Classifications

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  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1816—Erythropoietin [EPO]
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  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
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  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
  • A61K38/1866—Vascular endothelial growth factor [VEGF]
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  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/196—Thrombopoietin
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  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • A61K38/27—Growth hormone [GH], i.e. somatotropin
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  • 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
  • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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  • 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
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
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  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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  • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
• • A—HUMAN NECESSITIES
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  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/10—Growth factors
  • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/28—Vascular endothelial cells

Definitions

• the present invention relates to the use of at least one growth factor in isolated form for the cultivation of primarily differentiated cells, for the locally specific and / or directed differentiation of adult cells and / or for the regeneration of bones, tissues and / or endocrine organs.
• EGF Epidermal Growth Factor
• VEGF Vascular Endothelial Growth Factor
• HGF ' ⁇ epatocyte Growth Factor
• tissue extracts such as from the pituitary or hypothalamus, are particularly suitable for causing cell proliferation in hepatocytes (see, for example, US Pat. No. 6,008,047).
• Such animal or occasionally human extracts have already been added to cell cultures.
• communicable viral diseases such as BSE, swine or sheep viruses
• the expansion of animal or human tissue extracts is problematic in laboratory operation or in clinical use.
• the use of such extracts documents the lack of knowledge of the actually relevant factors and their potential for use and effectiveness.
• an inductive growth behavior of cells means a significant innovation, especially for therapeutic or biotechnological processes.
• Such a growth behavior, supported by a 3D carrier matrix should not only allow growth in the sense of settlement or structural remodeling, but should actually allow directed de novo formation from an induction nucleus.
• Such processes take place in ontogeny and build on a previously existing system.
• LIF Leukemia Inhibitory Factor
• CNTF iliary Neurotropic Factor
• GDNF Glial Derived Neurotrophic Factor
• NGF nerve growth factor
• TPO thrombopoietin
• EPO erythropoietin
• GH growth hormone
• LIF leukemia inhibitory factor
• CNTF Ciliary Neurotropic Factor
• the invention therefore relates to a method for the multiplication and differentiation of cells in vitro, in which the growth process of the cells through the use of the growth factors TPO and / or EPO and / or GH, in particular HGH and / or somatostatin and / or LIF and / or CNTF initiated or scheduled and structurally directed.
• TPO is also known as c-Mpl ligand, mpl ligand, megapoietin or megacariocyte growth and development factor and has not been used in the culture of z.
• B adult hepatocytes or other primary cells, except for platelets or their precursors.
• TPO is essential for the development and proliferation of megacariocytes and platelets and thus for the formation of blood platelets.
• TPO is consumed as a 332 amino acid protein in the liver and kidneys.
• additional growth factors can be "transforming growth factor beta” (TGF beta), prostaglandins, granulocyte-macrophage-stimulating factor (GM-CSF), "growth hormone releasing hormones” (GHRH), “thyrotropin-releasing homones” ( TRH), “Gonadotropin-releasing hormones” (GnRH), “Corticotropin-releasing hormones” (CRH), dopamine, "Antidiuretic hormones” (ADH), oxytocin, prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and / or vasopressin be used.
• TGF beta transforming growth factor beta
• prostaglandins granulocyte-macrophage-stimulating factor
• GM-CSF granulocyte-macrophage-stimulating factor
• GHRH growth hormone releasing hormones
• TRH thyrotropin-releasing homones
• TRH Gonadotropin-releasing hormone
• somatostatin and or TGF beta and / or prostaglandins are also suitable for terminating the growth process according to the invention.
• the individual concentrations of the growth factors in solution are usually about 1 to about 100 ng / ml, preferably about 10 to about 50 ng / ml, in particular about 10 to about 20 ng / ml. With local coatings, however, the concentrations of the growth factors can also be a multiple thereof.
• GHRH growth hormone releasing hormones
• TRH thyrotropin-releasing homones
• GnRH gonadotropin-releasing hormones
• CHC corticotropin-releasing hormones
• somatostatin dopamine
• ADH antidiuretic hormones
• oxytocin somatostatin
• Prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and / or vasopressin can also be used for the structural processes.
• one or more nerve regeneration factors preferably “nerve growth factor” (NGF) and / or one or several vascular regeneration factors, preferably “Vascular Endothelial Growth Factor” (VEGF) and / or “Plateled Derived Growth Factor” (PDGF), are used.
• NGF nerve growth factor
• VEGF Vascular Endothelial Growth Factor
• PDGF Platinum Derived Growth Factor
• endothelialization of the cells In the presence of endothelial cells, endothelialization of the cells and thus optimal hemocompatibility can also be achieved.
• the growth factors mentioned are generally commercially available, but can also be produced by genetic engineering using methods known to those skilled in the art. They include not only the naturally occurring growth factors, but also derivatives or variants with essentially the same biological activity.
• TPO can be purchased from CellSystems GmbH, St. Katharinen.
• TPO can be purchased from CellSystems GmbH, St. Katharinen.
• the use of human TPO is preferred.
• the production and characterization of TPO and its variants is described, for example, in EP 1 201 246, WO95 / 21919, WO95 / 21920 and WO95 / 26746.
• Suitable variants of TPO are, for example, the TPO derivatives described in WO95 / 21919 or the allelic variants or species homologues described in WO95 / 21920 or the pegylated TPO described in WO95 / 26746 and EP 1 201 246, without being restricted thereto.
• pegylated TPO is taken to mean TPO derivatives which are bound to an organic polymer, such as, for example, polyethylene glycol, polypropylene glycol or polyoxyalkylene.
• Derivatives of TPO which have a sequence identity of less than 100% and nevertheless have the activity of TPO, as preferably described in EP 1 201 246, are also understood as further variants of TPO.
• TPO derivatives usually have a sequence identity of at least 70%, preferably at least 75%, in particular at least 80% and above all at least 85% in comparison to human TPO inclusive their fragments with TPO activity.
• a particularly preferred TPO activity in the sense of the present invention is the acceleration of the proliferation, differentiation and / or maturation of megakaryocytes or megakaryocyte precursors in platelet-producing forms of these cells by TPO or its variants.
• EPO is also called the embryonic form of TPO and its variants are e.g. B. in EP 0 148 605, EP 0 205 564, EP 0 209 539, EP 0 267 678 or EP 0411 678.
• growth factor is consequently not only limited to the naturally occurring forms according to the present invention, but also includes non-naturally occurring forms or variants or derivatives.
• growth factor according to the present invention includes not only growth promoters, but also growth inhibitors, such as. B. somatostatin, TGF beta and / or prostaglandins. Such growth inhibitors are particularly suitable for suppressing or inhibiting the growth of changed cells, such as. B. tumor cells, by using them simultaneously or sequentially, for example, using hydrogels or slow-release materials, highly concentrated, locally.
• the growth process according to the invention is carried out in a culture suitable for the respective cells.
• the cell aggregates that may form during the growth process can be comminuted and, if necessary, encapsulated and optionally frozen using a suitable device.
• a grid with z. B. a cutting mesh structure of, for example, 500 ⁇ m in size, which means that new daughter aggregates of, for example, hepatocytes can be formed again and again. This can advantageously be done in a completely closed system.
• contactless, automatically or manually controlled pump systems can be used, the z. B. consist of piston pumps or generate magnetically or by compressed air compression of hoses, directed currents.
• the shear stress in a perfused bioreactor can lead to spontaneous confluence of the endothelial cells on the surfaces of the aggregates, which can be advantageous for further use.
• Suitable materials known to the person skilled in the art are suitable for the encapsulation, into which, for example, structured shapes or spaces are integrated which enable an in situ growth structure or enlargement.
• the capsule can be dispensed with and, for example, endothelialization and thus optimal hemocompatibility can be achieved in the presence of endothelial cells.
• the growth process of the cells is initiated locally or terminated and structurally controlled, preferably by means of a biological matrix.
• the biological matrix is treated here, for example, with one of the growth factors mentioned or with a combination of the growth factors mentioned as a mixture or sequentially. This also enables 3D regeneration and / or artificial control of tissue repair or tissue cultivation even in adult cell systems.
• the biological matrix is usually an implant, e.g. B. a stent, a patch or a catheter, a graft, e.g. B. a skin graft, and / or a carrier germ material for the growth of cells, for example a so-called slow-release material, for example a hydrogel, for example based on fibrin and / or polymers, such as, for.
• an implant e.g. B. a stent, a patch or a catheter
• a graft e.g. B. a skin graft
• a carrier germ material for the growth of cells
• a so-called slow-release material for example a hydrogel, for example based on fibrin and / or polymers, such as, for.
• a bone replacement material such as tricalcium phosphate, an allogeneic, autologous or xenogeneic acellularized or non-acellularized tissue, such as a heart valve, venous valve, arterial valve, skin, vessel, aorta, tendon, comea, cartilage, Bones, tracea, nerve, miniscus, intervertebral disk, ureters, urethra or bladder (see, for example, EP 0 989 867 or EP 1 172 120), a matrix such as a laminamine, collagen IV and / or Matrigel Matrix, preferably a feeder layer, such as collagen I, 3T3 and / or MRC-5 feeder layer, or a collagen flow.
• a bone replacement material such as tricalcium phosphate, an allogeneic, autologous or xenogeneic acellularized or non-acellularized tissue, such as a heart valve, venous valve, arterial valve, skin, vessel, aorta, tendon, come
• the biological matrix with cells preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and / or fiber tissue, e.g. with adult progenitor cells from the bone marrow, pre-colonized by methods known to those skilled in the art.
• tissue-specific cells preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and / or fiber tissue, e.g. with adult progenitor cells from the bone marrow, pre-colonized by methods known to those skilled in the art.
• adult cells in particular adult cells, i.e. H. primarily differentiated cells, which preferably no longer have an embryonic or fetal phenotype, and particularly preferably human adult cells.
• adult progenitor cells tissue-specific cells, preferably osteoblasts, fibroblasts, hepatocytes and / or smooth muscle cells.
• growth inhibitors such as somatostatin, TGF beta and / or prostaglandins
• the hydrogels or slow-release materials already mentioned which contain at least one of the growth contain or are enriched with hibitors and are applied locally or in the vicinity of the changed cells.
• the method according to the invention is therefore particularly suitable for locally specific and / or directed multiplication, structural growth and subsequent differentiation of adult cells and / or for the regeneration of bones, tissues and / or endocrine organs, e.g. of heart valves, venous valves, arterial valves, skin, vessels, aortas, tendons, comea, cartilage, bones, tracea, nerves, miniscus, intervertebral disk, ureters, urethra or blisters.
• heart valves venous valves, arterial valves, skin, vessels, aortas, tendons, comea, cartilage, bones, tracea, nerves, miniscus, intervertebral disk, ureters, urethra or blisters.
• the method according to the invention can also be used for local application in vivo, in which the growth factors mentioned are used either alone or in combination as a mixture or sequentially or in combination with the biological matrices or support structures mentioned, for example for tissue regeneration, such as, for. B. liver regeneration, heart muscle regeneration or for wound healing in the skin area, e.g. in diabetic ulcers, or gingiva.
• tissue regeneration such as, for. B. liver regeneration, heart muscle regeneration or for wound healing in the skin area, e.g. in diabetic ulcers, or gingiva.
• acute liver failure can be applied locally or systemically via a port using a catheter.
• the growth factors mentioned can thus z. B. be applied before, during or after liver resection or removal of liver tissue to support liver regeneration.
• the growth factors can be injected directly into the knee joint.
• the growth factor (s) can thus act directly on the formation of a new cartilage structure via the synovial fluid.
• the present invention also relates to the use of the growth factors TPO and / or EPO and or GH and / or somatostatin and / or LIF and / or CNTF for the manufacture of a medicament for the treatment of the regeneration of bones, cartilage, tissues and / or endocrine organs, for example parenchymatous and / or non-parenchymatous organs, in particular of heart muscle, heart valves, venous valves, arterial valves, skin, Vessels, aorta, tendons, comea, cartilage, bone, tracea, nerves, miniscus, intervertebral disk, liver, intestinal epithelium, ureters, urethra or blisters, or for the treatment of degenerative diseases and / or to support the wound healing process , in particular in Crohn's disease, ulcerative colitis and / or in the skin area, preferably in diabetic ulcers or gingiva and / or for the treatment of liver diseases, in particular cirrhosis of the liver
• TGF beta transforming growth factor beta
• GM-CSF granulocyte-macrophage-stimulating factor
• GHRH growth hormone releasing hormones
• TRH thyrotropin-releasing homones
• GnRH Gonadotropin-releasing hormones
• CHL Corticotropin-releasing hormones
• ADH Antidiuretic hormones
• oxytocin prolactin, adrenocorticotropin
• beta-celltropin beta-celltropin
• lutrotropin and / or vasopressin used or additionally one or more nerve regeneration factors, preferably "nerve growth factor” (NGF) and / or one or more vascular regeneration factors, preferably "Vascular Endothelial Growth Factor” (VEGF) and / or “Plateled Derived Growth Factor” (PDGF).
• NGF nerve regeneration factors
• VEGF Vascular Endothelial Growth Factor
• PDGF Platinum Derived Growth Factor
• a biological matrix or carrier structure containing at least one of the growth factors TPO, EPO, GH, in particular HGH, somatostatin, LIF and / or CNTF as an inductive substrate for 3-D growth and / or regeneration within a multiplication phase or after a multiplication phase for differentiation or used for growth arrest can be applied to a stent in combination with a so-called slow-release material, as described above by way of example.
• Another object of the present invention is therefore also a biological see matrix or carrier structure containing at least one of the growth factors thrombopoietin (TPO), erythropoietin (EPO) growth factor (GH), in particular "human growth hormone” (HGH), somatostatin, "leukemia inhibitory Factor “(LIF) and / or” Ciliary Neurotropic Factor “(CNTF), the biological matrix or carrier structure also including at least one of the growth factors TGF beta, prostaglandin, GM-CSF, GHRH, TRH, GnRH, CRH, dopamine, ADH , Oxytocin, prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and / or vasopressin and optionally additionally one or more nerve regeneration factors, preferably "nerve growth factor” (NGF) and / or one or more vascular regeneration factors, preferably "vascular endothelial growth factor” (VEGF) and
• the biological matrix or carrier structure represents, for example, an implant, a transplant and / or a carrier material for the growth of cells, the biological matrix or carrier structure being a stent, a catheter, a skin, a hydrogel, a bone substitute material, an allogeneic, auto- log or xenogeneic, acellularized or non-acellularized tissue, a synthetic tissue, a feeder layer or a flow such as e.g. B. a flow of collagen, laminin and / or fibronectin with or without a synthetic or other basic structure, such as. B. plastic or a biological matrix. Exemplary embodiments have already been described above.
• the biological matrix or carrier structure is preferably already populated with tissue-specific cells, progenitor cells, bone marrow cells, peripheral blood, fatty tissue and / or fiber tissue or is already prepared for in vivo colonization or inductive remodeling in vitro.
• the biological matrix or support structure can also be coated with a (bio) polymer layer which contains at least one of the growth factors mentioned.
• a (bio) polymer layer z.
• the present invention also relates to a method for producing a biological matrix or carrier structure according to the invention, in which an optionally activated biological matrix or carrier structure is coated with at least one of the growth factors TPO, EPO, GH, in particular HGH, somatostatin, LIF and / or CNTF , wherein said matrix or carrier structure optionally with at least one of the growth factors TGF beta, prostaglandin, GM-CSF, GHRH, TRH, GnRH, CRH, dopamine, ADH, oxytocin, prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and / or vasopressin and if necessary additionally with one or more nerve stimulation generation factors, preferably NGF and / or one or more vascular regeneration factors, preferably VEGF and / or PDGF can be coated.
• Activation of the biological matrix or support structure can be carried out, for example, by means of plasma ionization, e.g. using hydrogen peroxide, or by means of laser activation.
• biodegradable (bio) polymer layer that contains the growth factor (s) mentioned.
• a biodegradable (bio) polymer layer that contains the growth factor (s) mentioned.
• fibrin, plasma, blood, collagen and / or polylactides are suitable for this.
• the biological matrix or carrier structure can be pre-populated in vitro with cells, preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and / or fiber tissue.
• the present invention also extends to a device for carrying out the method according to the invention, a perfused bioreactor, in particular in the form of a closed system, being preferred.
• a phase-pure beta tricalcium phosphate is with a microporosity of z. B.> 15 microns prepared as granules and shaped into a shape of a 3-D defect according to a patient's needs. This usually happens in a sintering process. The material is then treated with plasma ionization so that the surfaces are activated and the construct is placed in a solution with thrombopoietin, erythropoietin and / or growth hormone (GH) and thus coated in small quantities in a defined manner. Alternatively, incubation in a solution without prior surface activation or coating with a biodegradable (bio) polymer layer that contains these growth factors can take place. Here e.g. Fibrin, plasma, collagen and / or polylactides can be used.
• This construct is then either immediately introduced into a defect or pre-colonized in vitro with tissue-specific cells, precursor cells or bone marrow cells. This ensures that the in vivo wound healing process is anticipated in vitro and thus a shorter reintegration time can take place after implantation in vivo (e.g. after 7 days).
• a combination with factors of nerve regeneration (NGF) or vascular regeneration (VEGF, PDGF) is possible.
• NGF nerve regeneration
• VEGF vascular regeneration
• PDGF vascular regeneration
• a biological matrix (allogeneic or autologous heart valve with and without acellularization, a synthetic carrier structure made of plastics that comes close to the physiological microenvironment of the cardiovascular target tissue with regard to the chemical composition of the collagens and their spatial arrangement) is precoated with thrombopoietin and erythropoietin as growth factors.
• the material is then treated with plasma ionization (e.g. using hydrogen peroxide, H 2 0 2 ), whereby sterilization is achieved at the same time, so that activation of the surfaces occurs and the construct in a solution with thrombopoietin, erythopoietin and / or Growth hormone (GH) is inserted and thus coated in small quantities in a defined manner.
• plasma ionization e.g. using hydrogen peroxide, H 2 0 2
• GH Growth hormone
• incubation in a solution without prior surface activation or coating with a biodegradable (bio) polymer layer that contains these growth factors can take place.
• a biodegradable (bio) polymer layer that contains these growth factors can take place.
• fibrin, plasma, blood, collagen or polylactide can be used here.
• This construct is then either immediately brought to the place of use (cardiac flap position, as a patch or vascular replacement) or pre-colonized in vitro with tissue-specific cells, precursor cells or bone marrow cells.
• the result of this is that the in vivo wound healing process is anticipated in vitro and thus a shorter reintegration time can take place after implantation in vivo (for example after 7 days).
• a combination with factors of nerve regeneration (NGF) or vascular regeneration (VEGF, PDGF) is possible, but not absolutely necessary.
• NGF nerve regeneration
• VEGF vascular regeneration
• PDGF vascular regeneration
• In vivo and in vitro there is integration of the blood-forming and stem cell-rich bone marrow as well as the accelerated differentiation of fibroblasts and smooth muscle cells and an accelerated absorption of the carrier matrix and a replacement by normal cardiovascular tissue. Due to the recruitment competence and the inductive character, a location-specific integration was carried out.
• Urological constructs can be produced in a corresponding manner.
• a mixed liver cell population from a biopsy or a subsectate is mixed with TPO and / or EPO and / or growth hormone, e.g. HGH, in a concentration of 10-50 ng / ml, treated by adding to the medium supernatant.
• the seed cell density is 10,000 cells / cm.
• the cells are treated with 0.005% collagenase and 0.01% trypsin with the addition of 2 g / 1 albumin or autologous serum (10-20%) for 5 h.
• the cells are then suctioned off and washed three times in culture medium (Williams E (Williams et al. (1971) Exptl. Cell Res., 69, 106) with 2 g / l albumin and then brought to sedimentation in a collagen-coated petri dish.
• the differentiation of the cells can be achieved by overlaying with an extracellular matrix.
• the cells can be prevented from sedimentation while moving and come together for aggregation.
• the cells can be guided in a corresponding device over a grid with a cutting mesh structure of 500 ⁇ m in size, so that new daughter aggregates can always be created. This can be done in a completely closed system.
• non-contact pump systems no crushing by peristaltic systems, but directional flows or piston pumps generated magnetically or by compressed air compression of hoses - automatically or manually.
• the cells can then be encapsulated and frozen. Structured forms and spaces can be integrated into the capsule structure, which enables an in situ growth structure and enlargement.
• the capsule can be dispensed with, and the presence of the endothelial cells in this system and the targeted addition of these cells achieve endothelialization and thus optimal hemocompatibility.
• the shear stress in a perfused bioreactor leads to a spontaneous confluence of the endothelial cells on the surfaces of the aggregates.
• they can e.g. be frozen in the bags already ideally used for culture.
• Soft tissue muscle patches, nerves, tendons
• collagen tiles or tiles such as laminin, fibronectin with or without a synthetic or other basic structure, such as e.g. B. plastic or a biological matrix, or spatially defined structures (tubes in nerves, tendons) according to the above be put.
• These collagen tiles or structures are shaped, coated with TPO, EPO and / or growth hormone (GH) and implanted or pre-colonized with cells of the target tissue (e.g. tendocyen, neurons).
• a biological matrix (allogeneic or autologous heart valve with and without acellularization, a synthetic carrier structure made of plastics that comes close to the physiological micro-environment of the target tissue with regard to the chemical composition of the collagens and their spatial arrangement) is precoated with thrombopoietin and erythropoietin as growth factors.
• the material is treated with plasma ionization (e.g. using hydrogen peroxide, H 2 0 2 ), which at the same time achieves sterilization so that the surfaces are activated and the construct is dissolved in a solution with thrombopoietin, erythopoietin and / or growth hormone is inserted and thus coated in small quantities in a defined manner.
• plasma ionization e.g. using hydrogen peroxide, H 2 0 2
• H 2 0 2 hydrogen peroxide
• incubation in a solution without prior surface activation or coating with a biodegradable (bio) polymer layer that contains these growth factors can take place.
• a biodegradable (bio) polymer layer that contains these growth factors can take place.
• fibrin, plasma, collagen and / or polylactide can be used here.
• This construct is then either immediately brought to the place of need (abdominal wall, heart muscle, skeletal muscle as a patch) or pre-colonized in vitro with tissue-specific cells, precursor cells or bone marrow cells. This ensures that the in vivo wound healing process is anticipated in vitro and thus a shorter reintegration time can take place after implantation in vivo (e.g. after 7 days).
• a combination with factors of nerve regeneration (NGF) or vascular regeneration (VEGF, PDGF) is possible, but not absolutely necessary.
• the blood-forming and stem cell-rich bone marrow is integrated in vivo and in vitro, as is an accelerated differentiation of Fibroblasts and smooth muscle cells and an accelerated absorption of the carrier matrix and a replacement by normal cardiovascular tissue. Due to the recruitment competence and the inductive character, a location-specific integration was carried out. This can be further promoted by moving to the external sides with keratin cysts (abdominal muscles), Schwann cells and / or fibrous tissue already in vitro.
• EPO is administered to the patient systemically and / or topically by application to the resection area in conjunction with a polymer.
• the polymer can be a biopolymer such as e.g. Fibrin (from e.g. fibrin glue), polymerized plasma, polymerized blood or bio-adhesives, e.g. Mussel adhesive. But it can also be synthetic or biological gels or hydrogels.
• the EPO can also be placed in flow-control bleeding (e.g. collagen flow, tamponade, woven and knitted fabrics).
• EPO can also be used for liver regeneration in chronic liver diseases such as cirrhosis, fibrosis, hepatitis. Thereby a therapeutic effect with regard to the liver parenchyma can be achieved for the first time.
• the absorption in the regional vascular area can be optimized by the administration of pegylated (PEG) compounds, so that the regional administration in the area of inflammation can have a systemic effect and thus initiate the wound healing process.
• PEG pegylated
• anemia can be seen as a positive prognostic factor in patients with Crohn's disease.
• anemia was an independent concomitant disease, or that it was due to consumption through absorption problems.
• Our results show that the wound healing disorder is a lack of endogenous EPO.
• Crohn's disease can be treated very selectively by exogenous administration of EPO.
• Other areas of application are also in the area of ulcerative colitis.
• EPO e.g. collagen fleece
• EPO can be given in a similar way for all other wound healing requirements, for example in the muscle area after sports injuries, muscle diseases, bone injuries, soft tissue injuries and generally to improve wound healing and tissue regeneration, for example after operations, acute and chronic diseases.

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