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EP0537167A1 - Procede utilise pour recouvrir des surfaces de protheses avec des cellules de mammiferes - Google Patents

Procede utilise pour recouvrir des surfaces de protheses avec des cellules de mammiferes

Info

Publication number
EP0537167A1
EP0537167A1 EP91909071A EP91909071A EP0537167A1 EP 0537167 A1 EP0537167 A1 EP 0537167A1 EP 91909071 A EP91909071 A EP 91909071A EP 91909071 A EP91909071 A EP 91909071A EP 0537167 A1 EP0537167 A1 EP 0537167A1
Authority
EP
European Patent Office
Prior art keywords
prosthetic
cells
conduit
prosthetic surface
platelets
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP91909071A
Other languages
German (de)
English (en)
Other versions
EP0537167A4 (en
Inventor
David R. Knighton
Vance Fiegel
Marco Cavallini
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Curative Technologies Inc
Original Assignee
Curative Technologies Inc
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 Curative Technologies Inc filed Critical Curative Technologies Inc
Publication of EP0537167A1 publication Critical patent/EP0537167A1/fr
Publication of EP0537167A4 publication Critical patent/EP0537167A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A61F2/062—Apparatus for the production of blood vessels made from natural tissue or with layers of living cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/04—Metals or alloys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses

Definitions

  • This application is a continuation-in-part of U.S. Patent Application Serial No. 513,284 filed April 17, 1990.
  • This invention relates to methods of coating a prosthetic surface with mammalian cells.
  • the invention relates to methods for treating the prosthetic surface with a substance causing directed growth of endothelial cells, and contacting the treated prosthetic surface with a tissue or physiological fluid containing mammalian cells, such that the mammalian cells coat the prosthetic surface.
  • Patency refers to vascular grafts in which blood flow is unhindered either by solid masses formed from blood constituents (clots) or by tissue infiltration and hyperplasia from cellular and tissue constituents associated with the graft.
  • Endothelial cells are cells that line the cavities of the heart and the lumens (cavities or channels) within blood vessels, including capillaries. The presence of an endothelial lining produces a non-thrombogenic surface, which might be an ultimate requirement of a biomedically compatible and therefore biomedically useful vascular graft. Recent studies have shown the importance of chemotactic and mitogenic agents in regulating the activities of endothelial cells. Chemotaxis is the directed locomotion of cells along a chemical gradient. Mitogenic agents (mitogens) are those agents that cause cell populations to proliferate, i.e. agents that cause cell division. Chemotactic and/or mitogenic agents may be produced by, among other sources, blood platelets, macrophages, neural tissues, fibroblasts, and endothelial cells.
  • PDGF platelet-derived growth factor
  • the present invention provides a method of coating a prosthetic surface with mammalian cells, comprising placing against the prosthetic surface a composition comprising a substance that causes directed growth of endothelial cells.
  • the method further comprises contacting the prosthetic surface, having the composition placed against it, with tissue or a physiologic fluid containing mammalian cells, under suitable conditions for the mammalian cells to coat the prosthetic surface.
  • the substance may be an isolate of blood or a synthetic product having the bioactivity of an isolate of blood.
  • the substance further may be chemotactic for endothelial cells.
  • the endothelial cells may be capillary endothelial cells.
  • the isolate may comprise a platelet releasate product where the platelet releasate product constitutes the materials released by platelets in the platelet release reaction or a fraction of the materials released by platelets in the platelet release reaction.
  • the platelets may be activated by an activator such as thrombin, adenosine diphosphate, collagen, cell disruption such as freeze thaw or other known means of activation.
  • the composition which contains the product of platelet activation may be substantially free of blood or plasma contaminants or of platelet ghosts or other material found in platelets but not released by platelets upon activation.
  • the composition may contain an isolate of blood comprising a macrophage releasate product where the macrophage releasate product constitutes the materials released by macrophages in the macrophage release reaction or a fraction of the materials released by macrophages in the macrophage release reaction.
  • the substance may be produced by polypeptide synthesis, recombinant DNA techniques, or other well-known methods of synthesis.
  • the prosthetic surface may be porous, and may have pore sizes averaging in the range of about 0.1 to about 250 micrometers ( "um” or “u") .
  • a porous prosthetic surface may be composed of any known biomedically useful polymer or silicon-based material, which is illustrated by dacron, polytetrafluorethylene, polymers of lactide-glycolide, polyglactin, polydioxanone, polyurethane or silicon-based material.
  • the prosthetic surface may be nonporous and may be composed of any biomedically compatible nonporous material, which is illustrated by stainless steel, titanium, cobalt chrome alloys, silicon-based material, or any of the polymers listed above.
  • the prosthetic surface may be the surface of tissue, an organ, or an aggregate of animal cells or genetically altered cells, where the prosthetic member is a piece of tissue, an organ for transplantation or an aggregate of animal cells or genetically altered cells for cell implantation.
  • the prosthetic surface may be a container, such as a polymer, having inside such tissue, organ or cell implant.
  • Cell implants might be useful for treating a variety of conditions including diabetes, hemophilia, Parkinson's Disease, immune or blood disorders, etc.
  • the coating of such surfaces whether it be the tissue, organ, cell implant itself or a container having the same inside, may be useful to prevent macrophage or other degradation of such surface and also useful to induce vascularization of such tissue, organ or cell implant.
  • the prosthetic member may be a conduit where the prosthetic surface is the lumenal surface or the outer surface of the conduit.
  • the conduit may be a duct implant, as illustrated by urinary ducts, kidney tubules, lymphatic ducts, bile ducts, pancreatic ducts, indwelling catheter, shunts, drains, or other known biomedical or anatomical ducts.
  • the conduit may be a vascular implant.
  • the composition containing the substance causing directed growth of endothelial cells may be placed against the prosthetic surface by coating the prosthetic surface with the composition.
  • the composition may be placed against the prosthetic surface by impregnating or coating a mandril with the composition and by placing the impregnated or coated mandril against the prosthetic surface.
  • the prosthetic surface of the conduit may be contacted with tissue containing mammalian cells, which are illustrated by endothelial cells, smooth muscle cells, fibroblasts, or other cells known to be useful for maintaining the patency and/or clinical usefulness of duct or vascular implants.
  • tissue containing mammalian cells which are illustrated by endothelial cells, smooth muscle cells, fibroblasts, or other cells known to be useful for maintaining the patency and/or clinical usefulness of duct or vascular implants.
  • the prosthetic surface of the conduit may be contacted with such tissue by implanting the conduit into the retroperitoneal tissue or connective tissue of a mammal. If the conduit is a vascular implant, the conduit may, in addition, be contacted with such tissue by grafting the conduit to replace or bypass one or more blood vessels.
  • the prosthetic surface of the conduit may be contacted with physiological fluid containing mammalian cells such as endothelial cells, either iri vivo (within the body) or in_ vitro.
  • physiological fluid containing mammalian cells such as endothelial cells, either iri vivo (within the body) or in_ vitro.
  • the lumenal surface of the conduit may be contacted with the physiological fluid by immersing the conduit in the physiological fluid _in vitro or by passing the fluid through the lumen in. vitro.
  • the prosthetic surface may be the surface of a stent, artificial joint, urological implant, patch, web, or other known forms of prostheses.
  • the invention further comprises a coated prosthetic surface prepared as described above, and a method of treatment of mammals comprising implanting the prosthetic device.
  • Figure 1 shows a longitudinal sectional view of the relative arrangements of the Teflon sheath, Hydron solution, and PTFE tube in an assembled
  • Figure 2 shows a plot of 280 nm absorbance versus time for the chromatographic separation run in which fraction 1 was isolated
  • Figure 3 shows ability of PDGF-BB and PDGF-AB to induce chemotaxis in rabbit wound capillary endothelial cells.
  • Figure 4 shows binding of 125I-PDGF-BB to receptors of rabbit wound capillary endothelial cells.
  • Figure 5 shows the effect of PDGF-BB on rabbit wound capillary endothelial cells proliferation.
  • a method of coating a prosthetic surface with mammalian cells employing a composition comprising a substance causing directed growth of endothelial cells is provided.
  • the substance may be isolated from blood in the form of the product of platelet activation.
  • the substance may be the materials released by platelets ("platelet releasate product") in the platelet release reaction, as described in Knighton et al., Annals of Surgery 196:379-388 (1982), incorporated herein in its entirety by reference thereto, and as described below in Example 1.
  • the product of platelet activation may comprise a fraction of the materials obtained upon platelet activation.
  • the biological activities of platelet releasate product were measured in a corneal implant assay designed to reveal ability to cause directed growth of endothelial cells (Example 2), and an endothelial cell chemotaxis assay (Example 3). Platelet releasate product, whether the materials released by platelets in the platelet releasate reaction or a fraction thereof, displayed positive responses in these assays. Isolation of a platelet releasate product comprising a fraction of the materials released by platelets in the platelet release reaction (hereinafter "traction 1") is shown in Example 4.
  • Platelet releasate product was negative in a rabbit wound endothelial cell mitogenic assay, adapted with minor modifications from the assay described in Takahara et. al. , Cell 49:415-422 (1987). Proliferation of endothelial cells in response to platelet releasate product was measured in this assay by monitoring cell numbers. On the other hand, platelet releasate product is mitogenic for mouse 3T3 fibroblasts. The fibroblast mitogenic assay (FMA) is described in Example 5.
  • FMA fibroblast mitogenic assay
  • catheter sheaths were coated with each of the above-described fraction 1- or placebo-containing Hydron solutions.
  • the catheter sheaths also referred to herein as "mandrils", are manufactured of Teflon and are available from Jelco, Tampa, FL 33607.
  • the mandrils were coated by dipping in the appropriate fraction 1- or placebo-containing Hydron solution, dried two hours under vacuum, recoated with the same solution, and dried under vacuum overnight.
  • the sheaths were then placed within 2.5 cm long, 2.0 mm internal (lumenal) diamter polytetrafluoroethylene (PTFE) tubes (commercially available from Impra, Inc., empe AZ, or other suppliers).
  • the PTFE tubes have a pore size of approximately 90 u.
  • the animals were divided into three groups: A) a control group for the surgical technique (12 rats), B) a control group, placebo-treated for the study of the in vivo ingrowth of the endothelial cells (12 rats) and C) the study group given fraction 1 (12 rats) .
  • Control Group for the Surgical Technique (12 rats)
  • inferior vena cava utilized in the recipient animals as a by-pass procedure along the abdominal aorta.
  • the inferior vena cava was chosen to match the available PTFE grafts in length and diameter.
  • the vena cava was harvested by carefully dividing between ligatures all the branches from the origin of the iliac vessels to the confluence of the left renal vein.
  • the infrarenal abdominal aorta was prepared for end-to-side by-pass anastomoses with the harvested veins.
  • Each anastomosis was performed with a standard running technique, under dissecting scopy (magnification 6x) and with 10/0 monofilament sutures.
  • the aorta within the anastomoses was occluded by transverse small metallic clips.
  • the animals were evenly assigned to 6 implantation schedules (4 animals per schedule,
  • Teflon sheath and wire were iri situ revascularized end-to-side to the underlying abdominal aorta.
  • the technique was tne same as that utilized for the vein in the control group.
  • the aorta within the anastomoses was occluded by small metallic clips.
  • Platelet Release Reaction 60 ml whole- blood was aeseptically obtained from a source in 6 ml of acid citrate dextrose anti-coagulant (hereinafter ACD), or 1 ml ACD per 10 ml of whole blood.
  • ACD acid citrate dextrose anti-coagulant
  • the blood was mixed well with ACD by inverting and rolling the syringe. ⁇ nti-coagulated blood samples were kept on ice until used in further processing.
  • the anti-coagulated blood was transferred to two sterile, siliconized 50 ml conical-bottom centrifuge tubes, evenly splitting the sample between tubes. The tubes were then centrifuged at 135 x g for 20 minutes at about 4 C. Upon completion of the centrifugation cycle, the rotor was allowed to coast to a stop. No braking was applied. The uppermost laysr of the cenLriluged sample, pl ⁇ Lt-leL-ricii plasma (hereinafter PRP) , was carefully transferred with a sterile pipette to another sterile, siliconized centrifuge tube. Drawing only 4-5 ml at a time minimized losses due to red blood cell contamination of the PRP. A platelet count of the PRP was then conducted using methods well known in the art.
  • PRP pl ⁇ Lt-leL-ricii plasma
  • the PRP was centrifuged at 750 x g for 10 minutes at about 4°C. The supernatant was discarded, being careful not to dislodge the platelet pellet. Using a sterile pipette, the pellet was resuspended by aspirating and expelling into buffer containing 0.05 M HEPES (N-2-hydroxyethyl piperazine-n-2 ethane sulfonic acid), 0.03 M dextrose, 0.004 M KC1 , 0.1 M NaCl , pH adjusted to approximately 6.5 at 28 C (hereinafter platelet buffer) to an approximate concentration
  • HEPES N-2-hydroxyethyl piperazine-n-2 ethane sulfonic acid
  • 0.03 M dextrose 0.03 M dextrose
  • 0.004 M KC1 0.004 M KC1
  • 0.1 M NaCl pH adjusted to approximately 6.5 at 28 C
  • the resulting platelet suspension was then activated with purified thrombin.
  • thrombin Preferably, about 1 unit of thrombin per ml of platelet suspension was added to the platelet suspension and mixed.
  • the platelets and thrombin were allowed to incubate at room temperature for about 10 minutes. After incubation, the resulting platelet aggregate was broken up by aspirating and expelling the suspension with a sterile pipette.
  • the platelet suspension may be activated with other activators that cause the platelets to release their contents.
  • Other activators include collagen, preferably 6-100 ug of monomer collagen per ml of buffer containing 10% platelets, ADP, preferably 2-10 u molar in said buffer, epinephrine, preferably 25-450 u molar in said buffer, and arachidonic acid, preferably 35-50 u molar in said buffer.
  • PRP can be activated with thrombin or otherwise before centrifugation.
  • the resulting supernatant was centrifuged at 950 x g for about 5 minutes at about 4°C, thereby removing the released platelet ghosts and any fibrin contained in the suspension. The pellet formed by such centrifugation was discarded after the supernatant was extracted. After removal of the platelet ghosts and fibrin, the remaining supernatant constitutes platelet releasate in platelet buffer, herein designated platelet releasate product.
  • the extract is frozen in 4 ml aliquots for storage or immediately used.
  • Platelet releasate product also may be prepared from platelets obtained from a blood bank or other source. Pheresis platelet concentrate may be obtained from a blood bank and immediately processed. One unit of platelets will yield approximately 200 mis of PRP.
  • the concentrate may be processed to produce the activated platelet suspension in the same manner as the anti-coagulated patient blood sample is processed above, except that the first platelet pellet obtained from PRP is centrifuged three additional times at
  • the platelet suspension is activated as described above and centrifuged at 950 x g for 10 minutes at about 4 C.
  • the supernatant is extracted and centrifuged at 10,000 x g for 15 minutes at about 4 C to remove residual platelets and any fibrin.
  • the pellet is discarded after the supernatant is extracted.
  • the supernatant which is the platelet releasate product is frozen in 4 ml aliquots for storage or immediately used.
  • PRP produced from banked platelets can be directly activated before centrigugation.
  • polymer solution 1% v/v polyethyleneglycol in 70% v/v ethanol should be prepared (hereinafter polymer solution) .
  • Polymer solution is mixed 1:1 v/v with test sample.
  • a piece of plastic autoclave bag is taped onto a flat surface, making sure it is taut. The surface is then wiped off with an alcohol prep, and allowed to dry. 20 ul of the 1:1 mixture is dropped onto the plastic.
  • the polymer pellets are then dried under vacuum for approximately 2 hours, or until dry.
  • the corneal implant assay is conducted on a 4-6 lb. New Zealand White Rabbit.
  • Anesthetic is prepared by mixing 1:1 v/v Ketamine hydrochloride
  • Ketaset commercially available as Ketaset from
  • Anesthetic is injected into the gluteus maximum or gastrocnemeus using a 23 gauge needle, gently rubbing the area after injection.
  • the rabbit is properly anesthetized when it cannot resist being rolled onto its back, usually in 10-15 minutes.
  • the rabbit is placed on a sterile drape. 3-5 drops of proparacine hydrochloride 0.5%, commercially
  • a canal is gently made through the cornea towards the capillary bed, stopping approximately 2 mm from the capillary bed.
  • a "pocket” is made for the polymer pellet by moving the tip of a probe side to side, taking care not to move the probe forward as the pellet should not be closer than 1 mm to the capillary bed.
  • a polymer pellet is lifted off the plastic using forceps and placed on the eye at the point of incision. With a spatula, the pellet is pushed through the canal and into the pocket.
  • Neosporin Opthalmic Solution from Burroughs Wellcome Co., Research Triangle Park, NC 27709, are put into each eye to minimize the possibility of infection.
  • One rabbit is used for each sample to be tested (i.e., 2 pellets of same sample per rabbit, one in each eye). Eyes are checked on days 3, 5 and 7 for any direct growth of capillaries towards the pellet and graded according to the method of Gimbrone et al., J. Natl. Cancer Inst. 5_2_:413-427 (1974), and Banda et al. , U.S. Patent No. 4,503,038, both of which are incorporated in their entirety by reference. Pictures of eyes are taken on day 7 to record capillary growth.
  • Endothelial Cells may be prepared as described in copending application Serial No. 337,284 filed April 13, 1989, the disclosure of which is incorporated herein by reference.
  • Centrifuge cells at 1400 rpm (approx. 450g on Mistral 3000 centrifuge) for 10 minutes at R.T. or 1,000 rpm (approx. 250 g on Beckman GPR) .
  • Flask 2 0.2% LA-M199 HBSS EC2(1X) size (cm feed media (ml) wash (ml) (ml)
  • CLEANING CHAMBERS The following procedure is used to remove residual proteins, etc., from the chemotaxis chambers and gaskets. (Source: Terri Superdock, 118:61, 2/21/89) .
  • a platelet pheresis preparation was obtained from the University of Minnesota blood bank and processed substantially as described in Example 1. The centrifuged platelets were resuspended in platelet buffer at
  • the chromatographic column was a Mono-Q anion exchange column, 1.0 cm (ID) X 5.0 cm (available from Pharmacia Co., Piscataway, NJ, serial number 7393041).
  • the protein concentration of the releasate solution was estimated from the 280 n U.V. absorbance, and 5-10 mg of total protein was loaded on the column.
  • the column was run as follows:
  • the flow rate for time intervals 1, 2, 3, 4 and 6 was 1.0 ml/min.
  • the flow rate for time interval 5 was 0.5 ml/min.
  • the chromatographic system was a Beckman System Gold (modified), with individual components as follows: a) Pump: Beckman Model 126, b) Detector: Beckman Model 167, variable wavelength, U.V./visible, c) Controlling computer: IBM Model PS 2/50, d) Printer: Epson Model 100, e) Fraction collector: Gilson Model 201.
  • the absorbance at 280 nM U.V. of the fractions collected from the column is shown in Fig. 2. Fractions collected in the time interval of 32-39 min. were tested in the chemotaxis assay described in Example 3, above. Results were as follows: 29
  • MEDIA CHANGE/DAY 3 Three days after initiating the microtiter plates, the media needs to be changed to 0.8% NCS/DMEM to continue. 1. Examine the plates under the microscope to determine if the fibroblasts have grown to confluency. (There should be no gaps between cells). If cells are confluent, continue. If cells 31
  • the FMA microtiter plates are labeled with radioactive thymidine to demonstrate mitogenic activity.
  • TCA trichloroacetic acid
  • Vials are capped tightly and shaken back and forth vigorously a few times to expose filter completely to the cocktail and disclodge, potential air bubbles.
  • Program No. 1 is programmed as follows: B-LS1701 B-2800
  • Units of l/ED-50 represents the dilution of the platelet releasate sample which results in a 50% stimulation of mitrogenic activity in fibroblast 3T3 cells. For example, if a 0.25 to 1:4 dilution of the sample gave 50% stimulation, the l/ED-50 would be 4 units. Similarly, a 1:8 dilution would give an l/ED-50 of 8 units. DESCRIPTION OF ALTERNATIVE EMBODIMENTS
  • the substance causing directed growth may be derived from other means of platelet activation such as platelet disruption involving freeze/thaw or heat treatment of platelets or may be derived from any isolate of blood, or any other material, which causes directed growth of endothelial cells.
  • the substance may be a synthetic product having the bioactivity of an isolate of blood.
  • Platelet derived growth factor is a cationic glycoprotein of about 32,000 molecular weight sourced from platelets by release reaction or freeze/thaw or heat treatment. PDGF may also be isolated from multiple tissues and cultured cells, including macrophages, smooth muscle cells, tumor cells and endothelial cells.
  • the predominant form of PDGF spcr -t-ed by platelets ia a hetercdimer consisting of an A chain and a B chain, and a less dominant form (about 20%) being the homodimer of the B chain.
  • the following examples will show that the homodimer of B chain (“PDGF-BB”) is chemotactic and nonmitogenic 35
  • Recombinant human PDGF-BB ( "rhPDGF-BB” ) was obtained commercially (Upstate Biotechnology, Inc., Lake Placid, NY 12946, Catalog #01-105: rhPDGF-BB expressed in S ⁇ cerevisiae) and recombinant human PDGF-AB ( "rhPDGF-AB” ) was also obtained commercially from the same source (Catalog #01-109: rhPDGF-AB expressed in E ⁇ coli) .
  • rhPDGF-BB and rhPDGF-AB obtained in lyophilized form was rehydrated to 5.0 ug/ml in PBS (containing 1.0 mg/ml HSA) . The rehydrated dimers are stored at -20° C until use.
  • Rehydrated rhPDGF-BB and rhPDGF-AB were assayed according to the endothelial cell chemotaxis assay of Example 3.
  • Figure 3 shows the concentration in ng/ml of rhPDGF-BB and rhPDGF-AB respectively, the rehydrated dimers being further diluted in Ml 99 containing 0.2% by volume lactalbumin.
  • rhPDGF-BB induces chemotaxis of rabbit wound capillary endothelial cells (RWCEC), while rhPDGF-AB marginally induces such chemotaxis.
  • RWCEC rabbit wound capillary endothelial cells
  • RWCEC Rabbit wound capillary endothelial cells
  • FBS fetal bovine serum
  • the cells are washed, 1 ml per well, with phosphate-buffered saline (#450-1300, Gibco Laboratories, Grand Island, NY) containing 1.0 mg/ml human serum albumin (NDC 0944-0490-01, Baxter Healthcare Corp., Glendale, CA) (PBS-HSA) cooled to 4° C and all subsequent operations are performed with precooled reagents and at 4 C.
  • PBS-HSA human serum albumin
  • the 24-well dish is placed onto a rocker platform for 30 minutes. After 30 minutes, the cells are washed again with PBS-HSA and incubated an additional 30 minutes. Following the wash step, 200 ul of PBS-HSA is added to each well along with 25 ul of the appropriate dilution of 125I-PDGF-BB (#IM.213, Amersham Corp.,
  • PDGF-BB for determination of nonspecific binding, 37 while the other wells will receive the material being tested for competitive activity (in duplicate) such as PDGF-AA (#01-109, Upstate Biotechnologies, Inc., Lake Placid, NY) or PDGF-AB (#01.-110, Upstate Biotechnologies, Inc., Lake Placid, NY).
  • the dishes are then incubated for 1 hour at 4 C on the rocker platform with constant rocking. Following the incubation each well is washed 3 times with 1.0 ml of cold PBS-HSA and the cell associated radioactivity is extracted by the addition of 250 ul of 0.5% by volume.
  • Triton X-100 (#X-100, Sigma Chemical Co., St. Louis, MO) in PBS.
  • the plates are allowed to sit overnight at room temperature and the radioactivity of each sample is determined in a gamma counter (Gamma 5500, Beckman Instruments, Inc., Irvine, CA) .
  • the dishes are thoroughly mixed and 100 ul of each well is counted and the total multiplied by 2.5 to get the total amount of radioactivity per well.
  • the specific binding is determined by subtracting the mean of the duplicates of the samples containing 125I-PDGF-BB + a 100 fold excess of PDGF-BB from the mean of the duplicates of the samples containing only I-PDGF-BB for each concentration of 125 I-PDGF-BB tested. If performing Scatchard plots, the number of cells per well is determined by counting four replicate wells in the same dish. The cells are removed with trypsin-EDTA (per the RWCEC proliferation assay) and enumerated in a hemocytometer as described in the RWCEC culture procedure.
  • the Kd (dissociation constant) of the receptor and the receptor number per cell can be calculated by the method of Scatchard (Scatchard, G., The Attraction of Proteins For Small Molecules an Ions, ?> ⁇ .r.. NY Acad. Sci. 51:660-672 (1949)).
  • Figure 4 shows the presence of a high affinity receptor for PDGF-BB with a Kd of 0.1-0.3 nM and a receptor number of 38
  • RWCEC Media 199
  • FBS fetal bovine serum
  • the cells are cultured at 37 degrees C in a 5% CO- atmosphere.
  • the next day (Day 1) the media in three wells is replaced with 1 ml of M199 containing 2.5% by volume FBS and three wells receive 1 ml of M199 containing 10% by volume FBS. The remaining
  • Trypsin/EDTA trypsin/0.53 mM ethylenediaminetetraacetic acid
  • Trpsin/EDTA trypsin/0.53 mM ethylenediaminetetraacetic acid
  • Figure 5 shows that rhPDGF-BB is not mitogenic as measured by the above assay for rabbit wound capillary endothelial cells. Concentrations were prepared with rehydrated dimers in M199 containing 2.5% by volume FBS.
  • Example 9 rhPDGF-BB was bioassayed according to the corneal implant assay of Example 2. The following results show significant angiogenesis for rhPDGF-BB. Concentrations were prepared with rehydrated dimers in PBS (containing 1 mg/ml HSA) .
  • the substance may comprise materials released from macrophages or a fraction of the materials thereof.
  • Macrophages undergo a "macrophage release reaction" in the sense that, under some conditions (for example, stimulation by endotoxin) , macrophages release materials capable of causing growth of endothelial cells and blood vessels Ln_ vivo and in_ vitro, as disclosed in Thakral et. al., J. Surgical Research 26:430-436 (1979), which is herein incorporated in its entirety by reference thereto.
  • the prosthetic surface may, depending on the material and the desired use, exhibit pore sizes other than approximately 90 um. For example, pore sizes averaging in the range of about 0.1 um to about 250 um, as well as other pore sizes compatible with the materials used and the intended use, may be employed.
  • porous prosthetic surfaces may be composed of any known biomedically useful polymer, such as dacron, polymers of lactide-glycolide, polyglactin, polydioxanone, or polyurethane. Porous silicon-based materials may also be used for prosthetic surfaces. Other biomedically useful materials for porous prosthetic surfaces will be known to those skilled in the art. Prosthetic surfaces also may be fashioned from any biomedically compatible (i.e., tolerated by the body without clinically unacceptable adverse effects) nonporous material, such as stainless steel, titanium, cobalt chrome alloys, silicon-based materials, and other known materials.
  • the prosthetic surface could be the lumenal surface or the outer surface of a conduit-type prosthetic member, depending on the intended use.
  • the conduit prosthetic member could be a vascular implant, as described in the preferred embodiment.
  • the conduit prosthetic member could be a duct-type implant, as illustrated by urinary ducts, kidney tubules, lymphatic ducts, bile ducts, pancreatic ducts, indwelling catheters, shunts, drains, or other known biomedical or anatomical ducts.
  • the prosthetic surface may be the surface of a stent, artificial joint, urological implant, patch, web, or other known forms of prostheses.
  • the composition may comprise the substance causing directed growth, plus a Hydron/polyethyleneglycol carrier.
  • a Hydron/polyethyleneglycol carrier may comprise the substance causing directed growth, plus a Hydron/polyethyleneglycol carrier.
  • Hydron/polyethyleneglycol will be known to those skilled in the art, depending on the intended use.
  • Useful carriers other than Hydron and/or polyethyleneglycol will also be known to those skilled in the art.
  • the composition may be used to coat a mandril or other object, which is then placed against the prosthetic surface as described in the preferred embodiment.
  • the mandril or other object may be impregnated with the composition (for example, by soaking an absorbent mandril in the composition), or the prosthetic surface itself may be coated with the composition.
  • the prosthetic surface in contact with the composition may be placed in an environment containing mammalian cells by implantation into retroperitoneal tissue of a rat, as described in the preferred embodiment.
  • the prosthetic surface may be implanted into other appropriate mammalian tissues.
  • the prosthetic surface may be contacted with physiological fluid such as various body fluids in vivo or such 42 as various formulations of cell culture media (iri vitro contact) known to those skilled in the art.
  • a conduit prosthetic member could be contacted with the physiological fluid by immersing the conduit in the fluid or by passing the fluid through the lumen of the conduit, _in. vivo or in. vitro.
  • the mammalian cells within the tissue or physiological fluid may be any cells useful for maintaining the patency and/or clinical usefulness of duct or vascular implants. These could include, without limitation, endothelial cells, smooth muscle cells, and/or fibroblasts.
  • the prosthetic surface is brought into contact with the composition, then contacted with mammalian tissue or physiological fluid containing mammalian cells that will coat the prosthetic surface. It is at that point that the prosthetic member could be grafted or implanted to its final location as a functioning prosthetic device. In an alternative embodiment, the prosthetic member, with the prosthetic surface in contact with the composition, could be grafted or implanted directly to its final location, where mammalian cells inherently in the vicinity of the final location would be induced to coat the prosthetic surface.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Inorganic Chemistry (AREA)
  • Pulmonology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Prostheses (AREA)
  • Materials For Medical Uses (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Un procédé utilisé pour recouvrir une surface de prothèse avec des cellules de mammifères consiste à placer contre la surface de prothèse une composition comprenant une substance qui provoque la croissance de cellules endothéliales, et à mettre la surface de prothèse contre laquelle est placée ladite composition, en contact avec du tissu ou un liquide physiologique, lesquels contiennent des cellules de mammifères, dans des conditions adaptées pour que les cellules de mammifères recouvrent la surface de prothèse. Cette invention concerne aussi une prothèse dont la surface est recouverte de cellules de mammifères comme décrit ci-dessus, ainsi qu'un procédé de traitement des mammifères par implantation de la prothèse décrite ci-dessus.
EP19910909071 1990-04-17 1991-04-16 Coating prosthetic surfaces with mammalian cells Withdrawn EP0537167A4 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US51328490A 1990-04-17 1990-04-17
US513284 1990-04-17
US65056191A 1991-02-01 1991-02-01
US650561 1991-02-01

Publications (2)

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EP0537167A1 true EP0537167A1 (fr) 1993-04-21
EP0537167A4 EP0537167A4 (en) 1993-04-28

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EP19910909071 Withdrawn EP0537167A4 (en) 1990-04-17 1991-04-16 Coating prosthetic surfaces with mammalian cells

Country Status (7)

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EP (1) EP0537167A4 (fr)
AU (1) AU656725B2 (fr)
CA (1) CA2082176A1 (fr)
FI (1) FI925083A0 (fr)
IL (1) IL97896A (fr)
NZ (1) NZ237832A (fr)
WO (1) WO1991016009A1 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5192312A (en) * 1991-03-05 1993-03-09 Colorado State University Research Foundation Treated tissue for implantation and methods of treatment and use
GB9116036D0 (en) * 1991-07-25 1991-09-11 Univ Leicester Preparing grafts for implantation
US5272074A (en) * 1992-04-23 1993-12-21 Mcmaster University Fibrin coated polymer surfaces
AU665813B2 (en) * 1992-05-11 1996-01-18 Sulzer Medizinaltechnik Ag Process and apparatus for producing endoprostheses
EP1452153A1 (fr) * 1994-03-14 2004-09-01 Cryolife, Inc Tissu traité pour l'implantation et une méthode de préparation
FR2722974B1 (fr) * 1994-07-29 1997-04-25 Marie Therese Zabot Procede de modification de la surface interne des protheses synthetiques utilisees en chirurgie vasculaire
US5609631A (en) * 1995-04-28 1997-03-11 Rubens; Fraser D. Fibrin D-domain multimer coated prostheses and methods for their production
GB2337255B (en) * 1995-08-03 2000-03-15 Secr Defence Biomaterial
AT407484B (de) 1997-11-12 2001-03-26 Bio Prod & Bio Eng Ag Arzneimittel zur förderung der wundheilung
WO2002058718A2 (fr) * 2001-01-26 2002-08-01 Genetix Pharmaceuticals, Inc. Methodes et compositions permettant de stimuler l'angiogenese
DE10138564B4 (de) * 2001-08-06 2007-07-12 Lamm, Peter Wilhelm, Dr. Verfahren zur Devitalisierung natürlicher Organe und/oder zur Bereitstellung extrazellulärer Matrices zum "Tissue-Engineering"
US9393267B2 (en) * 2009-02-19 2016-07-19 University Of Southern California Gel delivery system for tissue repair

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4546500A (en) * 1981-05-08 1985-10-15 Massachusetts Institute Of Technology Fabrication of living blood vessels and glandular tissues
US4589881A (en) * 1982-08-04 1986-05-20 La Jolla Cancer Research Foundation Polypeptide
DE3521684A1 (de) * 1985-06-18 1986-12-18 Dr. Müller-Lierheim KG, Biologische Laboratorien, 8033 Planegg Verfahren zur beschichtung von polymeren
US5108923A (en) * 1986-04-25 1992-04-28 Collaborative Research, Inc. Bioadhesives for cell and tissue adhesion
CA1340581C (fr) * 1986-11-20 1999-06-08 Joseph P. Vacanti Neomorphogenese chimerique d'organes par implatation cellulaire controlee, utilisant des matrices artificielles

Also Published As

Publication number Publication date
WO1991016009A1 (fr) 1991-10-31
IL97896A0 (en) 1992-06-21
EP0537167A4 (en) 1993-04-28
FI925083A7 (fi) 1992-11-09
IL97896A (en) 1995-05-26
AU656725B2 (en) 1995-02-16
AU7792591A (en) 1991-11-11
CA2082176A1 (fr) 1991-10-18
NZ237832A (en) 1994-05-26
FI925083A0 (fi) 1992-11-09

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