WO2009020650A2 - Materials and methods for treating and managing wounds and the effects of trauma - Google Patents

Materials and methods for treating and managing wounds and the effects of trauma

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Publication number
WO2009020650A2
WO2009020650A2 PCT/US2008/009540 US2008009540W WO2009020650A2 WO 2009020650 A2 WO2009020650 A2 WO 2009020650A2 US 2008009540 W US2008009540 W US 2008009540W WO 2009020650 A2 WO2009020650 A2 WO 2009020650A2
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Prior art keywords
cells
material
injury
traumatic
implantable
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PCT/US2008/009540
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French (fr)
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WO2009020650A3 (en )
Inventor
Helen Marie Nugent
Stephen A. Bollinger
Elazer R. Edelman
Shai Schubert
Yin Shan Ng
Tham Sjin Robert Tjin
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Pervasis Therapeutics, Inc.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L24/00Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0005Ingredients of undetermined constitution or reaction products thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L24/00Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001Use of materials characterised by their function or physical properties
    • A61L24/0015Medicaments; Biocides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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
    • A61L27/3804Materials 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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3808Endothelial cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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
    • A61L27/3804Materials 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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813Epithelial cells, e.g. keratinocytes, urothelial cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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
    • A61L27/3886Materials 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 comprising two or more cell types
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54Biologically active materials, e.g. therapeutic substances
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/25Peptides having up to 20 amino acids in a defined sequence
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404Biocides, antimicrobial agents, antiseptic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/41Anti-inflammatory agents, e.g. NSAIDs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/45Mixtures of two or more drugs, e.g. synergistic mixtures
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2400/00Materials characterised by their function or physical properties
    • A61L2400/06Flowable or injectable implant compositions

Abstract

Disclosed herein are materials and methods suitable for treating traumatic injury sites resulting from wounds, surgery or other tissue disruption. Traumatic injury sites can be treated by contacting a surface of a traumatic injury at or adjacent or in the vicinity of an area of injury or damage with an implantable material. The implantable material comprises a biocompatible matrix and cells and is in an amount effective to treat the traumatic injury site. A composition comprising a biocompatible matrix and cells engrafted therein or thereon can be used to treat the traumatic injury site. The composition can be a flexible planar material or a flowable composition.

Description

MATERIALS AND METHODS FOR TREATING AND MANAGING WOUNDS AND THE EFFECTS OF TRAUMA

Background of the Invention

[0001] Trauma includes wounds, injuries or disorders caused by an externally applied physical force. Exemplary traumas include changes to tissues, cells, organs or other body parts resulting from controlled purposeful interventions such as surgical manipulation, incision or other reparative procedures, or from unintended physical force, for example, from an injury. Examples of injuries resulting in trauma include surgical interventions, incisions, motor vehicle accidents, falls, drowning, gunshots, fires, burns, stabbing and other physical assault. Trauma kills more people between the ages of 1 and 44 than any other disease or illness. More than 100,000 people in the United States of all ages die from trauma each year.

[0002] Each year in the United States, 1.1 million burn injuries require medical attention. Approximately 45,000 of these require hospitalization, and roughly half those burn patients are admitted to a specialized burn unit. Each year, approximately 4,500 of these people die. Further, up to 10,000 people in the United States die each year of burn-related infections, most frequently from burn-related pneumonia.

[0003] Trauma-induced changes to tissues, cells, organs or other body parts resulting from controlled or unintended forces can further induce trauma-associated disease conditions, including, for example, ulceration, diabetic ulcers, pressure ulcers, venous ulcers, fibrosis, chronic obstructive pulmonary disease and colitis. Complications following initial traumatic injuries may occur long after the initial incident, often when the patient is in an intensive care unit (ICU). Many ICU patients face similar medical problems regardless of the reason for their admission to the unit. The leading cause of death in ICUs are multiple organ system dysfunction or multiple organ failure, in which several of the body's organs fail at once, and adult respiratory distress syndrome, in which the lungs in particular fail. In both multiple organ system dysfunction and multiple organ failure conditions, the organs of the body are ravaged by the patient's own immune system, leading to severe, debilitating, and uncontrolled inflammation. [0004] Current treatments for treating surgical interventions, traumatic injuries, burns and other wounds are limited and often have adverse consequences. Treatment options vary with age, health, and the severity of the injury or wound. One objective of the present invention is to provide methods and materials for the treatment of traumatic injuries resulting from externally applied physical forces including surgical interventions, burns and other wounds, to provide an adjunct therapy to traditional surgical or trauma interventions to promote repair and regeneration of injured tissue and to enhance the quality of life for surgery, trauma and burn patients.

Summary of the Invention

[0005] The present invention exploits the discovery that traumatic injuries, including controlled and unintended injuries such as open and closed wounds, surgical manipulation, incision or other reparative procedures and burns can be treated effectively by administration of a cell-based therapy to the site of a traumatic injury, including a site of a wound, surgical incision or burn. As disclosed herein, an implantable material comprising cells, preferably endothelial cells, epithelial cells or cells having an endothelial-like or epithelial-like phenotype, can be used to treat and manage wounds, surgical incisions, burns and other traumatic injuries when the material is situated at or near a wound, surgical incision, burn or other traumatic injury. This discovery permits the clinician to intervene in the treatment of a wound, surgical incision, burn or other traumatic injury for which there have heretofore been limited treatment options. Specifically, the present invention provides an adjunct therapy to traditional trauma and surgical interventions to promote repair and regeneration of injured tissue. [0006] In one aspect, the invention is a method of treating a traumatic injury in an individual in need thereof. The method comprises contacting with an implantable material a traumatic injury at, adjacent to or in the vicinity of an area of damage or injury, wherein said implantable material comprises a biocompatible matrix and cells and further wherein said implantable material is in an amount effective to treat the traumatic injury in said individual. [0007] In various embodiments, the biocompatible matrix is a flexible planar material or a flowable composition. The cells can be endothelial cells, epithelial cells, endothelial-like cells, epithelial-like cells, non-endothelial cells or non- epithelial cells, stem cells, endothelial progenitor cells or analogs thereof. The cells can be a co-culture of at least two different cell types. The implantable material can further include a tissue sealant. The implantable material can be applied to a surface of the traumatic injury.

[0008] In additional embodiments, the traumatic injury is an open or closed wound selected from the group comprising an incision, an incised wound, a laceration, an abrasion, a puncture wound, a penetration wound, a deep wound, a gunshot wound, a contusion, a hematoma or a crushing injury. The traumatic injury can be a burn, a diabetic ulcer, a pressure ulcer or a venous ulcer. The traumatic injury can be a pathological response to a primary traumatic injury and the pathological response can be fibrosis, a stricture, an adhesion, a contracture, a keloid, or a hypertrophic scar.

[0009] In certain embodiments, the implantable material regulates inflammation, smooth muscle cell proliferation and/or migration, fibroblast proliferation and/or migration, keratinocyte proliferation and/or migration, collagen deposition and/or accumulation, tissue remodeling, scar formation, re- epithelialization, neovascularization, extracellular matrix formation and/or degradation, reduces the incidence of dehiscence, reduces pain associated with traumatic injury, or reduces healing time of the traumatic injury.

[0010] In another aspect, the invention is a method of providing an adjunct therapy to a primary therapeutic intervention of a traumatic injury in an individual in need thereof. The method includes contacting with an implantable material a site at, adjacent to or in the vicinity of an area of a primary therapeutic intervention, wherein the implantable material includes a biocompatible matrix and cells and further wherein the implantable material is in an amount effective to treat the primary therapeutic intervention in the individual. According to various embodiments, the primary therapeutic intervention is suturing, stapling, ablation or debridement. [0011] In a further aspect, the invention is a composition suitable for the treatment or management of a traumatic injury. The composition includes a biocompatible matrix and cells. The composition is in an amount effective to treat or manage the traumatic injury.

[0012] In various embodiments, the biocompatible matrix is a flexible planar material or a flowable composition. The flowable composition can further include an attachment peptide wherein the cells are engrafted on or to the attachment peptide. The cells can be endothelial cells, epithelial cells, endothelial-like cells, epithelial-like cells, non-endothelial cells or non-epithelial cells or analogs thereof. The cells can be a co-culture of at least two different cell types. The implantable material can further include a tissue sealant, a second therapeutic agent, an agent that inhibits infection or an anti-inflammatory agent.

Brief Description of the Drawings

[0013] Figures IA and IB are representative cell growth curves according to an illustrative embodiment of the invention.

[0014] Figures 2A and 2B are representative graphs depicting data of fibroblasts treated according to the fibroblast migration assay according to an illustrative embodiment of the invention.

[0015] Figure 3 is a representative graph depicting wound healing data obtained from wounds treated according to an illustrative wound healing assay according to an embodiment of the invention.

[0016] Figure 4 is a representative graph depicting the percentage of open area present in the scratch area at 0 hours and at 24 hours for three different endothelial cell donor strains according to an illustrative smooth muscle cell migration assay according to an illustrative embodiment of the invention.

Detailed Description of the Invention

[0017] As explained herein, the invention is based on the discovery that a cell- based therapy can be used to treat, ameliorate, manage and/or reduce the effects of acute and chronic wounds, burns and other traumatic injuries and/or as an adjunct therapy to treat the effects of traditional therapies used to treat such traumatic injuries, including surgery, suturing, ablation, debridement, surgical closure devices and other critical care therapies. The cell-based therapy also can be used to regulate the stages of traumatic injury healing, including hemostasis, inflammation, cell adhesion, migration and transformation, proliferation and remodeling of injured tissues, to reduce the pathological response to tissue injury, and to regulate clinical sequelae associated with traumatic injury healing, including regulation of pain, wound closure and integrity, healing time and scar formation. The teachings presented below provide sufficient guidance to make and use the materials and methods of the present invention, and further provide sufficient guidance to identify suitable criteria and subjects for testing, measuring, and monitoring the performance of the materials and methods of the present invention.

[0018] As used herein, trauma and/or traumatic injuries include any external stimulus resulting in external or internal injury or damage to a subject. Trauma and/or traumatic injuries include changes to tissues, cells, organs or other body parts resulting from controlled purposeful interventions such as surgical manipulation, incision or other reparative procedures, or from unintended physical force such as a blunt force. Trauma and/or traumatic injuries also specifically include minor and severe wounds such as open wounds and closed wounds. Open wounds include, for example, incisions or incised wounds, lacerations, abrasions, puncture wounds, penetration wounds and gunshot wounds. Closed wounds include, for example, contusions, hematoma and crushing injuries. Traumatic injuries further include secondary traumatic injuries such as damage and diseases resulting from a primary traumatic injury. Exemplary secondary traumatic damage and disease include ulceration, fibrosis, chronic obstructive pulmonary disease, colitis, multiple organ system dysfunction and multiple organ failure.

[0019] When used in an effective amount, the cell-based therapy of the present invention, an implantable material comprising cells engrafted on, in and/or within a biocompatible matrix and having a preferred phenotype, produces factors positively associated with traumatic injury healing, including factors that control or regulate hemostasis, inflammation, proliferation and remodeling of the tissues at the traumatic injury site. For example, when used in an effective amount, the cells of the implantable material, when engrafted in or within a biocompatible matrix and having a preferred phenotype, can produce quantifiable amounts of heparan sulfate (HS), heparan sulfate proteoglycans (HSPGs), nitric oxide (NO), transforming growth factor-beta (TGF-β), fibroblast growth factors (FGFs) including fibroblast growth factor 2 (FGF2), matrix metal loproteinases (MMPs) and/or tissue inhibitors of matrix metalloproteinases (TIMPs) and vascular endothelial growth factor (VEGF).

[0020] For example, heparan sulfate (HS) regulates the proliferation and migration of smooth muscle cells and fibroblasts. Unregulated proliferation and migration of smooth muscle cell and fibroblast contribute to scar formation and/or delayed healing.

[0021] Additionally, nitric oxide (NO) is critical to wound collagen accumulation and acquisition of wound mechanical strength. NO regulates the proliferation and migration of fibroblasts, controls collagen synthesis in wound fibroblasts and controls the formation of new stroma during the proliferation and remodeling phases of traumatic injury repair. NO also regulates the proliferation and migration of smooth muscle cells. Unregulated proliferation and migration of smooth muscle cell and fibroblast contribute to scar formation and/or delayed healing. NO further regulates the recruitment and activity of macrophages, controlling the early inflammatory phase of traumatic injury repair. Additionally, NO regulates the proliferation and migration of keratinocytes, for example, at wound margins.

[0022] Transforming growth factor-beta (TGF-β) is involved in regulating various phases of traumatic injury healing including inflammation, re- epithelial ization, angiogenesis and the production of extracellular matrix during the remodeling phase of wound healing. TGF-β, when provided locally or systemically in chronic or impaired wounds, regulates healing of the traumatic injury site. Specifically, TGF-β is involved in the control of the proliferation and migration of fibroblasts and the regulation of the production of extracellular matrix by regulating the activity of various collagen gene promoters. TGF-β is also involved in the regulation of the degradation of extracellular matrix by regulating the expression, activity and relative levels of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). TGF- β also regulates the proliferation and migration of smooth muscle cells. Unregulated proliferation and migration of smooth muscle cells and fibroblasts contribute to scar formation and/or delayed healing. TGF-β is also involved in the chronic, maturation phase of wound healing, regulating the production of glycosaminoglycans, fibronectin and collagen and the expression of matrix-degrading enzymes.

[0023] Fibroblast growth factor 2 (FGF2) is involved in the regulation of angiogenesis and mitosis of fibroblasts, which, when their activity is regulated, control the level of collagen deposition in the dermis. FGF2 is also mitogenic for keratinocytes, regulating the proliferation and migration of keratinocytes into the traumatic injury site.

[0024] Additionally, vascular endothelial growth factor (VEGF) is involved in traumatic injury healing and attracts circulating neutrophils and monocytes to the traumatic injury site to regulate the inflammatory response. VEGF controls the permeability of blood vessels at the traumatic injury site and regulates endothelial cell proliferation and migration. In addition to regulating angiogenesis, VEGF also plays a role in vasculogenesis by recruiting endothelial progenitor cells from the bone marrow for endothelial vessel formation. Further, VEGF regulates the activity of pericytes to coat and stabilize the vasculature.

[0025] Accordingly, administration of an effective amount of the implantable material of the present invention can be used either alone or as an adjunct to surgical or critical care interventions to treat, ameliorate, manage and/or reduce the effects of wounds, surgical incisions, burns and other traumatic injuries or the traditional therapies used to treat such traumatic injuries, for example, suturing, ablation, surgical closure devices or debridement, by providing a targeted supply of therapeutic factors in vivo in an amount sufficient to induce and/or manage, for example, hemostasis, inflammation, proliferation and remodeling of injured tissues. [0026] Traumatic injury healing is a complex and dynamic biological process that results in the restoration of anatomical continuity and function following damage or injury to tissue. There are four basic responses that generally occur following an injury: hemostasis, inflammation, proliferation and remodeling.

[0027] The normal or controlled traumatic injury repair response experienced by most patients following injury is characterized by a healing response resulting in scar formation and/or ongoing scar remodeling. The pathological response to tissue injury, on the other hand, stands in sharp contrast to the normal repair response. In the pathological response, excessive healing, characterized by excessive deposition of collagen and other connective tissues results in an altered anatomical or tissue structure and possibly loss of function at the traumatic injury site or in the vicinity of the traumatic injury site. Exemplary excessive healing conditions include but are not limited to fibrosis, strictures, adhesions and contractures. Fibrosis further includes the conditions of keloids and hypertrophic scars in the skin. Contraction is a normal part of the healing process. However, if excessive, contraction can become pathologic, forming a structure known as a contracture. Deficient healing, in contrast to the characteristics of excessive healing present in, for example, fibrosis, is characterized by an insufficient deposition of connective tissue matrix and results in a tissue weakened to the point where it is subject to possible dehiscence or rupture. Failure to heal, malapposition of edges and chronic non-healing ulcers are additional examples of deficient healing. [0028] Dermal wound healing is characterized by three responses: connective tissue matrix deposition, contraction and epithelialization. Simple wounds can be closed by suture, tape or staples and can heal by primary intention. The main mechanism of healing during primary intention is connective tissue matrix deposition characterized by collagen, proteoglycan and attachment protein deposition to form a new extracellular matrix. In contrast, wounds that remain open heal mainly by contraction; the interaction between cells and matrix results in movement of new tissue towards the center of the wound. The underlying mechanisms responsible for contraction are not fully understood, but there appears to be a complex interaction between contractile fibroblasts sometimes referred to as myofibroblasts and the matrix components. Some work has indicated that nerve growth factor and IL-8 can modulate the contraction response. Additionally, during epithelialization, epithelial cells around the margin of the contracting wound or in residual skin appendages such as hair follicles and sebaceous glands lose contact inhibition and begin to migrate into the wound area. As migration proceeds, cells in the basal layers of the wound site begin to proliferate to provide additional epithelial cells at the site of contraction. [0029] Acute traumatic injuries normally heal in a very orderly and efficient manner characterized by four distinct, but overlapping phases: hemostasis, inflammation, proliferation and remodeling. The normal healing response begins the moment the tissue is injured. As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β). Following hemostasis, neutrophils enter the traumatic injury site and begin the critical task of phagocytosis to remove foreign materials, bacteria and damaged tissue. As part of the inflammatory phase, macrophages appear and continue the process of phagocytosis and release additional PDGF and TGF-β. Once the remaining foreign material, bacteria and damaged tissue is removed from the traumatic injury site, fibroblasts migrate into the traumatic injury site to begin the proliferative phase, characterized by deposition of new extracellular matrix. The new collagen matrix then becomes cross-linked and organized during the final remodeling phase.

[0030] The healing cascade begins immediately following injury when the platelets come into contact with exposed collagen. As platelet aggregation proceeds, clotting factors are released resulting in the deposition of a fibrin clot, comprised of cross-linked fibrin, and also fibronectin, vitronectin and thrombospondin, at the site of injury. The fibrin clot serves as a provisional matrix and sets the stage for the subsequent events of healing. Platelets not only release the clotting factors needed to control the bleeding and loss of fluid and electrolytes, but they also provide a cascade of chemical signals, known as cytokines or growth factors, that initiate the healing response. The two most important signals are PDGF and TGF-β. [0031] Platelet-derived growth factor initiates the chemotaxis of neutrophils, macrophages, smooth muscle cells and fibroblasts and also stimulates the mitogenesis of the fibroblasts and smooth muscle cells. TGF-β attracts macrophages and stimulates the macrophages to secrete additional cytokines including fibroblast growth factor (FGF), PDGF, tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-I). In addition, TGF-β further enhances fibroblast and smooth muscle cell chemotaxis and modulates collagen and collagenase expression. The net result of these redundant signals is a vigorous response of the matrix producing cells to ensure a rapid deposition of new connective tissue at the injury site during the proliferative phase that follows the inflammatory phase.

[0032] Neutrophils are the next predominant cell marker in the traumatic injury site within twenty-four hours after injury. The major function of the neutrophil is to remove foreign material, bacteria and non-functional host cells and damaged matrix components that may be present in the traumatic injury site. Bacteria give off chemical signals, attracting neutrophils, which ingest them by the process of phagocytosis. During bacterial protein synthesis a waste product represented by a tri-peptide called f-Met-Leu-Phe is released which in turn attracts inflammatory cells. Neutrophils will engorge themselves until they are filled with bacteria, forming a laudable pus in the injury site.

[0033] The mast cell is another marker of cell interest in traumatic injury healing. Mast cells release granules filled with enzymes, histamine and other active amines responsible for the characteristic signs of inflammation around the traumatic injury site. The active amines are released from the mast cell, causing surrounding vessels to become leaky and allow the speedy passage of the mononuclear cells into the injury area. In addition, fluid accumulates at the traumatic injury site and the characteristic signs of inflammation begin: rubor (redness), calor (heat), tumor (swelling) and dolor (pain).

[0034] By forty-eight hours after injury, fixed tissue monocytes become activated to become wound macrophages. These specialized wound macrophages are perhaps the most essential inflammatory cells involved in the normal healing response. Inhibition of macrophage function will delay the healing response. Once activated, these wound macrophages also release PDGF and TGF-β, which in turn further attract fibroblasts and smooth muscle cells to the traumatic injury site. These highly phagocytic macrophages are also responsible for removing nonfunctional host cells, bacteria-filled neutrophils, damaged matrix, foreign debris and any remaining bacteria from the traumatic injury site. The presence of wound macrophages is a marker that the inflammatory phase is nearing and end and that the proliferative phase is beginning. Lymphocytes come into the traumatic injury area at a later stage but are not considered to be major inflammatory cells involved in the healing response; their precise role in the traumatic injury healing process remains unclear.

[0035] As the proliferative phase progresses, TGF-β released by the platelets, macrophages and T lymphocytes becomes a critical signal. TGF-β is considered to be a master control signal that regulates a host of fibroblast functions. TGF-β has a three-pronged effect on extracellular matrix deposition. First, it increases transcription of the genes for collagen, proteoglycans and fibronectin thus increasing the overall production of matrix proteins. At the same time, TGF-β decreases the secretion of proteases responsible for the breakdown of the matrix and it also stimulates the protease inhibitor, tissue inhibitor of metalloproteinases (TIMP). Other cytokines considered to be important are interleukins, fibroblast growth factors and tumor necrosis factors.

[0036] As healing progresses, several other important biological responses are activated. The process of epithelialization is stimulated by the presence of epidermal growth factor (EGF) and TGF-α produced by activated wound macrophages, platelets and keratinocytes. Once the epithelial bridge is complete, enzymes are released to dissolve the attachment at the base of the scab resulting in removal. Due to the high metabolic activity at the traumatic injury site, there is an increasing demand for oxygen and nutrients. Local factors in the traumatic injury microenvironment such as low pH, reduced oxygen tension and increased lactate actually initiate the release of factors needed to bring in a new blood supply. This process is called angiogenesis or neovascularization and is stimulated by vascular endothelial cell growth factor (VEGF), fibroblast growth factor 2 (FGF2) and TGF- β. Epidermal cells, fibroblasts, macrophages and vascular endothelial cells produce these factors. One interesting signaling pathway involves the role of low oxygen tension that in turn stimulates the expression of a nuclear transcription factor termed "hypoxia-inducible factor" (HIF) by vascular endothelial cells. The HIF in turn binds to specific sequences of DNA that regulate the expression of VEGF thus stimulating angiogenesis. As new blood vessels enter the traumatic injury repair area and the oxygen tension returns to a normal level, oxygen binds to HIF and blocks its activity leading to a decreased synthesis of VEGF.

[0037] As the proliferative phase progresses, the predominant cell in the traumatic injury site is the fibroblast. This cell of mesenchymal origin is responsible for producing the new matrix needed to restore structure and function to the injured tissue. Fibroblasts attach to the cables of the provisional fibrin matrix and begin to produce collagen. As the collagen matures and becomes older, more and more of these intramolecular and intermolecular cross-links are placed in the molecules. This important cross-linking step gives collagen its strength and stability over time.

[0038] Dermal collagen on a per weight basis approaches the tensile strength of steel; in normal tissue it is a strong and highly organized molecule. In contrast, collagen fibers formed in scar tissue are much smaller and have a random appearance. Scar tissue is always weaker and will break apart before the surrounding normal tissue. The regained tensile strength in a wound will never approach normal. In fact, the maximum tensile strength that a wound can ever achieve is approximately 80% of normal skin. [0039] Finally, in the process of collagen remodeling, collagen degradation also occurs. Specific collagenase enzymes in fibroblasts, neutrophils and macrophages clip the molecule at a specific site through all three chains, and break it down to characteristic three-quarter and one-quarter pieces. These collagen fragments undergo further denaturation and digestion by other proteases. [0040] Fibrosis is characterized by excessive proliferation of fibroblasts, excessive deposition of collagen and the replacement of normal structural elements of the tissue by distorted, non-functional and excessive accumulation of scar tissue. This is perhaps the most significant biological marker for fibrosis. Many clinical problems are associated with excessive collagen deposition and scar formation. For example, keloids and hypertrophic scars in the skin, tendon adhesions, transmission blockage following nerve injury, scleroderma, Crohn's disease, esophageal strictures, urethral strictures, capsules around breast implants, liver cirrhosis, atherosclerosis and fibrotic non-union in bone.

[0041] Chronic non-healing dermal ulcers such as diabetic ulcers, pressure ulcers and venous ulcers, contribute significantly to the morbidity and even mortality of many patients. Diabetic ulcers are often caused by a combination of neuropathy, poor circulation and a compromised immune system. Pressure ulcers are a serious and frequent occurrence among the immobile and debilitated patients that often results from pressure that cuts off circulation to the area. Venous ulcers develop in large part due to venous hypertension and improper valve functioning that results in ischemia and tissue damage. Spinal cord injury patients are particularly vulnerable to pressure ulcer formation.

[0042] Excessive infiltration of these ulcers by neutrophils appears to be a significant biological marker. The over-abundant neutrophil infiltration is responsible for the chronic inflammation characteristic of non-healing ulcers. The neutrophils release significant amounts of enzymes such as collagenase (MMP-8) responsible for the destruction of the connective tissue matrix. In addition, the neutrophils release an enzyme called elastase that is capable of destroying important healing factors such as PDGF and TGF-β. Another marker of these chronic ulcers is an environment containing excessive reactive oxygen species that further damage the cells and healing tissues. These chronic ulcers will not heal until the chronic inflammation is reduced.

[0043] In order for the traumatic injury healing process to take place, the phases of the healing response must progress in normal course. If the injured tissue is unable to mount an effective immune response, chronic inflammation may result. Further, injured tissue may experience excessive fibroblast proliferation and extracellular matrix deposition, resulting in fibrotic lesions. For example, if too much collagen is deposited in the traumatic injury site by proliferating fibroblasts, normal anatomical structure is lost, function is compromised and fibrosis results. If, on the other hand, fibroblasts are inhibited, an insufficient amount of collagen is deposited at the traumatic injury site, resulting in a weak wound prone to dehiscence. The materials and methods of the present invention can be used to control the phases of the healing response and control the physiologic response of tissues to wounds and other traumatic injuries.

[0044] The materials and methods of the present invention can be used in connection with any of the above-described injuries to promote healing. The implantable material of the present invention is able to supply to the traumatic injury site multiple cell-based products in physiological proportions under physiological feed-back control. Local delivery of multiple compounds by these cells in a physiologically-dynamic dosing provide more effective regulation of the processes responsible for maintaining healing traumatic injury sites and diminishing the clinical sequel associated with traumatic injuries.

[0045] Additionally, the materials and methods of the present invention can be used as an adjunct therapy in conjunction with any one or more of numerous other wound, surgical, critical care or other traumatic injury interventions undertaken to treat or manage a traumatic injury, including, for example, suturing, stapling, ablation, surgical closure devices and debridement. In addition, the materials and methods of the present invention can be used as an adjunct therapy in connection with any other critical care or surgical intervention resulting in chronic or acute traumatic injury. The materials and methods of the present invention can be used in conjunction with these or other therapies to increase effectiveness and promote healing.

Implantable Material

[0046] General Considerations: The implantable material of the present invention comprises cells engrafted on, in and/or within a biocompatible matrix. Engrafted means securedly attached via cell to cell and/or cell to matrix interactions such that the cells meet the functional or phenotypical criteria set forth herein and withstand the rigors of the preparatory manipulations disclosed herein. As explained elsewhere herein, an operative embodiment of implantable material comprises a population of cells associated with a supporting substratum, preferably a differentiated cell population and/or a near-confluent, confluent or post-confluent cell population, having a preferred functionality and/or phenotype. [0047] Complex substrate specific interactions regulate the intercellular morphology and secretion of the cells and, accordingly, also regulate the functionality and phenotype of the cells associated with the supporting substratum. Cells associated with certain preferred biocompatible matrices, contemplated herein, may grow and conform to the architecture and surface of the local struts of matrix pores with less straining as they mold to the matrix. Also, the individual cells of a population of cells associated with a matrix retain distinct morphology and secretory ability even without complete contiguity between the cells. Further, cells associated with a biocompatible matrix may not exhibit planar restraint, as compared to similar cells grow as a monolayer on a tissue culture plate.

[0048] It is understood that embodiments of implantable material likely shed cells during preparatory manipulations and/or that certain cells are not as securely attached as are other cells. All that is required is that implantable material comprises cells associated with a supporting substratum that meet the functional or phenotypical criteria set forth herein.

[0049] That is, interaction between the cells and the matrix during the various phases of the cells' growth cycle can influence the cells' phenotype, with the preferred inhibitory phenotype described elsewhere herein correlating with quiescent cells (i.e., cells which are not in an exponential growth cycle). As explained elsewhere herein, it is understood that, while a quiescent cell typifies a population of cells which are near-confluent, confluent or post-confluent, the inhibitory phenotype associated with such a cell can be replicated by manipulating or influencing the interaction between a cell and a matrix so as to render a cell quiescent-like.

[0050] The implantable material of the present invention was developed on the principals of tissue engineering and represents a novel approach to addressing the above-described clinical needs. The implantable material of the present invention is unique in that the viable cells engrafted on, in and/or within the biocompatible matrix are able to supply to the traumatic injury site multiple cell-based products in physiological proportions under physiological feed-back control. As described elsewhere herein, the cells suitable for use with the implantable material include endothelial, endothelial-like, non-endothelial cells or analogs thereof. Local delivery of multiple compounds by these cells in a physiologically-dynamic dosing provide more effective regulation of the processes responsible for maintaining healing traumatic injury sites and diminishing the clinical sequel associated with wounds, burns, surgical incisions and other traumatic injuries.

[0051] The implantable material of the present invention, when wrapped, deposited or otherwise contacted with the surface of a traumatic injury site serves to reestablish homeostasis. That is, the implantable material of the present invention can provide an environment which mimics supportive physiology and is conducive to manage and/or promote healing a site of a wound, burn, surgical incision or other traumatic injury.

[0052] For purposes of the present invention, contacting means directly or indirectly interacting with an interior or exterior surface or volume of a traumatic injury site as defined elsewhere herein. In the case of certain preferred embodiments, actual physical contact is not required for effectiveness. In other embodiments, actual physical contact is preferred. All that is required to practice the present invention is deposition of the implantable material at, adjacent to or in the vicinity of a traumatic injury site in an amount effective to treat the traumatic injury site. In the case of certain traumatic injuries, a traumatic injury site can clinically manifest on an interior surface of an anatomical location, for example, on an interior or exterior surface or volume of an injured tissue or organ. In the case of other traumatic injuries, a traumatic injury site can clinically manifest on an exterior surface, for example, trauma resulting in abrasion or disruption of the epithelial tissue of the skin. In some traumatic wounds or injuries, a traumatic wound or injured site can clinically manifest on both an interior surface and an exterior surface of the anatomical location. The present invention is effective to treat any of the foregoing clinical manifestations.

[0053] For example, endothelial cells can release a wide variety of agents that in combination can inhibit or mitigate adverse physiological conditions associated with acute complications due to wounds, burn or other traumatic injuries. As exemplified herein, a composition and method of use that recapitulates normal physiology and dosing is useful to treat and manage traumatic injury healing. Typically, treatment includes placing the implantable material of the present invention at, adjacent to or in the vicinity of the wound, burn or other traumatic injury. When wrapped, wrapped around, deposited, or otherwise contacting a wound site, the cells of the implantable material can provide growth regulatory compounds to the traumatic injury site, for example within the traumatic injury site. It is also contemplated that, while inside or outside the traumatic injury site, the implantable material of the present invention comprising a biocompatible matrix or particle with engrafted cells provides a continuous supply of multiple regulatory and therapeutic compounds from the engrafted cells to the traumatic injury site. [0054] Cell Source: As described herein, the implantable material of the present invention comprises cells. Cells can be allogeneic, xenogeneic or autologous. In certain embodiments, a source of living cells can be derived from a suitable donor. In certain other embodiments, a source of cells can be derived from a cadaver or from a cell bank. [0055] In one currently preferred embodiment, cells are endothelial cells. In a particularly preferred embodiment, such endothelial cells are obtained from vascular tissue, preferably but not limited to arterial tissue. As exemplified below, one type of vascular endothelial cell suitable for use is an aortic endothelial cell. Another type of vascular endothelial cell suitable for use is umbilical cord vein endothelial cells. And, another type of vascular endothelial cell suitable for use is coronary artery endothelial cells. Yet another type of vascular endothelial cell suitable for use is saphenous vein endothelial cells. Yet other types of vascular endothelial cells suitable for use with the present invention include pulmonary artery endothelial cells and iliac artery endothelial cells. [0056] In another currently preferred embodiment, suitable endothelial cells can be obtained from non-vascular tissue. Non-vascular tissue can be derived from any anatomical structure or can be derived from any non-vascular tissue or organ. Exemplary anatomical structures include structures of the vascular system, the renal system, the reproductive system, the genitourinary system, the gastrointestinal system, the pulmonary system, the respiratory system and the ventricular system of the brain and spinal cord. [0057] In another embodiment, endothelial cells can be derived from endothelial progenitor cells or stem cells. In other preferred embodiments, cells can be non-endothelial cells that are allogeneic, xenogeneic or autologous and can be derived from vascular, neural or other tissue or organ. Cells can be selected on the basis of their tissue source and/or their immunogenicity. Exemplary non-endothelial cells include epithelial cells, neural cells, secretory cells, smooth muscle cells, fibroblasts, stem cells, endothelial progenitor cells, cardiomyocytes, keratinocytes, secretory and ciliated cells. The present invention also contemplates any of the foregoing which are genetically altered, modified or engineered. [0058] In another currently preferred embodiment, cells are epithelial cells. In a particularly preferred embodiment, such epithelial cells are obtained from gastrointestinal tissue, tracheal-bronchial-pulmonary tissue, genito-urinary tissue, lymphatic tissue and/or glandular tissue, or another epithelial cell source. According to various embodiments, the epithelial cells are squamous cells, cuboidal cells, columnar cells and/or transitional tissue.

[0059] In a further embodiment, two or more types of cells are co-cultured to prepare the present composition. For example, a first cell can be introduced into the biocompatible implantable material and cultured until confluent. The first cell type can include, for example, endothelial cells, epithelial cells, neural cells, secretory cells, smooth muscle cells, fibroblasts, stem cells, nerve stem cells, endothelial progenitor cells, keratinocytes, a combination of endothelial cells and keratinocytes, a combination of smooth muscle cells and fibroblasts, any other desired cell type or a combination of desired cell types suitable to create an environment conducive to growth of the second cell type. Once the first cell type has reached confluence, a second cell type is seeded on top of the first confluent cell type in, on or within the biocompatible matrix and cultured until both the first cell type and second cell type have reached confluence. The second cell type may include, for example, epithelial cells, neural cells, secretory cells, smooth muscle cells, fibroblasts, stem cells, nerve stem cells, endothelial cells, endothelial progenitor cells, keratinocytes or any other desired cell type or combination of cell types. It is contemplated that the first and second cell types can be introduced step wise, or as a single mixture. It is also contemplated that cell density can be modified to alter the ratio of the first cell type to the second cell type.

[0060] To prevent over-proliferation of smooth muscle cells or another cell type prone to excessive proliferation, the culture procedure and timing can be modified. For example, following confluence of the first cell type, the culture can be coated with an attachment factor suitable for the second cell type prior to introduction of the second cell type. Exemplary attachment factors include coating the culture with gelatin to improve attachment of endothelial cells. According to another embodiment, heparin can be added to the culture media during culture of the second cell type to reduce the proliferation of the first cell type and to optimize the desired first cell type to second cell type ratio. For example, after an initial growth of smooth muscle cells, heparin can be administered to control smooth muscle cell growth to achieve a greater ratio of endothelial cells to smooth muscle cells.

[0061] In a preferred embodiment, a co-culture is created by first seeding a biocompatible implantable material with smooth muscle cells to create structures, for example, but not limited to, structures that mimic the size and/or shape of the traumatic injury site. Once the smooth muscle cells have reached confluence, endothelial cells, epithelial cells, endothelial-like cells, epithelial-like cells, or non- endothelial cells are seeded on top of the cultured smooth muscle cells on the implantable material to create a simulated structure.

[0062] All that is required of the cells of the present composition is that they exhibit one or more preferred phenotypes or functional properties. As described earlier herein, the present invention is based on the discovery that a cell having a readily identifiable phenotype when associated with a preferred matrix (described elsewhere herein) can facilitate, restore and/or otherwise modulate cell physiology and/or homeostasis associated with the treatment of traumatic injury site generally.

[0063] For purposes of the present invention, one such preferred, readily identifiable phenotype typical of cells of the present invention is an ability to inhibit or otherwise interfere with smooth muscle cell proliferation and/or migration. Smooth muscle cell proliferation can be determined using an in vitro smooth muscle cell proliferation assay and smooth muscle cell migration can be determining using an in vitro smooth muscle cell migration assay, both of which are described below. The ability to regulate smooth muscle cells proliferation and/or migration is referred to herein as the inhibitory phenotype.

[0064] One other readily identifiable phenotype exhibited by cells of the present composition is that they are able to regulate fibroblast proliferation and/or migration and collagen deposition and/or accumulation. Fibroblast activity and collagen deposition activity can be determined using an in vitro fibroblast proliferation, in vitro fibroblast migration and/or an in vitro collagen accumulation assay, each of which are described below. The ability to regulate fibroblast proliferation and/or migration is also referred to herein as the inhibitory phenotype.

[0065] An additional readily identifiable phenotype exhibited by cells of the present composition is that they are able to increase keratinocyte proliferation and/or migration. Keratinocyte proliferation can be determined using an in vitro keratinocyte proliferation assay and keratinocyte migration can be determined using an in vitro keratinocyte migration assay, each of which are described below. The ability to regulate keratinocyte proliferation and/or migration is also referred to herein as the inhibitory phenotype.

[0066] Another readily identifiable phenotype exhibited by cells of the present composition is that they are anti-thrombotic or are able to inhibit platelet adhesion and aggregation. Anti-thrombotic activity can be determined using an in vitro heparan sulfate assay and/or an in vitro platelet aggregation assay, described below.

[0067] A further readily identifiable phenotype exhibited by cells of the present composition is the ability to restore the proteolytic balance, the MMP-TIMP balance, the ability to decrease expression of MMPs relative to the expression of TIMPs, or the ability to increase expression of TIMPs relative to the expression of MMPs. Proteolytic balance activity can be determined using an in vitro TIMP assay and/or an in vitro MMP assay described below.

[0068] In a typical operative embodiment of the present invention, cells need not exhibit more than one of the foregoing phenotypes. In certain embodiments, cells can exhibit more than one of the foregoing phenotypes. [0069] While the foregoing phenotypes each typify a functional endothelial cell, such as but not limited to a vascular endothelial cell, a non-endothelial cell exhibiting such a phenotype(s) is considered endothelial-like for purposes of the present invention and thus suitable for use with the present invention. Cells that are endothelial-like are also referred to herein as functional analogs of endothelial cells; or functional mimics of endothelial cells. Thus, by way of example only, cells suitable for use with the materials and methods disclosed herein also include epithelial cells, stem cells or progenitor cells that give rise to endothelial-like or epithelial-like cells; cells that are non-endothelial or non-epithelial cells in origin yet perform functionally like an endothelial or epithelial cell, respectively, using the parameters set forth herein; cells of any origin which are engineered or otherwise modified to have endothelial-like or epithelial-like functionality using the parameters set forth herein.

[0070] Typically, cells of the present invention exhibit one or more of the aforementioned functionalities and/or phenotypes when present and associated with a supporting substratum, such as the biocompatible matrices described herein. It is understood that individual cells attached to a matrix and/or interacting with a specific supporting substratum exhibit an altered expression of functional molecules, resulting in a preferred functionality or phenotype when the cells are associated with a matrix or supporting substratum that is absent when the cells are not associated with a supporting substratum.

[0071] According to one embodiment, the cells exhibit a preferred phenotype when the basal layer of the cell is associated with a supporting substratum. According to another embodiment, the cells exhibit a preferred phenotype when present in confluent, near confluent or post-confluent populations. As will be appreciated by one of ordinary skill in the art, populations of cells, for example, substrate adherent cells, and confluent, near confluent and post-confluent populations of cells, are identifiable readily by a variety of techniques, the most common and widely accepted of which is direct microscopic examination. Others include evaluation of cell number per surface area using standard cell counting techniques such as but not limited to a hemacytometer or coulter counter. [0072] Additionally, for purposes of the present invention, endothelial-like cells include but are not limited to cells which emulate or mimic functionally and phenotypically the preferred populations of cells set forth herein, including, for example, differentiated endothelial cells and confluent, near confluent or post- confluent endothelial cells, as measured by the parameters set forth herein.

[0073] Thus, using the detailed description and guidance set forth below, the practitioner of ordinary skill in the art will appreciate how to make, use, test and identify operative embodiments of the implantable material disclosed herein. That is, the teachings provided herein disclose all that is necessary to make and use the present invention's implantable materials. And further, the teachings provided herein disclose all that is necessary to identify, make and use operatively equivalent cell-containing compositions. At bottom, all that is required is that equivalent cell- containing compositions are effective to treat, manage, modulate and/or ameliorate a traumatic injury site in accordance with the methods disclosed herein. As will be appreciated by the skilled practitioner, equivalent embodiments of the present composition can be identified using only routine experimentation together with the teachings provided herein.

[0074] In certain preferred embodiments, endothelial cells used in the implantable material of the present invention are isolated from the aorta of human cadaver donors. Each lot of cells is derived from a single donor or from multiple donors, tested extensively for endothelial cell purity, biological function, the presence of bacteria, fungi, known human pathogens and other adventitious agents. The cells are cryopreserved and banked using well-known techniques for later expansion in culture for subsequent formulation in biocompatible implantable materials.

[0075] Cell Preparation: As stated above, suitable cells can be obtained from a variety of tissue types and cell types. In certain preferred embodiments, human aortic endothelial cells used in the implantable material are isolated from the aorta of cadaver donors. In other embodiments, porcine aortic endothelial cells (Cell Applications, San Diego, CA) are isolated from normal porcine aorta by a similar procedure used to isolate human aortic endothelial cells. Each lot of cells can be derived from a single donor or from multiple donors, tested extensively for endothelial cell viability, purity, biological function, the presence of mycoplasma, bacteria, fungi, yeast, known human pathogens and other adventitious agents. The cells are further expanded, characterized and cryopreserved to form a working cell bank at the third to sixth passage using well-known techniques for later expansion in culture and for subsequent formulation in biocompatible implantable material.

[0076] The human or porcine aortic endothelial cells are prepared in T-75 flasks pre-treated by the addition of approximately 15 ml of endothelial cell growth media per flask. Human aortic endothelial cells are prepared in Endothelial Growth Media (EGM-2, Lonza Biosciences, Basel, Switzerland). EGM-2 consists of Endothelial Cell Basal Media (EB M-2, Lonza Biosciences, Basel, Switzerland) supplemented with EGM-2 singlequots, which contain 2% FBS. Porcine cells are prepared in EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin. The flasks are placed in an incubator maintained at approximately 37°C and 5% CO2 / 95% air, 90% humidity for a minimum of 30 minutes. One or two vials of the cells are removed from the -160°C to -1400C freezer and thawed at approximately 37°C. Each vial of thawed cells is seeded into two T-75 flasks at a density of approximately 3 x 103 cells per cm2, preferably, but no less than 1.0 x 103 and no more than 7.0 x 103; and the flasks containing the cells are returned to the incubator. After about 8-24 hours, the spent media is removed and replaced with fresh media. The media is changed every two to three days, thereafter, until the cells reach approximately 85-100% confluence preferably, but no less than 60% and no more than 100%. When the implantable material is intended for clinical application, only antibiotic-free media is used in the post-thaw culture of human aortic endothelial cells and manufacture of the implantable material of the present invention.

[0077] The endothelial cell growth media is then removed, and the monolayer of cells is rinsed with 10 ml of HEPES buffered saline (HEPES). The HEPES is removed, and 2 ml of trypsin is added to detach the cells from the surface of the T- 75 flask. Once detachment has occurred, 3 ml of trypsin neutralizing solution (TNS) is added to stop the enzymatic reaction. An additional 5 ml of HEPES is added, and the cells are enumerated using a hemocytometer. The cell suspension is centrifuged and adjusted to a density of, in the case of human cells, approximately 2.0 - 1.75 x 106 cells/ml using EGM-2 without antibiotics, or in the case of porcine cells, approximately 2.0 - 1.5O x IO6 cells/ml using EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin.

[0078] Biocompatible Matrix: According to the present invention, the implantable material comprises a biocompatible matrix. The matrix is permissive for cell growth and attachment to, on or within the matrix. The matrix is flexible and conformable. The matrix can be a solid, a semi-solid or flowable porous composition. For purposes of the present invention, flowable composition means a composition susceptible to administration using an injection or injection-type delivery device such as, but not limited to, a needle, a syringe or a catheter. Other delivery devices which employ extrusion, ejection or expulsion are also contemplated herein. Porous matrices are preferred. The matrix also can be in the form of a flexible planar form. The matrix also can be in the form of a gel, a foam, a suspension, a particle, a microcarrier, a macrocarrier, a microcapsule, or a fibrous structure. A preferred flowable composition is shape-retaining. A currently preferred matrix has a particulate form. The biocompatible matrix can comprise particles and/or microcarriers and/or macrocarriers and the particles and/or microcarriers and/or macrocarriers can further comprise gelatin, collagen, fibronectin, fibrin, laminin or an attachment peptide. One exemplary attachment peptide is a peptide of sequence arginine-glycine-aspartate (RGD).

[0079] The matrix, when implanted on a surface of a traumatic injury site, can reside at the implantation site for at least about 7-90 days, preferably about at least 7-14 days, more preferably about at least 14-28 days, most preferably about at least 28-90 days before it bioerodes. [0080] One preferred matrix is Gelfoam® (Pfizer, Inc., New York, NY), an absorbable gelatin sponge (hereinafter "Gelfoam matrix"). Another preferred matrix is Surgifoam® (Johnson & Johnson, New Brunswick, NJ), also an absorbable gelatin sponge. Gelfoam and Surgifoam matrices are porous and flexible surgical sponges prepared from a specially treated, purified porcine dermal gelatin solution. [0081] According to another embodiment, the biocompatible matrix material can be a modified matrix material. Modifications to the matrix material can be selected to optimize and/or to control function of the cells, including the cells' phenotype (e.g., the inhibitory phenotype) as described above, when the cells are associated with the matrix. According to one embodiment, modifications to the matrix material include coating the matrix with attachment factors or adhesion peptides that enhance the ability of the cells to regulate smooth muscle cell and/or fibroblast and/or keratinocyte proliferation and migration, to decrease abnormal collagen deposition, to decrease scar formation, to decrease fibrosis, to increase TIMP production, to optimize the proteolytic balance (the MMP/TIMP balance), to decrease inflammation, to decrease pain associated with the traumatic injury, to decrease healing time, to increase heparan sulfate production, to increase prostacyclin production, and/or to increase FGF2, TGF-Bi and nitric oxide (NO) production.

[0082] According to another embodiment, the matrix is a matrix other than Gelfoam. Additional exemplary matrix materials include, for example, fibrin gel, alginate, gelatin bead microcarriers, polystyrene sodium sulfonate microcarriers, collagen coated dextran microcarriers, PLA/PGA and pHEMA/MMA copolymers (with polymer ratios ranging from 1-100% for each copolymer). According to one embodiment, a synthetic matrix material, for example, PLA/PGA, is treated with NaOH to increase the hydrophilicity of the material and, therefore, the ability of the cells to attach to the material. According to a preferred embodiment, these additional matrices are modified to include attachment factors or adhesion peptides, as recited and described above. Exemplary attachment factors include, for example, gelatin, collagen, fibronectin, fibrin gel, and covalently attached cell adhesion ligands (including for example RGD) utilizing standard aqueous carbodiimide chemistry. Additional cell adhesion ligands include peptides having cell adhesion recognition sequences, including but not limited to: RGDY, REDVY, GRGDF, GPDSGR, GRGDY and REDV.

[0083] That is, these types of modifications or alterations of a substrate influence the interaction between a cell and a matrix which, in turn, can mediate expression of the preferred inhibitory phenotype described elsewhere herein. It is contemplated that these types of modifications or alterations of a substrate can result in enhanced expression of an inhibitory phenotype; can result in prolonged or further sustained expression of an inhibitory phenotype; and/or can confer such a phenotype on a cell which otherwise in its natural state does not exhibit such a phenotype as in, for example but not limited to, an exponentially growing or non-quiescent cell. Moreover, in certain circumstances, it is preferable to prepare an implantable material of the present invention which comprises non-quiescent cells provided that the implantable material has an inhibitory phenotype in accordance with the requirements set forth elsewhere herein. As already explained, the source of cells, the origin of cells and/or types of cells useful with the present invention are not limited; all that is required is that the cells express an inhibitory phenotype.

[0084] Embodiments of Implantable Materials: As stated earlier, implantable material of the present invention can be a flexible planar form or a flowable composition. When in a flexible planar form, it can assume a variety of shapes and sizes, preferably a shape and size which conforms to a contoured surface of a traumatic injury site when situated at or adjacent to or in the vicinity of a traumatic injury site. Examples of preferred configurations suitable for use in this manner are disclosed in co-owned international patent application PCT/US05/43967 filed on December 6, 2005 (also known as Attorney Docket No. ELV-002PC), the entire contents of which are herein incorporated by reference.

[0085] Flowable Composition: In certain embodiments contemplated herein, the implantable material of the present invention is a flowable composition comprising a particulate biocompatible matrix which can be in the form of a gel, a foam, a suspension, a particle, a microcarrier, a macrocarrier, a microcapsule, macroporous beads, or other flowable material. The current invention contemplates any flowable composition that can be administered with an injection-type delivery device. For example, a delivery device such as a percutaneous injection-type delivery device is suitable for this purpose as described below. The flowable composition is preferably a shape-retaining composition. Thus, an implantable material comprising cells in, on or within a flowable-type particulate matrix as contemplated herein can be formulated for use with any injectable delivery device ranging in internal diameter from about 18 gauge to about 30 gauge and capable of delivering about 50 mg of flowable composition comprising particulate material containing preferably about 1 million cells in about 1 to about 3 ml of flowable composition.

[0086] According to a currently preferred embodiment, the flowable composition comprises a biocompatible particulate matrix such as Gelfoam® particles, Gelfoam® powder, or pulverized Gelfoam® (Pfizer Inc., New York, NY) (hereinafter "Gelfoam particles"), a product derived from porcine dermal gelatin. According to another embodiment, the particulate matrix is Surgifoam™ (Johnson & Johnson, New Brunswick, NJ) particles, comprised of absorbable gelatin powder. According to another embodiment, the particulate matrix is Cytodex-3 (Amersham Biosciences, Piscataway, NJ) microcarriers, comprised of denatured collagen coupled to a matrix of cross-linked dextran. According to a further embodiment, the particulate matrix is CultiSpher-G (Percell Biolytica AB, Astorp, Sweden) microcarrier, comprised of porcine gelatin. According to another embodiment, the particulate matrix is a macroporous material. According to one embodiment, the macroporous particulate matrix is CytoPore (Amersham Biosciences, Piscataway, NJ) macrocarrier, comprised of cross-linked cellulose which is substituted with positively charged N,N,-diethylaminoethyl groups.

[0087] According to alternative embodiments, the biocompatible implantable particulate matrix is a modified biocompatible matrix. Modifications include those described above for an implantable matrix material.

[0088] Related flowable compositions suitable for use to manage the development and/or progression of healing in traumatic injury sites in accordance with the present invention are disclosed in co-owned international patent application PCT/US05/43844 filed on December 6, 2005 (also known as Attorney Docket No. ELV-009PC), the entire contents of which are herein incorporated by reference.

[0089] Tissue Sealant: In certain embodiments contemplated herein, the implantable material of the present invention is a tissue sealant or tissue adhesive composition comprising a biocompatible tissue sealant matrix which can be in the form of a planar matrix, a gel, a foam, a suspension, a particle, a microcarrier, a macrocarrier, a microcapsule, macroporous beads, or other material having sealant or adhesive properties. According to one embodiment, the biocompatible matrix material of the implantable material is a biocompatible material having sealant or adhesive properties. According to another embodiment, the biocompatible matrix material is modified to provide or improve its sealant or adhesive properties. According to a further embodiment, a sealant or adhesive material, for example a sealant or adhesive carrier composition, is added to the implantable material to provide or improve the implantable material's sealant or adhesive properties. According to this embodiment, the sealant or adhesive material can be added prior to or coincident with administration of the implantable material to the traumatic injury site. The current invention contemplates any tissue sealant or adhesive composition and/or implantable material composition that has sealant, adhesive or absorptive properties and that, upon application to a traumatic injury, can maintain contact with the traumatic injury and does not significantly diminishing the viability and/or functionality of the implanted or engrafted cells.

[0090] Preparation of Implantable Material: Prior to cell seeding, the biocompatible matrix is re-hydrated by the addition of water, buffers and/or culture media such as EGM-2 without antibiotics at approximately 37°C and 5% CO2 / 95% air for 12 to 24 hours. The implantable material is then removed from their rehydration containers and placed in individual tissue culture dishes. The biocompatible matrix is seeded at a preferred density of approximately 1.5-2.0 x 105 cells (1.25-1.66 x 105 cells /cm3 of matrix) and placed in an incubator maintained at approximately 370C and 5% CO2 / 95% air, 90% humidity for 3-4 hours to 24 hours to facilitate cell attachment. The seeded matrix is then placed into individual containers (Evergreen, Los Angeles, CA) or tubes, each fitted with a cap containing a 0.2 μm filter with EGM-2 and incubated at approximately 37°C and 5% CO2 / 95% air. Alternatively, 3 seeded matrices can be placed into 150 mL bottle. The media is changed every two to three days, thereafter, until the cells have reached near-confluence, confluence or post-confluence. The cells in one preferred embodiment are preferably passage 6, but cells of fewer or more passages can be used. [0091] Cell Growth Curve and Confluence: A sample of implantable material is removed on or around days 3 or 4, 6 or 7, 9 or 10, and 12 or 13, the cells are counted and assessed for viability, and a growth curve is constructed and evaluated in order to assess the growth characteristics and to determine whether confluence, near confluence or post-confluence has been achieved. Representative growth curves from two preparations of implantable material comprising porcine aortic endothelial cell implanted lots are presented in FIGS. IA and IB. In these examples, the implantable material is in a flexible planar form. Generally, one of ordinary skill will appreciate the indicia of acceptable cell growth at early, mid- and late time points, such as observation of an increase in cell number at the early time points (when referring to FIG. IA, between about days 2-6), followed by a near confluent phase (when referring to FIG. IA, between about days 6-8), followed by a plateau in cell number once the cells have reached confluence as indicated by a relatively constant cell number (when referring to FIG. IA, between about days 8- 10) and maintenance of the cell number when the cells are post-confluent (when referring to FIG. IA, between about days 10-14). For purposes of the present invention, cell populations which are in a plateau for at least 72 hours are preferred. [0092] Cell counts are achieved by complete digestion of the aliquot of implantable material such as with a solution of 0.5 mg/ml collagenase in a CaCl2 solution in the case of gelatin-based matrix materials. After measuring the volume of the digested implantable material, a known volume of the cell suspension is diluted with 0.4% trypan blue (4: 1 cells to trypan blue) and viability assessed by trypan blue exclusion. Viable, non-viable and total cells are enumerated using a hemacytometer. Growth curves are constructed by plotting the number of viable cells versus the number of days in culture. Cells are shipped and implanted after reaching confluence.

[0093] For purposes of the present invention, confluence is defined as the presence of at least about 4 x 105 cells/cm3 when in a flexible planar form of the implantable material (1.0 x 4.0 x 0.3 cm), and preferably about 7 x 105 to 1 x 106 total cells per aliquot (50-70 mg) when in a flowable composition. For both, cell viability is at least about 90% preferably but no less than 80%. If the cells are not confluent by day 12 or 13, the media is changed, and incubation is continued for an additional day. This process is continued until confluence is achieved or until 14 days post-seeding. On day 14, if the cells are not confluent, the lot is discarded. If the cells are determined to be confluent after performing in-process checks, a final media change is performed. This final media change is performed using EGM-2 without phenol red and without antibiotics. Immediately following the media change, the tubes are fitted with sterile plug seal caps for shipping.

[0094] Shelf-Life of Implantable Material: The implantable material of the present invention comprising a confluent, near-confluent or post-confluent population of cells can be maintained at room temperature in a stable and viable condition for at least two weeks. Preferably, such implantable material is maintained in about 45-60 ml, more preferably about 50 ml per implantable material, of transport media with or without additional FBS or VEGF. Transport media comprises EGM-2 media without phenol red. FBS can be added to the volume of transport media up to about 10% FBS, or a total concentration of about 12% FBS. However, because FBS must be removed from the implantable material prior to implantation, it is preferred to limit the amount of FBS used in the transport media to reduce the length of rinse required prior to implantation. VEGF can be added to the volume of transport media up to a concentration of about 3-4 ng/mL.

[0095] Crvopreservation of Implantable Material: The implantable material of the present invention can be cryopreserved for storage and/or transport to the implantation site without diminishing its clinical potency or integrity upon eventual thaw. Preferably, implantable material is cryopreserved in a 15 ml cryovial (Nalgene®, Nalge Nunc Int'l, Rochester, NY) in a solution of about 5 ml CryoStor CS-10 solution (BioLife Solutions, Oswego, NY) containing about 10% DMSO, about 2-8% Dextran and about 20-75% FBS or human serum. Cryovials are placed in a cold iso-propanol water bath, transferred to an -800C freezer for 4 hours, and subsequently transferred to liquid nitrogen (-1500C to -165°C). [0096] Cryopreserved aliquots of the implantable material are then slowly thawed at room temperature for about 15 minutes, followed by an additional approximately 15 minutes in a room temperature water bath. The material is then washed about 3 times in about 200 - 250 mL saline, lactated ringers or EBM. The three rinse procedures are conducted for about 5 minutes at room temperature. The material may then be implanted. [0097] To determine the bioactivity of the thawed material, following the thaw and rinse procedures, the cryopreserved material is allowed to rest for about 48 hours in about 10 ml of recovery solution. For porcine endothelial cells, the recovery solution is EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin at 37°C in 5% CO2; for human endothelial cells, the recovery solution is EGM-2 with or without antibiotics. Further post-thaw conditioning can be carried out for at least another 24 hours prior to use and/or packaging for storage or transport.

[0098] Immediately prior to implantation, the transport or cryopreservation medium is decanted and the implantable material is rinsed 2-3 times in about 250- 500 ml sterile saline (USP). The medium in the final product contains a small amount of FBS to maintain cell viability during transport to a clinical site if necessary. The FBS has been tested extensively for the presence of bacteria, fungi and other viral agents according to Title 9 CFR: Animal and Animal Products. A rinsing procedure is employed just prior to implantation, which decreases the amount of FBS transferred preferably to between 0-60 ng per implant, but preferably no more than 1-2 μg per implant.

[0099] The total cell load per human patient will be preferably approximately 1.6-2.6 x 104 cells per kg body weight, but no less than about 2 x 103 and no more than about 2 x 106 cells per kg body weight. [0100] Evaluation of Functionality and Phenotype: For purposes of the invention described herein, the implantable material is further tested for indicia of functionality and phenotype prior to implantation. For example, conditioned media are collected during the culture period to ascertain levels of heparan sulfate, transforming growth factor-βi (TGF-βi), fibroblast growth factor 2 (FGF2), tissue inhibitors of matrix metal loproteinases (TIMP), and nitric oxide which are produced by the cultured endothelial cells. In certain preferred embodiments, the implantable material can be used for the purposes described herein when total cell number is at least about 2, preferably at least about 4 x 10s cells/cm3 of implantable material; percentage of viable cells is at least about 80-90%, preferably >90%, most preferably at least about 90%; heparan sulfate in conditioned media is at least about 0.23-1.0, preferably at least about 0.5 microg/mL/day; TGF-βι in conditioned media is at least about 200-300 picog/mL/day, preferably at least about 300 picog/ml/day; FGF2 in conditioned media is below about 200 picog/ml, preferably no more than about 400 picog/ml; TIMP-2 in conditioned media is at least about 5.0 - 10.0 ng/mL/day, preferably at least about 8.0 ng/mL/day; NO in conditioned media is at least about 0.5 - 3.0 μmol/L/day, preferably at least about 2.0 μmol/L/day.

[0101] Heparan sulfate levels can be quantified using a routine dimethylmethylene blue-chondroitinase ABC digestion spectrophotometric assay. Total sulfated glycosaminoglycan (GAG) levels are determined using a dimethylmethylene blue (DMB) dye binding assay in which unknown samples are compared to a standard curve generated using known quantities of purified chondroitin sulfate diluted in collection media. Additional samples of conditioned media are mixed with chondroitinase ABC to digest chondroitin and dermatan sulfates prior to the addition of the DMB color reagent. All absorbances are determined at the maximum wavelength absorbance of the DMB dye mixed with the GAG standard, generally around 515-525 nm. The concentration of heparan sulfate per day is calculated by multiplying the percentage heparan sulfate calculated by enzymatic digestion by the total sulfated glycosaminoglycan concentration in conditioned media samples. Chondroitinase ABC activity is confirmed by digesting a sample of purified 100% chondroitin sulfate and a 50/50 mixture of purified heparan sulfate and chondroitin sulfate. Conditioned medium samples are corrected appropriately if less than 100% of the purified chondroitin sulfate is digested. Heparan sulfate levels may also be quantitated using an ELISA assay employing monoclonal antibodies.

[0102] TGF-βi; TIMP, and FGF2 levels can be quantified using an ELISA assay employing monoclonal or polyclonal antibodies, preferably polyclonal. Control collection media can also be quantitated using an ELISA assay and the samples corrected appropriately for TGF-βi TIMP, and FGF2 levels present in control media.

[0103] Nitric oxide (NO) levels can be quantified using a standard Griess Reaction assay. The transient and volatile nature of nitric oxide makes it unsuitable for most detection methods. However, two stable breakdown products of nitric oxide, nitrate (NO3) and nitrite (NO2), can be detected using routine photometric methods. The Griess Reaction assay enzymatically converts nitrate to nitrite in the presence of nitrate reductase. Nitrite is detected colorimetrically as a colored azo dye product, absorbing visible light in the range of about 540 nm. The level of nitric oxide present in the system is determined by converting all nitrate into nitrite, determining the total concentration of nitrite in the unknown samples, and then comparing the resulting concentration of nitrite to a standard curve generated using known quantities of nitrate converted to nitrite.

[0104] The earlier-described preferred inhibitory phenotype is assessed using the quantitative heparan sulfate, TGF-Bi, TIMP, NO and/or FGF2 assays described above, as well as quantitative in vitro assays of smooth muscle cell proliferation and migration, fibroblast proliferation, migration and collagen deposition activity, keratinocyte proliferation and migration, and inhibition of thrombosis as follows. For purposes of the present invention, implantable material is ready for implantation when one or more of these alternative in vitro assays confirm that the implantable material is exhibiting the preferred inhibitory phenotype.

[0105] To evaluate inhibition of thrombosis in vitro, the level of heparan sulfate associated with the cultured endothelial cells is determined. Heparan sulfate has both anti-proliferative and anti-thrombotic properties. Using either the routine dimethylmethylene blue-chondroitinase ABC digestion spectrophotometric assay or an ELISA assay, both assays are described in detail above, the concentration of heparan sulfate is calculated. The implantable material can be used for the purposes described herein when the heparan sulfate in the conditioned media is at least about 0.23-1.0, preferably at least about 0.5 microg/mL/day. [0106] Another method to evaluate inhibition of thrombosis involves determining the magnitude of inhibition of platelet aggregation in vitro associated with platelet rich-plasma or platelet concentrate (Research Blood Components, Brighton, MA). Conditioned media is prepared from post-confluent endothelial cell cultures and added to aliquots of the platelet concentrate. A platelet aggregating agent (agonist) is added to the platelets seeded into 96 well plates as control.

Platelet agonists commonly include arachidonate, ADP, collagen type I, epinephrine, thrombin (Sigma-Aldrich Co., St. Louis, MO) or ristocetin (available from Sigma- Aldrich Co., St. Louis, MO). An additional well of platelets has no platelet agonist or conditioned media added, to assess for baseline spontaneous platelet aggregation. A positive control for inhibition of platelet aggregation is also included in each assay. Exemplary positive controls include aspirin, heparin, indomethacin (Sigma- Aldrich Co., St. Louis, MO), abciximab (ReoPro®, Eli Lilly, Indianapolis, IN), tirofiban (Aggrastat®, Merck & Co., Inc., Whitehouse Station, NJ) or eptifibatide (Integrilin®, Millennium Pharmaceuticals, Inc., Cambridge, MA). The resulting platelet aggregation of all test conditions are then measured using a plate reader and the absorbance read at 405 nm. The platelet reader measures platelet aggregation by monitoring optical density. As platelets aggregate, more light can pass through the specimen. The platelet reader reports results in absorbance, a function of the rate at which platelets aggregate. Aggregation is assessed as maximal aggregation between 6-12 minutes after the addition of the agonist. The effect of conditioned media on platelet aggregation is determined by comparing maximal agonist aggregation before the addition of conditioned medium with that after exposure of platelet concentrate to conditioned medium, and to the positive control. Results are expressed as a percentage of the baseline. The magnitude of inhibition associated with the conditioned media samples are compared to the magnitude of inhibition associated with the positive control. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits thrombosis by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control.

[0107] When ready for implantation, the planar form of implantable material is supplied in final product containers, each preferably containing a 1 x 4 x 0.3 cm (1.2 cm3), sterile implantable material with preferably approximately 5-8 x 105 or preferably at least about 4 x 105 cells/cm3, and at least about 90% viable cells (for example, human aortic endothelial cells derived from a single cadaver donor) per cubic centimeter implantable material in approximately 45-60 ml, preferably about 50 ml, endothelial growth medium (for example, endothelial growth medium (EGM- 2), containing no phenol red and no antibiotics). When porcine aortic endothelial cells are used, the growth medium is also EBM-2 containing no phenol red, but supplemented with 5% FBS and 50 μg/ml gentamicin.

[0108] In other preferred embodiments, the flowable composition (for example, a particulate form biocompatible matrix) is supplied in final product containers, including, for example, sealed tissue culture containers modified with filter caps or pre-loaded syringes, each preferably containing about 50-60 mg of flowable composition comprising about 7 x 105 to about 1 x 106 total endothelial cells in about 45-60 ml, preferably about 50 ml, growth medium per aliquot.

[0109] Administration of Implantable Material: The implantable material of the present invention when in a flowable composition comprises a particulate biocompatible matrix and cells, preferably endothelial cells, more preferably vascular endothelial cells, which are about 90% viable at a preferred density of about 0.8 x 104 cells/mg, more preferred of about 1.5 x 104 cells/mg, most preferred of about 2 x 104 cells/mg, and which can produce conditioned media containing heparan sulfate at least about 0.23-1.0, preferably at least about 0.5 microg/mL/day, TGF-βi at at least about 200-300 picog/ml/day, preferably at least about 300 picog/ml/day, and FGF2 below about 200 picog/ml and preferably no more than about 400 picog/ml; TIMP-2 in conditioned media is at least about 5.0 — 10.0 ng/mL/day, preferably at least about 8.0 ng/mL/day; NO in conditioned media is at least about 0.5 — 3.0 μmol/L/day, preferably at least about 2.0 μmol/L/day; and, display the earlier-described inhibitory phenotype.

[0110] For purposes of the present invention generally, administration of the implantable material is localized to a site in the vicinity of, adjacent to or at a wound, burn or other traumatic injury site. The site of deposition of the implantable material is at a traumatic injury site. As contemplated herein, localized deposition can be accomplished as follows.

[0111] In a particularly preferred embodiment, the implantable material is administered locally to an exterior or cutaneous surface of the patient's body at or near a traumatic injury site using a suitable syringe, needle or other suitable local administration method. According to one embodiment, a sealant, bandage or other barrier can be applied coincident with or following administration of the implantable material to maintain adequate hydration and/or retention of the implantable material during the course of treatment.

[0112] In another preferred embodiment, the flowable composition is first administered percutaneously to a traumatic injury site within the patient's body, entering the patient's body near the traumatic injury site and then deposited on an interior or exterior surface or volume of the traumatic injury site using a suitable needle, catheter or other suitable percutaneous delivery device. Alternatively, the flowable composition is delivered percutaneously using a needle, catheter or other suitable delivery device in conjunction with an identifying step to facilitate delivery to a desired location of the traumatic injury site. The identifying step can occur prior to or coincident with percutaneous delivery. The identifying step can be accomplished using physical examination, ultrasound, and/or CT scan, to name but a few. The identifying step is optionally performed and not required to practice the methods of the present invention. [0113] Preferably, the implantable material is deposited near a traumatic injury site, either at the traumatic injury site to be treated, or adjacent to or in the vicinity of the traumatic injury site. The composition can be deposited in a variety of locations relative to a traumatic injury site, for example, at the site of damage or injury, surrounding the site of damage or injury or adjacent to the site of damage or injury. According to a preferred embodiment, an adjacent site is within about 0 mm to 20 mm of the traumatic injury site. In another preferred embodiment, a site is within about 21 mm to 40 mm; in yet another preferred embodiment, a site is within about 41 mm to 60 mm. In another preferred embodiment, a site is within about 61 mm to 100 mm. Alternatively, an adjacent site is any other clinician-determined adjacent location where the deposited composition is capable of exhibiting a desired effect on a traumatic injury site in the proximity of the traumatic injury site.

[0114] In another embodiment, the implantable material is delivered directly to a surgically-exposed site within a patient's body at, adjacent to or in the vicinity of a traumatic injury site. In this case, delivery is guided and directed by direct observation of the site. Also in this case, delivery can be aided by coincident use of an identifying step as described above. Again, the identifying step is optional. [0115] According to another embodiment of the invention, the flexible planar form of the implantable material is delivered locally to a site within the patient's body at or near the traumatic injury site or at a surgically-exposed traumatic injury site or interior cavity at, adjacent to or in the vicinity of a traumatic injury site. In one case, at least one piece of the implantable material is applied to a desired site by applying the implantable material at or around the traumatic injury site. The implantable material need only be implanted in an amount effective to treat a damaged or injured site.

Examples

1. Proliferative Phase of Wound Healing

[0116] Fibroblasts are critical to the proliferative phase of wound healing. Following the inflammatory response, fibroblasts are the first cells to infiltrate the wound area and are primarily responsible for the deposition of new extracellular matrix at the wound area to restore structure and function to the injured or damaged tissue.

[0117] To evaluate fibroblast migration, the regulation of fibroblast migration associated with cultured endothelial cells is determined. Human foreskin fibroblast cells are sparsely seeded in 12 or 24 well tissue culture plates. The cells are grown to confluence and the media is then replaced with Dulbecco's modified Eagle's media (DMEM) containing 0.5% FBS and PS for 24 hours to growth arrest the cells. Cultures are scratched with a 250 ul sterile tip and washed twice with collection media. The following media conditions are then added to the wells: (1) Collection Media alone (CM) or conditioned media prepared from the implantable material (i.e., post-confluent endothelial cells grown on a matrix). After 7-48 hours, injury images are taken and the degree of migration determined by image analysis using either ImagePro or ImageJ software.

[0118] The effect of conditioned media on fibroblast migration is determined by comparing the percent open area within the scratch wound region per well immediately before the addition of conditioned media with that after 7-48 hours of exposure to conditioned media and to control media. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits or enhances fibroblast migration by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control. [0119] Figures 2A and 2B are representative graphs depicting data of fibroblasts treated according to the fibroblast migration assay described above. In particular, populations of human foreskin fibroblasts were grown to confluence, scratched with a 250 ul sterile tip and then treated with either collection media (CM- Control) or conditioned media from three different endothelial donor strains (EC- CM 1-3). Administration of the implantable material to the scratched population of fibroblasts resulted in improved migration of fibroblasts into the scratch region compared to control. The improved migration of fibroblasts into the scratch region indicates that the implantable material promotes the proliferative phase of wound healing and contributes to an enhanced therapeutic response to traumatic injury. Accordingly, administration of the implantable material to a site of injury or damage in an individual in need will improve the migration of fibroblasts into the site of injury or damage and contribute to an enhanced therapeutic response to the injury or damage in the treated individual.

2. Epithelialization Phase of Wound Healing [0120] Keratinocytes are critical to the epithelialization phase of wound healing. Following the migration of fibroblasts and the fibroblast's deposition of extracellular matrix, keratinocytes traverse the newly created extracellular matrix while secreting growth factors and basement membrane proteins. Keratinocytes and the basement membrane proteins they secrete enable epithelial cells to subsequently infiltrate and epithelialize the wound area to restore structure and function to the injured or damaged tissue.

[0121] To evaluate keratinocyte proliferation, the regulation of keratinocyte proliferation associated with cultured endothelial cells will be determined. Porcine or human keratinocytes are sparsely seeded in 24 or 96 well tissue culture plates in keratinocyte growth medium (KGM, Lonza Bioscience, Basel, Switzerland). The cells are allowed to attach for 24 hours. The media is then replaced with keratinocyte basal media (KBM) containing 0.2% FBS for 48-72 hours to growth arrest the cells. Conditioned media is prepared from post-confluent endothelial cell cultures, diluted 1 :1 with 2X keratinocyte growth media and added to the cultures. A positive control for keratinocyte growth is included in each assay. After three to four days, the number of cells in each sample is enumerated using a Coulter Counter or determined by colorimetric analysis after the addition of a dye.

[0122] The effect of conditioned media on keratinocyte proliferation is determined by comparing the number of keratinocytes per well immediately before the addition of conditioned media with that after three to four days of exposure to conditioned media, and to control media (standard growth media with and without the addition of growth factors). The magnitude of keratinocyte growth associated with the conditioned media samples are compared to the magnitude of keratinocyte growth associated with the positive control. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits or enhances keratinocyte proliferation by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control.

[0123] To evaluate keratinocyte migration, the regulation of keratinocyte migration associated with cultured endothelial cells is determined. Keratinocytes are sparsely seeded in 12 or 24 well tissue culture plates in keratinocyte growth medium (KGM). The cells are grown to confluence. The media is then replaced with Dulbecco's modified Eagle's media (DMEM) containing 0.5% FBS and PS for 24 hours to growth arrest the cells. Cultures are scratched with a 250 ul sterile tip and washed twice with collection media. The following media conditions are then added to the wells: (1) Collection Media alone (CM), or conditioned media prepared from the implantable material (i.e., post-confluent endothelial cells grown on a matrix). After 16-24 hours, injury images are taken and the degree of migration determined by direct visualization.

[0124] The effect of conditioned media on keratinocyte migration is determined by comparing the number of keratinocytes within the scratch wound region per well immediately before the addition of conditioned media with that after 16-24 hours of exposure to conditioned media and to control media. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits or enhances keratinocyte migration by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control.

[0125] It is expected that administration of the implantable material to the scratched population of keratinocytes will result in improved proliferation and migration of keratinocytes into the scratch region compared to control. The improved proliferation and migration of keratinocytes into the scratch region should indicate that the implantable material promotes the epithelialization phase of wound healing and, therefore, contributes to an enhanced therapeutic response to traumatic injury. Accordingly, administration of the implantable material to a site of injury or damage in an individual in need will improve the proliferation and migration of keratinocytes into the site of injury or damage and contribute to an enhanced epithelialization phase and therapeutic response to the injury or damage in the treated individual.

3. Incisional Wound Healing

[0126] The rat model described by Warner et al. (Exp. MoI. Pathol., 2007 May 18.) will be studied to demonstrate treatment and management of traumatic injuries resulting from dermal incisions. Each animal will undergo two standardized hind limb dermal incisions. The animals will be maintained similarly. On each animal, one incision will receive an effective amount of the implantable material at or near the incisional wound site post-incision; the other incision will receive either no treatment or sham treatment with a biocompatible material. Healing time, hemostatis, epithelialization, inflammation, collagen accumulation, wound size over time and scar formation will be monitored over time by visual observation, measurement of wound contraction, ultrasound, MRI, CT scan or by sacrificing the animals and examining the incisional wound sites microscopically and histologically. It is expected that hind limb incisions treated with the implantable material will display decreased healing time, improved epithelialization, controlled fibroblast proliferation, controlled collagen accumulation, reduced incidence of dehiscence and/or reduced scar formation at the incisional wound site.

4. Excisional Wound Healing

[0127] To evaluate wound healing, the regulation of normal wound healing associated with cultured endothelial cells was evaluated in rats using the rat excisional model described by Wanda A. Dorsett-Martin (Wound Rep. Reg., 2004;12:591-599). Each animal underwent two dermal excisional wounds, which were covered with a breathable and transparent medical dressing. Wounds received either sham treatment or implantable material. Eleven days after injury, images were taken and the degree of healing was determined by ImagePro software analysis. The effect of implantable material on normal wound healing was determined by comparing the non-healed area of sham treatment with that of the implantable material.

[0128] Healing time, hemostatis, epithelialization, inflammation, collagen accumulation, wound size over time and scar formation will be monitored over time by visual observation, measurement of wound contraction, ultrasound, MRI, CT scan or by sacrificing the animals and examining the excisional wound sites microscopically and histologically. It is expected that excisions treated with the implantable material will display decreased healing time, improved epithelialization, controlled fibroblast proliferation, controlled collagen accumulation, reduced incidence of dehiscence and/or reduced scar formation at the excisional wound site.

[0129] Figure 3 represents a graph depicting wound healing data obtained from wounds treated according to the wound healing study described above. Two excisional wounds were made on the back of rats and either treated with implantable material or sham treatment. After 1 1 days, rats were sacrificed and images were taken. Administration of the implantable material to the excised wound site resulted in improved epithelialization and controlled healing of the wound region compared to control. Accordingly, administration of the implantable material to a site of injury or damage in an individual in need will improve the healing response including improved healing time, hemostatis, epithelialization, inflammation, collagen accumulation, wound size over time and scar formation at the site of injury or damage and contribute to an enhanced therapeutic response to the injury or damage in the treated individual.

5. Smooth Muscle Cell Proliferation and Migration

[0130] To evaluate smooth muscle cell proliferation, the regulation of smooth muscle cell proliferation associated with cultured endothelial cells is determined. Porcine or human aortic smooth muscle cells are sparsely seeded in 24 or 96 well tissue culture plates in smooth muscle cell growth medium (SmGM-2, Lonza BioScience, Basel, Switzerland). The cells are allowed to attach for 24 hours. The media is then replaced with smooth muscle cell basal media (SmBM) containing 0.2% FBS for 48-72 hours to growth arrest the cells. Conditioned media is prepared from the implantable material (i.e., from post-confluent endothelial cells grown on a matrix), diluted 1 :1 with 2X SMC growth media and added to the cultures. A positive control for inhibition of smooth muscle cell growth is included in each assay. After three to four days, the number of cells in each sample is enumerated using a Coulter Counter or determined by colorimetric analysis after the addition of a dye.

[0131] The effect of conditioned media on smooth muscle cell proliferation is determined by comparing the number of smooth muscle cells per well immediately before the addition of conditioned media with that after three to four days of exposure to conditioned media, and to control media (standard growth media with and without the addition of growth factors). According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits or enhances smooth muscle cell proliferation by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control.

[0132] To evaluate smooth muscle cell migration, the regulation of smooth muscle cell migration associated with cultured endothelial cells is determined. Porcine or human aortic smooth muscle cells are seeded in 12 well tissue culture plates in smooth muscle cell growth medium (SmGM-2, Lonza BioScience, Basel, Switzerland) and grown to confluence. The media is then replaced with Dulbecco's modified Eagle's media (DMEM) containing 0.2% FBS and penicillin and streptomyocin (PS) for 24 hours to growth arrest the cells. Cultures are scratched with a 250 ul sterile tip and washed twice with collection media (phenol red free EBM and 0.5% FBS). The following media conditions are then added to the wells: (1) Collection Media alone (CM), CM supplemented with 10% FBS (total 10.5% FBS) or conditioned media prepared from the implantable material (i.e., post- confluent endothelial cells grown on a matrix. After 16-24 hours, injury images are taken and the degree of migration determined by direct visualization.

[0133] The effect of conditioned media on smooth muscle cell migration is determined by comparing the number of smooth muscle cells within the scratch wound region per well immediately before the addition of conditioned media with that after 16-24 hours of exposure to conditioned media, and to control media (collection media with or without the addition of 10% FBS).

[0134] Figure 4 is a graph depicting the percentage of open area present in the scratch area at 0 hours and at 24 hours for three different endothelial cell donor strains. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits or enhances smooth muscle cell migration by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control.

6. Severe Venous Hemorrhage and Hepatic Injury Healing [0135] The swine model described by Pusateri et al. (J. Trauma, 2003 Sep;

55(3):518-26.) will be studied to demonstrate treatment and management of traumatic injuries resulting from severe venous hemorrhage and hepatic injury. Pigs will undergo standardized liver injuries. Two groups of animals will be maintained similarly, except the treatment group will receive an effective amount of the implantable material at or near the traumatic injury site(s) post-injury when venous hemorrhage and/or hepatic injury are evident. Healing time, hemostatis, inflammation, collagen accumulation and scar formation will be monitored over time by ultrasound, MRI, CT scan or by sacrificing the animals and examining the traumatic injury sites microscopically and/or histologically. It is expected that pigs treated with the implantable material will display improved hepatic regeneration, controlled fibroblast proliferation, controlled collagen accumulation, reduced incidence of dehiscence and/or reduced scar formation at the traumatic injury site.

7. Burn Healing

[0136] The swine burn model described by Cuttle et al. (Burns, 2006 Nov., 32(7):806-20.) will be studied to demonstrate treatment and management of traumatic injuries resulting from deep dermal partial thickness burn injuries. Pigs will undergo deep dermal partial thickness contact burns using water at 92 0C for 15 seconds. Two groups of animals will be maintained similarly, except the treatment group will receive an effective amount of the implantable material at or near the burn injury site post-injury when dermal burns are evident. Re-epithelialization, collagen accumulation and scar formation will be monitored over time by direct examination, ultrasound, MRI, CT scan or by sacrificing the animals and examining the burn injury sites microscopically and/or histologically. It is expected that pigs treated with the implantable material will display improved epithelialization, controlled fibroblast proliferation, controlled collagen accumulation and/or reduced scar formation at the burn injury site.

8. Adjunct Therapy

[0137] The rat model described by Warner et al. (Exp. MoI. Pathol., 2007 May 18.) will be studied to demonstrate adjunct treatment and management of primary critical care or surgical interventions of traumatic injuries resulting from dermal incisions. Each animal will undergo two standardized hind limb dermal incisions. The animals will be maintained similarly. On each animal, one incision will be closed with sutures and will receive an effective amount of the implantable material at or near the incisional wound site post-incision; the other incision will also be closed with sutures but will receive no treatment with a biocompatible material. Healing time, hemostatis, epithelialization, inflammation, collagen accumulation, wound closure, wound size over time and scar formation will be monitored over time by visual observation, measurement of wound contraction, ultrasound, MRI, CT scan or by sacrificing the animals and examining the incisional wound sites microscopically and histologically. It is expected that hind limb incision sutures treated with an adjunct administration of the implantable material will display decreased healing time, improved epithelialization, controlled fibroblast proliferation, controlled collagen accumulation, reduced incidence of dehiscence and/or reduced scar formation at the incised and sutured wound site.

[0138] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

ClaimsWhat is claimed is:
1. A method of treating a traumatic injury in an individual in need thereof, the method comprising:
contacting with an implantable material a traumatic injury at, adjacent to or in the vicinity of an area of damage or injury, wherein said implantable material comprises a biocompatible matrix and cells and further wherein said implantable material is in an amount effective to treat the traumatic injury in said individual.
2. The method of claim 1 wherein the biocompatible matrix is a flexible planar material.
3. The method of claim 1 wherein the biocompatible matrix is a flowable composition.
4. The method of claim 1 wherein the cells are endothelial, epithelial, endothelial-like, epithelial-like, non-endothelial or non-epithelial cells, stem cells, endothelial progenitor cells or analogs thereof.
5. The method of claim 1 wherein the cells comprise a co-culture of at least two different cell types.
6. The method of claim 1 wherein the implantable material further comprises a tissue sealant.
7. The method of claim 1 wherein the traumatic injury is an open or closed wound comprising an incision, incised wound, laceration, abrasion, puncture wound, penetration wound, deep wound, gunshot wound, contusion, hematoma or crushing injury.
8. The method of claim 1 wherein the traumatic injury is a burn.
9. The method of claim 1 wherein the traumatic injury is a diabetic ulcer, a pressure ulcer or a venous ulcer.
10. The method of claim 1 wherein the traumatic injury is a pathological response to a primary traumatic injury.
1 1. The method of claim 10 wherein the pathological response comprises fibrosis, a stricture, an adhesion, a contracture, a keloid, or a hypertrophic scar.
12. The method of claim 1 wherein the implantable material is applied to a surface of the traumatic injury.
13. The method of claim 1 wherein the implantable material regulates inflammation associated with said traumatic injury.
14. The method of claim 1 wherein the implantable material regulates smooth muscle cell proliferation and/or migration.
15. The method of claim 1 wherein the implantable material regulates fibroblast proliferation and/or migration.
16. The method of claim 1 wherein the implantable material regulates keratinocyte proliferation and/or migration.
17. The method of claim 1 wherein the implantable material regulates collagen deposition and/or accumulation.
18. The method of claim 1 wherein the implantable material regulates tissue remodeling.
19. The method of claim 1 wherein the implantable material regulates scar formation.
20. The method of claim 1 wherein the implantable material regulates re- epithelialization.
21. The method of claim 1 wherein the implantable material regulates neovascularization .
22. The method of claim 1 wherein the implantable material regulates extracellular matrix formation and/or degradation.
23. The method of claim 1 wherein the implantable material reduces the incidence of dehiscence.
24. The method of claim 1 wherein the implantable material reduces pain associated with the traumatic injury.
25. The method of claim 1 wherein the implantable material reduces healing time of the traumatic injury.
26. A method of providing an adjunct therapy to a primary therapeutic intervention of a traumatic injury in an individual in need thereof, the method comprising:
contacting with an implantable material a site at, adjacent to or in the vicinity of an area of a primary therapeutic intervention, wherein said implantable material comprises a biocompatible matrix and cells and further wherein said implantable material is in an amount effective to treat the primary therapeutic intervention in said individual.
27. The method of claim 26 wherein the primary therapeutic intervention comprises suturing, stapling, ablation or debridement.
28. A composition suitable for the treatment or management of a traumatic injury, the composition comprising a biocompatible matrix and cells, wherein said composition is in an amount effective to treat or manage the traumatic injury.
29. The composition of claim 28 wherein the biocompatible matrix is a flexible planar material.
30. The composition of claim 28 wherein the biocompatible matrix is a flowable composition.
31. The composition of claim 30 wherein the flowable composition further comprises an attachment peptide and the cells are engrafted on or to the attachment peptide.
32. The composition of claim 28 wherein the implantable material further comprises a tissue sealant.
33. The composition of claim 28 wherein the cells are endothelial, epithelial, endothelial-like, epithelial-like, non-endothelial or non-epithelial cells, stem cells, progenitor cells or analogs thereof.
34. The composition of claim 28 wherein the cells comprise a population of cells selected from the group consisting of near-confluent cells, confluent cells and post- confluent cells.
35. The composition of claim 28 wherein the cells are at least about 80% viable.
36. The composition of claim 28 wherein the cells are not exponentially growing cells.
37. The composition of claim 28 wherein the cells are engrafted to the biocompatible matrix via cell to matrix interactions.
38. The composition of claim 28 wherein the cells comprise a co-culture of two or more different cell types.
39. The composition of claim 28 wherein the composition further comprises a second therapeutic agent.
40. The composition of claim 28 wherein the composition further comprises an agent that inhibits infection.
41. The composition of claim 28 wherein the composition further comprises an anti-inflammatory agent.
PCT/US2008/009540 2007-08-08 2008-08-08 Materials and methods for treating and managing wounds and the effects of trauma WO2009020650A3 (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9238090B1 (en) 2014-12-24 2016-01-19 Fettech, Llc Tissue-based compositions
WO2016054592A1 (en) * 2014-10-03 2016-04-07 Cytori Therapeutics, Inc. Use of regenerative cells in mitigating burn progression and improving skin graft incorporation and healing
US9463203B2 (en) 2001-12-07 2016-10-11 Cytori Therapeutics, Inc. Methods of using regenerative cells in the treatment of cartilage defects
US9486484B2 (en) 2008-08-19 2016-11-08 Cytori Therapeutics, Inc. Methods of using adipose tissue-derived cells in the treatment of the lymphatic system and malignant disease
US9492483B2 (en) 2001-12-07 2016-11-15 Cytori Therapeutics, Inc. Methods of using regenerative cells to treat a burn
US9597395B2 (en) 2001-12-07 2017-03-21 Cytori Therapeutics, Inc. Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions
US9849149B2 (en) 2001-12-07 2017-12-26 Cytori Therapeutics, Inc. Methods of using regenerative cells in the treatment of erectile dysfunction

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003007873A2 (en) * 2001-07-19 2003-01-30 Prochon Biotech Ltd. Plasma protein matrices and methods for their preparation
US20030091543A1 (en) * 2001-10-26 2003-05-15 Klein Matthew B. Therapeutic cell preparation grafts and methods of use thereof
WO2003076604A2 (en) * 2002-03-06 2003-09-18 University Of Cincinnati Surgical device for skin therapy or testing
WO2006062871A2 (en) * 2004-12-08 2006-06-15 Pervasis Therapeutics, Inc. Materials and methods for minimally-invasive administration of a cell-containing flowable composition
WO2006116357A1 (en) * 2005-04-21 2006-11-02 Massachusetts Institute Of Technology Materials and methods for altering an immune response to exogenous and endogenous immunogens, including syngeneic and non-syngeneic cells, tissues or organs

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003007873A2 (en) * 2001-07-19 2003-01-30 Prochon Biotech Ltd. Plasma protein matrices and methods for their preparation
US20030091543A1 (en) * 2001-10-26 2003-05-15 Klein Matthew B. Therapeutic cell preparation grafts and methods of use thereof
WO2003076604A2 (en) * 2002-03-06 2003-09-18 University Of Cincinnati Surgical device for skin therapy or testing
WO2006062871A2 (en) * 2004-12-08 2006-06-15 Pervasis Therapeutics, Inc. Materials and methods for minimally-invasive administration of a cell-containing flowable composition
WO2006116357A1 (en) * 2005-04-21 2006-11-02 Massachusetts Institute Of Technology Materials and methods for altering an immune response to exogenous and endogenous immunogens, including syngeneic and non-syngeneic cells, tissues or organs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BELLO Y M ET AL: "Tissue-engineered skin. Current status in wound healing" AMERICAN JOURNAL OF CLINICAL DERMATOLOGY, ADIS, US, vol. 2, no. 5, 1 January 2001 (2001-01-01), pages 305-313, XP009098074 ISSN: 1175-0561 *
EHRENREICH M ET AL: "Update on tissue-engineered biological dressings" TISSUE ENGINEERING 200609 US, vol. 12, no. 9, September 2006 (2006-09), pages 2407-2424, XP002510284 ISSN: 1076-3279 *
HUTMACHER D W ET AL: "MATRICES FOR TISSUE-ENGINEERED SKIN" DRUGS OF TODAY / MEDICAMENTOS DE ACTUALIDAD, J.R. PROUS SS.A. INTERNATIONAL PUBLISHERS, ES, vol. 38, no. 2, 1 January 2002 (2002-01-01), pages 113-133, XP009060963 ISSN: 0025-7656 *
METHE H ET AL: "Matrix adherence of endothelial cells attenuates immune reactivity: Induction of hyporesponsiveness in allo- and xenogeneic models" FASEB JOURNAL 200705 US, vol. 21, no. 7, May 2007 (2007-05), pages 1515-1526, XP002510285 ISSN: 0892-6638 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9511094B2 (en) 2001-12-07 2016-12-06 Cytori Therapeutics, Inc. Methods of using regenerative cells in the treatment of stroke and related diseases and disorders
US9511096B2 (en) 2001-12-07 2016-12-06 Cytori Therapeutics, Inc. Methods of using regenerative cells to treat an ischemic wound
US9463203B2 (en) 2001-12-07 2016-10-11 Cytori Therapeutics, Inc. Methods of using regenerative cells in the treatment of cartilage defects
US9849149B2 (en) 2001-12-07 2017-12-26 Cytori Therapeutics, Inc. Methods of using regenerative cells in the treatment of erectile dysfunction
US9492483B2 (en) 2001-12-07 2016-11-15 Cytori Therapeutics, Inc. Methods of using regenerative cells to treat a burn
US9504716B2 (en) 2001-12-07 2016-11-29 Cytori Therapeutics, Inc. Methods of using adipose derived regenerative cells to promote restoration of intevertebral disc
US9597395B2 (en) 2001-12-07 2017-03-21 Cytori Therapeutics, Inc. Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions
US9872877B2 (en) 2001-12-07 2018-01-23 Cytori Therapeutics, Inc. Methods of using regenerative cells to promote epithelialization or neodermis formation
US9486484B2 (en) 2008-08-19 2016-11-08 Cytori Therapeutics, Inc. Methods of using adipose tissue-derived cells in the treatment of the lymphatic system and malignant disease
WO2016054592A1 (en) * 2014-10-03 2016-04-07 Cytori Therapeutics, Inc. Use of regenerative cells in mitigating burn progression and improving skin graft incorporation and healing
US9238090B1 (en) 2014-12-24 2016-01-19 Fettech, Llc Tissue-based compositions

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