaged or infected wounds were discarded. Each strip was placed between the clamps of a 1101 Instron™ tensometer and the amount of force (Newtons) required to brake the strip was recorded. Tension was applied at 20 mm/min., and all measurements were done in a blind fashion.
The efficacy of PSE as a wound healing enhancer was tested in a model of rat surgical wound. Measured parameters included wound breaking strength 14 days post wounding, as well as histological examination of the scars.
As shown in Fig. 5 (experiments 1 and 2) , the fibrin matrix or the PSE alone (200 μl) had no effect on wound strength. However, combination of PSE (200 μl) and fibrin matrix resulted in a 46% increase in wound strength. The dose-activity relationship was deduced from experiments 3 and 4 (Fig. 6). In experiment 3, there was a dose-dependent increase in wound strength, from 0 to 250 μl of PSE (regression breaking strength/dose, r=0.51, P=0.013). Doses higher than 250 μl PSE failed to further increase wound breaking strength (experiment 4), and the 1 ml dose slightly reduced the stimulatory effect.
4.4 Histological evaluation On the day of the sacrified one strip of each wound from experiment 2 was fixed in 10% formaldehyde, and later embedded in paraffin. Four-micrometer thick sections were stained with Hematein-Phloxin-Safran™ (HPS) and examined under light microscopy (Optiphot 2™ microscope, from Nikon).
Morphological evaluation includes aspect of the epidermis and of the cells within the scar, whereas morphometric evaluation includes thickness of the epi¬ dermis surrounding the scar, width of the scar at the level of both superficial and reticular dermis and cel¬ lularity, by counting fibroblasts per microscopic field, magnification x 750.
Finally, some 4 μm-thick sections were stained with Sirius Red F3BA™ and examined under polarized light to evaluate the aspect of collagen within the scar.
Histological evaluation of the scars was con¬ ducted on wounded skin samples from experiment 2. At the epidermis level, the epithelium surrounding the scar was pluristratified, fully differentiated and keratinized in all treatment groups. Moreover, epi¬ dermis thickness was identical in all treatment groups (Fig. 7). At the dermis level, most of the cells within the scar were fibroblasts surrounded by colla- gen. When stained with Sirius Red, collagen fibers within the scar were shown to be unorganized and of smaller diameter when compared to those of normal der¬ mis. Although scars did not differ qualitatively between treatment groups, scars from PSE/fibrin treated wound were wider and contained more cells than control and PSE alone treated wounds (Fig. 7). Positive sig¬ nificant correlations were found between wound strength and morphometric parameters of experiment 2 as illus¬ trated in Table 1, wherein the coefficients of correla¬ tion were calculated from 23 observations.
Table 1
Correlation between morphometric parameters and wound strength
CELLULARITY SCAR WIDTH (cell number) (mm)
WOUND BREAKING r = 0.56 r = 0.59 STRENGTH (N) P = 0.005 P = 0.004
CELLULARITY r = 0.68 (cell number) P < 0.001
Thus, PSE increased wound' breaking strength in vivo when administered in a fibrin matrix. This effect was associated with increased scar width and cellular- ity. In all observed histological sections, no sign of inflammation, of cheloϊd or of tumoral foci could be detected.
In accordance with the present invention, there is described the manufacturing technology, the analyti¬ cal content of one whole blood-derived growth factor extract, and its applications to wound healing when applied in combination with a fibrin matrix.
The association between a seric and a platelet extract yielded a preparation containing at least sig¬ nificant amounts of PDGF, TGFβ 2, IGF-I, II and IGFBPs. Although growth factors were only characterized by immunoreactivity in accordance with one embodiment of the present invention, numerous reports confirm that platelets and serum are two major sources of growth factors. Platelets α granules are the richest in vivo source of TGFβ and PDGF. Porcine platelets contain two isotypes, TGFβ 1 and β 2. Human and porcine TGFβ 1 have total sequence identity and TGFβ 2 has approxi¬ mately 70% homology with TGFβ 1. All TGFβs are natu- rally found as a latent, high molecular weight, inac¬ tive complexes in platelets. In the extracts of the present invention, the measurement of non-extracted and acid-extracted platelet extracts yielded the same amount of TGFβ 1 and β 2, indicating that TGFβ 1 and β2 are present in our extracts in its active form of about 25 KD. Since activation of TGFβ in the latent complex has been shown to occur by treatment with acid, urea or heat in vitro, it was concluded that the heating step of the PE preparation was responsible for this activa- tion.
PDGF is a disulfide-linked dimer with a molecu¬ lar weight 30-32 KD. The subunits of the dimer are two related polypeptides designated the A and B chains. Although human platelet PDGF has been shown to consist of PDGF-AB and PDGF-BB, porcine platelet PDGF consists primarily of PDGF-BB homodimers. Because of its higher affinity for the type B PDGF receptor, PDGF-BB is more potent than PDGF-AB in stimulating cell proliferation in vitro. However, in vivo data suggest that PDGF AB and BB have equal potency as wound healing enhancers.
Both PDGF and TGFβs are known as stimulators of wound healing. They both induce extracellular matrix synthesis, granulation tissue formation and increase wound breaking strength in a variety of animal models. Both recombinant TGFβ 2 and PDGF-BB are currently tested in clinical trials as therapeutic agents for the healing of chronic ulcers with promising initial results.
In addition to PDGF and TGFβ, platelet α gran- ules have been shown to contain a number of agents that might play a role in the process of wound healing. Among these substances are Platelet Factor 4 (PF4) , a platelet-derived Endothelial Cell Growth Factor (pdECGF), an Epidermal Growth Factor-like protein (EGF), and traces of IGF-I, IGF-II and IGFBP3.
Insulin-like growth factors I and II are two related peptides of approximately 7500 KD and of iden¬ tical sequence in human and pig. In serum, they circu¬ late in large amount, tightly bound to specific, high affinity binding proteins. IGF-I is the most important mediator of the biological actions of growth hormone and possesses strong systemic and local anabolic actions. Both IGFs and IGFBPs are present in wound fluid in the course of wound healing. When adminis- tered alone or in combination with IGFBPs, IGF-I has been shown to stimulate wound strength and granulation tissue formation in models of normal or impaired wound healing.
Thus, the analytical results showed that the process of extraction in accordance with the present invention successfully recovered IGFs and IGFBPs from serum, and PDGF and TGFβ from platelets. In addition, other bioactive substances, such like PF4, pdECGF or EGF might also be present and might participate in the biological activity of PSE.
The process of the present invention for the manufacture of PSE is new, simple and inexpensive. It involves a few number of steps that can be performed in less than 24 hours to produce a mixture made of at least 5 important growth factors for wound healing (PDGF, TGFβ, IGF-I, IGF-II, IGFBPs).
Moreover, the porcine origin of the blood, in accordance with one embodiment of the present inven- tion, guaranties: 1) a total absence of potential transmission of human viral agents (hepatitis, HIV, etc.); 2) the production of growth factors nearly 100% identical to their human counterparts and, 3) a reli¬ able, largely available and highly reproducible source of raw material.
The in vitro and in vivo studies showed that the platelet/serum extracts of one embodiment of the present invention have positive effects on wound heal¬ ing, mediated by at least two different mechanisms, cell proliferation and collagen synthesis.
In cell culture, both the platelet and serum extracts of the present invention stimulated fibroblast proliferation in a dose dependent manner, and coadmin- istration of both extracts resulted in a synergic effect. Since SE and PE contained IGFs and PDGF, respectively, it is suggested that the synergy was due to interactions between these two growth factors. This synergy was first reported in cultured 3T3 fibroblasts, then confirmed in vivo in animal models of wound heal- ing. It has been explained by the complementary roles of PDGF and IGFs in the cell cycle, PDGF being a compe¬ tence factor and IGFs being a progression factor.
PSE induced a dose-dependent increase of por¬ cine primary cultured fibroblasts (PPCF) or 3T3 up to 10 μl/ml, while the stimulatory effect was inhibited at doses equal or above 50 μl/ml. In parallel, PSE was not cytotoxic at doses up to 100 μl/ml and stimulated protein (collagen) synthesis in a dose dependent fash¬ ion between 0 and 100 μl/ml. Based on these results, the hypothesis of a dual mode of action for PSE was raised: at low dose (0-10 μl/ml), it would stimulate preferentially fibroblast proliferation; at higher doses (> 50 μl/ml), the effect on extracellular matrix synthesis would be predominant and would overcome that on cell proliferation.
In vivo studies were conducted using a fibrin matrix as carrier. PSE alone failed to increase wound breaking strength, whereas PSE in fibrin did. This indicated that the fibrin sealant was necessary to maintain growth factors in contact with the wound for a sufficient time. This type of matrix is safe, natural and biodegradable. No sign of fibrin was detected by histological observation 14 days post administration, suggesting that the fibrinolysis was complete at that time. When applied in combination with the fibrin glue, the PSE of the present invention stimulated wound breaking strength in a surgical model in a dose depend¬ ent fashion from 0 to 250 μl of extract, with no fur¬ ther increase at higher doses. Histological observa- tion of the treated wound revealed increased cellular- ity and wider scars. This suggested that the increase of wound strength was due to an increased number of fibroblasts, synthetising larger amounts of collagen. Moreover, the absence of inflammation, of transformed cells and of cheloϊds indicated that the treatment was safe.
These in vitro and in vivo studies suggest that the platelet/serum extract of the present invention is a powerful stimulator of granulation tissue formation. Its potential therapeutical applications include the treatment of chronic ulcers and surgical wounds, and the regeneration of injured soft tissues.
According to the in vi tro and in vivo results, SE and PE of the present invention might be used alone or advantageously in combination (PSE) because they work synergically. In vivo, they have to be adminis¬ tered topically in a matrix slowly releasing them, such as fibrin glue, collagen, polymers among others.
If fibrin is selected as a carrier, for a 6 cm long surgical wound, the bioactive formulation is 50 to 1000 μl of a preparation made of 10 to 1000 IU/ml thrombin, 20 to 200 mg/ml fibrinogen, and 50 to 500 μl/ml SE (0.1 to 5%), PE (108 to 1012 platelet equiva¬ lent/ml buffer) or PSE (108 to 1012 platelet equiva- lent/ml SE 0.1 to 5%) in presence of CaCl2 (1 to 100 mM final) .
Recently, a randomized, double blind and pla¬ cebo clinical study reported significant improvements in diabetic ulcers treated with an homologous human platelet lysate derived from pooled human platelets. Moreover, a cost-efficacy analysis conducted for this study revealed that the treatment was associated with a 38% decrease in the medical costs when compared to a conventional therapy. Although the composition of the PSE differs from that of the human platelet lysate (thrombin activated platelet wound healing formula) developed by Curative Technologies Inc., the results of this study illustrate the therapeutical potential and the advantages of growth factor extracts over recombi- nant growth factors.
The present invention will be more readily un¬ derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope. EXAMPLE I Preparation of Platelet Extract
Three liters of platelet-rich-plasma (PRP) hav¬ ing a platelet concentration of 464 X 109 platelets/L was used. The amount of graphite to add was determined using the following formula:
Q = K.DX [PRP] . V. 10-9 where K is an empirically determined constant of 0.082, Dx is a ratio of the density of the neutral support with respect to the density of carbon (1 for graphite), [PRP] is the platelet concentration per liter of PRP (464 X 109), V is the PRP volume in L (3), and Q is the amount of graphite to add in g, thus Q=114 g of graph- ite.
This graphite is previously washed in water and carefully mixed with the three liters of PRP. After a 2 min. mixing, the platelet-adsorbed-graphite mixture is filtered under atmospheric pressure through a espe- cially design high capacity filtering membrane.
After 10 min. ' of filtration, the adsorbed platelet are washed twice with three liters of phos- phosaline buffer pH 7.4, then the washed filtrate is transferred in a beaker. The membrane is rinsed with 1 L of distilled water and the temperature of the washed filtrate in the beaker is raised to 100°C for 1 min.
The washed filtrate in the beaker is filtered and/or centrifuged; the supernatant is lyophilized. The lyophilisate constitutes the solid platelet extract (PE) which can be reconstituted.
In vitro bioassays were performed using porcine primary cultured fibroblasts (PPCF) and 3T3-Swiss albino cells (3T3) as previously described in sections 3.1, 3.1.1, 3.2, 3.2.1 and 3.3 of the description. Results
The following were measured according to the procedure described above in the disclosure. PDGF = 2.0 μg/1 GFβ! = 0.0 μg/1 TGFβ2 = 5.6 ng/1
IN VITRO BIOASSAYS
Proliferation assay M.T.T. test (cell count) (d.o. 570 nm)
Cell Dosage Mean S.E.M. % Mean S.E.M. % type proliferation oxydation
PPCF control 75858 763 100 0.21 0.017 100
1 ul/ml 83998 3239 111 0.23 0.035 111
10 ul/ml 102259 1855 135 0.26 0.025 126
100 ul/ml 141723 3416 187 0.29 0.038 140
3T3 control 220900 3186 100
1 ul/ml 230704 10642 104
10 ul/ml 284342 937 129
100 ul/ml 281076 12321 127
EXAMPLE II Preparation of Platelet Extract The procedure as described in Example I was carried out, except that carbure of silicium was used instead of graphite. This material has a density con¬ stant of 3.23, which resulted in a Q value of 335g of carbure of silicium used.
EXAMPLE III
Preparation of Platelet Extract
The procedure as described in Example I was carried out, except that nitrure of silicium was used instead of graphite. This material has a density con¬ stant of 3.44, which resulted in a Q value of 357g of nitrure of silicium used. EXAMPLE IV Preparation of Platelet Extract Procedure
Three liters of platelet-rich-plasma (PRP) hav- ing a platelet concentration of 464 X 109 platelets/L was used. The amount of carbon to add was' determined using the following formula:
Q = K[PRP]V where K is an empirically determined constant of 0.082, [PRP] is the platelet concentration per liter of PRP (464 X 109), V is the PRP volume in L (3), and Q is the amount of carbon to add in g, thus Q=114 g of carbon. This carbon is previously washed in 500 ml of picopure water, agitated vigorously and then centrifuged for 2 min. at 2000g using a Rotor™ JA10 (Beckman™ J2-21).
The precipitated carbon is kept and carefully mixed with the three liters of PRP. After a 2 min. mixing, the platelet-adsorbed-carbon mixture is fil- tered through a Whatman filter paper 2V™. The fil¬ tered PPP is discarded.
After the filtration, the adsorbed platelet are washed twice with 450 ml of phosphosaline buffer pH 7.4, then the washed filtrate is transferred in a beaker. The adsorbed platelet are resuspended in picopure water (>18.2 megaohms) to obtain a final con¬ centration of 10 pq/ml and the temperature of the beaker is cooled to about 0-4°C using an ice bath.
The adsorbed platelet are subjected to a first ultra-sound treatment with a sonicator (Kontes™ 40- watt; output control:60; monitor scale:20) for about 45 sec, a pause of 30 sec. and a second ultra-sound treatment for about 45 sec.
The washed filtrate in the beaker is centri- fuged for 5 min. at 5000g; the precipitate is resus- pended in a minimum quantity of picopure water and cen¬ trifuged again under the same conditions. Both super- natants are combined, frozen in liquid nitrogen and then lyophilized. The lyophilisate constitutes the solid platelet extract (PE) which is reconstituted in picopure water (>18.2 megaohms) to obtain a ' final con¬ centration of 1010 pq/ml, centrifuged for 30 min. at 10,000g. The supernatant is filtered first through 0.8 μm and 0.2 μm filters (Nalgene, Rochester, NY). The preparation is aliquoted and kept at -80°C.
In vitro bioassays were performed using porcine primary cultured fibroblasts (PPCF) and 3T3-Swiss albino cells (3T3) as previously described in sections 3.1, 3.1.1, 3.2, 3.2.1 and 3.3 of the description.
Results
The following are measured according to the procedure described above in the disclosure.
PDGF = 4.5 μg/1 TGFβi = 0.870 μg/1
TGFβ2 = 46.0 ng/1
IN VITRO BIOASSAYS
Proliferation assay M .T.T. test ( cell count ) (d .o. 570 nm)
Cell Dosage Mean S.E.M. % Mean S.E.M. % type proliferation oxydatiόn
PPCF control 76982 1706 100 0.21 0.017 100
1 ul/ml 84941 1658 110 0.26 0.025 122
10 ul/ml 103249 3076 134 0.27 0.013 130
100 ul/ml 205656 12731 267 0.41 0.033 195
3T3 control 225163 5149 100
1 ul/ml 239922 878 107
10 ul/ml 268620 7321 119
100 ul/ml 446137 5564 198 EXAMPLE V Preparation of Platelet Extract Procedure
The procedure as described in Example IV is carried out, except that activated carbon Darco™ G-60 (Aldrich) was used instead of carbon.
In vitro bioassays were performed using porcine primary cultured fibroblasts (PPCF) and 3T3-Swiss albino cells (3T3) as previously described in sections 3.1, 3.1.1, 3.2, 3.2.1 and 3.3 of the description.
Results
The following are measured according to the procedure described above in the disclosure. PDGF = 3.16 μg/1 TGFβi = 0.873 μg/1 TGFβ2 = 5.8 ng/1
IN VITRO BIOASSAYS
Proliferation assay M . T. T . test ( cell count ) ( d..o. 570 nm)
Cell Dosage Mean S.E.M. % Mean S.E.M. % type proliferation oxydation
PPCF control 73117 2345 100 0.21 0.017 100
1 ul/ml 68599 2354 94 0.22 0.014 107
10 ul/ml 75689 4188 104 0.21 0.023 102
100 ul/ml 159688 5937 218 0.41 0.039 194
3T3 control 237384 5019 100
1 ul/ml 246642 9229 104
10 ul/ml 256782 7338 108
100 ul/ml 427903 2352 180 EXAMPLE VI Preparation of Platelet Extract Procedure Three liters of platelet-rich-plasma (PRP) hav¬ ing a platelet concentration of 300 to 500 X 109 plate¬ lets/L are centrifuged for about 20 min. at 1500g (BRAKE=6; Rotor™ JA10; Beckman™ J2-21). The precipi¬ tated platelets are resuspended in phosphosaline buffer (PBS) to obtain a final concentration of 109 platelet equivalent per ml (pq/ml), which corresponds to the first washing step.
The resuspended platelets are centrifuged for a second time at the conditions mentioned above. The precipitated platelets are resuspended in picopure water (>18.2 megaohms) to a final concentration of 10*" platelet equivalent per ml (pq/ml).
The solution is cooled to about 0 to about 4°C in a ice bath and is subjected to a shock treatment consisting of two ultra-sound treatment with a sonica- tor (Kontes™ 40-watt; output control: 60; monitor scale: 20) for 45 sec. with a pause of 30 sec. in between.
The treated solution is frozen using liquid nitrogen and lyophilized overnight. The lyophilized platelets are reconstituted in picopure water to obtain a final concentration of 10^ pq/ml. The membrane fragments are eliminated by ultracentrifugation or by filtration through 0.8 μm, 0.45 μm and 0.2 μm filters (Nalgene, Rochester, NY). The preparation is aliquoted and kept at -80°C.
In vi tro bioassays were performed using porcine primary cultured fibroblasts (PPCF) and 3T3-Swiss albino cells (3T3) as previously described in sections 3.1, 3.1.1, 3.2, 3.2.1 and 3.3 of the description. Results
The following are measured according to the procedure described above in the disclosure. PDGF = 14.2 μg/1 TGFβi = 3.9 μg/1 TGFβ2 = 26.7 ng/1
IN VITRO BIOASSAYS
Proliferation assay M . T. T . test ( cell count) (d.. o . 570 nm)
Cell Dosage Mean S.E.M. % Mean S.E.M. % type proliferation oxydation
PPCF control 74174 1644 100 0.18 0.009 100
1 ul/ml 80234 4050 108 0.24 0.023 130
10 ul/ml 113888 1062 154 0.35 0.037 191
100 ul/ml 219769 7233 296 0.34 0.010 184
3T3 control 208538 5320 100
1 ul/ml 229108 7577 110
10 ul/ml 269219 9914 129
100 ul/ml 388493 10111 186
EXAMPLE VII Preparation of Serum Extract
A 300 ml of whole serum was 'first acidified with 18.2 ml of formic acid (88% formic acid, Fisher Scientific, Neapan, Ontario) at room temperature. After 5 min. of agitation at room temperature, 700 ml of pure acetone was added. The agitation was continued for another 5 min., then the suspension was filtered through a Whatman filter paper 2V™.
The filtrate was collected and slowly trans¬ ferred in a beaker containing 1400 ml of pure acetone constantly agitated. The order in the addition is of extreme importance. The precipitate is filtered through a Whatman filter paper 2V™, washed three times with 500 ml of pure acetone, then three times with 500 ml of ether, then dried. The solid mass is 1.7 g and is reconsti- tuted in 170 ml of picopure water (18.2 Ohm) (1% w/v), filter-sterilized through 0.22 μm filters (Nalgene, Rochester, NY) and aliquoted under laminar flux (Canadian Cabinets Co, Ltd., V6 MW99.C30™). This solution of pH 6.5 and 1% of final concentration, con- stitutes the serum extract (SE).
EXAMPLE VIII
Preparation of Serum Extract Procedure A 300 ml of whole serum was centrifuged for 15 min. at 3000g, the supernatant is kept and centrifuged for a second time under the same conditions. The cen¬ trifuged supernatant is acidified at pH 2 with 88% for¬ mic acid (Fisher Scientific, Neapan, Ontario) at room temperature.
The acidified serum is cooled at 4°C in a cold room. A quantity of 75% acetone is added and agitated magnetically for an hour to precipitate the proteins. The suspension is filtered through a Whatman filter paper 2V™.
The filtrate was collected arid slowly trans¬ ferred in a beaker containing an amount of pure acetone to obtain a final concentration of 90% acetone. After an incubation of one hour, the suspension is filtered through a Whatman filter paper 2V™.
The precipitate is washed twice with pure ace¬ tone, and then a third time with anhydride ether, then dried using a lyophilizer.
The solid mass is reconstituted in picopure water (>18.2 megaohms) to obtain a final concentration of 1%, filter-sterilized through 0.22 μm filters (Nalgene, Rochester, NY) and aliquoted under laminar flux (Canadian Cabinets Co, Ltd., V6 MW99.C30™) and kept at -80°C.
In vitro bioassays were performed using porcine primary cultured fibroblasts (PPCF) and 3T3-Swiss albino cells (3T3) as previously described in sections 3.1, 3.1.1, 3.2, 3.2.1 and 3.3 of the description.
Results
The following are measured according to the procedure described above in the disclosure. IGFII = 47.9 μg/1 IGFI = 16.9 μg/1 TGFβi = 1.98 μg/1 TGFβ2 = 0.027 μg/1
IN VITRO BIOASSAYS
Proliferation assay M . T.T. test ( cell count) ( d.. o . 570 nm)
Cell Dosage Mean S.E.M. % Mean S.E.M. % type proliferation oxydation
PPCF control 74087 1761 100 0.21 0.017 100 1 ul/ml 0.20 0.006 93 10 ul/ml 0.17 0.007 84
100 ul/ml 61368 3120 83 0.11 0.006 54
3T3 control 266384 1062 100
1 ul/ml 296206 5188 111
10 ul/ml 432394 1906 162
100 ul/ml 507700 18724 191
EXAMPLE IX
Preparation of Serum Extract Procedure
The procedure as described in Example VIII is carried out, except that the acidified serum is cooled at 0°C instead of 4°C. Also, the first precipitation using acetone is conducted such that the acetone is added dropwise until a final acetone concentration of 72% is achieved, the second acetone addition is effected under the same conditions until a final ace¬ tone concentration of 75% is achieved, and a third ace¬ tone addition is effected under the same conditions until a final acetone concentration of 90% is achieved.
Results
The following are measured according to the procedure described above in the disclosure. IGFII = 356 μg/1 IGFI = 88 μg/1 TGFβi = 21.5 μg/1 TGFβ2 = 0.290 μg/1
IN VITRO BIOASSAYS
Proliferation assay M.T.T. test (cell count) (d.o. 570 nm)
Cell Dosage Mean S.E.M. % Mean S.E.M. % type proliferation oxydation
PPCF control 74437 2317 100 0.21 0.017 100
1 ul/ml 0.17 0.017 83
10 ul/ml 0.16 0.008 77
100 ul/ml 67771 3727 91 0.16 0.007 74
3T3 control 260270 4083 100
1 ul/ml 310622 16617 119
10 ul/ml 391146 6134 150
100 ul/ml 449006 6863 173
EXAMPLE X Preparation of Serum Extract
The procedures as described in Example VII and VIII are carried out, except that formic acid is replaced by the following acid mixture, trifluoroacetic acid, chlorydric acid and phosphorous acid. EXAMPLE XI Preparation of Final Extract
The PE of Example I, II, III, IV, V or VI is reconstituted with SE of Example VII, VIII, IX, or X to obtain a final concentration of 10^ platelet per ml without neutralization. The mixture is centrifuged at 10,000g for 15 min. at +4°C, then filter sterilized through 0.22 μm filters and aliquoted under laminar flux. EXAMPLE XII
Preparation of Final Extract
The PE of Example I, II, III, IV, V or VI is reconstituted with SE of Example VII, VIII, IX, or X to obtain a final concentration of 10^ platelet per ml. The mixture is neutralized using a Amberlite IRA- 400(OH)™ resin, centrifuged at 10,000g for 15 min. at +4°C, then filter sterilized through 0.22 μm filters and aliquoted under laminar flux.
While the invention has been described in con- nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia¬ tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be ap¬ plied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WE CLAIM:
1. A process for the preparation of platelet- extract, which comprises the steps of: a) concentrating a platelet-rich-plasma prepared from whole blood by passing through a neutral support for adsorbing the platelets or by cen- trifuging whole blood at about lOOOg to about 3000g; and b) releasing the concentrated platelets of step a) by ultra-sound shock.
2. A process for the preparation of serum-extract, which comprises the steps of: a) precipitating a concentrated plasma prepared from whole blood by passing through a neutral support for adsorbing the platelets or by cen- trifuging whole blood at about lOOOg to about 3000g, a platelet-poor-plasma or a serum by adding an amount of acetone solution to obtain an acetone concentration of about 68 to about 95% at a temperature of about -15 to 5°C; thereby obtaining a solid plasma or serum pre¬ cipitate; and b) the serum precipitate of step a) is reconsti¬ tuted in an appropriate buffer to obtain a serum-extract of a concentration of about 0.5 to 6% of protein weight/volume.
3. A process for the preparation of platelet- serum-extract, which comprises the steps of: a) concentrating a platelet-rich-plasma prepared from whole blood by passing through a neutral support for adsorbing the platelets or by cen- trifuging whole blood at about lOOOg to about 3000g; b) releasing the concentrated platelets of step a) by ultra-sound shock; c) precipitating the concentrated plasma of step a), a platelet-poor-plasma or a serum by adding an amount of acetone solution to obtain an ace¬ tone concentration of about 68 to about 95% at a temperature of about -15 to 5°C; thereby obtaining a solid plasma or serum precipitate; d) the plasma or serum precipitate of step c) is reconstituted to a concentration of about 0.5 to 6% of protein weight/volume and mixed to the released platelets of step b) to obtain a platelet-serum-extract of a concentration which substantially has a physiological concentration to give a maximum synergy in promoting wound healing.
4. The process of claim 3, wherein the precipita¬ tion step c) is conducted using a first amount of ace¬ tone solution to obtain an acetone concentration of about 68% to about 72% and the resulting supernatant is precipitated with a second amount of acetone solution to obtain an acetone concentration of about 84% to about 95%, wherein the second precipitate is collected to form the serum precipitate.
5. The process of claim 4, wherein the plasma or serum precipitate of step c) is reconstituted in an appropriate buffer.
6. The process of claim 3, wherein the neutral support of step a) is selected from the group consist- ing of carbon, activated carbon, graphite and charcoal, SiC and Si3N4-
7. The process of claim 6, wherein the amount of neutral support used in step a) is determined by the following formula:
Q = K.DX [PRP] . V. 10-9. wherein:
K is an empirically determined constant ranging from 0.010 to 1.000;
Dx is a ratio of the density of the neutral sup¬ port with respect to the density of carbon; [PRP] is the platelet concentration per liter of PRP;
V is the PRP volume in L; and Q is the amount of neutral support in g.
8. The process of claim 7, wherein k is 0.082.
9. The process of claim 3, wherein the ultra-sound shock of step b) consists of two ultra-sound treatment with a sonicator for about 5 sec. to 10 min. with a pause of at least 30 sec. in between.
10. The process of claim 3, wherein after step a) a further step is added, which comprises: a' ) filtering the adsorbed platelet-support mixture through a filtering membrane.
11. The process of claim 3, wherein said whole blood is of human, porcine, bovine, ovine or caprine origin.
12. A pharmaceutical composition for promoting wound healing, which comprises an effective concentra- tion of platelet-extract prepared according to the process of claim 1 in association with a pharmaceuti¬ cally acceptable carrier.
13. A pharmaceutical composition for promoting wound healing, which comprises an effective concentra¬ tion of serum-extract prepared according to the process of claim 2 in association with a pharmaceutically acceptable carrier.
14. A pharmaceutical composition for promoting wound healing, which comprises an effective concentra¬ tion of platelet-serum-extract prepared according to the process of claim 3 in association with a pharmaceu¬ tically acceptable carrier.
15. The pharmaceutical composition of claim 14, wherein said platelet-serum-extract concentration is between 10*-* to 1012 platelet equivalent/ml in combina¬ tion with 0.5 to 2% serum-extract/ml.
16. The pharmaceutical composition of claim 14, wherein said pharmaceutically acceptable carrier is selected from the group consisting of fibrin glue, collagen, collagen derivatives, polysaccharidic poly¬ mers, gelatins, ointments and solid dressings suitable for wound treatment.
17. Use of the composition of claim 14 for the promotion of wound healing of a patient.
18. The method of use of the platelet-serum-extract prepared according to the process of claim 3 for pro¬ moting wound healing of a patient.
19. Use of the composition of claim 14 for the production of a medicament for the promotion of wound healing of a patient.
PCT/CA1994/000601 1993-12-08 1994-11-01 Process for the preparation of serum and platelet growth factors extract WO1995015763A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU79885/94A AU7988594A (en) 1993-12-08 1994-11-01 Process for the preparation of serum and platelet growth factors extract

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US16289893A 1993-12-08 1993-12-08
US08/162,898 1993-12-08
US32302294A 1994-10-14 1994-10-14
US08/323,022 1994-10-14

Publications (1)

Publication Number Publication Date
WO1995015763A1 true WO1995015763A1 (en) 1995-06-15

Family

ID=26859148

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1994/000601 WO1995015763A1 (en) 1993-12-08 1994-11-01 Process for the preparation of serum and platelet growth factors extract

Country Status (2)

Country Link
AU (1) AU7988594A (en)
WO (1) WO1995015763A1 (en)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034614A1 (en) * 1996-03-20 1997-09-25 Theratechnologies Inc. Process for the preparation of platelet growth factors extract for wound healing compositions
US6110484A (en) * 1998-11-24 2000-08-29 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
WO2005105121A1 (en) * 2004-05-05 2005-11-10 Synthes Gmbh Use of platelets or platelet rich plasma (prp)
EP1848447A2 (en) * 2004-08-20 2007-10-31 Alberto Gorrochategui Barrueta Biological tissue regenerative agent and method for preparing and using same
US20110008458A1 (en) * 2009-07-09 2011-01-13 Orogen Biosciences, Inc. Process for removing growth factors from platelets
EP2389942A1 (en) * 2010-05-25 2011-11-30 GWO REI Biomedical Technology Corp. Virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF and preparation method thereof
US20120114760A1 (en) * 2004-07-02 2012-05-10 James Gandy Compositions for treating wounds and processes for their preparation
US8293530B2 (en) 2006-10-17 2012-10-23 Carnegie Mellon University Method and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8529960B2 (en) 2002-03-18 2013-09-10 Carnell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8529961B2 (en) 2006-10-17 2013-09-10 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8821858B2 (en) * 2005-02-09 2014-09-02 Gw Ip, Llc Lyophilized platelet rich plasma for the use in wound healing (chronic or acute) and bone or tissue grafts or repair
US10166258B2 (en) 2008-09-16 2019-01-01 Mayo Foundation For Medical Education And Research Compositions containing platelet contents
CN110464740A (en) * 2019-09-02 2019-11-19 广州准优生物科技有限公司 PRP product and preparation method, PRP and regulatory T cells compound formulation and application
CN115463249A (en) * 2022-08-11 2022-12-13 中南大学湘雅医院 Platelet-rich plasma-loaded hydrogel and preparation method thereof
US11604026B2 (en) 2019-03-14 2023-03-14 Terumo Bct Biotechnologies, Llc Lyophilization loading tray assembly and system
US11634257B2 (en) 2017-10-09 2023-04-25 Terumo Bct Biotechnologies, Llc Lyophilization container and method of using same
US11891620B2 (en) 2014-05-16 2024-02-06 Mayo Foundation For Medical Education And Research Cell culture media compositions for primary cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3953290A (en) * 1973-04-30 1976-04-27 Ab Kabi Polypeptides as vitro active cell growth enhancing factors and methods of use
JPS58159419A (en) * 1982-03-05 1983-09-21 Fujirebio Inc Separating and obtaining method of alpha1-acidic glycoprotein
EP0136093A2 (en) * 1983-08-29 1985-04-03 Yoshio Sakagami Anti-cancer factors
WO1986003122A1 (en) * 1984-11-29 1986-06-05 Curatech, Inc. Wound healing agents

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3953290A (en) * 1973-04-30 1976-04-27 Ab Kabi Polypeptides as vitro active cell growth enhancing factors and methods of use
JPS58159419A (en) * 1982-03-05 1983-09-21 Fujirebio Inc Separating and obtaining method of alpha1-acidic glycoprotein
EP0136093A2 (en) * 1983-08-29 1985-04-03 Yoshio Sakagami Anti-cancer factors
WO1986003122A1 (en) * 1984-11-29 1986-06-05 Curatech, Inc. Wound healing agents

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CAN.J.ANIM.SCI., vol. 71, no. 3, 1991, pages 953 - 957 *
CHEMICAL ABSTRACTS, vol. 100, no. 7, 13 February 1984, Columbus, Ohio, US; abstract no. 49215w, WILLIAMS ET AL: "INTERACTIONS OF ULTRASOUND WITH PLATELETS AND THE BLOOD COAGULATION SYSTEM" page 387; column 1; *
CHEMICAL ABSTRACTS, vol. 116, no. 11, 16 March 1992, Columbus, Ohio, US; abstract no. 102145g, SEDGWICK ET AL: "EFFECT OF PROTEIN PRECIPITATING AGENTS ON THE RECOVERY OF PLASMA FREE AMINO ACIDS" page 360; column 1; *
DATABASE WPI Section Ch Week 8344, Derwent World Patents Index; Class B04, AN 83-804027 *
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.94074153 & CLIN ENDOCRINOL (OXF), (1993 NOV) 39 (5) 583-9 *
ULTRASOUND INTERACT.BIOL.MED.,(PROC.INT.SYMP.) 1980, 1983, pages 171 - 177 *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034614A1 (en) * 1996-03-20 1997-09-25 Theratechnologies Inc. Process for the preparation of platelet growth factors extract for wound healing compositions
AU725091B2 (en) * 1996-03-20 2000-10-05 Theratechnologies Inc. Process for the preparation of platelet growth factors extract for wound healing compositions
US6110484A (en) * 1998-11-24 2000-08-29 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
US6277394B1 (en) 1998-11-24 2001-08-21 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
US8529960B2 (en) 2002-03-18 2013-09-10 Carnell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
WO2005105121A1 (en) * 2004-05-05 2005-11-10 Synthes Gmbh Use of platelets or platelet rich plasma (prp)
US20120114760A1 (en) * 2004-07-02 2012-05-10 James Gandy Compositions for treating wounds and processes for their preparation
EP1848447A2 (en) * 2004-08-20 2007-10-31 Alberto Gorrochategui Barrueta Biological tissue regenerative agent and method for preparing and using same
EP1848447A4 (en) * 2004-08-20 2009-07-29 Barrueta Alberto Gorrochategui Biological tissue regenerative agent and method for preparing and using same
US8821858B2 (en) * 2005-02-09 2014-09-02 Gw Ip, Llc Lyophilized platelet rich plasma for the use in wound healing (chronic or acute) and bone or tissue grafts or repair
US8293530B2 (en) 2006-10-17 2012-10-23 Carnegie Mellon University Method and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US9364503B2 (en) 2006-10-17 2016-06-14 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8529961B2 (en) 2006-10-17 2013-09-10 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8529958B2 (en) 2006-10-17 2013-09-10 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8911789B2 (en) 2006-10-17 2014-12-16 Carnegie Mellon University Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US10925901B2 (en) 2008-09-16 2021-02-23 Mayo Foundation For Medical Education And Research Compositions containing platelet contents
US10166258B2 (en) 2008-09-16 2019-01-01 Mayo Foundation For Medical Education And Research Compositions containing platelet contents
US20110008458A1 (en) * 2009-07-09 2011-01-13 Orogen Biosciences, Inc. Process for removing growth factors from platelets
US8734854B2 (en) * 2009-07-09 2014-05-27 Orogen Biosciences Inc. Process for removing growth factors from platelets
US9511118B1 (en) 2009-07-09 2016-12-06 Pgfx Patent Holdings, Llc Process for removing growth factors from platelets
EP2389942A1 (en) * 2010-05-25 2011-11-30 GWO REI Biomedical Technology Corp. Virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF and preparation method thereof
US9034646B2 (en) 2010-05-25 2015-05-19 Zheng Yang Biomedical Technology Co., Ltd. Virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF and preparation method thereof
CN102985101B (en) * 2010-05-25 2014-07-30 国维联合科技股份有限公司 Virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF and preparation method thereof
CN102985101A (en) * 2010-05-25 2013-03-20 国维联合科技股份有限公司 Virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF and preparation method thereof
WO2011148326A1 (en) * 2010-05-25 2011-12-01 Gwo Rei Biomedical Technology Corp. Virally-inactivated growth factors-containing platelet lysate depleted of pdgf and vegf and preparation method thereof
US11891620B2 (en) 2014-05-16 2024-02-06 Mayo Foundation For Medical Education And Research Cell culture media compositions for primary cells
US11634257B2 (en) 2017-10-09 2023-04-25 Terumo Bct Biotechnologies, Llc Lyophilization container and method of using same
US11604026B2 (en) 2019-03-14 2023-03-14 Terumo Bct Biotechnologies, Llc Lyophilization loading tray assembly and system
US11609042B2 (en) 2019-03-14 2023-03-21 Terumo Bct Biotechnologies, Llc Multi-part lyophilization container and method of use
US11609043B2 (en) 2019-03-14 2023-03-21 Terumo Bct Biotechnologies, Llc Lyophilization container fill fixture, system and method of use
US11740019B2 (en) 2019-03-14 2023-08-29 Terumo Bct Biotechnologies, Llc Lyophilization loading tray assembly and system
US11747082B2 (en) 2019-03-14 2023-09-05 Terumo Bct Biotechnologies, Llc Multi-part lyophilization container and method of use
US11815311B2 (en) 2019-03-14 2023-11-14 Terumo Bct Biotechnologies, Llc Lyophilization container fill fixture, system and method of use
CN110464740A (en) * 2019-09-02 2019-11-19 广州准优生物科技有限公司 PRP product and preparation method, PRP and regulatory T cells compound formulation and application
CN115463249A (en) * 2022-08-11 2022-12-13 中南大学湘雅医院 Platelet-rich plasma-loaded hydrogel and preparation method thereof

Also Published As

Publication number Publication date
AU7988594A (en) 1995-06-27

Similar Documents

Publication Publication Date Title
EP1007061B1 (en) Process for the preparation of platelet growth factors extract for wound healing compositions
WO1995015763A1 (en) Process for the preparation of serum and platelet growth factors extract
US10272139B2 (en) Process, tube and device for the preparation of wound healant composition
US5605887A (en) Therapeutic fibrinogen compositions
CA2105652C (en) Reducing wound scarring with antibodies to growth factors
EP0090997B1 (en) Adhesive wound covering material comprising collagen
NAGELSCHMIDT et al. Effect of fibronectin therapy and fibronectin deficiency on wound healing: a study in rats
Mason et al. Some effects of a microcrystalline collagen preparation on blood
CA2031817C (en) Use of thrombospondin to promote wound healing
Uchiba et al. Recombinant human soluble thrombomodulin reduces endotoxin-induced pulmonary vascular injury via protein C activation in rats
JP3859176B2 (en) Compositions suitable for use as antidote to anticoagulants and their use
US8268362B2 (en) Medicinal product for the promotion of wound healing
JPS63218625A (en) Method and composition for treating bleeding affections
CA2100720C (en) Medicinal composition comprising tcf-ii
Cameron et al. Perioperative management of patients with von Willebrand’s disease
Cohler et al. Plasma fibronectin therapy and lung protein clearance with bacteremia after surgery
WO2017117995A1 (en) Chitosan-containing fibrin glue and use method thereof
Falcone et al. The effect of exogenous fibronectin on wound breaking strength
Pareti et al. Evaluation of the abnormal platelet function in von Willebrand disease by the blood filtration test
Allawi et al. Effect of homologous platelet rich fibrin matrix and injectable platelet rich fibrin on full thickness skin autograft healing in dogs
JPH07316066A (en) Wound healing agent
US6103691A (en) Utilisation of angiogenin and/or α1 -microglobulin and/or complement factor D in order to inhibit the secretion of proteins
Stemberger et al. Characterization of biomaterials for tissue repair
JPH0720873B2 (en) Wound healing agent
Rosenfeld Studies on normal human fibrinogen: the kinetics of its reaction with thrombin, its quantitative estimation, purification and stability

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA