WO2012038923A1 - Réponse anti-fibrotique provenant de cellules foetales à des implants et des systèmes de pose - Google Patents

Réponse anti-fibrotique provenant de cellules foetales à des implants et des systèmes de pose Download PDF

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Publication number
WO2012038923A1
WO2012038923A1 PCT/IB2011/054179 IB2011054179W WO2012038923A1 WO 2012038923 A1 WO2012038923 A1 WO 2012038923A1 IB 2011054179 W IB2011054179 W IB 2011054179W WO 2012038923 A1 WO2012038923 A1 WO 2012038923A1
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cells
tissue
fibrotic
fetal
biocompatible composition
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PCT/IB2011/054179
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English (en)
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Lee Ann Laurent-Applegate
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Centre Hospitalier Universitaire Vaudois
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells

Definitions

  • the present invention concerns a biocompatible composition for use in a method for preventing or treating a fibrotic-tissue response related to implant materials or delivery systems in a subject.
  • the invention also concerns methods for treating or preventing fibrotic- tissue defects related to implant materials or delivery systems in a subject.
  • Implant applications such as hip, knee and shoulder prostheses (along with all articulated joints), breast, muscle and dental implants, heart valves, vascular grafts and skin grafts have been successfully developed from different types of biomaterials including titanium, polymers and biological components and tissues such as collagens, hyaluronic acid, chitosan, pig intestine, tendon etc.. Nevertheless, even if there is an inert nature of the materials used to date, there is often implant dysfunction by lack of constructive interactions with the surrounding cells and tissues (biocompatibility). Moreover, the implantation of artificial devices or the transplantation of cells to promote tissue regeneration has been met with limited clinical success.
  • Fibrotic response related to implant materials resolves frequently in implant loosening and loss of organ function (i.e. hip, shoulder, and maxilla).
  • WO 98/54301 discloses method for forming a graft in heart tissue which comprises the transplantation of cells chosen from cardiomyocytes, fibroblasts, smooth muscle cells, endothelial cells and skeletal myoblasts.
  • the grafts are especially useful in treating scar tissue on the heart. Also provided is a method of isolating and culturing cardiomyocytes for use in such grafts.
  • WO 01/32129 discloses methods for the long-term augmentation and/or repair of skin defects (scars, skin laxness, skin thinning, and skin augmentation), cellulite, breast tissue, wounds and burns, urological and gastroesophageal sphincter structures, hernias, periodontal disease and disorders, tendon and ligament tears and baldness, by the injection or direct surgical placement/implantation of autologous cultured cells and/or cultured cell-produced extracellular matrix that is derived from connective tissue, dermis, fascia, lamina limba, stroma, adipose tissue, muscle, tendon, ligament or the hair follicle.
  • the corrective application is done on tissue proximal or within the area of the defect.
  • the method involves retrieving viable cells from the subject, a neonate or human fetus.
  • the corrective application involves the cells placed in a matrix, preferably comprised of autologous extracellular matrix constituents as a three-dimensional structure or as a suspension, prior to placement into a position with respect to the subject's defect.
  • a matrix preferably comprised of autologous extracellular matrix constituents as a three-dimensional structure or as a suspension, prior to placement into a position with respect to the subject's defect.
  • the preferable autologous extracellular matrix constituents are collected from culture and placed in a position with respect to the subject's defect.
  • US 2010/129414 (Medtronic Vascular, Inc.) describes methods for treating aneurysms, vascular occlusions, and vascular lesions.
  • the methods comprise the use of an implantable medical device which includes a bioactive agent substrate associated with its surface.
  • Liposomes are used to encapsulate the bioactive agent and are delivered either systemically or locally to the bloodstream.
  • a means for liberating the bioactive agents from the liposomes is used once an appropriate location is chosen and the liposomes have distributed themselves through the vasculature. Once liberated, the bioactive agent can be sequestered by the bioactive agent substrate associated with the implantable medical device, and slowly released to impart a therapeutic effect on the surrounding tissues.
  • Undifferentiated fetal cells have been disclosed in WO 03/068287 (Neocutis SA). Also disclosed are methods and compositions designed for treating a subject suffering from a skin condition, disorder or disease.
  • the compositions include undifferentiated fetal skin cells that are either integrated with a collagen matrix or a carrier.
  • the present invention concerns a biocompatible composition for use in a method for preventing or treating a fibrotic-tissue response related to implant materials or delivery systems in a subject.
  • the biocompatible composition of the invention comprises differentiated fetal cells products derived from 9 to 16 weeks gestation, preferably from 10 to 16 weeks and even more preferably from 12-14 weeks gestation.
  • the invention also concerns a method for treating a fibrotic-tissue defect related to implant materials or delivery systems in a subject, said method comprising placing into said implant materials or delivery systems at a site within or proximal to said fibrotic-tissue defect the biocompatible composition of the invention.
  • the method relates to the injection, implantation and/or attachment of a cultured three-dimensional differentiated fetal cell product or its derivatives or fragments.
  • Another object of the present invention concerns a method for preventing a fibrotic-tissue response related to implant materials or delivery systems in a subject, said method comprising the coating of said implant materials or delivery systems with the biocompatible composition of the invention.
  • FIG. 1 shows adult keratinocyte skin grafts attached and delivered on a "fetal-cell coated" matrix comprised of biocompatible horse collagen providing biocompatible evidence according to some of the embodiments described herein.
  • a) represents autologous cells and b) the matrix, implant with fetal cells.
  • Fig. 2. shows an actual sheet of human keratinocytes being enzymatically separated from the plastic tissue culture flask which needs a support for transfer to patient as described in Fig. 1 either in sheet form or individual cells.
  • Fig. 3. shows SEM of fetal cells on biomaterial surface showing biocompatible interaction of surfaces according to some of the embodiments described herein.
  • Fig. 4. shows that fibrous tissue is formed around implant material.
  • Biomaterial alone (PLA) inserted in bone defect.
  • Fibrous tissue formation with biomaterials alone solid white dense zone around circle-Inside circle very little to no bone growth observed. It depicts fibrotic response of implant materials in soft and hard tissues according to some of the embodiments described herein.
  • Fig 5. shows fetal cell delivery to bone defect in PLA promoting bone formation. Only bone formation throughout and no fibrous tissue formation depicting anti-fibrotic activity of fetal cells and/or their fragments according to some of the embodiments described herein.
  • Fig 6. depicts anti-fibrotic activity in soft tissues from skin to muscle seen following fetal cell delivery and compatibility with collagen foam matrix according to some of the embodiments described herein.
  • Fig 7. shows anti-fibrotic tissue accumulation in severely injured skin following fetal cell delivery and compatibility with collagen foam matrix according to some of the embodiments described herein. It is observed an inhibition of the fibrotic response with fetal skin cells and collagen matrix/implant.
  • Fig. 8. depicts stabilization of fetal cells and/or derivatives by lyophilization/freezing to maintain biological capacity and function according to some of the embodiments described herein. The biological activity is conserved following lyophilization.
  • Fig. 9. illustrates fetal muscle (16 weeks) and fetal skin (14 weeks) showing distinct cell morphology and staining. Fibroblast to myofibroblast formation is not seen over long-term culture.
  • Fig. 10 Shows that differentiated fetal skin fibroblasts at gestational age 12-14 weeks do not de-differentiate to myo-fibroblasts and are Alpha-SMA negative. Cells at this age of gestation interact with matrix surfaces with fibroblastic morphology. Undifferentiated fetal skin fibroblasts and mesenchymal stem cells are alpha-SMA positive and differentiate into myofibroblasts and interact with matrix material with a fibrotic response at the interface.
  • Fig. 11 Differentiated fetal skin cells, particularly at 12-14 weeks of gestation do not differentiate to myofibroblasts nor de-differentiate to other cell lineages such as osteoblasts when placed in osteogenic morphogens (media supplemented with growth factors to induce osteogenic differentiation) for 21 days nor into adipocytes when placed in adopogenic morphogens (media supplemented with growth factors to induce adipogenic differentiation) for 27 days.
  • Undifferentiated fetal cells ( ⁇ 9weeks) and MSCs have been shown to easily dedifferentiate into other cell lineages when placed in the same morphogen supplemented media.
  • Fig. 12. Shows early to late passage, cells do not differentiate into myofibroblasts even if they are in contact with a matrix or implant. The induction of differentiation in other cells (i.e. bone, muscle, cartilage,%) is limited.
  • Applicants have surprisingly found that using fetal cell products (live or dead), help render biomaterials compatible for adult cell and stem cell delivery (see Examples below). When implants or matrix are placed into the body, there is a reaction with the soft tissue especially but also with bone and cartilage, that fibrous tissue is formed. Applicants have demonstrated that the differentiated fetal cells products derived from 9 to 16, preferably from 10 to 16 weeks and even more preferably from 12-14 weeks gestation render the foreign object more acceptable by limiting and/or eliminating the fibrous tissue formation around the implant /delivery system.
  • embryonic stem cells use, for the most part, encapsulation procedures, which physically separate cells from host and confine the cells, to present cells for clinical situations to render them immunologically acceptable and to prevent tumor formation. Even these implants create a general fibrous, non-arranged tissue formation around the implants. In these situations, the use of differentiated fetal cells products derived from 9 to 16, preferably from 10 to 16 weeks and even more preferably from 12-14 weeks gestation in parallel to also limit and/or eliminate this unwanted fibrotic response is proposed.
  • a or "an” means “at least one” or “one or more.”
  • the term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components.
  • biomaterial means a natural or synthetic material, including metals, ceramics and polymers devoid of deleterious effects when in contact with cells or biological tissues.
  • the biomaterial support is selected from the group consisting of polymeric support comprising olefin polymers, fluorine polymers, polystyrene, polyacrylic polymers, polyesters polymers, polyurethane polymers, silicon polymers, cellulose polymers, epoxy polymers, silicone-based polymers, synthetic hydrogels, polycarbonates; biocompatible metallic supports comprising titanium and titanium alloys, nitinol, zirconia, stainless steel and cobalt chromium, alumina-zirconia composites; and/or biocompatible ceramics comprising porcelain, hydroxyapatite, and mixtures thereof.
  • polymeric support comprising olefin polymers, fluorine polymers, polystyrene, polyacrylic polymers, polyesters polymers, polyurethane polymers, silicon polymers, cellulose polymers, epoxy polymers, silicone-based polymers, synthetic hydrogels, polycarbonates
  • biocompatible metallic supports comprising titanium and titanium alloys, nitinol, zirconia, stainless steel
  • biocompatible shall mean any material that does not cause injury or death to the animal or induce an adverse reaction in an animal when placed in intimate contact with the animal's tissues.
  • Adverse reactions include, but are not limited to, inflammation, infection, fibrotic tissue formation, cell death and thrombosis.
  • Biodegradable refers to a polymeric composition that is biocompatible and subject to being broken down in vivo through the action of normal biochemical pathways. Bioresorbable and biodegradable may be used interchangeably, however they are not coextensive. Biodegradable polymers may or may not be reabsorbed into surrounding tissues. Bioresorbable polymers are biodegradable, and therefore, are capable of being cleaved into biocompatible byproducts through chemical- or enzyme-catalyzed hydrolysis.
  • Non-biodegradable refers to a polymeric composition that is biocompatible and not subject to being broken down in vivo through the action of normal biochemical pathways.
  • differentiated fetal cells mean differentiated cells compared to undifferentiated fetal cells according to WO 03/068287 (Neocutis SA). Contrary to the present invention the term “undifferentiated” is used in WO 03/068287 describe an immature or primitive cell.
  • undifferentiated fetal skin cells include those that can differentiate into dermal fibroblasts and epidermal keratinocytes. Differentiated cells are those that when placed in differentiation media specific for another cell type or into a different micro-environment, will not de-differentiate into a different cell lineage easily.
  • fetal skin fibroblasts are placed into osteogenic differentiation media, they will not become a whole population of osteoblasts or if they are placed in adipogenic media they will not become a whole population of adipocytes and if the same cells are placed into a 3D matrix in association with bone, will not de-differentiate into a whole population of osteoblasts because of the change in
  • Undifferentiated cell populations such as fetal cells less than 9 weeks and MSC's can be induced into other cell lineages, such as osteogenic or adipogenic by adding specific growth factors for 14-21 days.
  • the terms "differentiated fetal cells products derived from 9-16 weeks gestation" mean that the cell populations are derived from specific tissue i.e. skin, bone, cartilage, muscle and have morphology and surface markers of specific tissue types. Cells below 9 weeks of gestation are MSC's by definition and constitute undifferentiated cell populations.
  • appropriate culture conditions is a medium for culturing cells containing nutrients that promote proliferation.
  • the nutrient medium may contain any of the following in an appropriate combination and in the appropriate concentrations: isotonic saline, buffer, amino acids, serum or serum replacement, and other exogenously added factors. Those skilled in the art will recognize that any commonly employed culture conditions can be used.
  • cell line refers to a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and sufficient space. We do not make established cell lines but primary cell lines.
  • primary cell line refers to an established cell culture with limited passage numbers.
  • clone refers to a subset population of cells usually developed from a single selected cell.
  • cell bank refers to harvesting biopsies from donor fetal tissue; growing the fetal tissue and proliferating fetal cells to a high concentration under appropriate culture conditions; trypsinizing the tissue and cells of the resulting cultures to allow their suspension; pooling the suspended cells to make a generally uniform suspension of cells from the culture; gently mixing with a cryoprotectant; sealing aliquots of the cell suspension in ampoules; and freezing the aliquots (e. g. , by decreasing the temperature of the ampoule by 1 °C/min until- 80 C and then transferred to -160 °C approximately 24 hours later, or in programmed cycles in a automatic, calibrated Nano-Freezer for full cycle freezing to 165° C).
  • This ultra-cold temperature bank preserves the cells such that they stop aging, thereby allowing them to retain the function and activity they had on the day they were collected.
  • treating includes (1) preventing the condition, i. e. avoiding any clinical symptoms of the condition, (2) inhibiting the condition, that is, arresting the development or progression of clinical symptoms, and/or (3) relieving, repairing or reversing the condition, i. e. causing regression of clinical symptoms.
  • condition refers to physiological states that can be prevented or treated by administration of the biocompatible composition comprising fetal cells products of the invention as described herein.
  • subject (as in treatment of "a subject") or “patient” is intended to refer to a mammalian individual afflicted with, prone to, or suffering a condition, defect, disorder or disease (as specified herein).
  • This term includes both humans and animals.
  • the subjects can be, e. g., humans, non-human primates, wildlife, dogs, cats, horses, cows, pigs, sheep, rabbits, rats, or mice.
  • wildlife includes any mammals, birds, amphibians or fish that are not domesticated. Examples of such wildlife include, but are not limited to, badgers, beavers, lions, tigers, bears, hawks and deer.
  • a three dimensional matrix means any matrix selected from a collagen matrix or PLA, PLGA, PEG, chitosan, elastin, hydrogel including for example HA (hyaluronic acid), silicone, chitosan or a mixture thereof.
  • the matrix provides a three dimensional space to assure proper coverage and delivery of fetal cells or fetal products to or also in association with an additional implant material.
  • collagen refers to a polypeptide compound, which is hydrophilic in nature that is subject to degradation by extracellular enzymes. Because, this substance is well studied, many key parameters can be controlled. Collagen is a weak antigen, thereby resulting in minimal rejection potential.
  • a preferred collagen used in the methods and uses of the invention is horse collagen.
  • Implants can be made of artificial or natural materials. Medical implants are man-made devices and the surface of implants that contact the body might be made of a biomedical material such as titanium, silicone, polymers, apatite, biofoams and biogels. Some implants can have associated bioactive eluting drugs such as implantable capsules or drug-eluting stents. Implant materials can be associated with specific tissue-type differentiated fetal cells for anti-fibrotic response.
  • PLA polylactic acid
  • PLGA polylactic glutamic acid
  • hydroxyapatite hydroxyapatite
  • TCP tricalcium phosphate
  • Materials include titanium, ceramic.
  • a titanium hip prosthesis can contain other materials such as a ceramic head and polyethylene acetabular cup.
  • Materials include silicon elastomers, polyurethane foam, collagens, Hydrogels of various viscoelastic properties.
  • - Muscle implants soft tissue mass restructure using materials such as silicon and collagens.
  • - Cornea and retina implants include silicone, gels, PLA
  • - Skin implants include, HA gels, collagens, silicone.
  • implantable medical devices include, but are not limited to stents, catheters, prostheses in general, sutures, implantable pacemaker, micro-particles, probes and vascular grafts.
  • the implantable medical devices can be used individually or in combination.
  • delivery systems mean any metallic or regularly used orthopedic, trauma, maxillo-facial natural or synthetic implant materials, hydrogels, silicones or grafts providing a means to transfer differentiated fetal cells or differentiated fetal cell products according to the invention alone or in association with an implant to treat tissue or render implant.
  • This new finding of the anti-fibrotic activity with the differentiated fetal cell derived from 9 to 16, preferably from 10 to 16 weeks and even more preferably from 12-14 weeks gestation delivery is of crucial importance. Also, using differentiated fetal cell products derived from 9 to 16, preferably from 10 to 16 weeks and even more preferably from 12-14 weeks gestation (live or dead), help render biomaterials compatible for adult cell and stem cell delivery (see Examples below). Implants and devices can be associated with the presence of inflammation and accumulation of poorly organized fibrotic tissue depending on implant/device materials, surface degradation properties and terminal sterilization procedures. Stimulation of rapid new tissue around implants/device can determine the optimal functional outcome.
  • Differentiated fetal cell derived from 9-16 weeks, preferably from 10 to 16 weeks and even more preferably 12-14 weeks gestation can aid in rapid tissue repair and adaptation around implants/devices to avoid inflammation responses.
  • Another role of the differentiated fetal cell at early gestation (9-16 weeks, preferably from 10 to 16 weeks and even more preferably 12-14 weeks) is the inhibition of myofibroblast formation and/or activation during tissue repair and thus inhibits fibrosis, especially when associated with an implant/device.
  • Myofibroblasts play a major role in fibrotic conditions characterized by excess production of extracellular matrix and the expression of alpha-SMA (smooth muscle actin).
  • fetal and adult fibroblasts due to the de-differentiation of adult fibroblasts to myofibroblast morphology and their intrinsic differences in contractile capacity, are key elements for why fetal fibroblasts are not capable of a fibrotic response and one of the main mechanisms that there is no scar tissue during fetal wound healing (Moulin et al., J Cell Physiology, 188:211-222, 2001).
  • Expression of alpha-SMA is shown to be expressed in embryonic development in early mesenchymal cell populations (Clement et al., J of Cell Science, 120:229-238, 2006).
  • fetal skin cells of 12-14 weeks or between 10-16 weeks or 9-16 weeks are cultured in media only including fetal serum as growth factor with biomaterials, matrix and implants (i.e. PLA, collagen sponge, HA gels, plastics), they do not de-differentiated to myofibroblasts nor express alpha-SMA (see Figure 10). These cells do not cause constriction or consolidation around material.
  • Adult mesencymal stem cells do not associate or integrate with matrix, implant material and de-differentiate into myofibroblast phenotype forming accumulation at the matrix/implant interface. These cells have also a phenotype of myofibroblasts and are altered in their growth (apoptotic in nature).
  • Differentiated fetal cells at 9-16 weeks of gestation can be given either fresh or lyophilized (see the biological activity equivalence in Example 3 and Figure 8 below). Stabilization of differentiated fetal cells through lyophilization processes provides an interesting alternative for implant coating to "traditional drugs" to date.
  • fetal tissue is pre-immunoincompetent and associated with a reduced capacity to evoke an immunological response in the recipient of such cells.
  • the integration of the differentiated fetal cells at 9-16 preferably at 10 to 16 weeks and even more preferably at 12-14 weeks of gestation with collagen or hydrogels can occur by mixing, combining, pipetting, seeding, plating, or placing the cells within the collagen or hydrogel.
  • integrated or “integrated with” is used to describe any means of blending, particularly those relating to adding cells to a matrix.
  • the term includes, but is not limited to mixing, combining, pipetting, seeding, plating or placing.
  • the collagen matrix of the construct is a horse collagen matrix and in another hydrogel of HA composition.
  • the biocompatible composition comprising differentiated fetal cells products at 9 to 16, preferably from 10 to 16 weeks and even more preferably from 12-14 weeks of gestation according to the invention may be prepared by harvesting biopsies from donor fetal tissue; developing cell lines from the fetal tissue; growing the fetal tissue and proliferating fetal cells to a high concentration to create a cell bank from which grafts are derived; and integrating the grafts with a collagen matrix or within a hydrogel.
  • the biocompatible composition comprising differentiated fetal cells products at 9 to 16 weeks, preferably at 10 to 16 weeks and even more preferably at 12-14 weeks of gestation according to the invention may be prepared by obtaining fetal cells;
  • proliferating the fetal cells proliferating the fetal cells; and integrating the differentiated fetal cells or differentiated fetal cell products with a collagen matrix or within a hydrogel.
  • the present invention concerns a biocompatible composition for use in a method for preventing, limiting or treating a fibrotic-tissue response related to implant materials or delivery systems in a subject.
  • the biocompatible composition of the invention comprises differentiated fetal cells products derived from 9-16 weeks gestation.
  • said biocompatible composition of the invention consists in differentiated fetal cells products derived from 9-16 weeks gestation preferably from 10-16 weeks of gestation and even more preferably differentiated fetal cells products derived from 12-14 weeks gestation.
  • differentiated fetal cells products consist of differentiated fetal cells, derivatives or fragments thereof.
  • Derivatives of differentiated fetal cells can include growth factors, cytokines, proteins, DNA, RNA, cell walls, or whole cell products. Fragments of differentiated fetal cells products are defined as for example cell organelles, cell walls and cell membranes.
  • Said fetal cells are live or death cells that are differentiated.
  • Differentiated fetal cells come from specific tissues such as fetal skin fibroblasts from fetal skin dermis, fetal bone cells from fetal long bones, fetal cartilage cells from endplates of bone or articular joints, fetal tendon cells from fetal tendon etc.
  • fetal skin fibroblasts from fetal skin dermis
  • fetal bone cells from fetal long bones
  • fetal cartilage cells from endplates of bone or articular joints
  • fetal tendon cells from fetal tendon etc.
  • the differentiated fetal cells derived from 9-16 weeks gestation preferably from 10-16 weeks of gestation and even more differentiated fetal cells derived from 12-14 weeks gestation are originated from one single organ donation.
  • the differentiated fetal cells of the invention may be originated from an in vitro culture.
  • Differentiated fetal cells of the invention or fragments thereof are preferably selected from skin, cartilage, bone, muscle, disc, lung or tendon cells.
  • Said differentiated fetal cells are generally fresh or lyophilized fetal cells.
  • the biocompatible composition comprising differentiated fetal cells product derived from 9-16 weeks gestation and preferably differentiated fetal cells products derived from 10 to 16 weeks and even more preferably from 12-14 weeks gestation further comprises a three dimensional matrix (as described above) made of any synthetic or natural materials.
  • the three dimensional matrix wherein differentiated fetal cells products according to the invention are integrated is selected from a collagen matrix or PLA, PLGA, PEG, elastin, hydrogel including HA, silicone, chitosan or a mixture thereof.
  • the three dimensional matrix used in the present invention may be biodegradable or nonbiodegradable depending on its field of application. Obviously the selected matrix is not substantially toxic and shall be pharmaceutically acceptable.
  • Biological properties that can be expressed by three-dimensional tissue and/or cells and growth factors associated include but are not limited to prevention and/or reduction of tissue fibrotic response, tissue remodeling, promotion of epitheliazation, tissue growth,
  • vascularization and/or angiogenesis it is also the object of the present invention to provide a method for promoting tissue remodeling, epitheliazation, tissue growth,
  • tissue repair with fibrotic response is prevented, normal tissue repair is not blocked but rather enhanced for epitheliazation, tissue growth, vascularization and/or angiogenesis as vessels can migrate.
  • implant materials that create fibrotic-tissue response are selected from dental implants, dental augmentation, hip, shoulder, knee, articular joint implants, breast implants, muscle implants implantable medical devices, or any soft tissue including skin and muscle.
  • Other soft tissue could be heart, lung, brain, kidney, pancreas etc (see the definition above).
  • delivery systems that create fibrotic-tissue response are selected from metallic or regularly used orthopedic, trauma, maxillo-facial natural or synthetic implant materials, hydrogels, chitosans, silicones or grafts.
  • the "biocompatible composition" of the invention may contain one or more differentiated fetal cells from 9-16 weeks gestation and preferably from 10 to 16 weeks and even more preferably differentiated fetal cells derived from 12-14 weeks gestation along with other chemical components including, but not limited to, traditional drugs, physiologically suitable carriers and excipients.
  • the biocompatible composition of the invention may include one or more differentiated fetal cells differentiated fetal cells derived from 9-16 weeks gestation and preferably from 10 to 16 weeks and even more preferably differentiated fetal cells derived from 12-14 weeks gestation and/or one or more fetal proteins that have been stabilized, along with other chemical components including, but not limited to, traditional drugs, physiologically suitable carriers and excipients.
  • biocompatible compositions of the present invention may be manufactured by processes well known in the art, e. g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • active agents may also be included in the "biocompatible composition" of the invention, e. g., anti-inflammatory agents, analgesics, antimicrobial agents, antifungal agents, antibiotics, vitamins, antioxidants.
  • composition is meant a nontoxic, but sufficient, amount that provides the desired effect at a reasonable benefit/risk ratio attending any medical treatment.
  • the desired effect can be alleviation or prevention of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • the differentiated fetal cells derived from 9-16 weeks gestation and preferably from 10 to 16 weeks and even preferably differentiated fetal cells derived from 12-14 weeks gestation used to make the three-dimensional biocompatible composition of the invention may include differentiated fetal skin cells, for skin grafts and differentiated fetal muscle cells, for muscle grafts; and differentiated fetal bone cells, for bone grafts, differentiated fetal tendon cells for tendon grafts, differentiated fetal cartilage for cartilage grafts and more specifically differentiated fetal articular cartilage for articular cartilage grafts.
  • the invention provides a three-dimensional cutaneous tissue allograft construct containing differentiated fetal cells derived from 9-16 weeks gestation preferably from 10-16 weeks of gestation and even more preferably differentiated fetal cells derived from 12-14 weeks gestation integrated with a collagen matrix (e. g., horse collagen).
  • a collagen matrix e. g., horse collagen
  • the differentiated fetal cells derived from 9-16 weeks gestation and preferably from 10 to 16 weeks and even more preferably differentiated fetal cells derived from 12-14 weeks gestation may be fetal skin cells such as those that are able to differentiate into dermal fibroblasts and epidermal keratinocytes under appropriate culture conditions. Integration can be
  • This construct can then be used to treat a subject suffering from a fibrotic-tissue response defect, disorder or disease.
  • biopsies from fetal tissue are obtained immediately following pregnancy interruption in accordance with the procedures and policies of the Ethics committee of the CHUV. Collected donor tissue is ideally between 12-16 weeks gestation. The fetal tissue is divided into small fragments in multiple 10 cm tissue culture plates, and is grown in Dulbecco's MEM (DMEM) tissue culture media with glutamine. Fetal serum is then added to the plates. When cell growth advances, e. g., after about one week, the tissue and cells are trypsinized. Some of the plates are then frozen into individual units, e. g. in liquid nitrogen. Fetal cells are then centrifuged and resuspended to produce a generally uniform suspension of cells from the culture.
  • DMEM Dulbecco's MEM
  • the cells are gently mixed with a cryoprotectant (for example DMEM, 5ml + fetal calf serum, 4 ml + dimethylsulfoxide, 1 ml (DMSO)).
  • a cryoprotectant for example DMEM, 5ml + fetal calf serum, 4 ml + dimethylsulfoxide, 1 ml (DMSO)
  • the fetal cell suspensions are then sealed in aliquots, and frozen, e. g. in liquid nitrogen.
  • the aliquots are frozen at the rate of 1 C/min until they reach a temperature of -80 C and then transferred to - 160 C approximately 24 hours later.
  • Aliquots of homogeneous banked cells can then be used for any desired purpose such as production of the three-dimensional tissue allograft construct and/or the biocompatible composition of the invention, or for use in the manufacture of a medicament for treating a fibrotic-tissue response defect in a subject in need thereof, by unsealing an ampoule of the fetal cell bank, thawing the contents, and transfer into ordinary cell culture medium.
  • biopsies from fetal donor tissue can be obtained and cell lines developed as described above.
  • the fetal tissue can be divided into small fragments in multiple 10 cm tissue culture plates, which have been prepared with grid incisions made by a scalpel.
  • DMEM tissue culture media with glutamine and 10% fetal serum is added to the plates.
  • the fetal cells are expanded in high concentration to create a cell bank. Fetal cells are then frozen in a mixture of DMSO, DMEM and fetal serum until they are needed.
  • Fetal cells from passages 5-10 are prepared at a concentration of about 5.3 x 10 cells/ml. This concentration may vary depending on the type of fibrotic-tissue response defect and whether the patient is an adult or child. For example, the concentration of cells can range from
  • fetal proteins are then stabilized and are incorporated into a carrier (three dimensional matrix), either alone or in combination with the fetal cells.
  • Differentiated fetal cells derived from 9-16 weeks gestation and preferably from 10-16 weeks of gestation and even more preferably differentiated fetal cells derived from 12-14 weeks gestation are prepared from 1 cm2 of skin from abdominal tissue.
  • the fetal tissue is divided into small fragments in multiple 10 cm tissue culture plates, and is grown in Dulbecco's MEM (DMEM) tissue culture media with glutamine. Fetal serum is then added to the plates. When cell growth advances, e. g., after about one week, the tissue and cells are trypsinized. Some of the plates are then frozen into individual units, e. g. in liquid nitrogen. Fetal cells are then centrifuged and resuspended to produce a generally uniform suspension of cells from the culture.
  • DMEM Dulbecco's MEM
  • the cells are gently mixed with a cryoprotectant (for example DMEM, 5ml + fetal calf serum, 4 ml + dimethylsulfoxide, 1 ml (DMSO)).
  • a cryoprotectant for example DMEM, 5ml + fetal calf serum, 4 ml + dimethylsulfoxide, 1 ml (DMSO)
  • the fetal cell suspensions are then sealed in aliquots, and frozen, e. g. in liquid nitrogen.
  • the aliquots are frozen at the rate of 1 C/min until they reach a temperature of -80 °C and then transferred to - 160 °C approximately 24 hours later.
  • Aliquots of homogeneous banked cells can then be used for any desired purpose such as production of the three-dimensional tissue allograft construct and/or the biocompatible composition of the invention, or for use in the manufacture of a medicament for treating a fibrotic-tissue response defect in a subject in need thereof, by unsealing an ampoule of the fetal cell bank, thawing the contents, and transfer into ordinary cell culture medium.
  • Another object of the invention is to provide a method for treating a fibrotic-tissue defect related to implant materials or delivery systems in a subject, said method comprising placing into said implant materials or delivery systems at a site within or proximal to said fibrotic- tissue defect the biocompatible composition of the invention as described above.
  • a further object of the invention is a method for limiting and/or preventing a fibrotic-tissue response related to implant materials or delivery systems in a subject, said method comprising the coating of said implant materials or delivery systems with the biocompatible composition of the invention.
  • Another aim of the invention is a method of treating or preventing a subject suffering from a fibrotic-tissue response disorder, the method comprising applying to said subject the biocompatible composition of the invention.
  • said fibrotic-tissue response disorder is preferably originated from implant materials or delivery systems or grafts.
  • biocompatible composition of the invention in the
  • manufacture of a medicament for the treatment or prevention of a fibrotic-tissue response disorder preferably originated from implant materials or delivery systems or grafts.
  • differentiated fetal cells i.e. derived from 9-16 weeks gestation, preferably from 10-16 weeks of gestation and even more preferably differentiated fetal cells derived from 12- 14 weeks gestation seem to be immunologically accepted by host, one organ donation is necessary for cell bank creation and testing.
  • this invention presents significant biological advantages, for example this invention provides a single, traceable process for the treatment of hundreds of thousands of patients.
  • Keratinocyte autografts are traditionally delivered as thin sheets of cells (cultured onto mouse fetal fibroblasts or foreskin fibroblasts treated with MMC or irradiation to act as feeder layers) (Figure 2) which have to be attached to a gauze with ⁇ 12 titanium clips and/or fibrin glue to be able to transfer these preparations to the patient. Even still, the preparations are very fragile as is evidenced by the photo (see Figure 1-2) and are dependent of the culture time for cells to form an entire cell sheet for delivery.
  • Adult cells do not normally adhere to biomaterials such as collagens and foams readily. This is why they have not been used to date to stabilize the keratinocyte cultures.
  • Fetal cells adhere readily with implant materials such as collagen and polymers such as PLA (see Figure 3, SEM of fetal cells on PLA surface showing full attachment).
  • Matrix coating with fetal skin cells to provide delivery for all types of autografting techniques with cells such as skin, cartilage, bone, cornea.
  • Fetal cells render matrix "biocompatible" to adult cells and with an anti-fibrotic nature. This is not the case for the matrix alone (i.e. adult keratinocytes do not attach readily).
  • Fibrous tissue can be formed such as a capsule around implant material
  • fetal cells associated with matrix/implant can reduce or eliminate fibrotic tissue response. This can be seen in muscle and skin soft tissues (see Figure 6) and in large surface skin restructuring (see Figure 7). Importantly, biological activity of fetal cells can be stabilized by freezing/lyophilizing cells and/or fragments (see Figure 8) by comparing angiogenesis activity.
  • MSC Fetal mesenchymal skin cells
  • Differentiated fetal skin cells of 12-14 weeks (or 9-16 weeks) gestation are differentiated cells that are difficult to dedifferentiate and do not cause a fibrotic response when associated with matrix or with implant materials.
  • fetal skin cells of 12-14 weeks or between 9-16 weeks are cultured with biomaterials, matrix and implants (i.e. PLA, collagen sponge, HA gels, plastics), they do not de-differentiated to myofibroblasts nor express alpha-SMA. These cells do not cause constriction or consolidation around material.
  • Adult mesenchymal stem cells do not associate or integrate with matrix, implant material and de-differentiate into myofibroblast phenotype forming accumulation at the matrix/implant interface.
  • Differentiated fetal skin cells at 9-16 preferably 12-14 weeks of gestation do not de-differentiate easily and are not with the morphology of a myofibroblast phenotype ( Figures 11 and 12).
  • These cell populations that have been developed in a specific dedicated cell bank are very different from fetal mesenchymal skin cells and mesenchymal stem cells in the above mechanisms. Expression of alpha-SMA is shown to be expressed in embryonic development in early mesenchymal cell populations (Clement et al., J of Cell Science, 120:229-238, 2006).

Abstract

La présente invention concerne une composition biocompatible destinée à être utilisée dans une méthode de prévention ou de traitement d'une réponse de tissu fibrotique liée à des matériaux d'implant ou à des systèmes de pose chez un sujet. L'invention porte en outre sur des méthodes de traitement ou de prévention de défauts de tissu fibrotique liées à des matériaux d'implant ou à des systèmes de pose chez un sujet.
PCT/IB2011/054179 2010-09-22 2011-09-22 Réponse anti-fibrotique provenant de cellules foetales à des implants et des systèmes de pose WO2012038923A1 (fr)

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FR2999083A1 (fr) * 2012-12-12 2014-06-13 Univ Nantes Pansement contenant des fibroblastes et des keratinocytes foetaux

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WO2014090961A1 (fr) * 2012-12-12 2014-06-19 Universite De Nantes Pansement contenant des fibroblastes et des keratinocytes foetaux
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