EP2040766A2 - Système d'implant intégré (sii) biocompatible, biodégradable et bioactif comprenant une matrice polymère poreuse stérile biocompatible et un gel, avec intégration in situ de la structure matricielle tridimensionnelle - Google Patents

Système d'implant intégré (sii) biocompatible, biodégradable et bioactif comprenant une matrice polymère poreuse stérile biocompatible et un gel, avec intégration in situ de la structure matricielle tridimensionnelle

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Publication number
EP2040766A2
EP2040766A2 EP07725905A EP07725905A EP2040766A2 EP 2040766 A2 EP2040766 A2 EP 2040766A2 EP 07725905 A EP07725905 A EP 07725905A EP 07725905 A EP07725905 A EP 07725905A EP 2040766 A2 EP2040766 A2 EP 2040766A2
Authority
EP
European Patent Office
Prior art keywords
cells
matrix
implant system
gel
integrated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07725905A
Other languages
German (de)
English (en)
Inventor
Manuel Eduardo Young Anze
Cristian Andrés ACEVEDO GUTIÉRREZ
Fernando Antonio Albornoz Marquez
Caroline Ruth Weinstein Oppenheimer
Alexis Roobins Aceituno Alvarez
Donald Irving Brown Gonzalez
Sergio Miguel Tapia Murua
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fundacion Instituto De Seguridad Del Trabajo
Universidad Tecnica Federico Santa Maria USM
Universidad de Valparaiso
Original Assignee
Fundacion Instituto De Seguridad Del Trabajo
Universidad Tecnica Federico Santa Maria USM
Universidad de Valparaiso
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fundacion Instituto De Seguridad Del Trabajo, Universidad Tecnica Federico Santa Maria USM, Universidad de Valparaiso filed Critical Fundacion Instituto De Seguridad Del Trabajo
Publication of EP2040766A2 publication Critical patent/EP2040766A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers

Definitions

  • BIOCOMPATIBLE BIODEGRADABLE AND BIOACTIVE
  • COMPRISING A BIOCOMPATIBLE STERILE POROUS POLYMERIC MATRIX AND A GEL INTEGRATING IN SITU THE TRIDIMENSIONAL MATRIX STRUCTURE
  • the present invention is related to an Integrated Implant System easy to use and handle for being applied in surgical grafting. It is constituted as a gel-matrix-cells integrated system, a feature that allows providing implants in a brief time period, for covering a great skin extension to be treated, with a successful acceptance in patients with burns, chronic damage or wound skin, needing of a skin grafting.
  • Skin is the largest organ of the body, covering the body's outer surface area. Skin is composed of two main layers: the surface epithelia or epidermis, containing epidermal cells as keratinocytes, and the underlying connective tissue layer or dermis, containing dermal cells as fibroblasts.
  • the protection against injury and dehydration acting as a barrier against infections, perceiving or detecting environmental stimuli, excreting diverse substances, regulating the body temperature, and helping to maintain the hydric balance.
  • Substantially intact and healthy skin is needed , not only for the body welfare, but for its survival.
  • the skin health and integrity may be affected by congenital or acquired pathological conditions, either acute or chronic, for which the natural skin repair and regeneration processes may be insufficient. These conditions include burns, wounds, ulcers, infections, congenital diseases and/or abnormalities. Therefore, a non-appropriate condition and state of the skin is extremely harmful, since it affects the health condition and the organism existence. Those patients that have been affected by burns in a vast surface area of their bodies often require an immediate and extensive replacement of skin. In addition, some less life threatening conditions can be present, however considered as chronic conditions of skin, for instance as occurs for passive hyperemia, the diabetic ulcers or decubitus ulcer, which may result in more severe conditions, if no treatment is applied, particularly since those patients exhibiting these conditions suffer an underlying pathology. On the other hand, the morbidity and mortality drop-off in this kind of patients depends of the appropriate and effective restoration of the skin structure and function.
  • Skin substitutes may be used for treating these or other conditions.
  • the desirable properties for skin substitutes are availability and handling, low rejection degree, high adherence degree, highly independent from a skin donor, relative easiness for being produced from a skin biopsy of a minimal size, and the feature of a cost-effective production and use.
  • Several methods for elaborating skin substitutes have been assayed, which satisfy some or all these requirements, but with varied success degrees.
  • US 2003/0170892 describes a device comprising a cell- free biocompatible reticulated matrix, wherein a first (layer) of dermal cells are cultured, and over these cells, epidermal cells are grown. Further, it describes the method for producing said device, useful for applications in skin wounds, in therapeutic treatment and/or for the in vitro assay of human skin. Providing similar structures and functions to a normal healthy skin, as a barrier function.
  • the described implant corresponds to a device comprising cells grown in the surface and in clearly differentiated layers.
  • the required time for applying the same is longer than 24 hours, considered from the moment when the dermal cells culture is available for being seeded over the matrix .
  • CN 1562392 describes a method for preparing an artificial skin in an active bi-layer supported on a collagen sponge, which is cultured in a bioreactor with a fibroblasts suspension for generating the dermis and on top of this, epidermal cells are seeded.
  • Structural differences with the corresponding graft of the present invention can be clearly distinguished since this document mentions a layer type of graft exhibiting cells on the surface, wherein the seeding method is less efficient, more complex, more expensive and takes longer time for reaching similar results.
  • the same differences regarding the present invention are observed for the patent application CN 1468634, wherein an artificial skin implant with a double film is described, with superficial growth of epidermal cells over a fibroblasts-PGA structure.
  • US 6,733,530 describes a device wherein autologous keratinocytes are grown over a biocompatible substrate, which is pre-seeded with allogenic or autologous dermal fibroblasts, which can be applied over an artificial skin substrate grafted to the receptor patient.
  • the layered and superficial structure of the device, and the method for preparing the same, described in this document, makes the present invention a different device with clear advantages regarding the applications of the same.
  • a fibrin cell support to form cell cultures. It is possible to establish cell cultures on this support, such as keratinocytes cultures. In a disclosed embodiment, it is possible to incorporate cells in the fibrin gel structure.
  • the above description considerably differs from the present invention device, since the present invention corresponds to a gel -matrix-cells integrated device, which exhibits better handling characteristics as it comprises a biocompatible support available as the porous matrix, which contributes to device strength, resistance and flexibility.
  • the presence of a biocompatible support does not form part of what is described in the document, since the fibrin gel is used in combination with a preliminary treatment of the wound with artificial skin (IntegraTM) .
  • the product is applied as a paste or spray on the wound, which implies great difficulties for fixing the fibrin gel to the wound and for being properly accepted by the grafted patient. Accordingly, combined additional or supplementary treatments are needed, in order to allow the graft fixing to the wound, since the fibrin gel slides from the application site. In the present invention no fixing or additional treatments are required in order to maintain the device on the application site and to start the regeneration of the damaged skin.
  • the incorporation of polymers these function only as fibrin carriers, furthermore these are not incorporated into the fibrin gel, as in the present invention. The above provides a different support with a closer in situ integration of the matrix-gel, corresponding to one of the present invention contributions.
  • the patent application WO02078721 describes a system for providing an autologous in situ implant, wherein a keratinocytes and fibroblasts suspension containing fibrin, are mixed with a thrombin, fibrinogen and collagenase solution.
  • the reference differs from the present invention, as it is not related to an integrated device comprising a biocompatible support as a porous matrix.
  • the strength, handling and adherence properties are improved and optimized, regarding the technical solution disclosed in the document WO02078721.
  • the main purpose of the skin substitutes is to save the patient's life, providing a coating or barrier, which prevents the skin dehydration and infection.
  • a second objective is to allow for a functional healing, which additionally should be cosmetically acceptable.
  • Different cell types and material combinations, which have been evaluated for producing cutaneous substitutes, are nowadays commercially available. Most of said commercial products are formed by allogenic cells-containing matrix, generally from neonatal foreskin, which exhibits the advantage of containing a higher number of stem cells for keratinocytes, potential mitogenic properties, an exacerbated metabolism and minimal antigenicity (1 ' 2) .
  • EPICEL ® is composed by autologous keratinocytes arranged on a paraffined gauze, which is a non biodegradable material. Said cells are obtained from a full thickness biopsy from the injured subject, the cells are subsequently grown over a layer of murine fibroblasts, which have previously been irradiated. Thus, stratified keratinocytes layers are achieved, containing from two to eight layers' 3 '. In this system, rejection is totally avoided and the cells are permanently incorporated into the tissue. However, it is fragile and with poor cosmetic ability of maintaining an acceptable appearance in the patients.
  • This device can be used combined with HyaffTM a support containing dermis fibroblasts, wherein the fibroblasts can be autologous or allogenic.
  • HyaffTM a support containing dermis fibroblasts, wherein the fibroblasts can be autologous or allogenic.
  • These products correspond to devices wherein cells can be grown within their matrix, not only in the surface, nevertheless, these do not correspond to an in situ integrated system formed by matrix-gel cells as the one of the present invention.
  • Celaderm ® is a commercially available product, this product contains metabolically active foreskin-derived heterologous keratinocytes which however are unable of proliferating. This product has been used for treating chronic ulcer; and as a further advantage, it can be • cryo-preserved (6) . Some available studies reveal the effectiveness of this product in burns, however not compared with autografting (7> . This product exhibits significant differences regarding the present invention, as it does not involve an integrated system.
  • Dermagraft ® an approved substitute for treating diabetic foot ulcer. In this case, fibroblasts are obtained from neonatal foreskin and cultured on a polyglactine matrix, during approximately three weeks. In this term, cells secrete matrix proteins, providing a tridimensional in situ matrix which serves as a dermis substitute. The product is delivered cryo-preserved and requires thawing and washing to be used (8) .
  • Transcyte ® originally known as Dermagraft-TC ®
  • Said product is prescribed as a temporary cover or coating for burn wounds which have been split by surgery, as an alternative for cadaver skin (9fl0> .
  • LSE allogenic product
  • the LSE corresponds to a cutaneous substitute composed by a collagen matrix with viable fibroblasts and a stratified epidermal layer.
  • One difference regarding the present invention is that it corresponds to an integrated device without forming stratified or differentiated epithelia.
  • the OrCel ® product is very similar to the previous one with the difference that the dermal matrix is a porous, crosslinked type II collagen sponge, instead of a gel.
  • the sponge is non symmetrical, in a way that a side is covered by a layer of an acid soluble atelocollagen gel, for sealing the macroscopic pores.
  • Fibroblasts are grown on or within the porous side of the collagen sponge, while the keratinocytes are cultured in the non-porous side, covered by the gel of this collagen matrix. The cells seeded matrix is kept submerged for inhibiting differentiation and stratification of keratinocytes.
  • the time and ratio for the fibroblasts and keratinocytes seeding are designed for controlling the cellular density and the cytokines expression in the final product. It has been seen that the co-culture of fibroblasts and keratinocytes exhibits synergic effects over the production levels of some cytokines and growth factors' 12"131 .
  • the PolyActiveTM product uses polyethylene oxide/polybutylphtalate (PEO/PBT) and may use the own patient fibroblasts for the dermis, and the own patient keratinocytes cultured for the epidermis.
  • IntegraTM which uses a collagen-glycosaminoglycan matrix
  • GAG providing a non cellular or acellular dermal component, and which may use a thin autograft or other devices containing cells.
  • tissue engineering discipline intended to the generation of a solution for burned patients, or with disability scars or cutaneous ulcers, is an area of great scientific interest.
  • Numerous scientific publications can be found, wherein products are described, which contain autologous or heterologous cells, in combination with some different nature matrix.
  • the market known and existing products exhibit differences and disadvantages regarding the present invention.
  • the main differences are that said devices do not correspond to an in situ integrated device with a fibrin gel and a biocompatible polymer.
  • the content of bovine nature components can be found; therefore, it should be considered that these products soon will no longer be used, due to the appearance of prion contamination cases in USA, the need of long incubating or preparation periods, and the differentiation degree
  • a clear disadvantage of the devices for grafting is the need of adhesives for fixing the implants onto the injuries.
  • Mechanical devices or organic polymers can be used as attachers (stapples or gauze) , wherein the main objective of these is to achieve the graft take, being this understood as the fixing of the device to the damaged skin, achieving the placement in the site to be treated.
  • Lyophilized pigskin has been used to immediate treatment of patients after the occurrence of a traumatic event. This is a transitory solution, since it only allows extending the patient life, while it is possible to apply definitive solutions.
  • solutions have been developed, on the base of materials that help for burns healing. However, they are cell- free solutions, which unlikely can be compared with the complexity and with the contribution and efficiency in the wound healing, which implies an integrated system with cellular components and a biocompatible matrix developed as part of the present invention.
  • the present invention provides an integrated implant system, being this understood as an intimate link among cells-gel-support, wherein can be combined more than one type of dermal or epidermal, autologous, allogenic, xenogenic or chimeric cells, exhibiting favourable: handling characteristics, time for obtaining the same, application availability, adherence, almost no rejection by the patient, better cellular development and easy conservation. Further, it is informed an easy and fast process for obtaining these integrated implants, wherein the cells are incorporated in the support matrix, providing a device for immediately being applied or conserved.
  • Figure 1 shows a photography of the biocompatible cell free, microporous matrix, which is used in the present invention as the support for the integrated implant.
  • Photograph A corresponds to the polymerized matrix and
  • photograph B corresponds to the cross-linked matrix.
  • Figure 2 corresponds to a table wherein different assays are identified, which are carried out in rabbits (column 1) .
  • the animal was surgically prepared, and then the integrated implant of the present invention was applied with different amounts of cells per implant area unit (column 2) .
  • the ratio between keratinocytes (column 3) and fibroblasts (column 4) that are seeded in the implant varies, with the purpose of observing the differences in the wounds response as function of different proportions of skin cells of the implant .
  • Figure 4 Presents a plot showing the kinetics of cellular proliferation in an integrated implant system (IIS) like the one of the present invention and the proliferation in a monolayer system within a culture flask, for fibroblasts, at 4xlO 4 cell/cm 2 , as the initial concentration.
  • IIS integrated implant system
  • the high proliferation up to 300% in the first 48 hours, turns this development in an effective tool at 24 hours, wherein the growth is of 200%, an advantageous situation for the treatment of critical patients.
  • the present invention corresponds to an integrated implant system (IIS) , wherein into a matrix or porous support a gel or polymer components is incorporated and get absorbed into the matrix and in situ clots.
  • IIS integrated implant system
  • the present invention corresponds to an integrated implant system (IIS) , wherein into a matrix or porous support are incorporated the components of a gel or polymer with an active agent or with the desired cells, which are absorbed in the inner part of the matrix, coagulating in situ.
  • IIS integrated implant system
  • the active agent can be selected from the group consisting of: plants, animals or microorganisms natural extracts; chemicals, pharmaceuticals, cosmetics or polymers, their salts or derivatives; macromolecules or microorganisms.
  • IIS applications may be many and multiple, depending on the presence of active agents and/or cells. In the case it contains cells, the applications will depend of the features and the cell type to be applied in the IIS, the type of porous matrix and the polymer type to be integrated. Even, the SII could be used as a carrier, for therapeutic evaluation, for safety or efficiency of natural, chemical, pharmaceutical, cosmetic products, polymers or macromolecules. Nevertheless, in a preferred modality the application will be related to treating, curing, or delivering a benefit for pathologies o for chronic or acute injuries, affecting the skin of a mammal, preferably humans. Additionally, the invention comprises a method for preparing the IIS from isolated cells, which will be integrated into the matrix forming the gel -matrix- cells IIS.
  • the applications will be intended to problems related with burns, wounds, ulcers, infections, surgeries, diseases and/or congenital abnormalities of the skin.
  • the main objective and application of the invention is to be used as a surgical skin implant for burned people, chronic diseases or for regenerative treatments.
  • the IIS can be used on a non damaged surface, a minimally damaged surface, or a surface that has surgically been prepared, and which for other reasons requires an IIS graft.
  • the IIS is composed by a biocompatible cell -free reticulated matrix, known as support or scaffold, providing an easy to handle support.
  • Said scaffold may be acquired from those commercially available or may be prepared according to a production protocol, as is described below in the present invention or by other production protocol for preparing biocompatible polymers.
  • a matrix composed of a full length natural or synthetic protein, or a polypeptide, as well as inorganic or organic polymers or their mixture For example, a lyophilized collagen sponge can be used, either alone or combined with a carbohydrate
  • the collagen can be bovine tissue collagen, from bovine tendon, or from other bovine sources (bone or muscle) , other xenogenic sources
  • proteins such as elastin or reticulin, or natural or synthetic amino acid polymers, may also be used.
  • One preferred embodiment for the matrix used in the present invention is composed by gelatin-chitosan-hyaluronic acid.
  • the matrix can be commercially available or it can be obtained according to the following process.
  • a matrix containing a concentration between about 0.5 to 5%, preferably between about 1 to about 3% of gelatin is used, a concentration between about 0.5 to 3%, preferably between about 1 to about 2% of chitosan is used, and a concentration between about 0.5 to 2%, preferably between about 0.8 to about 0.5% of hyaluronic acid is used, and preferably these components are used in a weight ratio of 7:2:1, respectively.
  • the polymeric matrix may be prepared according to the following state of the art derived procedure (Haifeng L. et al . 2004) :
  • a gelatin solution (1% w/v) is mixed with a chitosan (2% w/v) solution, in 1% v/v acetic acid solution, together with a hyaluronic acid (0,01% w/v) solution.
  • Said mixture is homogenized by stirring at 5O 0 C, during 30 minutes, subsequently the mixture is poured in a container or device for shaping it, for example a Petri dish until the desired height.
  • the plate containing the mixture is cooled at 4°C, until a gel is formed, which may subsequently be frozen at -2O 0 C, during 8 hours, or at - 80 0 C during 6 hours.
  • Said frozen gel is carefully immersed in liquid nitrogen, during 2 to 5 minutes, and finally is lyophilized during 24-48 hours.
  • the lyophilized polymer is submerged in 20 mL of a 50 mM 2 -morpholine-ethane sulfonic acid (MES) solution, 90% ethanol , during 30 minutes at room temperature.
  • MES mM 2 -morpholine-ethane sulfonic acid
  • the cross-linking is achieved submerging the obtained matrix in 20 mL of a cross-linking solution which is composed by 50 mM MES, 30 mM 1-ethyl- (3 , 3-dimethyl- aminopropyl) carbodiimide (EDC) and 8 mM N- hydroxysuccinimide (NHS), in a 1:9 water : ethanol mixture.
  • EDC 1-ethyl- (3 , 3-dimethyl- aminopropyl) carbodiimide
  • NHS N- hydroxysuccinimide
  • the formed IIS is a biocompatible, cells- free, reticulated matrix, and which provides an easy to handle support, for a cells population to be integrated, previous to the implanting, comprising the components of a polymer or gel which is in situ polymerized.
  • said matrix may comprise the above indicated components, and may be obtained according to the above disclosed embodiments.
  • the cells used for being integrated within the matrix, embedded therein in order to form one embodiment of the SII may be obtained from the patient to be treated (autologous) , may be obtained from other human subject (allogenic), or may be obtained from other species (xenogenics) , or obtained from many other sources (chimeric) .
  • the cells used in the development of the IIS are autologous skin cells, either from dermis and/or epidermis.
  • epidermal cells which may be selected, for example, from keratinocytes, melanocytes, immunocytes, stem cells or others; and/or dermal cells, selected, per example, from fibroblasts, endothelial cells, immunocytes, nervous cells, myocytes, stem cells or others.
  • the cells which will form the IIS may be grown as a pure culture or as a mixed culture. Once a suitable number of cells are obtained, the cellular populations are harvested for their inclusion within the matrix. In the embodiments of the invention, the cells may be integrated into the matrix, in solutions containing a concentration of up to 8 x 10 6 , preferably between 2 X 10 2 to 4 X 10 6 , more preferably between 3.5 X 10 2 to 4 X 10 6 , and the most preferred about 1 X 10 5 cells/mL.
  • the used cells are dermal and/or epidermal cells
  • the ratio of the cells used for inoculating the matrix is comprised within the range of about 20:1 to 1:20 of dermal cells : epidermal cells, in a preferred manner in ratios from 1:1 to 1:10, more preferably between 1:2 and 1:5, considering the usual ratios as from 1:2 to 1:4.
  • the IIS is used as a temporary skin substitute.
  • the matrix may be seeded with cells having non autologous genotypes .
  • the invention is intended as a method to prepare said IIS for surgical grafting on skin wounds.
  • a biocompatible and porous matrix as those previously described, one of the gel components is deposited, therefore incorporating it easily and quickly into the matrix, after which the next component is added causing the in situ gel formation.
  • Said gelified matrix is incubated under appropriate conditions for forming the IIS of the invention.
  • a desired cells suspension is deposited, contained in one of the gel component, in order to easily and quickly incorporate the cells into the matrix, after which the next component is added, causing the in situ gel formation.
  • Said inoculated and gelified matrix is incubated under proper conditions for obtaining the integrated implant system (IIS) of the invention.
  • IIS integrated implant system
  • the IIS comprises dermal cells and/or epidermal cells population cultured under suitable conditions and which are obtained in the conventional manner, as described in the state of the art, which are deposited as a part of a cells suspension in a thrombin solution, onto the matrix. Then, the integration of said cellular suspension within the matrix is carried out, preferably through the use of a thrombin and fibrinogen system, in such a way that a fibrin polymer is produced in situ resulting in an gel like environment, which allows the cells to get embedded within the matrix.
  • a gel with adhesive properties such as the combinations of cyanoacrylate esters/water or amine, gelatin-resorcinol/aldehyde, natural bioadhesives/enzymes .
  • this IIS Once incubated this IIS, it is considered in condition or ready for being surgically grafted to the patient, which can be carried out during the first day, i.e. in a period no longer than 24 hours, in one embodiment of the invention, from the moment when the desired cells, available in a culture, are integrated into the matrix.
  • the availability of cultured cells highly reduces the need of a skin donor, in order to complete the closing of the extended and full thickness skin wounds.
  • the cells suspension has a concentration of up to 8 x 10 s cells/mL, preferably between 2 X 10 2 to 4 X 10 6 , more preferably between 3,5 X 10 2 to 4 X 10 6 , and in a preferred modality about 1 X 10 5 , wherein the cells are suspended in a thrombin solution at a concentration comprised between 25 to 750 NIH/mL (NIH: Enzymatic Activity Units) , more preferably 125 to 500 NIH/mL and in a preferred manner about 250 NIH/mL.
  • NIH Enzymatic Activity Units
  • the described fibrinogen solution can be used at a concentration comprised between 10 to 90 mg/mL, preferably between 10-50 mg/mL, preferably 15-30 mg/mL and more preferably about 20 mg/mL.
  • a fibrinogen solution at a ratio of 50 to 500 ⁇ L/cm 2 , preferably between 50-250 ⁇ L/cm 2 and more preferably of about 100 ⁇ L/cm 2 is applied onto the matrix, producing the fibrin gel formation, which gets incorporated, i.e. it gets integrated, into the matrix, providing a IIS wherein matrix and gel are closely integrated in the matrix depth.
  • the thrombin: fibrinogen ratio may fluctuate from 1:0.5 to 1:5, preferably 1:0.5 to 1:2.5 and in a more preferred ratio 1:1.
  • suspended cells can be added in said solution, in such a way that when the fibrinogen is added, a fibrin gel is formed, wherein the gel gets integrated into the matrix and together to this the applied cells, such that in the IIS, the components are closely related, in the full depth of the matrix, the gel, the cells with the matrix, as can be appreciated in figure 3.
  • the gel-matrix-cells IIS is submerged in a suitable media, which can be selected from DMEM/F-12, DMEM, RPMI or MEM, among others; preferably it is submerged in DMEM/F-12 culture media.
  • the IIS and the method of the invention exhibit a great contribution and advantage, since they incorporate both the main skin cellular populations, i.e. fibroblasts and keratinocytes .
  • both cells types are integrated, simultaneously, within the gel- matrix-cells IIS.
  • said cells are autologous and the keratinocytes are not differentiated, therefore, they have a great potential for proliferation.
  • the cells are included, i.e. embedded within the IIS.
  • the IIS performs as a carrier system, adapts to the shape of the wound, with an appropriate adjustment, which does not require of methods, devices or additional adhesives for fixing the IIS to the skin wound to be treated.
  • it is 100% biodegradable, having been identified the in vitro ability of the cells integrated into the IIS of moving to the IIS surroundings (data not shown) .
  • the carrier components are non- inert and exhibit antibacterial, chemotactic and proangiogenic properties, helping to an earlier wound healing.
  • the IIS does not require of additional supports neither of adhesives, which is highly beneficial since with a single and direct application the graft "starting" can be achieved, i.e. it gets adhered almost immediately, without the need of additional components or products for adhering it.
  • the integrated implant system Once the integrated implant system has been grafted to the patient, and once the biodegradable matrix has been reabsorbed by the body, the cells get organized in order to form a functional skin tissue.
  • the device comprises many of the properties and structures that are found in the normal non-damaged skin, and functions as in the usual way as does a non damaged skin, in order to protect the subject from the fluids loss and against bacterial infections.
  • the integrated implant system establishes a basement membrane, and maintains the same anatomical configuration for the layers or cell populations in the same way as usually occurs in the non-damaged skin.
  • EXAMPLE 1 Construction of the Integrated Implant System (IIS) .
  • di -hydrate calcium chloride About 3 to 12 g of di -hydrate calcium chloride are dissolved in 1 liter of sterile milli-Q water (Millipore) . Sodium chloride is added until a solution with an osmolarity comprised between 280 -320 mOsm/L, is obtained. Preferably 4,5 g of di-hydrate CaCl2 and 6,1 g of NaCl in 1 liter of water (300 mOsm/L) , are used.
  • 20 mg of a lyophilized product is used, with a minimal concentration of 65% (13 mg) .
  • Thrombin solution In 1 mL of sterile calcium chloride and sodium chloride solution, between 250 and 500 NIH thrombin are dissolved. Preferably, 1 mg of a lyophilized product with an activity of 258 NIH/mg-lyophilized product (258 NIH) is used.
  • Integrated implant system preparation The IIS preparation is always carried out in a sterile environment.
  • a polymeric matrix portion is provided, which is either commercially available or prepared according to the above indicated process, in an appropriate size for the required application.
  • the matrix portion is sterilized, through methods usually known in the state of the art, as for instance, through irradiation, UV treatment or with and alcohol solution, without being restricted to said options.
  • the matrix portion is submerged for a suitable time period, preferably for about 1 hour, in an aqueous alcohol solution, wherein said alcohol corresponds to a 50% to 90% alcohol solution in sterile Milli-Q water, preferably a 70% aqueous alcohol solution, wherein the alcohol is selected from the group consisting of ethanol, propanol and iso-propanol .
  • the matrix is removed and dried over a sterile absorbent paper.
  • the matrix is placed in a suitable sterile container (for example, a Petri dish or other sterile container with appropriate dimensions for the selected matrix portion) and over the matrix portion a thrombin solution is spread at a ratio of about 100 ⁇ L solution, to be applied over about 1 cm 2 of the polymer. Subsequently, the absorption of the solution is allowed, which usually takes place within the first three minutes, preferable before 2 minutes. Then, about 100 ⁇ L/cm 2 of fibrinogen is added over the matrix containing the absorbed solution. Subsequently, the clot formation is visually verified, which means that the IIS can be immediately used or conserved for later applications .
  • a suitable sterile container for example, a Petri dish or other sterile container with appropriate dimensions for the selected matrix portion
  • EXAMPLE 2 Construction of the Integrated implant system (IIS) containing cells.
  • IIS Integrated implant system
  • an IIS containing the desired cells integrated into the matrix-gel system it is possible to prepare an IIS containing the desired cells integrated into the matrix-gel system.
  • the matrix is prepared as described above and additionally a cellular suspension is prepared, for which the cells from a cell culture obtained through trypsinization, are centrifuged in order to obtain a pellet, which is suspended in 1 mL of a CaCl 2 and NaCl -containing thrombin solution.
  • a polymeric matrix portion is provided, which can be either commercially available or prepared according to the above-indicated process, in an appropriate size.
  • the matrix is submerged during a suitable time period, preferably for about 1 hour, in an aqueous alcohol solution, wherein said alcohol corresponds to a 50% to 90% alcohol solution in sterile Milli-Q water, preferably it corresponds to an aqueous 70% alcohol solution, wherein the alcohol is selected from the group consisting of ethanol, propanol and iso-propanol .
  • the matrix is removed and dried over sterile absorbent paper.
  • the dried matrix is submerged in DMEM/F12 (10% FBS) media, for about 18 to 30 hours, preferably during less or about 24 hours.
  • DMEM/F12 (10% FBS) media for about 18 to 30 hours, preferably during less or about 24 hours.
  • the sterility is checked through microscopy observation, by the media turbidity and the appearance of change in color.
  • the polymer is removed, and once more dried over sterile absorbent paper.
  • the matrix is placed in a suitable sterile container, for example, a Petri dish or other sterile container with appropriate dimensions for the selected matrix portion.
  • a suitable sterile container for example, a Petri dish or other sterile container with appropriate dimensions for the selected matrix portion.
  • a thrombin and cells solution are spread, containing a maximum of up to 8 x 10 6 , preferably between 2 X 10 2 to 4 X 10 6 , more preferably between 3.5 x 10 2 and 3.5 x 10 6 cells/mL of final solution, preferably a suspension containing about 2 x 10 5 cells/ mL of solution, for being applied onto about 1 cm 2 of the matrix. Then, the absorption of the cellular suspension is allowed, which generally occurs within the first three minutes, preferably before 2 minutes. Subsequently, over the matrix containing the absorbed suspension, 100 ⁇ L/cm 2 of fibrinogen are added. Later, the clot formation is visually verified, and the obtained implant system is submerged in DMEM/F-12 (10% FBS) culture media.
  • the cells suspension corresponds to allogenic cells, autologous cells or stem cells, wherein the cells come from a cellular culture for each of said cellular types, and which have been isolated from biopsies obtained according to standard procedures described in the state of the art.
  • said cellular cultures correspond to an autologous fibroblasts cellular culture, or to an autologous keratinocytes cellular culture, or to a stem cell culture, or a mixture thereof, in a calcium chloride (30 mM) and sodium chloride (100 mM) solution.
  • the cells suspension contains an autologous fibroblasts and autologous keratinocytes combination.
  • the cell cultures in the IIS show that during the first 50 hours of culture, an outstanding increase of the proliferation is achieved, in regard to the monolayer system ( Figure 4) .
  • the latter is a relevant advantage of the present invention, insofar as 24 hours from the assembly and seeding of the IIS, it is possible to proceed to implanting it onto the cutaneous lesions, wherein the IIS comprises cells in an increasing and active curve of a maximum proliferation ability, which may provide better qualities and lower recovery time in the lesion to which the IIS is applied.
  • Rabbits were surgically prepared, in order to receive the IIS, this corresponds to the extraction of circular zones 2.5 cm in diameter of dermis and epidermis from the animal's dorsal area.
  • the animals treated with the IIS showed very low infections incidence and when these occurred, they shared the feature of spontaneously disappearing. This may mean that the IIS rapidly reconstitutes the functional skin systems that provide recovery qualities to the damaged area .
  • Curran MP Plosker GL. Bilayered bioengineered skin substitute (Apligraf) : A review of its use in the treatment of venous leg ulcers and diabetic foot ulcers. Bio Drugs 2002 / 16:439-55. 12. Maas-Szabowski N, Stark H-J 7 Fusenig NE. Keratinocyte growth regulation in defined organotypic cultures through Il-l induced KGF expression in resting fibroblasts. J Invest Dermatol 2000 ; 14 : 1075-84. 13. Stark H-J, Maas-Szabowski H, Smola H, et al .
  • Organotypic keratinocyte- fibroblast co-cultures In vitro skin equivalents to study the molecular mechanisms of cutaneous regeneration. In: Horch RE, Munster AM, Achauer BM (eds) . Cultured Human Keratinocytes and Tissue Engineered Skin Substitutes. Stuttgart, Germany: Georg Thieme Verlag, 2001.

Abstract

La présente invention concerne un système d'implant intégré sous forme de système intégré gel-matrice-cellules, qui permet de fournir rapidement des implants afin de couvrir une grande surface de peau à traiter, avec un taux d'acceptation satisfaisant chez les patients soufrant de brûlures, de détérioration chronique ou de blessures cutanées nécessitant une greffe de peau.
EP07725905A 2006-06-07 2007-06-07 Système d'implant intégré (sii) biocompatible, biodégradable et bioactif comprenant une matrice polymère poreuse stérile biocompatible et un gel, avec intégration in situ de la structure matricielle tridimensionnelle Withdrawn EP2040766A2 (fr)

Applications Claiming Priority (2)

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CL200601397 2006-06-07
PCT/EP2007/005060 WO2007141028A2 (fr) 2006-06-07 2007-06-07 Système d'implant intégré (sii) biocompatible, biodégradable et bioactif comprenant une matrice polymère poreuse stérile biocompatible et un gel, avec intégration in situ de la structure matricielle tridimensionnelle

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115137868A (zh) * 2021-03-30 2022-10-04 西安德诺海思医疗科技有限公司 一种重组胶原蛋白贴敷料及其制备方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9259445B2 (en) 2006-06-07 2016-02-16 Universidad Tecnica Federico Santa Maria Integrated implant system (IIS) biocompatible, biodegradable and bioactive, comprising a biocompatible sterile porous polymeric matrix and a gel, integrating in situ the tridimensional matrix structure
AU2016343292B9 (en) * 2015-10-19 2017-07-20 Skin Tissue Engineering Pty Ltd Porous matrix with incorporated cells

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2657352B1 (fr) * 1990-01-25 1993-08-13 France Etat Armement Nouveau produit biologique de remplacement du tissu conjonctif, a structure composite a base de collagene, et procede pour sa preparation.
US5891558A (en) * 1994-11-22 1999-04-06 Tissue Engineering, Inc. Biopolymer foams for use in tissue repair and reconstruction
GB9704749D0 (en) * 1997-03-07 1997-04-23 Univ London Tissue Implant
US20030095993A1 (en) * 2000-01-28 2003-05-22 Hanne Bentz Gel-infused sponges for tissue repair and augmentation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007141028A3 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115137868A (zh) * 2021-03-30 2022-10-04 西安德诺海思医疗科技有限公司 一种重组胶原蛋白贴敷料及其制备方法
CN115137868B (zh) * 2021-03-30 2023-10-20 西安德诺海思医疗科技有限公司 一种重组胶原蛋白贴敷料及其制备方法

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